Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.Since the first report of wheat transformation by  Transformation of cereal crops is a powerful research tool for gene discovery and function to investigate genetically controlled traits and is fast becoming a key element in the process of varietal improvement. It provides key underpinning knowledge to inform and short-cut conventional breeding strategies. For specific crops, it also enables the introduction of novel genes directly into locally-adapted germplasm and the creation of new genetically modified varieties. As testament to this, a total of 81 million Ha of approved GM crops, mainly for herbicide tolerance or insect resistance, were planted in 2004 [Agrobacterium, including a reduction in transgene copy number, the stable integration with fewer rearrangements of long molecules of DNA with defined ends and the ability to generate lines free from selectable marker genes [Agrobacterium tumefaciens to deliver DNA although the ability to routinely transform wheat in this way is currently restricted to a few, well-resourced public and commercial laboratories worldwide. This is partly due to the need for experienced personnel and expensive laboratory and plant growth infrastructure but also through a lack of clearly-written, complete, publicly-available protocols. There are several research papers and patents describing specific improvements to methodologies but these fail to provide a step-by-step guide to the transformation process as a whole.Wheat was among the last of the major crops to be transformed with the first fertile transgenic plants being reported using particle bombardment little over a decade ago -6. Advaner genes ,11-14. TAgrobacterium strains, resident Ti plasmids and binary vectors used and consider the importance of additional virulence genes. The various inoculation and co-cultivation conditions are discussed and finally the key steps to control the overgrowth of Agrobacterium cells and the selection of regenerating transformed plants are described. We then provide a detailed protocol for the transformation of freshly isolated immature embryos and regeneration of fertile plants in 9–12 weeks.We have compared the published literature under headings that describe the main variables in the transformation process. First, we consider the relatively narrow range of wheat genotypes that have been successfully transformed, the choice of explant and the pre-treatments that were carried out. Second, we compare the et al. [Immature embryos of Bobwhite, pre-cultured for between 1 and 6 days on CM4C medium, are the most commonly used explant -18, althet al.  report net al.  and it iet al. -21. A maAgrobacterium transformation of wheat. Air-drying pre-cultured immature embryos and embryogenic callus explants during Agrobacterium co-cultivation increased T-DNA-delivery and suppressed Agrobacterium cell growth which in turn facilitated better plant cell recovery [Agrobacterium inoculation caused a marked increase of GUS transient expression in pre-cultured rice calli [Various explant pre-treatment steps have been evaluated in attempts to improve T-DNA delivery or tissue-culture response in particular varieties. Osmotic and desiccation treatments have been evaluated and incorporated into protocols based on particle bombardment -26, and recovery . The samrecovery  and sugarecovery . Osmoticce calli  but a plce calli .Agrobacterium strains to transform plant cells is defined by their chromosomal and plasmid genomes which between them must encode all the machinery necessary for attachment and DNA-transfer. The Agrobacterium strains that have been successfully used for wheat transformation are based on only two chromosomal backgrounds, LBA4404 (Ach5) and C58 but these have been used with a wide range of Ti and binary plasmids. Some strains, notably AGL0 and AGL1 have been engineered to contain the so-called hypervirulent Ti plasmid, pTiBo542 harbouring additional vir genes originating from the Agrobacterium strain A281 which in its oncogenic form possesses a broad host range and a induces large, rapidly appearing tumours [Agrobacterium strains demonstrated that AGL0, a hypervirulent strain containing a disarmed pTiBo542 plasmid [vir genes from this plasmid have been widely adopted for monocot transformation vectors . The we plasmid . Further plasmid  enhanced plasmid . Althoug vectors -18. Ther plasmid ,39. Thers roseus  and Norw soybean .Agrobacterium infection process is divided into two stages: first, a short period, typically a few minutes to a few hours  or nptII (aph3'II) which encode phosphotransferase enzymes that confer tolerance to the aminoglycoside antibiotics such as kanamycin, neomycin, paromomycin, G418 and hygromycin. A second system utilizes the bar gene which confers tolerance to glufosinate ammonium-based herbicides such as PPT, Basta, Bialaphos etc. A third system is based on the aroA:CP4 genes conferring tolerance to glyphosate-based herbicides such as Roundup. The use of 0.02 mM glyphosate on regenerating meristems has been reported to reduce the number of plants escaping selection to zero [NptII, bar and aroA:CP4 have been successfully used by different groups to produce transgenic wheat plants but it is not possible to draw direct comparisons between selection systems because often a visual marker was also used in conjunction with chemical selection. For example, in several studies, the GUS reporter gene has been used in addition to the conventional selectable marker to help optimise the identification of transformants [hpt and GFP, along with hygromycin selection, has been used to identify early events in the transformation process [After the co-cultivation period, infected explants progress in a series of tissue culture steps on media designed to inhibit the growth of  to zero . NptII, formants ,21,45. A process .Agrobacterium, the total length of time, from isolation of the original explant to the transfer of young plants to soil, is typically 12–16 weeks depending on the length of pre-culture and the number of selection steps. A shortened protocol taking only 7–11 weeks, achieved mainly by reducing the selection step to one week, has also been reported [bar/glyphosate selection with a GUS assay to confirm T-DNA integration and expression and takes approximately 12 weeks.In wheat transformation via Agrobacterium-mediated plant transformation. Compared to rice and maize, progress with wheat has been slower but as described here, robust methods for the transformation of wheat using Agrobacterium now exist. There is scope to further optimise the media components and pH and to investigate the ideal virulence gene complement. Current bottlenecks limiting throughput include the labour-intensive steps of embryo isolation and transfers between media. Unlike biolistics, Agrobacterium suspensions can be manipulated by liquid handing robots and this combined with the use of callus cultures and the automation of transfer steps would enable a higher throughput which even at low efficiency would allow significantly more transgenic lines to be producedThe advantages arising from simple molecular integrations of single copy DNA fragments with defined ends have driven research into Agrobacterium tumefaciens strain AGL1 [. The binary vector pAL156 contains a single T-DNA incorporating the bar gene conferring Basta resistance and a modified uidA (GUS) gene with an intron within the open reading frame to prevent its expression in Agrobacteium itself. Both the bar and uidA genes are driven by the maize ubiquitin1 promoter plus ubiquitin1 intron [bar gene is located next to the left border, and uidA is adjacent to the right border. A helper plasmid pAL154 provides replication functions for pAL156 in trans and also contains the 15 kb Komari fragment [vir genes. Other Agrobacteium strains and plasmid combinations may also be appropriate in our protocol but have not yet been tested.This method was developed for the winter wheat cultivar Florida but with minor modifications has also been used to successfully transform the spring wheat varieties Fielder and Cadenza. It utilises the super-virulent ain AGL1  containiain AGL1 , . The b1 intron . The barfragment ,47 supplAgrobacterium tumefaciens; 2. induction of embryogenic callus and regeneration of shoots and roots; 3. application of a herbicide selection system to allow only the transgenic plantlets to survive. The average efficiency of transformation  is approximately 1%. The protocol takes 9–12 weeks from the isolation of immature embryos to the potting of putative transgenic plantlets to soil , 0.5 kg PG mix/m3 . Although other soil formulations may also be suitable.1.2 Grow wheat plants in environmentally controlled growth rooms for approximately 11 weeks to provide immature seeds.-2s-1 photosynthetically active radiation (PAR).1.3 Growth rooms are maintained at 18–20°C day and 14–15°C night temperatures with a relative air humidity of 50–70% under a 16 h photo-period provided by banks of 400 W High Temperature Quartz Iodine lamps  which give light intensity ~700 μmolm-2s-1 PAR at 300 mm from the lights.1.4 Before transferring to these conditions, winter wheat varieties are vernalised from seed for 8 weeks at 4–5°C with a 12 hour photoperiod provided by 70 W fluorescent lamps giving approximately 150 μmolm1.5 The water is supplied by an automated flooding system, but seedling-stage plants are initially top watered individually for a few weeks .Agrobacterium liquid cultures by adding ~200 μl of a standard glycerol inoculum to 10 ml MG/L [2.1 Initiate  ml MG/L   for 12–24 hours (to reach an OD >1 (Abs = 600 nm)).Agrobacterium culture at 4500 g for 10 minutes and resuspend in 4 ml single-strength inoculation medium (see 6.2.2) supplemented with 200 μM acetosyringone for each 10 ml culture.2.3 Pellet the 2.4 Replace the cultures back on the shaker until required, but they should be used within 3 hours.Agrobacterium strain and binary vectors used. For the AGL1 strain used in this protocol, carbenicillin (200 mg/l) is used and pAL154/156 combinations are selected with kanamycin (100 mg/l) which is the selectable marker on pAL156.Note, The antibiotics used depend on the selectable markers in the 3.1.1 Collect ears at approximately 12–16 days post-anthesis, a few seeds can be opened at the time of collection to determine the size and texture of the embryos, which should be 0.8 – 1.5 mm in length and translucent in appearance.3.1.2 Surface sterilise by rinsing in 70% (v/v) aqueous ethanol for 1 minute then 15 minutes in 10% (v/v) Domestos bleach solution (Lever) with gentle shaking. Rinse with sterile distilled water at least three times.Note, due to asynchronous development, only half or two thirds of the seeds on any one ear will be suitable, the seeds nearest to the peduncle are generally younger and smaller.3.2.1 Isolate the embryos from the seed under a stereo microscope in a sterile environment using a sharp scalpel.3.2.2 Remove and discard the embryo axis first then isolate the remaining portion of the embryo which is now referred to as the scutellum.3.2.3 Plate scutella with the axis side (now removed) down onto semi-solid inoculation medium in 55 mm Petri dishes, about 50 scutella per plate.Agrobacterium tumefaciens, as described below, before isolating embryos for the next plate.3.2.4 It is important to inoculate each plate of 50 scutella with Agrobacterium suspension from the shaker, add 60 μl 1% Silwet to make a final concentration of 0.015% and pour the whole 4 ml over a batch of 50 plated scutella.4.1 Take the resuspended 4.2 Incubate for 1–3 hours at room temperature while preparing more scutella for inoculation as described in 3.2.4.3 Transfer the scutella without blotting, keeping the ex-axis side down, onto fresh inoculation medium in 55 mm dishes. Allow to co-cultivate in the dark at 22–23°C for 2–3 days.5.1 After 2–3 days co-cultivation, transfer all scutella to induction medium Table  and cont5.2 After 18 days, transfer embryogenic calli to RDZ medium Table , and inc5.3 After 3 weeks, transfer embryogenic calli to selection medium RPPT  and autoclaved at 121°C for 20 min :4NO3 ,16.5 g/l NH3 ,19.0 g/l KNO2PO4 (Fisher Scientific UK),1.7 g/l KH4·7H2O (Fisher Scientific UK),3.7 g/l MgSO2·2H2O (Fisher Scientific UK).4.4 g/l CaClNote, Dissolve each component in distilled water separately before mixing. Autoclave at 121°C for 20 min and store at 4°C.6.2.4 L7 Microsalts (×1000):4 (Fisher Scientific UK),15.0 g/l MnSO3BO3 (Fisher Scientific UK),5.0 g/l H4·7H2O (Fisher Scientific UK),7.5 g/l ZnSO0.75 g/l KI (Fisher Scientific UK),2MoO4·2H2O ,0.25 g/l Na4·5H2O ,0.025 g/l CuSO2·6H2O (Sigma-Aldrich).0.025 g/l CoCl4 may have various hydrated states which will alter the required weight. For MnSO4·H2O, add 17.05 g/l, for MnSO4·4H2O, add 23.22 g/l, for MnSO4·7H2O, add 27.95 g/l. Prepare 100 ml microsalt stock solution at a time. Filter sterilise, and store at 4°C.Note, MnSO6.2.5 MS Vitamins (-Glycine) (×1000):0.1 g/l Thiamine HCl (Sigma-Aldrich),0.5 g/l Pyridoxine HCl (Sigma-Aldrich),0.5 g/l Nicotinic acid (Sigma-Aldrich).Prepare 100 ml at a time. Filter sterilise, and store at 4°C.6.2.6 Vitamins/Inositol (×200):40.0 g/l Myo-Inositol (Sigma-Aldrich),2.0 g/l Thiamine HCl (Sigma-Aldrich),0.2 g/l Pyridoxine HCl (Sigma-Aldrich),0.2 g/l Nicotinic acid (Sigma-Aldrich),0.2 g/l Ca-Pantothenate (Sigma-Aldrich),0.2 g/l Ascorbic acid (Sigma-Aldrich).Filter sterilize and store at -20°C in 10 ml aliquots.6.2.7 Supplements• Acetosyringone  , Dissolve in 70% ethanol to give 10 mg/ml or 50 mM stock solution. Filter sterilise, aliquot and store at -20°C.• 2,4-Dichlorophenoxyacetic acid  (Sigma-Aldrich), 1 mg/ml in ethanol/water (dissolve powder in ethanol then add water to volume). Filter sterilise, and store at -20°C in 1 ml aliquots.• Zeatin mixed isomers (10 mg/ml) (Sigma-Aldrich), Dissolve powder in small volume 1 M HCl and make up to volume with water, mix well/vortex. Filter sterilise, and store at -20°C in 1 ml aliquots.• Picloram (1 mg/ml) (Sigma-Aidrich), Dissolve picloram in water, filter sterilise and store at -20°C in 2 ml aliquots.• Timentin (300 mg/ml) , Dissolve Timentin (Ticarcillin/Clavulanic (15:1)) in water, filter sterilise and store at -20°C in 1 ml aliquots.• PPT (10 mg/ml)(Glufosinate Ammonium) , Dissolve in water, mix well/vortex, filter sterilize, and store at -20°C in 1 ml aliquots.• Silwet L-77 (1% v/v) , Dissolve in water, filter sterilize, and store at 4°C in 0.5 ml aliquots.The author(s) declare that they have no competing interests.


ALAD) single-nucleotide polymorphisms (SNPs) to determine if these SNPs could modify the relationship between blood lead (PbB) and some renal parameters. This is a cross-sectional study of 276 lead-exposed workers in Vietnam. All workers were measured for PbB, urinary retinol-binding protein (URBP), urinary α1-microglobulin (Uα1m), urinary β2-microglobulin (Uβ2m), urinary N-acetyl-β-d-glucosaminidase (NAG), urinary aminolevulinic acid (ALAU), serum α1-microglobulin (Sα1m), serum β2-microglobulin (Sβ2m), and urinary albumin . The six SNPs were Msp and Rsa in exon 4, Rsa39488 in exon 5, HpyIV and HpyCH4 in intron 6, and Sau3A in intron 12. Analysis of covariance (ANCOVA) with interaction of PbB × SNPs were applied to examine modifying effect of the SNPs on the association of renal parameters and PbB, adjusting for potential confounders of age, gender, body mass index, and exposure duration. HpyCH4 was found to be associated with certain renal parameters. For HpyCH4 1-1, an increase of 1 μg/dL PbB caused an increase of 1.042 mg/g creatinine (Cr) Uα1m, 1.069 mg/g Cr Uβ2m, 1.038 mg/g Cr URBP, and 1.033 mg/g Cr Ualb, whereas in HpyCH4 1-2, an increase of 1 μg/dL PbB resulted in an increase of only 1.009 mg/g Cr Uα1m, 1.012 mg/g Cr Uβ2m, 1.009 mg/g Cr URBP, and 1.007 mg/g Cr Ualb. HpyCH4 SNP appeared to modify the lead toxicity to kidney with wild-type allele being more susceptible than variants. The mechanism for this effect is not clear. Further studies are needed to confirm this observation.We examined six newly identified polymorphisms in the δ-aminolevulinic acid dehydratase ( ALAD) polymorphism studied was the Msp single-nucleotide polymorphism (SNP) in exon 4. Since it was first reported . The differences achieved only borderline significance after adjusting for potential confounders. Msp-2 carriers than for Msp 1-1 homozygotes in a sample of 89 lead workers. A linear relationship was observed between patella lead concentration, using X-ray fluorescence method, and serum uric acid when patellar lead passes 15 μg/g bone mineral for Msp-2 carriers, but the similar linear relation can be seen only when patella lead was > 101 μg/g bone mineral for the Msp 1-1 group ] representing proximal tubule injury are considered good alternatives because early lead-induced nephropathy usually involves damage of proximal tubule cell (1-microglobulin (Uα1m) can be a useful indicator of renal impairment. A study of 128 lead workers (2-microglobulin (Uβ2m) and urinary retinol-binding protein (URBP). Another study of environmental lead exposure in children showed that URBP is increased by lead with good dose–effect and dose–response relations with PbB  to test for linkage disequilibrium among the 46 SNPs; 6 SNPs were shown not to be in linkage disequilibrium. In this study we examined these 6 SNPs  and examined their association with certain renal functions among a group of lead-exposed workers in Vietnam.To date, 111 SNPs have been reported in the National Center for Biotechnology Information website . But wheThe study population consisted of 323 workers from a battery factory in Hai Phong City, Vietnam. All workers from the production line as well as in the division of management and quality control were recruited into this study. Of these 323 workers, 246 were occupationally exposed to lead, and the remaining 77 were not directly exposed to lead. Although these 77 workers were not directly exposed to lead, many had a previous history of exposure. Some were still exposed, albeit less than the 246 group. Therefore, these workers were also included in the study. Signed consent was obtained for each worker before blood and urine samples were taken for subsequent analysis. The workers also filled out a questionnaire (in Vietnamese) with the help of a Vietnamese interviewer. After completing the questionnaire, a spot urine sample and 10 mL of blood were collected from each worker during the medical examination. Twenty of the exposed lead workers were not present during the study period, and 17 did not want to participate. Of the 77 workers who were not directly exposed to lead, 10 were unwilling to give their urine and blood for analysis. Because the study was strictly on a voluntary basis, the workers’ decisions in not giving urine and/or blood were respected. Hence, the 47 workers were excluded in the study, giving a response rate of 88.2% (276 of 323).Information gathered included age, years of education, detailed occupational history, and current and previous smoking habits. Alcohol intake was also documented carefully, as it could confound the findings. Actual amount of alcohol consumption per day  and number of years of drinking were noted. Six workers gave a history of diabetes mellitus and/or hypertension. These workers were excluded from the study because diabetes mellitus and hypertension are known to affect renal functions.We obtained blood samples by venipuncture with lead-free disposable syringes and stored the samples in heparinized lead-free polypropylene tubes. PbB was analyzed using atomic absorption spectrophotometry  with a graphite furnace. External quality control was performed yearly under the National External Quality Assurance Scheme (England) and Inter-laboratory Comparison Programme (Canada). We obtained spot urine samples, and those used for analysis of ALAU, URBP, Uα1m, Uβ2m, urinary albumin , and NAG were buffered (pH 7.2). Serum and urine samples were stored at –30°C until analysis. We analyzed all samples within 1 month of arrival in Singapore at our laboratory and adjusted all urine parameters for variability in urine flow using urinary creatinine that was expressed per gram of creatinine. Urine creatinine concentrations were determined using standard laboratory techniques. Ualb, ALAU, URBP, Uα1m, Uβ2m, and NAG were measured by enzyme-linked immunosorbent assay using commercially available polyclonal antibodies or test kits . The details of these tests have been reported previously . NAG wasALAD gene were selected for analyses in our study. They are MspI and RsaI polymorphisms, which span a 248-bp region of the genomic ALAD sequence, as well as Rsa39488 (232 bp), HpyIV (234 bp), HpyCH4 (213 bp), and Sau3AI (282 bp) polymorphisms. The products of interest were determined through amplification by polymerase chain reaction (PCR), using primers designed with Primer3 . The amplification cycles were performed on the PTC-100 MJ Thermal Cycler  and optimized to ensure specific amplification of the products of interest. Each PCR reaction was performed in a final volume of 25 μL, which included 10 ng of pure genomic DNA, 1.0 μM each primer, 200 μM each dNTP, 1× Taq buffer, 1.5 mM MgCl2, and 1.0 U of Taq DNA polymerase , and topped up to the required volume with sterile distilled water. We used the restriction fragment length polymorphism method to locate the site of mutation; the fragments were determined on a 2.0% agarose gel  and visualized on the Biorad Gel Doc 2000 system.Six polymorphisms located on the We determined that 28 people did not have any genotype measurements and had to exclude them from the analysis, leaving a sample of 248 workers. The data set of these 248 workers was used in all subsequent analyses. For some of the biologic samples, we were not able to analyze all the parameters because these samples ran out before we could complete the test.1-microglobulin (Sα1m), and serum β2-microglobulin (Sβ2m) went through logarithmic transformation. NAG went through square root transformation. The transformed variables all follow normal distribution with an insignificant p-value from the one-sample K-S test.The distributions of variables were checked individually with histogram and one-sample Kolmogorov-Smirnov (K-S) test for normality. Necessary transformation was undertaken to achieve normal distribution for all important variables. In the end, ALAU, Uα1m, Uβ2m, Ualb, URBP, serum αa) Is there any difference in the mean PbB concentrations and renal parameters between genotypes of each SNP after adjusting for known confounders? b) Within each single SNP, is the effect of PbB concentration on the renal parameters similar across genotype subgroups? Analysis of covariance  and PbB concentration (x-axis) to identify linearity and extreme points.Statistical analysis addressed two issues:  ANCOVA;  was the p < 0.05 (two-sided).We addressed the first issue using ANCOVA to adjust for age, exposure duration, body mass index (BMI), PbB (where appropriate), and gender. For the second issue, an interaction term of PbB × SNP was included while adjusting for the known confounders. Multiply linear regression equation models were used in the generation of regressions lines for Msp, Sau3A, HpyIV, and HpyCH4, the wild-type allele is more predominant, whereas for Rsa and Rsa39488, the variant allele is more prevalent. The basic characteristics of the measurement variables are shown in HpyIV variant group had higher mean ALAU and Ualb levels than the group with the wild-type genotype . The wild-type genotype group for the Sau3A marker (1-1) had lower ALAU levels compared with those of the variant group (0.93 vs. 1.08 mg/g Cr) but higher NAG concentrations (2.93 vs. 2.53 U/g Cr). Only Rsa39488 subgroups had significant differences in PbB, with means of ALAD 1-1, ALAD 1-2, and ALAD 2-2 being 21.87 μg/dL, 20.43 μg/dL, and 25.54 μg/dL, respectively. The group with the Rsa39488 variant genotype, 2-2, had much higher PbB levels than the 1-1 and 1-2 genotype groups  were male and 64 (25.8%) were female, with a mean age of 39  years. The range of exposure duration was 1–41 years. Demographic features by SNP are summarized in e groups .Multiple linear regression equation models were constructed for each of the six SNPs. Of these six SNPs, only HpyCH4 showed significant association with URBP, Uα1m, and Uβ2m in the multiple regression analysis. The increment in renal parameters corresponding to unit increase in PbB concentration is higher for HpyCH4 1-1 homozygotes than for HpyCH4 1-2 workers. For Uα1m, each increase of 1 μg/dL PbB corresponds to an increase of 0.016 log Uα1m (anti-log 1.042 mg/g Cr) for HpyCH4 1-1 workers, whereas for HpyCH4 1-2 workers the increase is 0.004 (anti-log 1.009 mg/g Cr). The same trend is shown in the relations of Uβ2m, URBP, and Ualb to PbB concentrations. The contrast between HpyCH4 1-1 and HpyCH4 1-2 is 1.069 versus 1.012 mg/g Cr, 1.038 versus 1.009 mg/g Cr, and 1.033 versus 1.007 mg/g Cr for Uβ2m, URBP, and Ualb, respectively.For ALAD SNPs, this study is the first to address more than one SNP. In addition to Msp in exon 4, we also examined Rsa SNP in exon 4, Rsa39488 in exon 5, HpyCH4 and HpyIV in intron 6, and Sau3A in intron 12. It has been shown by linkage analyses that these six SNPs are not in linkage disequilibrium.We did not find any correlation of lead with Sα1m or Sβ2m, which is consistent with other studies . In our Although we determined some positive findings with Rsa, Rsa39488, HpyIV, and Sau3A, our findings were not consistent. Therefore, these SNPs are not discussed further; we concentrate our discussion on HpyCH4 and its association with renal function. HpyCH4 is a SNP that involves a G/C transversion in gene position 12,916 in intron 6. This SNP was first reported by Several studies have shown that URBP , Uα1m C, Uβ2m N, and uriOur study found that a newly identified ALAD polymorphism, HpyCH4 in intron 6, was able to modify the association of PbB concentrations with certain renal parameters. For HpyCH4 1-1 homozygotes, 1 μg/dL PbB caused an increase of 1.042 mg/g Cr Uα1m, 1.069 mg/g Cr Ub2m, 1.038 mg/g Cr URBP, and 1.033 mg/g Cr Ualb, whereas in HpyCH4 1-2 heterozygotes, an increase of 1 μg/dL PbB resulted in an increase of only 1.009 mg/g Cr Uα1m, 1.012 mg/g Cr Uβ2m, 1.009 mg/g Cr URBP, and 1.007 mg/g Cr Ualb. These findings suggest that the HpyCH4 1-2 variant could be more resistant to the effects of lead toxicity on some renal functions than HpyCH4 1-1.We showed that in the relationship between PbB and some renal parameters –4, two rFor the Msp polymorphism, the differences noted in relation to the susceptibility of effects of lead have been attributed to the differential binding abilities of the ALAD isozymes. However, the HpyCH4 SNP, located in the intron region of the ALAD gene, technically does not have any protein-coding functions involved. Some limitations are inherent in this study. It is possible that individuals with certain ALAD polymorphisms may be more susceptible to the effects of lead toxicity on the renal system and thus would have been excluded from the workforce even before this study was conducted . But these susceptible workers will need to have obvious clinical renal diseases to be removed from the workforce. This is first time that factory workers in Vietnam were tested with these renal tests. Therefore, it is highly unlikely that we are dealing with a healthy worker effect. In Vietnam, most factories are state owned, as is this factory. Workers work in a factory all their lives  parameters than workers with the HpyCH4 1-1 genotype. The sample size of our study is also not large. There were only 25 workers with HpyCH4 1-2 compared with 191 workers with HpyCH4 1-1. Cadmium is also known to affect some of the renal parameters that were measured in this study . It has HpyCH4 SNP located in intron 6 may have modifying effects on the relationship between lead exposure and renal function, with individuals carrying the HpyCH4 1-1 genotype being more susceptible to lead toxicity of the kidneys. The mechanism for this is unclear. Further studies are needed to confirm this observation.In conclusion,


Leishmania in dogs, using histological (HE), immunohistochemical (IHC) and polymerase chain reaction (PCR) techniques.We compared skin biopsy samples from different anatomical regions for detecting The sensitivity was 82.8 percent for PCR, 62.1 percent for IHC and 44.8 percent for HE. These methods do not appear to depend on the clinical status of the animal or the anatomical source of the skin sample; there is no "best region" for any method. However, PCR was more effective than IHC and HE for ear and nose skin samples whereas IHC was better than HE for nose samples. There was weak agreement between PCR and HE for all tissue samples; good agreement between PCR and IHC for ear and abdomen samples, and weak agreement for nose; and optimal agreement between IHC and HE for ear and abdomen and good agreement for nose samples.The PCR on ear skin could be the best procedure for diagnosing canine visceral leishmaniasis. The good agreement between PCR and IHC indicates that IHC can be used as an alternative method. Finally, tissue samples from ears, nose and abdomen, particularly ears and nose, are potentially useful for diagnosing canine visceral leishmaniasis independently of the animal's clinical status. Leishmania infantum (syn. L. chagasi) , where it constitutes a significant public health problem. The disease in this region is basically a zoonosis; humans are only incidental hosts in the life cycles of the various pathogenic parasite species . In Amerchagasi) , and domentified ,12,21. Centified .Leishmania species and their diverse clinical manifestations, and by the fact that each parasite species has a unique epidemiological pattern. Since a combined risk exists for both canine and human infections in areas where ZVL is endemic, there is a need for sensitive and specific diagnostic techniques AGGGGCGTTCT [G/C]CGAA-3' and 5'- [G/C] [G/C] [G/C) (A/T]CTAT [A/T]TTACACCAACCCC-3'). A 120-base pair (bp) PCR product was generated . Reactions were carried out in volumes of 10 μl containing 1.0 μg of DNA, 0.2 mM dNTPs, 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 5 pmol of each primer and 1 U Taq polymerase. The conditions for PCR amplification were as follows: initial denaturation at 94°C for 5 min, 30 cycles consisting of denaturation at 94°C for 1 min, annealing at 65°C for 1 min and extension at 72°C for 1 min, and final extension at 72°C for 5 min. The reaction mixtures were cycled in a Perkin-Elmer GenAmp 9600 thermocycler. In all assays, a positive control containing L. chagasi (MHOM/BR/1972/BH46 strain) genomic DNA and a negative control without DNA were included. Following amplification, the samples were submitted to electrophoresis on 6% polyacrylamide gel and silver-stained. The DNA of bacteriophage φX 174 cleaved by HAE III was used as a molecular marker .Initially, the samples were washed in ethanol then xylol to dissolve the paraffin, then DNA was extracted from a skin specimen measuring approximately 1.0 mmp value of less than 0.05 was considered significant. As a measure of agreement, index Kappa (κ) was also evaluated between PCR × IHC, PCR × HE and HE × IHC. We accepted κ <0.4 as weak agreement, 0.7> κ >0.4 as good agreement and κ > 0.7 as optimal agreement. The sensitivity of the test was determined using optical microscopy (OM) as the gold standard  for HMA, WLT and WLT have made substantial contributions to conception and design, analysis and interpretation of data


FLGO sheets with a starting C/O ratio of 2.5 are reduced by SCW gasification of glycerol to rFLGO with a C/O ratio of 28.2, above those reported for hydrazine-based methods. Additionally, simultaneous glycerol gasification resulted in the concurrent production of H2, CO, CH4 and valuable hydrocarbons such as alkylated and non-alkylated long chain hydrocarbon (C12–C31), polycyclic aromatic hydrocarbons (PAH), and phthalate, phenol, cresol and furan based compounds.A sustainable and effective method for de-oxygenation of few-layer graphene oxide (FLGO) by glycerol gasification in supercritical water (SCW) is described. In this manner, reduction of FLGO and valorization of glycerol, in turn catalyzed by FLGO, are achieved simultaneously. The addition of glycerol enhanced FLGO oxygen removal by up to 59% due to the in situ hydrogen generation as compared to the use of SCW only. Physicochemical characterization of the reduced FLGO (rFLGO) showed a high restoration of the sp Graphenoperties ,4, make operties ,6,7. Theoperties .2-conjugated graphene network disrupted by oxygenated functionalities, such as hydroxyl and epoxide groups in basal planes and carboxylic and carbonyl groups on the edges [Oxidative exfoliation methods are top-down chemical approaches based on conventional methods initially intended for the oxidation of graphite, i.e., Brodie , Staudenhe edges ,16,17, ahe edges ,18 and che edges ,14 GO rehe edges . Chemicahe edges . Howeverhe edges ,21. Commhe edges ,24,25,26he edges ,31,32,33he edges  reductio2 network. In fact, the removal of oxygenated groups can be improved by increasing water pressure and temperature to near- or supercritical conditions (374 °C and 22.1 MPa). At supercritical conditions, water density, ionic product and dielectric constant are reduced while its diffusivity increase, which make it a highly reactive and homogeneous medium for reactions of heterolytic (ionic) bond cleavage [N,N-dimethylformamide (DMF) [N-methyl-pyrrolidone (NMP) [2 [Hydrothermal methods, employing reductant-free superheated water at low temperatures (160–220 °C), have achieved a partial removal of oxygenated groups, reaching a C/O atomic ratio reduction from 2–4 in the starting GO  to 5.3–5cleavage . Supercrcleavage ,37,38 ancleavage ,41,42,43de (DMF) , N-methyne (NMP) , or CO2 (NMP) [2 , have be(NMP) [2  observed2CO3, H2SO4, K3PO4, K2CO3, KOH or NaOH have been used to improve the carbon to gas conversion and the yield to hydrogen by means of the water-gas shift reaction . Heterogeneous catalysts based on transition metals such as Ni, Ru, Pt, Co, Cu or Zn [In this communication, an unexplored sustainable approach for GO reduction via supercritical water gasification (SCWG) of glycerol is reported by the first time. Glycerol is a valuable green compound generated as a by-product of the biodiesel production process (at the ratio of 1 ton per 9 tons of biodiesel), which presents low toxicity, very low price, large availability and renewability . The proCu or Zn ,70,71,72Cu or Zn , offeredIn this work, the sustainable reduction of few-layer graphene oxide sheets (FLGO) with a starting C/O ratio of 2.5 is studied by using glycerol in SCW at 400 and 500 °C. In this manner, reduction of FLGO and valorization of glycerol, in turn catalyzed by FLGO, are addressed simultaneously.2 adsorption, thermogravimetric analysis (TGA) and X-ray diffraction (XRD), respectively  analysis based on Nectively .2 physisorption shown in 0 = 0.45, and evolved from H2 type in the case of FLGO to a combination of H2 and H3 types for reduced samples; both correspond to complex pore structures in which network effects are important. The drop after a saturation plateau in the desorption branch at p/p0 = 0.55–0.6 was greater in the case of H2 type hysteresis, and it is attributed to pore blocking in pore necks [2 to a clean graphene surfaces due to oxygen groups removal. rFLGO samples showed an inner interlayer stacking more accessible to gas than starting FLGO where oxygenated functional groups cause blockage of the pores. In FLGO, microporosity (26% of the BET surface area) and narrow mesoporosity (<5 nm) are mainly ascribed to the accessible cuneiform pores between the graphene layers (slit type pores). On the other hand, G/W-rFLGO samples presented a remarkable increase in surface area, but lower than that achieved with SCW only. As will be further discussed, the largest reduction of GO by glycerol gasification produced higher graphene layer restacking and folding of the starting FLGO with consequent loss of porosity. 2 g−1 and 0.5 cm3 g−1 of micropore surface area and total pore volume, respectively, compared to 11 m2 g−1 and 0.035 cm3 g−1 in the starting FLGO (see 2 g−1) [Based on the results of Nre necks . Furtherre necks . The BETFLGO see . In addie 2 g−1) .−1 under N2 atmosphere. A 25 wt. % weight loss was registered before 300 °C for FLGO, and it is attributed to the release of water molecules intercalated between graphene oxide layers (up to 100 °C) and the evolution of labile oxygenated groups to CO and CO2 from 150 °C onwards [The thermal stability of the samples was analyzed by TGA.  onwards . After 3 onwards .d-spacing = d002 = 0.336 nm) shifted to 10.7° in FLGO, corresponds to a d-spacing of 0.830 nm commonly attributed to GO. Supercritical reduction recovered the (002) peak at 25.7–26.3° at the expense of shifted (002) driven by the approach of graphene layers after the removal of intercalated oxygenated groups. The contribution of two peaks is observed in some XRD patterns ) × 100%) [2 physisorption (see cL/d002 + 1) from 4 in starting FLGO (cL = 3.1 nm) to ca. 10 layers in rFLGO (cL = 3.4–4.4 nm).XRD patterns of the FLGO compared to those of the rFLGO are depicted in ectively . This stectively . The gra × 100%) . As can  × 100%) ,79,80,81tion see . The red2 graphene network restoration.TEM images in 2 graphitic carbon (C=C) and sp3 hybridized carbon (C–C) are combined in an asymmetric peak at 284.5 eV [2-carbon recovery and the inherent appearance of the π-π* transition. Consequently, there were remarkable increases of the C/O ratio for W-rFLGO and G/W-rFLGO. In addition, SCW treatment and to an even further extent glycerol SCWG removed both the in-plane oxygenated groups (hydroxyls and epoxides) and out-plane groups (carbonyls and carboxyls). Focusing on the temperature effect, at 500 °C higher C/O ratios were achieved with and without glycerol. Contrasting now the composition values obtained by EDX, oxygen contents were somewhat lower than those calculated by XPS, since the latter is a surface technique and oxygen is more abundant on the surface of the graphene/graphite stacks. The difference in oxygen content is greater when there is an excessive restacking of the reduced graphene-oxide layers  were used to quantify the atomic composition of FLGO and rFLGO samples. Three components were deconvoluted by Gaussian/Lorentzian (40:60) fitting from XPS C1s high-resolution spectra as shown in 284.5 eV , C–O bon2, CO, CO2 and CH4 in varying proportions. As expected, higher gas formation was obtained at higher temperature (500 °C), due to the increased production of H2, CO2 and CH4. On the contrary, CO formation decreased at 500 °C. Increased gas production was originated by SCWG of glycerol. The product distribution was also determined by the WGS reaction, which leads to additional H2 and CO2 by CO consumption [4, in turn, is produced by pyrolysis of glycerol intermediates or by methanation reactions (CO + 3H2 ↔ CH4 + H2O) [2 and CO2 due to H2 consumption in the reduction of the FLGO. On the contrary, the methanation reaction towards the formation of CH4 would not be favored initially. Glycerol gasification (and hence hydrogen production) could be improved by using shorter residence times, lower concentrations of glycerol in the feed and/or higher operating temperatures [Gas composition after reaction was analyzed by gas chromatography to evaluate the gas obtained in reduction of FLGO in water or glycerol/water under supercritical conditions, as shown in sumption . In addiperature 0 °C, duesumption . CH4, in4 + H2O) . Heterog4 + H2O) ,66,67,69eratures ,66,70, b2 and CO2 as a result of FLGO gasification and evolution of oxygen-containing functional groups in FLGO [4 were also measured, latter indicates that methanation would not be favored under these conditions. Note that SCW in the absence of FLGO did not produce gas. Hence, H2 produced from glycerol and, presumably, the self-generated overpressure would improve FLGO reduction, contributing to the differences between G/W-rFLGO and W-rFLGO observed in the physicochemical characterization and C/O ratios obtained.In tests with SCW only (leading to W-rFLGO), gas formation was much lower than when glycerol was used and gas products were mainly H in FLGO ,87. Low 2 atmosphere enhanced the GO reduction [2 consumption in FLGO reduction simultaneous with SCWG of glycerol was ca. 0.41 mmol per mmol−1 of oxygen initially present in of FLGO at both temperatures , and therefore there was a H2 surplus, especially at 500 °C. This surplus could potentially be used to develop a process that couples FLGO reduction with hydrogen production, which would imply a further improvement towards larger scale operation in a continuous reactor than reduction in SCW alone, although residence times should be shortened in order to make the operation viable. In addition to the gases from glycerol and FLGO gasification, liquid products were collected  [3 during 30 min at boiling temperature to eliminate metal particles  and generate oxygenated surface groups, which have a relevant importance in its subsequent unzipping [2·g−1. In 3 (purity ≥ 99.0%), and 69 mL of H2SO4 (96%) were stirred together in an ice bath. 13.8 g of KMnO4 was slowly added to the mixture under vigorous stirring to obtain a ratio of KMnO4 to as prepared MWCNT according to the optimum oxidation ratio (OR) obtained in a previous work OR = KMnO4/MWCNT = 10  [4, temperature was kept below 20 °C during mixing and then the solution was stirred at 30 ± 5 °C for 2 h and at room temperature overnight. After oxidation, a brownish thick paste was formed. Then, 120 mL of deionized water was slowly added while maintaining the temperature below 70 °C. Subsequently, the solution was stirred for 60 min and was diluted with 300 mL of deionized water. A volume of 14 mL of H2O2 (33%) was added dropwise, turning the solution colour to yellowish brown. Material exfoliation was carried out by sonication for 4 h. The product was washed by centrifugation at 9500 rpm with HCl (10%) and deionized water until neutral pH. GO-based suspension with a concentration of 13.2 mg/mL was separated from unreacted MWCNT and coarse particles as supernatant at 1000 rpm and cycles of 10 min. Finally, FLGO were dried at 65 °C overnight. After the oxidation/exfoliation method FLGO powder was completely free of impurities , as confirmed by inductively coupled plasma-optical emission spectrometry (ICP-OES) (not shown).A suspension of FLGO sheets was obtained by unzipping of MWCNT previously produced by catalytic decomposition of methane in a rotary bed reactor ,92. Brie.1:31.6) ,92. Prionzipping . These cFor the reduction method presented here, FLGO sheets from MWCNT unzipping were used; however, this reduction method may also be applied for any graphene oxide material, including those obtained by the conventional graphite oxidation process. To perform reduction reactions a stainless steel microbomb batch reactor was used (a detailed diagram is shown in the 3/H2O 1:1 v/v. rFLGO were separated from the liquid products by centrifugation at 3800 rpm and then dried at room temperature under an inert gas flow (N2 oxygen free). After solvent removal by evaporation in N2 at room pressure and temperature, liquids were analyzed by gas chromatography-mass spectrometry (GC-MS).In a typical run, 0.130 g of dried FLGO were dispersed in a proper volume of glycerol/water solution  and placed in the stainless steel tee before attaching it to the rest of the reactor. The reactor was purged with He and closed at ambient pressure to ensure an oxygen free atmosphere to start the experiment. Volumes of 4 and 2.9 mL of glycerol/water solution guaranteed supercritical water conditions during reaction at 400 and 500 °C, respectively, resulting in a pressure of 230 bar. The corresponding water pressure in the system was calculated using data from National Institute of Standards and Technology (NIST) . During d-spacing), d002, or mean crystallite size along c axis (transverse to the graphene planes), cL. The mean interlayer spacing was evaluated from the position of the (002) peak applying Bragg’s Law [K = 0.89 [n, was calculated as (cL/d002) + 1 [X-ray diffraction (XRD) of FLGO and rFLGO were acquired in a Bruker D8 Advance Series 2 diffractometer . The angle range scanned was 4–55° using a counting step of 0.02° and a counting time per step of 4 s. XRD data were fitted using the structure analysis software TOPAS  including subtraction of a Chebyshev polynomial background and peak deconvolution by fitting to a split pseudo-Voigt functions. The graphite peak fit gave information on structural parameters such as interlayer spacing  + 1 .hv = 1253.6 eV) and under vacuum (<5 × 10−9 Torr). Adventitious carbon is removed by argon sputtering . Analyser pass energy of 50 eV was used for survey scans and 20 eV for scans of C1s region. For calibration purposes, the C1s binding energy of the graphitic peak (BE) was referenced at 284.5 eV. A survey scan (1 sweep/200 ms dwell) was acquired between 1200 eV and 0 eV. The CASA XPS data processing software allowed smoothing, Shirley type background subtraction, peak fitting and quantification.X-ray photoelectron spectroscopy (XPS) analyses were carried out with an ESCAPlus OMICROM system equipped with a hemispherical electron energy analyser. The spectrometer was operated at 15 kV and 15 mA (225 W), using a non-monochromatic MgKα X-ray source  using a Tecnai F30 (FEI) microscope, equipped with a cannon of 300 KeV and a maximum resolution of 1.5 Å. In addition, an energy dispersive X-ray (EDX) analyzer Röntec XFlash Si(Li) coupled to a Hitachi S-3400N scanning electron microscope (SEM) was used to study the bulk composition of the samples.−1 within a temperature range from room temperature to 1000 °C under a flow rate of 50 mL min−1 of N2.Thermogravimetric analysis (TGA) was carried out in a NETZSCH TG 209 F1 Libra thermobalance . TGA profiles were obtained from a sample amount of 8 mg and using a heat rate of 5 °C minGas chromatography with mass spectrometry analysis of the liquid product was performed in a Varian Star 3400/Saturn 2000 GC-MS equipped with a non-polar HT-5 (25 m × 0.32 mm) column from SGE . The products were identified by comparison with the spectrum found in the instrument library. Gas products were analysed in a Perkin Elmer Clarus 500 gas chromatograph fitted with a thermal conductivity detector (TCD). The GC-TCD was equipped with a Carboxen 1010 plot capillary column (30 m × 0.53 mm).2-conjugated graphene network was restored through the removal of basal oxygenated groups. In addition, H2, CH4, CO2 and CO gases were recovered. The H2 produced by glycerol gasification enhanced the FLGO reduction in SCW, achieving greater oxygen removal than the recently reported process of reduction by SCW or SCF. Simultaneous glycerol gasification could be used to reduce FLGO with the concurrent production of H2, CO, CH4 and valuable hydrocarbons.A sustainable and effective method to reduce GO-based materials using simultaneous glycerol gasification in SCW was demonstrated. Glycerol is a byproduct of biodiesel production and can revalue through use in continuous processes as reduction reactions in supercritical conditions. In this manner, reduction of FLGO and valorization of glycerol have jointly been achieved, obtaining G/W-rFLGO by SCWG of glycerol at 500 °C with a C/O ratio of 28.2, well above those of the traditionally hazardous hydrazine-based methods (C/O ~ 10.3–11.5). In this way, it was demonstrated that the sp


ATGs) in the wheat pathogenic fungus Fusarium graminearum. We identified twenty-eight genes associated with the regulation and operation of autophagy in F. graminearum. Using targeted gene deletion, we generated a set of 28 isogenic mutants. Autophagy mutants were classified into two groups by differences in their growth patterns. Radial growth of 18 Group 1 ATG mutants was significantly reduced compared to the wild-type strain PH-1, while 10 Group 2 mutants grew normally. Loss of any of the ATG genes, except FgATG17, prevented the fungus from causing Fusarium head blight disease. Moreover, subsets of autophagy genes were necessary for asexual/sexual differentiation and deoxynivalenol (DON) production, respectively. FgATG1 and FgATG5 were investigated in detail and showed severe defects in autophagy. Taken together, we conclude that autophagy plays a critical role in growth, asexual/sexual sporulation, deoxynivalenol production and virulence in F. graminearum.Autophagy is a conserved cellular recycling and trafficking pathway in eukaryotic cells and has been reported to be important in the virulence of a number of microbial pathogens. Here, we report genome-wide identification and characterization of autophagy-related genes ( Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schweinitz) Petch) is a homothallic filamentous ascomycete fungus and the causal agent of Fusarium head blight (FHB) or head scab disease of wheat, barley, rice and other small grain cereals worldwide3. Damage from head scab results in reduced yield, discolored, shrived “tombstone” kernels, contamination with mycotoxins, and reduction in seed quality4. F. graminearum produces Trichothecene mycotoxins, such as nivalenol (NIV) and deoxynivalenol (DON) and an estrogenic mycotoxin, zearalenone (ZEN). Contamination of cereals and feeds with these mycotoxins sporadically causes food and feed-borne intoxication in man and farm animals6. FHB is one of the most economically important diseases of grain cereals3 and is not controlled well by any current strategies. It is therefore important to understand the infection mechanisms of F. graminearum to guide development of more durable control strategies against FHB.Ciba Foundation Symposium on Lysosomes7. Autophagy is required for maintaining the homeostasis of eukaryotic cells and plays an important role in normal development and differentiation8. To date, 38 ATG genes (AuTophagy-related Genes) have been identified in Saccharomyces cerevisiae, and the biological properties of most of the corresponding Atg proteins have now been characterized10. Autophagy can be divided into three main types based on recognized mechanisms and functions– macroautophagy, microautophagy, and chaperone-mediated autophagy12. Macroautophagy is generally referred to simply as autophagy, and is the most well characterized process of the three processes, used for sequestration and degradation of cytosolic components in a process that uses specialized cytosolic double membrane vesicles called autophagosomes, which ultimately fuse with the lysosome/vacuole, releasing the contents of the vesicle and subsequently breaking down, as proteolysis occurs14. Chaperone-mediated autophagy degrades soluble proteins that contain a motif biochemically related to the pentapeptide KFERQ15. Although autophagy can be nonspecific, there are many selective forms of autophagy in S. cerevisiae, including the cytoplasm-to-vacuole (Cvt) targeting pathway, mitophagy, pexophagy, and other specific forms that target specific organelles. In filamentous fungi, autophagy has been shown to be involved in vegetative growth, asexual/sexual differentiation, environmental stresses, and virulence22. In the rice blast fungus Magnaporthe oryzae, a set of 22 isogenic mutants differing by a single component of the predicted autophagic machinery of the fungus showed that autophagy is necessary for rice blast disease17. The Mgatg8 mutant impaired in autophagy arrests conidial cell death and this renders M. grisea non-pathogenic23. Liu et al. have independently shown that the autophagy genes, MgATG1, MgATG4, MgATG5, MgATG8 and MgATG9 are required for pathogenesis in M. oryzae26. In the corn smut fungus Ustilago maydis, autophagy is also involved in pathogenicity27. Recently, Yanagisawa and colleagues have analyzed the function of Aoatg1 and detected the Cvt pathway in Aspergillus oryzae28. More recently, in the endophytic fungus Harpophora oryzae, it has been shown that autophagy is required for vegetative growth, sporulation and virulence29. Selective autophagy may also be significant in pathogenesis, because it has been shown that Atg26-mediated pexophagy is necessary for appressorium-mediated plant infection in the hemi-biotrophic plant pathogenic fungus Colletotrichum orbiculare16. Similarly, He et al. revealed that Atg24-assisted mitophagy in foot cells is necessary for proper asexual differentiation and efficient conidiogenesis in M. oryzae30.The term “autophagy” was first used by Christian de Duve in 1963 on the occasion of the FgATG15 and FgATG8, have been functionally characterized in F. graminearum31. FgATG15 is involved in fungal growth, aerial hyphae production, conidia production and germination and important for lipid turnover and plant infection31. FgATG8 is related to linear growth rate, formation of aerial mycelium, use of storage lipid droplets, growth over an inert plastic surface, infection and formation of reproductive structures20. To further understand the biological roles of autophagy in morphogenesis and plant infection, we identified all 26 ATG genes, except the previously reported FgATG8 and FgATG15, in the genome of F. graminearum. We generated targeted deletion mutants of 28 ATG genes and demonstrated that loss of any of the ATGs, except FgATG17, prevents the fungus from causing head blight disease. Moreover, we observed that autophagy is important for vegetative growth, asexual/sexual differentiation and DON production. We conclude that autophagy plays a critical role in growth, sporulation, deoxynivalenol production and virulence in F. graminearum.Recently, two autophagy-related genes, F. graminearum genome database using S. cerevisiae functional annotations as a guide and, in this way, we defined a set of 28 ATG genes, which are described in detail in Table 8, but can be conceptually divided into several steps, based on studies in yeast. According to molecular analysis of a battery of autophagy-related genes38, the predicted genes involved in autophagy of F. graminearum could be functionally separated into those that putatively play a role in the induction of autophagy , vesicle nucleation , autophagosome expansion , docking and fusion, and recycling . By reference to Atg proteins specific for selective autophagy in yeast40, FgATG11, FgATG20, FgATG23, FgATG24, FgATG26, FgATG27, FgATG28, FgATG33 and FgATG37 may be required for mitophagy, pexophagy, or the Cvt pathway in F. graminearum. However, homologs of yeast ATG19, ATG21, ATG25, ATG30, ATG31, ATG32, ATG34, ATG36, ATG38, ATG39, ATG40 and ATG41 were not found in F. graminearum . These included ∆Fgatg1, ∆Fgatg3, ∆Fgatg4, ∆Fgatg5, ∆Fgatg6, ∆Fgatg7, ∆Fgatg8, ∆Fgatg9, ∆Fgatg10, ∆Fgatg11, ∆Fgatg14, ∆Fgatg15, ∆Fgatg20, ∆Fgatg22, ∆Fgatg23, ∆Fgatg24, ∆Fgatg29 and ∆Fgatg33  cm and (5.02 ± 0.03) cm after incubation for 3 days on PDA at 25 °C, respectively, which was significantly smaller than (6.68 ± 0.09) cm of the PH-1 strain. In Group 2 mutants, no significant difference in growth rate was observed compared to the wild-type strain. These mutants included ∆Fgatg2, ∆Fgatg12, ∆Fgatg13, ∆Fgatg16, ∆Fgatg17, ∆Fgatg18, ∆Fgatg26, ∆Fgatg27, ∆Fgatg28 and ∆Fgatg37  showed significantly decreased development of aerial mycelium compared to PH-1  × 104 macroconidia per milliliter from such cultures. Conidiation of ∆Fgatg1, ∆Fgatg5, ∆Fgatg8, ∆Fgatg9, ∆Fgatg11, ∆Fgatg13, ∆Fgatg14, ∆Fgatg15, ∆Fgatg16, ∆Fgatg20 and ∆Fgatg24 was significantly reduced  × 104 macroconidia per milliliter, a decrease of 44.52% compared to PH-1. Conidiation of ∆Fgatg1, ∆Fgatg5, ∆Fgatg8, ∆Fgatg9, ∆Fgatg13, ∆Fgatg14 and ∆Fgatg16 was only1.25%, 1.10%, 0.68%, 7.01%, 3.17%, 0.85% and 0.53% that of the wild-type strain, respectively.We next examined conidiogenesis of the wild-type strain and 11 ced Fig. . The ∆FgATG mutants on self-mating carrot agar cultures. The wild-type strain PH-1 produced abundant perithecia after two-week self-fertilization. By contrast, ∆Fgatg1 and ∆Fgatg5 mutants completely failed to form any perithecia under the same culture conditions , PH-1 caused typical scab symptom on inoculated and nearby spikelets. The rate of infected spikelets inoculated with most ATG mutants was significantly reduced compared to PH-1 (P < 0.01), while FgATG17 in Group 2 was fully virulent  was performed. The results showed that DON production between PH-1 and the ∆Fgatg17 mutant had no significant difference  milligrams DON per kilogram inoculated wheat kernels. The levels of DON production in the all tested mutants, including ∆Fgatg2, ∆Fgatg3, ∆Fgatg4, ∆Fgatg7, ∆Fgatg8, ∆Fgatg12, ∆Fgatg13, ∆Fgatg15, ∆Fgatg16, ∆Fgatg20, ∆Fgatg22, ∆Fgatg24 and ∆Fgatg26, were significantly reduced (P < 0.05) , (4.90 ± 1.30), (1.89 ± 0.33) and (4.38 ± 2.47) milligrams DON per kilogram inoculated wheat kernel powder, respectively, which was significantly less than that in PH-1 infections  is known to be an important virulence determinant in MoATG1 and MoATG5 are necessary for conidiation, normal development and pathogenicity in the rice blast fungus M. oryzae25. To determine whether FgATG1 and FgATG5 can functionally complement defects in ∆Moatg1 and ∆Moatg5 mutants, the full length coding sequence of FgATG1 (under the control of the MoATG1 native promoter) and FgATG5 (under the control of the MoATG5 native promoter) were transformed into ∆Moatg1 and ∆Moatg5 mutants, respectively. The complementation transformants, ∆Moatg1/FgATG1 and ∆Moatg5/FgATG5, were identified. Phenotypic analysis showed that ∆Moatg1 and ∆Moatg5 mutants cultured on CM plates for 12 days formed sparse aerial mycelium, but ∆Moatg1/FgATG1 and ∆Moatg5/FgATG5 strains produced dense aerial hyphae, which were identical to the wild-type strain Guy11 (data no shown). Compared to (3.42 ± 0.41) × 107 conidia per plate produced by the wild-type strain Guy11, conidiogenesis in ∆Moatg1 and ∆Moatg5 mutants was significantly reduced (P < 0.05), only produced (7.73 ± 1.57) × 104 and (5.35 ± 1.14) × 105 conidia respectively. The ∆Moatg1/FgATG1 strain produced (2.62 ± 0.38) × 107 conidia per plate approximately 76.6% of that of the wild-type strain  × 107 conidia per plate, approximately 65.5% of that of the wild-type strains  for 24 h, we observed that GFP-FgAtg8 was expressed and dispersed throughout the cytoplasm and GFP-FgAtg8 signals were localized in the punctate structures close to vacuoles in both PH-1 and ∆Fgatg1 and ∆Fgatg5 mutants , a clear full-length GFP-FgAtg8 band (40 kDa) and a free GFP band (26 kDa) were detected in both PH-1 and ∆Fgatg1 and ∆Fgatg5 mutants by immunoblotting with an anti-GFP antibody , TRI6 (transcription regulator) and TRI10 (transcription regulator) genes in mycelium cultured for 24 h were significantly decreased in ∆Fgatg20 and ∆Fgatg24 mutants (data not shown). The data suggest that reduction of DON biosynthesis of ATG mutants is associated with the low expression of the TRI genes in these mutants. Since deoxynivalenol (DON) was an important virulence determinant in F. graminearum, and DON is necessary to suppress plant defense enabling the pathogen to break through the rachis node54, the reduction of DON production in these mutants may be a cause of their loss of virulence or may be associated with the relative lack of infection ability of the mutants.Autophagy is a conserved process from budding yeasts to mammalian cells, and a major cellular pathway for degradation of long-lived proteins and cytoplasmic organellesH-1 Fig. . Howeverain Fig. . This sudia Fig. . Also, wked Fig. . SporulaM. oryzae autophagy genes can be classified into those predicted to be required for nonselective autophagy and those necessary for pexophagy, mitophagy, or the Cvt pathway17. Loss of any of the 16 genes necessary for nonselective macroautophagy leads to M. oryzae being unable to cause rice blast disease, but the 6 genes necessary only for selective autophagy are dispensable for appressorium-mediated plant infection17. However, in the present study, deletion of any of the genes required presumptively for pexophagy, mitophagy, or the Cvt pathway  and genes necessary for the nonselective macroautophagy  in F. graminearum leads to the significant reduction in virulence  are involved in regulating vegetative growth, aerial mycelium formation, asexual/sexual sporulation, DON production and virulence in F. graminearum.In summary, we conclude that autophagy-related genes  plates at 25 °C to assess mycelial growth and colony characteristics. Conidiation assays of all strains were performed after growing 4 days in 1% mung bean liquid (MBL) medium (10 g mung beans boiled in 1 L water for 20 min). Cultures in PDB (PDA without agar) were used for genomic DNA isolation. Complete medium (CM) and minimal medium without the nitrogen source (MM-N) were used for autophagy assays.The wild-type 63. All PCR primers used in this study were listed in Table The DNA cassettes used for the gene deletions were constructed as described previously64.For the vegetative growth of each colony, 5-mm plugs cut from the edge of a 3-day-old colony of each strain were placed on PDA plates and incubated at 25 °C. After 3 days, the diameter of each strain was measured and recorded. For the conidiation assay, five 5-mm plugs of each strain from the edge of 3-day-old colony were inoculated in 20 mL 1% MBL. After 4 d cultivation in a 180 rpm-shaker, conidia of PH-1 were harvested by filtering through cheesecloth and directly counted using a haemocytometer. The conidia of mutants were harvested using the same method and then centrifuged at 8000 rpm for 10 min. The harvested conidia of mutants were re-suspended in 1 mL sterile distilled water and subsequently counted with the haemocytometer. Each experiment with three replicates was independently repeated three times. For self-crossing assays, perithecium formation was assayed on carrot agar (CA) medium as previously describedF. graminearum ATG mutants produced few conidia, mycelial plugs taken from the PDA plates were applied for pathogenicity assays. Agar plugs with mycelium from 3-day-old PDA plates of PH-1 or mutants were scraped off with dentiscalprum, and the middle spikelet of flowering wheat heads of the susceptible cultivar Jimai33 was inoculated. Symptomatic spikelets and quantification of infected spikelets among the whole wheat heads were determined after incubation for 14 days.Since the After immersion in water for 12 h, 50 g wet wheat kernels were sterilized for 3 times and inoculated with ten mycelial plugs (5 mm in diameter) of each strain from the edge of 3-day-old colony and incubation at 25 °C for 25 days. The inoculated wheat kernels were dried at 37 °C for 24 h, and then broken into powder with juicer. The powder was delivered to the company named Pribolab biological engineering co., LTD in Qingdao which performed the DON production assays.TM HRP-conjugated Goat Anti-Mouse IgG (H + L)  was applied to immunoblot analysis at a 1:10000–1:50000 dilution. FDTM FDbio-Femto Ecl chemiluminescent substrate  was used for antigen antibody detections.A piece of agar blocks with mycelia of each tested strain was introduced into 20 mL of liquid CM medium. The suspension was shaken at 25 °C, 180 rpm for 24 h, and then transferred into the MM-N medium in the presence of 2 mM PMSF (phenymethylsulfonyl fluride) for 4 h, 8 h and 12 h. Hyphae of each sample were harvested, washed with sterile distilled water, ground into powder in liquid nitrogen, and then suspended in the protein lysis buffer (50 mM Tris-Cl (pH = 7.4), 0.15 M NaCl, 1 mM EDTA, 1% Triton × 100) added 1% 100 × Protease Inhibitor Cocktail for Fungal/Yeast Cell . The lysate was centrifuged at 13200 rpm for 20 min at 4 °C after lysis for 30 min. Afterwards, 50 μL supernatant was mixed with isovolumetric 2 × protein loading buffer and boiled for 5 min and then cooled on the ice immediately. 10–15 μL of each sample was taken for loading on 10% SDS-PAGE gels. GFP-Tag (7G9) Mouse mAb  as primary antibody was used at a 1:5000 dilution. HiSecMoatg1 and Moatg5 mutants respectively were constructed as described previously63.The pCB1532-FgATG1 and pCB1532-FgATG5 vectors for the complementation of 18.Hyphae expressing the GFP-Atg8 fusion protein were cultured in CM medium at 25 °C, 180 rpm for 24 h, and then transferred into the MM-N medium in the presence of 2 mM PMSF for 4 h at 25 °C in a 180 rpm shaker. CMAC (7-amino-4-chloromethycoumarin)  was used for vacuole staining. Photographs were taken under a confocal laser scanning microscopy. Transmission electron microscopy was implemented as described previouslyAll data generated or analysed during this study are including in this published article (and its Supplementary Information files).Supplementary Information


The tree shrew, as a primate-like animal model, has been used for studying high brain functions such as social emotion and spatial learning memory. However, little is known about the excitatory synaptic transmission in cortical brain areas of the tree shrew. In the present study, we have characterized the excitatory synaptic transmission and intrinsic properties of pyramidal neurons in the anterior cingulate cortex (ACC) of the adult tree shrew, a key cortical region for pain perception and emotion. We found that glutamate is the major excitatory transmitter for fast synaptic transmission. Excitatory synaptic responses induced by local stimulation were mediated by AMPA and kainate (KA) receptors. As compared with mice, AMPA and KA receptor mediated responses were significantly greater. Interestingly, the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) in tree shrews was significantly less than that of mice. Moreover, both the ratio of paired-pulse facilitation (PPF) and the time of 50% decay for fast blockade of NMDA receptor mediated EPSCs were greater in the tree shrew. Finally, tree shrew neurons showed higher initial firing frequency and neuronal excitability with a cell type-specific manner in the ACC. Our studies provide the first report of the basal synaptic transmission in the ACC of adult tree shrew. The tree shrew has a well-developed brain and central nervous system. Cumulative evidence has shown that the tree shrew is an ideal animal model for brain diseases of humans. Molecular phylogeny and whole genome sequencing analysis studies suggest that tree shrew has a close affinity to primates –4. Tree Both animal and human studies have consistently demonstrated that the anterior cingulate cortex (ACC) plays important roles in many major brain functions such as awareness, emotion, memory, and pain –15. HoweGlutamate is the major excitatory neurotransmitter for synaptic transmission in the central nervous system. Glutamatergic synaptic transmission is mainly mediated by three kinds of gate ionotropic receptors, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), kainate (KA) and N-methyl-D-aspartate (NMDA) receptors , 16–18. In the present study, by combing whole-cell patch recording, pharmacology blocking, and two-photon calcium imaging observation, we investigated the composition characteristics of basal synaptic transmission in the ACC pyramidal neurons of tree shrew. Using mice as a control, we found the AMPA and KA receptor mediated postsynaptic responses were enlarged, but the presynaptic glutamate release probabilities were lower in tree shrews. The global calcium signals and intrinsic neuronal excitability of pyramidal neurons were also found higher in tree shrews. Our studies provide the first report for the basic electrophysiological characteristics of glutamatergic synaptic transmission and neuronal properties in the ACC pyramidal neurons of tree shrews.Experiments were performed with adult male tree shrews (10–12 months old) and male C57BL/6 mice (6–8 weeks old). Tree shrews were purchased from Kunming Institute of Zoology in China. All tree shrews and mice were maintained on a 12 h light/dark cycle with food and water provided ad libitum. Animal care, as well as all experiments, was conducted in accordance with the European Community guidelines for the use of experimental animals (86/609/EEC). All performed research protocols were approved by the Ethics Committee of Xi’an Jiaotong University.2 and 5% CO2), ice cold cutting artificial cerebrospinal fluid (ACSF) containing the following (in mM): 252 sucrose, 2.5 KCl, 6 MgSO4, 0.5 CaCl2, 25 NaHCO3, 1.2 NaH2PO4 and 10 glucose, pH 7.3–7.4. After cooling in the ACSF for a short time, the whole brain was trimmed for an appropriate part to glue onto the ice-cold stage of a vibrating tissue slicer . Slices were transferred to a submerged recovery chamber containing oxygenated (95% O2 and 5% CO2) ACSF (in mM): 124 NaCl, 4.4 KCl, 2 CaCl2, 1 MgSO4, 25 NaHCO3, 1 NaH2PO4, and 10 glucose at room temperature for recording at least 1 h later.Coronal brain slices (300 μm) at the level of the ACC were prepared using standard methods , 24–26. 2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na3-GTP . AMPA and kainate receptor mediated EPSCs were induced by repetitive stimulations at 0.02 Hz, and neurons were voltage clamped at −60 mV in the presence of AP5 (50 μM) for AMPA currents and both AP5 and GIKI 53655 (100 μM) for KA currents. NMDA receptor mediated EPSCs were pharmacologically isolated in Mg2+-free ACSF containing CNQX (20 μM) and glycine (1 μM), and neurons were voltage-clamped at −20 to −30 mV and induced by repetitive stimulations at 0.05 Hz. The patch electrode internal solution (in mM) 112 Cs-Gluconate, 5 TEA-Cl, 3.7 NaCl, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na3-GTP and 5 QX-314  were used for recording NMDA receptor mediated EPSCs and AMPA, KA, NMDA receptors mediated I-V curves. For miniature EPSCs (mEPSCs) recording, TTX (1 μM) was added in the perfusion solution. The current-clamp configuration was used recording action potentials (APs) for a single spike (current injection of 100 pA/5 ms) and five spikes at 5, 10, 20, and 50 Hz (current injection five times of 100 pA/5 ms at different frequencies). Picrotoxin (100 μM) was always present to block GABAA receptor mediated inhibitory synaptic currents in all experiments. Access resistance was 15–30 MΩ and monitored throughout the experiment. Data were discarded if access resistance changed 15% during an experiment. Data were filtered at 1 kHz, and digitized at 10 kHz using the digidata 1440A.Whole cell recordings were performed in a recording chamber on the stage of a BX51W1 (Olympus) microscope equipped with infrared differential interference contrast (DIC) optics for visualization. EPSCs were recorded from layer II/III neurons with an Axon 200B amplifier (Molecular Devices), and the stimulations were delivered by a bipolar tungsten stimulating electrode placed in layer V/VI of the ACC. The recording pipettes (3–5 MΩ) were filled with a solution containing (in mM) 145 K-gluconate, 5 NaCl, 1 MgClTo identify the morphological properties of the pyramidal cells in the tree shrew, 0.5% biocytin was added into the recording solution for the labeled patched neurons. After recording, the brain slices containing biocytin labeled cells were immediately fixed with 4% paraformaldehyde in 0.1 M PB  for 4 h at room temperature. Then the slices were transferred to 30% sucrose overnight at 4 °C temperature. After thoroughly washing with PBS, all slices were immunostained with FITC conjugated avidin  for 2 h at room temperature. The immunofluorescence labeled neurons were imaged with a confocal microscope  using the appropriate filter for FITC. Each section was imaged through the depth scan and collapsed stack using z projection generated a two-dimensional reconstruction of the labeled neurons. The photomicrograph was assembled by the software of Adobe Photoshope. Only brightness and contrast were adjusted.+ salt (200 μM) and Alexa594 K+ salt (20 μM) were separated into green and red channels by a dichroic mirror and emission filters , and detected by a pair of photomultiplier tubes  at 800 nm. To obtain time series of fluorescent signals from global soma images, images were collected with the following parameters , 3130, 3n = 8 neurons/4 tree shrews; Fig. To test whether the excitatory synaptic transmission is mediated by glutamate, we bath applied an AMPA/Kainate (KA) receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione . EPSCs were rapidly and largely reduced by CNQX. Small residual EPSCs persisted in the presence of CNQX 10 min after perfusion. These EPSCs were blocked by following application of NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid   compared with mouse  = 8.24, P < 0.01, two-way ANOVA), indicating that the basal excitatory responses are potentiated in tree shrew. However, the I-V curves (−70 to +50 mV) were not different between tree shrew and mouse  = 0.13, P = 0.72, two-way ANOVA)  and current-voltage curves (I-V curves) were recorded in ACC neurons. Picrotoxin (100 μM) and AP5 (50 μM) were bath applied for recording AMPA receptor mediated EPSCs. As shown in Fig. VA) Fig. .Fig. 3Thn = 13 neurons/4 mice, F = 0.26, P = 0.61, two-way ANOVA). Furthermore, the I-V curves were also not different between tree shrew and mouse  = 0.49, P = 0.48, two-way ANOVA)  and CNQX (20 μM). As the results shown in Fig. VA) Fig. . Our resn = 6 neurons/3 tree shrews). These results suggest that KA receptors mediate a relatively small component of the excitatory non-NMDA receptor mediated synaptic transmission in the ACC of tree shrew.In addition to AMPA receptors, KA receptors have been found to play roles in synaptic transmission in the ACC , 23, 32.n = 11 neurons/4 tree shrews, P < 0.05; Fig. n = 9 neurons/3 mice; F = 18.80, P < 0.01, two-way ANOVA)  Fig. .I-V) relationship in KA receptor mediated EPSCs. The I-V curve of KA receptor can reflect the calcium permeability and the subunit composition of channels cyclohepten-5,10-imine maleate (MK-801), a non-competitive NMDA receptor antagonist with activity-dependent manner, has been widely reported to estimate the glutamate release probability [n = 8 neurons/3 tree shrews; mouse: 4.97 ± 0.69 min, n = 8 neurons/3 mice, P < 0.05). Taken together, these results indicate that the rate of presynaptic glutamate release in the ACC of tree shrews is slower as compared with that in mice.The blocking rate of NMDA receptor mediated responses by (+)-5-methyl-10,11-dihydro-5bability , 35, 36.2+ imaging observation, we recorded the global Ca2+ signals in the ACC pyramidal neurons of tree shrew. After 30 min diffusion of Alexa594 and Cal-520, the neuronal morphology was well labeled  and frequencies  dependent manners. Interesting, we found the Ca2+ signals were significantly larger in tree shrew than mouse  = 4.25, P < 0.05, n = 6 neurons/3 tree shrews and n = 7 neurons/3 mice; frequency: F = 8.92, P < 0.01, n = 5 neurons/3 tree shrews and 5 neurons/3 mice; two-way ANOVA)  Fig. .Fig. 7Thn = 65 neurons/22 tree shrews) showed a larger membrane capacitance (Cm) , smaller membrane resistance (Rm)  and faster charge-discharge time (Tau) , suggesting that the tree shrew pyramidal cells have a larger membrane surface and higher electrical responses capability. By analyzing single AP at the threshold, we found the half width  and decay time  were smaller in tree shrews than mice. The decay slope  was larger in tree shrews as well. These results indicate that the spike of pyramidal cell is more narrow and sharp in tree shrews than mice. However, although the resting membrane potential (RMP) and the threshold membrane potential (Vthreshold) were not different, the rheobase (the minimum current required to evoke an AP) was higher in tree shrews compared to mice. Taken together, the present results suggest that, although a stronger current input is needed to initiate the spike (maybe due to the larger surface membrane and capacitance), pyramidal cells in tree shrews will spike more intensely than in mice. The hypothesis were further confirmed after injection of increased step current, in which the spike number of tree shrew neurons was not different with mouse neurons in face of weak inputs, but was significantly larger in face of stronger inputs  = 38.94, P < 0.001, two-way ANOVA)  Fig. .Table 1Sn = 65 neurons/22 tree shrews; mouse: RS 36.7%, IM 43.3%, IB 20.0%, total n = 60 neurons/17 mice)  (AHP without ADP), intermediate (IM) (AHPs with ADP), and intrinsic bursting (IB) (the ADP will trigger bursting spikes) neurons. In our previous studies, IM and IB cells showed the higher membrane excitability than RS cell, and the population distribution of them were increased in neuropathic pain mice . IB cellCortical synaptic transmission and plasticity are critical for sensory and cognitive processes in mammals. However, there is limited information about cortical synaptic transmission and plasticity obtained from primate animal models. Recent cumulative evidence has shown that the tree shrew is a potentially useful primate-like animal model for human brain diseases –4. In thGlutamatergic synaptic transmission plays important roles in both physiological and pathological conditions to play important roles in the ACC , 10. In Postsynaptic KA receptors contribute to fast synaptic responses in pain related cortical areas , 18, 39.Both presynaptic and postsynaptic glutamate transmissions contribute to synaptic plasticity in the ACC , 35, 40.2+ imaging observation, our recent studies have characterized the properties of postsynaptic calcium signals in the pyramidal neurons of ACC in mice. We also reveal the dynamic change of Ca2+ ion in the induction phase of LTP in the ACC of mice [2+ signals in the ACC neurons of tree shrews and that the summation of calcium signals induced by repetitive stimulation is larger in tree shrew neurons as compared with mouse ACC.Calcium signals are thought to be critical for synaptic transmission and plasticity in the ACC , 13, 41. of mice . In the We also identified three main types of pyramidal cells  in the ACC of adult tree shrews, which are similar with cell types reported in mouse ACC , 38. We Animal models have been useful for the investigation of different physiological and pathological mechanisms of brain diseases. Cumulative studies have consistently indicated that ACC and related cortical areas play vital roles in many brain functions, including pain perception, fear memory, and anxiety , 19, 21.


Balancing panicle-related traits such as panicle length and the numbers of primary and secondary branches per panicle, is key to improving the number of spikelets per panicle in rice. Identifying genetic information contributes to a broader understanding of the roles of gene and provides candidate alleles for use as DNA markers. Discovering relations between panicle-related traits and sequence variants allows opportunity for molecular application in rice breeding to improve the number of spikelets per panicle.indica and tong-il varieties, exhibited the largest average number of favorable alleles and highest average number of spikelets per panicle, suggesting that the favorable allele combination found in this cluster was beneficial in increasing the number of spikelets per panicle.In total, 142 polymorphic sites, which constructed 58 haplotypes, were detected in coding regions of ten panicle development gene and 35 sequence variants in six genes were significantly associated with panicle-related traits. Rice cultivars were clustered according to their sequence variant profiles. One of the four resultant clusters, which contained only Favorable alleles identified in this study can be used to develop functional markers for rice breeding programs. Furthermore, stacking several favorable alleles has the potential to substantially improve the number of spikelets per panicle in rice.The online version of this article (10.1186/s12863-017-0591-6) contains supplementary material, which is available to authorized users. ABERRANT PANICLE ORGANIZATION 1 (APO1) [ABERRANT PANICLE ORGANIZATION 2 (APO2) [GRAIN NUMBER 1a (GN1a) [DENSE AND ERECT PANICLE 1 (DEP1) [GRAIN NUMBER, PLANT HEIGHT, AND HEADING DATE 8 (GHD8) [HEADING DATE 1 (HD1) [FLORAL ORGAN NUMBER 1 (FON1) [SHORT PANICLE 1 (SP1) [LAX PANICLE 1 (LAX1) [MONOCULM 1 (MOC1) [In rice, the number of spikelets per panicle (SPP) is an important agronomic characteristic that has a strong impact on yield. SPP is determined by several panicle-related traits, such as the number of primary branches per panicle (PB), the number of secondary branches per panicle (SB), and panicle length (PL). Improving the balance between panicle-related traits is needed to optimize SPP . Panicle1 (APO1) , ABERRAN2 (APO2) , GRAIN Na (GN1a) , DENSE A1 (DEP1) , GRAIN N8 (GHD8) , HEADING 1 (HD1) , FLORAL  1 (SP1) , LAX PAN1 (LAX1) , and MON1 (MOC1) . A bette1 (MOC1) .GHD7 that were not identified in an earlier study were detected using General Linear Model (GLM) association analysis [HD1 were significantly associated with flowering date in rice.Candidate gene association analysis investigates the relation between polymorphic sites in genes which are involved in specific phenotypes and phenotypic variations, thereby facilitating identification of causative sequence variants for particular characteristics leading to phenotypic variation. Candidate gene association analysis was previously used to identify novel beneficial polymorphic sites related to important agronomic traits. For example, some functional mutations of analysis , 14 and analysis  found thFunctional markers that are directly derived from polymorphic sites within the genes responsible for variations in the target trait can be used directly for marker-assisted breeding as the most effective marker. Prior to identifying the favorable alleles that can be used as resource of functional marker, genetic information about genes, such as level of DNA polymorphism and linkage disequilibrium (LD), are required for successful marker development .The objectives of this study were to (1) identify novel sequence variants in the coding regions of panicle development genes which were previously reported to control panicle development and investigate genetic information, (2) identify sequence variants associated with panicle-related traits, including PL, PB, and SB, and (3) find combinations of favorable alleles for each trait that could contribute to increasing SPP in rice.japonica (N = 22), indica (N = 18), and tong-il (N = 5)  method . To idenhPrimer3  . Neutrality tests were conducted for calculating Tajima’s D [Coding and non-coding regions of 10 genes, including RGSP-1.0 . MultiplRGSP-1.0  and furtRGSP-1.0  , and the MOC1-G allele was observed in ten varieties, including eight varieties of indica and two tong-il. A total length of coding region of LAX1 is 649 bp and only one SNP (T-G) which caused change of amino acid was detected at 349 bp  ranged from 1.44 × 10−3 (GN1a) to 6.16 × 10−3 (GHD8). π for APO1, APO2, DEP1, and GHD8 were higher in indica than in japonica varieties, whereas π for FON1, GN1a, HD1, and SP1 were higher in japonica than in indica.Eight representative varieties were sequenced to find sequence variant in coding region of Hd (haplotype diversity) for eight of the ten genes  ranged from 0.533 (APO1) to 0.812 (GHD8) . Eleven of the twelve polymorphisms, located at 40, 57, 537, 546, 627, 765, 795, 850, 951, 1005, and 1188 bp, were in complete LD  and had seven favorable alleles on average. Cluster C contained four indica and three tong-il varieties and had the largest number of favorable alleles . The lowest average number of favorable alleles (4.5) was found in Cluster D. The average SPP value for each cluster was proportional to the number of favorable alleles, and SPP for Cluster C was higher than for other clusters  . Deviation from neutrality was represented using Tajima’s D statistic. Significantly positive estimates of Tajima’s D were detected only for APO2, FON1, and HD1. No significant deviation from neutrality was detected in the other genes, indicating that population subdivision events or balancing selection occurred at those loci during evolutionary and breeding processes  Fig. . By contSCM2 allele of APO1, which was isolated from ‘Habataki’, a high-yielding Japanese indica variety, increased SPP. ‘Koshihikari’ near isogenic lines (NILs) introgressed with SCM2 showed significantly increased spikelet numbers and grain yield, indicating that the allele could improve yield in japonica varieties [APO1–3 haplotype, which was identical to SCM2, was detected only in indica and tong-il accessions. Tong-il type varieties, which were derived from crosses between indica and japonica varieties developed in Korea, exhibited longer panicles, thicker neck nodes, and higher numbers of total rachis branches than japonica varieties [APO1–3 was introgressed from indica to tong-il type and was artificially selected as a beneficial haplotype during cross breeding between subspecies. In addition, similar patterns were detected in APO2–3, FON1–4, and SP1–5. With the exception of ‘Hanmaeum’, which exhibited the lowest SPP among the tong-il type varieties, all tong-il type varieties carried the same indica-derived haplotypes  and allelic variants were assessed to identify beneficial sequence polymorphisms with the potential to improve SPP. To reduce the risk of false positive association, we evaluated population structure with admixture model. Estimated Q matrix was used as covariate for the GLM association analysis. In addition, we only used common sequence variants to detect true association, removing minor sequence variants less than MAF 5%.APO1 coding region were significantly associated with SB. Of these, only two non-synonymous SNPs located at 40 bp and 765 bp, and two INDELs located at 850 bp and 1188 bp position led to amino acid sequence changes and were thus likely to affect SB phenotypic variation.Five, fourteen, and sixteen polymorphic sites in coding region were significantly associated with PL, PB, and SB, respectively. Some of these sites, however, could not directly affect phenotype. These polymorphic sites can be interpreted as a result of strong LD with trait-related sequence variants . For exaHD1 coding region, four polymorphic sites, including two large INDEL regions of 129 bp and 33 bp, and a small 4 bp INDEL causing a frameshift, were significantly associated with PB. The 4 bp INDEL was also associated with SB under natural long-day conditions in the experimental field in Suwon, South Korea (37°N latitude). These results were consistent with previous reports revealing that large INDELs and small frameshift-inducing INDELs led to partial or complete loss of HD1 function [HD1 were also significantly associated with PB. These SNPs could be attributed to change of HD1 protein function as they substituted several amino acid sequence. However, it was unclear whether phenotypic variation was due to altered protein function from amino acid substitutions or to strong LD (r2 = 0.91) with an 2 bp INDEL inducing loss of function, located at 1000 bp Additional file 2:General information for total accessions used in this study. (XLSX 16 kb)Additional file 3:Primers used in this study. (PDF 22 kb)Additional file 4:FASTA format files containing coding sequences for 9 genes. (ZIP 19 kb)Additional file 5:MOC1 and LAX1 coding region. (PDF 420 kb)Results of sequence analysis of Additional file 6:Summary of statistics for four traits of 45 varieties. (XLSX 10 kb)Additional file 7:Haplotypes of four genes. (PDF 197 kb)Additional file 8:Plots of Evanno’s delta K (∆K). (PDF 115 kb)


In chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations.We performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens.IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p < 0.001), and the complex karyotype (p = 0.003). A high risk as assessed by FISH analysis was associated with mutations affecting TP53 (p = 0.012), BIRC3 (p = 0.003), and FBXW7 (p = 0.003) while the complex karyotype was significantly associated with TP53, ATM, and MYD88 mutations . By multivariate analysis, the multi-hit profile (≥2 mutations by NGS) was independently associated with a shorter time to first treatment (p = 0.004) along with TP53 disruption (p = 0.040), IGHV unmutated status (p < 0.001), and advanced stage (p < 0.001). Advanced stage (p = 0.010), TP53 disruption (p < 0.001), IGHV unmutated status (p = 0.020), and the complex karyotype (p = 0.007) were independently associated with a shorter overall survival.Mutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated At diagnosis, an extensive biologic characterization including NGS and karyotype analyses using novel mitogens may offer new perspectives for a better refinement of risk stratification that could be of help in the clinical management of CLL patients.The online version of this article (doi:10.1186/s13045-016-0320-z) contains supplementary material, which is available to authorized users. IGHV) [TP53, NOTCH1, and SF3B1 gene mutations have a negative impact on the time to first treatment (TTFT) and overall survival (OS) [Chronic lymphocytic leukemia (CLL) displays a heterogeneous clinical course –3, some IGHV)  and specIGHV) –14, and val (OS) –17. Thesval (OS) , 19, andval (OS) , 21 helpval (OS) .NOTCH1, SF3B1, BIRC3, and TP53 mutations, the latter having a negative prognostic impact that was similar to TP53 clonal mutations [Next-generation sequencing (NGS) techniques documented that, besides the aforementioned genes, a number of previously unidentified genes may be mutated in CLL and that the disruption of putative core cellular pathways represents an important mechanism promoting disease progression and drug resistance –26. NGS utations –29 as deThus, NGS is becoming of age for usage in clinical practice, and indeed, over 50 % of CLL patients were shown to carry mutations in one or more genes , 31, potTo assess whether an extended mutational screening by NGS at diagnosis could allow for a refinement of our capability to predict TTFT and OS, we designed a CLL-specific gene panel, covering hotspots or complete coding regions of 20 genes more frequently mutated in CLL. We performed NGS of these 20 genes using a resource-efficient platform in 200 consecutive newly diagnosed patients representing over 90 % of CLL incident cases in our region. By correlating mutational data obtained by an extensive genetic/cytogenetic characterization with clinic-biological parameters and outcome, we were able to show that NGS screening was an independent prognostic factor for TTFT and that complex karyotype was a strong predictor of an inferior survival in this patient population.The study cohort consisted of 200 consecutive untreated CLL patients diagnosed and followed between 2007 and 2014. All patients were diagnosed according to NCI criteria . Only paInterphase FISH was performed on PB samples obtained at diagnosis using probes for the following regions: 13q14, 12q13, 11q22/ATM, and 17p13/TP53  as described . Each paCytogenetic analysis was performed on the same samples used for FISH analysis using CpG-oligonucleotide DSP30  plus IL2  as described . The comIGHV genes were amplified from genomic DNA and sequenced according to standard methods with the cutoff of 98 % homology to the germline sequence to discriminate between mutated (<98 %) and unmutated (≥98 %) cases, as reported [reported .ATM, BIRC3, BRAF, CDKN2A, PTEN, CDH2, DDX3X, FBXW7, KIT, KLHL6, KRAS, MYD88, NOTCH1, NRAS, PIK3CA, POT1, SF3B1, TP53, XPO1, ZMYM3) starting from genomic DNA from PB samples, according to HaloPlex Target Enrichment System . Diluted libraries were linked to Ion Sphere Particles, clonally amplified in an emulsion PCR and enriched using Ion OneTouch emulsion PCR System . Exon-enriched DNA was precipitated with magnetic beads coated with streptavidin. Enriched, template-positive Ion Sphere Particles were loaded in one ion chip and sequenced using Ion Torrent PGM . Sequencing data were aligned to the human reference genome (GRCh37). Data analysis and variant identification were performed using Torrent Suite 3.4 and Variant Caller plugin 3.4.4  [NGS analysis was performed on the same samples used for FISH and cytogenetic analyses. In all samples, the percentage of CLL cells was over 90 % as assessed by flow cytometry analysis. Agilent HaloPlex Target Enrichment kit  was used to produce libraries of exonic regions from 20 genes  in the NOTCH1 gene, 15 (7.5 %) in the SF3B1 gene, 10 (5.0 %) in the ATM gene, 8 (4.0 %) in the BIRC3 gene, 7 (3.5 %) in the MYD88 gene, 7 (3.5 %) in the PTEN gene, 6 (3.0 %) in the FBXW7 gene, 5 (2.5 %) in the POT1 gene, 5 (2.5 %) in the BRAF gene, 5 (2.5 %) in the ZMYM3 gene, and 19 (9.5 %) cases in the remaining 9 genes  were significantly more frequent among patients with 2 or more mutations while a trend was observed for BIRC3 mutations (p = 0.059) and mutations of genes less frequently mutated in CLL (p = 0.057)  cases. One hundred thirty-six mutations were found in these 84 patients; 114 missense mutations, 7 nonsense mutations, 14 frameshit deletions, and 1 frameshit insertion. Mutations were detected with a frequency ranging from 5.0 to 96.7 % of the reads. Sixteen cases (8.0 %) showed mutations in the p = 0.010), IGHV unmutated status (p = 0.009), intermediate high-risk cytogenetics by FISH analysis (p < 0.001), and the complex karyotype , BIRC3 (p = 0.003), and FBXW7 (p = 0.003) while the complex karyotype was significantly associated with TP53, ATM, and MYD88 mutations ; and complex karyotype. A shorter TTFT was also observed for TP53-, NOTCH1-, ATM-, and BRAF-mutated patients. By multivariate analysis (Table p = 0.004) along with TP53 disruption (p = 0.040), IGHV unmutated status (p < 0.001), and advanced stage (p < 0.001).The median follow-up for the 200 CLL patients was 52.3 months. In univariate analysis Table , the occTP53 mutations, and with advanced stage, CD38 positivity, IGHV unmutated status, TP53 disruption, and complex karyotype. In multivariate analysis, advanced stage (p = 0.010), IGHV unmutated status (p = 0.020), TP53 disruption (p < 0.001), and the complex karyotype (p = 0.007) independently predicted a worse outcome  was independently associated with a shorter TTFT along with TP53 disruption, IGHV unmutated status, and advanced stage.We then analyzed the correlation between the mutational status and outcome. A shorter TTFT was observed in those patients with mutations by NGS and with mutations involving Given the complexity of CLL genetic landscape, we suggest that not only the presence of clones or subclones  but alsoTP53 and NOTCH1 genes, with the multi-hit profile, with IGHV unmutated status, with TP53 disruption, and with the complex karyotype. However, by multivariate analysis, only TP53 disruption was independently associated with a worse outcome along with advanced stage, IGHV unmutated status, and the complex karyotype.When considering OS, a poorer outcome was associated with the presence of mutations by NGS, with mutations in IGHV mutational status and TP53 disruption was previously demonstrated [Whereas the strong independent impact on TTFT and OS of nstrated , 45, 46,nstrated , 34, befnstrated , and in nstrated . We may nstrated , 50.TP53 disruption, and IGHV assessment, may identify those patients that are at higher risk of progression and therefore need a stricter follow-up whereas karyotyping could represent along with TP53 disruption the best genetic predictor of OS. However, some issues need to be better defined before the introduction of the extensive NGS approach into the routine clinical practice: (i) which genes and how many genes should be included in the work-up panel for an efficient and affordable routine applicability, (ii) what cutoff for mutational analysis should be considered clinically relevant, and (iii) how to develop a standardized methodology ensuring reproducibility of the results [Altogether, our data suggests that NGS may play an important role in the definition of the risk of disease progression and therefore could be useful in the diagnostic work-up of CLL patients as an efficient, sensitive, and affordable technique for routine screening of mutations. Indeed, NGS analysis, in combination with clinical stage,  results .