The UV/Vis study in DMSO, DMSO + water, and DMSO + PBS buffer (pH = 7.2) confirmed their molecular sustainability in liquids. Their in vitro anticancer activity against breast cancer cell lines such as MCF-7 and MDA-MB-231 makes them interesting for in vivo analysis. Their stronger DNA binding activity (DBA) compared with free ligand suggested them as a good DNA binder. DBA was further confirmed by physicochemical studies such as surface tension and viscosity of complex + DNA which inferred the disruption of DNA and intercalation of complexes, respectively. Their % binding activity, % disruption of DNA base pairs (DNABP), and % intercalating strength are reported in this paper for the first time for better understanding of DNA binding mechanism. Along with this, their scavenging activity (SA) determined through DPPH free radical and the results indicate good antioxidant behaviour of complexes.A new series of Pd (II) complexes of methyl substituted benzylamine ligands (BLs) has been synthesized and characterized via spectroscopic techniques such as UV/Vis. FTIR, LCMS,  From the last few decades, the transition metal complexes with different amine ligands have drawn a significant interest in exploring anticancer activities, specifically related to solid tumor chemotherapies –4. The b in vitro antitumor activity on breast cancer cell lines MCF-7 and MDA-MB-231 which expressed effective anticancer potential. Since DNA is a primary molecular target of anticancer drugs and ascertains an extent of drug's chemotherapeutic potential, the DBA of synthesized complexes have chosen to investigate their anticancer nature  . UV/Vis in DMSO: λmax [ε (dm3mol−1cm−1)] = 275 (2569), 330 (664), 370 (477) nm, in DMSO: water (1 : 1): λmax [ε (dm3mol−1cm−1)] = 270 (2921), 340 (358) nm, in DMSO: phosphate buffer (1 : 1): λmax [ε (dm3mol−1cm−1)] = 265 (2252), 340 (410) nm.Yield: 0.1810 g, 74.76%. Elemental analysis, found: C, 55.74; H, 5.26; N, 8.13%. Calcd for C16H20N2Pd: C, 55.79; H, 5.33; N, 8.19%. IR (KBr): υmax/cm−1 3293 and 3210 (NH2), 1493 and 1449.7 , 744.64 (mono substituted Ph), 1099 (C–N), 437.42 (Pd–N). 1H NMR  δ 2.086 , 3.932 , 2.404 , 7.21–7.23 , 7.08–7.10  and 7.12–7.20 . 13C NMR  δ 47.674 (C1), 138.36 (C2), 137.23 (C3), 129.04 (C4), 127.90 (C5), 125.48 (C6), 128.15 (C7) and 20.98 (C8). +ve ESI-MS: m/z 346.05 [M + 1] . UV/Vis in DMSO: λmax [ε (dm3mol−1cm−1)] = 275 (2509), 340 (595), 370 (486) nm, in DMSO: water (1 : 1): λmax [ε (dm3mol−1cm−1)] = 270 (2745), 340 (344) nm, in DMSO: phosphate buffer (1 : 1): λmax [ε (dm3mol−1cm−1)] = 265 (2086), 340 (412) nm.Yield: 0.1722 g, 63.26%. Elemental analysis, found: C, 55.74; H, 5.26; N, 8.13%. Calcd for C16H20N2Pd: C, 55.88; H, 5.29; N, 8.21%. IR (KBr): υmax/cm−1 3257 and 3210 (NH2), 1453 and 1355 , 756.46 (mono substituted Ph), 1000.7 and 1036.1 (C–N), 492.6 (Pd–N). 1H NMR  δ 2.09 , 3.93 , 2.31 , 7.33–7.35 , 7.18–7.20  and 7.25 . 13C NMR  δ 47.67 (C1), 139.11 (C2), 127.89 (C3), 128.76 (C4), 138.23 (C5), 129.11 (C6), 128.14 (C7) and 20.29 (C8). +ve ESI-MS: m/z 346.9 [M + 1] . UV/Vis in DMSO: λmax [ε (dm3mol−1cm−1)] = 280 (2208), 340 (447), 370 (368) nm, in DMSO: water (1 : 1): λmax [ε (dm3mol−1cm−1)] = 265 (2770), 340 (286) nm, in DMSO: phosphate buffer (1 : 1): λmax [ε (dm3mol−1cm−1)] = 265 (2276), 340 (371) nm.Yield: 0.1785 g, 74.20%. Elemental analysis, found: C, 55.74; H, 5.26; N, 8.13%. Calcd for CCell viability was estimated colorimetrically using 2-(3-diethylamino-6 diethylazaniumylidene-xanthen-9-yl)-5-sulfobenzenesulfonate, SRB (sulforhodamine B) as standard assay with high reproducibility .2 incubator.Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from NCI, USA, and grown in minimal essential medium (MEM). Eagles media were supplemented with 10% heat inactivated fetal bovine serum , 2 mM L-glutamine, and 1 mm sodium pyruvate (Hyclone) in humidified COCytotoxicity of Pd (II) complexes was determined by SRB assay where E5000 cells were seeded into each well of a 96-well clear flat bottom polystyrene tissue culture plate and incubated for 2 h in MEM. An additional 190 mL cell suspension was added in each well containing 10 mL test sample in 10% DMSO with 10 mL Adriamycin (doxorubicin) as a positive drug control. Each experiment was carried out in 3 replicate wells. After an incubation of 48 h, 100 mL of 0.057% SRB solution (w/v) was added in each well. Then 200 mL of 10 mM Tris base solution (pH = 10.5) was added into each well and shaken smoothly. The cell viability was assayed by absorption at 510 nm with a microplate reader. The experiments were repeated thrice with 3 replicates each time and 99% reproducibility was obtained.µM DNA stock solution and used for absorption titration, viscosity, surface tension, conductivity, and zeta potential measurements.CT-DNA  was used as received. Tris-HCl buffer  was prepared in Milli-Q water for 50 −1cm−1, a molar absorption coefficient at 260 nm [µM complex solutions were prepared in 10% DMSO in Tris buffer for Pd(BLs)2 to which the DNA stock solutions were added. Their ri = [complex]/[DNA] at 0.2, 0.6, 1, 1.4, and 1.8 were calculated by absorption titration. Before recording UV spectra the Pd(BLs)2 + DNA solutions were incubated for 15 min at rt to enable sufficient and reproducible DNA binding with complexes. Further their binding strength, an intrinsic binding constant (Kb) with CT-DNA, was obtained by monitoring a change in DNA absorbance on increasing Pd(BLs)2 concentrations and calculated with the following equation . A plot of [DNA]/(εa − εf) versus [complex] produced a slope of 1/(εb − εf) and a Y intercept equal to 1/Kb(εb − εf); Kb is the ratio of slope to the Y intercept  confirming their molecular mass. In UV study, the absorption spectrum consists of a band at 400 nm and may be assigned as 1A1g → 1A2g (dxy → dx2−y2) transition occurring with Pd complexes  [2 . The overall patterns of spectra for complexes solution were found to be similar to the different mediums to ensure their stability. Thus our synthesized Pd (II) complexes having UV/Vis absorption from 265 to 270 nm and 1H NMR coupling constant between 5 and 10 MHz have confirmed their trans geometry 2 . Two douPd(BLs)2 . The arogeometry . in vitro studies on different cell lines have been reported, suggesting their cytotoxicity up to some extent [ in vitro [ in vitro anticancer study of some novel Pd complexes using BLs as ligands for better anticancer activity. The complexes were tested in vitro against MCF-7 and MDA-MB-231 human breast tumor cell lines by colorimetric microculture 2-(3-diethylamino-6-diethylazaniumylidene-xanthen-9-yl)-5-sulfobenzenesulfonate (SRB) assay and compared with Adriamycin (ADR) and cisplatin [µM Pd(BLs)2 have been illustrated in Figures 50, TGI, and LC50 in µM have inferred 50% growth inhibition, resultant total growth inhibition, and a net loss of 50% cells after treatment, respectively  complexes, many of thee extent –33. In sin vitro –33. It sisplatin . The MCFectively .50 value less than 10 µM depicts the anticancer activity with respect to ADR and cisplatin, where the closer GI50 values of Pd2MBA, Pd3MBA, and Pd4MBA from standards make them interesting for testing against other cancerous cell lines.The GIThe DBA explains the anticancer nature of the complex or the drug and can be analyzed by the absorption spectral study. In light of this, the Pd (II) complexes were mixed with CT-DNA at certain concentration and shown the changes in absorbance. Generally, the hypochromism effect in UV study reveals all synthesized complexes intercalative binding strength attributed to an interaction with DNA bases , 36. SimThe decrease in UV absorption predicted the strong interactions of Pd2MBA, Pd3MBA, and Pd4MBA, whose intercalating strengths depend on size and electron densities of interacting aromatic rings with an amine group –38. TheiComparative % Binding Affinity of Complexes. For a better understanding of their binding potential, a % binding affinity (BA) with DNA has been calculated through well known spectrophotometric method at a characteristic wavelength of 260 nm. The % BA was calculated as per the following equation (equation : (4)%  BThe all Pd (II) complexes show approximately the same binding activity at each concentration indicating the same binding potential of each complexes. The same binding potential can further be explained by the same geometry of the all complexes. Therefore, it can be understood that the Pd complexes with the same geometry may equally participate when they interacted with DNA.Along with spectrophotometric study, the physicochemical study, such as viscosity, surface tension, conductivity, and zeta potentials, is also an effective method for better understanding of DBA. Thus, we have mathematically established a comparison of their physicochemical data that presumes their binding affinity with DNA at structural level.1) Study of Structural Disruption of CT-DNA. Initially, the complex entertains the DNA and attacks on its base pairs; therefore it weakens the intermolecular forces of bases which results in the disruption of base pairs. Since, it is an intermolecular force disruption and such disruption can be measured by the analysis of surface tension, therefore, we have analysed and compared the surface tensions of DNA with and without complexes. Generally, the decrease in surface tension infers the force disruption and we have also obtained such results where we compared the surface tension of pure DNA and DNA + complexes. For instance, the surface tension values of the DNA solution with increasing amounts of Pd (II) complexes  have decreased as compared to pure DNA ((pure DNA .A decrease in surface tension of Pd (II) complexes + DNA inferred the weakening of intermolecular interaction of DNA bases and has lost interaction with complexes.Complex Contributions on Disruption of DNA Base Pairs. Surface tension is an important analysing aspect for the disruption of DNA base pairs; therefore, for the advanced study we have also measured and compared the % disruption of DNA base pairs. The % complex contribution was calculated on the disruption of DNA %  di2) Study on Intercalation Phenomenon of Complexes. For the classical intercalative mode, the viscosity of the DNA solution is increased due to an increase in overall DNA length while it is decreased for a partial nonclassical intercalation process causing a bend or kink in the DNA helix that reduces its effective length concomitantly [η/η0)1/3  were plotted against 1/R   .2, an increase in viscosity of DNA was observed, proving the Pd(BLs)2 as DNA intercalators (On increasing concentration of Pd(BLs)calators . For ethComplex Contributions towards Intercalation with DNA Base Pairs. Apart from the above discussion, we have also established the % binding affinity with respect to viscosity (η) using two different methods. In both methods the % complex on the intercalation with DNA was calculated:3) Conductivity and Zeta Potential. The DNA and complexes are charge molecules; therefore their conductivity and zeta potential have also been considered as interaction parameters as well as key factors for an analyzing conformational behaviour of an isolated DNA chain with the complexes. A DNA solution of 50 µM had 3426 µS conductivity while, with increasing amounts of complexes, the decrease in zeta potential as well as conductivities has been found activity . Therefosolution . Thus, a2O2 proposed the facts responsible for their mode of mechanism [• as per standard procedure for the Pd(BLs)2 [2 has been determined in a concentration-dependent mode in comparison with the DPPH• absorption at 517 nm [• free radical's absorption at 517 nm with DMSO was 0.906 and for Pd(BLs)2. From 50 to 250 µM with an interval of 50 µM, complexes have expressed a decrease in absorption 2 , 46. Thet 517 nm –50. The sorption  that chaThe 55.47, 28.812, and 24.24% for Pd2MBA, Pd3MBA, and Pd4MBA, respectively, inferred Pd2MBA, a strong antioxidant among them. Thus, the antioxidant activities of Pd complexes have inferred their significance in medicinal sciences.2 could not express the anticancer activity, their DBA and SA refer them as anticancer compounds having some modification. The closer GI50 values as compared to control drugs such as doxorubicin and cisplatin make the synthesized complexes more interesting to test against other cancerous cell lines. The DBA analysis with viscosity, surface tension, zeta potential, and conductance has inferred strong intercalating as well as DNA disruption and intercalating nature of the complexes. The quantitative evaluation of % DNABP disruption and % intercalating strength advances the DNA binding mechanism. The complexes have shown significant free radical scavenging activities acting as antioxidants and could be used for medicinal purposes. Thereby, our study could have potential in better understanding of medicinal aspects of the complexes.Since the synthesized Pd(BLs)The supplementary material contains UV/Vis spectra of complexes in different solvent and spectra of DNA binding with and without complexes.


Oil content, fatty acid composition and the distribution of vitamin-E-active compounds of selected Turkish seeds that are typically by-products of the food processing industries , were determined. The oil content of the samples ranged from 16.9 to 53.4 g/100 g. The dominating fatty acids were oleic acid  in the range of 52.5 to 68.4 g/100 g and linoleic acid  with 48.1 to 56.3 g/100 g, while in linseed oil mainly α-linolenic acid (53.2 g/100 g) and in fennel seed oil mainly 18:1 fatty acids (80.5 g/100 g) with petroselinic acid predominating. The total content of vitamin-E-active compounds ranged from 20.1 (fennel seed oil) to 96 mg/100 g (apple seed oil). The predominant isomers were established as α- and γ-tocopherol. Large amounts of different seeds are discarded yearly as by-products of food processing ,2. TheseThe samples were collected by hand in May and August in 2007 from plants growing at several locations of Turkey. Seeds and kernels were obtained from fruits by hand-processing and then transferred to laboratory in polypropylene bags under cool conditions. Then they were stored in glass jars at 6°C until analysis. Detailed information related to the samples is given in Petroleum ether (40–60) was of analytical grade . Heptane and tert-butyl methyl ether were of HPLC grade . Tocopherol and tocotrienol standard compounds were purchased from CalBiochem .The oil content was determined according to the method ISO 659:1998 . About 2n-heptane, 50 μg of sodium methylate  was added, and the closed tube was agitated vigorously for 1 min at room temperature. After addition of 100 μL of water, the tube was centrifuged at 4500 g for 10 min and the lower aqueous phase was removed. Then 50 μL of HCl ) was added, the solution was shortly mixed, and the lower aqueous phase was rejected. About 20 mg of sodium hydrogen sulphate  was added, and after centrifugation at 4500 g for 10 min, the top n-heptane phase was transferred to a vial and injected in a HP5890 gas chromotograph , with a capillary column, CP-Sil 88 . The temperature program was as follows: From 155 °C; heated to 220 °C (1.5 °C/min), 10 min isotherm; injector 250 °C, detector 250 °C; carrier gas 36 cm/s hydrogen; split ratio 1:50; detector gas 30 mL/min hydrogen; 300 mL/min air and 30 mL/min nitrogen; manual injection volume less than 1 μL. The peak areas were computed by the integration software, and percentages of fatty acid methyl esters (FAME) were obtained as weight percent by direct internal normalization.The fatty acid composition was determined following the ISO standard ISO 5509:2000 . In brien-heptane was directly used for the HPLC. The HPLC analysis was conducted using a Merck-Hitachi low-pressure gradient system, fitted with a L-6000 pump , a Merck-Hitachi F-1000 fluorescence spectrophotometer , and a ChemStation integration system . The samples in the amount of 20 μL were injected by a Merck 655-A40 autosampler  onto a Diol phase HPLC column 25 cm × 4.6 mm ID  used with a flow rate of 1.3 mL/min. The mobile phase used was 99 mL n-heptane + 1 mL tert-butyl methyl ether [For determination of vitamin-E-active compounds, a solution of 250 mg of oil in 25 mL of The results for oil content and fatty acid composition of the seeds are shown in et al. [Results for the fatty acid composition showed that the oils can be divided into two groups with one group predominant in 18:1 fatty acids and the other group high in linoleic acid (18:2). Only in linseed oil was α-linolenic acid predominating with amounts of 53.2 g/100 g, while its content in the other samples was between 0.1 and 1.0 g/100 g, only. The highest contents of oleic acid were found in apricot (sweet) (68.3 g/100 g), chufa (62.4 g/100 g) and apricot (bitter) (57.8 g/100 g) seed oils. Fennel (dulce) seed oil is characterized by a high content of 18:1 fatty acids (80.5 g/100 g) with petroselinic acid as predominant fatty acid. The used GLC-method was not able to separate petroselinic acid from oleic acid, but from literature it is known that petroselinic acid is predominant in members of the family Apiaceae. From a nutritional point of view this high content of oleic acid favors these seed oils for human nutrition. The content of linoleic acid of the oils ranged from 12.0 g/100 g (fennel seed oil) to 56.3 g/100 g (cotton seed oil), with half (six) of the selected seed oils comprising of at least 50 g/100 g linoleic acid. The relatively low content of saturated fatty acids such as palmitic acid or stearic acid with amounts below 10 g/100 g is interesting from a nutritional point of view. Only chufa seed oil (17.2 g/100 g) and cottonseed oil (24.2 g/100 g) had higher amounts of nutritionally unfavourable saturated fatty acids. The fatty acid composition found in the present investigation for apricot seed oil was similar to the fatty acid composition published by Dubois et al.  with 4.4et al. [et al. [The results from Dubois et al.  also con [et al.  publishe [et al.  with 28.et al. [et al. [.Reiter et al.  determin [et al. .et al. [et al. [In linseed oil, the major fatty acids were reported as 5.5 ±1.5 g/100 g palmitic acid, 3.5 ±1.2 g/100 g stearic acid, 22.1 ±5 g/100 g oleic acid, 20.5 ±1.5 g/100 g linoleic acid and 47.5 ±5.6 g/100 g α-linolenic acid . Also, Set al.  reported [et al.  determinPalmitic acid and linoleic acid were the dominant fatty acids, constituting 70–80 g/100 g of the total fatty acids in the apple fruit . As seenet al. [For peanut oil Dubois et al.  found 11et al. .et al. [In cottonseed oil, 21.0 and 27.2 g/100 g palmitic acid, 1.8 and 2.2 g/100 g stearic acid, 16.9 and 16.9 g/100 g oleic acid, 50.5 and 56.7 g/100 g linoleic acid and 0.9 and 1.2 g/100 g α-linolenic acids were determined [et al.  determinIn vegetable oils four different derivatives of tocopherols and tocotrienols , respectively, can be found as vitamin-E-active compounds differing in the methylation of the chroman ring. Vitamin-E-active compounds have two functions: On one side they stabilize the oil against oxidative deterioration during storage or heat-treatment and on the other side they have some biological effects to humans. The main biochemical function of the tocopherols is believed to be the protection of polyunsaturated fatty acids against peroxidation ,23. The In general the antioxidant activity increases for tocopherols and tocotrienols in the order α to δ, whereas the biological activity is opposite to the antioxidant activity ,29. AnotDue to the antioxidant properties the content and the composition of vitamin-E-active compounds is an important feature for the assessment of seed oils. The total content of vitamin-E-active compounds in the different seeds varied between 20.1 (fennel) and 94.0 mg/100g (quince) . In compApiaceae are characterized by a higher content of tocotrienols, such as dill (Anethum graveolens) (10.2 mg/100 g α-tocotrienol) or coriander (Coriandrum sativum L.) (23.1 mg/100 g γ-tocotrienol) [All the oils investigated exhibited differences in their tocopherol contents. The predominant tocopherol compounds in the different seeds oils were α- and γ-tocopherol. Additionally in some oils lower amounts of α-tocotrienol, β-tocopherol, plastochromanol-8 and δ-tocopherol were found. The main vitamin-E-active compound of fennel seed oil was γ-tocotrienol with 18.2 mg/100 g. Also seed oils of other members of the family trienol) .The α-tocopherol group comprised of apple (golden) (51.4 mg/100 g), apple (starking) , quince (49.6 mg/100 g) and chufa (68.5 mg/100 g) seed oil. Except for chufa seed oil, these oils also contained higher amounts of β-tocopherol with an average amount of about 30 mg/100 g ranging between 27.8 (quince seed oil) and 34.3 mg/100 g (apple seed oil (starking)). γ-tocopherol was predominant in linseed oil (39.0 mg/100 g), apricot seed oil , apricot seed oil , pear seed oil (55.6 mg/100 g), and cottonseed oil (48.7 mg/100 g).Among theseed oils in this group only cottonseed oil contained considerably high amount of α-tocopherol (36.2 mg/100 g) while for the other seed oils γ-tocopherol accounted for between 72% and 93% of the total vitamin-E-active compounds. In linseed oil remarkable amounts of plastochromanol-8 were found (13.5 mg/100 g). Peanut oil neither belongs to the α-tocopherol nor to the γ-tocopherol group. The oil showed the lowest total amount of vitamin-E-active compounds of all investigated oils with almost equal amounts of α-tocopherol (14.9 mg/100 g) and γ-tocopherol (16.9 mg/100 g).Linseeds, apricot seeds, pear seeds and peanut seeds contained more than 30 g/100 g oil making them interesting as valuable sources for oil production. Due to the lower oil content the other seeds can be interesting for oil processing if the raw material also contains protein with valuable amino acid composition, such as soybeans. The value of the oil depends on the fatty acid composition and the composition of vitamin-E-active compounds. Here the oils can be divided into two groups, one predominant in oleic acid with contents higher than 50 g/100 g  and one group mainly comprised of linoleic acid . Linseed oil is characterized by a high amount of α-linolenic acid and fennel seed oil contained higher amounts of 18:1 fatty acids with petroselinic acid as predominant fatty acid which is usable in technical applications. Especially the fatty acid composition of apricot seed oil with low amount of saturated fatty acids, high amounts of oleic acid and moderate amounts of polyunsaturated fatty acids seems to be an interesting sources as edible oils for human nutrition. The composition of vitamin-E-active compounds of the different oils is characterized by either high amounts of α-tocopherol together with moderate amounts of β-tocopherol or by high amounts of γ-tocopherol. The predominant vitamin-E-amount in fennel seed oil is γ-tocotrienol and peanut oil contains more or less equal amounts of α- and γ-tocopherol. High sources of vitamin-E-active compounds with more than 70 mg/100 g are apricot seed oil, apple seed oil, cottonseed oil and quince seed oil.In summary it can be concluded that raw materials such as seeds from fruit and vegetable processing are interesting and valuable sources for the production of vegetable oils usable as suppliers of fatty acids and vitamin-E-active compounds in human nutrition or technical applications.


The aim of this study was to evaluate the effects of caffeine on physiological responses to submaximal exercise, with a focus on blood lactate concentration ([BLa]).-1) completed four trials on an electromagnetically-braked cycle ergometer. Each trial consisted of a six-stage incremental test (3 minute stages) followed by 30 minutes of passive recovery. One hour before trials 2–4, participants ingested a capsule containing 5 mg∙kg-1 of either caffeine or placebo . Trials 2 and 3 were designed to evaluate the effects of caffeine on various physiological responses during exercise and recovery. In contrast, Trial 4 was designed to evaluate the effects of caffeine on [BLa] during passive recovery from an end-exercise concentration of 4 mmol∙L-1.Using a randomised, single-blind, crossover design; 16 endurance-trained, male cyclists , but did not affect the time-course of [BLa] during recovery (p = 0.604). Caffeine reduced ratings of perceived exertion  and heart rate  during exercise, with the effect on the latter dissipating as exercise intensity increased. Supplement × exercise intensity interactions were observed for respiratory exchange ratio (p = 0.004) and minute ventilation (p = 0.034).Relative to placebo, caffeine increased [BLa] during exercise, independent of exercise intensity (mean difference: 0.33 ± 0.41 mmol∙LThe results of the present study illustrate the clear, though often subtle, effects of caffeine on physiological responses to submaximal exercise. Researchers should be aware of these responses, particularly when evaluating the physiological effects of various experimental interventions. SimilE)  during exercise (3). The effect is strange because, although the increase in [BLa] can often be explained by a concomitant caffeine-induced increase in performance ,14,22, t-1 (55.6 ± 5.0 ml∙kg-1∙min-1), respectively. Participants were instructed to maintain their normal diet throughout the testing period, to follow the same diet for 24 hours prior to each trial, to avoid food and drink in the hour before each trial, and to refrain from strenuous exercise for 24 hours before each trial. Participants were provided with a list of dietary sources of caffeine and asked to refrain from consuming these for 24 hours prior to each trial. A questionnaire was used to establish normal daily caffeine intake.Sixteen endurance-trained , competi-1 and ending with a [BLa] > 4 mmol∙L-1. The duration of each increment was 3 minutes, and a 30 s break was provided at the end of each stage to enable [BLa] to be evaluated. To limit any effect of cadence on the [BLa] response to exercise  ≥ 8 mmol∙L-1.All trials were completed at approximately the same time of day in a laboratory which was thermostatically controlled at 19°C. On arrival at the laboratory, and after approximately five minutes of seated rest, a resting blood sample (~ 5 ml) was drawn from a branch of the basilic vein and collected in a lithium-heparin tube . At the same time, a blood sample was obtained from the earlobe via capillary puncture for the evaluation of [BLa] via an automated analyser . In Trial 1, participants subsequently had their body fat content evaluated using a four-site skinfold protocol , before exercise , particiexercise . After f-1 of caffeine , or placebo . After supplementation, participants rested for 40 minutes before the same blood sampling procedure was repeated. In trials 2 and 3, using a single-blind balanced randomised crossover design, participants completed subsequently a six-stage submaximal incremental test with Stage 3 set at the power output required to elicit the gas exchange threshold (GET) and Stage 6 set at the power output required to elicit the onset of [Bla] accumulation (OBLA); the latter equating to a [Bla] of 4 mmol∙L-1  close to 4 mmol∙L-1. If, on the other hand, caffeine produced the end-exercise [BLa] closest to 4 mmol∙L-1 then Trial 4 became a placebo supplemented trial, with the protocol adjusted accordingly based on the results of the previous placebo trial. In effect, Trial 4 was designed to evaluate the effects of caffeine on [BLa] clearance during passive recovery from a fixed starting [BLa] of approximately 4 mmol∙L-1. The recovery kinetics of [BLa] were evaluated by fitting mono-exponential models . Before analysis, plasma samples were thawed, transferred to a separating flask, and made up to 2 mL with HPLC grade water. Following the addition of an internal standard, samples underwent solvent extraction using a chloroform/IPA mix (85%/15%). Each sample was extracted twice and the organic phase was removed each time. The organic phase from both extracts were subsequently combined, evaporated to dryness under nitrogen, and re-suspended in HPLC grade water. Analysis of caffeine content was carried out by reverse phase HPLC using a C18 column  with a mobile phase of 80% water/20% methanol, a flow rate of 1.5 mL∙mint-tests. The effects of supplementation on the time constants of the exponential models used to describe the recovery kinetics of [BLa] were also determined using a paired-samples t-test. Goodness of fit of the exponential models was evaluated using F tests. The effects of caffeine on physiological responses during the incremental tests, including the effects on [BLa] during recovery, were evaluated using two-way (supplement × exercise intensity or time) ANOVAs with repeated measures on both factors. α was set at 0.05 for all analyses. Violations to assumptions of sphericity were adjusted using the Greenhouse-Geisser correction factor. Significant interactions were followed-up using post hoc tests with Bonferroni adjustments for multiple comparisons. The above analyses provided 95% confidence limits for all estimates.All statistical analyses were conducted using the Statistical Package for the Social Sciences . Measures of centrality and spread are presented as means ± standard deviation. Differences between plasma samples in caffeine content were evaluated using a one-way repeated measures analysis of variance (ANOVA). The effects of caffeine supplementation on resting measures of [BLa] were evaluated using a two-way (supplement × time) repeated measures ANOVA; with effects on the other physiological measures, at rest and during the last 30 s of recovery, determined using paired-samples -1. Subject compliance with caffeine restriction prior to each trial was confirmed by the fact that in all non-caffeine supplemented conditions, plasma caffeine concentrations were low (0.25 ± 0.28 μg·mL-1), whereas values were high (3.90 ± 2.23 μg·mL-1) following caffeine.Mean habitual caffeine consumption of the participants was estimated as 225 ± 135 mg∙dF = 15.87; p < 0.001)  from pre-supplementation values of 1.24 ± 0.37 and 1.12 ± 0.40 mmol∙L-1 for caffeine and placebo trials respectively, to corresponding post-supplementation (pre-exercise) values of 1.07 ± 0.41 and 0.90 ± 0.34 mmol∙L-1. However, there was no effect of supplementation  = 1.30; p = 0.263) and no supplement × time interaction  = 0.25; p = 0.624). The effects of caffeine on [BLa] during exercise and recovery are presented in F = 10.13; p = 0.006) in [BLa] throughout the incremental tests of 0.33 ± 0.41 mmol∙L-1 (95% likely range: 0.11 to 0.55 mmol∙L-1); and there was a significant effect of exercise intensity on [BLa]  = 181.57; p < 0.001). However, there was no significant supplement × exercise intensity interaction  = 0.72; p = 0.466). In contrast, [BLa] decreased significantly over time during recovery  = 213.28; p < 0.001), but there was no effect of supplementation on the recovery process  = 0.28; p = 0.604), and no supplement × time interaction  = 1.74; p = 0.181). The recovery kinetics of [BLa] were very well described by the mono-exponential models ; however, time constants were not significantly different (t(15) = 0.53; p = 0.604) between the caffeine (τ = 14.8 ± 6.7 minutes) and the placebo (τ = 14.3 ± 6.0 minutes) conditions.Blood lactate concentration decreased significantly (t(15) = 3.36; p = 0.004) reduction in heart rate at rest  and during incremental exercise  = 14.49; p = 0.002). However, the effect of caffeine on heart rate dissipated as exercise intensity increased  = 2.49; p = 0.039). Post hoc tests revealed significant reductions in heart rate during the first three incremental stages only (t(15) = 0.88; p = 0.391) between caffeine and placebo conditions  = 167.84; p < 0.001), and RPE was significantly lower  = 8.81; p = 0.010) following caffeine supplementation than following placebo . However, there was no supplement × exercise intensity interaction  = 0.77; p = 0.48)  .F = 305.54; p < 0.001) and, in comparison with placebo, caffeine supplementation significantly increased (t(15) = 2.51; p = 0.024) -1; 95% likely range: 0.01 to 0.11 L·min-1; -1; 95% likely range: 0.03 to 0.11 L·min-1; F = 3.49; p = 0.082) , and no supplement × exercise intensity interaction  = 0.879; p = 0.499) (There was a significant effect of exercise intensity on = 0.499) .F = 161.98; p < 0.001) and, in comparison with placebo, caffeine significantly increased (t(15) = 2.25; p = 0.040) RER at rest  = 2.17; p = 0.162), there was a significant supplement × exercise intensity interaction  = 5.24; p = 0.004)  = 1.23; p = 0.240) of caffeine on RER at the end of the post-exercise recovery period  . Neverthy period .F = 186.30; p < 0.001), and, in comparison with placebo, there was a significant increase (t(15) = 2.44; p = 0.027) in -1; 95% likely range: 0.2 to 3.6 L∙min-1; F = 0.39; p = 0.543), following caffeine supplementation. Although there was a significant supplement × exercise intensity interaction  = 3.36; p = 0.034)  = 2.93; p = 0.011) at the end of the post-exercise recovery period  , post hoF = 65.53; p < 0.001) from starting values (grand means) of 23.9 ± 3.4 breaths∙min-1 to end-exercise values of 32.8 ± 5.3 breaths∙min-1; however, relative to placebo, there was no effect of caffeine supplementation on BF either at rest  = 0.79; p = 0.387). Similarly, there was no supplement × exercise intensity interaction  = 1.67; p = 0.152).Breathing frequency increased progressively throughout the incremental tests , with no corresponding change in It is difficult to resolve the mechanisms by which caffeine influenced the interaction effect on RER; however, it is possible that the effect was, at least in part, mediated by the corresponding supplementation × intensity interaction on increase ,7 or no increase –20. In tprimates  and is as likely , by phoss likely . A caffe& Wetter , who obsThe caffeine-induced reduction in resting heart rate observed in the present study is of a magnitude similar to that previously reported . Moreove1 adenosine receptors  during exercise; an effect which appears to be independent of exercise intensity and which, though easily explained by a stimulation of glycolysis, clearly requires more research to clarify. In addition, caffeine reduced resting heart rate and increased resting


Ciona savignyi were investigated in a laboratory setting. The progression of metamorphic changes were tracked under various temperature, photoperiod, substrate, larval density, and vessel size regimes. Metamorphosis was maximised at 18 °C, 12:12 h subdued light:dark, smooth polystyrene substrate, and 10 larvae mL−1 in a twelve-well tissue culture plate. Eliminating the air-water interface by filling culture vessels to capacity further increased the proportion of metamorphosed larvae; 87 ± 5% of larvae completed metamorphosis within 5 days compared to 45 ± 5% in control wells. The effects of the reference antifouling compounds polygodial, portimine, oroidin, chlorothalonil, and tolylfluanid on C. savignyi were subsequently determined, highlighting (1) the sensitivity of C. savignyi metamorphosis to chemical exposure and (2) the potential to use C. savignyi larvae to screen for bioactivity in an optimised laboratory setting. The compounds were bioactive in the low ng mL−1 to high µg mL−1 range. Polygodial was chosen for additional investigations, where it was shown that mean reductions in the proportions of larvae reaching stage E were highly repeatable both within (repeatability = 14 ± 9%) and between (intermediate precision = 17 ± 3%) independent experiments. An environmental extract had no effect on the larvae but exposing larvae to both the extract and polygodial reduced potency relative to polygodial alone. This change in potency stresses the need for caution when working with complex samples, as is routinely implemented when isolating natural compounds from their biological source. Overall, the outcomes of this study highlight the sensitivity of C. savignyi metamorphosis to environmental variations and chemical exposure.In this study, the effects of environmental variables on larval metamorphosis of the solitary ascidian  Ciona savignyi Herdman (Cionidae) is a Japanese native with an expanding invasive range that currently includes Argentina, British Columbia, California, New Zealand, Puget Sound, and Spain  or subdued (10 ± 5 µmol m−2 s−1) light:dark. Substrate type  and texture (smooth or rough) were assessed using unlined wells (polystyrene) or wells lined with acrylic discs (35-mm diameter) or glass cover slips . Untreated unlined wells, acrylic discs, and glass cover slips comprised smooth treatments. Sandblasted acrylic and sanded (800-grit sandpaper) glass and polystyrene comprised rough treatments; it should be noted that these surface treatments likely resulted in different feature sizes for each of the three substrate types and that the walls of the wells remained untreated in all cases.The temperature, photoperiod, and substrate preferences of larvae were assessed in three separate experiments. Firstly, the effects of exposing larvae to 15, 18, 21, and 25 °C were evaluated. For the photoperiod experiment, larvae were exposed to the following lighting regimes: 24:0, 12:12, 0:24 h intense  filled with 10-mL aliquots of 2.5 larvae mL−1 RSW. Default parameters were 18 °C, 12:12 h subdued light:dark, and unlined smooth wells; three replicates were performed in all cases (n = 3). Culture waters were renewed with RSW every other day and settled larvae were scored according to metamorphic stage  were performed in six-well tissue culture plates  were filled with 25-, 10-, or 5-mL aliquots of larval suspension, respectively. Four larval densities  were tested in each of the three vessel sizes, yielding twelve vessel size × larval density treatments in total (n = 3). Vessels were held at 18 °C, 12:12h subdued light to dark and culture waters were renewed every other day. After 5 days, the number of larvae that had completed stage E of metamorphosis were counted , six-well plates, and twelve-well plates . The temperature, photoperiod, and experimental end-point matched those used for the vessel size and larval density experiments but culture waters were not renewed for the duration of this experiment.Fill volume was assessed using twelve-well plates and a larval density of 10 larvae mL−1 RSW; held at 18 °C, 12:12 h subdued light to dark. Concentrations of ethanol and dimethyl sulfoxide (DMSO) evaluated were 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, and 40 µL mL−1; controls contained no solvent (n = 3). Larvae that had completed stage E of metamorphosis were counted after 5 days.The sensitivity of larvae to two common carrier solvents was assessed in twelve-well plates with 7.1 mL aliquots of 10 larvae mL−1) and portimine , concentrations tested against the larvae were 0.001, 0.005, 0.01, 1, 5, and 10 µg mL−1. Stock solutions prepared in 20% (v/v) ethanol , RSW (portimine), or 20% (v/v) DMSO  were added to wells to yield the desired test concentrations; control wells contained solvent only (n = 3).Five reference compounds, polygodial , portimine , oroidin (ENZO Life Sciences), chlorothalonil , and tolylfluanid (Sigma-Aldrich), were screened using the protocol outlined for carrier solvent. With the exception of polygodial  were subsequently included in eight independent experiments performed over a 1-year period, allowing repeatability  and intermediate precision  to be estimated. In a separate experiment assessing the potential for interactions among complex mixtures of natural compounds, the potency of pure polygodial was compared to that of polygodial enriched with an environmental extract. The extract was produced by eluting 10 L seawater that ∼400 g of green-lipped mussels, Perna canaliculus Gmelin, had been cultured in for 1 day through a 20-cm3 column of Diaion HP20 resin . The column was flushed with 50 mL ethanol, and the resulting extract dried and re-suspended in 1 mL 20% (v/v) ethanol. Polygodial stock solutions prepared in 1 mL of 20% (v/v) ethanol or 1 mL of extract were added to wells to yield final polygodial concentrations of 2.5, 5, 10, and 50 ng mL−1 (n = 3).Polygodial treatments  calculated , and slowed by exposure to constant intense light , photoperiod , and substrate  datasets were driven by the latter stages of metamorphosis. Mean absolute PRC coefficients revealed a strong treatment response for stages E (4.1 ± 0.9) and F (5.1 ± 1.5), compared to weak effects for stages A (0.0002 ± 0.0001), B (0.0005 ± 0.0003), C (0.017 ± 0.011), and D (0.27 ± 0.14).Larvae responded to variations in temperature, photoperiod, and substrate. Daily metamorphic progress was comparable for larvae reared at 15, 18, or 21 °C but hindered at 25 °C  and vessel size  significantly influenced metamorphosis. The proportion of larvae reaching Stage E within 5 days was enhanced above 10 larvae mL−1 for Petri dishes or 5 larvae mL−1 for six- and twelve-well plates. Overall, the greatest proportion of Stage E larvae were observed in twelve-well plates . In wells filled to capacity, 87 ± 5% of larvae completed stage E of metamorphosis within 5 days compared to 45 ± 5% in control wells.Both density .No detectable effects on larval metamorphosis were observed when carrier solvent was added to the vessels at or below 20 µL mLEC50 = 4.5ng mL−1), portimine (EC50 = 1.0ng mL−1), oroidin (EC50 = 1.1μg mL−1), chlorothalonil (EC50 = 0.1μg mL−1), and tolylfluanid (EC50 = 0.3μg mL−1) potently inhibited metamorphosis; dose-response curves closely fit the data (R2 > 0.9). The polygodial positive controls from the eight independent experiments had negative dose-dependent effects on metamorphosis, with mean reductions in the proportions of larvae reaching stage E varying by 14 ± 9% within experiments  and 17 ± 3% between experiments .Polygodial  was little affected by variations in temperature, photoperiod, or substrate. It is not clear why the later stages of metamorphosis were more sensitive to environmental variations but the lecithotrophic nature of C. savignyi may be partly responsible. Energy reserves within the larvae may become depleted later in metamorphosis . This phenomenon has been reported previously for other ascidians .Filling wells to capacity further increased the proportion of Stage E larvae observed after 5 days. When wells were not filled to capacity, some ns e.g., , and is ns e.g., . BecauseC. savignyi metamorphosis highlighted the potential to use C. savignyi larvae to screen for bioactivity under an optimised laboratory setting. The natural antifouling agent polygodial (−1 to high µg mL−1 ranges. Tolerance of C. savignyi larvae for the carrier solvents ethanol (20 µL mL−1 max.) and DMSO (10 µL mL−1 max.) facilitated screening these compounds, which, with the exception of portimine, have only limited water solubility. The results reported here meet or exceed potency estimates previously determined for these compounds against other organisms. For example, polygodial is effective against fungal pathogens at approximately 1 µg mL−1 .Click here for additional data file.


To evaluate the cyclic fatigue resistance and the force generated by OneShape files during preparation of simulated curved canals.Six OneShape files (the test) and six ProTaper F2 files (the control) were subject to the bending ability test. Another thirty files of each type were used to prepare artificial canals (n = 60), which were divided into 3 groups according to respective curvatures of the canals . The numbers of cycles to fatigue (NCF) as well as the positive and negative forces that were generated by files during canal preparation were recorded. The scanning electron microscopy was applied to detect the fracture surfaces.P < .05), 60° (P < .05) and 75° (P < .01). No significant difference was found at 30°. OneShape files presented a higher NCF in both 60° and 90° canals than the control (P < .01). No significant difference of NCF was found between OneShape and ProTaper files in 30° canals. During the preparation of 30° canals by both files, the negative forces were dominant. With the increase of the curvature, more positive forces were observed. When the OneShape Files were compared with the control, significant different forces were found at D3 and D2 (P < .05) in 30° canals, at D2 (P < .05), D1 (P < .01) and D0 (P < .01) in 60° canals, and at D4 and D3 (P < .01) in 90° canals.Compared with ProTaper F2 files, the bending loads of OneShape files were significantly lower at deflections of 45°(OneShape files possessed a reliable flexibility and cyclic fatigue resistance. According to the assessments of the forces generated by files, OneShape instruments performed in a more fatigue-resistant way during curved canal preparation, compared with the ProTaper F2 files. Nickel-titanium (NiTi) instruments, essentially owning to their superelastic characters, have become an elementary implement in root canal preparation , 2. In sIn most cases, the longitudinal axial stress subject to files during canal preparation was considered as resistance resulting from the apical compressive force, which was also named as the positive force (PF) , 10. ConThe single-file system, a new concept for canal preparation, has been introduced recently. OneShape , a typical single-file instrument, is fabricated with the conventional austenite 55-NiTi alloy, motor-driven in continuous rotation. Bürklein , 13 and in vitro study was to evaluate the cyclic fatigue resistance of OneShape instruments, and to assess the positive/negative force generated during preparation of curved canals.The purpose of this i.e. 30°, 60° and 90°. The configuration of the curved canal was based on the Arc Length Formula, i.e. s = (π×r×θ)/180. S is the arc length, π is pi (≈3.14), r is the radius of the circle, θ is the central angle of the arc in degrees. The arc length (s) is set at 4 mm in the apical portion of the canal, the 3 canal curvatures (θ) was then defined and made into 30°, 60° and 90°  and thirty-six ProTaper F2 instruments  (the control) were used in this study. The simulated curved canals were custom-made in plastic blocks  with the inner diameter of 0.2 mm and the total length of 16 mm . The simulated canals (n = 60) were custom-made into 3 curvatures,  and 90° .For the OneShape group (the test), the file size was #25 with a constant 0.06 taper (0.25 mm tip diameter). For the ProTaper group (the control), the selected file was F2 file, with a 0.08 taper at its apical 3 mm, followed by a 0.06 taper from the 3 mm up to the coronal. The tip diameter of ProfTaper F2 file is 0.25 mm.The bending resistance test, according to the ISO 3630–1 , was applied to 6 new instruments of each group . The fili.e. 30°, 60° and 90°). Canals were initially explored with 15# K-file, lubricated with EDTA and flared by the ProTaper SX file . As recommended by the manufacturers, a 16:1 reduction handpiece powered by a torque-controlled motor  was applied. The torque and rotational speed was set at 4.0 Ncm, 400 rpm for OneShape, and 2.0 Ncm, 250 rpm for ProTaper F2. The speed of insertion was set at 3 mm/min. After the working length (16 mm) was reached, the files were kept rotating freely in the canals. The time was recorded when the fracture was detected. The NCF was calculated according to the time and rotation rate.Thirty OneShape files and thirty ProTaper F2 files were used to prepare sixty simulated curved canals, which were divided into three groups according to their respective arc curvature (i.e. when the files arrived at 5 mm from working length (D5), 4 mm from working length (D4), 3 mm from working length (D3), 2 mm from working length (D2), 1 mm from working length (D1) and the point of the full working length (D0).The detection of positive/negative force were performed with the Universal Test Machine, as described previously . In brieAfter the cyclic fatigue test, the fracture surfaces of fragments were observed under the scanning electron microscope  for the topographic features analysis.t-test  with the significance set at P < 0.05.Data were reported as the mean±standard deviation. The data were analyzed by the analysis of variance and vs. 6.58±1.28 N), 60° (14.00±5.59 vs. 6.17±2.64 N) (P < .05) and 75° (65.83±10.21 vs. 10.25±4.33 N) (P < .01). No significant difference was found at deflections of 30° (7.33±3.93 vs. 6.50±1.64 N)  .vs. 986.18±138.16) and 90° (257.04±82.28 vs. 649.88±183.76) (P < .01). No significant difference was found in group of 30° (2581.00±121.79 vs. 2554.80±254.17)  .vs. -1.05±0.50 N) (P < .05) and D2 (-1.15±0.24 vs. -1.60±0.57 N) (P < .05). In group of 60°  (P < .05), D1 (2.05±0.44 vs. -0.75±0.47 N) (P < .01) and D0 (1.45±0.60 vs. -0.30±0.26 N) (P < .01), respectively. In group of 90°  (P < .01) and D3 (0.85±0.34 vs. -0.05±0.42 N) (P < .01).As showed in p of 60° , the NF p of 90° , the NF The scanning electron microscopic topography of the fracture surfaces showed typical features of cyclic fatigue. Representative fracture surface images of both OneShape and ProTaper F2 files from the curved canals were lay out in i.e. 64 KHN) is close to that of dentin (i.e. 68 KHN), the plastic blocks were chosen to make simulated canals for this study [in vitro experiment is to make a comparable and stable working field to assess the performance of the objective files. ProTaper F2 files were selected as the control because (1) both OneShape and ProTaper F2 files are made of a conventional NiTi alloy, (2) they have the same tip size, (3) they have the similar triangular cross-section design in the tip area and (4) they are applied in continuous clockwise rotation.Simulated root canals made of plastic blocks were used in the present study, which is similar to previous experiments , 18. It is study . The reaThe bending ability test was to evaluate the flexibility of the files, which plays a significant role in resisting cyclic fatigue. The flexibility of ProTaper F2 has been studied elsewhere. The results of the present study were similar to those reported by Viana  and Neccet al. [i.e. the plastic block of this study vs. stainless steel block). Nevertheless, the results of NCF suggested that OneShape files possess better cyclic fatigue resistance than ProTaper F2. A Unique technique of electropolishing procedure applied to the manufacture of OneShape files offered an explanation [et al [The cyclic fatigue resistance of ProTaper F2 has been reported previously , 24, whiet al. , probabllanation . This prlanation , 27. Morn [et al , the relIn the present study, the files are driven into the canals in a ramp-like pattern to obtain the smooth and complete depth-stress curve, and longitudinal axial stress was assessed separately as positive force and negative force. Acting as the resistance of the canal wall against files, the increased positive force can lead to longitudinal defects of instruments, accelerating the occurrence cyclic fatigue. On the other hand, the sudden occurrence of screw-in/threading-in effect, which was not observed in present study, could result in increased torque . HoweverDue to the resistance of the simulated canals increased along with the curvatures, the negative force decreased along with the increase of the canal curvature . For ProScanning electron microscopic observations indicated that both the OneShape and ProTaper F2 showed the typical fracture surfaces of ductile type, consisting of the crack initiation area, fatigue striations, and a fast fracture zone with several dimples and micropores.OneShape files possessed a reliable flexibility and cyclic fatigue resistance. According to the assessments of the forces generated by files, OneShape instruments performed in a more fatigue-resistant way during curved canal preparation, compared with the ProTaper F2 files.


Burkholderia genus exhibiting the highest strain diversity and accounting for 36.36% of all the 22 strains. Three isolates among the 22 strains showed higher carboxymethyl cellulase (CMCase) activity, and isolate ME27-1 exhibited the highest CMCase activity in liquid culture. The strain ME27-1 was identified as Paenibacillus terrae on the basis of 16S rRNA gene sequence analysis as well as physiological and biochemical properties. The optimum pH and temperature for CMCase activity produced by the strain ME27-1 were 5.5 and 50°C, respectively, and the enzyme was stable at a wide pH range of 5.0–9.5. A 12-fold improvement in the CMCase activity (2.08 U/mL) of ME27-1 was obtained under optimal conditions for CMCase production. Thus, this study provided further information about the diversity of cellulose-degrading bacteria in the subtropical region of China and found P. terrae ME27-1 to be highly cellulolytic.From different natural reserves in the subtropical region of China, a total of 245 aerobic bacterial strains were isolated on agar plates containing sugarcane bagasse pulp as the sole carbon source. Of the 245 strains, 22 showed hydrolyzing zones on agar plates containing carboxymethyl cellulose after Congo-red staining. Molecular identification showed that the 22 strains belonged to 10 different genera, with the  Usually, cellulases are mainly composed of three types of synergistic enzymes: endoglucanases EC 3.2.1.4) that hydrolyze the exposed cellulose chains of the cellulose polymer, exoglucanases  that act to release cellobiose from the reducing and nonreducing ends, and .4 that h Trichoderma, Aspergillus, Penicillium, Phanerochaete, and Fomitopsis have been widely studied in recent years, researchers have also been paying attention to various bacteria that produce cellulases because of their fast growth, expression of multienzyme complexes, and resistance to extreme environments [ Clostridium, Cellulomonas, Cellulosimicrobium, Thermomonospora, Bacillus, Ruminococcus, Erwinia, Bacteriodes, Acetovibrio, Streptomyces, Microbispora, Fibrobacter, and Paenibacillus have been observed to produce different kinds of cellulase when incubated under anaerobic or aerobic conditions [Numerous microorganisms that are able to degrade cellulose have been isolated and identified. However, many studies have put more emphasis on fungi because the cellulases that they produce are abundant and easy to extract, and some of the fungal cellulases have been used as commercial cellulase . Althougronments –8. Bactenditions , 9, 10. Acinetobacter anitratus was observed in the late logarithm phase [ Brevibacillus sp. DUSELG12 and Geobacillus sp. DUSELR7 on days 10 and 7, respectively [Several studies have been carried out to investigate the carboxymethyl cellulase (CMCase) activity of aerobic bacteria. For instance, a maximum CMCase activity (0.48 U/mL) ofhm phase . Rastogiectively . Bacillus subtilis AS3 exhibited a maximum CMCase activity of 0.43 U/mL [ Streptomyces viridobrunneus SCPE-09 and Pseudomonas sp. HP207, respectively [With regard to studies on optimization of cellulase production by aerobic bacteria, Deka et al. used response surface methodology and found that.43 U/mL . Furtherectively , 16. Thu Burkholderia sp. being the most ubiquitous. Furthermore, a bacterial strain ME27-1, producing CMCase at 2.08 U/mL after optimization of culture conditions, was isolated and identified.In this study, diverse aerobic bacteria capable of hydrolyzing cellulose were isolated from the subtropical region of China, withThe soil samples used in this study were collected from Maoer Mountain (Guilin City), Longgang (Chongzuo City), Dawang Ridge (Baise City), Huaping (Guilin City), Shankou Halodrymium (Beihai City) Natural Reserves, a starch factory in Fangchenggang City, a bagasse compost at the experimental farm of Guangxi University (Nanning City) in Guangxi Zhuang Autonomous Region, China, and Baima Snow Mountain Natural Reserve in Yunnan Province, China. The samples were taken from organic-rich soil.3, 2; MgSO4·7H2O, 0.5; NaCl, 0.5; FeSO4·7H2O, 0.01; KH2PO4, 1.0; yeast extract, 0.4; pulp, 5 (containing 80% water); and agar, 15.0; pH 5.0) and incubated at 28°C. Subsequently, single colonies were picked using an inoculating needle and inoculated onto Mandels and Reese medium [2PO4, 2.0; (NH4)2SO4, 1.4; MgSO4·7H2O, 0.3; CaCl2, 0.3; yeast extract, 0.4; FeSO4·7H2O, 0.005; MnSO4, 0.0016; ZnCl2, 0.0017; CoCl2, 0.002; CMC-Na, 5.0; and agar, 15.0; pH 5.0). After incubation at 28°C for 48 h, all the plates were stained with 1% (w/v) Congo-red solution for 15 min and discolored with 1 M NaCl for 15 min [The soil sample suspensions were inoculated on Czapek's medium  containie medium  containir 15 min . The deg2PO4, 1.5; Na2HPO4·7H2O, 2.5; (NH4)2SO4, 1.5; MgSO4·7H2O, 0.3; CaCl2, 0.1; FeSO4·7H2O, 0.005; MnSO4, 0.0016; ZnCl2, 0.0017; and CoCl2, 0.002; pH 7.0). The bacterial isolates were precultured overnight in general bacteria medium  at 28°C and 180 rpm. Subsequently, 2 mL of the culture was inoculated into 250 mL conical flask containing 50 mL of basal medium with 10 g/L of CMC-Na as the sole carbon source and incubated at 28°C and 180 rpm for 60 h.The modified Mandels medium  used for CMCase production by the isolates contained the following components (in g/L: KHμL of crude enzyme (appropriately diluted) mixed with 250 μL of 2% (w/v) CMC for determining the CMCase activity; 500 μL of enzyme mixed with 1 mL of Avicel  for determining the Avicelase activity; and 500 μL of enzyme mixed with 50 mg of Whatman number 1 filter paper (1.0 × 6.0 cm) in 1 mL of buffer for determining the FPase activity. The buffer used for dissolving or resuspending the substrates was 100 mM sodium citrate buffer (pH 5.5). The mixtures were incubated at 50°C for 30 min for CMCase assay and for 1 h for Avicelase and FPase assay, respectively. Then, the reactions were stopped by adding 1 mL of DNS reagent for CMCase assay and 3 mL of DNS reagent for Avicelase and FPase assay, respectively. All the mixtures were heated in boiling water for 5 min for color development. Subsequently, 200 μL of each sample was transferred to 96-well microplate and the absorbance was measured at 540 nm [μmol of reducing sugars equivalent to glucose per minute during the reaction.Enzyme production during cultivation was assayed at 12 h intervals up to 3 days. The cultures were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatants were collected as crude enzyme for enzyme assay. CMCase, Avicel cellulase (Avicelase), and filter-paper cellulase (FPase) activities were determined using the 3,5-dinitrosalicylic acid (DNS) method . The reat 540 nm , 22. Oneβ-glucosidase was measured by using p-nitrophenyl-β-D-glucopyranoside (p-NPG) as substrate. The enzyme activity was determined by detecting the amount of p-nitrophenol (p-NP) produced from p-NPG [β-glucosidase activity was defined as the amount of enzyme liberating 1 μmol of p-NP per minute.The activity of om p-NPG . One uniμL mixtures were composed of 1 μL of bacterial culture as template DNA, 12.5 μL of 2 × Taq PCR Master Mix , 100 mM KCl, 3 mM MgCl2, and bromophenol blue, purchased from Tiangen Biotech, Beijing, China), 1 μL of each primer (10 μM), and 9.5 μL of double-distilled H2O. The PCR procedure employed was as follows: primary denaturation for 5 min at 94°C; 30 cycles of denaturation at 94°C for 30 s; annealing at 55°C for 30 s, and extension at 72°C for 100 s; and an additional reaction for 10 min at 72°C. The PCR products were detected on 0.8% agarose gel to confirm its purity, quantity, and size. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd., China, for sequencing.The CMC-degrading isolates were cultivated in general bacteria medium at 28°C for 24 h. The culture was directly used for the amplification of bacterial 16S rRNA gene by PCR . The 25 http://blast.ncbi.nlm.nih.gov/Blast.cgi) and aligned with similar sequences by using CLUSTX program. The phylogenetic tree was constructed by applying the neighbor-joining method using MAGA4.1 program based on Kimura-2 parameters with 1000 replicates of bootstrap values [The 16S rRNA gene sequences were compared with other 16S rRNA gene sequences available in GenBank by using the BLASTN program , and Gram staining were evaluated [The effect of initial pH and temperature on CMCase production by the strain ME27-1 was determined by cultivating the strain in 50 mL of basal medium containing 10 g/L of CMC-Na at various pH  and temperatures (26–34°C) for 60 h at 180 rpm.4)2SO4, NH4NO3, NaNO3, KNO3, NH4Cl, urea, soybean, yeast extract, tryptone, and beef extract. The carbon sources were used at a concentration of 10 g/L, instead of the carbon source in the basal medium. Furthermore, different concentrations  of optimal carbon source were examined. Similarly, the effect of nitrogen sources was also studied with an initial concentration of 1.5 g/L.The effect of carbon and nitrogen sources on cellulase production by the strain ME27-1 was determined by using 11 different carbon sources , Avicel, soluble starch, and wheat bran which was chopped into 80 mesh size) and 10 different nitrogen sources as below:  on enzyme production was tested. All media were in pH 8.0. All the flasks were incubated at 28°C. The CMCase activity was detected at an interval of 12 h.μL of crude CMCase supernatant was incubated with 250 μL of CMC-Na  at 50°C and different pH , respectively. To observe the effect of temperature, CMCase was incubated with 2% CMC-Na at a pH of 5.5 and temperature ranging from 30 to 75°C with an interval of 5°C. The maximum CMCase activity obtained at different pH and temperatures was considered to be 100%.To determine the optimal pH, 250 The effect of pH on the stability of CMCase was studied by mixing the crude enzyme with different buffers  with pH ranging from 3.0 to 10.0. The CMCase activity of the crude enzyme after incubating at 4°C for 24 h at different pH was detected. To study the thermostability of the CMCase produced by the strain ME27-1, the crude enzyme was preincubated at different temperatures  for 1 h. The residual CMCase activity was detected. The maximum CMCase activity obtained at pH 3.0–10.0 or temperature 30–75°C was considered to be 100%. All the enzyme assays were carried out in triplicate.All the DNA sequences of the partial 16S rRNA genes of the 22 strains reported in this study have been deposited into the GenBank database under the accession numbers from KF536877 to KF536898.A total of 245 cellulose-degrading aerobic bacterial strains were isolated from different natural reserves in the subtropical region of China, which were cultured in agar medium containing sugarcane bagasse pulp as the sole carbon source. Out of these strains, 22 isolates showed hydrolyzing zones on agar plates containing CMC-Na after Congo-red staining . The hydβ-glucosidase activities of all the 22 bacterial strains were also undetectable.Among the 22 isolates, only three isolates  were found to produce measurable CMCase after liquid cultivation, and isolate ME27-1 showed the highest CMCase activity (0.17 U/mL) after incubation for 60 h in basal liquid medium containing 10 g/L of CMC-Na . The CMCCongo-red staining has been widely used in many studies for screening cellulose-degrading microorganisms. Although Teather and Wood described the relationship between the diameter of hydrolyzing zone and log enzyme concentration, this correlation could not represent the enzyme-producing ability of the microorganisms . In the β-glucosidase. In a study carried out by Rastogi et al., Brevibacillus sp. DUSELG12 and Geobacillus sp. DUSELR7 were found to produce a maximum FPase activity of 0.027 and 0.043 U/mL on days 7 and 8, respectively [In general, aerobic bacteria produce low levels of Avicelase, FPase, and The DNA fragments containing partial 16S rRNA genes of the 22 isolates were amplified and sequenced. The sequences obtained were matched with those available in GenBank, which revealed maximum identity of these isolates and allowed identification of these cellulose-degrading bacterial strains . Burkholderia (36.36%), Bacillus (13.65%), Citrobacter (13.65%), Arthrobacter (9.10%), Enterobacter (4.54%), Chryseobacterium (4.54%), Pandoraea (4.54%), Paenibacillus (4.54%), Dyella (4.54%), and Pseudomonas (4.54%). The phylogenetic tree of the 22 strains was constructed by using MAGA4.1 program . Morphological tests revealed that the cells of the strain ME27-1 were rod-shaped, endospore-forming, Gram-positive, and 0.8 × 1.9–3.2 μm in size. The appearance of the colony on the TSA medium was cream-colored, moist, irregular, swollen, and pigment-free. The biochemical properties of the strain ME27-1 are listed in  P. terrae [ P. terrae.The strain ME27-1, with higher CMCase activity, was thoroughly examined. The partial 16S rRNA gene (1309 bp) from the strain ME27-1 showed a maximum identity of 99% with that of P. terrae, although other species of Paenibacillus have been found to produce cellulase. Some CMCase genes cloned from Paenibacillus polymyxa GS01, Paenibacillus barcinonensis, Paenibacillus xylanilyticus KJ-03, and Paenibacillus cookii SS-24 have been expressed in Escherichia coli and Saccharomyces cerevisiae [ Paenibacillus curdlanolyticus B-6, Paenibacillus campinasensis BL11, Paenibacillus sp. B39, and P. polymyxa have been purified [To our knowledge, till date, no study has reported about CMCase production byrevisiae –35. On tpurified –39. Paenibacillus sp. In a previous study, P. curdlanolyticus B-6 was cultivated for enzyme production at pH 7.0 and 37°C [ P. polymyxa were 5.5 and 37°C, respectively [ P. terrae was 30°C, which is similar to that observed for optimal CMCase production by the strain ME27-1 [The best incubation conditions were pH 8.0 and 28°C Figures . The CMCand 37°C . Furtherectively . Yoon et S. viridobrunneus SCPE-09 [ Cellulophaga lytica LBH-14, while Kumar et al. reported that high CMCase production by P. polymyxa was obtained when using mango peel as substrate [ Cellulomonas sp. and Cellulosimicrobium cellulans [Various cellulosic materials have been used to induce microorganisms to produce cellulase. When fructose and glucose were used as the sole carbon source, no CMCase activity was detected. Wheat bran induced the highest CMCase activity, which was about 2.5-fold higher than that observed in the basal medium containing CMC-Na . The opt SCPE-09 . Gao et ubstrate , 40. In ellulans .4Cl as the sole nitrogen source (4Cl in the medium was observed to be 3 g/L (Furthermore, maximum CMCase activity was noted when using NHn source , and thebe 3 g/L . Many re Pseudomonas sp. HP207 and S. viridobrunneus SCPE-09 was observed after 24 and 48 h of incubation, respectively, which is much earlier than that noted for the strain ME27-1 [ C. lytica LBH-14 was obtained after 72 h of incubation, whereas that of Brevibacillus sp. DUSELG12 and Geobacillus sp. DUSELR7 was noted after days 9 and 7, respectively [In addition, use of an inoculum size of 2% resulted in maximum CMCase activity after incubation of the strain for 60 h . There hn ME27-1 , 16. Howectively .The optimum pH and temperature of CMCase produced by strain ME27-1 were found to be 5.5 and 50°C, respectively Figures . The CMC S. viridobrunneus SCPE-09 and P. cookii SS-24, with an optimal pH of 5.0 and 5.1 and an optimal temperature of 50° and 55°C, respectively [ P. campinasensis BL11, P. polymyxa GS01, Paenibacillus sp. B39, and Bacillus mycoides S122C were observed at neutral or alkaline conditions [Similar results were observed for cellulases produced byectively , 35. Hownditions , 46, 47.nditions . P. terrae ME27-1 produced CMCase activity of 2.08 U/mL under the optimized cultivation conditions, which was a 12-fold improvement in the CMCase production. This yield of CMCase production was higher than most of the aerobic bacterial strains but less than some of aerobic bacterial strains that have been exploited previously ( P. terrae ME27-1 was lower than that by several anaerobic bacterial strains, for example, Clostridium papyrosolvens CFR-703, C. thermocellum YM4, C. thermocopriae JT3-3 (When measured at the optimal pH and temperature of CMCase,eviously . Howeverae JT3-3 . Some an P. terrae ME27-1 was lower than that by most aerobic fungal strains while it was higher than that by anaerobic fungal strains (Furthermore, the CMCase produced by strains . The CMC Burkholderia was found to be the most prevalent and predominant. The strain ME27-1, identified to be P. terrae, showed the highest CMCase activity among the 22 strains isolated, and after optimization of the cultivation conditions, the enzyme activity was significantly improved to 2.08 U/mL. This bacterial species has been rarely found to produce cellulase. Thus, this study revealed the diversity of cellulose-degrading bacteria in the subtropical region of China and found that P. terrae ME27-1 was a good CMCase producer.Ten genera of bacteria hydrolyzing cellulose were isolated from different natural reserves in the subtropical region of China, and the genus


Gossypium raimondii (D genome) sequence-based SSR markers to identify polymorphism between two upland cotton strains 0–153 and sGK9708. A total of 851 polymorphic primers were finally selected and used to genotype 196 recombinant inbred lines (RIL) derived from a cross between 0 and 153 and sGK9708 and used to construct a linkage map. The RIL population was evaluated for fiber quality traits in six locations in China for five years. Stable QTLs identified in this intraspecific cross could be used in future cotton breeding program and with fewer obstacles.The identification of quantitative trait loci (QTLs) that are stable and consistent across multiple environments and populations plays an essential role in marker-assisted selection (MAS). In the present study, we used 28,861 simple sequence repeat (SSR) markers, which included 12,560 t sub-genome comprised 44 QTLs, whereas 14 clusters in the Dt sub-genome that included 59 QTLs for fiber quality were identified. Four chromosomes, including chromosome 4 (c4), c7, c14, and c25 were rich in clusters harboring 5, 4, 5, and 6 clusters respectively. A meta-analysis was performed using Biomercator V4.2 to integrate QTLs from 11 environmental datasets on the RIL populations of the above mentioned parents and previous QTL reports. Among the 165 identified QTLs, 90 were identified as common QTLs, whereas the remaining 75 QTLs were determined to be novel QTLs. The broad sense heritability estimates of fiber quality traits were high for fiber length (0.93), fiber strength (0.92), fiber micronaire (0.85), and fiber uniformity (0.80), but low for fiber elongation (0.27). Meta-clusters on c4, c7, c14 and c25 were identified as stable QTL clusters and were considered more valuable in MAS for the improvement of fiber quality of upland cotton.The map covered a distance of 4,110 cM, which represents about 93.2 % of the upland cotton genome, and with an average distance of 5.2 cM between adjacent markers. We identified 165 QTLs for fiber quality traits, of which 47 QTLs were determined to be stable across multiple environments. Most of these QTLs aggregated into clusters with two or more traits. A total of 30 QTL clusters were identified which consisted of 103 QTLs. Sixteen clusters in the AMultiple environmental evaluations of an intraspecific RIL population were conducted to identify stable QTLs. Meta-QTL analyses identified a common chromosomal region that plays an important role in fiber development. Therefore, QTLs identified in the present study are an ideal candidate for MAS in cotton breeding programs to improve fiber quality.The online version of this article (doi:10.1186/s12864-016-2560-2) contains supplementary material, which is available to authorized users. Gossypium) is a well-known and highly important industrial crop that has been grown in more than 80 countries located in tropical and subtropical regions [Gossypium comprises approximately 45 diploid species and five tetraploid species. Two tetraploid species, G. hirsutum and G. barbadense, and two diploid species, G. herbaceum and G. arboreum have been extensively cultivated around the world, with G. hirsutum covering >90 % of the total world production and is generally referred to as upland cotton [G. barbadense has superior fiber quality attributes that subsequently gives it a 30–50 % price advantage over upland cotton [G. barbadense restricts its production to specific regions around the world. To fulfill the global requirements of the growing human population and the recent advancement in spinning technology justify the need for increased cotton fiber yield and improved cotton fiber traits. Fiber quality traits and yield components are quantitative traits that are negatively correlated [Cotton  is prestigious blessing that breaks the linkage among these traits, as it directly selects genetic markers that are tightly linked to quantitative trait loci (QTLs) other than the conventional procedure of indirectly selecting strains with superior phenotypic performance for breeding. Recent developments in field of molecular markers have allowed plant breeders to identify and evaluate complex agronomical traits. The construction of a molecular genetic map is a foundation for the genetic dissection of important economical and agronomical traits, MAS, and map-based cloning . The firore QTLs , 23, 24.G. raimondii [G. arboreum [G. hirsutum [G. arboreum (A genome) and G. raimondii (D genome) about 1–2 million years ago [t sub-genome of upland cotton compared to that in the At sub-genome, thus suggesting that it may play an important role in fiber developments [G. raimondii BAC-end sequences has revealed that these D genome-based primers are widely distributed and suitable for whole-genome mapping. Therefore, because of the importance of the Dt sub-genome in determining fiber quality traits [G. raimondii) sequence-based SSR primers [http://www.cottonmarker.org/) to construct an intraspecific linkage map. Previously, Sun et al. [2, F2:3 and RIL populations in 4 environments. We added 603 primers to our published genetic map and identified QTLs for fiber quality in 11 environments, including four previously reported environments [6:8) population of upland cotton. Furthermore we conducted a meta-analyses with Biomercator V4.2 [2, F2:3 and RIL population [Recently physical genome drafts of aimondii , 26 G. aarboreum  and G. hhirsutum , 29 havelopments –32. A hilopments  with SSRy traits , we usedn et al.  reportedronments   contained 3 SDRs, 6 chromosomes  comprised 2 SDRs, whereas the remaining 11 chromosomes  harbored only 1 SDR.Segregation distortion is a common occurrence in plants , includiG. hirsutum physical map of various chromosomes is presented in Fig. t and Dt sub-genomes of G. hirsutum. In the At sub-genome (c1–c13), 1.76 GB corresponded to 1,635 cM, whereas in the Dt sub-genome (c14–c26) 774 Mb was equivalent to 2,327 cM.Loci collinearity between linkage map and the t sub-genome chromosomes, whereas 107 QTLs were localized to the Dt sub-genome chromosomes. QTLs positions with their observed phenotypic variance (PV) and nearest loci are listed in Additional file A total of 165 QTLs for five fiber traits were identified on 24 chromosomes using the composite interval mapping method ., Forty qFL-C4-2, was identified in three environments, explaining 5.8–8.1 % of the observed PV. Two QTLs on c7, qFL-C7-1 and qFL-C7-2 were also identified in 4 environments described in our previous report [qFL-C7-1 was stable and identified in 3 environments, explaining 5.8–12.1 % of the observed PV. Three QTLs on c14, qFL-C14-1, qFL-C14-2 and qFL-C14-3 were identified in 8, 6, and 3 environments, explaining 8.1–13.1 %, 7.1–11.5 %, and 6.3–8.1 % of the observed PVs, respectively. The QTL qFL-C14-2 was also identified in our previous report [qFL-C16-1 was identified in three environments, explaining 5.7–7.5 % of the observed PV. The QTL on c18, qFL-C18-3 was identified in a single environment in our previous report [qFL-21-1 was identified in seven environments, explaining 8.7–23.6 % of the observed PV. The QTL on c23, qFL-C23-2 was identified in a single environment in our previous report [qFL-C25-2, qFL-C25-3, qFL-C25-4, qFL-C25-5, and qFL-C25-6 were respectively identified in 4, 6, 5, 5, and 3 environments, explaining 5.2–10 %, 6.8–9.4 %, 6.8–11.8 %, 6.5–10.5 % and 8.6–10.6 % of the observed PVs, respectively. Two QTLs, qFL-C25-2 and qFL-C25-3, were also previously identified in four environments [In total, 31 QTLs for FL were detected on 11 chromosomes, including c4, c6, c7, c14, c16, c18, c21, c22, c23, c24, and c25 . Cluster, C4-m-cluster-2 from 73 to 93 cM interval was located between markers DPL0451 and CIR218 . The cluster on Cons.c7, C7-m-cluster-1, from the 20–36 cM interval was localized between markers NAU5303 and NAU3918 . Cluster, C7-m-cluster-2 from the 40–58 cM interval was located between markers BNL1597 and NAU2186 . Cluster, C7-m-cluster-3 from 60 to 72 cM interval was situated between markers NAU1085 and CIR238 . The cluster on Cons.c14, C14-m-cluster-1, from 0 to 15 cM interval was localized between markers SWU14174 and SWU14188 . Cluster, C14-m-cluster-2 from 20 to 36 cM interval was localized between markers BNL3099 and COT099 . C14-m-cluster-3 from 38 to 54 cM interval was between markers NAU3393 and PGML0989  and C14-m-cluster-4 from the 58–78 cM interval was between markers DPL0354 and BNL3033 .In the meta-analysis, a total of 38 meta-cluster regions on 11 chromosomes were identified, which included c4, c5, c7, c12, c13, c14, c15, c16, c20, c23, and c25  were identified in the At sub-genome, whereas 107 were identified in the Dt sub-genome. Previous comparative meta-analyses conducted by Rong et al. [t sub-genome chromosomes, and gene expression among homologous pairs were not uniform [t sub-genome in an inter specific backcross inbred line population. In the present study a higher number of loci were mapped to the Dt sub-genome. This observation might be due to the presence of more SSR markers that were developed from the D genome sequence [1 population. However we also observed that some At sub-genome chromosomes also have more loci than its homologous counterparts in Dt sub-genome chromosomes. This unequal distribution of loci indicates the presence of active regions with more recombination frequencies in the upland cotton genome [The distribution of QTLs was not uniform in the Ag et al. , Lacape g et al.  and Saidg et al.  have ind uniform , 47. Yu  uniform  also obssequence , althougsequence  in theirn genome , 43.G. arboreum) produces spinnable fibers, whereas the D genome (G. raimondii) lacks this characteristic. After polyploidization, transposable elements tend to be more active, especially in the Dt sub-genome, compared to that in the At sub-genome. Furthermore, the Dt sub-genome also has a higher mutation rate than the At sub-genome [t sub-genome harbored more QTLs than the At sub-genome. However, the additional of novel markers for the At Sub-genome may improve the assessment of the contribution of each sub-genome in fiber quality traits.Comparison of the tetraploid cotton genome with its ancestors shows that only the A genome  at the Cotton Fiber Quality Inspection and Testing Center of Ministry of Agriculture, Anyang, China. The fiber quality traits included FE, FL, FM, FS and FU. These observed phenotypic data were analyzed by using the software SPSS20.0 . For ANOVA, we used the SAS statistical software . To calculate broad sense heritability the following equation was used2G is genotypic variance, σ2G*E is genotype * environment variance, and σ2E is variance of error.Where σet al. [2O, 1 μL 10× buffer (with 1.5 mL Mg+), 0.5 μL dNTPs (10 mM), 0.5 μL each primer, 0.15 μL of Taq polymerase (500U) and 1.2 μL of genomic DNA (30 ng/μL). PCR amplification conditions comprised of an initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 57 °C for 30s and an extension at 72 °C for 60s followed by a final elongation at 72 °C for 5 min, and then held at 4 °C until analysis. PCR products were electrophoresed on an 8 % non-denatured polyacrylamide gel and silver staining was used for visualization of bands.Young leaves were collected from each line and stored at −80 °C. Genomic DNA from the parents and 196 RILs was extracted using a modified CTAB method as described by Paterson et al. . PCR ampA total of 28,891 primers pairs, including 12,560 SWU primers (D genome sequence-based), were used to detect polymorphisms between the two parents. Approximately 851 polymorphic primers were selected and used in genotyping 196 recombinant inbred lines. All loci were named according to their respective primer names. In the case of multiple loci generated by single primer pair that showed a different segregation pattern from that of the main band, a suffix of a/b/c was used after the primer name to differentiate loci according to increasing molecular size. The details of the primers used in the present study are listed in Additional file http://www.cottongen.org/). Small groups that were mapped to the same chromosome were recalculated to combine these into one group. A minimum LOD score of 6 was used to combine these groups. In the case of c20 and c23, an LOD score of 5 was used to combine small linkage groups into one. The G. hirsutum fasta sequence was downloaded from http://www.cottongen.org/ and used to check co-linearity of loci between the linkage map and the G. hirsutum physical map.A linkage map was constructed using JoinMap 4.0  with a l56G. hirsutum genome, and QTL analysis was performed using the composite interval mapping method as earlier described.Windows QTL Cartographer 2.5  was usedwww.cottonqtldb.org. We used the high-density consensus map [2,F2:3, and RIL populations of same parents [Meta-analysis was performed with Biomercator 4.2  as descrnsus map  as a ref parents , and 635 parents , 38 wereFor meta-analyses, two separate input files were prepared, a map file and a QTL file. The map file contained distances between markers on each chromosome, and the QTL file contained 12 columns, where each row represented a single QTL in a given environment, i.e., QTL name, trait name, trait ontology, experiment place, year, chromosome name, linkage group name, LOD score, observed PV value (R2), most likely position of the QTL, CI start position and CI end position. First both files were loaded into the software and checked for map connectivity. Then QTLs were projected on a consensus map and meta-analyses were performed for each trait. Four models were thus generated, each with an Akaike information criterion (AIC) value. The model with lowest AIC value was selected and used for the identification of mQTL position, whereas QTL clusters were determined manually. The QTLs within the region of 20 cM on the consensus map were considered as part of same cluster as earlier defined by Said et al. .


Cellulosimicrobium sp. MTCC 10645. The bacterium produced thermo-alkali stable xylanase in a basal medium supplemented with wheat bran (1% w/v), which was optimally active at pH 7.0 and 50°C. The xylanase was stable at temperature 50°C for 1 h and retained up to 86% of the activity. The xylanase was stable in a broad pH range of 6.0–11.0 for 1 h at 50°C. Metal ions Ca+2, Hg+2, and Pb+2 were inhibitory for xylanase retaining 72.3%, 35.07% and 36.7% relative activity at 10 mM concentration, whereas Fe+2, Cu+2, Mn+2, Na+2, Co+2, and Zn+2 were inducers at concentrations of 5 mM and 10 mM. The enzyme exhibited greater binding affinity exclusively for xylans but not for avicel, CMC, cellobiose, starch, or p-nitrophenyl xylopyranoside. Parachloromercuric benzoate and iodoacetamide were found stimulatory, while potassium permanganate, cysteine, and cystine markedly reduced the activity. The xylanase dose of 2.0 U/g dry weight pulp of 10% consistency gave optimum bleach boosting of kraft pulp at pH 8.0 and temperature 50°C for 5 h reaction time.The most promising application of xylanases (E.C. 3.2.1.8) is in the prebleaching of kraft pulp. The present paper reports bleaching effects of a thermoalkali stable xylanase from  MTCC 10645.The most promising application of xylanases E.C. 3.2.1.8) is in the prebleaching of kraft pulp. The pulp and paper industry is modifying its pulping, bleaching, and effluent treatment technologies to reduce the environmental impact of mill effluents. Prebleaching of kraft pulps with xylanases lowers chlorine charges, which reduce chloroorganic discharges .8 is in . Thus releaching –7. The pleaching . The occleaching . Xylanasleaching –14. The Cellulosimicrobium sp. MTCC 10645 was isolated from the compost sample collected at Kolhapur . The culture was purified and maintained on xylan agar (pH 7.0) supplemented with 0.1% oat spelt xylan. The xylanase production was carried out in 250 mL Erlenmeyer flasks, each containing 18 mL basal salt solution supplemented with 10 g of wheat bran (1 : 1.8 moisture ratio) in static conditions [nditions . Each flμmol of reducing sugars equivalent to xylose per minute under the assay conditions . Total soluble protein was measured using bovine serum albumin as a standard [Xylanase activity was determined by measuring the release of reducing sugars from birchwood xylan (1% w/v) using the dinitrosalicylic acid method . One unistandard .+2, Hg+2, Fe+2, Cu+2, Mn+2, Na+2, Co+2, Pb+2, and Zn+2 at 1 mM, 5 mM, and 10 mM concentration on enzyme activity was determined by incubating the enzyme with respective compounds at room temperature for 1 h followed by determination of residual activities under standard assay conditions. Substrate specificity of the xylanase was studied by using 1% xylan, cellobiose, starch, carboxy methyl cellulose (CMC), p-nitrophenyl xylopyranoside, and avicel as substrates. Effect of additives like parachloromercuric benzoate, iodoacetamide, potassium permanganate, cysteine, and cystine (0.5 mM) was tested on xylanase activity.The cell free supernatant (75 mL) was precipitated using fractional (35–80%) ammonium sulphate saturation. The precipitate was dialysed and the protein thus obtained was treated as partially purified enzyme and used for further experiments. The thermal stability was determined by incubating the equal volume of enzyme solution at temperatures ranges between 30°C–80°C for 4 h. Optimal pH for xylanase activity was determined using different buffers 50 mM sodium phosphate , 50 mM Tris HCl , 50 mM carbonate bicarbonate buffer (pH 10.0) and 50 mM glycineNaOH buffer (pH 11.0). The pH stability was determined by incubating the equal volume of enzyme solution with different buffers ranging from 6.0–11.0 at 50°C and the residual activities were determined after 1 h. The effect of various metal ions solution of chloride salts of either CaCellulosimicrobium sp. MTCC 10645 at 2 U/g, 5 U/g, 10 U/g, and 25 U/g oven dried pulp of consistency 3.0%, 5% and 10% for variable time intervals 3 h and 5 h. The chemical characteristics of Kraft pulp: kappa number (T 236 cm-85), release of reducing sugars, release of phenolic compounds (A237 nm and 280 nm), and hydrophobic compounds (A465 nm) were determined [The enzymatic bleaching studies were performed at pH 8.0 at 50°C, unless otherwise mentioned. The optimization of enzyme dose, pulp consistency and reaction time was carried out by treating the pulp with different doses of xylanase from termined , 18, 19.Cellulosimicrobium sp. and deposited with Microbial Type Culture Collection no. 10645 at IMTECH, Chandigarh, India. Xylanase production was monitored in Cellulosimicrobium sp. MTCC 10645 grown for 7 days in static conditions on wheat bran. Xylanase activity appeared after day 1 and reached maximum (960 U/mL) after day 2 at pH 7.0 and 40°C. The test organism was isolated from compost sample collected at Kolhapur . It was identified as Cellulosimicrobium sp. MTCC 10645 was partially purified at 80% (NH4)2SO4 saturation with a yield of 52.03% and a purification fold of 16.0. The partially purified xylanase from Cellulosimicrobium sp. MTCC 10645 was optimally active at 50°C and pH 7.0  were purified, named xyl I and III which did not showed activity towards glycans such as starch, pachyman, and avicel  but showed one twentieth activity towards carboxymethyl cellulose [Aspergillus fumigatus AR1 [Thermomonospora sp. [The xylanase was active on oat spelt and birchwood xylans but not on avicel, CMC, cellobiose, starch, or a viride . Two endellulose . Additivatus AR1 . Iodoacepora sp. .Thermomyces lanuginosus CBS 288.54 was significantly greater with increasing time [The xylanase dose for biobleaching of kraft pulp at 50°C was optimized as 2.0 U/g of moisture free pulp . Reducining time . Cellulosimicrobium sp. MTCC 10645, the release of light absorbing material and reducing sugars increased with increase in consistency. Release of absorbing materials at 237 nm, 280 nm, and 465 nm can be correlated with release of lignin. The absorbance measured was highest at 280 nm wavelength as compared to 237 nm and 465 nm at 10% pulp consistency. At 237 nm wavelength, the absorbance measured was 0.215, 0.231, and 0.256 at 3%, 5%, and 10% consistency of pulp, respectively. At 0% pulp consistency, the absorbance measured was 0.211 at 237 nm wavelength. At 465 nm the absorbance measured was comparatively less 0.218 at 10% pulp consistency. The reducing sugars released were 0.75 mg/g, 1.15 mg/g, 1.65 mg/g, and 2.161 mg/g of pulp at 0%, 3%, 5%, and 10% consistency, respectively  in the bleaching of kraft pulp and found that, xylanase P (at an enzyme dose 5 U/g pulp) increased the brightness of kraft pulp by 5.1% while xylanase from Thermomyces lanuginosus (at an enzyme dose 5 U/g pulp) increased 2.1% [Bacillus sp. NCIM 59 on unbleached bagasse pulp [Aspergillus indicus, Aspergillus flavus, and Aspergillus niveus increased brightness of 42.0–45.0 ISO units from 19.83 ISO units and reduced kappa number of 5.0–6.8 from 18.60 of the kraft pulp [In the present study, the brightness increased from 30.13% (brightness of unbleached pulp) to 70.45% after the treatment of the kraft pulp with xylanase 25 U/g of pulp with a retention time of 5 h at 10% consistency. The kappa number was reduced from 14.90 (kappa number of unbleached pulp) to 1.62. Li et al. reported that when wheat straw pulp was treated with the xylanase isolated from .93% ISO . Madlalaaft pulp .Cellulosimicrobium sp. MTCC 10645.To the best of our knowledge, this is the first paper on the thermo-alkali stable xylanase production from