Rice panicle architecture is a key target of selection when breeding for yield and grain quality. However, panicle phenotypes are difficult to measure and susceptible to confounding during genetic mapping due to correlation with flowering and subpopulation structure. Here we quantify 49 panicle phenotypes in 242 tropical rice accessions with the imaging platform PANorama. Using flowering as a covariate, we conduct a genome-wide association study (GWAS), detect numerous subpopulation-specific associations, and dissect multi-trait peaks using panicle phenotype covariates. Ten candidate genes in pathways known to regulate plant architecture fall under GWAS peaks, half of which overlap with quantitative trait loci identified in an experimental population. This is the first study to assess inflorescence phenotypes of field-grown material using a high-resolution phenotyping platform. Herein, we establish a panicle morphocline for domesticated rice, propose a genetic model underlying complex panicle traits, and demonstrate subtle links between panicle size and yield performance. Panicle architecture is an important determinant of crop yield and a target of selection by plant breeders. Here, Crowell et al. combine image-based phenotyping with high-density array-based genotyping to perform a genome-wide association study revealing a number of candidate genes linked to panicle variation in rice. Oryza sativa), a staple crop for billions of people, optimizing rice panicle size and structure represents a challenge for breeders attempting to improve yield potential and maximize grain quality1O. sativa panicle architecture have been relatively subtle. Seeds are born on long primary branches that sometimes iterate into secondary and tertiary branches2346Zea mays), where inflorescences have been selected for extreme divergence into a branchless female cob and a highly branched male tasselOryza rufipogon and Oryza nivaraAs the bearers of grain, grass inflorescences have been the target of selection for thousands of yearsOsLIGULESS1 (OsLG1) locus was recently identified as a domestication gene and controls the shift from open to closed paniclesDENSE AND ERECT PANICLE 1 (DEP1) within high-yielding Chinese rice varieties boosts yield potential by pleiotropically reducing panicle internode length (NL), while increasing both primary and secondary branch numberGrain Number 1a (GN1a) significantly increases secondary panicle branching, grain count and yieldMany genes have been cloned relating to rice inflorescence development6O. sativa comprises two major varietal groups (sometimes referred to as subspecies), Indica and Japonica, which can be further divided into five subpopulations 14151617181916The inbreeding nature of rice and multiple origins of domestication have led to the formation of deep subpopulation structure, which has partitioned genetic and phenotypic variation in the species. In this study, we performed GWAS using phenotypes collected with a high-resolution panicle phenotyping platform, PANorama16fastStructureK means  analysis, we confirmed that the top three PCs account for the aus, indica and tropical japonica subpopulations and explain ∼30% of the genetic variation within our panel  heritability by calculating additive+dominance (AD) heritabilityAs the diversity panel comprises inbred accessions and does not contain heterozygous alleles, it was not possible to calculate true heritabilities for each phenotype; instead, we estimated narrow sense (r=0.4. Increases in width traits such as rachis thickness and exsertion thickness were positively correlated with increased primary branch length (PBL) and primary branch number (PBN). Internode number (NN) and PBN, which estimate meristematic divisions, were positively correlated , NL was weakly correlated with rachis length (RL)  , suggestgth (RL)  also hadgth (RL) .tropical japonica subpopulation had the highest average RL (17 cm) and PBN (11), whereas the aus subpopulation had the largest average PBL (11 cm). Historically, many of the highest-yielding varieties have been bred within the indica subpopulation28indica outperformed both aus and tropical japonica in several components of yield as follows: panicle weight, total grain weight and grain number. Interestingly, indica accessions generally had intermediate-sized panicles, but distinctly had the smallest average NL. Despite varying distributions among phenotypes within the subpopulations, all phenotypic and genetic relationships between panicle traits and yield components were largely the same in the Indica and Japonica varietal groups , which confounds association studies by reducing mapping resolution and increasing type I error1517japonica . FurtherP<1 × 10−6 or larger) . In addition, many were from PBN traits (∼130 SNPs), which generally showed improved quantile–quantile plots with the use of the HD covariate  . Only onhickness . When HDhickness , suggesthickness . The eff tropics . Many siovariate .These results confirm established pleiotropic relationships between flowering time and inflorescence architecture in rice3839To compare association results across many traits, we constructed ‘association networks' using the programme CytoscapeP<1 × 10−6 or larger); only 18 SNPs showed a P<1 × 10−7 and the most significant panicle trait association was for PBL s.d.  were the result of linkage or pleiotropy, we constructed association networks using every phenotype in the panel. We observed 92 regions of the genome with significant SNPs for more than 1 trait; 10 regions had associations for 8 or more panicle traits . In mosth traits .aus (mentioned above) was associated with overall size traits such as RL, PL and width traits  within the mixed model: PBL, PBN, RL or NL  grown in a second environment (see Methods section). By subdividing traits and mapping with nested panicle phenotypesa priori candidate genes  MADS Box gene (OsMADS31)NARROW LEAF1 (NAL1)28515253OsKS gene family fell directly underneath the most significant GWAS SNPs for both maximum NL and 1,000GW  are responsible for the phenotypes we observe, nor to distinguish whether multiple genes were acting combinatorially to generate a ‘synthetic' QTL of larger effectInterestingly, the traits associated with RIL–QTL were sometimes different than the traits associated with GWAS–QTL in the same region. For example, we identified several RIL–QTL for an agronomic trait that overlapped with a GWAS–QTL for a panicle trait, or vice versa . To charlations) . Within  1,000GW , well wisynthase . HoweverO. sativa, many studies have evaluated large panels of diverse germplasm to increase GWAS mapping resolution1617193056Although panicles are the grain-bearing organs in rice, breeders have an incomplete picture of the genetic architecture underlying panicle development in different subpopulations and of the relationships between panicle traits and yield performance. Previous GWAS and QTL studies have collected only a limited number of panicle phenotypes and/or assessed plants grown in controlled environments1819a priori candidate genes from pathways known to regulate rice plant architecture , a trait linked to rice yield performance18Gene expression levels have been shown to directly affect flowering time in riceSEMIDWARF1 (SD1) and optimized vegetative architecture without drastically changing panicle phenotypestropical japonica into indica varieties63Within the public breeding community, there have been two major initiatives over the past 70 years to boost yield by optimizing independent phases of rice development. The first occurred during the Green Revolution, when breeders successfully leveraged a large-effect allele of indica and tropical japonica, it is not surprising that the New Plant Type initiative successfully generated large panicle phenotypes but failed to achieve desired combinations of sink and source traits that generate high-yielding varieties63per se will probably involve managing a highly interactive network or trait complex. This will require integrating quantitative tools and strategies, such as those used in this study, into model-based crop improvement pipelines incorporating genomic selection. That being said, targeted introgression of key genomic regions encompassing specific combinations of beneficial alleles in the form of a complex or ‘synthetic' QTL holds great promise as a strategy for coordinately improving the suites of traits that are essential for resilience and yield improvementGiven the quantitative nature of panicle development that we detected within and between NAL1-OsKS1 megalocus on chromosome 4 is of particular interest for rice breeding because it is rich in allelic variation and multiple studies have demonstrated yield improvement using introgression of Japonica alleles into Indica varieties285152NAL1 is hypothesized to have been a target of selection during rice domestication28515253OsKS1, which is tandemly linked with two of its homologues (OsKS2 and OsKS3) in the region and catalyses an early step in gibberellin biosynthesis, with mutant alleles leading to dwarfing phenotypes in both vegetative and reproductive tissueOsMADS31, which is ubiquitously expressed throughout panicle development and the first stages of seed formationThe The phenotyping methods and mapping resolution presented in this study provide us with the ability to hypothesize the existence of numerous complex QTL that merit further dissection using expression QTL mappingO. sativa was recently genotyped for 700,000 SNPs using an HDRAaus, 84 indica, 79 tropical japonica, 11 admixed Japonica, 3 temperate japonica and 2 admixed accessions). Detailed information regarding accessions is located within A collection of 1,568 accessions representing the five major subpopulations in Indica) and Azucena (Japonica), followed by single-seed descent in the greenhouse at Institut de Recherche pour le Développement in Montpellier, France. Both IR64 and Azucena were included the diversity panel used for GWAS  and the command chooseK.py was used to identify the model complexity that maximized the marginal likelihood (K=8). r2 between SNPs, which was calculated using the --r2 --ld-window 99999 --ld-window-r2 0 command in PLINKThe PC analysis was conducted using the svd function in Rindica, aus and tropical japonica subpopulations  at the International Rice Research Institute in Los Baños, Philippines in a randomized block design under flooded paddy conditions. Each replication consisted of a two-row plot 4.6 m in length, with 0.2 m between plants and 0.3 m between rows. Panicle traits, HD and booting date were collected on all 242 accessions within the panel. All other yield components were collected on 136 randomly sampled accessions from the ulations . Detaileet al.n=15 panicles per RIL) using the PANorama imaging protocol described belowThree plant replicates for each of the 168 RILs used for QTL mapping were grown in the Guterman greenhouse in Ithaca, New York, during summer 2012 using a pseudo-randomized block design that accounted for extreme plant height differencessourceforge.net/panorama1.Following the PANorama imaging protocolP-values for Pearson's correlation coefficients were calculated with a two-sided t-test using the cor.test function in Ret al.P-values for genetic correlation coefficients between BLUPs were calculated with a two-sided t-test using the cor.test function in RHistograms, boxplots, correlations and GWAS analyses were constructed using phenotypic grand means for each variety. EMMAX was used to calculate the linear mixed model and significance levels within the GWAS modelFor GWAS runs incorporating HD as a covariate:For GWAS runs incorporating a panicle trait as a covariate:Y and X represent the phenotype and SNP genotype vectors, respectively; P is a matrix containing the residuals of the first three PCs; HD represents a vector of the HD phenotype; and PAN represents a vector of the panicle phenotype used within the run . For genotypic and environmental random effects, respectively, μ∼N and ɛ∼N, where K is an identity by state kinship matrix accounting for pairwise relatedness between accessions. SNP marker filtering  and identity by state matrix calculations were performed using PLINKIndica and Japonica subspecies rather than in the individual subpopulations, to maximize our power to detect loci. We noted that certain traits were more susceptible to confounding than others, especially when performing GWAS across subpopulations using the entire panel of accessions. To correct for these issues, we systematically diagnosed the quantile–quantile plots for every trait–subpopulation–covariate combination and used logarithmic transformations on non-normal phenotypes .Using R/QTL (version 1.24.9), QTL mapping was performed as described in Crowell P<1 × 10−5, pairwise r2-values were calculated between surrounding SNPs that (1) fell within 150 kb and (2) had a P<1 × 10−3; any two SNPs meeting this criteria that also shared an r2≥0.3 were clumped into bins. All significant SNPs within the study were used in the construction of bins, regardless of the traits with which they shared associations. In addition, any bins sharing overlapping borders after using the PLINK clump command were collapsed into a single bin. Singleton, significant SNPs (<1 × 10−5) were discarded if no other SNP within the LD window was<2.5 × 10−4. To construct association networks, traits and their corresponding bins were treated as nodes within the programme CytoscapeSignificant SNPs were binned together into peaks using a sliding window based on the decay of a LD using the PLINKa priori candidate genes in http://ricefrend.dna.affrc.go.jp/) using the settings displayed alongside the HyperTree in r2-values for candidate gene zoom-ins were constructed using HaploviewA list of 319 candidate genes was assembled using a literature review and BLAST searches for candidate gene homologues . Single How to cite this article: Crowell, S. et al. Genome-wide association and high-resolution phenotyping link Oryza sativa panicle traits to numerous trait-specific QTL clusters. Nat. Commun. 7:10527 doi: 10.1038/ncomms10527 (2016).Supplementary Figures 1-94, Supplementary Tables 1-6, Supplementary Note and Supplementary References


MSX1 gene which is predicted damaging by multiple scores. This variant is in exon 2, a highly conserved region of the gene. Previous sequencing has suggested that mutations in MSX1 may account for ~2% of NSCLP. Our analysis further supports evidence that a proportion of NSCLP cases arise through monogenic coding mutations, though further work is required to unravel the complex interplay of genetics and environment involved in facial clefting.Nonsyndromic Cleft Lip and/or Palate (NSCLP) is regarded as a multifactorial condition in which clefting is an isolated phenotype, distinguished from the largely monogenic, syndromic forms which include clefts among a spectrum of phenotypes. Nonsyndromic clefting has been shown to arise through complex interactions between genetic and environmental factors. However, there is increasing evidence that the broad NSCLP classification may include a proportion of cases showing familial patterns of inheritance and contain highly penetrant deleterious variation in specific genes. Through exome sequencing of multi-case families ascertained in Bogota, Colombia, we identify 28 non-synonymous single nucleotide variants that are considered damaging by at least one predictive score. We discuss the functional impact of candidate variants identified. In one family we find a coding variant in the  If NSCLP is entirely multifactorial, the contribution of rarer variants may be largely polygenic and mediated by numerous variants of very small individual effect. In this case, causal genes may only be detectible through the analysis of large numbers of patients using, for example, burden testsExome sequences of twelve individuals from seven multi-case families (CL1-CL7) with NSCLP phenotypes were obtained. All experimental protocols were approved by the Research Ethics Committee at the Universidad de La Sabana, Bogota; informed consent was obtained for all participants and research was conducted in accordance with the Declaration of Helsinki. Families included between two and six individuals with isolated NSCLP . Most inDNA samples were extracted from blood collected at Operation Smile, Bogota, Colombia and exomes were captured using the Agilent SureSelect v5 (51 Mb) kit and sequenced on a HiSeq 2000. Read depth coverage statistics for all 12 exome sequences are given in et al.et al.To understand the spectrum of potentially damaging variation, we considered the list of 865 genes previously implicated in any form of CLP phenotype presented by Pengelly et al.We filtered the lists of variants  found inWe excluded variants found in homopolymer/repeat regions that can arise through misalignment between the sequenced reads and reference sequence. Any variants with read depth of <10 or in genes considered to be ‘highly mutable’WNT7A, MSX1, CLPTM1 and EVC2, ranked 9, 10, 11 and 23 respectively) have been previously identified as containing variants implicated in NSCLP phenotypes. Family CL1 has the 9th ranked variant in the WNT7A gene. Members of the WNT gene family have previously been associated with NSCLP phenotypes2223WNT3A, WNT5A, WNT9B, and WNT11 have been established as candidatesth ranked variant, found in family CL4, is in the MSX1 gene, and considered damaging by SIFT, PolyPhen-2 and MutationTaster and has high GERP++ and CADD scores. Variants in this gene have been strongly implicated in NSCLP in several studies. Jezewski et al.MSX1, in which the p.P260T is located, has been found to be highly conserved with significantly fewer sequence variants compared with exon 1 of this small (two exon) geneMSX1 as a candidate is established through a cleft palate and foreshortened maxilla phenotype in knockout miceMSX1 in NSCLP4282930MSX1 microsatellite alleles were analysed and an increased risk of CLP was observed with CA polymorphisms in the geneMSX1 mutation in a family with clefting and tooth agenesis showed a familial pattern of segregating MSX1 mutationsMSX1 promotes growth and inhibits differentiation. Mutations in MSX1 can cause primary or secondary facial clefting in mouse modelsThe 10th ranked variant (from family CL1) is in the CLPTM1 gene  which is situated at 19q13.3. A balanced translocation is this region was found in a multi-case CLP familyde novo deletion of 0.8 Mb in this region associated with CLP, but not encompassing CLPTM1, has been reportedCLPTM1 or other genes in this locus is uncertain.The 11rd ranked variant is in the EVC2 gene (family CL2) and belongs to the same two megabase chromosomal region as MSX1 (4p16). Ingersoll et al.EVC2. Mutations in EVC2 can lead to Weyers acrofacial dysostosisThe 23et al.TBX22, PVRL1, IRF6, P63 and MSX1, can also contribute to ~10% of NSCLP. Scapoli et al.IRF6 gene, which cause VWS, have been firmly implicated in some NSCLP caseset al.IRF6 was associated with the most severe complete bilateral NSCLP phenotype. This suggests multi-case families with bilateral clefts are the most likely to be segregating single gene mutations. This strategy is supported by Vieira et al.Linkage, candidate gene association and genome-wide association (GWAS) have been applied to investigate numerous multifactorial diseases, including NSCLP. As a result of these studies more than 11 genes and gene regions are now known or likely to have an etiologic role in NSCLPMSX1 gene shared by affected family members in CL4 in which the proband has a bilateral CLP phenotype. Direct sequencing of coding regions has shown rare mutations in MSX1 may account for ~2% of NSCLP. The identified MSX1 variant is present at low frequency in the ExAC database (http://exac.broadinstitute.org/). Functional studies and analyses of larger cohorts of multi-case NSCLP families are required to establish a possible role for this and other rare variants identified in NSCLP phenotypes. Variants identified here also include candidates in the WNT7A (family CL1) ,CLPTM1 (family CL1) and EVC2 genes (family CL2) which should be considered as targets for analysis in additional families.In database . ExAC coFor investigations aiming to resolve the genetic factors underlying NSCLP in multiple case families, exome sequencing presents a relatively cost-effective approach in which sequencing a small number of affected family members can identify candidate underlying genetic variation. NSCLP provides a particular challenge for genetic studies, with incomplete penetrance and environmental factors hindering the identification of aetiological variance2Exome sequencing yields thousands of variants per individual and identification of candidate variants can only be achieved following extensive filtering. We have undertaken filtering to identify variants predicted as damaging by restricting analysis to a list of 865 genes which have been previously associated with any condition involving CLP. Such an approach risks missing causal variants in novel genes not previously linked to NSCLP, but facilitates practicable data interpretation by virtue of the greater prior probability that they are associated with NSCLP. The composite score based rank using PhyloP, GERP++. CADD and logit  has beenIn silico protein modelling approaches may also be used to improve throughput, however these require the prior determination of protein structure, which has not been reported in the majority of genes discussed herein. Overall, it is clear that functional validation is a significant bottleneck in NGS studies, and one not readily assuaged.Whilst predictive tools are essential for the prioritisation of variants discovered in next generation sequencing (NGS) studies, ultimately functional validation of the effects of variants on protein function is required to confirm their impact. Given the volume of potentially pathogenic variants being identified in NGS studies, routine functional validation is infeasible. The limitations of exome sequencing include lack of coverage outside gene coding regions thereby excluding regulatory variants, which may influence risk. Technical limitations include poor coverage of some coding regions thereby missing potential causal variants. Whole genome sequencing offers a solution to these coverage issues, but at higher cost and considerably increased analytical complexity. Given the extent of the missing heritability in CLP, it is likely non-coding regions of the genome play a significant role; whole genome sequencing may therefore provide a valuable tool as sequencing costs continue to drop.In this study we have limited our analyses to 865 genes with a known/suspected involvement in CLP phenotypes. Whilst this will prevent us from identifying novel aetiological genes, 7 families would be underpowered to identify novel causal genes reliably. Large cohort studies are required in order to identify novel CLP genes; to this end we have made our WES data available in MSX1 gene in family CL4 which is a strong candidate for causality. Deleterious variants in at least three additional genes may be implicated in NSCLP phenotypes in some of the other families. Although NSCLP is primarily a complex multifactorial phenotype, our study adds to the growing body of evidence that Mendelian sub-forms exist and these are best studied in multi-case families particularly where there are more severe phenotypic features such as bilateral clefting.In conclusion, we have undertaken exome analysis in seven Colombian families with NSCLP phenotypes. We find a deleterious variant in the How to cite this article: Pengelly, R. J. et al. Deleterious coding variants in multi-case families with non-syndromic cleft lip and/or palate phenotypes. Sci. Rep.6, 30457; doi: 10.1038/srep30457 (2016).


Here, we analyze the movements of mitochondria in ganglion cell dendrites in the intact retina. We find that whereas during development 30% of mitochondria are motile at any time, as dendrites mature, mitochondria all but stop moving and localize stably to synapses and branch points. Neither spontaneous nor sensory-evoked activity and Ca2+ transients alter motility of dendritic mitochondria; and pathological hyperactivity in a mouse model of retinal degeneration elevates rather than reduces motility. Thus, our findings indicate that dendritic mitochondria reach stable positions during a critical developmental period of high motility, and challenge current views about the role of activity in regulating mitochondrial transport in dendrites.Mitochondria move throughout neuronal dendrites and localize to sites of energy demand. The prevailing view of dendritic mitochondria as highly motile organelles whose distribution is continually adjusted by neuronal activity via CaDOI:http://dx.doi.org/10.7554/eLife.11583.001 Inside the cells of animals and plants, compartments called mitochondria play several important roles including supplying chemical energy for cellular processes. The mitochondria inside nerve cells are produced in the main body of each cell, and must travel down a long nerve fiber called the axon and the branch-like extensions called dendrites to reach the sites where they are most needed. As the nerve cells form, dendritic branches grow and retract as the connections between different nerve cells – known as synapses – form and disappear. Later on, the dendrites and synapses become more stable, but it is not clear if the amount that the mitochondria move also changes.Faits et al. used microscopy to study the movement of mitochondria in the developing dendrites of ganglion cells in the eyes of mice. The experiments show that early on in the development of nerve cells, the mitochondria are very mobile. However, as the synapses become more stable later on, the mitochondria become almost motionless. The movement of another type of cell compartment to the dendrites is unaffected, which suggests that this decline in movement is specific to mitochondria.Next, Faits et al. studied mutant mice that suffer from degeneration of part of the eye called the retina. These mice have ganglion cells that display higher levels of spontaneous activity than normal and their synapses continue to form and disappear later in development. The experiments show that the mitochondria in the ganglion cells remain mobile in the adult mutant mice. Faits et al.’s findings challenge the prevailing views of mitochondria in dendrites, and suggest that mitochondria reach stable positions during a critical period in the development of the retina.Further studies should reveal how the decline in the movement of mitochondria is regulated, which may help us to understand how differences in the movement of mitochondria can lead to the degeneration of nerve cells in some human diseases, such as dominant optic atrophy.DOI:http://dx.doi.org/10.7554/eLife.11583.002 In neurons, mitochondrial biosynthesis occurs in the soma, but sites of energy use and Ca2+ influx are dispersed across axonal and dendritic arbors , raising the question whether physiologically occurring activity patterns in intact circuits exert a similar influence.Neuronal activity is thought to control the movements of dendritic mitochondria. In particular, increases in intracellular Cain Miro1 . However2+, we find that although elevation of external K+ reduces motility of dendritic mitochondria in the retina as it does in cultured neurons, neither spontaneous waves of activity during development, nor sensory-evoked activity at maturity alter the motility of dendritic mitochondria in RGCs. Finally, pathologically elevated RGC activity, a common feature of retinal degeneration, restores motility in mature retinas to developmental levels.Here, we analyze how development and neuronal activity shape the distribution and dynamics of mitochondria in dendrites of ganglion cells in the intact retina. We find that mitochondrial density in retinal ganglion cell (RGC) dendrites reaches near-mature levels before most synapses are formed. In the adult retina, mitochondria are enriched at synapses and branch points of RGC dendrites. Mitochondria localize to synapses early, whereas their accumulation at branch points occurs gradually during circuit development. The motility of mitochondria changes fundamentally across development. During the period of dendrite growth and synaptogenesis approximately 30% of mitochondria are in motion at any time. By contrast, we observed no movements of dendritic mitochondria in mature circuits. This drastic decline in motility is cargo-specific, as peroxisomes remain motile in mature RGC dendrites. Using simultaneous two-photon imaging of mitochondrial movements and intracellular CaWe used RGCs to study how development and neuronal activity influence the distribution and dynamics of dendritic mitochondria. In the mature retina, photoreceptors translate changes in photon flux into changes in glutamate release , and bipTo analyze the distribution and dynamics of mitochondria in RGC dendrites, we performed confocal and two-photon imaging in flat-mounted explants, which leave the circuitry of the retina intact . We have2+ signaling, and assess its changes across development, we biolistically labeled RGCs with mitochondrially targeted yellow fluorescent protein (mtYFP), the postsynaptic density protein 95 fused to CFP (PSD95-CFP), and a red cytosolic fluorophore (tdTomato). Others and we previously showed that PSD95-CFP localizes specifically to BC synapses on RGC dendrites (i.e. excitatory synapses)  . We labeynapses) : P9 (earynapses) . Similarynapses) . This syynapses) . Thus, mThy1-mtCFP-P) labeled with mtYFP and mtCFP, respectively (data not shown). Interestingly, the movement patterns - including duration of runs, speed during uninterrupted motion, and duration of short pauses - of motile mitochondria at P15 were not significantly different from those observed at P9  on RGCs biolistically or transgenically  exhibited synchronous global Ca2+ transients across their dendritic arbors. The frequency and correlation of these transients was indistinguishable from that of spike bursts in MEA recordings  elicits bursts of action potentials in RGCs . As wavecordings , indicatal waves . To testovements , revealefraction  nor the 2+ elevations were shown to stop moving mitochondria via the adaptor protein Miro1, which uncouples mitochondria from kinesin motors upon Ca2+ binding. Miro1 is readily detected in Western blots of the retina from at least P6 onwards and its expression reaches mature levels by P15 (2+-binding-deficient mutant (Miro1KK) does not alter the motility of dendritic mitochondria. Thus, a lack of Miro1 expression or function is unlikely to account for the lack of effect of physiologic activity patterns on the movements of mitochondria in RGC dendrites.In cultured neurons, Cas by P15 . In addi+ concentrations to elicit changes in mitochondrial motility. We therefore tested the effects of this manipulation in the developing retina. Elevating external K+ to 30 mM tonically increased the concentration of Ca2+ in RGC dendrites and, similar to observations in cultured neurons, decreased the motile fraction of mitochondria  exhibited local dendritic Cal stores . Like glmotility .2+ transients in the developing retina do not regulate mitochondrial motility in RGC dendrites, irrespective of Miro1 expression, and that tonic elevations of Ca2+ upon application of high K+ solution may engage different mechanisms to physiological transients.Together these results suggest that physiologically occurring activity patterns and CaThy1-mtCFP-P retinas every 15 min on a two-photon microscope. In the intervals between image acquisitions, retinas were either kept in darkness (P21 – no stim) or presented a full-field white noise stimulus with alternating contrast levels . MEA recordings confirmed that this stimulus robustly elevates the firing rates of RGCs  and Oligomycin (an ATP synthase inhibitor) to P9 and P21 retinas. Fast (0.9 fps) and long-interval (15 min) time-lapse imaging revealed no significant changes in the motility of mitochondria in developing or mature RGC dendrites .-/-Crx mice, a model of Leber congenital amaurosis, retinal waves are preserved. But, from P15 on RGCs exhibit rhythmic hyperactivity as a result of enhanced input from BCs . By contrast, stationary mitochondria remain in place for long periods of time. Unlike transitions between runs and pauses in the motile state, transitions between motile and stationary state are rare (+/-Opa1 mice) results in shortened mitochondria, which accumulate in proximal RGC dendrites (+/-Opa1 mice age (>1 yr), the number of excitatory synapses and branches of RGC dendrites declines, highlighting the importance of mitochondrial fusion for dendritic and synaptic integrity   . Togethet shown) , these o2+ and mitochondria in the intact retina, we find that neither patterned spontaneous activity during development  approved by the Animal Studies Committee of Washington University School of Medicine and performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. -mtCFP-P  and Crx- . Tissue was prepared in ice-cold mouse Artificial Cerebrospinal Fluid (mACSF) buffered with HEPES  or sodium bicarbonate . Retinas used for biolistic transfection were incubated at 33°C in an O2-perfused chamber overnight. Retinas used for light stimulus and MEA experiments were dissected under infrared illumination (excitation wavelength >900 nm) and incubated at 33°C in a light-tight chamber for approximately 2 hr before experiments. Live retinal tissue was constantly perfused at 1 ml/min with 33°C mACSF bubbled with O2  or 95% O2 / 5% CO2 (two-photon experiments) during light stimulation and imaging. In pharmacologic experiments, 5 µM DhβE (Tocris Bioscience) or 4 µM Antimycin A (Enzo Life Sciences) and 10 µM Oligomycin (Sigma-Aldrich) were added to mACSF. To raise extracellular K+ concentrations to 30 mM, KCl was substituted for NaCl in mACSF .Eyes were removed from mice deeply anesthetized with COA gene gun was used to transfect RGCs in the ex vivo retinal flat-mount preparations as described previously . BrieflyMulti-electrode array (MEA) recordings of RGC action potentials were acquired as described previously . BrieflyFixed and live retinas were imaged on Olympus Fv1000 laser scanning confocal and two-photon microscopes using 60x 1.35 NA oil-immersion (fixed retinas), 60x 1.1 NA water-immersion, and 20x 0.95 NA water-immersion objectives (live retinas). Images were processed and analyzed using ImageJ (NIH), Amira (FEI), and software written in MATLAB (MathWorks).To analyze mitochondrial and synaptic distributions, retinas were fixed in 4% paraformaldehyde for 30 min at RT. Synapses were identified and dendrites skeletonized using custom MATLAB software described previously . Briefly2+ waves were acquired at 0.9 frames per second (fps). Motile fractions were calculated as (motile organelles)/(motile organelles + stationary organelles). We refer to the motile fraction as the fraction moving during 1 min of observation, whereas instantaneous motile fraction is used to denote the fraction moving between consecutive frames in 0.9 fps imaging series. Kymographs were generated using the ImageJ Multiple Kymograph plugin. Mitochondrial speeds and peroxisomal speeds and mitochondrial run and pause durations were calculated using custom MATLAB software and organelle positions tracked manually using the ImageJ MTrack2 plugin.Time-lapse recordings of mitochondrial and peroxisomal motility, and CaMitochondrial displacement during light stimulation of P21 retinas was computed using custom MATLAB software. mtCFP signal was masked in Amira to include only mitochondria in contiguous dendritic branches. mtCFP intensity was binarized and MATLAB software was used to identify regions of connected pixels as individual mitochondria. Images at t = 30 min were subtracted from images at t = 0 min; mitochondria identified at 0 min that did not have at least 20% overlap with a mitochondrion at 30 min was counted as a displaced mitochondrion. Percent displacement was calculated as 100 x (displaced mitochondria)/. Stimulated retinas were shown light stimulus patterns of alternating high- and low-contrast (mean intensity 5000 R*/rod/s) periods lasting 60 s each for a total of 15 min. Image stacks of mtCFP signal throughout RGC dendrites were collected between light stimulations. Unstimulated retinas were kept in darkness for 15 min between image stacks. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Richard Aldrich (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing.Thank you for submitting your work entitled "Dendritic mitochondria reach stable positions during circuit development" for consideration by The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:The authors quantify mitochondrial location and trafficking rates in dendrites during retinal ganglion cell development in situ and find that trafficking stops upon neuron maturation. The timing of localization of mitochondria to synapses and dendrite branch points differs during development. The authors also find that calcium waves reported previously to halt mitochondrial trafficking via Miro1 do not do so under more physiological conditions. Mitochondrial motility was retained during development in a model of retinal degeneration , where synaptogenesis between bipolar and retinal ganglion cells is elevated and prolonged. The conclusions of these well controlled experiments performed under fairly physiological conditions contrast with those from several prior papers using isolated cultured neurons and would have substantial impact on the field.Essential revisions:1) Mutations in Opa1 leading to DOA indicate that mitochondrial fusion is essential for retinal ganglion cells, likely involving dendrites (see point 2 below), possibly to mitigate mitochondrial damage or to support mitophagy by balancing mitochondrial fission. As mitochondrial fusion would demand some degree of motility, assessing mitochondrial trafficking after applying mitochondrial stress would be important. Assessing effects of mitochondrial toxins such as Rotenone, Oligomycin or Antimycin A applied to the retinal cultures at P9 and P21 and determining if and in what way the stressors alter movement may yield insight into the apparent lack of motility despite the essential role of mitochondrial fusion. Perhaps the motility involved in fusion is on a much slower time scale than the motility involved in trafficking at P9. Some mention of the essential role of Opa1 particularly in retinal ganglion cells and how this mitochondrial fusion requirement relates to the level of motility studied here would strengthen the Discussion.2) Furthermore, the two papers below that support the necessity of mitochondrial dynamics to maintain dendritic mitochondria should be cited and discussed. The first pathological changes in a mouse model of DOA with mutant OPA1 (defective fusion) actually affect dendrites before impairing axons and cell bodies in retinal ganglion cells :2942-51 and Williams PA, et al. Opa1 is essential for retinal ganglion cell synaptic architecture and connectivity. Brain 2012;135:493-505).-/-Crx mice? It would be important to assess the extent of calcium influx change to test/corroborate the important conclusion of the authors that physiological levels of calcium transients do not halt mitochondrial transport.3) Do calcium transients increase along with the retinal wave increase in RGCs from  1) Mutations in Opa1 leading to DOA indicate that mitochondrial fusion is essential for retinal ganglion cells, likely involving dendrites (see point 2 below), possibly to mitigate mitochondrial damage or to support mitophagy by balancing mitochondrial fission. As mitochondrial fusion would demand some degree of motility, assessing mitochondrial trafficking after applying mitochondrial stress would be important. Assessing effects of mitochondrial toxins such as Rotenone, Oligomycin or Antimycin A applied to the retinal cultures at P9 and P21 and determining if and in what way the stressors alter movement may yield insight into the apparent lack of motility despite the essential role of mitochondrial fusion. Perhaps the motility involved in fusion is on a much slower time scale than the motility involved in trafficking at P9. Some mention of the essential role of Opa1 particularly in retinal ganglion cells and how this mitochondrial fusion requirement relates to the level of motility studied here would strengthen the Discussion.As suggested, we performed additional live imaging experiments to test the effects of Antimycin A (a complex III inhibitor) and Oligomycin (an ATP synthase inhibitor) on mitochondrial motility in RGC dendrites. Toxin-mediated stress had previously been reported to have varied effects on mitochondrial transport, possibly depending on the neuron type and compartment (axon vs. dendrite) examined. Both at P9 and at P21 we found that Antimycin A and Oligomycin did not affect the motility of dendritic mitochondria in RGCs. These data are presented in a supplementary figure  in our r2) Furthermore, the two papers below that support the necessity of mitochondrial dynamics to maintain dendritic mitochondria should be cited and discussed. The first pathological changes in a mouse model of DOA with mutant OPA1 (defective fusion) actually affect dendrites before impairing axons and cell bodies in retinal ganglion cells :2942-51 and Williams PA, et al. Opa1 is essential for retinal ganglion cell synaptic architecture and connectivity. Brain 2012;135:493-505).+/-Opa1mice, which highlights the importance of mitochondrial fusion to dendritic and synaptic integrity, and cite the studies mentioned by the reviewers.In the Discussion section of our revised manuscript, we speculate that the rare mitochondrial displacements we observe in long-interval time-lapse imaging experiments at P21 may be associated with mitochondrial fission, or support mitochondrial fusion or mitophagy. In this context we also discuss the evidence obtained in -/- mice? It would be important to assess the extent of calcium influx change to test/corroborate the important conclusion of the authors that physiological levels of calcium transients do not halt mitochondrial transport.3) Do calcium transients increase along with the retinal wave increase in RGCs from Crx-/-Crxmice is greatly increased with RGCs firing sustained at approximately 6 Hz. These firing rates are too high to distinguish individual firing events using GCaMP6s . We tried to resolve firing events using a faster Ca2+ indicator: OGB-1. We simultaneously recorded spike trains (via patch clamp) and imaged dendritic Ca2+ signals (via 2-photon imaging) from individual RGCs in -/-Crxmice. As shown in the figure below (2+ imaging. Dendritic Ca2+ levels were reduced when hyperpolarizing currents were injected to suppress spiking (data not shown). We chose not to include these data in our revised manuscript as similar experiments have been performed in a variety of systems. Our results therefore amount to a confirmation of already described kinetics of Ca2+ indicators, and their inclusion, we feel, would distract from the novel findings of our study. We hope that the reviewers agree with this choice.Spontaneous activity in re below , however


TRPV1 is well known as a sensor ion channel that transduces a potentially harmful environment into electrical depolarization of the peripheral terminal of the nociceptive primary afferents. Although TRPV1 is also expressed in central regions of the nervous system, its roles in the area remain unclear. A series of recent reports on the spinal cord synapses have provided evidence that TRPV1 plays an important role in synaptic transmission in the pain pathway. Particularly, in pathologic pain states, TRPV1 in the central terminal of sensory neurons and interneurons is suggested to commonly contribute to pain exacerbation. These observations may lead to insights regarding novel synaptic mechanisms revealing veiled roles of spinal cord TRPV1 and may offer another opportunity to modulate pathological pain by controlling TRPV1. In this review, we introduce historical perspectives of this view and details of the recent promising results. We also focus on extended issues and unsolved problems to fully understand the role of TRPV1 in pathological pain. Together with recent findings, further efforts for fine analysis of TRPV1's plastic roles in pain synapses at different levels in the central nervous system will promote a better understanding of pathologic pain mechanisms and assist in developing novel analgesic strategies. Transient receptor potential vanilloid subtype 1 (TRPV1) is a well-known pain-mediating ion channel expressed in sensory neurons including dorsal root ganglionic (DRG) neurons, trigeminal ganglionic (TG) neurons, and vagal neurons. In response to various harmful stimuli, TRPV1 pore opens and cationic flux through the pore into the nerve terminal causes electrical depolarization which may lead to action potential generation. When propagated and transmitted into the brain, the signals finally result in the perception of pain. Because of its extreme polymodality compared to other known peripheral sensor molecules that allows TRPV1 to cover a large spectrum of pain qualities from chemical through thermal ones and of its famous activator capsaicin which has been traditionally utilized for pain research even before TRPV1 discovery, TRPV1 has garnered a great deal of attention as a peripheral pain-modulating target. Topical application of a TRPV1 modulator is currently the mainstream for TRPV1-targeting analgesic strategies while systemic approaches have been dropped due to a potential for hyperthermic adverse effect because body temperature regulation is perturbed by antagonism of vagal TRPV1.Although the initial report about TRPV1 finding suggested that it is specifically expressed in sensory neurons, its wider distribution in various regions including certain regions of the central nervous system (CNS) and nonnervous tissues has been surmised  exposure. Because the frequency but not the amplitude of EPSCs was increased, TNF-α seemed to elevate glutamate release by acting at presynaptic terminals. This perfusion with TNF-α ex vivo may represent an inflammatory or neuropathic pain state in vivo. Simultaneous treatment of capsazepine reversed this TNF-α effect: the changes were limited to numbers of sEPSC frequencies. Consequently, the results of this study provide multiple implications: the primary action site of the acute TNF-α effect is presynaptic sensory neurons; TRPV1 had been presumed to be a major effector of TNF-α action, at least in the periphery [α and TRPV1 via its G-protein-coupled receptor (GPCR) activation, as a part of its painkilling mechanisms [Gone through simple traditional observations where spontaneous excitatory postsynaptic currents (sEPSCs) were facilitated by the presynaptic actions of capsaicin to understand the circuitry under normal conditions , 37, reseriphery , 39, andchanisms –42.2+ indicator protein is driven by the promoter of Pirt , a pan-DRG/TG marker [2+ levels in cell bodies and peripheral and central terminals can be discerned in DRG and TG neurons, even under ex vivo conditions surrounded by other tissue types or buried in complex synaptic circuits. They also created a cheek mechanical hyperalgesia model using CCI of the infraorbital nerve, which is the major branch of the maxillary (V2) TG nerve. Accordingly, the trigeminal subnucleus caudalis (Vc), which is analogous to the spinal dorsal horn in terms of the sensory synaptic circuit, was observed for presynaptic TRPV1 functions.More recently, the Wei and Dong labs revisited the presynaptic role of TRPV1 from the viewpoint of descending excitatory modulation when they tried to explain secondary hyperalgesia that occurs from neighboring but uninjured receptive field under neuropathic conditions . They deG marker . With th2+ fluorescence level due to GCaMP3 was analyzed as a surrogate measure for excitability, both V2 and V3 central terminals in Vc exhibited larger Ca2+ increases than under normal conditions. Furthermore, these elevated sensitivities were commonly observed in terminals from superficial through deep laminae, suggesting that not only injured but also adjacent undamaged nerve fibers became hyperactive and that this situation consequently led to secondary hyperalgesia and allodynia. Based on their previous observations, Wei and Dong's group hypothesized that the rostral ventromedial medulla (RVM) in the brainstem relays 5-hydroxytryptamine  dependent excitatory input to uninjured nerves [2+ signals and hypersensitive behaviors. Since 5-HT receptor-mediated TRPV1 facilitation was confirmed in the central presynaptic terminals, the descending excitatory axons from RVM seem to constitute axoaxonal contacts. Collectively, TRPV1 in the presynapse of sensory neurons that covers the undamaged regions participates in secondary pain amplification through a descending facilitation mechanism  TG nerve territories. When intracellular Cad nerves , 45. Indechanism .2+ concentration and the reduction of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activities, similar to other typical LTD processes. Accordingly, LTD caused less excitability and reduced GABA release of the GABAergic interneurons, and therefore the secondary projection neurons  received less inhibitory input, resulting in enhanced relay of pain signals to higher brain areas  activation for elevated TRPV1 expression were raised by studies on DRG neurons [α axis. Even if this prostaglandin-mediated mechanism is true, the mechanism can only sensitize but not activate TRPV1. By this sensitization, heat threshold for TRPV1 activation might descend around the core body temperature. In addition, while only ERK itself has been shown to participate, there has not been sufficient clarification of the further downstream signal in this pathway. Moreover, it also needs to be explored in the presynapse whether the releases of neurotransmitter vesicles are facilitated simply by increased intracellular Ca2+ ions through TRPV1 opening-induced depolarization, or other unknown molecular downstream signals are involved.Although TRPV1 is known as a major heat sensor in the body, its thermosensory functions and related thermoregulatory feedback mechanisms are unlikely to be exerted in the spinal cord because this region only experiences a limited range of temperatures, close to the core body temperature. Evidence mentioned above suggests that pharmacological antagonism for TRPV1 in the spinal cord does not cause thermal effects. However, that situation leads to another question: What conditions else activate TRPV1 there? Inflammatory peptides including TNF-u et al. , and neu neurons , 54. Ext neurons , but it The 5-HT3 receptor is a major receptor that receives descending excitatory input from RVM . InteresTransient receptor potential ankyrin subtype 1 (TRPA1) is comparable to TRPV1 in terms of the importance of covering pain modalities and transductory roles to initiate nociceptor depolarization in the periphery . If bothAs mentioned, Kim et al.  proposedStemming from the recent accomplishments in spinal TRPV1 research, unexplored mechanisms connecting newly uncovered TRPV1's roles and related hypotheses are being raised.Like pharmacological approaches, gene editing strategies often promote understanding of a mechanism and also offer therapeutic insight. A series of studies using local RNA interference techniques have given new lines of firm evidence for the roles of TRPV1 in pathologic pain progress and also for its action in the spinal cord –70. As tβ finally enhances excitability of the dorsal horn neurons by inducing phosphorylation of an N-methyl-D-aspartate receptor subunit. It is currently unexplored whether the presynaptic TRPV1 activation is linked with this process.Since TRPV1 expression is absent in glial components, it is not likely that the effects of pharmacological modulations of spinal TRPV1 have a direct link to glial contributions. However, in a follow-up study of Kim et al. , the Weiμ-opioid receptor-induced LTP in the spinal cord appears to occur in TRPV1-positive presynapses and it might be a future issue whether changes in TRPV1 activity in the central terminal are practically involved in opioid signaling [Nitric oxide (NO) is of general importance as a retrograde signal for modifying brain synaptic strength. In pain synapses, postsynaptic NO plays a central role for presynaptic activation of the guanylyl cyclase-cyclic guanosine monophosphate- (cGMP-) protein kinase G pathway , 76. Intignaling .Two of many reasons why peripheral TRPV1 has received much attention from industry in the decade since its gene discovery seem to be its polymodality integrating painful inputs with diverse qualities and its peripheral location. For the latter, a central advantage of targeting peripheral TRPV1 is that its ligand avoids the adverse effects of the CNS when it is designed to be BBB-impenetrable. However, assuming that the aim is CNS administration and considering the novel nociceptive roles of TRPV1 in the spinal circuit, one may need to conceive similar adverse situation as already observed or predicted for other CNS analgesic candidates because the target is possibly expressed in other central regions and may have differential actions.μ-opioid receptor is simultaneously activated TRPV1 activation contributes to facilitation of glutamatergic interneuronal activity, which offers GABAergic inhibition of descending excitatory ON cells in the RVM circuit, leading to a reduction of nociceptive transmission in the spinal cord [As mentioned above, the bulbospinal circuit receives descending input from periaqueductal gray (PAG) and confers descending excitatory inputs as well as inhibitory ones , 81. Earnal cord . Decreasnal cord . Because+ and Ca2+ channels [Exploring the mechanism of analgesic effects of acetaminophen, researchers have found out that TRPV1 activation in the central nervous system is involved , 86. Whechannels .Other adverse situations due to the potential presence of TRPV1 in CNS regions might be possible. For example, whether inadvertent diffusion of an antagonist in the ventricular regions may enable access to untargeted areas and affect other brain functions including memory or mood needs to be carefully evaluated , 88, 89.+ channels, with or without their permeant blockade of TRPV1 itself. TRPV1-negative neurons should be inert to the blocking effects of these drugs since the drugs can be admitted only through TRPV1, which may allow avoidance of common adverse effects of the local anesthetics including numbness and motor defects via interfering nonpain pathways. Such approaches of specific application may be taken into consideration in the future for modulation of spinal cord TRPV1.Whether TRPV1 activation can gain a therapeutic advantage is conceivable. Specific delivery of local anesthetics to TRPV1-positive nociceptor is being considered as a novel analgesic strategy , 91. SomAmong TRPV1-targeting pain therapies, only topical capsaicin treatment is currently available in clinics. The rationale is based on the functional incapacitation of the peripheral sensory terminals by agonist-induced TRPV1 desensitization and mitochondrial permeability transition which leads to terminal ablation , 92. TheIn the early stage, more attention was given to the sensory involvement of TRPV1 in the peripheral terminal of the nociceptor neurons to harmful environments and to its contribution to neurogenic inflammation , 95. How


Triticum aestivum), durum wheat (T. durum), and barley (Hordeum vulgare) plants exposed to various abiotic stress factors  is provided. Possible roles of dehydrin proteins in an acquisition and maintenance of an enhanced frost tolerance are analyzed in the context of plant developmental processes . Quantitative and qualitative differences as well as post-translational modifications in accumulated dehydrin proteins between barley cultivars revealing differential tolerance to drought and salinity are also discussed. Current knowledge on dehydrin role in wheat and barley response to major dehydrative stresses is summarized and the major challenges in dehydrin research are outlined.Dehydrins as a group of late embryogenesis abundant II proteins represent important dehydration-inducible proteins whose accumulation is induced by developmental processes (embryo maturation) as well as by several abiotic stress factors . In the review, an overview of studies aimed at investigation of dehydrin accumulation patterns at transcript and protein levels as well as their possible functions in common wheat ( Abiotic stress factors – cold, frost, drought, salinity – severely limit plant growth and development as well as the final yield in crops including cereals from the tribe Triticeae. Low temperatures, drought, and salinity represent stress factors associated with plant cell dehydration. Dehydration stress factors induce profound cellular response aimed at an elimination of water loss. Plant cell response to dehydration includes an accumulation of osmotically active compounds including hydrophilic proteins such as dehydrins. Several physiological studies focused on plant stress response have reported a positive relationship between the level of accumulation of dehydrin transcripts or proteins and plant stress tolerance . More toTriticum aestivum), durum wheat (T. durum) and barley (Hordeum vulgare) exposed to cold, frost, drought, and salt stress factors.The aim of the study lies in a summary of the research focused on dehydrin transcript and protein accumulation in wheat and barley plants exposed to various abiotic stresses with a dehydrative component – cold, drought, salinity. We have tried to summarize recent results gained on cultivated common wheat (T. aestivum) complete genome sequencing is still in progress . Barley (H. vulgare) represents the relatively most tolerant cultivated Triticeae species with respect to dehydrative stresses, especially drought and salinity  out of five dehydrin structural types (nKm type (Table 1). The largest group of dehydrins in barley (10 out of 13 Dhn genes) as well as in common wheat belong to YnSKm type which encompass basic dehydrins induced by strong dehydration stresses  as well as by abscisic acid (ABA) due to an occurrence of several ABRE elements in their promoters  in common wheat are induced by both cold and drought as well as by ABA  up to 0.25 mM while an inhibition by SA levels higher than 0.4 mM (n type dehydrins with multiple copies of K-segment such as barley DHN5 (K9) and wheat WCS120 (K6), a cryoprotective activity has been reported  in common wheat and are induced predominantly by cold , gibberellins (GA), and SA  while the remaining three dehydrin proteins DHN5 (K9), DHN8 (SK3), and DHN13 (KS) revealed significant differences with respect to YnSKm type dehydrins as well as each other  in durum wheat  for DHN5  and cryoprotective functions as reported for Kn type dehydrins with multiple copies of K-segment such as WCS120 (K6) in common wheat (9) in barley (2 protein of 227 amino acids (in vitro study on wild-type and mutated forms of durum wheat DHN5 with respect to their protective effects on lactate dehydrogenase (LDH) and β-glucosidase activities under dehydration stress  and acquired frost tolerance in a set of 30 barley cultivars including both spring and winter growth habits of a wide range of lethal temperature of 50% of the sample (LT50) values  and proteins  can be considered a reliable marker of plant frost tolerance under controlled conditions. Under field conditions, the quantitative relationships between dehydrin protein relative accumulation and frost tolerance seem to be more complex and the evaluation of the sum of all dehydrin proteins on the immunoblots rather than a single dehydrin protein seem to provide more reliable results  values . At tran) values . There h results . There h samples .Arabidopsis thaliana. In A. thaliana, a negative effect of vernalization-induced SOC1 (suppressor of overexpression of constans1) factor on CBF (C-repeat binding factor) transcription factors has been described resulting in an inhibition of CBFs and a downstream Cor (Cold-regulated) gene expression in vernalized plants. A direct physical interaction of SOC1 protein with CBF promoter regions resulting in an inhibition of CBF gene expression has been found  on the expression of CBFs and downstream Cor genes (Dynamics of WCS120 accumulation under a long-term cold treatment (16 weeks) has been studied in a high frost-tolerant winter wheat Mironovskaya 808 . Under cen found . In einkor genes ; however∘C for 1 h), there has been found an accumulation of high-molecular Dhn5 as well as several low-molecular Dhn transcripts of YnSKm type  in barley  accessions sampled at two sites in Israel and Jordan, there has been found an enhanced expression of several Dhn transcripts  revealing quantitative differences with respect to differential drought tolerance  in barley flag leaf under terminal drought. Relative expression levels of Dhn3 and Dhn9 revealed positive correlations with chlorophyll a and b contents, osmotic adjustment, plant biomass and grain yield, and negative correlations with malondialdehyde and electrolyte leakage levels. In two barley cultivars with a differential tolerance to drought, a Czech spring variety Amulet and Syrian landrace-derived cultivar Tadmor, both quantitative and qualitative differences in low-molecular dehydrin proteins have been found when the plants were subjected to a decreased field water capacity  is most tolerant being able to grow up to 250 mM NaCl, common wheat (T. aestivum) is less tolerant and durum wheat (T. durum) is the least salt-tolerant crop  in transgenic thaliana . A pleiobidopsis . Expressild-type . HoweverWCS120 transcript and protein during cold acclimation of common wheat have revealed a much faster induction of WCS120 transcript upon cold reaching a peak at ca 2 days after an onset of cold treatment while WCS120 protein accumulation reached a peak at ca 21 days after an onset of cold  and plant maximum acquired frost tolerance in plants subjected to cold (Dehydrins have been studied in many plants during the past two decades . Cereals of cold . Similar of cold . Therefo to cold  and even to cold . In fielThere have already been published several studies on dehydrins in wheat and barley plants. Dehydrins have been well characterized at protein sequence, promoter structure and short-term transcript induction. However, a long-term dynamics of transcripts and proteins with respect to an acquisition of an enhanced stress tolerance as well as dehydrin protein post-translational modifications, cellular localization and their physico-biochemical functions in stress-treated plants remain largely unresolved and represent currently the main challenge to understand dehydrin role in plant stress response.Klára Kosová has outlined the idea and prepared the text of a brief summary on current state of dehydrin research in stress-treated wheat and barley plants. Pavel Vítámvás and Ilja T. Prášil contributed to preparation, drafting, critical reading, and publication of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purkinje cell pathology is a common finding in a range of inherited and acquired cerebellar disorders, with the degree of Purkinje cell injury dependent on the underlying aetiology. Purkinje cells have an unparalleled resistance to insult and display unique regenerative capabilities within the central nervous system. Their response to cell injury is not typical of most neurons and likely represents both degenerative, compensatory and regenerative mechanisms. Here we present a pathological study showing novel and fundamental insights into Purkinje cell injury, remodelling and repair in Friedreich’s ataxia; the most common inherited ataxia. Analysing post-mortem cerebellum tissue from patients who had Friedreich's ataxia, we provide evidence of significant injury to the Purkinje cell axonal compartment with relative preservation of both the perikaryon and its extensive dendritic arborisation. Axonal remodelling of Purkinje cells was clearly elevated in the disease. For the first time in a genetic condition, we have also shown a disease-related increase in the frequency of Purkinje cell fusion and heterokaryon formation in Friedreich's ataxia cases; with evidence that underlying levels of cerebellar inflammation influence heterokaryon formation. Our results together further demonstrate the Purkinje cell’s unique plasticity and regenerative potential. Elucidating the biological mechanisms behind these phenomena could have significant clinical implications for manipulating neuronal repair in response to neurological injury. Purkinje cells represent the sole output neuron of the cerebellar cortex and thus changes in their function have significant impact on the function of the cerebellum as a whole. Purkinje cell pathology is a common finding in a range of inherited and acquired cerebellar disorders, with the degree of Purkinje cell injury dependent on the underlying aetiology. The inherited ataxias are a group of genetically heterogeneous conditions that share common clinical features that give rise to progressive ataxia, with cerebellar injury and varying degrees of degeneration of other grey matter regions. Despite advances in detection of precise genetic causes, no therapy has been shown to prevent or slow the progression of disability in these disorders.FXN gene [Friedreich ataxia (FRDA) is the commonest autosomal recessive ataxic condition, with 95 % of cases caused by a homozygous GAA.TCC tri-nucleotide repeat expansion mutation within intron 1 of the FXN gene  leading FXN gene , 46. PatFXN gene . NeuropaFXN gene . HypoxicFXN gene . NeverthFXN gene , 39.Purkinje cells have a fairly unique and unparalleled resistance to axonal injury within the CNS . Their rUnderstanding whether Purkinje cell axon remodelling and/or fusion represent mechanisms by which cerebellar functions can be maintained in genetic cerebellar disease has important therapeutic consequences. With the potential to protect and rescue neuronal cells and restore homeostatic balance during neurodegeneration, understanding the circumstances in which they occur may lead to techniques to manipulate these mechanisms therapeutically. With this in mind, using post-mortem cerebellum tissue, our aims were to quantify the extent of Purkinje cell injury and structural plasticity in FRDA, a condition typically associated with Purkinje cell preservation, in order to explore whether plasticity and fusion might contribute to Purkinje cell survival.BRAIN UK at the University of Southampton, Southampton, UK and The UK Multiple Sclerosis Tissue Bank at the Imperial College, London, UK. The majority of cases pre-dated genetic testing for FRDA and information regarding the GAA.TCC tri-nucleotide repeat expansion lengths for each FRDA case were not available. As a result, patients had been clinically diagnosed as having FRDA and diagnosis had been confirmed during neuropathological autopsy examination. Neuropathological reports at post-mortem included: axonal loss and prominent gliosis in the dorsal columns (more marked in the gracile than the cuneate fasciculus), long tracts and spinocerebellar tracts of the spinal cord; neuronal loss in the dorsal nucleus of Clarke; gliosis within the white matter of the cerebellum; neuronal shrinkage and loss in the dentate nucleus with consequent atrophy of the superior cerebellar peduncles; and marked depletion and gliosis of the gracile and cuneate nuclei, vestibular nuclei and solitary tract of the medulla. In the periphery, neuronal loss in the dorsal root ganglia was documented and peripheral nerves also showed demyelination and axonal degeneration. For several FRDA cases, stained spinal cord and DRG post mortem sections were available. Thus where possible, representative images of highly specific FRDA lesions in the spinal cord, DRG were taken , Bielschowsky silver staining or Luxol fast blue with Cresyl violet.28K (1:500)  and HLA-DP DQ DR (1:800) . Sections were deparaffinised in Clearene, dehydrated in 100 % ethanol, hydrated in distilled water, and immersed in 3 % hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity, rinsed and microwaved in sodium citrate buffer  or EDTA buffer  as appropriate and rinsed in phosphate-buffered saline (PBS). Non-specific binding was blocked with Vectastain blocking serum (20 min). After addition of the primary antibody, sections were incubated overnight at 4 °C. The sections were then rinsed in PBS before incubation for 20 min with secondary antibody  and 20 min with VectaElite ABC Complex  followed by a 10-min incubation with 3,3′-diaminobenzidine (DAB) and 0.01 % H2O2. Sections were washed in water, immersed in copper sulphate DAB enhancer (4 min), counterstained with haematoxylin, dehydrated, cleared and mounted. Images were acquired using an Olympus IX70 microscope coupled with Image-Pro Plus software.Cerebellar sections were immuno-stained with antibodies to rabbit anti-Calbindin-D28K (1:500) , mouse anti-SMI-34   and rat anti-myelin basic protein (MBP) (1:100) . Non-specific binding was blocked with 10 % normal goat serum diluted in PBS containing 0.1 % triton. Sections were incubated at 4 °C overnight with primary antibodies. Sections were then washed in PBS and incubated for 30 min in the dark with Alexa Fluor 555, goat anti-mouse (1:500), Alexa Fluor 488, goat anti-rabbit (1:500) or Alexa Fluor 555, goat anti-rat (1:500) , before being washed in PBS and mounted in Vectashield medium containing the nuclear dye 4′6′-diamidino-2-phenylindole (DAPI) . Sections were imaged using either: 1) a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope with Leica Application Suite Advanced Fluorescence software and Volocity 3D image software ; or 2) a Nikon C1 confocal microscope and EZ viewer software.Sections were deparaffinised, hydrated and washed as above. An antigen retrieval step was performed through microwaving in sodium citrate buffer . Purkinje cells were labelled by single or double immunofluorescence using rabbit anti-Calbindin-DThe total number of Purkinje cells was counted in FRDA and control sections, scanning the entire length of the Purkinje cell layer. Purkinje cells were identified based on Calbindin positivity and their specific location. To account for the difference in section sizes and plane of cutting, the length of the Purkinje cell layer was traced and measured in each section using Image J software to obtain the number of Purkinje cells per standardized unit length (mm). For these sections, the length of Purkinje cell layer was measured and the number of Purkinje cells in each area counted.The total number of axonal spheroids and Purkinje cell perikarya positive for SMI-34 were counted in FRDA and control sections, scanning the entire Purkinje cell and granular layers.Using Image J software, the outline of each Purkinje cell perikaryon (where the nucleus was clearly visible) was traced and the area within subsequently calculated. The total number of Purkinje cells with an abnormal perikaryal morphology were also counted in FRDA and control sections, scanning the entire length of the Purkinje cell layer. To investigate changes in dendritic arborisation, concentric circles (with increasing diameter of 20 μm) were aligned on the centre of each Purkinje cell perikaryon and the number of times each circle transected a dendrite was counted. Subsequently, the number of transections per mm at each distance was calculated. As the dendritic tree of Purkinje cells is not spherical, only section orientations that resulted in a full display of Purkinje cell arbor to be visualised were used for quantification.28K positive thickened axons , thickened recurrent collaterals  and thickened arciform fibres (hypertrophic axons with a minimum 90° turn back towards the Purkinje cell layer). Both Calbindin-D28K positive axonal branching  and terminal axonal sprouting (a network of fine nodular/branched processes projecting from the intact axon) were also counted [Specific changes in Purkinje cell axonal morphology were counted in both FRDA and control sections, scanning the entire length of the Purkinje cell and granular layers. Changes included the number of Calbindin-D counted , 43. For28K were viewed using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope. Each section was scanned along the entire length of the Purkinje cell layer, situated between the granular cell layer and molecular layer, for Purkinje cell bodies containing two separate nuclei. At least 2000 Purkinje cells from each patient sample were examined, allowing for the determination of the frequency of bi-nucleate Purkinje cells. All bi-nucleate Purkinje cells were confirmed on the confocal microscope by obtaining serial sections throughout the whole Purkinje cell body. All Z-stack and 3-dimensional imaging was created using both Leica Application Suite Advanced Fluorescence software and Volocity 3D image software .For identification of bi-nucleate Purkinje cell heterokaryons, cerebellar sections immuno-labelled for Calbindin-D2 was subsequently calculated.All HLA-DP DQ DR positive cells were counted, using at least 18 randomly assigned fields per case, within the white matter and grey matter regions of the cerebellum. The number of HLA-DP DQ DR positive cells per mmP <0.05 to represent statistical significance. Data between two groups were analysed using either unpaired t-tests or Mann-Whitney U tests. Statistical comparisons for over two groups were analysed using either one-way or two-way analysis of variance (ANOVA) with post hoc testing between groups where appropriate. Pearson’s correlation was used to analyse relationships between two sets of data.The analysis was performed using GraphPad Prism . For all tests, values are expressed as the mean ± SE from at least five independent cases, taking 28K. Purkinje cells were readily identified based on their unique morphology and location; their perikaryon situated within the Purkinje cell layer positioned between the granular cell and molecular layers  was used to further characterise Purkinje cell axonal morphology and injury [28K and SMI-34   Fig. .Fig. 2Abp <0.05) . Dendritic arborisation of Purkinje cells is a critical determinant of neuronal connectivity and health [p <0.05)  Fig. . This su05) Fig. ; a propo05) Fig. . Both th05) Fig.  and the 05) Fig.  were sigd health , 34. To 05) Fig. .Fig. 3Anp <0.05)  Table .Fig. 4Pu28k co-labelling, we also observed significant changes in Purkinje cell axonal myelination within the granular layer, with evidence of myelin fragmentation and loss localised to areas of axonal hypertrophy and axonal torpedoes . Using epifluorescence microscopy, we examined ≥2000 Purkinje cells from each case . The location of each suspected bi-nucleate Purkinje cell was noted and subsequently scanned using confocal microscopy acquiring 0.20 μm serial sections throughout the entire Purkinje cell soma   Fig. .Fig. 5Anp <0.05). All other bi-nucleate Purkinje cells contained one large Purkinje cell-like nucleus and a second smaller nucleus with compact chromatin .As previously reported , two distin Fig.  and d. UBoth experimentally in animal models  and humap <0.05)  Fig. . No corrHere we present a pathological study showing novel and fundamental insights into Purkinje cell injury, remodelling and spontaneous repair in FRDA. We provide evidence of significant injury to the Purkinje cell axonal compartment with relative preservation of the perikaryon and its extensive dendritic arborisation. Axonal remodelling of Purkinje cells was clearly apparent. We have also shown a considerable increase in the frequency of bi-nucleate Purkinje cell heterokaryon formation in FRDA cases. This is the first time in humans that Purkinje cell fusion and heterokaryon formation in the brain has been reported in a genetic condition. We also provide evidence that heterokaryon formation is influenced by underlying levels of cerebellar inflammation associated with the disease. Our results together demonstrate the Purkinje cell’s unique plasticity and regenerative potential in response to damage.28k demonstrating that abnormal axon terminals were of Purkinje cell origin. The resulting synaptic detachment of Purkinje cell axon terminals from degenerating neurons within the dentate nucleus would likely result in a retrograde change and a lack of synaptic contact with the efferent target may lead to trans-neuronal degeneration of the Purkinje cell. However, contrary to pathological nomenclature, recent reports suggest grumose degeneration may actually be a beneficial phenomenon in which Purkinje cells escape retrograde atrophy by producing new axonal sprouts that establish contact with small surviving neurons where they form reparative grumose clusters [Atrophy of the dentate nucleus, which receives inhibitory GABA-ergic inputs from Purkinje cells originating within the cerebellar hemispheres, is the most significant change seen within the central nervous system of patients with FRDA , 31, 49.clusters .Indeed, there were marked changes to the Purkinje cell axonal compartment, with abnormal axonal phosphorylation and abundant axonal thickening/formation of axonal torpedoes within the granular cell layer. It is thought in disease states that abnormal accumulation of the axonal structural components leads to morphological changes within the axon, and depending on the underlying disease mechanism, the accumulated material can differ , 57. TheThe typical response of most neurons to axonal injury is a cell body reaction involving a complex sequence of molecular and genetic changes that initially sustain compensatory and regenerative responses, but eventually become regressive leading to atrophy or cell death . HoweverRemodelling of the Purkinje cell axon can be induced by extrinsic inhibitory cues such as myelin-associated molecules. For example, blocking of myelin-associated neurite growth inhibitory protein (Nogo-A) binding to it receptor complex situated on the axonal membrane, can induce prolific spouting in Purkinje cells from both the corticofugal axon and the intra-cortical plexus , 14. Nog-nucleate cells. A key observation in this study was evidence of Purkinje cell fusion in diseased FRDA brains through detecting bi-nucleate Purkinje cell heterokaryons; the frequency of these cells in FRDA being significantly higher than that previously reported in control cases [Spino-olivo-ponto-cerebello-nigral atrophy [Of major relevance to ataxia is the potentially neuro-regenerative process of BMDCs migrating into the CNS and fusing with, in particular, Purkinje cells within the cerebellum. A process in which the two distinct cells fuse together to become a single cell, resulting in transfer and amalgamation of nuclear material, to form bi-nucleate or monool cases . The pheol cases , 10, 11 ol cases , Alzheimol cases  and Spin atrophy .Purkinje cells are generated only during early brain development and in contrast to other neurons there is no evidence of their generation after birth . Under nin vitro [Little is known concerning the regulatory mechanisms behind controlling Purkinje cell fusion in response to injury; however, inflammatory cues are known to play a physiologically significant role. Fusion between Purkinje cells and BMDCs occurs ordinarily at low levels, but the incidence of fusion is substantially increased by chronic inflammation , 21. Thein vitro , the cerin vitro , 31. Intin vitro . It is hin vitro . Hence, Whether the observed changes to Purkinje cells in our FRDA cohort are solely a response to depleted cellular frataxin levels and/or loss of the dentate nucleus; or alternatively, at least in part, a result of secondary damage caused by hypoxic-ischemic injury (due to either cardiomyopathy or pulmonary complications as reported in our FRDA cases) cannot be fully determined. As yet, the precise functional significance of Purkinje cell plasticity in response to stress/injury is also unknown. Nevertheless, it is likely to be closely linked to the central role Purkinje cells play in maintaining cerebellar function. It is tempting to speculate the reasons for such phenomena; survival of Purkinje cells in FRDA may be functionally irrelevant as neuronal loss within the dentate nucleus blocks corticonuclear transmission. However, these fairly unique changes in response to axonal injury may allow the Purkinje cell to convey its output information through nearby intact neurons, or be a neuroprotective process to prevent a further cascade of disabling neurodegenerative changes, as would likely result from a loss of Purkinje cells, within the cerebellar milieu. More research into these mechanisms are warranted and elucidating the biological pathways behind both neuronal plasticity and cell fusion in disease could have significant clinical implications in manipulating neuronal repair in response to injury.


Solution-phase catalysis using molecular transition metal complexes is an extremely powerful tool for chemical synthesis and a key technology for sustainable manufacturing. However, as the reaction complexity and thermal sensitivity of the catalytic system increase, engineering challenges associated with product separation and catalyst recovery can override the value of the product. This persistent downstream issue often renders industrial exploitation of homogeneous catalysis uneconomical despite impressive batch performance of the catalyst. In this regard, continuous-flow systems that allow steady-state homogeneous turnover in a stationary liquid phase while at the same time effecting integrated product separation at mild process temperatures represent a particularly attractive scenario. While continuous-flow processing is a standard procedure for large volume manufacturing, capitalizing on its potential in the realm of the molecular complexity of organic synthesis is still an emerging area that requires innovative solutions. Here we highlight some recent developments which have succeeded in realizing such systems by the combination of near- and supercritical fluids with homogeneous catalysts in supported liquid phases. The cases discussed exemplify how all three levels of continuous-flow homogeneous catalysis  must be matched to locate viable process conditions. If then a compatible process scheme can be designed around this, an integrated continuous-flow process may be realized  12,13].,13.12,132) as transport medium for continuous-flow applications is particularly attractive to establish practically ‘solvent-free’ processes for asymmetric synthesis.Multiphasic systems consisting of a molecular catalyst in a product-separable fluid phase or on the surface of a solid support proved to be viable strategies in this regard . Phase bOn the molecular level, SLPs represent a particularly gentle immobilization technique because it immobilizes the solvent and not the catalyst. Therefore, modification of the catalyst itself is not required in most cases  and similar mechanisms and kinetics as in bulk solution prevail in the SLP. It is also beneficial to the macroscale process scheme because the bulk properties of the catalyst material are dominated by the solid support, and thus the solvent is kept entirely on the mesoscale where it permits homogeneous catalytic turnover . Challen2.et al. [3)3], which makes SLPs the oldest by-design approach to continuous-flow multiphasic homogeneous catalysis in the open literature. Ethyleneglycol solutions of RhCl3-hydrate were supported on porous silicates by wet impregnation with MeOH, and drying yielded free-flowing powders containing the ethyleneglycol catalyst solution in the pores of the support material [3)2CO] immobilized in butylbenzylphthalate on silica gel  in SLP in great detail. The supported catalyst materials were analysed by multiple techniques including differential scanning calorimetry (DSC), microscopy, porosimetry, gas adsorption and IR spectroscopy. Intrinsic reaction rates, activation energies and diffusion effects were experimentally measured and fitted by theory  and CF3SO3H in imidazolium ILs on fumed amorphous silica have been analysed for component interactions by multiple techniques 2 in thin films of BMIM NTf2 on alumina introduced by physical vapour deposition showed that multi-component systems can be surface-analysed under carefully chosen conditions + in different imidazolium ILs was deposited on porous polyvinylfluoride by wet impregnation. The materials were used for the continuous hydrogenation of propylene and ethylene, with olefins and hydrogen fed from different sides of the membrane, respectively. Various IL anions were screened, and activity trends could be related to gas solubilities. Dimerization of ethylene with a [NiCl2(Pcy3)]2/EtAlCl2 catalyst in chloroaluminate ILs on polyethersulfone membranes was also reported in the same year  was used as catalyst with 25 wt% IL-loading and applied in neat liquid substrate. At 100°C, the SILP system achieved nearly three times the rate of the unsupported bulk biphasic system (TOF=3900 h−1 versus 1380 h−1), which was attributed to enhanced catalyst accessibility in SILP. However, at conversions more than 50% leaching of IL into the liquid product phase entrained up to 2% of the catalyst per run. The hydrogenation SILP catalyst containing [Rh(nbd)(PPh3)2] PF6 in BMIM PF6 on silica did not suffer from such strong depletion effects, due to the cationic nature of the active catalyst and because the liquid substrate/product phase remains unpolar throughout the reaction. No metal could be detected in the product phase (less than 0.03 ppm), and the SILP catalyst could be recycled for at least 18 times without apparent deactivation in batch mode. Electron microscopy confirmed the absence of rhodium clusters more than 6 Å, and variation of hydrogen pressure revealed first-order kinetics in accordance with molecular catalysis in the SILP.Mehnert reported on solution-phase applications of SILP catalysts based on Rh-catalysed olefin hydroformylation  and hydr6 containing [Rh(nbd)(PPh3)2] PF6 at various loadings (method (II)). The materials were analysed by different techniques and shown to afford much increased reaction rates (TOF 2880 h−1) in the hydrogenation of 1-hexene as compared to the same catalyst in SILP on oxidic support materials such as silica, titania, zirconia or alumina, and also activated carbon. The high thermal conductivity as well as the open channel structures of the CNT was used as explanation for this effect. The materials were recycled five times without loss of activity and undetectable rhodium leaching into the organic phase.Serp reported on functionalized CNT as tailored support materials for hydrogenation catalysts in SILP . Post-sy6 on silica was reported by Hagiwara et al. [6 on a polyimide nano-membrane [Ring-closing metathesis and cross-metathesis catalysed by Grubbs’ catalyst in HMIM PFa et al. . Six reca et al. . Ring-clmembrane . StepwisCatalytic hydroamination of phenylacetylene with rhodium, palladium, copper and zinc complexes in SILP physisorbed on silylated diatomaceous earth (amorphous silica) using heptane as solvent was reported by Müller and co-workers . Selectiet al. reported on palladium complexes immobilized in BMIM PF6 physisorbed on silica [−1 and batch-wise recycling was demonstrated for five runs. Decreasing activity could be restored by washing the SILP catalyst with aqueous NaOH. However, the formation of small amounts of homogeneous palladium species partitioning into the reaction medium under reaction conditions cannot be fully excluded.Hagiwara n silica . At 150°6 were used on poly (method (II)) for the asymmetric C=O hydrogenation of methyl-acetoacetate. At 60°C, activities of the SILP catalysts were up to three times higher than the bulk IL-organic biphasic media with identical levels of enantioselectivity (97% ee). The catalysts were re-used once in batch mode, though catalyst retention or leaching was not quantified.Vankelecom and co-workers described the first chiral organometallic complexes in a SILP catalyst on a polar polymer as support . Enantioee were achieved with Rh-BINAP and K2CO3 in a tetra-alkylphosphonium carboxylate IL physisorbed on silica when used with hexane as solvent. Although no comparison of the same system in neat IL without support was reported, it was speculated that intensified substrate-catalyst interactions induced by the thin films of the SILP could be responsible for this effect. The hexane phase did not exhibit residual activity but recycling of the SILP catalysts was not performed.Müller and co-workers  reported2 extraction yielded superior catalyst performance than freeze-drying from aqueous solutions. Performing catalysis in neat substrate, leaching issues could not be fully resolved (up to 9% per run), and stability was rather poor during batch-wise recycling experiments (complete deactivation from fifth cycle). Asymmetric versions were also demonstrated, but without recycling experiments.Allylic substitution with Pd-catalysts in SILP on chitosan as support has been reported . Drying 3 was performed yielding good enantioselectivities in the range of 65–96% ee, reaching 314 TON. Catalyst leaching up to 1% was observed even with hexane as mobile phase.Dioos & Jacobs reported on continuous liquid-phase application of chiral Cr-salen complexes in SILP physisorbed on silica . Asymmet2, the Lewis-acid promoted condensation of methylpyruvate and phenyl-TMS-ethene was at least 60 times faster than in CH2Cl2 with complete retention of enantioselectivity (82% ee). However, hydrolysis of the TMS-ether resulted in about 10% lower chemoselectivities. Adsorbing the IL on either imidazolium-functionalized or plain silica, this side reaction was effectively suppressed, and the SILP catalyst combined high activity with good enantioselectivity when applied in a biphasic system with Et2O. While neutral copper catalysts leached out to 19%, an imidazolium-modified ligand afforded enhanced retention significantly. However, deactivation was still noticeable after the fifth recycling.Hardacre co-workers  reportedThe above examples show that a wide variety of homogeneous catalytic systems, including highly enantioselective ones, are compatible with SILP-type environments and that there is scope for activity improvement due to short diffusion pathways and large interfacial areas. A clear limitation of their application with liquid phases, however, is the effectiveness of catalyst retention; non-volatile, highly functionalized substrates and products require the use of good solvents which then often dissolve some of the IL film and leach out the immobilized catalyst. While volatility discrimination has been shown to be an effective separation barrier for continuous gas-phase application of thermally robust SILP catalysts (see §4c), polarity discrimination alone is clearly less effective. Unfortunately, the thermal process window of gas-phase processes excludes stereoselective transformations of complex molecules, one of the key skills of molecular transition metal catalysis. A gas-like solvent with liquid-like solvation power at mild temperatures would extend the potential of SILP catalysts to continuous-flow catalysis for fine chemical and pharmaceutical production. As shown in the following, SCFs show great promise in fulfilling precisely this role.(e)2, CO or O2. They are, however, unable to dissolve ILs [2 in particular is attractive due to its mild critical conditions (Tc=31°C), and has been shown to exert some interesting effects on ILs, including reductions of melting point, interfacial tension, bulk viscosity and increased gas solubility [2 as mobile phase [2 as flow medium. Despite this promising perspective, the combination of SLP systems with scCO2 has been achieved only recently.The SCFs combine the transport properties of gases with the solvent properties of liquids for a wide variety of organic molecules . In compolve ILs  or otherolve ILs , and areolve ILs . Thus, tlubility . A numbelubility . For exale phase . In contle phase , substrain silica (method (III)) as highly active catalysts for the selective aerobic oxidation of alcohols. Application with scCO2 allowed efficient oxidation of non-volatile alcohols at 75°C. Batch-wise recycling was attempted, but practical difficulties of material loss prevented more than three repetitive runs.Pagliaro and co-workers  describe2 [2 on dehydroxylated silica (method (I)) proceeded with even higher rates than in bulk-IL/scCO2 media, and the system was very stable for at least 40 h  higher syngas pressures decreased the rate (in accordance with the intrinsic reaction kinetics), while at high loadings (44 wt%) higher syngas pressure increased the hydroformylation rate. In conjunction with phase behaviour observations it was concluded that the reaction proceeded best in an expanded liquid substrate phase rather than in a single supercritical phase, and the IL film thickness became gas limiting at high loadings .2 (2 on dehydroxylated silica (method (I)) proceeded with good rates for at least 9 h (TON>10 000), although slight loss of activity over time was observed. Ruthenium leaching levels were in the range of 10 ppm in the self-metathesis product. In the best case, 6 g of substrate per hour could be converted using a 9 ml reactor.Cole-Hamilton and co-workers  extended2 . Self-me2 at 40°C using a flexible, fully automated set-up [ee were achieved, and 65 h of continuous operation were demonstrated, although at slightly reduced selectivity (70% ee) after 10 h on stream [−1 h−1. Rhodium leaching levels were below the detection limit of 1 ppm as judged by inductively coupled-optical emission spectroscopy (ICP-OES) of both the product fractions and the spent SILP catalyst. Fine-tuning of the support surface to avoid accumulation of trace water in the SILP turned out to be a crucial parameter for preventing progressive deactivation of the active species. Using either a water scavenger or a perfluoroalkyl-functionalized silica support increased catalyst stability to more than 80 h on stream to reach greater than 140 000 TON.The group of Leitner  developed set-up . At quann stream . Remarka2 immobilization strategy with the bulk IL-scCO2 approach in the asymmetric hydrogenation of the enol ester 1-(trifluoromethyl)vinyl acetate using the same molecular catalyst and IL , TON of 70, 400, and robust enantioselectivity (80–84% ee), which even slowly increased over time. Although Rh/Xyl-QUINAPHOS was the most active catalyst under batch conditions it also provided the best stability and highest productivity in continuous-flow application, highlighting the importance of long-term stability experiments for the identification of the most suitable catalytic system.Leitner and co-workers  comparedt and IL . Whereas5.From the examples discussed in this contribution it becomes evident that SLPs represent a versatile and promising approach to using molecular catalysts in continuous flow. The concept of SLPs appears particularly attractive for ILs, because despite being good media for organometallic catalysis with extremely low volatility they suffer from high viscosity and cost. In SILP, the effective use of both solvent and catalyst in SILP catalysts is enhanced as compared to bulk-IL systems, because the catalyst is very close to a large interface in the ultra-thin film on the surface of the solid material. However, experience also shows that the combination of SLP materials with liquid flow media is bedevilled by progressive loss of the surface film through dissolution or physical abrasion under continuous operation.in situ extraction of products from the catalyst surface may also be used to continuously re-adjust the equilibria of thermodynamically unfavourable reactions, thus overcoming equilibrium limitations [The combination of SILP catalysts with compressible gases in the form of SCFs or expanded liquid phases offers particularly promising possibilities of broadening the window of applicable reactions and increasing overall process efficiency. Recovery of products from the mobile phase, free of any organic solvent, may be conveniently achieved by depressurisation or temperature swings . Excitinitations . Online itations –192. Anoitations –195. Theitations .2 and IL/scCO2 systems [It is important to note that this is not restricted to organometallic catalysis, but enzymatic catalysis has also been efficiently carried out continuously in SILP/scCO systems . For exa systems . In view