Setaria italica (L.) Beauv) is one of the earliest domesticated grains, which has been cultivated in northern China by 8,700 years before present (YBP) and across Eurasia by 4,000 YBP. Owing to a small genome and diploid nature, foxtail millet is a tractable model crop for studying functional genomics of millets and bioenergy grasses. In this study, we examined nucleotide sequence diversity, geographic structure, and levels of linkage disequilibrium at four nuclear loci  in representative samples of 311 landrace accessions across its cultivated range. Higher levels of nucleotide sequence and haplotype diversity were observed in samples from China relative to other sampled regions. Genetic assignment analysis classified the accessions into seven clusters based on nucleotide sequence polymorphisms. Intralocus LD decayed rapidly to half the initial value within ~1.2 kb or less.Foxtail millet ( Since the inception of the human agropastoral transition, many crops have been domesticated and served as important food sources. In East Asia, foxtail millet, known in Chinese as ‘xiao mi’ or ‘su’ is one of the most important and earliest domesticated grain crops. The earliest archeological evidence of domesticated foxtail millet was found at the Nanzhuangtou 11.5–11.0 YBP) and Donghulin (11.0–9.5 YBP) in the North China Plain , 3. Worl–11.0 YBPBrachypodium distachium has been sequenced as a means of providing more ready access to the large genomes of Triticeae species such as barley (Hordeum vulgare L.), rye , and wheat (Triticum aestivum L.) [Panicum virgatum L.), pearl millet (Pennisetum glaucum (L.) R.Br.), and napiergrass (Pennisetum purpureum Schumach.) [http://www.phytozome.net/Setariaitalica), short generation time makes foxtail millet a readily tractable system to rapidly explore the genetic basis of resistance to abiotic stress, agronomic traits, and phenotypic variation in Paniceae grasses that have applications as bioenergy crops [Setaria genome research is the release of the first assembled reference genome of cultivated and wild foxtail millet [There is also renewed interest in foxtail millet as a model system for bioenergy research –9. Just ivum L.) , foxtailhumach.) . With angy crops –12. A mal millet , 14, whil millet . Severall millet ; identifl millet ; identifl millet .Both association studies  and genoLevels of nucleotide sequence diversity and geographic structure are important in association studies , 23, subApplying a collection of 311 foxtail millet accessions across its historical range of cultivation, we used resequencing data from four loci to: (1) document the nucleotide diversity within and among geographic regions; (2) examine the genetic structure; (3) investigate the extent of intralocus linkage disequilibrium (LD). Based on this information, the application of association studies in foxtail millet was addressed.A total of 311 foxtail millet landrace accessions were sampled. China, Central Asia, and European accessions constitute a large portion of the total sample because these regions have been the major regions of cultivation and/or putative domesticated centers . Seeds fTPI1), glyceraldehyde 3-phosphate dehydrogenase (G3PDH), Alcohol dehydrogenase 1 (ADH1), the intergenic spacer 1 (IGS1) region of ribosomal DNA. Because the IGS1 region is a portion of nuclear ribosomal DNA that occurs as tandem array, which is subjected to concerted evolution [IGS1 among geographic regions but did not include it in estimates of average diversity. The PCR and sequencing primers for ADH1, G3PDH, IGS1 and TPI1 were designed from Zea mays. Primer information is detailed in Oryza sativa and Zea mays, thus these primers have potential utility across the Poaceae.Genomic DNA was extracted from young leaf tissue from a single individual of each accession using the modified CTAB method . We examvolution , we comp2+), 2.5 mM of dNTP, and 1 unit of Taq DNA polymerase. PCR reactions (except G3PDH) used the following cycling conditions: 94°C for 3 min; 35 cycles ; a final extension of 10 min at 72°C, the annealing temperature of G3PDH is 56°C. Amplified products were purified with EasyPure PCR Purification Kit , and were directly sequenced on an ABI  3730. The majority of PCR amplicons were directly sequenced. Amplicons from accessions that were determined to be heterozygous within a locus were cloned using pEASY kit . Not all samples were sequenced successfully, thus the sample size of final data matrix for ADH1, G3PDH, IGS1, TPI1 are 296, 285, 293, and 289, respectively.Polymerase chain reaction (PCR) was performed in a total volume of 25 μl containing 50 ng of template DNA, 10 μM of each primer, 2.5 μL 10 × PCR buffer , and edited sequences were aligned using Clustal X , and werθ = 4Neμ, based on the number of segregating sites (θw) [θπ) [T [T was tested by 10,000 replicate coalescent simulations.Descriptive statistics for nucleotide sequence diversity and tests of neutrality were estimated using tools from the libsequence C++ software library . Insert/es (θw)  and the w) [θπ) . We also[θπ) [T , a test KST* and Snn methods of Hudson [Snn (nearest-neighbor statistic) is a powerful statistic for detecting genetic differentiation using sequence-based analysis over a wide range of sample size and levels of variation [Snn is expected to be near one. KST* measures genetic differentiation using resequencing data and can be more powerful for detecting population structure than SNP frequency-based estimates of FST, especially in the case of high haplotype diversity [The sample was partitioned into nine geographic regions: China (CH), Central Asia (CA), South Asia (SA), Near East (NE), Korea and Japan (KJ), Southeast Asia (SEA), Europe (ER), North America (NA), and Africa (AF). We tested the extent of geographic structure in and among these partitions of the sample using f Hudson , 50 applf Hudson . Snn  in the model was systematically varied from 1 to 10, with the median likelihood of each K value estimated from the 20 runs. We used the △K method [Genetic differentiation was investigated using genetic assignment in STRUCTURE 2.3.4 , 53. BurK method  represenK method .ρ = 4Ner, where Ne is the effective population size and r is the recombination rate per generation [B [Rm) [Multiple methods were applied to estimate levels of LD in the data: a parametric estimate of the recombination rate: neration , 57; a cneration ; and intation [B , a summa[B [Rm)  were alsr2 [http://www r-project.org) was used to plot LD versus distance.LD between pairs of polymorphic sites was calculated based on squared correlation in allele frequency, r2 , betweenr2 . The staADH1, G3PDH, IGS1, and TPI1 are 875, 1390, 970, and 870 bp, respectively, and the combined total length of aligned sequence for four loci is 4105 bp , respectively. None of the Tajima’s T values show significant deviation from expectations for a panmictic population evolving under neutrality. Nucleotide diversity estimated by the number of pairwise differences among haplotypes (θπ) ranges from 2.86–10.05 × 10−3 for the four loci, with on average of 6.98 × 10−3  for all four loci. KST*statistics in locus of IGS1 (P < 0.05) also demonstrates significant geography structure  by physical distance between sites in foxtail millet are shown in r2 against distance in base pairs between SNPs  also suggests similar pattern of intralocus LD.Plots of squared allele frequency correlations  with average diversity θw = 4.04 × 10−3, θπ = 3.63 × 10−3. The level of genetic diversity is lower than wild green foxtail (Setaria viridis (L.) P. Beauv.), where θw = 5.9 × 10−3 [Oryza sativa) (θπ = 3.04 × 10−3) [−3) [Foxtail millet has relatively low diversity for the three single copy genes ( × 10−3) . There w−3) [−3) , Liu et −3) [−3) . Then th−3) [−3) . Many ge−3) [−3) , SSR mar−3) [−3) , 35, tra−3) [−3) , which cS. viridis [θπ) for loci G3PDH and TPI1, Europe for locus ADH1, and Central Asia for locus IGS1 showed. Europe has the highest haplotype diversity for loci ADH1 and IGS1, China for locus G3PDH, and Central Asia for locus IGS1. All the four regions except Near East contain high nucleotide diversity, haplotype diversity and private SNPs for certain locus, which should be major regions for germplasm conservation. Korea and Japan (KJ) have rarely been included in previous studies, high diversity  was revealed in this study. The Snn test indicates significant geographic structure for four loci studied (P = 0). The ADH1 locus also show significant population structure with KST* (P < 0.05). Given that the number of loci sampled is limited, the geographic structure of foxtail millet populations will require further exploration, but current data suggest a high degree of differentiation among geographic regions.Cytological and genetic studies indicated that the wild ancestor of this crop is  viridis , 67–69.  viridis , 70–72. Arabidopsis. thaliana (within 10 kb) [Boechera stricta (within ~ 10 kb or less) [O. sativa) (< 1 kb) [Generally speaking, LD decays more rapidly in outcrossing than selfing species  because n 10 kb)  and Boecor less) ; more ra(< 1 kb) , and ver(< 1 kb) –78. FoxtOur results show that low levels of LD in inbreeding organisms are not exceptional and could be explained by the following hypotheses. First, low LD may be resulted from a species-wide scale of sampling, which incorporates the entire history of polymorphism and recombination within a species over thousands of generations , 75, 77.There are also some previous studies about linkage disequilibrium of foxtail millet. Wang et al. reported similar level of LD as ours (extends to 1 kb) applied 9 gene loci . HoweverS1 Fig(TIF)Click here for additional data file.S1 TableGG = geographic group.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


MASP1, encoding lectin complement pathway enzymes MASP-1 and MASP-3, have thus far been reported to cause 3MC1 syndrome. Only one previously reported mutation affects both MASP-1 and MASP-3, while the other mutations affect only MASP-3.3MC1 syndrome is a rare autosomal recessive disorder characterized by intellectual disability, short stature and distinct craniofacial, umbilical, and sacral anomalies. Five mutations in MASP1. We also measured complement lectin and alternative pathway activities in an affected individual’s serum.We evaluated six unrelated individuals with 3MC1 syndrome and performed Sanger sequencing for all coding exons of We found two novel splice site mutations, c.1012-2A > G in one and c.891 + 1G > T in two probands, and three novel missense mutations, c.1451G > A (p.G484E), c.1657G > A (p.D553N), and c.1987G > T (p.D663Y). Missense mutations affect only MASP-3, while splice site mutations affect both MASP-1 and MASP-3. In a proband who is homozygous for c.891 + 1G > T, we detected a total lack of lectin complement pathway activity and a 2.5-fold lower alternative pathway activity. The phenotype observed in patients whose both MASP-1 and MASP-3 are affected and in those whose only MASP-3 is affected does not appear to be different. We observed structural brain abnormalities, neonatal tooth, a vascular anomaly and a solid lesion in liver as novel phenotypic features of 3MC1 syndrome.Novel mutations and additional phenotypic features expand the genotypic and phenotypic spectrum of 3MC1 syndrome. Although patients with MASP-1 dysfunction in addition to disrupted MASP-3 have an altered complement system, their disease phenotype is not different from those having only MASP-3 dysfunction.The online version of this article (doi:10.1186/s13023-015-0345-3) contains supplementary material, which is available to authorized users. Craniosynostosis, radioulnar synostosis, genital and vesicorenal anomalies, caudal appendage, and umbilical hernia/ omphalocele or periumbilical depression are also often seen in this syndrome. 3MC syndrome has been named by lumping Michels, Malpuech, Mingarelli and Carnevale syndromes, which were originally reported to be separate entities with overlapping features , 2.MASP1 (MIM 600521) and COLEC11 (MIM 612502), which both encode proteins playing important roles in the lectin complement pathway, were found to be responsible for 3MC syndrome [MASP1 and COLEC11 under 3MC1 (MIM 257920) and 3MC2 (MIM 265050) syndromes, respectively. The MASP1 gene is located on chromosome 3q27-28 and gives rise to three alternative splice products playing roles in the lectin pathway of the innate immune system: MASP-1, MASP-3, and MAp44 [Mutations in two genes, syndrome . While t1 MIM 60021 and COC1 MIM 25920 and 3nd MAp44 . In thisSix unrelated probands clinically diagnosed with 3MC syndrome and their parents were included in this study. Written informed consent was obtained from all participants. The study was approved by the Institutional Review Board at the University of Miami.MASP1 gene was performed on all patients and mutated exons were analyzed for cosegregation in their unaffected parents (primer sequences are available on request). A touchdown PCR protocol was used to amplify target DNA region. The resulting amplicons were cleaned up using Sephadex G-25 beads . An ABI PRISM 3730 DNA analyzer (Applied Biosystems) and Big Dye Terminator Cycle Sequencing V3.1 Ready Reaction Kit (Life Technologies) were used to elucidate the DNA sequence. Variants were named according to NM_139125.3.Genomic DNA was extracted from peripheral leukocytes according to standard protocols. Sanger sequencing for all coding exons of the http://www.fruitfly.org/) as a splice site prediction program. Missense variants were analyzed for having a pathogenic prediction score at least in two of the following tools: PolyPhen2, SIFT, and MutationTaster. PhyloP and GERP scores were used to evaluate the levels of conservation of the regions in which the mutations were located [To evaluate the implication of splice site mutations, we used The Berkeley Drosophila Genome Project (PDGP) , patent ductus arteriosus (PDA) and pulmonary hypertension were detected on echocardiogram.th centile), height of 58 cm (<3th centile), and head circumference of 39 cm (<3th centile). Anterior fontanelle was 3 × 4 cm, and posterior fontanelle was still palpable as 1 × 1 cm. In addition to typical facial features of 3MC syndrome, clitoromegaly of 1.5 cm, anterior ectopic anus, prominent coccyx with a sacral pit, and a presacral capillary malformation of 2 × 3 cm were observed. She was hypotonic with normal deep tendon reflexes. Head control was not achieved. Although cell culture from peripheral blood lymphocytes was unsuccessful, interphase FISH study with centromeric probes showed two signals for X chromosome and none for Y. 17-hydroxyprogesteron, androstenedione, cortisol levels, and plasma renin activity were normal. The patient underwent a successful PDA ligation operation, but unfortunately died 2 months later due to pneumonia.Patient 6 was noticed to have bilateral cleft lip and palate, and clitoromegaly at birth. Echocardiography revealed a secundum ASD with a wide PDA. A diagnostic work up including a head ultrasound, serum electrolytes, detailed biochemical screening such as liver enzymes, and renal function tests, were all within normal limits. Abdominal ultrasound showed a heterogeneous hypoechoic solid lesion of 38 × 24 mm, containing serpiginous tubular structures, in the anterior segment of the right lobe of the liver; the left renal agenesis was also observed. On examination at 6 months of age, she had a weight of 3200 g  long term follow ups were available. Patient 2 showed normal developmental milestones from birth to 15 years old and did not experience any significant health problem.Patient 3 presented with recurrent bronchiolitis episodes and her weights, heights and head circumferences were in lower limits of normal at the age of 6. She had normal cognitive functions.rd centile), weight was 21.7 kg (3–10th centile), and head circumference was 52 cm (25–50th centile). A WISC-R IQ score was 98. At age 12, she was evaluated for short stature, and was found to have growth hormone deficiency. Growth hormone treatment was administered until age 17. At 22 years of age, her height was 153 cm (3–10th centile), weight was 40 kg (<3th centile), and head circumference was 54.5 cm (3–10th centile). She was working as a clerk in a state institution, and did not report additional health problems.Patient 5 underwent an operation for excision of the cystic tumor, tethered cord, and bone repair because of her lumbosacral anomalies at the age of 2. Her neuromotor development was on target except for a mild speech delay. Audiometric evaluation revealed moderate conductive hearing loss on the right, due to chronic otitis media. At 9 years and 2 months of age, her height was 122.5 cm  and ExAC databases (http://exac.broadinstitute.org/). All missense mutations were predicted to be disease causing when tested using in silico prediction tools  and were located in very conserved regions. According to an in silico protein structure analysis (HOPE) [The other three mutations were missense , located in exon 12. None of these was present as homozygous variation in the EVS (s (HOPE) , of thess (HOPE) . All varIn the present report we tested the serum levels of MASP-1, MASP-3 and MAp44 of patient 3, who is homozygous for c.891 + 1G > T. For all three proteins we did not detect a signal. We further found that the patient was MBL deficient, i.e. MBL level was below 100 ng/ml .In the literature there have been reports on the involvement of MASP-1 and MASP-3 on the alternative pathway activity of serum , 21. We MASP1 mutations cause 3MC syndrome in two unrelated families [The MBL-associated serine proteases (MASPs), MASP-1, MASP-2 and MASP-3, are proteins playing crucial roles in the lectin complement pathway . In addifamilies , 22.MASP1 mutations. Three missense variants found in this study affect only the SP domain of MASP-3 and support the earlier observation that the disruption of MASP-3 with normal MASP-1 and MAp44 is sufficient to cause 3MC syndrome (Fig. MASP1, MASP-1, MASP-3 and MAp44 [MASP1 which gave rise to no MASP-1, MASP-3 or MAp44 in plasma of the patient. In the present report we describe that similarly we cannot measure any MASP-1, MASP-3 or MAp44 in the plasma of patient 3, who is homozygous for c.891 + 1G > T.Here we report five additional in vivo, and they noted that this occurred in the absence of detectable mature factor D [It was previously reported in mice that MASP-1 was the protease responsible for activating the proenzyme profactor D to the active enzyme Factor D, which is essential for activities of the alternative pathway of the complement system . This fifactor D . Furtherfactor D . Commentfactor D  and repofactor D . Rabbit factor D , the actfactor D , 28. In MASP1 protein products.The 3MC1 syndrome is a rare disorder that affects many different tissues and organs (Table MASP1 mutation in the literature was reported to have anterior chamber anomalies [MASP1 mutations. A number of eye abnormalities, such as blepharophimosis, ptosis, lower eyelid eversion, anterior chamber abnormalities, have been reported in 3MC patients [Although only one patient with nomalies , 4, Petenomalies . Moleculpatients . The majOther clinical features not reported in this syndrome until this study include a vascular anomaly and a solid lesion in liver, absent olfactory bulbs, small pituitary, neonatal tooth, and severe narrowing of foramen magnum. These additional features might be coincidental and need further studies for conclusion. In this study, MRI studies revealed cranial and spinal morphological abnormalities in patients 1 and 5 and a neurosurgical operation was performed on patient 5 for symptomatic spinal abnormality. We thus recommend imaging studies for detecting cranial and spinal anomalies in patients with 3MC1 syndrome.3MC syndrome includes overlapping features of four syndromes, which were considered to be separate entities previously. Regarding the previous nosology, when we evaluate the patients presented here, Michels or Malpuech syndrome would be the most probable diagnosis for patients 1, 4 and 5, Carnavale syndrome for patient 2, Michels or Carnavale syndrome for patient 3.MASP1, 10 patients had mutations in COLEC11. While comparison of phenotypic features of patients with COLEC11 and MASP1 mutations would be speculative because of the limited number of patients, craniosynostosis and intellectual disability were reported in 27.2 and 28.5 % of patients with MASP1 mutations, respectively, while they were observed in 60 and 70 % of patients with COLEC11 mutations, respectively. Conversely, hearing loss, cleft lip/palate, genital anomalies, vesicorenal anomalies, and caudal appendages were reported to be more common in patients with MASP1 mutations, compared to those with COLEC11 mutations [Up to date, there have been 26 3MC patients reported, including our 6 patients, whose causative mutations are identified. Sixteen  of them had mutations in utations , 22.MASP1 mutations and phenotypic features expand the genotypic and phenotypic spectrum of 3MC1 syndrome. Patients with disrupted MASP-1 and MASP-3 have altered complement system, while their phenotype doesn’t appear to be different from those with only disrupted MASP-3. We confirm that an alternative pathway activity is present in the MASP-1, MASP-3 and MAp44 deficient serum from a patient and that lectin pathway activity in serum from this patient is lacking.Novel


The dose/rate for OC99 was established from a pilot study conducted in six bled dogs. Subsequently twenty-four dogs were randomly assigned to one of four groups (n = 6 per group) and administered: 0.0, 0.065, 0.325, or 0.65 g/kg of OC99 combined with 10 mL/kg lactated Ringers solution administered in conjunction with 20 mL/kg Hextend IV over 60 minutes. The administration of 0.325 g/kg and 0.65 g/kg OC99 produced plasma hemoglobin concentrations of 0.63 ± 0.01 and 1.11 ± 0.02 g/dL, respectively, improved systemic hemodynamics, enhanced tPO2, and restored lactate and base excess values compared to 0.0 and 0.065 g/kg OC99. The administration of 0.65 g/kg OC99 significantly elevated pulmonary artery pressure. Plasma hemoglobin concentrations of OC99 ranging from 0.3 to 1.1 g/dL, in conjunction with colloid based fluid resuscitation, normalized clinical surrogates of tissue oxygen debt, improved tPO2, and avoided clinically relevant increases in pulmonary artery pressure.We determined the dose-dependent effects of OC99, a novel, stabilized hemoglobin-based oxygen-carrier, on hemodynamics, systemic and pulmonary artery pressures, surrogates of tissue oxygen debt , and tissue oxygen tension (tPO Multle shock , 5, 6.2) carrying blood substitutes (O2 therapeutics) were developed to address concerns regarding the availability, storage, transport, and safety of the human blood supply [2 carriers (HBOCs) and gas-carrying fluids have evolved with most demonstrating the ability to improve hemodynamics, restore tissue oxygen tension, reduce transfusion requirements, and delay definitive care compared to the administration of nonoxygen carrying crystalloids or colloids [2 species, effects that have been primarily attributed to nitric oxide scavenging and extravasation, high P50, and hyperoxygenation [2 can be maintained providing plasma Hb concentrations (Hbp) do not exceed 1.4 g/dL [Oxygen  in hemorrhaged dogs was identified [2 without producing pulmonary hypertension.We investigated the acute dose response characteristics of a low concentration (6.5 g/dL) of highly purified PBH solution  on systemic and pulmonary arterial blood pressures, tissue oxygenation, and the metabolic correlates of oxygen debt in an experimental model of controlled hemorrhagic shock in dogs. OC99 dose selection was based upon pilot experiments wherein the Hbentified . We hypoAll procedures were reviewed and approved by the Institutional Animal Care and Use Committee at QTest Labs and complied with federal guidelines for the care and use of laboratory animals and were conducted according to the principles in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, Eighth Edition  and the Animal Welfare Act as amended.Thirty-three young mature male Beagle dogs were acclimated to the animal facility during a seven-day quarantine period. All dogs were determined to be in good health based upon a physical examination, body temperature, heart and respiratory rate, general appearance, auscultation of the thoracic cavity, and palpation of the abdominal cavity. Dogs were individually housed in cages and exercised daily during the in-life phase of the study. The environmental conditions of the animal room were a light/dark cycle set to maintain approximately 12 hours of light and 12 hours of dark each day and a room temperature and relative humidity of 64 to 74°F and 30 to 60%, respectively. The room temperature and humidity were recorded hourly and monitored daily. The dogs were provided commercial dry dog food and water ad libitum.2) partial pressure (PaCO2) between 38 and 48 mm Hg. Anesthesia was initially maintained with 1.5% isoflurane in room air. Core body temperature ranged from 37 to 39°C and was maintained with temperature-controlled warm air blankets. The dogs were initially placed in right lateral recumbency, skin electrodes were attached, and a catheter introducer was placed in the left jugular vein through which a thermodilution catheter was introduced and positioned with the thermistor in the main pulmonary artery. The left carotid artery was isolated and a dual tipped pressure sensing catheter  was positioned in the ascending aorta and left ventricle, respectively. These catheters permitted the determination of core body temperature (°C), pulmonary artery pressure (mm Hg), cardiac output (L/min) via thermodilution , and aortic pressure . The left or right femoral artery and vein were isolated and catheterized in order to permit hemorrhage and blood sampling, respectively. Once instrumented the dog was repositioned in dorsal recumbency for the remainder of the experiment. A 5 to 6 cm midline incision was made through the skin and linea alba and an intestinal loop of gut (jejunum) was exposed. A 20-gauge needle was used to establish a tract between the serosa and muscular layers of the intestine for probe placement and determination of intestinal mucosa tissue-oxygen tension (mm Hg) via optical fluorescence . The dogs were stabilized for approximately 30–45 minutes following instrumentation and administered 100 units/kg/h IV heparin in order to facilitate hemorrhage and blood sampling. Baseline data were recorded and blood samples obtained for determination of blood pH, gases  and biochemical data . Each dog was then hemorrhaged to a mean arterial pressure ranging from 30 to 40 mm Hg by controlled bleeding . Hemorrhaged blood was collected into a heparinized collection bag and weighed. Arterial lactate and base excess and their ratio were monitored every 15 minutes during bleeding.Vascular catheters were placed in the cephalic and saphenous veins for administration of anesthetic drugs  and treatments (saphenous vein), respectively. Dogs were administered butorphanol, 0.25 mg/kg IV, and anesthetized with propofol, 3.5 to 6 mg/kg IV, to facilitate orotracheal intubation. All dogs were mechanically ventilated with a volume-cycled ventilator  in order to maintain the arterial blood carbon dioxide (COp (g/dL), pH, PO2 (mm Hg), PCO2 (mm Hg), lactate (mmol/L), BE (mEq/L), venous blood oxygen saturation (SvO2: %), and intestinal mucosa tPO2 (mm Hg) were determined immediately before (baseline) and at 5 (except CO), 10, 20, and 30 minutes after each infusion. Additional data were obtained at 60 and 90 minutes after the last infusion.A pilot potency assay was performed in six anesthetized and hemorrhaged dogs weighing 10 ± 0.3 kg to determine the plasma hemoglobin concentration (g/dL) that produced a pulmonary artery pressure >25 mm Hg. We considered, a priori, a mean pulmonary artery pressure >25 mm Hg to be hypertensive . Six dogA controlled, dose response study was performed in 24 dogs weighing 10.7 ± 0.2 kg. Six dogs were assigned to one of four treatment groups (6 dogs per group) using a twofold randomization process. Dogs were randomly selected from the colony of enrolled animals. The dogs were bled to a predetermined degree of oxygen debt (oxygen debt ≈ 110 mL/kg) that predicated a 50% mortality based upon metabolic surrogates . Subsequently, each dog was randomly assigned to receive one of the four treatments. Randomization lists (dog and group) were generated using statistical software . All test treatments were administered  for 60 minutes through the catheter placed in the saphenous vein.The test articles included OC99 and two non-oxygen carrying control solutions: Lactated Ringer's Solution  and 6% HEX. OC99 is a highly purified and polymerized  bovine Hb dissolved in Ringer's Acetate solution to a concentration of 6.5 ± 0.5 g/dL. OC99 contains greater than 85% tetramers and has a shelf life of 12 months at 25°C . Six dogp, pH, PO2, PCO2, arterial lactate (La), base excess (BE), and SvO2. The COP was determined at 3 time points: baseline (before hemorrhage), the end of hemorrhage immediately before fluid resuscitation, and at the end of treatment. Hemodynamic parameters were recorded at baseline, immediately before fluid resuscitation, and every 20 minutes after the start of treatment. Data included an electrocardiogram (ECG), heart rate , mean right-atrial  and pulmonary-artery  systolic (SAP), diastolic (DAP), and mean (MAP) arterial pressures (mm Hg), and left ventricular end systolic  and end diastolic pressures .Hematological and biochemical data were determined from arterial and venous whole blood samples collected before hemorrhage, every 15 min during bleeding and at predetermined times for the duration of the 3-hour experiment. The blood samples were analyzed for HCT and Hbp concentration, tissue oxygenation tPO2 arterial lactate, base deficit, and SvO2. All statistical tests were performed using computer software . Statistical analyses were performed separately for each parameter. All data are reported as the mean ± the standard error of the mean (SEM). The analysis of variance for repeated measures (ANOVA) included dose, time, and the interaction of dose and time as fixed effects, and the animal as a random effect. The model took into account the correlations in the postdosing averages that were present due to monitoring the same animals over multiple time points following a given dosing through the use of a covariance structure. If the F-value exceeded the critical value, post hoc (Holm-Sidak) tests were conducted within the ANOVA to test for significant differences among dose groups/time intervals. A Benjamini and Hochberg method was utilized to control for the false discovery rate for these tests across all time intervals. Bonferroni corrections were applied, when applicable, to the significance levels of multiple comparisons in order to control the overall error rate associated with each dose group comparisons at a given time interval to no more than 5%. The significant level was P < 0.05 and values below P < 0.1 level were considered as indication of a notable trend.The primary end-points of this study were changes in HbP < 0.05) increases in heart rate (HR: 123 ± 5 to 195 ± 16 bpm). The tPO2 was significantly decreased (31 ± 8 to 2 ± 3 mm Hg) after hemorrhage. Systemic and pulmonary vascular resistance values significantly increased after hemorrhage . Blood arterial lactate significantly increased (La: 1.4 ± 0.1 to 4.5 ± 0.5 mM/L) while arterial base excess (BE: −7 ± 1 to −13 ± 1 mM/L) and venous oxygen saturation  decreased significantly.Controlled hemorrhage (51 ± 3 mL/kg) produced anticipated hemodynamic and blood chemical changes consistent with the development of hypovolemic shock and oxygen imbalance. Mean systemic and pulmonary arterial blood pressure significantly decreased . Cardiac output (CO: 1.4 ± 0.1 to 0.4 ± 0.0 L/min) and left ventricular end diastolic pressure (LVEDP: 10 ± 1 to 1 ± 1 mm Hg) significantly decreased associated with significant  administered over 10 minutes were 0.84, 0.34, and 0.20 g/dL and 1.26, 0.48, and 0.27 g/dL, respectively. The infusion of OC99 produced Hbp concentration dependent increases in tPO2 in the intestinal mucosa. The greatest increases in tPO2 occurred at a Hbp concentration averaging 0.84 ± 0.13 g/dL. The administration of HEX infusions containing OC99 produced Hbp concentrations below 0.3 g/dL resulting in little or no change in PAP or tPO2  and when Hbp concentration was less than 1.1 g/dL. Subsequent dose response experiments were designed to encompass OC99 Hbp concentrations (0.3–1.1 gm/dL) that improved tPO2 without producing pulmonary hypertension.Infusion of OC99 produced dose and rate of administration dependent increases in Hbpressure . PulmonaTwenty-seven dogs were subjected to controlled hemorrhage. Two animals died during or immediately after hemorrhage and were not considered for analysis, while one dog did not meet entry criteria for lactate and base-deficit.P < 0.05) decreased and heart rate (210 ± 5 bpm) was significantly increased in all dogs. Systemic and pulmonary vascular resistance significantly increased after hemorrhage (2) significantly increased. Intestinal tPO2 significantly decreased and arterial LA and BE were significantly increased  produced acute significant changes in the magnitude and sustainability of hemodynamic and blood chemical values in all fluid treatment groups. Larger doses of OC99  normalized or improved systemic pressures, CO and biochemical markers of anaerobic metabolism , and tPO2 . Increasased PAP . The admased PAP .2 were independent of Hct and COP and dependent upon OC99 Hbp concentration  among groups . Increasntration . Posttre/kg OC99 . Dogs ad/kg OC99 .2 without producing significant increases in pulmonary artery blood pressure. The administration of 0.650 g/kg OC99 (plasma Hb ~1.0 g/dL) produced further increases in tPO2 and increased mean pulmonary arterial pressure to values greater than 25 mm Hg.We determined the effects of increasing circulating concentrations of a highly purified PBH (OC99) on hemodynamics, metabolic correlates of oxygen debt , and tissue oxygenation in a large animal model of controlled hemorrhagic shock. The administration of 0.325 g/kg of OC99 (plasma Hb ~0.6 g/dL) in conjunction with a colloid (Hextend) improved hemodynamics and tPO2, and survival [+2) limiting the ability of Hb to carry oxygen and facilitating the production of tissue injury by free radicals [2 decreases [Multiple studies conducted in various experimental animal shock  models have demonstrated the ability of various HBOC solutions to improve hemodynamics, blood oxygen content, oxygen delivery, tPOsurvival , 16–21. survival . Oxidatiradicals . Vasoconradicals , 23. Hypecreases . Additioecreases . Systemiecreases , 24.p, and enhanced tissue oxygen tension [2 [p concentration and their subsequent efficacy and safety. This information combined with knowledge of the increased sensitivity of the pulmonary vasculature to the vasoconstrictive effects of HBOC solutions support the opinion that HBOC solutions should be considered as drugs and that their administration should be guided by the Hbp concentration, pharmacokinetics, and the setting (anemia versus hemorrhage) in which they are administered [Experimental studies in anesthetized dogs and swine that have been bled to a mean systemic arterial pressure of 50 mm Hg and conscious hemorrhaged and anemic hamsters implanted with window-chambers in their cheek pouches in order to visualize the microcirculation indicate that the administration of a 12-13 g/dL HBOC solution failed to return cardiac output and oxygen delivery to baseline values and reduced functional capillary density (the numbers/length of capillaries per unit area of tissue with visible RBC transit), respectively, despite an increase in blood oxygen content , 25, 26.nsion [2 . These snistered .2. Importantly, dogs administered 0.325 and 0.650 g/kg OC99 demonstrated increases in tPO2 to values previously demonstrated, in skeletal muscle and gut [2 leading to better recovery after hemorrhage compared to control dogs. Notably, the administration of 0.325 g/kg OC99 did not produce clinically relevant increases in PAP  while 0.65 g/kg OC99 resulted in a pulmonary vasopressor effect by the end of the dosing period . Experimental studies in hemorrhaged swine and newborn lambs suggest that the pulmonary vasopressor effect is caused by cell free Hbp and NO dysregulation and that breathing low levels (30–80 ppm) of NO or administration of various NO donors  is palliative [We utilized a validated hypovolemic hemorrhagic acute shock model designed to produce severe oxygen imbalance, resulting in the accumulation of oxygen debt resulting in a mortality >50% within 2 to 3 hours , 30. Our and gut , 32, to lliative , 34. Dislliative .p concentrations ranging from approximately 0.4 g/dL and 1.1 g/dL, respectively, and that Hbp values ≤0.8 g/dL do not produce a significant increase in pulmonary artery pressure. The data support and extend earlier studies investigating the systemic, pulmonary, and microcirculatory effects of HBOCs by identifying that Hbp concentrations greater than 1.1 g/dL may cause systemic and pulmonary vasoconstriction limiting increases in tPO2 [Our studies indicate that the administration of 0.325 and 0.650 g/kg OC99 produced Hb in tPO2 , 36. Altp concentrations augment tissue oxygen tension without producing adverse systemic or pulmonary vascular consequences. The administration of OC99 in conjunction with standard colloid-based fluid resuscitation produced dose-dependent increases in Hbp concentration, systemic and pulmonary artery pressures, oxygen delivery, tPO2, and normalized clinical surrogates of tissue oxygen debt in a dog model of severe controlled hemorrhagic shock. These effects were independent of Hct and COP and suggest that OC99 can be administered as an adjunct to standard replacement fluid therapy to improve tissue oxygenation following hemorrhage. The data also suggest that pulmonary artery pressure is a more sensitive indicator of the response to cell-free Hbp concentration than systemic pressure and should be monitored in order to avoid the potentially detrimental effects of vasoconstriction.In conclusion, relatively low OC99 Hb


Plasmodium falciparum PfMDR1 transporter to anti-malarial drug resistance acquisition. In this study, the possible involvement of two related MDR proteins, PfMDR2 and PfMDR5, to emerging drug resistance is investigated by a reverse genetics approach.Membrane-associated ATP binding cassette (ABC) transport proteins hydrolyze ATP in order to translocate a broad spectrum of substrates, from single ions to macromolecules across membranes. In humans, members from this transport family have been linked to drug resistance phenotypes, e.g., tumour resistance by enhanced export of chemotherapeutic agents from cancer cells due to gene amplifications or polymorphisms in multidrug resistance (MDR) protein 1. Similar mechanisms have linked the P. falciparum parasites lacking the Pfmdr2 (PfΔmdr2) or Pfmdr5 (PfΔmdr5) gene. Plasmodium lactate dehydrogenase activity was used as read-out for sensitivity to artemisinin (ART), atovaquone (ATO), dihydroartemisinin (DHA), chloroquine (CQ), lumefantrine (LUM), mefloquine (MQ), and quinine (QN). Differences in half maximal inhibitory concentration (IC50) values between wild type and each mutant line were determined using a paired t-test.A homologous double crossover strategy was used to generate PfΔmdr2 and PfΔmdr5 clones were capable of asexual multiplication. Upon drug exposure, PfΔmdr2 showed a marginally decreased sensitivity to ATO (IC50 of 1.2 nM to 1.8 nM), MQ (124 nM to 185 nM) and QN (40 nM to 70 nM), as compared to wild type (NF54) parasites. On the other hand, PfΔmdr5 showed slightly increased sensitivity to ART (IC50 of 26 nM to 19 nM).Both Pfmdr2 and Pfmdr5 are dispensable for blood stage development while the deletion lines show altered sensitivity profiles to commonly used anti-malarial drugs. The findings show for the first time that next to PfMDR2, the PfMDR5 transport protein could play a role in emerging drug resistance.Both The online version of this article (doi:10.1186/s12936-015-0581-y) contains supplementary material, which is available to authorized users. MDR1 gene amplifications or polymorphisms [Plasmodium falciparum has been shown to contain 16 ABC family members in its genome [ATP binding cassette (ABC) transporters are membrane-bound proteins that translocate a wide variety of substrates, including sugars, peptides, inorganic ions, proteins, and drugs across membranes by consumption of ATP, even against high concentration gradients ,2. Theseorphisms -7. Baseds genome , some ofPlasmodium falciparum causes the most dangerous type of malaria, a life-threatening disease that affected 198 million individuals with an estimated 584,000 fatalities in 2013, especially African children under five [P. falciparum mdr1 and mrp1 genes have been associated with, e.g., artemisinin, chloroquine, lumefantrine, mefloquine, and quinine resistance [Pfmdr6 were shown to alter drug sensitivity to artesunate and piperaquine [PfMDR2 [in vitro pyrimethamine resistance, which however, depended on dhfr mutation status [P. falciparum drug resistance acquisition. Although the contribution of the MDR family in emerging drug resistance seems evident, as of yet no studies have assessed the effect of Pfmdr deletion lines on altered drug sensitivity.der five ,11. Wherder five . Furthersistance . In addieraquine , while a [PfMDR2  was linkn status . These fPfmdr2 and Pfmdr5 were targeted for gene deletion. Subsequently, the involvement of two P. falciparum genes, Pfmdr2 and Pfmdr5, on drug sensitivity to seven well-established anti-malarial drugs, including artemisinin (ART), atovaquone (ATO), dihydroartemisinin (DHA), chloroquine (CQ), lumefantrine (LUM), mefloquine (MQ), and quinine (QN), belonging to four different drug classes , was studied [Here, using a reverse genetics approach, both  studied .Plasmodium falciparum wild type (NF54) and mutant lines PfΔmdr2 and PfΔmdr5 were cultured in a semi-automated system as described previously [in vitro in 5% haematocrit in RPMI medium supplemented with human serum (complete medium), which was changed twice daily. Human red blood cells were refreshed weekly and obtained from the Dutch national blood bank .eviously ,27. BriePlasmodium falciparum mdr2 and mdr5 genes were stably deleted according to a homologous double crossover strategy [Pf52 construct in which the homologous regions were replaced with mdr2 and mdr5 target regions (TRs), respectively [P. falciparum NF54 genomic DNA (gDNA) using PfuUltra II Fusion HS DNA Polymerase  with primers (P) P9 and P10 for the 5′ target region and primers P11 and P12 for the 3′ TR of mdr2, respectively. Similarly, primers P13-P16 were used to generate TRs for mdr5 -Pf52 construct using BssHII plus BsiWI and XmaI plus NheI restriction enzymes, respectively. This resulted in two deletion constructs, pHHT-FRT-(GFP)-Pfmdr2 and pHHT-FRT-(GFP)-Pfmdr5. Transfection and selection were performed as previously described [mdr mutant lines. Both lines were subsequently transfected with the pMV-FLPe construct in order to excise heterologous DNA (hdhfr::gfp selectable marker) flanked by flippase recognition target (FRT) sites using enhanced flippase (FLPe) recombinase [PfΔmdr2 and PfΔmdr5 were derived for downstream analysis.strategy ,29. Deleectively . These rescribed , resultiombinase . Followimdr2 and mdr5, respectively, were used to analyse wild type and mutant DNA using LR-PCR for correct double homologous crossover integration. The LR-PCR started with an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 43.5-50°C  for 30 sec, elongation at 60°C for 15 min [mdr genes . Denaturation at 94°C for 2 min was followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 52°C for 45 sec and elongation at 60°C for 30 sec, followed by a final elongation step at 60°C for 2 min.Genotype analysis of the parasite mutants was performed using Expand Long Range dNTPack  (LR-PCR). Mixed asexual blood stage gDNA from wild type and deletion parasites was isolated using the QIAamp DNA Blood Mini Kit . Primers P1 and P2 plus P5 and P6  method as described previously [50) values were determined by non-linear regression fitting of dose–response inhibition curves with variable slopes using GraphPad Prism version 5.03 . For each anti-malarial drug, Hill slopes were fixed to the average of all non-ambiguous slopes for each mdr mutant and their wild type control. Comparisons for significant differences between the obtained logIC50-values of wild type and mutant groups were carried out by a paired t-test. Finally, the plotted curves represent the combined averages of each individual anti-malarial assay per line, where the top of each curve fit was set to 100%.ART, ATO, CQ, DHA, LUM, MQ, and QN were all purchased from Sigma-Aldrich Chemie B.V. . ART and QN were dissolved in methanol, ATO, DHA, LUM, and MQ were dissolved in DMSO, CQ was dissolved in complete medium (described above) and serial dilutions of all drugs were stored at −20°C and thawed prior to use. Anti-malarial sensitivity assays were performed in at least three independent consecutive experiments using a slightly modified eviously . In shoreviously , the 96-P. falciparum MDR transport proteins, PfMDR2 and PfMDR5, were studied by a reverse genetics approach. In order to generate stable Pfmdr2 and Pfmdr5 gene deletion asexual stage mutants, a homologous double crossover strategy was used [Pfmdr2 and pHHT-FRT-(GFP)-Pfmdr5 deletion constructs  and Pfmdr5 (PfΔmdr5) genes. LR-PCR using primers (P) P1 and P2 flanking the target regions resulted in the expected amplification products of 5,571 bp and 2,553 bp for gDNA from NF54 wild type and PfΔmdr2 parasites, respectively  and altered sensitivity to any of the drugs was assessed compared to wild type (NF54). No difference in sensitivity to ART, CQ, DHA, and LUM was observed for the PfΔmdr2 mutant  marginally decreased, as is shown by an increase in average IC50 from 1.2 nM (95% CI 0.9–1.5 nM) (wild type) to 1.8 nM (95% CI 1.3–2.5 nM)   and QN (n = 4), both belonging to the aryl-amino alcohol class of anti-malarials [50 of 124 nM (95% CI 86–180 nM) against MQ in wild type to 185 nM (95% CI 145–234 nM) in PfΔmdr2 parasites (p = 0.004), and an IC50 of 40 nM (95% CI 27–60 nM) in wild type to 70 nM (95% CI 57–85 nM) in PfΔmdr2 parasites for QN (p = 0.022)  from an average of 26 nM (95% CI 19–36 nM) in wild type to 19 nM (95% CI 14–27 nM) in PfΔmdr5 parasites, highlighting ART as being the single compound showing an increase in sensitivity following mdr deletion in this study  the anti-malarial is a substrate for the wild type transporter; and, ii) the wild type MDR protein is located on the plasma membrane, which in the case of mutant lines would result in accumulation of the compounds within the parasite followed by its demise.Increasing P. falciparum mdr2 induced some decrease in sensitivity to the anti-malarials ATO, MQ and QN. As PfMDR2 has been reported to localize to the plasma membrane of the parasite [PfMDR2 expression on the food vacuole has also been proposed [PfMDR2 might prevent accumulation of these drugs in the digestive vacuole, resulting in decreased sensitivity of PfΔmdr2 to MQ and QN as shown here. In contradiction to these results, it has been reported that MQ and potentially QN prevent uptake of host cell hemoglobin by inhibiting endocytosis [P. falciparum to sequester these drugs in the food vacuole [Pfmdr2 deletion. While LUM belongs to the same drug class as MQ and QN [PfΔmdr2 mutant does not show the same trend in sensitivity to LUM as compared to the latter drugs. This might indicate that LUM is not a substrate for Pfmdr2 or (less likely) it has a different site of action, which currently is undetermined. ATO exerts its anti-malarial activity by inhibiting the parasite’s oxygen consumption in mitochondria [PfMDR2 has not been shown, further research may link PfMDR2 deletion to decreased ATO sensitivity. Moreover, it would be interesting to test several drug combinations to observe synergistic effects. These findings indicate that PfMDR2 may be involved in drug sensitivity either directly or indirectly via drug substrates.In contrast to the hypothesis above, deletion of parasite , deletinproposed ,35. MQ aproposed  and therocytosis ,38 and t vacuole . HoweverQ and QN , the PfΔchondria . While mP. falciparum mdr5, only sensitivity to ART was altered. In concordance with the hypothesis, parasites lacking PfMDR5 became slightly more sensitive to ART, suggesting that this drug is a possible substrate for the PfMDR5 transport protein. As PfMDR5 has previously been shown to localize to the parasite’s plasma membrane [PfΔmdr5 versus wild type parasites to DHA, the active metabolite of artemisinin derivatives [Upon deletion of membrane , lackingivatives , highligPlasmodium falciparum does not require PfMDR2 and PfMDR5 to progress through the asexual multiplication cycle. Additionally, deletion of PfMDR2 resulted in a minor decreased sensitivity phenotype to ATO, MQ and QN. Therefore, P. falciparum might increase its resistance to these anti-malarials by modulating PfMDR2 in order to prevent these drugs from reaching their proposed target sites. Moreover, the PfMDR5 deletion mutant became slightly more sensitive upon exposure to ART, suggesting that over-expression or enhanced activity of this protein might contribute to ART resistance.


Triticum aestivum Wangshuibai, germplasms T. monococcum TA2026 and TA2033. A total of 1,233,792,170,880 and 263,040 clones were picked and arrayed in 384-well plates. On the basis of genome sizes of 16.8 Gb for hexaploid wheat and 5.6 Gb for diploid wheat, the three libraries represented 9.05-, 2.60-, and 3.71-fold coverage of the haploid genomes, respectively. An improved descending pooling system for BAC libraries screening was established. This improved strategy can save 80% of the time and 68% of polymerase chain reaction (PCR) with the same successful rate as the universal 6D pooling strategy.We have constructed three bacterial artificial chromosome (BAC) libraries of wheat cultivar  Wheat is one of the most important food crops in the world . The lacTriticeae species including T.urartu, Ae.speltoides and Ae. tauschii , N for clone numbers, I for insert size, and GS for genome size [Rht-1 and another 18 SSR markers (BamHI and HindIII). All of the primer pairs except BARC61 recovered at least one positive pool . For the descending pooling strategy, the positive clone relative to marker BARC95 should theoretically be in TP17 as described in the Section Material and Methods. As shown in 17 of descending pools showed a positive band. The same results were observed when using marker WMC413. These perfect matches between the two different pooling strategies confirmed the high efficiency of the descending pooling strategy , an indigenous Chinese cultivar with a high level of scab resistance, and two more diploid wheat Triticummonococcum accessions, TA2026 and TA2033, were used for library construction. TA2026 and TA2033 were kindly provided by Dr. B.S. Gill . These two types of wheat harbor the powdery mildew resistance genes pm2026 [mlm2033 [Common wheat Wangshuibai  buffer using a Bio-Rad model 422 Electro-Eluter .Nuclei were isolated from 20 to 30 g etiolated young leaves, as described by Peterson et al.  and Zhan [et al. . High-mo−1, whereas two rounds of size selection reduced the concentration to 2–3 ng·μL−1. To absorb excess water from the DNA solution, 30% PEG 8000 (polyethylene glycol 8000) was employed. The minimal DNA concentration used for ligation was 3 ng·μL−1.Then, to exclude the small fragments trapped in large fragments, two rounds of size selection were conducted. The concentration of recovered DNA from 5 to 6 μg HMW DNA following one size selection was 10 ng·μLTo optimize the ligation conditions, we established two ligation reactions. One tube was incubated at 16 °C for 24 h followed by 4 °C for another 2 days (3-day ligation) and the other was incubated at 16 °C for 24 h only (1-day ligation). Then the ligation products were transformed into the complement cells, respectively. The insert size and clone number (transformation efficiency) were checked and compared between the two methods. The experiments were repeated three times with three different patches of DNA.−1 chloramphenicol, 0.55 mol/L Isopropyl β-d-Thiogalactoside (IPTG) and 80 μg of X-Gal/mL [–1 chloramphenicol. All 384-well plates were incubated at 37 °C overnight until the media became turbid. Two copies of all clones were made, one for storage and another for descending-pool preparation. Clones in 384-well plates were stored at −80 °C.The eluted DNA was ligated to pIndigoBAC-5 vectors . The mole ratio of vector to insert DNA was 5–10:1. The ligation products were introduced into ElectroMAX™ DH10B™ cells  with a Gene Pulser Xcell™ Total System  at 1.7 kV/cm, 200 Ω in a 0.1-cm cuvette . Transformed cells were spread on LB Petri plates containing 12.5 μg·mLX-Gal/mL . White c–1 chloramphenicol. Plasmids were subsequently isolated and digested with the restriction enzyme NotI, which spanned the insert site. The digested plasmids were separated by PFGE. Band size was estimated on the basis of the Low Range PFG Marker . Clones without insert DNA were considered empty clones.Clones were cultured overnight in 3 mL liquid LB media containing 12.5 μg·mLndhA, rbcL , trnC, hondria, ) that we–1 chloramphenicol. After overnight incubation at 37 °C, the membranes were soaked sequentially in 10% sodium dodecyl sulfate (SDS) for 3 min, then in denaturation solution  for 5 min, and then in neutralizing solution  for 5 min twice. The membranes were then soaked in 2× SSPE buffer  for 5 min. After air-drying, the filters were baked for 2 h at 80 °C in an oven, and then were pre-hybridized with sheared salmon sperm DNA (0.1 mg·mL−1) for 2 h at 65 °C in a buffer containing 40 mM Na2HPO4, 10 mM NaH2PO4, 1% bovine serum albumin (BSA), and 7% SDS, before addition of 32P labeled [Clones were double-spotted onto Hybond-N+  membranes with a 384-pin Microplate replicator . The membranes were incubated with the colony side up on the LB agar media containing 12.5 μg·mL labeled  probes. To reduce the workload of BAC library screening, we designed a descending-pool system. The simple pooling procedure is shown in For screening, PCR was first performed with the tertiary DNA. When a positive signal was obtained for the pool, the DNAs of the individual secondary pools constituting the tertiary pool were amplified. Then the DNAs of the corresponding primary pools and individual clones were extracted for PCR screening.2+, 5 nmol dNTPs, 1 U Taq DNA polymerase , and 5 pmol forward and reverse primers, respectively. PCR was performed using the following procedure: three minutes at 94 °C followed by 36 cycles of 30 s at 94 °C, 30 s at TM for annealing and 30–60 s at 72 °C for extension with a final extension step of 5 min at 72 °C. Genomic DNA was used as positive control. PCR products were separated by electrophoresis on 1%–2% (w/v) agarose gel or by 8% polyacrylamide gel electrophoresis.Markers used in library screening are listed in To estimate the genome coverage by PCR, lanes showing target bands comparable to positive controls were assigned as positive pools. The number of positive pools was equal to the number of positive clones of the corresponding marker ,28.et al. [H; average insert size 102 kb) were arrayed in a grid containing 48 layers × six plates/layer. The six plates of each layer were arranged in a 2 × 3 matrix . The x-axis was parallel to the row line of the plates, the y-axis was vertical to the row line and parallel to the row line of the plates, the z-axis was vertical to the x- and y-axis. Thus, the initial clone located at the near top-left corner was  as shown by the black point in The Six Dimensional (6D) pools were made as described by Klein et al. . A total3 matrix . As eachz-axis, y-axis, and x-axis, respectively. RP was constructed as follows: BAC clones in row  were pooled into RPyz − 1+ . To keep the number of clones in each pool constant (2304 clones per pool), wrapping occurred. That is, when y + z > 49, then 48 was subtracted to get RPy z − 49+  . CP was made as well as RP. BACs in the same column  were pooled into CPxz − 1+ , and wrapped as the same as RP. Clones in column (x) and row (y) of all 48 planes were in DPxy − 1+ . Forty-eight was subtracted when x + y > 49 to give the correct pool number. All pools were started from the plane or line that contained the initial point. That is, clone  was in PPz, FPy, SPx, RPyz − 1/(49)+ , CPxz − 1/(49)+  and DPx y − 1/(49)+ . Each kind of pool has 48 members, thus there were 6D 288 pools.PP, FP, and SP were defined as a set of clones in a plane sharing the same values of The same 288 384-well plates were also constructed for descending pools. Plasmids of column pools were isolated and mixed as super pools.Plasmids of all pools were extracted and diluted as described above and used as templates for PCR amplification. Two SSR markers, WMC413 and BARC95, were used to screen 288 6D pools and 48 descending pools that were constructed from the 288 384-well plates. For descending pools, we amplified the tertiary pools only. The secondary pool was based on the assumption that lanes showing target bands comparable to positive controls were assigned as positive pools.The BAC libraries of three disease-resistant germplasms, Wangshuibai, TA2026 and TA2033, represent a new resource for wheat genome research. The three libraries are being employed in genetic map saturation, cloning of disease resistance, and agronomic traits genes ,46,47,48


This study was designed to investigate whether inhalational exposure of concentrated PM2.5 in a genetically susceptible animal model would result in abnormalities in energy metabolism and exacerbation of peripheral glycemic control.Prior experimental and epidemiologic data support a link between exposure to fine ambient particulate matter  for 5–8 weeks via a whole body exposure system. Glucose tolerance, insulin sensitivity, oxygen consumption and heat production were evaluated. At euthanasia, blood, spleen and visceral adipose tissue were collected to measure inflammatory cells using flow cytometry. Standard immnunohistochemical methods, western blotting and quantitative PCR were used to assess targets of interest.KKay mice, which are susceptible to Type II DM, were assigned to either concentrated ambient PM2.5 exposure influenced energy metabolism including O2 consumption, CO2 production, respiratory exchange ratio and thermogenesis. These changes were accompanied by worsened insulin resistance, visceral adiposity and inflammation in spleen and visceral adipose depots. Plasma adiponectin were decreased in response to PM2.5 exposure while leptin levels increased. PM2.5 exposure resulted in a significant increase in expression of inflammatory genes and decreased UCP1 expression in brown adipose tissue and activated p38 and ERK pathways in the liver of the KKay mice.PM2.5 exposure impairs energy metabolism, concomitant with abnormalities in glucose homeostasis, increased inflammation in insulin responsive organs, brown adipose inflammation and results in imbalance in circulating leptin/adiponectin levels in a genetically susceptible diabetic model. These results provide additional insights into the mechanisms surrounding air pollution mediated susceptibility to Type II DM.Concentrated ambient PM As a co2.5 exposure has been previously demonstrated to result in an increase in macrophages in visceral fat depots with a shift to a pro-inflammatory phenotype characterized by an increase in F4/80 macrophages and a pro-inflammatory “M1 phenotype” typified by an increase in TNFα, IL-6 and a decrease in IL-10, MgI1 gene expression in a diet-induced obesity model[2.5 exposure over long durations in C57BL/6 mice, accompanied by increase in excess oxidative and nitrosative stress[2.5 accelerates diabetes development in a genetically susceptible model and if PM2.5 influences parameters of energy metabolism. In this study, we examined the hypothesis that inhalation of concentrated PM2.5 would result in abnormalities in energy metabolism and exacerbate peripheral glycemic control through inflammatory mechanisms in a genetically susceptible mouse model of Type II DM.Type II DM and its complications are widely believed to result from unbridled inflammatory pathways in insulin-responsive tissues such as the liver, adipose tissue, skeletal muscle and vasculature-5. AdipoKKay mice (5-week-old) were purchased from Jackson Laboratories . All mice were maintained at 21°C on a 12-h light/12-h dark cycle with free access to water and food. The protocols and the use of animals were approved by and in accordance with the Ohio State University Animal Care and Use Committee.2.5 for 6 h/d, 5 d/wk from December 28 2011 to February 28, 2012, for a total of 5weeks or 8weeks, in an exposure system . This exposure system is a mobile trailer versatile aerosol concentration enrichment system which allows concentration of ambient particles 10 ~ 12 folds. Animal exposure and monitoring of the exposure environment and ambient aerosol were performed as previously described[2.5 refers to concentrated ambient particles in this study.KKay mice were exposed by inhalation to either filtered air (FA) or concentrated ambient PM2.5 in the exposure chambers, samples were collected on Teflon filters  and weighed before and after sampling in a temperature- and humidity-controlled weighing room using a Mettler Toledo Excellence Plus XP microbalance. Weight gains were used to calculate exposure concentrations. Elemental composition was determined by high-resolution inductively coupled plasma-mass spectrometry (ICSP-MS) .To calculate exposure mass concentrations of PM2.5, mice were fasted for intra-peritoneal glucose tolerance testing (IPGTT) or insulin tolerance test (ITT) as previously described. Briefly, mice were fasted overnight and dextrose (2 mg/g body weight) was injected intra-peritoneally for IPGTT. Blood sample was collected from the vena caudalis and blood glucose measurement was conducted with an Contour Blood Glucose Meter  at baseline, and 30, 60, 90, and 120minutes after the dextrose injection. When ITT was performed, insulin (0.5 U/kg) was administered by intra-peritoneal injection after 4.5hours fasting. Blood glucose measurement was conducted in the same way as IPGTT with the same Contour Blood Glucose Meter at baseline, and 30, 60, 90, and 120minutes after insulin injection.Before and subsequent to the exposure to FA or PMAdiponectin and leptin levels were determined by the mouse adiponectin kit  or leptin quantification kit  following the manufacturer’s instructions.2 production, respiratory exchange ratio and heat production were measured simultaneously using a computer-controlled, open-circuit Oxymax/CLAMS System . Each mouse was measured individually in a resting state at 22°C in the presence of food and water. Measurements were taken for a 24-h period, including a 12-h light cycle and a 12-h dark cycle. Data were normalized to body weight.The mice were isolated in a semi-sealed cage, and the inner air was aspirated at a constant volume/min. Oxygen consumption, COProtein levels were determined by western blot. Tissues of brown adipose and liver were homogenized with M-PER Mammalian protein extraction reagent  on ice. Equal quantities of proteins from these tissues were loaded and separated by 10% SDS-PAGE. Following transfer to immobilon-P polyvinylidene difluoride (PVDF) membrane and blocking with 5% nonfat milk, the blots were incubated with different primary antibodies: UCP1 , P-AKT (phosphorylation at Ser473)/AKT, PI3K, P-AMPK (phosphorylation at Thr172)/AMPK, MAPK pathway proteins . The immunoblots were then incubated with a secondary antibody conjugated with horseradish peroxidase and visualized with enhanced chemiluminescence, and the autoradiograph was quantitied by densitometric analysis with ImageJ software. β-actin was used as control reference.Epididymal white adipose tissue (epiWAT) was excised, minced, and digested with collagenase type II, and the stromal vascular fraction isolated as described previously. Spleens were isolated, homogenized and suspended in PBS. Bone marrow derived cells were collected by flushing the femur and tibia with PBS. These cells were centrifuged at 500 × g for 5 min. Whole blood was centrifuged at 500 × g, 4°C for 5 min and plasma was collected. The remaining blood cells and the resulting pellets were re-suspended in 1X red blood cell lysis buffer , at room temperature for 3 minutes followed by addition of 1 X PBS and centrifugation. Then, blood cells, spleen cells and bone marrow derived cells were stained with anti-CD11b, anti-7/4 and anti-Gr-1, stromal vascular fraction was stained with anti-CD11c and F4/80, both followed by incubation at room temperature for 45 minutes. Cells were subsequently washed with 1 X PBS and re-suspended in 1% neutral buffered formalin and run by flow cytometry . Data was analyzed using BD FACS Diva software . All antibodies were purchased from Biolegend, Miltenyi Biotec, or BD Bioscience.6/well and treated with anti-CD3  at a concentration of 1 μg/ml for different period. Cells were collected at the end of incubation. Total RNA from brown adipose tissue or splenic cells was isolated with Trizol  according to the manufacturer’s protocol. cDNA was reversely transcribed using High Capacity cDNA Transcription kit . Quantitative polymerase chain reaction (qPCR) was performed in duplicate using the lightcycler 480. “No template”, cDNA negative controls were included for each gene set in all PCR reactions to detect contamination. The expression level for each gene was calculated using the ΔCt method relative to β-actin. The sequences of all primers are listed in Table RT-PCR was performed using RNA extracted from different tissues of the experimental mice. Splenic cells from 8-week exposed KKay mice were seeded in one 24-well plate with 1.0 X 103 25, CaCl22.5, MgCl2 1, KH2PO41.2, and D-glucose 11. After careful removal of adherent connective tissue, the artery was cut into 2 mm-length ring segments. The ring was suspended between two stainless bins in a 5-ml chamber on a Multi Myograph  as previously described. Krebs solution in the bathing chamber was constantly bubbled with 95% O2 - 5% CO2 and maintained at 37°C (pH 7.4). Following 60 mins equilibration, each ring was stretched to 5 mN, a determined optimal resting tone for the development of isometric contraction. After artery contractility was tested, phenylephrine  was used to contract the rings, followed by dose response of acetylcholine (30 nM-10 μM) in aorta. The rings were then rinsed in pre-warmed, oxygenated Krebs solution several times until a stable resting tone returned and finally equilibrated for 60 mins. The resting tone was readjusted to 5mN if necessary. And then, insulin (0.1U/ml – 10 U/ml) -induced relaxation was tested.After the mice were sacrificed by isoflurane inhalation, vascular function of thoracic aorta was evaluated as previously. Briefly2/5% CO2 and maintained at 37°C (pH 7.4). The intraluminal pressure was monitored, and vessel diameter was recorded by a light-inverted microscope with video camera monitored with the automated software Myo-View software . For testing endothelial responsiveness, phenylephrine (5 μM) was added to induce stable contraction at 100 mmHg intraluminal pressure and various doses of acetylcholine was used to test endothelial response at constant shear. The same setting was used for all the groups. After washing the vessel at the end of this intervention, graded doses of phenylephrine was tested to assess response. At the end of experiment, passive dilation was achieved by changing the bathing solution to Ca2+ free Krebs solution. Data was expressed as % of maximal KCl tone for phenylephrine and as % of phenylephrine tone (5 μM) for acetylcholine.For pressure myograph studies, function of segments of resistance mesenteric arteries (2nd order) was evaluated as described. Brieflyt test when 2 groups (PM and FA group) were compared. Concentration-relaxation curves were analyzed by two-way ANOVA followed by Bonferroni’s post-tests. P value of <0.05 was considered statistically significant.Data are expressed as means ± standard error of the mean unless otherwise indicated. Graphpad Prism software (Version 5) was used for Student’s 2.5 concentration at the study site in Columbus, OH, was 8.28 ± 0.65 μg/m3. The mean concentration of PM2.5 was 1.51 ± 0.38 μg/m3 in the filtered air chamber, and 102.9 ± 19.16 μg/m3 in the exposure chamber respectively. This represented 12.4-fold concentration over ambient levels  were analyzed, analysis data from the Dark phase showed significant decrement in respiratory exchange ratio  suggesting alterations in this key adipokine in response to PM2.5 exposure[We measured inflammatory biomarkers in the blood to see if PMexposure. There w2.5-exposed KKay mice. As shown in Figure 2.5 exposure increased CD11b+Gr-1low7/4hi cells, the inflammatory monocytes, in the peripheral circulation , Prdm16 (a master regulator of brown adipocyte specification and differentiation), CPT-1 M , Cidea (a lipid droplet-associated protein with a role in fat storage) and Elovl3 (an elongase enzyme important for elongation of monounsaturated or saturated very long chain fatty acids). However, no differences were observed except for the gene expression of Elovl3 was inhibited in response to PM2.5 exposure  and enhanced inflammatory cell infiltration. Macrophages in the adipose tissue have been shown to increase from 10-15% to 45-60% of total cellular content during obesity and may independently have contributed to expansion of VAT mass[2.5 inhalation as has been shown by us previously[2.5 could potentially be attributable to reduced adiponectin release. Leptin is another major adipokine from adipose tissue. Contrary to the remarkably decreased circulating leptin levels in response to PM2.5 exposure in C57BL/6 mice fed on regular chow[2.5 inhaled KKay mice. The mechanisms by which leptin expression is regulated need further study. The net action of leptin is to inhibit appetite, stimulate thermogenesis, enhance fatty acid oxidation, decrease glucose, and thus reduce body weight and fat through central mechanisms[2.5 exposure, our results raise the possibility of leptin resistance as an additional component and will need further study. Consistent with our findings, Bremer et al. demonstrated the novel observation that adipose tissue in subjects with nascent metabolic syndrome have increased levels of leptin as well as decreased levels of adiponectin and omentin-1, concomitant with increased adipokines such as IL-1, IL-6, IL-8, PAI-1 and MCP1[White adipose tissue is a major source of energy for the human body. The increased epididymal  fat mass in mice exposed to concentrated PMVAT mass. In addiVAT mass. In linechanisms. Recent and MCP1,29. Thus2.5 inhalation did not enhance inflammotary cytokines production in blood, it did result in increased circulating inflammatory monocytes (CD11b+/7/4hi/Gr-1low) accompanied by a corresponding reduction in bone marrow. We have previously demonstrated this same finding in our earlier studies and shown an important role for TLR4 in mobilization of inflammatory subsets of monocytes in response to PM2.5 exposure[+/CD11c+ is a widely used marker to label “classically activated” macrophages that have been demonstrated to play a pathophysiological role in high-fat diet-induced obesity[2.5 exposure, in excess of the effects of high-fat diet alone[2.5  demonstrated an “M1” profile with increased cytokines such as TNFα and IL-6, and reduced expression of IL-10 and N-acetyl-galactosamine specific lectin 1[fmsYFP), we further confirmed enhanced monocyte adhesion in microcirculation of VAT and accumulation in visceral fat[2.5 exposure further appears to be dependent on CCR2 , as it was abolished by genetic ablation of CCR2[+/CD11c+ in eWAT suggest mechanisms similar to those involved in diet mediated aggravation of the VAT infiltration via CCR2-dependent pathways[2.5 exposure[2.5-mediated up-regulation of pro-inflammatory genes in BAT and provided additional explanation for the inhibited energy metabolism. Together with the redirection of Th1/Th2 balance towards a Th1 polarized state in response to PM2.5 exposure (Figure A number of studies have highlighted the innate immune mechanisms as the critical role that is responsible for the pathophysiological abnormalities, including IR. In line with this, our results demonstrated that although PMexposure. This suexposure. It is wexposure. As a hoexposure. F4/80+/ obesity,32-34. Oet alone. Further of CCR2. Thus, tpathways. Except 2.5-exposed C57BL/6 mice fed on regular chaw by Zheng et al.[2.5 in the KKay mice. These differences likely represent differences in strains and genetic susceptibility, in addition to the complex interactions of diet and environmental signals. In keeping a line with our study, Jiao et al. have suggested that the increased hepatic ERK activity may contribute to increased liver glycogen content and decreased energy expenditure in obesity and may play a central role in hepatic glucose and lipid metabolism[p38 MAPK belongs to a family of evolutionarily conserved serine-threonine MAPKs that link extracellular signals to intracellular machinery regulating a plethora of cellular processes. Together with JNK, they are activated by environmental or genotoxic stress and described as stress-activated protein kinases-37. Difftabolism,40. Howetabolism. Therefo2.5 in modulating susceptibility to Type II DM and may have important implications for public health at a global scale.In summary, our results suggest that particulate air pollution exposure resulted in dysregulated metabolism and influenced IR likely through complex pathways involving the liver, visceral and brown adipose tissue in a genetic KKay diabetes model. These findings suggest an important role for PM2.5: Particulate matter <2.5 μm in aerodynamic diameter; Type II DM: Type II diabetes; UCP1: Uncoupling protein 1; eWAT: Epididymal white adipose tissue.BAT: Brown adipose tissue; FA: Filtered air; IR: Insulin resistance; ITT: Insulin tolerance test; IPGTT: Intra-peritoneal glucose tolerance testing; PM: Particulate matter; PMThe authors declare that they have no competing interests.2.5 exposure of the animals. The manuscript was written by CL and revised critically by SR, QS, LS, MP and JTD. All authors read, corrected and approved the manuscript.CL, YB, XX, AW, TW, ZY, SM and MM performed the experiments and contributed to acquisition of data. CL, ZY, and SM analyzed the data and interpreted the results. AW contributed to PM2.5 concentration to which mice were exposed at the study site. Figure S2. Effect of PM2.5 exposure on vascular function from KKay mice. A, Dose–response to phenylephrine, acetylcholine and intraluminal pressure in small mesenteric artery at the end of PM2.5 exposure. B, Dose response to acetylcholine and insulin in in aortic rings precontracted with phenylephrine at the end of PM2.5 exposure. *P < 0.05 compared to FA group. n = 5-8 per group.PMClick here for file


This work aimed at identifying heat protective mechanisms promoted by CO2 in Coffea arabica (cv. Icatu and IPR108) and Coffea canephora cv. Conilon CL153. Plants were grown at 25/20°C (day/night), under 380 or 700 μL CO2 L−1, and then gradually submitted to 31/25, 37/30, and 42/34°C. Relevant heat tolerance up to 37/30°C for both [CO2] and all coffee genotypes was observed, likely supported by the maintenance or increase of the pools of several protective molecules , activities of antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), and the upregulated expression of some genes . However, at 42/34°C a tolerance threshold was reached, mostly in the 380-plants and Icatu. Adjustments in raffinose, lutein, β-carotene, α-tocopherol and HSP70 pools, and the upregulated expression of genes related to protective  and antioxidant  proteins were largely driven by temperature. However, enhanced [CO2] maintained higher activities of GR (Icatu) and CAT (Icatu and IPR108), kept (or even increased) the Cu,Zn-SOD, APX, and CAT activities, and promoted a greater upregulation of those enzyme genes, as well as those related to HSP70, ELIPs, Chaperonins in CL153, and Icatu. These changes likely favored the maintenance of reactive oxygen species (ROS) at controlled levels and contributed to mitigate of photosystem II photoinhibition at the highest temperature. Overall, our results highlighted the important role of enhanced [CO2] on the coffee crop acclimation and sustainability under predicted future global warming scenarios.Modeling studies have predicted that coffee crop will be endangered by future global warming, but recent reports highlighted that high [CO According to recent projections, depending on the greenhouse gas emission scenarios air [CO2] could rise to between 421 and 936 μL CO2 L−1,  by 2100. These will be accompanied by a global surface warming between 0.3 and 1.7°C (best scenario) and 2.6–4.8°C , relative to 1986–2005 , 1chl, singlet oxygen (1O2)]. Furthermore, O2 reduction in photosystems (particularly in PSI) may occur, producing 2O2 and OH·, all of which can cause lipid peroxidation, bleaching of pigments , protein oxidation , enzyme inactivation, and DNA degradation   and C. canephora (22–26°C) , with large worldwide losses on suitable areas and productivity, although coffee production is believed to have already been affected by climate changes due to the occurrence of severe droughts and high temperatures , followed by an in-depth characterization of protective mechanisms, including dynamics of photosynthetic pigments, enzymatic and non-enzymatic antioxidant systems, quantification of raffinose family oligosaccharides (RFOs), and heat-shock protein pools, in addition to assessing the expression of selected genes with potential roles in heat acclimation.We previously demonstrated that both C. arabica L. (cvs. Icatu and IPR108) and C. canephora Pierre ex A. Froehner (cv. Conilon Clone 153—CL153), were evaluated. Plants, ca. 1.5 years of age, were transferred into two walk-in growth chambers  differing in air [CO2] supply: 380 μL CO2 L−1 (380-plants) or 700 μL CO2 L−1 (700-plants). Both groups of plants were then grown for 10 months in 28 L pots in a substrate consisting of a mixture of soil, peat, and sand , as optimized for coffee plants , RH (75%), and photoperiod (12 h) were provided to the plants, and permanently monitored along the whole period. The temperature was then gradually increased from 25/20°C up to 42/34°C in both growth chambers at a rate of 0.5°C day−1, with 7 days of stabilization at 31/25, 37/30, and 42/34°C for evaluations.The plant materials were collected from the same experiments described in Rodrigues et al. , therefo2 and stored at −80°C until analysis. Each biological replicate is a pool of leaves of each plant. Along the experiment the plants were maintained without restrictions in water, nutrients, and root development, the latter evaluated by visual examination at the end of the experiment after removing the plants from their pots  part of each plant. For biochemical evaluations, leaf material was collected after ca. 2 h of illumination from 6 to 8 plants of each genotype and used immediately or flash frozen in liquid NChr), representing the percent reduction in Fv/Fm at each temperature relative to the maximal Fv/Fm obtained during the entire experiment; (B) dynamic photoinhibition (PIDyn), representing the decline in Fv/Fm that is fully reversible overnight, being measured as the percent reduction in midday v/Fm at each temperature, relative to the maximal Fv/Fm from the entire experiment; (C) total photoinhibition . The Fv/Fm and v/Fm and The PSII photoinhibition indexes were calculated according to Werner et al. , and incs et al. .2). Sample processing and subsequent reverse-phase HPLC were carried out as optimized for coffee  5-μm Spherisorb ODS-2 column (250 × 4.6 mm). Detection was performed at 440 nm in an HPLC system  coupled to a diode-array  detector, and identification and quantification were performed using individual sugar standards. The de-epoxidation state, involving xanthophyll cycle components, was calculated as DEPS = (Zeaxanthin (Z) + 0.5 Antheraxanthin (A)) / (Violaxanthin(V) + A + Z).Carotenoids (Car) were assessed from four frozen leaf discs (each 0.5 cmChlorophylls (Chls) from the same samples were extracted in 80% acetone, and quantified spectrophotometrically according to Lichtenthaler .−1 cm−1 for calculations. Glutathione reductase  was assessed through the NADPH oxidation . Catalase  was assessed in whole-leaf extracts (prepared using 200 mg FW of leaf tissue), as described in Fortunato et al. , and a freshly made H2O2 standard curve (0–1 M) was used for quantification.Unless otherwise indicated, enzyme activities were determined in chloroplast extracts (obtained using 3–4 g FW of leaf tissue), as described previously  and α-tocopherol (TOC) determinations were performed on 100 and 200 mg FW of leaf tissue, respectively, based on HPLC methods, as described in detail in Fortunato et al. .−1) diluted in 100 mM carbonate buffer (pH 9.6). After incubating the plates at 37°C during 90 min, the coated plates were washed four times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS/T) and non-specific binding sites blocked by incubation with 300 μL of PBS/T containing 0.1% BSA (PBS/T/BSA) for 60 min at 37°C. After washing, 50 μL of the standard and samples were added and the plates incubated for 90 min at 37°C. Plates were then washed four times and 100 μL of mouse monoclonal anti-HSP70 (1/400) diluted in PBS/T were added. After 60 min on a shaker at 37°C, plates were washed four times and incubated with 100 μL of the diluted  secondary antibody  for 60 min at 37°C. Thereafter, plates were washed and 100 μL of ABTS  substrate were added. After 45 min at 37°C, the reaction was stopped with 50 μL of 1 N H2SO4 and the absorbance was determined at 405 nm using a microplate reader. HSP70 concentrations were detected by comparing sample absorbance with the absorbance of a reference purified HSP70 protein.For the heat shock protein 70 (HSP70) assays, 100 mg FW of frozen leaf tissues per plant were homogenized in 1 mL 200 mM Tris-HCl (pH 8.0), containing 20 mM β-mercaptoethanol, 2 mM dithiothreitol (DTT), 2% triton X-100, 4% (v/v) “Complete-protease inhibitor cocktail” with EDTA, 10% polyvinylpolypyrrolidone, and 10% glycerol. The homogenate was then centrifuged  and the supernatant was used for HSP70 quantification through Enzyme Linked Immunosorbent Assay (ELISA), using Flat-bottomed micro-ELISA plates  as described in Njemini et al.  with minTotal soluble protein contents of the enzyme extracts followed Bradford , using b2O as the eluent (containing 50 mg EDTA-Ca L−1 H2O) and a flow rate of 0.5 mL min−1. Another 20 μL aliquot of each sample was injected through a DionexCarboPac PA1 analytical column  coupled to a DionexCarboPac PA1 Guard (4 × 50 mm) at 20°C. Ultrapure water and 300 mM NaOH were used as eluents  at a 1 mL min−1 flow rate. A refractive index detector  was used for sugar detection. Sugars were quantified using specific standard curves.RFOs evaluation was performed in samples of 150 mg of powdered frozen material, following the HPLC method for soluble sugars described in Ramalho et al. , with soTotal RNA was isolated and quantified as described in Goulao et al. . One mic6), with all procedures performed as described in Goulao et al. (C. canephora sequences (http://www.coffee-genome.org/coffeacanephora) with Primer3 software. Primer length was set to 19–20 bp, with a GC content between 45 and 60% and a melting temperature  between 62 and 65°C. Amplicon length ranges were set to be between 80 and 150 bp. The probability of formation of hairpin structures and primer dimerization was subsequently checked using the Oligo Calculator algorithm. To determine the specificity of the primer pairs used in this study, melting/dissociation curve analysis was performed following the RT-qPCR experiment. A single peak in the obtained melting curve confirmed the specificity of the amplicon, and no signal was detected in the negative controls for all of the tested primers. All qRT-PCR reactions and relative gene expression were calculated after normalization with the reference genes ubiquitin-conjugating enzyme E2 (UbQ2) and eukaryotic initiation factor 4α(eLF-4).Genes related to the antioxidative system and other protection proteins were selected for the expression studies Tables , 6, witho et al. . PrimersP < 0.05) to evaluate the differences between the two growth atmospheric [CO2] and among the several temperature treatments, as well as their interaction, followed by a Tukey's test for mean comparisons, except when otherwise stated. Each ANOVA was performed independently for each of the studied genotypes. Overall, the [CO2] × temperature interaction for most parameters was significant. To the sake of simplicity we do not considered also the comparison between genotypes within each [CO2] and temperature.Data were analyzed using two-way ANOVAs (2 L−1 air) within each genotype. Data analysis was performed using the Relative Expression Software Tool (REST 2009), available at http://www.genequantification.de/rest-2009.html. A 95% confidence level was adopted for all tests.The relative expression ratio of each target gene was computed based on its real-time PCR efficiency and the crossing point (CP) difference of a target sample vs. a control  was mostly unresponsive to temperature and [CO2]  was manifested almost exclusively at 42/34°C  showed significantly higher values in the 380-plants of all genotypes , as well as in the 700-plants of C. arabica genotypes  in CL153 , carotenes, and total Car.At control temperature 25/20°C), enhanced  treatment caused marginal/occasional changes in pigment composition at high temperatures. Such changes were mostly restricted to 700-plants of IPR108, which at 37/30°C showed lower contents of neoxanthin, VAZ pool, DEPS, lutein, β- and total carotenes, total Car, and Chl (a + b) relative to their 380-counterparts. However, no significant differences associated with [CO2] treatments could be found at 42/34°C. In contrast to [CO2] treatment, increasing temperature remarkably impacted the C. arabica genotypes regardless of [CO2], as denoted by the increases in total Chls and total Car, reaching maximal values at 37/30°C or 42/34°C. The rise in total Car was related to increases in neoxanthin, Z and, especially, lutein and β-carotene .In types, CO treatmen2], as well as in CL153 380-plants. Furthermore, at 42/34°C most pigments (with the referred exceptions related to lutein and β-carotene) tended to lower concentrations than those observed at 37/30°C.Specifically regarding the photoprotective Z, moderately low values were maintained over the course of the experiment, but at 42/34°C significant Z increases  were observed in Icatu and IPR108 regardless of . However, at 42/34°C the CL153 700-plants displayed lower Chl content than 380-plants, resulting from slight divergent shifts promoted by temperature in these plant groups (Table a/b) ratio were similar between [CO2], but decreased at the two highest temperatures in all genotypes. Finally, the total Chl-to-total Car ratio, reflecting the light capture-to-dissipation pigment ratio, was quite stable until 37/30°C, but strongly decreased at 42/34°C in the C. arabica genotypes. No differences were found in this ratio in response to [CO2] over the entire experiment in all genotypes.A moderate rise in total Chl was observed in 2], although the values were maintained within the range usually observed for genotypes of both C. arabica and C. canephora . In IPR108, different patterns were observed related to [CO2], with activity increases in 700-plants , and decreases in 380-plants (non-significantly for CAT) in all enzymes.With temperature rise to 37/30°C, enzyme activities changed differently with respect to genotype and [COns Table . In CL152], Cu,Zn-SOD and GR activities remained invariant in parallel with an enhanced CAT activity (53% for 380 plants and 189% for 700-plants), whereas APX activity was remarkably reduced . Within C. arabica genotypes, Icatu displayed reductions in APX , GR and CAT (380-plants) and Cu,Zn-SOD (700-plants), in parallel with increases in both GR and CAT (700-plants) as well as in Cu,Zn-SOD (380-plants). In IPR108, the 700-plants showed increased activities of Cu,Zn-SOD (28%), APX (30%), GR (40%), and CAT, but only the activity of CAT varied significantly in response to [CO2] treatments.Pronounced changes in enzyme activities were further noted when temperature increased from 37/30 to 42/34°C. In CL153 plants, regardless of , with a 22% decrease in 380-plants and a 21% rise in 700-plants. In contrast, at 42/34°C and under both CO2 conditions, TOC pools increased in Icatu and IPR108, ranging from 89 (Icatu-700) to 257% (Icatu-380), with 380-plants presenting the highest contents.α-tocopherol (TOC) showed different trends between the two coffee species. Despite some fluctuations with rising temperatures, at 42/34°C TOC pools changed moderately in CL153 irrespective of [CO2] treatments. In CL153, significant increases were observed only for stachyose in the 700-plants from 31/25°C onwards. On the other hand, at 42/34°C, both stachyose and raffinose displayed greater contents in C. arabica plants, when compared to 25/20°C, particularly raffinose whose contents increased between 153% (IPR108-700) and 263% (Icatu-700). Also, in IPR108, both RFOs content were always higher in the 380-plants at supra-optimal temperatures.At 25/20°C the 700-plants showed similar (raffinose) or lower (stachyose) contents of RFOs than their 380-plant counterparts. Temperature rise prompted modifications in RFOs, with no clear relation with the [CO2] in C. arabica genotypes, but presented a higher value in CL153 700-plants. Rising temperature clearly promoted HSP70 accumulation: significantly higher HSP70 contents were already observed at 31/25°C in all genotypes, which were mostly maintained until 42/34°C (C. arabica plants) or until 37/30°C in CL153 (decreasing afterwards). In all genotypes, maximal HSP70 contents, relative to control temperature, were observed in CL153-380 at 31/25°C (208%), and in the 700-plants of Icatu at 37/30°C (360%) and IPR108 at 42/34°C (136%).The constitutive cell HSP70 pools (25/20°C) were similar between [CO2] and temperature  in all genotypes and both [CO2], although stronger in the 700-plants of Icatu and CL153 and 380-plants of IPR. Notably, CL153 700-plants showed the highest increases: ca. 33- (Chape 60), 55- (ELIP), 59- (HSP70), and 145- (Chape 20) fold.Several changes in gene expression patterns were imposed by enhanced  in Icatu, and at 42/34°C in CL153-700 and IPR-380 plants. TOC genes were mainly downregulated (except CL153 700-plants). Notably, IPR108 had the strongest global downregulation for the genes related to the antioxidative system, with the exception of APX Cyt at 42/34°C in 380-plants.Overall, the expression patterns of genes related to antioxidative system components (Dyn) and chronic (PIChr) PSII photoinhibition estimates , in contrast to what is often observed in other species even at moderately high temperature  were evident at 42/34°C ) observed in 700 plants at 42/34°C  under the exposure to supra-optimal temperatures  in C. arabica genotypes, similarly to what has been found in other species, where a relaxation of the antioxidant system was associated with a higher C-assimilation that ultimately led to a reduced energy load on the photosynthetic apparatus  driven by temperature. Increases in total Car resulted mostly from the moderate Z increases and the strong rises in lutein and β-carotene that have complementary actions, decreasing the formation of highly reactive species of Chl and oxygen, preventing PSs and membrane photodamage, and protecting the Cyt b6/f complex from photobleaching promoted by 1O2  quenches 3Chl* and 1O2, which are lipoperoxidation initiators , when compared to their respective 380-plants. In contrast, ess of CO, suggestC. arabica plants  ratio showed similar values between [CO2], but decreased in all genotypes at the two highest temperatures, especially at 42/34°C. Since net degradation of Chls was apparently absent, this Chl (a/b) reduction reflected preferential Chl b synthesis, suggesting functional readjustments with a higher proportion of light harvesting chlorophyll-protein complex from PSII (LHCII), which contains the majority of Chl b (with a Chl a/b ratio around 1.1–1.3)  might reflect some heat susceptibility given that this ratio has been shown to increase in heat-tolerant genotypes of other species   and non-enzyme  components, which altogether scavenge several ROS  Asada, . With th2 conditions), and Cu,Zn-SOD and CAT in IPR108 700-plants. In addition, in line with the observed heat tolerance until 37/30°C the 380-plants from Icatu presented similar  or higher (GR and CAT) activities than their 700-plants counterparts, compensating for the lower photosynthetic rates. Similarly, the IPR108 380-plants presented higher  or similar (GR and APX) enzyme activities than their respective 700-plants. Irrespective of genotypes and [CO2], FeSOD gene was not significantly upregulated under supra-optimal temperatures, and therefore With rising temperatures thermotolerance can be improved by increasing transcript and protein levels of ROS-scavenging enzymes , but the H2O2 control might have been achieved in extra-chloroplast compartments in good agreement with both increases in CAT activity in both [CO2] and upregulation of CAT and APX Cyt genes   gene expression at 42/34°C, which may have contributed to a lower impact on the photosynthetic performance in elevated [CO2]. At the two highest temperatures, the scavenging capability in IPR108 700-plants was not stronger than that of 380-plants (with the exception related to CAT activity), and a lower gene expression for the antioxidant enzymes and other protective proteins  was observed. Therefore, a better ROS control could be mostly prompted by the higher rates of electrons driven to C-assimilation, as also reported in grapevine (Vitis vinifera) under high [CO2] and increased temperature  Table . This reAPX Cyt expression, which was the most responsive gene to high temperature (in CL153). Since CAT was the only enzyme to present significant increased activities in the three genotypes in the 700-plants , this suggested a drift of H2O2 control from chloroplast APX to extra-chloroplast CAT  and possibly cytosolic APX. In fact, since H2O2 is capable of diffusing passively across membranes, extrachloroplastic scavenging systems are important H2O2 detoxification pathways  that co-operate in limiting 1O2 damages  in C. arabica plants regardless of [CO2]. However, this apparent discrepancy may be related to the observation that these final steps might be not limiting to TOC synthesis   and early light-induced protein (ELIP) from the chloroplast was also examined (Table Chape 20 and 60 showed a clear upregulation under elevated CO2 at 42/34°C (in CL153 and Icatu), under control temperature the expression did not significantly change, or even tend to decrease, with CO2 enrichment, as also observed by Vicente et al. , by the activities of antioxidant enzymes , and by the upregulated expression of some genes . Nevertheless, C. arabica plants seemed to be more responsive, although improved photosynthetic activity was promoted under high [CO2] in all genotypes , as well as raffinose content, constituted a common heat defense line in all genotypes. A consistently higher expression was observed in CL153 at the highest temperature, in agreement with its better tolerance to elevated temperatures. TOC and HSP70 seemed to be particularly relevant in C. arabica genotypes. The presence of RFOs, lutein, β-carotene, TOC, and HSP70, as well as the upregulated expression of genes related to protective proteins  and antioxidant enzymes , which should act in concert to control ROS formation and scavenging, were mostly driven by temperature increase. Nevertheless, it was noteworthy that the plants grown under enhanced [CO2] maintained higher activities of GR (Icatu), CAT (Icatu and IPR), and kept (or even increased) the activities of Cu,Zn-SOD, APX, and CAT. These differences between [CO2] were particularly clear in Icatu, with the 380-plants suffering from the greatest photosynthetic impact amongst the genotypes. Furthermore, the strongest upregulation of genes related to protective proteins found in the 700-plants of CL153 and Icatu likely contributed to strengthen their ability to maintain higher functional levels. The simultaneous gene upregulation of antioxidative enzymes and molecular chaperones at the two highest temperatures fully agrees with the known pivotal protective roles of these proteins, as well as to the observed cross-talk between HSPs/chaperones and other stress response mechanisms under abiotic stress conditions  to extra-chloroplast (CAT) taking into account the increases in CAT activities in all 700-plants (and CL153 380-plants) coupled with strong decreases in APX activity. The 700-plants showed higher photochemical energy use ability, which should constitute the major sink for electron transport, preventing electrons to react with O2 to produce ROS. Therefore, this should be perceived as a first line of defense against excessive energy load, constituting a primary mean of protection against oxidative conditions and photoinhibition, allowing acclimation to take place.Notably, our data also suggest a drift of H2] mitigated the heat impact through higher photosynthetic functioning, upregulation of protective molecules, as well as through higher activity of some antioxidant enzymes. Such high [CO2]-dependent effects on antioxidant defenses likely favored the maintenance of ROS at controlled levels and would in turn justify the lower heat impact on the photosynthetic components under elevated [CO2]  and UID/GEO/04035/2013 (GeoBioTec), as well through the grant SFRH/BPD/47563/2008 (AF) co-financed through the POPH program subsidized by the European Social Fund. Brazilian funding from CAPES , CNPq and Fapemig  are also greatly acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.