Zea mays genetic sequences that encode the enzymatic components of FAE and developed a heterologous expression system to evaluate their functionality. The ability of the maize components to genetically complement Saccharomyces cerevisiae lethal mutants confirmed the functionality of ZmKCS4, ZmELO1, ZmKCR1, ZmKCR2, ZmHCD and ZmECR, and the VLCFA profiles of the resulting strains were used to infer the ability of each enzyme component to determine the product profile of FAE. These characterizations indicate that the product profile of the FAE system is an attribute shared among the KCS, ELO, and KCR components of FAE.Fatty acids of more than 18-carbons, generally known as very long chain fatty acids (VLCFAs) are essential for eukaryotic cell viability, and uniquely in terrestrial plants they are the precursors of the cuticular lipids that form the organism’s outer barrier to the environment. VLCFAs are synthesized by fatty acid elongase (FAE), which is an integral membrane enzyme system with multiple components. The genetic complexity of the FAE system, and its membrane association has hampered the biochemical characterization of FAE. In this study we computationally identified  Two primary characteristics affect the chemo-physical properties of fatty acids that enable them to fulfill diverse biological functions, the length of the alkyl-chain, and the number and geometric isomerism of carbon-carbon double bonds in the alkyl-chain. In eukaryotic organisms, such as plants, fatty acids are initially assembled as fully saturated acyl-chains, and subsequently carbon-carbon double bonds are created in the alkyl chain by an aerobic process catalyzed by specific desaturases, which remove hydrogen atoms from adjoining carbon atoms . TherefoAll three fatty acid assembly systems utilize the same biochemical mechanism, iterative reaction cycles of condensation-reduction-dehydration-reduction. Apart from the distinct organelle localizations of the three assembly systems, three additional features distinguish the FAE system from FAS: 1) in the FAE system the pantetheine group that carries the intermediates of the growing fatty acyl chain is Coenzyme A (CoA); 2) the FAE system is an integral membrane-bound system localized to the endoplasmic reticulum; and 3) FAE initiates the elongation process by using pre-existing (C16 or C18) fatty acyl-CoAs.elovl1 and elovl4 null mutant mice that cannot produce VLCFAs, these components contribute a critical functionality to the skin’s normal function and structure [As exemplified by the lethal consequence of mutations that affect VLCFA biosynthesis, these acyl-chains participate in vital cellular functions, specifically associated with cellular sphingolipid pools –10. Thestructure ,13,14. Structure . The mettructure ,16.Plants have generated and maintain a large degree of biochemical and genetic redundancy within the FAE system. Two distinct non-homologous enzyme families catalyze the initial condensation reaction: 1) the Arabidopsis FAE1-like, 3-ketoacyl-CoA synthases (KCS-type enzymes); and 2) the ELONGATION DEFECTIVE-LIKEs (ELO-type enzymes). These two enzymes catalyze the same chemical reaction, creating a new C-C bond, and this reaction is thought to be the chain-length determining and possibly rate-limiting reaction of the FAE system ,18.gl8a (ZmKCR1) and gl8b (ZmKCR2), have been characterized in maize, with partially redundant functions [The second reaction of the FAE cycle is catalyzed by 3-ketoacyl-CoA reductase (KCR); two highly homologous KCR paralogs, unctions –21. In cunctions . The remunctions –26 and tunctions , 27–29.Saccharomyces cerevisiae to identify four additional catalytic components of the Zea mays FAE system using the strategy of genetic complementation of yeast mutant strains as an assay to identify gene functionality. The maize FAE system is not only of interest because of its importance to global agriculture, but because of the unique complexity that maize presents by the functional redundancy of the ELO, KCS, and KCR components. Biochemical characterization of the fatty acid profiles of the complemented yeast strains demonstrate that the maize ELO, KCS, and KCR paralogs contribute different fatty acid profiles, indicating that these components contribute to the product specificity of the overall FAE systems.In this study, we have utilized heterologous expression in ZmKCS  and ZmELO  were identified based on sequence homology to a characterized Arabidopsis KCS  [Zea mays inbred line B73. ZmKCR1 and ZmKCR2 were previously genetically identified and molecularly isolated as the Glossy8a and Glossy8b loci, respectively [ZmHCD; GenBank accession DR817981.1, GRMZM2G151087) was identified from the maize EST assembly at PlantGDB (www.plantgdb.org) based on its sequence homology to the yeast  [PASTICCINO2; AT5G10480) [ZmECR; GenBank accession AQK90310.1, GRMZM2G481843) was identified by its homology to the yeast  [CER10; AT3G55360) [ZmECR was independently shown to be encoded by the genetically defined Glossy26 locus, which is required for the normal deposition of cuticular waxes [ZmHCD and ZmECR cDNAs were isolated by RT-PCR from the Zea mays inbred line B73.With the exception of the KCR component –21, all 2g16280)  or yeastectively –21. The YJL097w)  and Arab5G10480) . The maiYDL015c)  and Arab3G55360) . ZmECR wArabidopsis thaliana, Zea mays, Solanum lycopersicum, Sorghum bicolor, Oryza sativa, Saccharomyces cerevisiae, and Homo sapiens. Homologs were identified by BLASTP [Neighbor-joining phylogenetic trees were constructed for each of the FAE components, and these included homologous sequences identified from y BLASTP  analysisy BLASTP . Sequency BLASTP , using tGAL1 promoter to control the heterologous expression of maize FAE components [ZmKCS, ZmELO, ZmHCD and ZmECR components were cloned into the high-copy episomal plasmids, pAG426 (URA3), pAG423 (HIS3) or pAG424 (TRP1)  [ZmKCR1 and ZmKCR2 were each cloned into pYX043 (an integrative yeast shuttle vector carrying the LEU2 marker), using the EcoRI-SalI and KpnI-XbaI restriction sites, respectively. All recombinant yeast shuttle vectors were confirmed by DNA sequencing, and were maintained in E. coli TOP10 or DH5α cells , propagated in Luria Bertani (LB) media with the appropriate antibiotics.All yeast expression cassettes used the galactose-inducible mponents . Using tbad, CA)  . This mutated construct, ZmECR(p.Ser283Cys)/pAG424, was used as a negative control in the yeast genetic complementation experiments. The transversion was corrected by QuikChange mutagenesis  using primer ECRcorrA2T  in SD media.Yeast cultures were grown according to standard procedures ,37. The BstXI-digested linearized plasmids. For the purpose of generating and maintaining the synthetically lethal scelo2, scelo3 double mutant strain, a ScELO3-expression vector was constructed by cloning the ScELO3 sequence with its native promoter sequence in the pAG416  plasmid backbone using In-Fusion cloning . Restriction sites and primers were designed to remove the promoter region of the vector backbone. The resulting plasmid (carrying PELO3-ELO3) was transformed into a diploid strain that was heterozygous mutant at both scelo2 and scelo3 loci. The resulting strain was sporulated, enabling the recovery of the scelo2, scelo3 double mutant, which episomally expressed PELO3-ELO3. This was genetically confirmed by the inability of the strain to grow on 5-FOA counter-selection media, and molecularly confirmed with PCR.Plasmids were transformed into yeast using a standard lithium acetate transformation protocol . HomologSporulation of diploid strains was performed according to Enyenihi and Saunders  with thehttps://www.biotek.com/products/software-robotics-software/gen5-microplate-reader-and-imager-software/), and statistical evaluations were carried out using JMP Pro 9.0 , using Student’s t tests to identify statistical rigor. Raw data is available at DOI 10.25380/iastate.7635962.Growth of all yeast strains were assessed in synthetic complete (SC) or SD media, supplemented with the appropriate auxotrophic need, and expression of maize genes was induced with galactose. Strains were grown in shake flasks or using a BioTek microplate reader , maintained at 30°C with constant shaking, and growth was monitored by measuring OD600 every 30 minutes. Doubling times were calculated using Gen5 data analysis software . The metabolite abundance data were evaluated using Tukey Honest Significant Difference (HSD) to evaluate statistical rigor.Between 5 and 10 mg of dry cell pellet and a known amount of nonadecanoic acid, as an internal standard, was homogenized with acid-washed glass beads , and treated with a solution of 10% (w/v) barium hydroxide at 110°C for 24-hours. Upon acidification, the saponified fatty acids were recovered by extracting with hexane. Following methylation and silylation, fatty acid methyl esters were analyzed by GC-MS or GC-FID ,41. StatZmKCS4), which is classified as belonging to the phylogenetically mixed α/βclade.ELO homologs. These candidates were further classified based on the occurrence of four conserved amino acid motifs  that have been experimentally characterized as being functionally important with yeast ELOs [ZmELO1. The other five candidates were not considered for further experimental validation because they only partially retain the yeast and human ELO-defined conserved motifs ELO homolast ELOs , the maiast ELOs . Based ud motifs .Glossy8a and Glossy8b [16YX3K) [3GXGX3AX3AX2G), which is commonly conserved in the broader short chain dehydrogenases/reductase superfamily of enzymes [Glossy8b –21), yeaGlossy8b  and ArabGlossy8b  KCRs, an[16YX3K) ,22 and t enzymes . In addi enzymes .These phylogenetic analyses establish that KCR enzymes divide into three separate clades, specific to monocotyledonous plants, dicotyledonous plants, and yeast and mammals. This finding therefore, indicates that the evolutionary history of this FAE component is ancient and precedes the divergence of Plantae from Fungi and Animalia. In addition, like maize, many plant species contain multiple KCR homologs, which appear to have arisen post-speciation, and while some of these duplications have retained the KCR catalytic function, as is the case for maize –21, in oSimilar sequence-based phylogenetic analyses using yeast, human and Arabidopsis sequences identified putative maize, rice, sorghum, and tomato HCD and ECR homologs . Apart fZmKCS4, ZmELO1, ZmKCR1, ZmKCR2, ZmHCD and ZmECR) were individually evaluated based on their ability to complement yeast strains lacking the respective endogenous function. The yeast mutant strains used in this study carry disruption knock-out alleles, which are either lethal  or nearr [sckcr ). Each mpromoter .ZmECR component, an additional control experiment was with the use of the ZmECR (p.Ser283Cys) mutant. This point mutation is at a residue that appears to be crucial, because the resulting strain is inviable, and suggests that residue p.Ser283 is critical to catalysis.As an additional control, all complemented strains were in parallel compared to strains that expressed maize FAE components in a wild-type yeast strain, which carried functional alleles of the endogenous yeast FAE components. In the case of the functional complementation with the ScELO1, ScELO2 and ScELO3) encode the enzyme that catalyzes the condensation reaction in the yeast FAE cycle, but only the latter two are crucial for VLCFA biosynthesis [scelo1, scelo2, or scelo3 knockout mutant alleles are viable, but the scelo2, scelo3 double mutant is lethal [ZmKCS4 and ZmELO1 to complement this deficiency, we evaluated the quantitative and qualitative changes in the VLCFA profiles of the complemented scelo2, scelo3 double mutant strains, and compared these to the wild-type and each scelo2 and scelo3 single mutant, in the presence and absence of the maize complementing gene  were also expressed individually in the wild-type background, resulting in the overexpression of each catalytic FAE functionality. In the case of the ZmKCS4 and ZmELO1 components, we also evaluated the effect of overexpression on the growth of the viable, individual scelo2 and scelo3 mutant strains , which accounts for >90% of the fatty acids in these strains, were only subtely affected by the genetic manipulations of FAE components , and these are illustrated in The fatty acid profiles of each yeast mutant strain that was rescued by the expression of individual maize FAE components were compared to the wild-type strains, profiling the detectable fatty acids, from 12 to 28 carbon chain length. As expected, changes in the profiles of the products of mponents ; the excZmKCS4 complementation of the scelo2, scelo3 double mutant strain induced a nearly 10-fold increase in the levels of VLCFAs as compared to the wild-type strain, and 20:0 accounts for the majority of the additional VLCFAs that accumulate . However, overexpression of ZmKCS4 in viable scelo2 or scelo3 single mutant strains, induced inverse effects on VLCFA accumulation between the two strains. Specifically, ZmKCS4 overexpression in the scelo2 background induced increased accumulation of VLCFAs as compared to the parental mutant strain; whereas in the scelo3 background, the yield of VLCFA is reduced. These differences are primarily due to the individual scelo2 or scelo3 mutations, as the composition of the VLCFA profiles remain unaltered by the overexpression of ZmKCS4.The expression of either ZmKCS4 or ZmELO1 induced two different effects on VLCFA profiles. The cumulate . This coZmELO1-complemented, scelo2, scelo3 double mutant is near identical to the wild-type and to the strain overexpressing ZmELO1 in a wild-type background , but the VLCFA profile of this strain is distinct from the wild-type, producing larger amounts of non-hydroxylated 22:0, 24:0, and 26:0 fatty acids. The ZmKCR2-rescued strain hyperaccumulates VLCFAs by 10-fold over the wild-type strain , but the resulting VLCFA profile resembles the wild-type strain. In contrast to the strains that are genetically complemented by the expression of either ZmKCR1 or ZmKCR2, the overexpression of either of these two maize components in a wild-type background produces only subtle quantitative or qualitative changes in the VLCFA profiles. The exception is the production of 28:0 by the ZmKCR2 overexpressing strain, which is a VLCFA product that is undetectable in the absence of the maize gene  or in the appropriate mutant background  generates VLCFA profiles that are indistinguishable from the wild-type strains . These rSeveral attributes of FAE that generates VLCFA molecules contribute to the inability to biochemically characterize this system. These include the fact that a) FAE is an integral membrane system associated with the ER, making its biochemical isolation and characterization more difficult; b) there has been confusion as to the chemical nature of the acyl substrate needed by FAE ; c) in min vivo properties of the individual FAE enzymatic components, as they impact the product profile of the FAE system. Collectively, the series of genetic complementation experiments validate the use of yeast as a heterologous expression platform for the characterization of the maize FAE system. Exemplary of this validation, the platform was used to functionally characterize the role of maize KCS4, ELO1, KCR1, KCR2, HCD and ECR components on determining the VLCFA product profile of the FAE system. This platform ultimately has the capability of evaluating the genetic diversity among the KCS, ELO, and KCR components, and providing a direct means to functionally evaluate how the FAE system is assembled and regulated to generate a diversity of end products. The experiments described herein indicate that there are two major enzymatic determinants that govern the product profile of the FAE system: the first and second reaction of the four-reaction cycle that constitutes the iterative biochemistry of fatty acid elongation. In maize, the first reaction can be catalyzed by up to 33 different enzymes (26 KCS type and 6 ELO type isozymes) and the second reaction can be catalyzed by two KCR isozymes. This diversity of component enzymes suggests that maize has the potential of expressing a large number of different FAE complexes differing in the KCS, ELO and KCR components.In this study we have demonstrated a yeast-based platform that can be used to characterize the fae1-mutant, which expresses a deficiency in the ability to sequentially elongate 18:1 to 20:1 and 22:1 during seed development [Exemplary of plants, the maize genome encodes two families of enzymes that may catalyze the formation of new carbon-carbon bonds during each FAE-catalyzed elongation cycle, the KCS-class of enzymes, and the ELO-type enzymes. The contribution of this potential biochemical redundancy to the product specificity of the plant FAE system is unknown. The archetypal KCS enzyme was identified by the analysis of the Arabidopsis elopment –56. Subselopment  studies elopment ,58,59. Oelopment  leading S. cerevisiae, which expresses three homologs [ELO2 and ELO3 are responsible for elongating 16- and 18-carbon fatty acids to chain-lengths of up to 26 carbons [In contrast to KCS, much less is known about the plant ELO family of enzymes. The ELO-type enzymes were initially identified in homologs ,49–51. GZmELO1 or ZmKCS4 can rescue the viability of the yeast strain that lacks both scelo2 and scelo3 functions. The fatty acid profiles of these rescued strains indicate that, whereas the expression of ZmELO1 reconstituted the VLCFA profile to that of the wild-type strain, the expression of ZmKCS4 generated a VLCFA profile that is distinct from the wild-type. This difference in the biochemical outcomes between the ZmELO1 and ZmKCS4 is also apparent when these maize genes are expressed in the wild-type yeast background, and in the scelo2 or scelo3 single mutant backgrounds. Both the individual scelo2 and scelo3 mutants are viable in the absence of the maize genes, but they generate different VLCFA profiles from the wild-type, and the expression of ZmKCS4 in these strains generates uniquely distinct VLCFA profiles.The expression of either ZmKCS4-expressing strains generate VLCFA profiles that are distinct from the “normal” yeast profiles, whereas the ZmELO1-expressing strains generate near wild-type VLCFA profiles, we suggest that the yeast FAE system better accommodates the latter enzyme. This can be rationalized by the fact that ELO homologs share greater structural similarity with the yeast FAE components, as compared to KCS, facilitating the interactions between the other three enzymatic components of the yeast FAE system. Assuming each KCS or ELO homolog may have different substrate specificities, this complexity provides plants with the ability to generate diverse VLCFA profiles. We further infer therefore, that interactions between the ELO/KCS components and the other three components are significant in determining the product profile of FAE.Collectively, these finding indicate that despite the large evolutionary structural differences between the maize and the yeast FAE proteins, the yeast system has sufficient flexibility to accommodate the plant components. However, because the GLOSSY8A (ZmKCR1) and GLOSSY8B (ZmKCR2) genes [glossy8a mutations reduce the seedling leaf cuticle, whereas glossy8b mutations have no such effect on this trait; however, the glossy8a, glossy8b double mutant is embryo-lethal [Indeed the expression of the two highly homologous maize KCR enzymes (encoded by the 2) genes –21), furo-lethal .sckcr [ZmKCR1-complemented strain expresses similar quantities of VLCFAs as the wild-type, but these products are of distinct chain-length distribution from the wild-type. The ZmKCR2-complemented strain in contrast, produces larger quantities of VLCFAs, but with much more subtle change in the VLCFA profile.The genetic complementation of the near-lethal sckcr  mutant sin planta chemotypes that were characterized with the glossy8a (zmkcr1) and glossy8b (zmkcr2) mutants of maize [glossy8a mutant that only expresses ZmKCR2 primarily affects the VLCFA-derived cuticular lipids, and does not affect the VLCFAs that are associated with the ceramide lipids, whereas the glossy8b mutant does not affect the deposition of cuticular lipids, but primarily reduces the VLCFAs associated with ceramides [A possible explanation for this variance maybe associated with different substrate specificties of each ZmKCR homolg. However, a more complex model may need to be invoked when one considers that the two ZmKCR homologs vary from each other by only 11 conservative substitutions among 326 residues . A potenof maize . Specifieramides ,21.In the context of the two types of FAE systems that can be envisioned in maize (one that utilizes KCS and one that utilizes ELO condensing enzymes), these KCR-associated attributes may indicate that ZmKCR1 and ZmKCR2 have differential product targeting effects by selectively preferring to associate with one or the other of the FAE systems. Based on the fact that the ELO-containing FAE system in yeast preferentially generates the VLCFAs that are used to assemble the ceramide moiety of sphingolipids , and if ZmHCD and ZmECR to complement the respective mutations in the yeast FAE components indicates that these maize enzymes can also associate with the other yeast FAE components and enable the successful completion of the VLCFA biosynthetic process. In contrast to the alterations of the VLCFA profiles that result from replacing the yeast ELO and KCR components with maize homologs, the replacement of the yeast HCD and ECR components with the maize homologs did not change the resulting VLCFA profiles. We conclude therefore that the enzyme components that catalyze the 3rd and 4th reactions of the FAE cycle are not significant determinants of the product specificity of the FAE system.The ability of Although the plant FAE system has previously been isolated as an apparent complex –63 due tS1 Figa, b)ZmKCS4 (n = 6), c, d)ZmELO1 (n = 6) where (◇) indicates the presence of the maintenance plasmid (PELO3-ELO3), e, f)ZmKCR1 and ZmKCR2 (n = 3), and g, h)ZmHCD (n = 8 for control n = 10 for complementing strain) and ZmECR (n = 8 for control n = 11 for complementing strain). Differing letters indicate statistically significantly different yields based on Tukey HSD . All strains were analyzed using GC-MS except ZmELO1, which was analyzed by GC-FID. Yeast strain is indicated under graphs, were (-/+) indicated the absence and presence of the maize gene respectively for a-d.Quantitative totals of FAS products and molar percentage of total FAS product pools for (PDF)Click here for additional data file.S2 Figa, c, e, g) WT with empty vector  and WT with ZmKCR1 (n = 4). a) Quantitative totals of FAS products; b) Molar percentage of total FAS product pools; c) Quantitative totals of VLCFAs; and d) Molar percentages of totally VLCFAs product pools. b, d, f, h) WT with empty vector (pYES2) and WT with ZmKCR2 (n = 5). b) Quantitative totals of FAS products; d) Molar percentage of total FAS product pools; f) Quantitative totals of VLCFAs; and h) Molar percentages of total VLCFA product pools.(PDF)Click here for additional data file.S3 Figa) Quantitative totals of FAS products; b) Molar percentage of total FAS product pools; c) Quantitative totals of VLCFAs; and d) Molar percentages of totally VLCFAs product pools. Strains were analyzed with replicates of n = 4 for WT with the empty vector (pYX043) and n = 5 for WT with ZmECR.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


This review deals with olefin carbonylation catalysed by Pd(II)-phosphine complexes in protic solvents. In particular, the results obtained in the carbonylation with ethene are reviewed. After a short description of the basic concepts relevant to this catalysis, the review treats in greater details the influence of the bite angle, skeletal rigidity, electronic and steric bulk properties of the ligand on the formation of the products, which range from high molecular weight perfectly alternating polyketones to methyl propanoate. It is shown that the steric bulk plays a major role in directing the selectivity. Particular emphasis is given to the factors governing the very active and selective catalysis to methyl propanoate, including the mechanism of the catalytic cycles with diphosphine- and monophosphine-catalysts. A brief note on the synthesis of methyl propanoate using a “Lucite” type catalyst in ionic liquids is also illustrated. A chapter is dedicated to the carbonylation of olefins in aqueous reaction media. The nonalternating CO-ethene copolymerization is also treated. The carbonylation of ethene in EtOH gave ethyl propanoate. No mention of other carbonylated products was reported [ et al. reported that cationic complexes of the general formula [Pd(PPh3)n(CH3CN)4-n](BF4)2 (n = 1–3) were active even at room conditions. In CH2Cl2 or CHCl3 as solvents, a high molecular weight alternating CO-ethene polyketone (PK) was obtained, whereas in MeOH the main product was methyl propanoate (MP) which formed together with alternating cooligomers of the general formula H(CH2CH2CO)nOMe. The corresponding neutral complex [PdCl2(PPh3)2] was ineffective, indicating that the vacancy of coordination sites, created by the dissociation of the weakly coordinating ligands, played a key role , in coreported ,5. In thkey role ,7. In thhemistry , many achemistry ,16,17,18hemistry ,20,21. Chemistry ,20,22,23hemistry .The carbonylation of ethene is of interest not only for making PKs, but also for the production of methyl propanoate (MP), an intermediate for the environmentally friendly synthesis of methyl methacrylate, a high-value monomer for the production of signs, coatings, adhesives, fibres, paints and so-called organic glasses.cfr.Although the carbonylation reactions may be carried out in aprotic solvents ,29,30,31(i)3 chain, give PKs with higher productivities and molecular weights than the others (BF4)2 (n = 1–3), the low molecular weight PK is formed through a cycle in which the termination occurs exclusively via methanolysis with re-generation of the Pd-H+ initiator, so that only one type of copolymer is formed, i.e., EK . The presence of β- and γ-chelates 1 and 2 was detected  could occur every ca. 105 CO insertions into the same bond, which accounts for the strictly alternating chain growth n+  [3)]+  Ethene does not insert, which is indicative that a coordination site is not easily available. Thus, in this case, methanolysis may occur intermolecularly [Chain transfer occurs via protonolysis with MeOH or methanolysis. Model reactions with CHnd group A 38]. T. T3OD haric bulk . The fac3CN, CO) . Howeverecularly . Intermoecularly   and dimer species (CH3CN)(dibpp)](TfO) was used as a Pd-H initiator , then the cycle is shifted to the other (1'→2'→3'→4'→1'').In 5 is very slow, which also favours the propagation process.In the hydride pathway, it has been found that protonolysis of the third-generation β-chelate at r.t. is significantly slower than that of a second generation β-chelate. This fact would imply that this termination becomes even slower as the chain growing goes on, thus favouring the formation of higher products. It was also found that protonolysis of the β-ester chelate i.e., temperature, pressure, monomer ratio, reaction time, solvent, counteranion, as well as the presence of promoters /PPh3/HCl system is also noteworthy. In MeOH, it was expected to give MP at a low rate, because the metal is coordinated by a monophosphine and a strongly coordinating chloride anion. Instead, it has been found that at 100 °C, 60 bar total pressure, in the presence of an excess of HCl , a PK is formed at a comparable rate of MP (TOF ca. 150 as (moles of ethene absorbed)(mol·Pd·h)−1, whereas when ethene:CO = 40:20, the main product is a PK of moderate Mn , the formation of MP being almost suppressed. It was suggested that the acid, in addition to stabilizing the Pd-H initiator, could also destabilize the β- and γ-chelates through protonation of the oxygen atoms coordinating the metal centre, thus easing the chain growing process  < dippf B. The fa+ initiator, which inserts ethene and then CO to give a Pd-acyl intermediate. At this point, further insertion of the olefin yields a keto β-chelate, which undergoes protonolysis with MeOH to giving DEK and a Pd-OCH3+ species, which re-generates the Pd-H+ through β-H abstraction. This initiator is also formed by methanolysis of the Pd-acyl intermediate, which gives MP.Steric factors apparently play a major role in the case of the dippf catalysis, which produces 3-pentanone (DEK) and MP at comparable rates, indicating that catalysis undergoes via a common intermediate involved in the slow step. The formation of DEK occurs exclusively through a Pd-H3-P,P,Fe coordination mode. It was suggested that the formation of the dative Fe-Pd bond may be the factor that controls the selective formation of the two products and that the κ3-P,P,Fe acyl species is the common intermediate. Since the two products are formed at comparable rates, it was concluded that the slow step of the two catalytic cycles may be the hapticity change of dippf from κ3-P,P,Fe to κ2-P,P, because both the methanolysis of the acyl intermediate or the insertion of ethene require cis-P atoms to occur  have shown the presence of a strong Pd-Os bond (Os-Pd 2.840(1) Å) and a square planar coordination geometry around the Pd centre with a P-Pd-P angle of 165° . For the competing methanolysis termination, the most likely route involves the formation of [Pd(CH3OH)(COCH3)(dmpe)], followed by a proton-transfer/reductive-elimination process mediated by the solvent. The overall barrier for this process is higher than that for ethene insertion, in line with the fact that this catalyst promotes the formation of a PK. Electronic bite angle effects on the rates of ethene insertion and ethanoyl methanolysis were estimated with catalysts with four electronically and sterically related ligands, (Me)2P(CH2)nP(Me)2 (n = 1–4). Steric effects were studied for larger tBu substituted ligands. It was found that ethene coordination and subsequent insertion into the Pd-ethanoyl bond are disfavoured by increasing the steric bulk and that the key intermediates in the methanolysis step are stabilized because of electronic effects caused by increasing the bite angle of the ligand. The combined effects explain which ligands favour the formation of PKs over that of MP  in MeOH  was allowed to react with CO in EtOH in the presence of NEt3, or with MeOH in the presence of Na(OCH3), in place of the expected carboalkoxy complex [Pd(COOR)Cl(dtbpx)]  /PPh3 in ethene hydromethoxycarbonylation, it has been found that a source of hydride, such as H2O, TsOH or H2, enhances the catalytic activity [trans-[Pd(COEt)(TsO)(PPh3)2], relevant to the hydride route, has been isolated   has been recovered after cooling [The carbomethoxy complex (PPh3)2]  relevant(PPh3)2] ,86,87, d(PPh3)2] . At high cooling . Moreovecis-[Pd(SO4)(PPh3)2], which turns into an active catalyst when used in combination with H2SO4 and PPh3. After catalysis and upon addition of LiCl, trans-[Pd(COEt)Cl(PPh3)2], related to the Pd-H catalytic cycle, has been isolated. Moreover, it has been found that cis-[Pd(SO4)(PPh3)2], dissolved in CD2Cl2/MeOH in a NMR tube, reacts with CO to give a Pd-COOMe complex, which neither inserts ethene, nor gives MP. In the presence of H2O and H2SO4 the carbomethoxy complex is unstable giving a Pd-H complex, which promotes catalysis to MP in the presence of CO and ethene [Other evidence in favour of the Pd-H route has been found using the precursor d ethene .cis-[Pd(H2O)2(PPh3)2](TsO)2·2(H2O) (I), trans-[Pd(COEt)(TsO)(PPh3)2] (II) and trans-[Pd(COOMe)(TsO)(PPh3)2] (III) has been studied by 1H and 31P{1H} NMR spectroscopy conditions that mime the catalytic ethene hydromethoxycarbonylation, i.e., in the presence of PPh3, H2O and TsOH. (I), in combination with two equivalents of PPh3, reacts with MeOH and CO  giving [Pd(COOMe)(TsO)(PPh3)3] (III'), which reacts at 20 °C with H2O in the presence of TsOH generating [PdH(PPh3)3](TsO) (IV) quantitatively. This hydride inserts ethene  yielding trans-[Pd(Et)(TsO)(PPh3)2] (V), which reacts with CO  with formation of [Pd(COEt)(PPh3)3](TsO) (II') and initiates catalysis to MP at 20 °C. II, in combination with PPh3 and TsOH, reacts at 20 °C with MeOH with quantitative formation of MP and IV and promotes catalysis starting from this temperature, under 6 bar of CO/ethene (1:1). In the absence of added PPh3, MP is formed in a stoichiometric amount, catalysis is not observed and decomposition to Pd metal occurs. Therefore, PPh3 is essential in order to stabilize hydride IV. III does not insert ethene even at 70 °C, a temperature well above the one at which catalysis occurs. All these experimental findings are in favour of the Pd-H route [NMR studies have provided further evidence in favour of the “hydride” mechanism . The rea-H route .2(PPh3)2]/PPh3/TsOH, phosphine degradation side reactions with formation of phosphonium cations such as EtPPh3+ and EtCOCH2CH2PPh3+, provide further evidence in favour of the hydride path (TfO)  ,35.2(PPh3)2], with which trans-[PdCl(COR)(PPh3)2] and trans-[PdCl(COOR')(PPh3)2] have been isolated after catalysis  with ethene in the presence of BQ have been studied by 31P{1H} NMR. BQ consumes the Pd-hydride and directs the catalysis toward a Pd-COOMe initiator leading to DMS (PPh3)2], used as catalyst precursor, is stable in the absence of BQ. As a matter of fact, it can be synthesized in a high yield by carbonylation of trans-[PdCl2(PPh3)2] in MeOH even at 70 °C . . et al. −1 at 90 °C, 4 MPa, CO:ethene = 1:1). It is worth noticing that the 13C-NMR analysis showed that the perfectly alternating PK presented approximately 85% keto-end groups and 15% acid-end groups (TfO) with the sulfonate analog of d-o-app having an o-MeO substituent group in each of the phenyl rings (32.2 kg PK(g·Pd·h)−1 at 90 °C, 60 bar, CO:ethene = 1:1) (BF4)2 (BF4)2 . Howeverc system .o-app-s catalyst, supported on the PK itself, is also active (21.3 kg PK(g·Pd·h)−1. Such a “gas-phase process” reduces at a minimum the volume of the solvent and avoids fouling drawback   precursors, which are inactive in MeOH. Under standard conditions, an exceptionally high productivity was achieved (28 kg PK(g·Pd·h)−1), which is the highest up to now reported in this solvent , which is also inactive in MeOH, unless used in combination with excess of H2SO4 (H2SO4:Pd = 1000–3000:1) [2(dppf)] and [PdCl2(dapp)] [2(dppf)] under standard conditions, up to 15.5 kg PK(g·Pd·h)−1 of a PK of moderately high molecular weight  was obtained. It is worth noticing that the cationic complex [Pd(TsO)(dppf)] in MeOH is only moderately active and yields MP, DMS and low molecular weight co-oligomers  ,120, whiligomers . Thus, t2(dapp)]. Under standard conditions, productivity as high as 20 kg PK(g·Pd·h)−1 is achievable. High molecular weight PKs are produced under high pressure at a relatively low temperature, without sacrificing the productivity too much −1, P 110 bar, CO:ethane = 1:1, AcOH 70%) [The influence of the operating conditions was investigated with the precursor [PdClcOH 70%) . 2(dppf)] in water-formic acid as a solvent for the copolymerization of ethene [2(dppp)] in this solvent together with aprotic solvents, such as 1,4-dioxane or nitromethane (TsO) and neutral precursors [PdCl2(P-P)]  in either protic or aprotic solvents. In particular, in MeOH or H2O-AcOH higher productivities as well as higher molecular weights were achieved for both the dppp-like catalysts with respect to the corresponding dppe-like catalysts  and the o-MeO modified catalysts are by far more productive than their counterparts and also yield higher molecular weight PKs. Unlike the neutral complexes, the 1H-NMR spectrum of the cationic o-MeO-dppp complex indicates no interaction between palladium and the ortho-hydrogen atoms of the aryl groups, thus providing valuable information on the catalysis resting states and intermediates and contributing to rationalize the catalytic activity. Aprotic solvents, such as CH2Cl2 or toluene, promote the formation of high molecular weight PKs, though in unsatisfactory yields  and [PdCl2(o-MeO-dppp)]. The highest productivity with 3 and 4 was obtained with 65 mol % and that of o-MeO-catalysts with 75 mol % H2O-AcOH. For the CO-ethene copolymerization, 3 yielded the highest productivity (12.20 kg PK(g·Pd·h)−1 under standard conditions), which was about three times higher than with 4 and higher than the one with the o-MeO-catalysts.Complexes 3 and 4 may be related with the different productivity. Moreover, since these complexes gave PKs of comparable Mn, it was suggested that the metal centre in both catalysts has comparable electrophilicity and, hence, capacity to promote the β-elimination of the Pd-alkyl species. Similar trends were obtained in CO-propene copolymerization, though the productivity was about one order of size lower. In the terpolymerzation, 3 and 4 gave PKs with a significantly lower content of propene, the o-MeO-dppe yielded the terpolymer with the lowest melting point −1)  nOH, n = 1, 3–8; h R = (CH2)3CH(CH2OH)2, in combination with HBF4 (Pd:B = 1:10) are highly active in the CO/ethene copolymerization under biphasic conditions (water-toluene). In the presence of potassium dodecylsulfate as an emulsifier and MeOH as an activator, the catalytic activity increased by a factor of about three (1672 g PK(g·Pd·h)−1, Mw = 46.5 kg·mol−1, Mw/Mn = 2.2 for catalyst e) under standard conditions. Also higher olefins could be successfully incorporated into the copolymerization with CO and the terpolymerization with ethene and CO. Compared to the sulfonate dppp-s catalyst, the water soluble catalyst just reported is more active in the 1-hexene incorporation in the terpolymerization of ethene with 1-hexene and CO  in combination with (+)-neomenthyldiphenylphosphine and TsOH under mild conditions   (1) dissolved in MeOH or immobilized on a PK as a support suspended in MeOH, toluene or heptane, in the presence of TsOH  has been studied for CO-ethene-styrene terpolymerization  (1) . 1 reactr et al. . 2 reactopolymer A. It wasting PKs .3 with CO or ethene depicted in 3 are in the ratio of ca. 50:1 MeOH in the presence of TsOH allowed to detect the species shown in 5a is a β-chelate with growing chains of different lengths, including nonalternating ones. This species was detected after cooling the NMR tube to r.t. These results confirm that β-chelates play a key role in the nonalternating process as first proposed by Drent [TsOH prevented decomposition to palladium black, BQ had a negative effect. The productivity and the incorporation of extra ethene were  of TsOH . Operandby Drent .1 in 2 (1:1) and the preformed precursor 2 in combination with B(C6F5)3 (Pd:B = 1:1.1) were tested for the nonalternating CO-ethene copolymerization. At 110 °C, CO:ethene = 5:60), the ethene extra insertion was relatively high (25%–27%), however the activity is rather low (21–32 g PK(mmol·Pd·h)−1 [Ligand ·Pd·h)−1 .3 chain may improve the productivity to higher molecular weight PKs. Cationic complexes are active in MeOH or in suspension, whereas the corresponding neutral complexes with coordinating anions, inactive in this solvent, turn to be exceptionally active in H2O-organic acid as a solvent. Water soluble catalysts with ligands d-o-app-s or d-p-app-s are also very active. Substitution of the phenyl groups with bulkier ones, such as tBu groups, causes a dramatic change in selectivity as MP formation is largely dominant. Several experimental findings point out that steric hindrance plays a key role in governing the selectivity of the carbonylation of ethene. On one hand, by carefully increasing the steric hindrance of catalysts suitable for the formation of PKs, the productivity increases, due to the destabilization of resting states preceding the insertion of the olefin, thus easing the rate determining step. However, further increase of the steric bulk inhibits more and more the insertion of the olefin, to the extreme case in which after the insertion of just one molecule of each monomer, fast methanolysis of the Pd-acyl intermediate occurs with selective formation of MP as in the case of the dtbpx based-catalyst. Other highly active and selective catalysts for the production of MP have been developed. Catalysts with unsymmetrical ligands having only one P(tBu)2 group are also very active and selective, which indicates that only one P(tBu)2 group is required to achieve excellent selectivity and high activity. The CO-ethene copolymerization, catalysed by cationic (P-P) complexes in MeOH, proceeds through both the Pd-H and Pd-OCH3 initiators, as unambiguously proven by NMR end group analysis, whereas the monocarbonylation to MP catalysed by monophosphine- or bulky diphosphine-catalysts occurs via a Pd-H initiator only, as proven by synthesis, spectroscopic characterisation of all the key intermediates of the catalytic cycle, as well as isotope labelling experiments and the use of model compounds as precursors. Catalysis with the highly active and selective complexes with the unsymmetrical ligands, having one P(tBu)2 group involves two geometric isomers for each of the catalytic intermediates in equilibrium as shown in 1, with the Pd-acyl moiety trans to the P(tBu)2 group is kinetically dominant over diastereoisomer 2 and dictates the chemoselective product forming step. Neutral complexes with P-O− anionic ligands, which maintain neutrality during catalysis, yield nonalternating PKs, with an excess of ethene in the polymer chain. The origin of the nonalternation has been investigated by experimental studies as well as theoretical ones. The extra insertion of ethene in the P-O− is facilitated because: (i) the formation of Pd-O chelates through coordination of the oxygen atom on the growing chain is less favourable than in the case of cationic P-P catalysts, which yield perfectly alternating PKs; (ii) the ratio of the binding affinities of CO and ethene to the palladium β-chelate is much smaller than in cationic catalysts (50:1 versus 104:1); (iii) the lower stability of the γ-chelate in the P-O− catalysts facilitates the decarbonylation of its open-chain analogue. Although important results have been achieved, room remains for further development of more efficient catalytic systems.The carbonylation of ethene catalysed by Pd(II)-phosphine complexes yields several products, ranging from high molecular weight PKs to MP. The activity and selectivity depend on the bite angle, skeletal rigidity, electronic and steric bulk properties of the ligand. Dppp type ligands are more suitable for the production of perfectly alternating PKs. Substituents at the phenyl rings or at the C


Surface modification of the fabricated composite nanofibers was illustrated using XRD, FTIR, and SEM analyses.In this study, the photodegradation of three pharmaceuticals, namely Ibuprofen (IBP), Naproxen (NPX), and Cetirizine (CIZ) in aqueous media was investigated under UV irradiation. The photocatalyst used in this work consists of surface functionalized titanium dioxide , solution pH (2–9), and irradiation time on the degradation efficiency. The results demonstrated that more than 99% degradation efficiency was obtained for IBP, CIZ, and NPX within 120, 40, and 25 min, respectively.2–NH2 composite nanofibers was much more efficient than with PAN/TiO2–NH2 composite nanofibers.Comparatively, the photocatalytic degradation of pharmaceuticals using PAN-CNT/TiO Nowadays, the world recognizes the importance of the continuous development on the area of synthesis and production of a variety of pharmaceutical drugs for both humans and animals. Large scales of different chemical compounds are used as medicinal products. These compounds classified as emerging pollutants, while their essential passage into the environment is pharmaceutical industries, excretory products of medically treated humans and animals, which followed by their inefficient removal in wastewater treatment plants , 2. Also4 composite photocatalyst and the results show that the composite photocatalyst degraded NPX drug rapidly within 20 min under visible light irradiation. The photocatalytic performances of composite stayed efficient up to fifth cycle, and this indicates to the excellent stability [The most considerably detected groups of pharmaceuticals in the environment are Non-steroidal anti-inflammatory drugs (NSAIDs). These are one of the widely available drugs in the world. NSAID group have a main common characteristic, which is the carboxylic aryl acid moiety, which provides their acidic properties. Ibuprofen (IBP) belongs to this family of pharmaceuticals, which is an analgesic drug highly used for the medicament of myoskeletal injuries, rheumatoid arthritis, and fever . A largetability , 11. Mortability . The degtability , studied2, Fe2O3, ZnO) under light exposure (UV-light or sunlight) to degrade the organic and inorganic contaminants. Most of the researches are now focusing on the photocatalytic techniques using composite nanofibers [2) is the most common semiconducting catalyst used due to its cheap cost combined with a high photocatalytic activity, nonhazardous compound and eco-friendly [2–NH2) under UV irradiation. The effect of operating conditions such as initial drug concentration, and solution pH were evaluated.The most conventional treatment processes applied at domestic wastewater treatment plants fail to remove completely pharmaceutical substances. Therefore, the treatment of the polluted water requires the application of effective techniques such as Advanced Oxidation Processes (AOPs) , 15. Amonofibers , 19. Manfriendly , 21. Thefriendly –24. In afriendly , 26. The2 Degussa P-25, 3-aminopropyltriethoxysilane (APTES), Glutaraldehyde (GA), polyacrylonitrile, PAN ; dimethylformamide (DMF), hydrochloric acid (HCI), sodium hydroxide (NaOH), Ibuprofen, Naproxen, and Cetirizine were purchased from sigma Aldrich and used as received. Multi-walled carbon nanotubes, MWCNTs  were synthesized and the procedure is described elsewhere [TiOlsewhere , 28. ThePAN MW = 50,000; d2–NH2 NPs. The composite nanofibers was then immersed in the crosslinking medium, which consisting of 100 mL distilled water and 2.5 wt% GA and placed in a mechanical shaker for 24 h at the room temperature. After the crosslinking process the composite nanofibers was washed with deionized water. The surface functionalization TiO2–NH2 NPs was suspended in 5 mL deionized water, afterward sonicated for 2 h and then added to the cross-linked composite nanofibers and mixed using mechanical shaker for 24 h. The PAN-CNT/TiO2–NH2 composite nanofibers was finally washed with deionized water and dried in air at the room temperature.A 10 wt% solution of PAN powder in DMF was prepared by mixing 1 g PAN with 9 mL DMF for 4 h at 50 °C. In parallel, 3 wt% surface activated MWCNTs were dispersed in DMF; the dispersion was stirred for 15 min, and then sonicated for 30 min. PAN solution was then added to the MWCNTs. The mixture was magnetically stirred for 15 min and then sonicated for 3 h. The electrospinning process was conducted at room temperature using a voltage of 25 kV, flow rate of 0.5 ml/h, and distance from needle tip to collector was 15 cm . The ele2 NPs attached to the surface of the composite nanofibers. The crystal phases of the composite nanofibers were evaluated by X-ray diffractometry  using Cu Kα radiation (λ  =  1.5406 Å). The concentration of IBP, NPX, and CTZ in the solution was measured using UV–Vis/NIR spectrophotometer  at maximum absorption wavelength (λmax) 222 nm, 229 nm and 232 nm for IBP, CTZ, and NPX, respectively.The morphology of the composites nanofibers was examined using Scanning Electron Microscopy . Fourier transform infrared spectroscopy  was used to confirm the presence of amino groups on the TiO2–NH2 and PAN-MWCNT/TiO2–NH2 was studied by recording the degradation of IBP, NPX, and CIZ using UV–Vis spectrophotometer. The photocatalysis experiments were performed by placing the composite nanofibers mat in a column (2 cm × 30 cm) and the drug solution of 100 mL was circulated at a flow rate of 7 mL/min under UV-light irradiation. 5 mL of the drug solution were taken at fixed intervals of time. The solution pH was adjusted between 2 and 9 by the addition of HCl or NaOH. The pH was measured using a pH meter . After each experiment, the composite nanofibers were washed many times using deionized water to be able to reuse. All experiments were duplicated to assure the consistency and reproducibility of the results. The photodegradation efficiency was calculated according to the following equation:Ci (mg/L) is the initial drug concentration and Ct (mg/L) is the drug concentration at time (t).A stock solution of 100 mg/L of each pharmaceutical was prepared by addition of the appropriate amount of the pharmaceutical to 500 mL deionized water. Different drugs concentration  at different pH starting from 2 to 9 were prepared using the stock solution. For detection and quantification purposes, the range of concentrations in this study is higher than those typically detected in the environment. Using a UV-A lamp (315–400 nm) of 40 Watts for the UV-light irradiation which, suitable for the photocatalytic process especially whose work with polymer based composite nanofibers. The photocatalytic activity of PAN nanofibers, and composite nanofibers of PAN/TiO2–NH2). This may be due to the increased number of active sites on PAN-MWCNT/TiO2–NH2 surface. Consequently, the increased generation of electron–hole pairs on the surface of the catalyst subsequently leading to higher amounts of reactive hydroxyl radicals that can be attributed to the better degradation observed. The increase in MWCNT/TiO2–NH2 loading provides more binding sites for substrate molecules to adsorb on the catalyst surface. In this study the fabricated composite nanofibers with 75% TiO2–NH2 NPs and 25% of MWCNT was used in all experiments.In order to determine the effect of the photocatalyst amount, a set of experiments were performed by varying the amount of the catalyst from 5 to 30 mg in a total volume of 100 mL and drug concentration of 5 mg/L at pH = 2. Figure 2–NH2, and PAN-MWCNT/TiO2–NH2 composite nanofibers to illustrate the effect of the catalyst (TiO2–NH2 and MWCNT) on the composite nanofiber. Figure 2–NH2 composite nanofibers showed the highest degradation efficiency for all tested contaminates, which is attributed to the high surface to mass ratio for the composite nanofibers. Additionally, CNTs can effectively generate a number of free electrons and holes, which leads to acceleration of the photocatalytic reaction to enhance the photocatalytic activity. The complete degradation was observed at 120 min for IBP, 40 min for NPX, and 25 min for CIZ.The photocatalytic degradation was carried under controlled condition using PAN nanofiber, PAN/TiO2–NH2), less active sites for the adsorption of hydroxyl radicals will be available. Hence, large amounts of adsorbed pharmaceuticals would have an inhibitory influence on the reaction between pharmaceuticals molecules and hydroxyl radicals due to the lack of any direct contact between them. On the other hand, most of the UV-light is absorbed by as the drug molecules as their concentration is increased, and the photons do not reach the surface of photocatalyst to generate hydroxyl radicals [The photocatalytic degradation efficiency of a certain drug depends on its concentration, nature and on the presence of other existing compounds in the water matrix. Figure radicals . In addi2–NH2 is significantly improved with decreasing pH value. The effect of pH on the degradation efficiency of pharmaceuticals drugs was studied by varying the solution pH from 2 to 9 at fixed catalyst amount of 15 mg and UV-light irradiation time of 120, 40, and 25 min for IBP, NPX, and CIZ, respectively. Figure 2–NH2 is protonated and electropositive [2–NH2 is electropositive and the concentration of H+ is excessive, which might lead to the reductions of OH·. As a result, the degradation efficiency of the pharmaceutical drugs was decreased. In neutral and alkaline media, the pharmaceutical drugs are electronegative due to ionization. Therefore, it can repel electronegative TiO2–NH2 and OH− [The photocatalytic abatement of IBP, NPX, and CIZ over TiOpositive , 32. Thepositive , which a and OH− .Fig. 4Ef2–NH2 composite nanofibers were used in sequential photocatalytic process to investigate the durability of the catalytic composite nanofibers. The composite nanofibers washed several times with deionized water and then dried in air after each process, in order to reuse it in other experiments. The Photodegradation efficiency of PAN-MWCNT/TiO2–NH2 composite nanofibers remained stable during the first four sequential cycles, then decreased by about 7% in the last cycle as shown in Fig. The catalyst re-usability examined by cycling experiments under controlled reaction conditions. The reuse of the composite nanofibers is important and has an economic necessity. The PAN-MWCNT/TiO2–NH2 composite nanofibers as shown in Fig. 2 NPs are attached to the surface of the PAN/MWCNT nanofibers due to the crosslinking process.Scanning electron microscope (SEM) analysis was performed to investigate the morphological of the electrospun PAN nanofibers and PAN-MWCNT/TiO2 and PAN-MWCNT/TiO2–NH2 composite nanofibers are shown in Fig. −1, carbonyl at 1650 cm−1 and C–H stretching at 2940 cm−1 [−1, which are attributed to nitrile, carbonyl, C–H stretching, and C–O–C stretching vibrations of the MWCNT, indicating the existence of PAN-CNT nanofibers [−1, is assigned to C ≡ N in the PAN/MWCNT-TiO2 composite nanofibers. The bands observed at 3159, 1520, and 1152 cm−1 are assigned to the aliphatic C–H bending vibration of the CH2 of polymeric chain [−1. After the crosslinking of PAN-MWCNT to TiO2–NH2 NPs, new peaks were observed in the range 3100–3700 cm−1 are assigned to N–H and O–H, and the bending vibrations of the amine group NH or NH2 observed at 1680 cm−1. The band observed at 912 cm−1 is assigned to N–O [The FTIR spectra of PAN, PAN-CNT, PAN/MWCNT-TiO940 cm−1 . The spenofibers . In addiic chain . The alid to N–O .Fig. 7FT2–NH2 composite nanofibers before and after photocatalysis as shown in Fig. In addition, XRD patterns were used to identify the crystal phases of the PAN-CNT/TiO2–NH2 NPs were successfully cross-linked to the PAN/MWCNT composite nanofibers. This study demonstrated that the fabricated composite nanofibers PAN-MWCNT/TiO2–NH2 is an effective photocatalyst for the degradation of IBP, NPX, and CIZ in aqueous solutions under UV-light. Operational parameters, such as initial drug concentration, catalyst amount, irradiation time, and solution pH were investigated. It was shown that the complete degradation can be achieved at low drug concentration (5 mg/L), acidic pH = 2, and at a low power intensity of the UV lamp (40 Watt). The complete photodegradation of IBP, NPX, and CIZ required 120, 40, and 25 min, respectively. The stability studies showed that the photodegradation efficiency of PAN-MWCNT/TiO2–NH2 composite nanofibers remained stable under the experimental conditions studied.TiO


After the publication of this work , an erro


Metarhiziumanisopliae", with DOI number 10.1590/1678-4685-gmb-2017-0363, published inthe journal Genetics and Molecular Biology, 41(4):843-857, on page 843, a footnoteinforming the current address of the following author was omitted: Claudia ElizabethThompson, Departamento de Farmacociências, Universidade Federal de Ciências da Saúde dePorto Alegre, Rua Sarmento Leite, 245, 90050-170 Porto Alegre, RS, Brazil.In the article "Molecular evolution and transcriptional profile of GH3 and GH20ß-N-acetylglucosaminidases in the entomopathogenic fungus


Various effects of dose rate, molar ratio of the monomer to the chain transfer agent, monomer concentration and reaction time on the AN polymerization behaviors were performed to improve the controllability of molecular the weight and molecular weight distribution of the obtained PAN. The feature of the controlled polymerization was proven by the first-order kinetics, linear increase of the molecular weight with the monomer conversion and a successful chain-extension experiment. The molecular weight and molecular weight distribution of PAN were characterized by size exclusion chromatography (SEC). 1H NMR and Matrix assisted laser desorption ionization/time of flight mass spectra (MALDI-TOF-MS) confirmed the chain-end functionality of PAN, which also was supported by the successful chain-extension experiments of original PANs with acrylonitrile and styrene as the second monomers respectively. Because of its remarkable and unique performance, including rigidity, tensile strength and chemical resistance, polyacrylonitrile (PAN) as the precursor for carbon fibers plays an important role in many industries, such as aerospace, automotive, sports goods and so on . It is wShindo et al. first reported the successful synthesis of PAN-based carbon fibers in 1961 ,8, have Compared with other LRP techniques, RAFT polymerization is an outstanding strategy with high functional-group tolerance and without any metal catalyst. Up to now, there have been only some successful examples of RAFT polymerization of acrylonitrile using cumyl dithiobenzoate (CDB) , 2-cyano60Co γ-ray irradiation, as a kind of environment-friendly energy source, can be used to initiate vinyl polymerization, and to prepare graft or cross-linked copolymers to modify the properties of polymers -malononitrile  as the matrix prepared in CDCl3 (20 mg/mL) and the cationizing agent sodium trifluoroacetate prepared in ethanol (10 mg/mL) were mixed in a ratio of 10/1 (v/v).The number-average molecular weight .An amount of 15.26 mg PAN .An amount of 9.60 mg PAN (sample in Mw/Mn) of the obtained PAN decreased with the decrease of [AN]0/[CPDN]0 molar ratio under the constant conditions of dose rate, monomer concentration and reaction time, indicating that these polymerizations developed towards a more controlled direction. Mw/Mn) decreased with the decrease of the polymerization rate. Comparing 1b and 4a  of polymers [0/[M]) as a function of polymerization time for 60Co γ-ray irradiation-initiated RAFT polymerization of AN at 1.8 kGy/h under the constant initial concentration of AN. These profiles of plots were approximately linear, indicating the stability of the number of the active centers during the polymerization process. The polymerization rate decreased with the decrease of the [AN]0/[CPDN]0 molar ratio. It could be observed that there was an inhibition period at the beginning of polymerization, perhaps because of the slow fragmentation of the intermediate radical  and molecular weight distribution (Mw/Mn) on the monomer conversions at different [AN]0/[CPDN]0 ratios. The Mn,GPC value of PAN increased linearly with the monomer conversion and the molecular weight distribution indices of PAN were kept under 1.4 in most cases. Furthermore, the molecular weight distribution decreased with the decrease of the [AN]0/[CPDN]0 molar ratios as a whole. All the results indicated that an appropriate increase of the CPDN molar concentration could make the polymerization behavior of AN become much more controllable.According to the RAFT polymerization mechanism, the [M]polymers ,16. Figu radical II ) as a function of time for 60Co γ-ray irradiation-initiated RAFT polymerization of AN with different dose rates (1.8 and 1.2 kGy/h) under the molar ratio of [AN]0/[CPDN]0 = 1000/1, were observed. The linear relationship between ln([M]0/[M]) and polymerization time meant that the number of active centers remains constant during the polymerization process. As expected, the rate of polymerization decreased in the order of 1.8 kGy/h > 1.2 kGy/h, indicating the relatively lower radical concentrations induced by 60Co γ-ray irradiation with the lower dose rate. There still existed an inhibition period at the beginning of polymerization. Mn,SEC) and molecular weight distribution (Mw/Mn) versus the monomer conversion under the molar ratio of [AN]0/[CPDN]0 = 1000/1. It could be seen that the Mn,SEC value of PAN increased linearly with monomer conversion and that molecular weight distribution indices of PAN were also kept under 1.4 in most cases. Meanwhile, molecular weight distribution indices broadened with the increase in dose rate as a whole, demonstrating that an appropriate decrease of the dose rate could make AN polymerize in a more controlled fashion.The effects of different dose rates on the RAFT polymerization of AN were also investigated. As presented in 1H NMR  and signals of CH3 (1.20–1.50 ppm) in the CPDN unit were clearly observed. The (a + b)/c/f/g integration ratio was close to 7/1/2/6, confirming the efficient chain-end functionality of PAN. Furthermore, the molecular weight of PAN could be calculated by comparing the integration ratio value of aromatic protons from end groups with CH2 protons from the polymer main chain. The number-average molecular weight  of PAN was near 2100 g/mol calculated by the 1H NMR spectrum corresponding to the Mn,th of 2000 g/mol. In m/z, 930.297 Da), corresponding to 12 repeat units of PAN with a sodium cation, is consistent with the theoretically calculated value . The difference of two adjacent peaks is 53.02 Da, equal to the mass of one acrylonitrile unit. The successful chain-extension experiments  to polymers (PAN or PAN-b-PS), strongly proving the living feature of the previous polymerization. In the NMR spectra of copolymers  polymerization at room temperature with 2-cyanoprop-2-yl 1-dithionaphthalate (CPDN) as the chain transfer agent was first applied to acrylonitrile (AN) polymerization. Various effects of dose rate, molar ratio of the monomer to the chain transfer agent, monomer concentration and reaction time on the AN polymerization behaviors were performed to improve the controllability of the molecular weight and molecular weight distribution of the obtained PAN. The feature of the controlled polymerization was proven by the first-order kinetics, linear increase of the molecular weight with the monomer conversion and a successful chain-extension experiment. This work provides a new “green” platform for preparing PAN-based carbon fibers using an environment-friendly energy source and benefits the optimization of the mechanical properties of PAN-based carbon fibers.


The obtained PdBC nanofibers were characterized by a series of analytical techniques. The results revealed that Pd nanoparticles were evenly dispersed on the surfaces of BC nanofibers. Then, the as-prepared PdBC nanofibers were mixed with laccase (Lac) and Nafion to obtain mixture suspension, which was further modified on electrode surface to construct novel biosensing platform. Finally, the prepared electrochemical biosensor was employed to detect dopamine. The analysis result was satisfactory, the sensor showed excellent electrocatalysis towards dopamine with high sensitivity (38.4 µA·mM−1), low detection limit (1.26 µM), and wide linear range (5–167 µM). Moreover, the biosensor also showed good repeatability, reproducibility, selectivity and stability and was successfully used in the detection of dopamine in human urine, thus providing a promising method for dopamine analysis in clinical application.Palladium nanoparticle-bacterial cellulose (PdBC) hybrid nanofibers were synthesized by  Dopamine (DA) is an important neurotransmitter, which plays pivotal roles in the central nervous, cardiovascular and hormonal systems . The abnAcetobacter xylinum, which usually forms netlike pellicles consisting of ultrafine nanofibers with widths less than 100 nm. BC possesses numerous unique properties, like high mechanical strength, high crystallinity, water absorbance, biocompatibility, polyfunctionality, and hydrophilicity [In order to improve the sensitivity and selectivity of biosensors, a variety of conductive materials or conductive nanomaterials have been added to the biosensing system, which mainly contain metal nanoparticles, metal oxide nanoparticles, carbon materials and conductive polymers. Metal nanoparticles have been widely applied in both chemical sensors and biosensors due to their high surface area, high mechanical strength, ultra-light weight, rich electronic properties, and excellent chemical and thermal stability . Howeverhilicity . Accordiin-situ chemical reduction method. The as-prepared PdBC nanofibers were further employed to modify electrode with Lac and Nafion to construct novel biosensing platform. The obtained biosensor showed excellent bioeletrocatalysis towards dopamine with high sensitivity, low detection limit and wide linear range. Moreover, the sensor demonstrated its practical application potential by detecting dopamine existing in human urine with satisfactory recovery. Our study offers the experimental and technological supports for exploiting high-efficient enzyme based biosensors for dopamine detection.In this work, we synthesized Pd nanoparticles-BC (PdBC) hybrid nanofibers by −1 was related to the stretching vibration of O-H, and the absorption peak at around 2895 cm−1 was ascribed to the stretching vibration of –CH2 [−1, 1107 cm−1, and 1055 cm−1 were due to the C-C stretching vibration, skeletal/vibrations and ring vibrations, respectively [Fourier transform infrared (FT-IR) spectra was employed to investigate the surface functional groups of BC and PdBC, and the result is shown in  of –CH2 . In addiectively . For theca. 40.2°, 46.6°, and 68.2° corresponded to the (111), (200), and (220) plane reflections of palladium, respectively [ca. 14.8°, 17.2°, and 22.9° were related to the (1–10), (110) and (200) crystallographic planes of bacterial cellulose, respectively [D is the crystallite size, K is Scherrer constant, γ is the wavelength of X-ray radiation, B is the full width at half maximum (FWHM) of peak and θ corresponds to the peak position . The average diameter of Pd nanoparticles was calculated to be ca. 4.7 nm. EDS and XRD characterizations jointly demonstrated the successful synthesis of palladium nanoparticles in the PdBC nanofibers.Energy dispersive X-ray (EDX) spectroscopy characterization was used to determine the Pd element existing in the PdBC nanofibers. As shown in ectively . Besidesectively . AccordiThe morphologies of PdBC and BC were observed by SEM and TEM. As can be seen in ct) of modified electrodes are proportional to the diameters of semicircles. Apparently, bare electrode (GCE) shows almost a straight line, indicating the negligible Rct value. However, an obvious semicircle occurred on the curve for GCE/Lac-Nafion, the Rct value is about 168 Ω, suggesting that the interface resistance was increased by the immobilization process of Lac, while the semicircle diameter of the curve for GCE/Lac-PdBC-Nafion was smaller than that of GCE/Lac-Nafion, and the Rct value also decreased to 78 Ω. This demonstrated that the PdBC nanofibers accelerated the electron transfer, weakened the interface resistance of modified electrode, which could be attributed to the good electron conductivity ability of palladium nanoparticles.Electrochemical Impedance Spectroscopy (EIS) characterization was utilized to compare the interface resistances of different modified electrodes, the result of Nyquist polt of impedance is shown in ca. 8.67 µA. This can be attributed to the highly efficient catalysis of Lac toward dopamine. It is noticeable that GCE/Lac-PdBC-Nafion shows the largest redox peak current values, which were increased to 11.59 µA and 9.88 µA, respectively. This demonstrated that the PdBC nanofibers can enhance the electrochemical response of modified electrode, maybe attributing to the good conductivity and the synergetic catalysis of palladium nanoparticles. The whole electrochemical reaction was a quasi-reversible cyclic process and the sensing mechanism of Lac on GCE/Lac-PdBC-Nafion is illustrated in The electrochemical responses of three modified electrodes, including bare GCE, GCE/Lac-Nafion, and GCE/Lac-PdBC-Nafion, were compared by comparing the cyclic voltammograms of these electrodes in pH 7.0 PBS containing 100 µM dopamine. The result is shown in −1, both of anodic peak and cathodic peak current values increased. As can be seen in Chronoamperometry was employed to investigate the analytical performance of as-prepared biosensor for detecting dopamine. Before the amperometric tests, some parameters like solution pH and applied work voltage were optimized. As shown in versus dopamine concentrations. The current values increased linearly with the ascent of dopamine concentration. The linear range was from 5 µM to 167 µM with a correlation coefficient (R2) of 0.997 (n = 7). The sensitivity was 38.4 µA·mM−1 and the detection limit was estimated to be 1.26 µM at a signal-to-noise of 3 (S/N = 3). RSD) was 3.7%, implying the acceptable reproducibility of the GCE/Lac-PdBC-Nafion. The RSD of the GCE/Lac-PdBC-Nafion for 15 times of successive detection of 100 µM dopamine was 2.3%, indicating excellent repeatability of the GCE/Lac-PdBC-Nafion. The selectivity experimental result is shown in The fabrication reproducibility of the GCE/Lac-PdBC-Nafion was investigated by successive detection of 100 µM dopamine by six modified electrodes prepared in the same way. The relative standard deviation . Nafion (5% w/w) was obtained from Shanghai Branch, Du Pont China Holding Co., Ltd. . Palladium chloride (PdCl2) and dopamine were purchased from Shanghai Aladdin Chemical Reagent Company . All of the chemicals were analytical grade and used without further purification. In addition, 0.1 M phosphate buffer solution  was used as a supporting electrolyte. All aqueous solutions were prepared with deionized water (DIW).Laccase  from −1 on a Nicolet iS10 FT-IR spectrometer . The chemical components of PdBC nanofibers were analyzed by an Energy dispersive X-ray spectroscopy  and a Powder D8 Advance X-ray diffraction . Thermogravimetric analysis (TGA) measurement was conducted using a Mettler Toledo analyzer  in air atmosphere, the temperature range was from ambient temperature to 700 °C with a heating rate of 10 °C/min. The morphologies of PdBC nanofibers were observed using a scanning electron microscope  and a high-resolution transmission electron microscope . Prior to scanning under the SEM, the samples were sputter coated with gold for 90 s to avoid charge accumulations. Nano Measurer software  was used to measure the diameters of the BC nanofibers and Pd nanoparticles. Electrochemical experiments were conducted at room temperature using a CHI 660E electrochemical workstation . A three-electrode cell with a glass carbon electrode (GCE) , a platinum wire auxiliary electrode and an Ag/AgCl reference electrode was used for electrochemical measurements. The electrolyte solution was bubbled with highly pure nitrogen for 15 min before electrochemical experiments and a nitrogen atmosphere was kept for the solution throughout the experiments except the amperometric experiments.Fourier transform infrared (FT-IR) spectra were recorded in the range of 500–4000 cmGluconacetobacter xylinum was kindly provided by Donghua Univerisity  and used to produce the bacterial cellulose pellicles. The bacterium was cultured on Hestrin and Schramm (HS) medium by staticing incubation. The medium was composed of 5% (w/v) glucose, 1.6% (w/v) bacto-peptone, 0.2% (w/v) citric acid, 0.2% (w/v) disodium hydrogen phosphate, 0.3% (w/v) potassium dihydrogen phosphate, and 0.03% (w/v) magnesium sulfate. These culture media were sterilized at 120 °C in autoclave for 2 h by autoclaving. Cells pre-cultured in a test tube containing a small cellulose pellicle on the surface of the medium were inoculated into a 100 mL Erlenmeyer flask containing 10 mL of the HS medium. The flask was incubated statically at 30 °C for 7 days. The synthesized cellulose were dipped into 1% sodium hydroxide solution for 2 h at 80 °C in order to eliminate the cells and components of the culture liquid, and then rinsed 3 times to pH 7 in deionized water.in-situ chemical reduction method. The as-prepared bacterial cellulose (BC) was disrupted by Ultrasonic Cell Disruption System for 10 min to disperse the BC. Then the BC was immersed in an aqueous mixture solution containing saturated PdCl2 metal salt at 40 °C for 24 h. After that, 0.01 M KBH4 solution was added to reduce the palladium salt to obtain Pd nanoparticles. The final products were rinsed and dried.The PdBC hybrid nanofibers were synthesized by Considering the optimal response and stability of modified electrode, among control-experiments, the concentration and mass ratio of Nafion, PdBC nanofibers and Lac were optimized. The preparation procedure of biosensors are described as follows: 1.5 mg of PdBC nanofibers were added into 5 mL of pH 7.0 PBS with the aid of ultrasonication and stirring to obtain PdBC suspension. Subsequently, 15 mg of Lac and 75 µL of Nafion solution (5 wt %) were added into the above PdBC suspension, by which the final mixture suspension was achieved. Eventually, 10 µL of the mixture was dropped on a freshly polished glass carbon electrode (GCE) surface to fabricate the biosensor, and the dried modified GCE was named GCE/Lac-PdBC-Nafion, which was stored at 4 °C for use.GCE/Lac-Nafion modified electrode was fabricated by similar methods with same amount of Lac for comparison experiments. All the modified electrodes were immersed into buffer for 30 min to remove impurities before electrochemical measurements.in-situ chemical reduction method. The Pd nanoparticles were observed to be evenly dispersed on the surfaces of BC nanofibers, which is beneficial for developing their electrocatalytic effect. A sensitive biosensor was fabricated by modifying a mixture containing PdBC nanofibers, Lac, and Nafion on electrode surface. The as-prepared biosensor showed great biological electrocatalysis towards dopamine with high sensitivity, low detection limit and wide linear range. Besides, the sensor was successfully used in dopamine detection in human urine. This novel electrochemical biosensor provided a promising method for dopamine analysis in clinical application.In summary, PdBC hybrid nanofibers were synthesized by


Saturated fatty acid (SFA) vs polyunsaturated fatty acid (PUFA) may promote nonalcoholic fatty liver disease by yet unclear mechanisms.To investigate if overeating SFA- and PUFA-enriched diets lead to differential liver fat accumulation in overweight and obese humans.Double-blind randomized trial (LIPOGAIN-2). Overfeeding SFA vs PUFA for 8 weeks, followed by 4 weeks of caloric restriction.General community.Men and women who are overweight or have obesity (n = 61).Muffins, high in either palm (SFA) or sunflower oil (PUFA), were added to the habitual diet.Lean tissue mass (not reported here). Secondary and exploratory outcomes included liver and ectopic fat depots.P = 0.50. SFA markedly induced liver fat content (50% relative increase) along with liver enzymes and atherogenic serum lipids. In contrast, despite similar weight gain, PUFA did not increase liver fat or liver enzymes or cause any adverse effects on blood lipids. SFA had no differential effect on the accumulation of visceral fat, pancreas fat, or total body fat compared with PUFA. SFA consistently increased, whereas PUFA reduced circulating ceramides, changes that were moderately associated with liver fat changes and proposed markers of hepatic lipogenesis. The adverse metabolic effects of SFA were reversed by calorie restriction.By design, body weight gain was similar in SFA (2.31 ± 1.38 kg) and PUFA (2.01 ± 1.90 kg) groups, SFA markedly induces liver fat and serum ceramides, whereas dietary PUFA prevents liver fat accumulation and reduces ceramides and hyperlipidemia during excess energy intake and weight gain in overweight individuals. We show the importance of dietary fat type on liver fat accumulation and ceramides during overfeeding-induced weight gain in 61 overweight men and women. NAFLD isistance . Furthersistance . PotentiA vs SFA –6. FurthA vs SFA , 8, and A vs SFA , 3.Bile acids (BAs) have broad effects on hepatic triglyceride and cholesterol metabolism and have been suggested to play a central role in the development of NAFLD . The chon-6 PUFA also increases inflammation, although this is based mainly on experimental studies and theoretical assumptions with little supporting data in humans .Here, overweight individuals were overfed with either SFA  or omega-6 PUFA (sunflower oil) with the hypothesis that PUFA would counteract the accumulation of liver fat during 2 months of controlled weight gain and that SFA-induced liver fat accumulation is a result of increased hepatic uptake of palmitate. We further aimed to investigate whether effects on liver fat accumulation may be related to changes in sphingolipids  or liver disease; pregnancy; lactation; alcohol abuse; claustrophobia; abnormal clinical chemistry test results; use of drugs influencing energy metabolism; use of omega-3 supplements or extreme diets; regular heavy exercise (>3 h/wk); intolerance to gluten, egg, or milk protein; and implanted metals. Subjects were instructed to maintain their habitual diet and physical activity level throughout the study. Subjects were fasted overnight (10 to 12 hours) and were discouraged from physical exercise and alcohol intake 48 hours before measurements.Overweight men and women were recruited by local advertising. Inclusion criteria included participants ages 20 to 55 years and body mass index (BMI) of 25 to 32 kg/mThe LIPOGAIN-2 study was a 12-week, double-blind, parallel-group, randomized trial in free-living subjects, carried out from August 2014 through June 2015 at the Uppsala University Hospital, Uppsala, Sweden. Subjects were randomized by computer-generated lists (generated by a statistician not otherwise involved in the project), stratified by sex, age, and BMI. Double-blinding was ensured by labeling, and the code was concealed from all investigators until study completion and statistical analyses of primary outcomes. During the first 8 weeks, subjects were overfed (weight gain), followed by 4 weeks of caloric restriction (weight loss). The trial was registered at www.clinicaltrials.gov as NCT02211612.Sixty-one participants were randomized to eat muffins containing either sunflower oil  or palm oil  during 8 weeks. Both oils were refined; fatty acid composition has been described . Body weLiver and pancreas fat content, liver volume, and volume of total body and visceral fat were assessed by MRI using a 1.5T Achieva clinical scanner , modified to allow arbitrary table speed. Collection and analyses of the MRI data were performed at one center under blinded conditions. A single operator, trained by an experienced radiologist, performed all measurements. The absolute amount of liver fat in liters was calculated by the multiplication of liver volume by liver fat content (%). Total body and visceral fat were quantified using a previously described automated image analysis algorithm . Total b11C]palmitate (361.35 ± 11.9 MBq). Regions of interest for liver, pancreas, and skeletal muscle tissue, as well as the blood pool in ascending aorta and portal vein, were outlined manually based on the anatomical information in a T1-weighted MR image acquired simultaneously with the PET scan. Myocardium and left-ventricular cavity were segmented automatically on PET images using the cardiac module in Carimas. Quantification of [11C]palmitate uptake in liver, pancreas, and skeletal muscle was performed using an image-derived input function based on average radioactivity concentrations in an ascending aorta and portal vein, corrected for radioactive metabolites, using the measured parent fraction remaining in plasma at 10, 20, 30, 40, and 50 minutes postinjection, as well as plasma to whole-blood radioactivity concentration at 5, 10, 20, 30, 40, and 50 minutes postinjection. Four different parameters were calculated based on the [11C]palmitate data:i)] was estimated using Patlak analysis for the 5- to 60-minute postinjection interval.The accumulation rate constant [influx constant /body weight (gram) × administered tracer (becquerel)] for the uptake windows of 30 to 35 minutes and 55 to 60 minutes postinjection.Standardized uptake values (SUVs) were estimated, adjusting radioactivity concentrations for intersubject differences in body weight and administered amount of . Levels of serum BAs and 7a-hydroxy-4-cholesten-3-one (C4), a BA synthesis marker, were determined by liquid chromatography–tandem mass-spectrometry using deuterium-labeled standards for C4 and BA . Unestere.g., for PET-MR) were analyzed with Mann-Whitney test. Data are given as means (SD) or medians [interquartile range (IQR)]. Correlations between variables are given as Pearson’s r or Spearman’s rho. A P value <0.05 was considered statistically significant. JMP version 13.1.0 was used; heatmaps were created using R. Primary outcome measure for the trial was change in lean tissue mass (reported in a separate manuscript); secondary outcome measures included changes in liver and pancreas fat, VAT, total body fat, palmitate uptake, and blood lipids. Sample size was determined using Lehr formula, based on our previous trial, using similar intervention and outcome measures . All authors reviewed and approved the final manuscript.P = 0.75), BMI , and sex distribution (12:18 vs 11:19 women/men) were similar between SFA and PUFA groups, respectively. By design, surplus energy intake and macronutrient distribution were also similar between groups, resulting in similar (P = 0.50) body weight gain . Baseline, 24-hour physical activity was similar (P = 0.32) between groups and did not change during intervention (data not shown).Sixty subjects (n = 30 in each group) completed the 8-week hypercaloric period; baseline characteristics are shown in P < 0.0001 for comparison between groups), whereas palmitate, the major fatty acid in the SFA muffins, increased in both plasma PL and adipose tissue in the SFA group during the intervention (P < 0.0001 for difference between groups). In addition, based on checklists/self-report, subjects in the SFA group consumed 96.8%, and subjects in the PUFA group consumed 97.1% of the provided muffins, and the 4-day weighed dietary records indicated no changes other than those desired/expected in fat type. Finally, cholesterol levels changed in accordance to what would be expected based on increased intakes of SFA and PUFA, respectively (vide infra). Thus, both key dietary biomarkers, as well as self-reported data, suggested excellent compliance to both diets.Changes in fatty acid composition in both plasma and adipose tissue reflected the assigned interventions, indicating high compliance . Linoleae.g., increasing LDL-cholesterol and apoB), whereas PUFA overfeeding reduced atherogenic blood lipids, despite weight gain (P = 0.004) in circulating lathosterol, a marker of cholesterol synthesis; this was, however, attenuated when corrected for total cholesterol, indicating no change in cholesterol synthesis, in line with unchanged PCSK9. Neither fasting serum total BAs nor their composition or other markers of cholesterol and BA synthesis  differed between diets (SFA overfeeding deteriorated the blood lipid profile (ght gain  28). Co. Coe.g.,P = 0.001 for between-group difference), despite similar body-weight gain (P = 0.035 for between-group difference). The change in liver fat was inversely correlated with change in circulating linoleate in both plasma  and adipose tissue  but directly associated with change in the circulating palmitate  and SCD activity index in CE . The changes in liver fat did not translate to significant changes in glucose tolerance or insulin sensitivity between groups  in the SFA group, whereas in the PUFA group, there was a 2% decrease  in SFA (0.49 ± 2.29%) and PUFA (0.46 ± 1.69%) groups (Pancreas fat accumulation was similar  groups .P = 0.17) in SFA (0.37 ± 0.29 L) and PUFA (0.26 ± 0.30 L) groups, as was the accumulation of total body fat  tended to be inversely associated with change in liver fat, but there was no association between hepatic palmitate net uptake rate and liver fat at baseline .In the exploratory subgroup study using PET-MRI, the change in hepatic palmitate uptake did not differ between groups, neither when expressed as SUVs at 30 or 60 minutes nor when assessing net influx rate Kntervals . Change P = 0.08) when adjusted for changes in C16-containing ceramides . Changes in circulating 16:1n-7 and the SCD index were positively correlated with changes in multiple ceramide species, whereas changes in circulating linoleate showed consistent, inverse associations (SFA- and PUFA-enriched diets had markedly different effects on serum ceramides (Cer), dihydroceramides (DiCer), glucosylceramides (GluCer), and lactosylceramides (LacCer). Overall, SFA increased, whereas PUFA decreased ceramide levels—changes that were already evident at 4 weeks . Change ciations .Although changes in adipose tissue ceramides were also generally different between SFA and PUFA groups, changes were less pronounced and with few exceptions, not statistically different between groups.P < 0.03; data not shown).SFA and PUFA overfeeding had differential effects, also on circulating sphingomyelin species: 16:0, 16:1, 18:0, and 18:1 species increased significantly more by SFA , and changes in 16:0 and 16:1 species were directly associated with changes in both liver and VAT fat levels  in both groups combined (data not shown).Changes in palmitate uptake in pancreas, skeletal muscle, or heart did not differ between groups . In bothi. Furthermore, similar results were observed for the individual C16 and C18 species .Changes in serum Cer, DiCer, GluCer, and LacCer were strongly, positively associated (r = 0.63 to 0.95), with changes in pancreatic (but not hepatic) palmitate uptake assessed as SUV at 30 and 60 minutes . Similarα) as a result of PUFA compared with SFA overfeeding , endothelial function , or free radical-induced lipid peroxidation (isoprostane 8-iso-prostaglandin F2rfeeding .P = 0.92) weight loss . Furthermore, both groups decreased similarly (P = 0.82) in waist circumference  and total body fat . All differences observed during weight gain between groups in blood lipids were abolished by weight loss. Likewise, no differences between groups were observed for liver enzymes or ceramides after weight loss (data not shown). In both groups combined, changes in ceramides and sphingolipids showed diverse effects in response to weight loss. Total serum BAs (P = 0.012) and FGF19  were higher in the SFA vs PUFA group after weight loss.Fifty-one subjects  completed the 4-week hypocaloric period , which caused similar  also after the intervention; ut NAFLD . Likewisut NAFLD  indicateut NAFLD , there aut NAFLD .n-6 PUFA in the form of linoleate (a precursor to arachidonic acid), there were no signs of increased inflammation or lipid peroxidation, as judged by established plasma markers of inflammation and oxidative stress compared with SFA. This finding accords with results from our previous trials  for the prevention of NAFLD and hyperlipidemia.


Cold Spring Harbor Molecular Case Studies, we have included the first publication  or request a custom report from ClinGen , which will also highlight variants in which the submitter has an outlier interpretation compared to other submitters (Cold Spring Harbor Molecular Case Studies.Clinical laboratories can obtain reports of their variant interpretation differences from the ClinVar Miner website (bmitters . Studiesbmitters . We encoThe author has declared no competing interest.


Escherichia coli, the bacterial adhesin FimH is responsible for the attachment of the bacteria to the (human) host by specifically binding to highly-mannosylated glycoproteins located on the exterior of the host cell wall. Adhesion represents a necessary early step in bacterial infection and specific inhibition of this process represents a valuable alternative pathway to antibiotic treatments, as such anti-adhesive drugs are non-intrusive and are therefore unlikely to induce bacterial resistance. The currently available anti-adhesives with the highest affinities for FimH still feature affinities in the nanomolar range. A prerequisite to develop higher-affinity FimH inhibitors is a molecular understanding of the FimH-inhibitor complex formation. The latest insights in the formation process are achieved by combining several molecular simulation and traditional experimental techniques. This review summarizes how molecular simulation contributed to the current knowledge of the molecular function of FimH and the importance of dynamics in the inhibitor binding process, and highlights the importance of the incorporation of dynamical aspects in (future) drug-design studies.Located at the tip of type I fimbria of  Escherichia coli bacteria live in symbiosis with their human hosts as part of the gut flora, several E. coli strains are pathogenic to humans : t: t30]: tAs shown in the examples, the incorporation of data on the dynamics behavior of the protein, the complex and the ligand in water, often originating from MD simulations, allows to describe and therefore understand the dynamics of the binding process. It thus complements the more traditional approaches such as crystallography and affinity measurements from which static pictures are obtained. Adhesion of pathogenic E. coli is mediated by multiple type 1 fimbria and thus FimH binding to MGP displayed on the host cells  and a dynamic modelling (as in MD) were applied together with different experimental techniques including SPR, ITC, X-ray crystallography or NMR.All different molecular simulation approaches have their own advantages and problems. Force-field based methods such as MD for example are computationally very efficient and can handle large systems, giving an  correct representation of the biological reality. In contrast, QM calculations are very time consuming and can handle only a fraction of the real system. However, force-field based methods suffer from serious shortcomings in e.g., the description of charge transfer, halogen bonding and polarizability (see for example  and refepriori performed experiments. Example are the study of the molecular origin of the increased entropic penalty in ITC measurements if the HM mannose sugar is replaced by a septanose leading to HS [In most studies on FimH, molecular simulation was used to understand functional details of the protein not decipherable by the ng to HS  or the dng to HS . Howeverng to HS ,92: the ng to HS  or MrpH ng to HS . A similng to HS .It is a necessity for the development of UTI or CD vaccines as well as for the development of treatment alternatives to antibiotics for these diseases to combine experimental and theoretical approaches in future research. Molecular simulation can help to predict possible conformations and interactions as well as affinities of ligands and can thus explain experimental results and/or allow for the design of new experiments.The use of molecular dynamics simulation in complement to crystallographic assays offers a powerful combination to study ligand binding. For instance, the application of molecular dynamics simulation in combination with quantum-chemical calculations have allowed to understand the molecular importance of the FimH tyrosine gate and the impact of mutation of its residues on binding affinity. The incorporation of dynamical information on the wild-type FimH lectin domain in the ligand-free and ligand-bound state into a structure-based rational drug design approach allowed for the identification of a previously unidentified and promising binding mode of the ligand in the FimH binding site, in which it is oriented towards the clamp loop. These two examples among others highlight the added value of molecular simulation in the drug design of inhibitor molecules, here targeting FimH.Further applications of molecular simulations techniques could be the identification of alternative binding positions for anti-adhesives on the FimH lectin domain, such as the recently identified EDTA binding site, or of compounds binding tightly to these positions. Such compounds might fix FimH in an off-path conformation and could thus abolish FimH receptor binding. In a similar fashion, simulation techniques can be expected to lead to the identification of key residues in the conformational change which could be then be targeted.Molecular simulation will allow to rationally design new anti-adhesives either being specific to a single FimH conformational state or to all states and will give further insight into the molecular details of FimH binding. Therefore, it will provide the necessary details allowing experimentalists to design and perform new and more precise experiments proving and complementing the concepts provided in-silico.It is our expectation that molecular simulation integrated with experimental techniques will lead to new routes for drug development not only for bacterial adhesins but for a variety of proteins involved in bacterial infection. In contrast to the currently used antibiotics, precision antimicrobial drugs will allow to specifically target selected bacterial strains and will thus constitute a valuable non-antibiotic alternative treatment.