Sleep loss and insufficient sleep are risk factors for cardiometabolic diseases, but data on how insufficient sleep contributes to these diseases are scarce. These questions were addressed using two approaches: an experimental, partial sleep restriction study (14 cases and 7 control subjects) with objective verification of sleep amount, and two independent epidemiological cohorts  with questions of sleep insufficiency. In both approaches, blood transcriptome and serum metabolome were analysed. Sleep loss decreased the expression of genes encoding cholesterol transporters and increased expression in pathways involved in inflammatory responses in both paradigms. Metabolomic analyses revealed lower circulating large HDL in the population cohorts among subjects reporting insufficient sleep, while circulating LDL decreased in the experimental sleep restriction study. These findings suggest that prolonged sleep deprivation modifies inflammatory and cholesterol pathways at the level of gene expression and serum lipoproteins, inducing changes toward potentially higher risk for cardiometabolic diseases. Short sleep duration, complaints of poor sleep quality and diagnosed sleep problems have in epidemiological studies been associated with metabolic disorders, which relate to low-grade chronic inflammation, including cardiovascular diseases, metabolic syndrome, obesity, and type 2 diabetes mellitus123Several previous studies have shown that experimental restriction of sleep to 4–5 h per night for 1–2 weeks activates immune responses569101213The duration of sleep restriction in previous studies has ranged from one to five days, which in the development of chronic diseases is a short period. In trying to understand the role of restricted sleep as a predisposing factor for such diseases, the key questions are: how do the short-term modifications in metabolism and inflammation develop when the duration of the sleep restriction is prolonged, and are these modifications such that could explain the association between restricted sleep and increased risk for e.g. atherosclerosis, as evidenced by the epidemiological studies?The assessment of circulating lipid profiles using nuclear magnetic resonance (NMR) spectroscopy goes beyond the typically measured total lipids, like total cholesterol and triglycerides, and allows detailed characterization of many lipoprotein features at the subclass level, including the size of the particles, which are important in lipid physiology and pathophysiology16181920The short-term effects of insufficient sleep were assessed in a highly controlled experiment where a group of volunteers restricted their sleep to 4 hours per night during 5 days . The results focusing on the immunological effects at the level of gene expression, cytokines, and CRP have been previously publishedPartial sleep loss was induced experimentally as 4 h sleep/night for 5 nights for 14 healthy young males in the SR study  23.2 ± 2.2 y, In the Cardiovascular Risk in Young Finns Study  37.7 ± 5.0 y, P < 0.05). Enrichment of biological processes among these genes was analysed using DAVID Functional Annotation ClusteringP values of the pathways <0.05) in subjects with SSI  in the SR study with SSI .P = 0.045, P < 0.001) reported previously (P < 0.05) also after including BMI as a covariate in the explanatory model.The GO cluster 5 , Cholesterol homeostasis and Sterol homeostasis (both with a borderline significance of P = 0.052)  . The genP < 0.05) in this cohort, supporting the suppressive effect of subjective sleep loss on this cholesterol transporter.Gene expression was measured in the YFS replication sample (N = 1407) from whole blood using Illumina microarrays to evaluate whether the expression of the genes found in the down-regulated pathways in DILGOM  was lower in subjects with SSI also in this sample. The ABCG1 finding replicated  as well as small VLDL particles (P < 0.05) was decreased after SR compared to BL, while there were no changes in the number of small and medium size HDL particles . These changes were also reflected by changes in LDL/VLDL structural protein apoB-100. ApoB-100 levels decreased (P < 0.005) whereas apoA-I  levels did not change.In the SR study , the number of small, medium, and large LDL particles (articles . Large HP < 0.05). SSI had an independent association to large HDL concentration also after adding BMI as a covariate in the model (P < 0.05). There was no significant difference in the levels of any of the LDL or VLDL subclasses, although there was a consistent trend for increase in both LDL and VLDL particles of all sizes (In the DILGOM sample (N = 414), the number of serum large HDL particles was lower among individuals with SSI (pointwise ll sizes .P < 0.005) in the YFS sample (N = 2077) (P < 0.005). No differences were observed in small or medium HDLs. For the epidemiological samples, age and gender were adjusted for in the linear regression model. The decrease in large HDL was independently associated with SSI (P < 0.01) also when probable self-reported obstructive sleep apnoea (OSA) was included in the model.The lower number of large HDL particles in subjects with SSI replicated ( = 2077) . In thisP < 0.05; In the SR study , molecular lipids were further analysed with MS-based lipidomics. Altogether 20 lipids were increased after SR in the sleep-deprived cases as compared to the controls . The incThe findings from the SR study suggested that cholesterol transport had declined and inflammation increased. An important transcriptional regulator of the RCT is the nuclear liver X receptor (LXR) signalling, which promotes RCT and decreases inflammation3031P < 0.05), and genes encoding for both IL-1b and TNF-a in the Young Finns replication sample  (Inflammation suppresses LXR activity via toll-like receptors (TLR)33347ctively)  and 3. Ictively) , MyD88 (ctively) . IncreasIn order to distinguish direct and indirect interactions of these immune, metabolic and sleep variables, we utilised undirected Gaussian graphical model where the variables are connected if and only if their partial correlation is significantly non-zero (using FDR multiple testing for the selection of edges) to visualise the dependencies as a networkP < 0.05 for all associations shown in the figure). The correlation analysis revealed significant positive associations with PE(38:3), PE(38:5e), PE(36:2e), ChoE(16:1) and B-cells. PE(38:3) was further negatively correlated with TNF-a, which was positively correlated with interleukins 18 and 1 and interferon-γ (IFNG). LXRA associated positively with B cells and TLR8, and negatively with ABCA1, NRIP1 and LXRB. TLR4 had a strong negative association with NPC1 and ABCA1  or phospholipid transfer protein (PLTP) in the experimental SR study. Furthermore, no changes were observed in the activity of lecithin-cholesterol acyltransferase (LCAT), the enzyme converting HDL unesterified cholesterol to cholesteryl ester, or the atheroprotective enzyme paraoxonase 1 (PON1) linked to antioxidative properties of HDL particles.The main finding of the present study was that restriction of sleep either experimentally or in natural living conditions modified lipoprotein metabolism and immune responses. The changes were detected at the level of transcriptome as well as in the circulating lipid profile. The transcriptional changes - the down-regulation of reverse cholesterol transport-related gene pathways - were similar in the relatively short term exposure of the experimental sleep restriction and in the epidemiological cohorts among subjects reporting insufficient sleep. Interestingly, the lipoprotein profile showed decreased LDL in experimental SR, resembling LDL changes in acute-phase responseThe classical view on the relationship between serum lipid levels and risk for cardiovascular events, formulated based on the findings of the Framingham studyCombining the knowledge of the epidemiologically verified increased risk for CVD associated with the low HDL/high LDL lipid profile and short/insufficient sleep, we expected to measure such lipid profiles in SR and in persons with SSI. This prediction did not prove quite correct. In SR we measured, using the NMR metabolomics, decreased levels of LDL while we found no significant changes in HDL levels. In the SSI subjects we found decreased HDL levels but not significant differences in LDL levels. The one week experimental SR may have been too short to affect the number of HDL particles, while in the epidemiological samples, the number of large HDL particles was lower among those who reported insufficient sleep. Since the nature of conditions that induce insufficient sleep is often persistent484950While some previous gene expression studies have indicated that cholesterol/lipid metabolism is regulated by sleep-wake cycle and sleep restriction could modify it852The results from the gene expression pathways were more consistent than those of the lipid profiles. The cholesterol pathways that were down-regulated in SR and SSI included members of the ATP-binding cassette families A and G (ABCA1 and ABCG1). ABCA1 and ABCG1 facilitate the efflux of free cholesterol and phospholipids from macrophage-foam cells to HDL particles. Of all cholesterol carried by HDL, the proportion originating from peripheral macrophages is low compared to liver (75%) and intestine (20%) produced HDL. However, this route of removal of cholesterol (macrophage RCT) has been regarded as an important (but not only) component in retaining cholesterol balance in arterial endothelium39Caveolin-1 (CAV1) is found in caveolae, cholesterol-rich membrane lipid formations that are involved in cholesterol traffic and homeostasis6062in vitro immunological challenge is increased after sleep restriction, as well as serum acute phase protein CRPPrevious studies indicate that the immune system is activated during experimental sleep restriction, and in persons who in epidemiological studies report short sleep duration , with acute phase protein production in the liver as an integral part of the host defence response. In addition to changes in protein synthesis, also lipid metabolism undergoes significant modifications in APR663966activity .To evaluate sleep in the epidemiological samples, we used subjective evaluation of sleep insufficiency, instead of sleep duration. While sleep duration as a measure of sleep has many advantages, in evaluation of sleep insufficiency it has one considerable disadvantage: it cannot differentiate sleep insufficiency-inducing short sleep from natural short sleepP = 2.5E-10 and β = −0.5 h in DILGOM, and P = 4.9E-93 and β = −0.5 h in YFS, 69In epidemiological studies sleep duration and self-reported sleep insufficiency have shown independent effects on cardiovascular outcomes While the participants of the experimental study were carefully screened not to have any (sleep) disorders, the participants in the epidemiological studies reported different medical conditions, including sleep apnoea, which is known to be associated to both insufficient sleep and cardiovascular diseases. The evaluation of the potential effects of OSA on our results revealed that although the subjects reporting symptoms of OSA reported also more sleep insufficiency, OSA did not explain the difference in HDL, as SSI was independently associated to lower level of large HDL.The main regulation of metabolism takes place in other tissues  than blood cells, which were used in the gene expression analyses. Thus it is unclear to which extent the results reflect changes in the actual metabolic processes. An additional source of variation lies in the differences in age and gender between the populations studied. The effects of these factors have been addressed by including them in the linear models used for statistical analyses.Sleep loss is often accompanied with circadian misalignment, also known to cause changes in the regulation of the immune system and metabolism and associate to cardiovascular diseases72In real life, people frequently experience consecutive cycles of sleep restriction and recovery sleep . Often recovery sleep between two periods of sleep restriction remains incomplete and induces a carry-over effectWe propose that sleep restriction arouses a host defence response that shares features with APR aroused by trauma and infection. These responses modulate lipid metabolism, possibly through LXRs, and in the long run may contribute to the increased risk of cardiovascular diseases.Sleep restriction-induced decrease in expression of genes in pathways related to reverse cholesterol transport from macrophages, in combination with inflammatory activation, may at least partly explain the increased risk for cardiovascular diseases established in epidemiological studies for persons with short/insufficient sleep.An experimental schedule simulating a working week with restricted sleep was executed at the Brain and Work Research Centre of the Finnish Institute of Occupational Health (FIOH). We have earlier reported changes observed in glucose metabolismDuring waking, main activities of the participants included training and testing of memory and motor tasks simulating office work tasks. These tests were conducted during the day at 10:00–12:30 and 14:30–15:40, and during the sleep restriction at 00:30–01:40. In the test room the illumination ranged from 150 to 400 lux and in the living room from 350 to 600 lux. Physical exercise or leaving the sleep laboratory was not allowed during the experiment. Polygraphy was measured continuously and EEG scored according to the Rechtschaffen-Kales manual75Prestudy mean (±s.d.) body mass index (BMI) was 23.2 (±2.4) for controls and 23.5 (±2.6) for cases. Standardised meals based on the Finnish nutrition recommendations for 18–30 years old normal-weighted men with low activitySaliva cortisol was assessed 10 times per day during the experiment, and only a modest, 16 min on average, delay in the circadian phase measured by the morning peak of cortisol (from mean ± s.d. 07:39 ± 0:14 in BL to 07:55 ± 0:11 in SR) was observed in the sleep-deprived casesThe Dietary Lifestyle and Genetic determinants of Obesity and Metabolic Syndrome (DILGOM) study was originally performed as an extension of the FINRISK 2007 study . The national, cross-sectional FINRISK surveys have been carried out every 5 years since 1972 to assess the risk factors of chronic diseases  and health behaviour in the working age population in Finland. The aim of the DILGOM study (N = 5024) was to characterise risk factors for metabolic and cardiovascular diseases in the Finnish population both at the epidemiological and at the genetic levelNMR metabolomics and genome-wide gene expression were measured for a subsample from Helsinki metropolitan area  is a Finnish prospective multi-centre cohortIn this cohort, subjective sleep insufficiency was assessed using two questions. Subjects reported their habitual sleep duration on a 10-level scale . They also evaluated their subjective sleep need (“How many hours of sleep do you need per day to feel well rested?”) on the same scale. A measure of subjective sleep insufficiency for each subject was formed by subtracting the sleep duration from the sleep need. Subjects (N = 2221 with SSI data) were grouped into three groups based on their level of SSI: no (or only mild) SSI , moderate SSI , and heavy SSI . Subjects sleeping more than 1 h over their subjective sleep need (N = 37) were not included in the analyses. Correlation of sleep insufficiency with sleep duration is shown in Possible self-reported OSA was estimated using questions addressing the frequency and quality of snoring, as described earlierGenome-wide gene expression analysis was performed for 518 individuals . Gene exStatistical analysis. All arrays were quantile normalised at the strip-level. Technical replicates were combined via a bead-count weighted average, and replicates with Pearson’s correlation coefficient <0.94 or Spearman’s rank correlation coefficient <0.60 were removed from further analysis. 472 subjects with gene expression and sleep sufficiency data were included in the final analysis. 35,420 probes were analysed.Subjects were grouped into two groups  based on their report of subjective sleep insufficiency. Linear regression was used to correlate RNA expression with SSI, adjusting for age and gender (R version 2.14).Genome-wide gene expression was detected using Illumina microarrays as described inThe concentrations and purity of the RNA samples were evaluated spectrophotometrically with NanoDrop . Samples were considered pure, if the 260/280 ratio was 1.8–2.2. The RNA isolating process was validated by analysing the integrity of RNA with the RNA 6000 Nano Chip Kit (Agilent).The expression levels were analysed with an Illumina HumanHT-12 version 4 Expression BeadChip (Illumina Inc.) containing 47,231 expression and 770 control probes. In brief, 200 ng of RNA was reverse-transcribed into cDNA and biotin-UTP-labelled using the Illumina TotalPrep RNA Amplification Kit (Ambion); 1500 ng of cDNA was then hybridised to the Illumina HumanHT-12 v4 Expression BeadChip. The BeadChips were scanned with the Illumina iScan system.http://www.r-project.org/) using the Bioconductor (http://www.bioconductor.org/) packages. The expression data was processed using nonparametric background correction, followed by quantile normalization with control and expression probes, using the neqc function in the limma package and log2 transformation. The expression analysis was successful for 1650 subjects.Statistical analysis. Raw Illumina probe data was exported from GenomeStudio and analysed in R . Linear regression was used to correlate RNA expression with SSI, adjusting for age and gender (R version 2.14).P < 0.05) in the DILGOM cohort were further analysed with Database for Annotation, Visualization and Integrated Discovery (DAVID) pathway analysis . Illumina probe IDs were converted to DAVID IDs using Gene ID Conversion Tool, removing all redundancies in the original IDs. DAVID Functional Annotation Tool was used for identifying enriched biological processes among the genes with lower or higher expression in the SSI group  compared to the whole human genomeThe genes with differing expression levels in the SSI group compared to subjects satisfied with their sleep amount .Statistical analysis. The concentrations of 135 serum metabolites were assessed with NMR spectroscopy from serum samples of the experimental SR study and the epidemiological cohorts. In the SR study, SR timepoint was compared to BL with paired In the epidemiological samples DILGOM and YFS, linear regression was used to correlate NMR metabolites with SSI, adjusting for age and gender (R version 3.0), similarly as was done in the gene expression analysis. In the YFS sample, the analyses were run also with self-reported probable OSA added in the model.Lipidomic analyses were performed on serum samples from the experimental SR study at VTT Technical Research Centre of Finland. Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (QTOFMS) was used with the established protocolt tests.Statistical analysis. BL-normalised values of the lipids in the SR timepoint were compared between cases and controls using 3H]cholesterol-labelled HDL proteoliposome discs as the substrateCETP activity was analysed as the transfer/exchange of radiolabelled [14C]cholesteryl oleate between exogenously added human LDL and HDL2, as described int tests between the two timepoints.Statistical analysis. The activities in the SR timepoint were compared to their BL using first 2-way repeated measures ANOVA with group (case vs control) and timepoint (BL vs SR) (R version 3.0), and then with paired Partial correlation network analysis using the undirected Gaussian graphical model  between cases and controls at SR timepoint for BL-normalised data. The sleep variable data used were those from the previous night before the blood sampling (second night of BL and fifth night of SR).52 variables from the SR study were selected for the network analysis and are listed in P < 0.05) between pairs of variables and thus imply direct interactions. The edge width is proportional to strength of dependency. The node colour corresponds to the significance and direction of regulation comparing cases versus controls. The yED graphical editorIn these networks non-missing edges denote non-zero partial correlations How to cite this article: Aho, V. et al. Prolonged sleep restriction induces changes in pathways involved in cholesterol metabolism and inflammatory responses. Sci. Rep.6, 24828; doi: 10.1038/srep24828 (2016).


The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution. Cyanobacteria1A large number of plant genes encoding plastid proteins originated via independent horizontal gene transfer (HGT) events from prokaryotes other than in vivo may provide clues about the evolutionary mechanisms that ultimately resulted in their organellar targeting. We accordingly screened for candidate proteins of non-photosynthetic function that were present exclusively in prokaryotes and plants, and whose plastidial localization in the later could not be explained by a strict functional requirement. A uniquely suited candidate for a case study was found to be polyphenol oxidase , a bacterial enzyme67We addressed this conundrum using a novel approach that is based on the idea that studying the ancestral (cytosolic) and modern  subcellular localization of HGT-derived proteins Arabidopsis thaliana  suggests that the plastidial targeting of PPO in plants is likely a consequence of selective pressures acting against the deleterious effects caused by a PPO enzyme functioning in the cytosol. Given that biological processes in eukaryotic cells are highly compartmentalized, selection against horizontally acquired genes encoding non-targeted heterologous proteins may represent a general route by which novel organellar protein versions can arise.We present here evidence indicating that an early common ancestor of land plants acquired a bacterial gene coding for PPO through an ancient HGT event during the plant colonization of land. Recapitulating the bacteria-to-plants transition of PPO in the model plant Chitinomonas koreensis, the plant pathogen betaproteobacterium Ralstonia solanacearum, the gammaproteobacterium Rheinheimera sp. and the cyanobacterium Gloeocapsa sp. Some bacterial PPOs possess only one of these domains, while PPOs of many other bacteria as well as all fungal PPOs lack recognizable DWL and FKDV domains , Physcomitrella patens (AAX69084) and V. carteri (XP_002956492) further illustrate a greater structural similarity among land plant and bacterial TYR domains  and Lycophyta (Selaginella moellendorffii), are all grouped together with bacteria and that PPO genes cannot be found in glaucophyte, red and green algal genomes, strongly suggest that a PPO-like gene was transferred from a bacterium to an early land plant ancestor circa 450–500 million years ago during the transition of plants to live on landPPO bacteria-to-plants HGT is the observation that, compared to the average of plant genes, which typically contain 5–6 introns15PPOs have substantially fewer or no introns (0–2 introns)The survey described above indicates that PPO proteins are only found in land plants, fungi and some bacteria. To analyse the phylogenetic relations between these proteins, we focused on the TYR domain. A Maximum Likelihood analysis of the relationships between PPO tyrosinase (PPO-TYR) domains of land plants, bacteria and fungi reveals that land plant PPO-TYR domains form a well-supported monophyletic group with the PPO-TYR of several bacteria . Protein domains . The PPO domains . AlthougPPO, we set to experimentally recreate its horizontal acquisition by land plants. Our aim was to generate a model to study the effect of incorporating a nuclear gene coding for a protein of prokaryotic origin (PPO) that initially lacked information for a defined subcellular localization and eventually evolved a plastidial destination. Given that inferred ancestral proteins and proteins having evolved in distant lineages may result inactive or cause a stress derived from maladapted cell components when introduced into a distantly related host18PPO gene from Solanum tuberosum that we previously confirmed to encode an active PPO enzymePPO gene was identified in silico using ChloroPA, for enzyme with Ancestral localization) and one retaining it   tag was fused to the C-terminal end of both PPO versions and the corresponding constructs were transiently expressed in Nicotiana benthamiana leaves. Laser-scanning confocal microscopy analyses showed that PPOA presents a cytosolic distribution pattern while PPOM is detected only in plastids (chloroplasts) (Having defined the bacteria-to-plants HGT of ization) . The recynthesis . To deteoplasts) , as expeAgrobacterium-mediated transformation with constructs for constitutive PPOA or PPOM expression. In agreement with the conclusion that PPO activity is not absolutely required for photosynthesis or any other primary plant function5620PPO genesPPO gene ab initio. After isolating independent homozygous lines containing a single T-DNA insertion in the nuclear genome, we selected three PPOA and three PPOM lines with different PPO gene-expression , we generated stably transformed Arabidopsis plants by pression  and actipression  levels. controls . The expcontrols , supporting time , which aA, PPOM, and untransformed plants. However, the presence of an active cytosolic PPO enzyme in PPOA lines had a strong quantitative impact on anthocyanin levels, which were doubled relative to PPOM plants or wild-type controls (A lines (A enzyme), a negative impact on plant growth occurs.At the metabolic level, the qualitative profile (i.e. number of HPLC peaks) of phenylpropanoid compounds such as flavonols  and anthcontrols , Anthocy(A lines . TogethePPO) for a case study, we have aimed to shed some light on this subject.Despite the relevant contribution of HGT-acquired prokaryotic proteins to the establishment of the plastid proteome1PPO is less parsimonious and would require, at least, multiple independent PPO losses in the glaucophyte, red algae and green algae lineages, as well as in the animal lineage. Furthermore, since the last common ancestor of the supergroup PlantaePPO gene was maintained for more than 700 million years only in the algal lineage leading to land plants to be then lost after the colonization of land. Interestingly, the land plant acquisition of PPO corresponds to the period when the phenylpropanoid biosynthetic pathway (a rich source of phenolic metabolites) emerged in land plants, also in part by the gain of horizontally acquired genes from soil bacteria or fungiOur results strongly suggest that an early common ancestor of land plants horizontally acquired a gene coding for PPO of bacterial origin, possibly as a consequence of life in close contact with soil bacteria . An earlPPO gene integrated by chance in a genomic location simultaneously conferring both transcriptional activation and plastid targeting or that the acquisition of a plastid localization signal preceded the transcriptional activation of the gene. Under these hypotheses, eventual mutations or genomic rearrangements resulting in the loss of the plastid-localization signal would lead to a disadvantageous phenotype and the cytosolic PPO would not be retained. In either case, the finding that PPOA triggers a stress response (as deduced from the accumulation of anthocyanins) that could eventually lead to reduced growth rates when compared with PPOM implies that directional selection would increase the frequency of PPOM relative to PPOA after the horizontal acquisition of the bacterial gene, ultimately favouring the fixation of the plastidial enzyme within the population.It has been established that translocation of genes from the essentially prokaryotic plastid genome to the nucleus involves post-insertional genomic rearrangements rather than random integration into a genomic location conferring instant functionality26272830In stark contrast to the common assumption that newly acquired genes conferring a strongly negative fitness effect will irreversibly undergo gene degeneration or loss, we propose that the fortuitous acquisition of organelle targeting sequences can provide an alternative fate to such genes. The argument is made that a horizontally acquired prokaryotic gene coding for a delocalized protein could have a deleterious effect that is only context-specific in a eukaryotic cell where different biological processes are highly compartmentalized. The targeting of the foreign protein to an organelle such as the plastid could alleviate the deleterious effect, hence allowing the gene to endure in the recipient genome and thereafter serve as raw material for the evolution of novel functions. The plausible generality of the proposed evolutionary mechanism is supported by the well-known fact that cytosolic localization of heterologous proteins is often deleterious to plant growth when compared with plastid-localized versions323334A fundamental goal in biology is to elucidate the forces leading to evolutionary change. Previous work has revealed the unexpected broad contribution of prokaryotic proteins acquired via independent HGT events to the plastid proteome126303637http://genome.jgi-psf.org/) and Phytozome (www.phytozome.net). To check the robustness of the gene sampling additional searches using PSI-BLASTExtensive BLASTTyrosinase domains of PPO proteins and other TYR-containing proteins were aligned with ClustalW46349http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi)http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml)Genes coding for proteins with tyrosinase domains were compared by examining their corresponding protein domain architectures using CDART The structural modelling was performed using the intensive modelling mode of the Protein Homology/analogy Recognition Engine (Phyre) Version 2.0 (Solanum tuberosum PPO (GenBank accession number: U22921) or a PPO version lacking localization signal were amplified from cDNA and cloned into plasmid pDONR207 using Gateway technology . The in silico detection of the subcellular localization signal was identified using ChloroPhttp://www.cbs.dtu.dk/services/ChloroP/). Sequences were then subcloned into plasmids pGWB405Nicotiana benthamiana leavesEscherichia coli. Constructs in pB7FWG2 were used for stably Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana plants (ecotype Col-0)M and 29 PPOA homozygous lines were isolated; from which three independent lines of each PPO version were selected for further characterization. Arabidopsis plants were grown on soil in a climate controlled growth chamber  under 8 h of light/16 h of dark photoperiod for 4 weeks. For phenylpropanoid profiling, Arabidopsis seeds were surface-sterilized and sown on Petri plates with sterile MS medium containing 1% agar and 30 g/L sucrose. When indicated, plates were supplemented with 5 μM norflurazon. After stratification for 3 days at 4 °C in the dark, the plates were incubated in growth chambers for 20 days at 22 °C under continuous light .Full-length cDNAs encoding E. coli cells strain Rosetta 2 (DE3)  with purified plasmids pET28-PPOA and pET28-PPOM, the activity of PPOA and PPOM was confirmed by growing the transformed E. coli cells for three days at 28 °C on LB agar plates supplemented with 34 μg/mL chloramphenicol, 25 μg/mL kanamycin, 0.5 mM IPTG, 40 μg/ml CuSO4, and 600 μg/ml chlorogenic acid, similarly to methods previously describedA and PPOM colonies were dark because the cells were able to produce melanin-like polymers.Following transformation of competent Subcellular localization of GFP fluorescence and chlorophyll fluorescence was determined with an Olympus FV 1000 confocal laser-scanning microscope  using an argon laser for excitation (at 488 nm) and a 500–510 nm filter for detection of GFP fluorescence and a 610–700 nm filter for detection of chlorophyll fluorescence.2.The surface area of rosette leaves was measured using Photoshop  and GraphPad Prism 5.0a  by analysing digital images (acquired from above) of 4-week-old plants and comparing them with size standard series ranging from 4 to 16 cm18V)] and 2 μg of total RNA. The relative mRNA abundance was evaluated via quantitative reverse transcription PCR (RT-qPCR) in a total reaction volume of 20 μl using LightCycler 480 SYBR Green I Master  on a LightCycler 480 Real-Time PCR System  with 0.3 μM of each specific sense and anti-sense primers. Three independent biological replicates of each sample and three technical replicates of each biological replicate were performed and the mean values were considered for further calculations. The normalized expression of PPO was calculated as describedUBC  as the endogenous reference gene. Information about primers used is described in detail in Leaf samples from 4-week-old plants were harvested and total RNA was extracted using the Maxwell 16 LEV simplyRNA Tissue Kit  according to the manufacturer’s instructions. Purified RNAs were quantified by spectroscopy using a NanoDrop apparatus  and RNA integrity was evaluated by agarose gel electrophoresis. The First Strand cDNA Synthesis Kit  was used to generate cDNA according to the manufacturer’s instructions, using 50 pmol of an anchored poly(dT) primer [d containing 10 mM L-DOPA. The reaction was measured with a SpectraMax M3 spectrophotometer  by the change in absorbance at 475 nm and 25 °C.Protein extracts were obtained from rosette leaves homogenized at a ratio of 50 mg of fresh weight to 1000 μl of 20 mM phosphate buffer (pH 6). The homogenate was centrifuged at 16.000 × 2O (9:1:10) extraction solvent containing 0.1 mg/mL of naringenin as an internal standard for quantification. Homogenates were incubated in the dark at 4 °C for 30 min with agitation (1000 rpm) and then centrifuged at 14,000 × g and 4 °C for 5 min. Supernatants were recovered, filtered and analysed by high-performance liquid chromatography (HPLC) using an Agilent 1200 series HPLC equipment  with a XSelect CSH C18  column . Flavonols and anthocyanins were determined at 320 and 520 nm, respectively.Phenylpropanoids  were purified and analysed as described previouslyr values) were calculated using the means of the surface area of rosette leaves, PPO mRNA abundance and PPO activity. Statistical analysis was performed using GraphPad Prism 5.0a .ANOVA followed by Newman-Keuls multiple comparison post-hoc tests were used to determine statistical significance for multiple groups. Pearson correlation coefficients .


To describe structural covariance networks of gray matter volume (GMV) change in 28 patients with first-ever stroke to the primary sensorimotor cortices, and to investigate their relationship to hand function recovery and local GMV change.Tensor-based morphometry maps derived from high-resolution structural images were subject to principal component analyses to identify the networks. We calculated correlations between network expression and local GMV change, sensorimotor hand function and lesion volume. To verify which of the structural covariance networks of GMV change have a significant relationship to hand function, we performed an additional multivariate regression approach.Expression of the second network, explaining 9.1% of variance, correlated with GMV increase in the medio-dorsal (md) thalamus and hand motor skill. Patients with positive expression coefficients were distinguished by significantly higher GMV increase of this structure during stroke recovery. Significant nodes of this network were located in md thalamus, dorsolateral prefrontal cortex, and higher order sensorimotor cortices. Parameter of hand function had a unique relationship to the network and depended on an interaction between network expression and lesion volume. Inversely, network expression is limited in patients with large lesion volumes.Chronic phase of sensorimotor cortical stroke has been characterized by a large scale co-varying structural network in the ipsilesional hemisphere associated specifically with sensorimotor hand skill. Its expression is related to GMV increase of md thalamus, one constituent of the network, and correlated with the cortico-striato-thalamic loop involved in control of motor execution and higher order sensorimotor cortices. A close relation between expression of this network with degree of recovery might indicate reduced compensatory resources in the impaired subgroup. As both cross-sectional and a few longitudinal observational studies have demonstrated, behavioral recovery from hemiparesis after ischemic stroke shows marked between-subject variability , 2. ThisActivation studies performed with fMRI have shown that successfully recovered subjects show almost normal cerebral patterns, exhibiting change during recovery from attention demanding controlled processing of motor performance in the subacute stage to more fluent and automatic processing in the late chronic stage . This suin vivo to a specific mechanism, e.g., axon sprouting, dendritic branching or synaptogenesis , to quantify gray matter volume (GMV) changes during recovery , 19. Whiogenesis . Requiriogenesis  delineatogenesis . An examogenesis , the acqAccompanying the imaging was an array of clinical, motor and sensory assessments performed regularly during the 9-month study. Of the behavioral assessments, picking small objects (PSO), a lateralized motor skill requiring a particular precision grip, showed the greatest variance over the 9-month trial period . ResponsA previous mass-univariate analysis of these TBM images yielded three findings: (i) most striking, impaired patients with chronic disturbed hand motor skills showed the most prominent GMV increase in the ipsilesional medio-dorsal (md) thalamus, including also the head of the caudate nucleus; (ii) all patients evidenced GMV decreases within the contralesional anterior cerebellum at a location typical of cerebellar diaschisis after sensorimotor cortical stroke; and (iii) patients showing fast recovery exhibited a slight GMV increase in the perilesional premotor cortex (PMC). These results stimulated several questions: Does the significant GMV increase of md thalamus in these patients represent an isolated, local effect or does it implicate an extended gray matter network involved in recovery after a sensorimotor cortical stroke? Does the extended network show a structural covariance pattern that discriminates among classes of recovery process? How does the network relate to the initial lesion pattern?These questions led to the hypotheses examined in the current study: the prominent GMV changes in the md thalamus relate to the dorsolateral prefrontal circuit of Alexander et al.  as propot-test: t(49) = 3.4, p < 0.12) and handedness according to the Edinburgh Handedness Questionnaire (unpaired two-tailed t-test: t(49) = 0.36, p < 0.30). The study received ethical approval from both research centers . All participants gave written informed consent before enrollment according to the Declaration of Helsinki. The same cohort was used for our previous publications  from January 01, 2008 through July 31, 2010. Inclusion criteria were (1) first-ever stroke, (2) clinically significant contralesional sensorimotor hand function impairment as leading symptom, and (3) inclusion of the pre- and/or post-central gyri within the ischemic lesion confirmed on acute diffusion-weighted (DWI) and fluid attenuated inversion recovery (FLAIR) MRI scans. Patients were excluded if they presented (1) aphasia or cognitive deficits that precluded understanding the study purposes or task instructions, (2) prior cerebrovascular events, (3) occlusion or stenosis >70% of the carotid arteries in MR–angiography, (4) purely subcortical stroke, and (5) other medical conditions interfering with task performance. We recruited 36 patients, seven of which dropped out . The final sample consisted of 29 patients . As a control group for the analyses of behavioral and clinical data, we recruited 22 healthy older adults  from the local community. Groups were matched for age  with extended measurements of clinical and behavioral data (see below). The same measurements were taken 3 months  and 9 months  after stroke. During each of these two visits, we acquired high-resolution anatomical imaging data. Patients were additionally seen at monthly intervals in-between these examinations to evaluate recovery of dexterous hand function.Clinical stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS) . Hand mo3 , fat saturation, 12 min total acquisition time. Identical prescription of MR images was achieved by use of the Siemens auto-align sequence that automatically sets up consistent slice orientation based on a standard MRI atlas.All patients underwent acute phase imaging at admission according to local stroke imaging protocols. This included a diffusion-weighted imaging (DWI) scan and T1-weighted (T1w) anatomical image. At 3 and 9 months after stroke, each patient underwent high-resolution T1w imaging using a 3D-MDEFT with following imaging parameters : repetitz-transformed longitudinal data using linear and non-linear curve fitting. At the group level, we then calculate a PCA of these curves to derive a number that summarizes each patient’s recovery relative to the whole cohort. The analysis of structural high-resolution imaging data was performed similarly. At the single-subject level, we calculated TBM maps that encode  local GMV change between 3 and 9 months after stroke, as previously described , slow  and impaired recovery . The principal component analyses of the PSO and TOR task, and NIH evaluation were performed with Matlab program, princomp . The PSO task yielded ten principal component time courses and variances (one per visit); the TOR task and NIH evaluation, three time courses and variances. Each produced 36 patient expression coefficients (or “scores”). The Kaiser–Guttmann criterion was used to select salient principal components . Furthermore, we calculated the overlap between each network cluster and subgroup lesion density maps.The PCA of the TBM images was performed on a subset of 28 patients representing the volume changes between months 3 and 9 (of the 29 patients retained for the study 1 had to be excluded because of MR motion artifacts). PCA was executed on the images data using in house software written in MATLAB based on the algorithm described by Alexander et al. and Moeller et al. , 45. ExtU test to compare pairs of subgroups against each other. Finally, we used robust (multiple) regression within the framework of the general linear model to test the relationship of network expression, clinical and structural variables to hand function recovery and their interaction across the whole patient cohort. The criterion for significance was set at p < 0.05, Bonferroni corrected for multiple comparisons.We used median and range for descriptive statistics. We first assessed the relationship of structural covariance component expression, clinical and structural variables, e.g., lesion volume and regional GMV change, using Pearson’s correlation coefficient in order to identify the network related to hand function recovery. Next, we assessed differences with respect to subgroups in network expression and behavioral variables. To do so, we first applied the Shapiro–Wilk test and inspected Q–Q plots for each variable to assess deviations from normality. We used then non-parametric tests to compare scalar variables where appropriate, i.e., the Kruskal–Wallis one-way analysis of variance by ranks to assess differences in the central tendency among any of the three subgroups, and the Mann–Whitney Clinical characteristics of the patient cohort are summarized in Table TBM) correlated with GMV reduction in the cerebellum contralateral to the affected hemisphere. The second component (PC2TBM) correlated with lesion size, GMV volume increase in the md thalamus, clinical (PC1NIHSS expression) and hand function specific recovery (PC1PSO expression). The fourth principal component correlated exclusively with PC1NIHSS expression. None of the other PCs surviving the Kaiser–Guttmann criterion correlated with any of the external variables.Table TBM correlated with our specific measure of hand function recovery, we focused further analysis on its critical clusters . Functional interpretation was done in the context of motor hand function, based on current literature. The expression of this network had a strong correlation with thalamic GMV change across the whole cohort  and specifically in PC1PSO (p < 0.0001). This group had also the largest GMV expansion in the medio-dorsal thalamus and the highest lesion volumes (both p < 0.05). Figure TBM expression and thalamic GMV change (panel A) and hand skill recovery as reflected by PSO (panel B), respectively. Considering all individuals, GMV change correlated with expression coefficients of the structural covariance network of PC2TBM (R = 0.72 and p < 0.5 after correction for multiple comparisons). PC2TBM expression could also distinguish between subgroups: When dividing patients into subgroups with positive versus negative network expression coefficients (without regard to recovery subgroup assignments), we found that the positive subgroup has significantly higher thalamus GMV change (median 1.35% with range 0.83–1.79%), whereas the negative subgroup shows no significant change .We further compared the patients subgroups presenting normal motor performance after 9 months (fast and slow recovery) with the subgroup that did not achieve normal performance (impaired recovery). As expected from the RFA, the latter group yielded the highest expression coefficients in PC1TBM and recovery , PC2TBM expression (p = 0.048) and lesion volume (p = 0.037). The significant intercept indicated residual variance not modeled by our predictors. We, therefore, investigated a reduced model that included PC1PSO as dependent variable, and only the significant predictors from the first model, i.e., PC2TBM expression, lesion volume and their interaction (PC2TBM expression × lesion volume) as independent variables. The interaction term significantly predicted PC1PSO scores  = 3.83, p < 0.001) over and above the other variables (both p > 0.1). The interaction term explained a significant portion of variance in hand function recovery  = 14.18, p < 1.6e−5). Full model parameters are summarized in the Table S3 in Supplementary Material.To further test the specificity of the association between PC2TBM appears to reflect a neuronal network caused by diaschisis from sensorimotor cortex  the unique relationship of PC1This study represents important progress following our recent paper on “Gray matter volumetric changes related to recovery from hand paresis after cortical sensorimotor stroke”  as it reTBM network distinguishes clearly within the study cohort since the subgroup with positive expression coefficients is associated with large GMV increases in the md thalamus between months 3 and 9, while the subgroup with negative expression coefficients did not exhibit a recognizable GMV change. The GMV increases in the former subgroup exceed the measurement uncertainty and are consistent with the few comparable studies, e.g., in the paper of Gauthier et al.  , which oTBM, representing possible sub-networks engaged in higher order sensorimotor information processing and spatial awareness (see below). In the PPC locally functional processed information, e.g., space and action perception, is transmitted via feedback loops to ventral PMC  faster than men depending on age, and upper limb kinesthetic asymmetries in contralateral reproduction of elbow movements, elicited by tendon vibration, were prevalent in males , 87.PSO and inversely were limited in the expression of the structural covariance network of PC2TBM. To conclude, our application of tensor-based morphology has shown it to be a powerful method for studying gray matter changes after stroke; it is capable of revealing both local changes and in associated extensive neural networks. Regarding its future use application, TBM will be potentially of interest in the study of targeted treatment effects in the long-term.As posited in Section “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.http://journal.frontiersin.org/article/10.3389/fneur.2015.00211The Supplementary Material for this article can be found online at Click here for additional data file.


A prevailing question remained: Does visual deprivation first induce a change in gene expression, followed by a shift in INCs, or vice versa? In the present study, we address this question by investigating the neuroanatomy and patterns of gene expression in post-natal day (P) 1 and 4 mice following bilateral enucleation at birth. Our results demonstrate a rapid reduction in dorsal lateral geniculate nucleus (dLGN) size and ephrin A5 gene expression 24-hours post-enucleation, with more profound effects apparent at P4. The reduced nuclear size and diminished gene expression mirrors subtle changes in ephrin A5 expression evident in P1 and P4 enucleated neocortex, 11 and 8 days prior to natural eye opening, respectively. Somatosensory and visual INCs were indistinguishable between P1 and P4 mice bilaterally enucleated at birth, indicating that perinatal bilateral enucleation initiates a rapid change in gene expression (within one day) followed by an alteration of sensory INCs later on . With these results, we gain a deeper understanding of how gene expression and sensory input together regulate cortical arealization and plasticity during early development.Functional sensory and motor areas in the developing mammalian neocortex are formed through a complex interaction of cortically intrinsic mechanisms, such as gene expression, and cortically extrinsic mechanisms such as those mediated by thalamic input from the senses. Both intrinsic and extrinsic mechanisms are believed to be involved in cortical patterning and the establishment of areal boundaries in early development; however, the nature of the interaction between intrinsic and extrinsic processes is not well understood. In a previous study, we used a perinatal bilateral enucleation mouse model to test some aspects of this interaction by reweighting sensory input to the developing cortex. Visual deprivation at birth resulted in a shift of intraneocortical connections (INCs) that aligned with ectopic  The mammalian neocortex is a complex structure in the central nervous system that contains a unique set of functional and anatomical properties. This structure is responsible for integration of naturally occurring cognitive demands in the everyday environment, including language, decision-making, motivation and sensori-motor processing. During development, sensory and motor areas are formed in the neocortex through a process called arealization. The mechanisms responsible for cortical area development, or arealization, have been debated for years, with two contradictory models initially emerging: the Protomap and Protocortex hypotheses –2. In thDuring early development, visual cortical map formation is driven by spontaneous retinal activity where retinal ganglion cells create excitatory potentials that propagate across the retina in the form of ‘waves’ . Specifiephrin A5 expression were observed at the somatosensory-visual (S-V) area border [ephrin A5 expression during the first postnatal week .An important feature of cortical arealization, or cortical patterning, is the establishment of precise intraneocortical connections (INCs), which serve as the primary network for cortical function. Neocortical gene expression is believed to drive initial arealization and targeting of INCs prior to eye opening, during preliminary developmental stages –19. Subsa border . Thus, ba border  we extenAll studies were conducted under a research protocol approved by the Institutional Animal Care and Use Committee at the University of California, Riverside. All experimental and breeding mice were of CD1 background, originally purchased from Charles River Laboratories International, Inc., Wilmington, Massachusetts, USA.Enucleation procedures were conducted just after birth (P0). A maximum of 1 hour of nursing was allowed prior to surgery. Pups were anesthetized with a mixture of ketamine  and xylazine , then placed on ice for 1 to 4 minutes prior to bilateral eye removal –22. A toin situ RNA hybridization experiments. Following dye crystal placement, hemispheres used for postmortem dye tracing were stored in 4% PFA at room temperature during the 6–10 weeks required for tracer transport. Hemispheres reserved for in situ RNA hybridization were dehydrated in methanol, and stored at -20°C until further processing.Experimental animals were subjected to an early sensory deprivation period (either 1 or 4 days), after which both enucleated and control mice were given a lethal dose of sodium pentobarbital (100mg/kg) and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (pH 7.4). Brains were removed from the skull, post-fixed for a 24-hour period and hemisected. One hemisphere of each brain was used for postmortem tracing and thalamic measures, and the opposite hemisphere used for in situ RNA hybridization were used to visualize gene expression. Ephrin A5  probe was used to identify the patterns of neocortical and dLGN gene expression at P1 and P4, in experimental and control murine offspring. Previously dehydrated hemispheres reserved for hybridization were rehydrated, embedded in gelatin-albumin and sectioned in the coronal plane at 100μm using a Vibratome. All sections were mounted in glycerol onto glass slides, cover slipped and photographed as described below following hybridization. A minimum of six replicate hemispheres for each age and condition was required for gene expression assays.Standard protocols and methods for non-radioactive free-floating To determine patterns of ipsilateral INC development in control and bilaterally enucleated P1 and P4 mice, single crystals of DiI and DiA  were placed in two discrete dye placement locations (DPLs) after overnight post-fixation with 4% PFA at -20°C: the parietal (putative somatosensory cortex) and occipital  lobes of postmortem neocortical tissue. Methods for dye crystal placement have been described in detail elsewhere . A dye pin situ RNA hybridization were imaged under a bright field scope with a digital high resolution Zeiss Axio camera using Axiovision software (Version 4.7) coupled to a Zeiss Stereo Discovery V12 stereomicroscope. ISH sections were analyzed both qualitatively and quantitatively through transcript density measures. Specifically, transcript density of ephrin A5 was quantified using ImageJ software. Digital images were first converted to a binary form with a distinct threshold being sustained across identical anatomical levels in all replicates (n = 6 control and n = 6 enucleates). Laminar boundaries for quantifying regions of interest (ROIs) were distinguished by identifying landmarks using DAPI staining that co-registered with in situ hybridization of ephrin A5, thus allowing us to adequately define ROIs in superficial, middle and deep cortical layers. ROIs were then defined by electronically positioning grids over static position in P1 and P4 cortex of control and enucleated mice. Individual measures of each area were taken to determine transcript density in the specific region. The presence of transcript signal was measured, and reported as area fraction of total ROI.All sections processed for P-value of less than 0.05 was chosen for statistical significance between groups.Values for ISH density are reported as the mean ± standard error. The mean for each control group and enucleated group was established from the 6 measurements in control tissue, and 6 measurements in enucleated tissue. Two-sample independent t-tests were conducted to compare gene expression density in the ROI across control and enucleated brains. A ephrin A5 expression was quantified in serial sections at a fixed magnification using ImageJ. Borders of the dLGN were drawn using ImageJ at a fixed magnification, with experimenter being blind to the condition when defining the borders of the nucleus. Borders were determined as follows: the ventral lateral border of the dLGN was marked by the intergeniculate leaf (IGL); the ventral medial border was marked by the external medullary lamina (EM); the dorsal border was differentiated by the cell sparse nature of the lateral posterior nucleus (LP); the dorsal lateral border determined from visualization of the brachium of the superior colliculus (BSC). Data is displayed as a percent change in baseline (control) measure . Two-sample independent t-tests were used to compare area of nuclear gene expression in control and enucleated cases. A P-value of less of than 0.05 was used to establish statistical significance.Thalamic nuclear (dLGN) area of P-value of less than 0.05 was chosen for statistical significance between groups.All images were captured with a digital high resolution Zeiss Axio camera using Axiovision software (Version 4.7) coupled to a Zeiss Axio Imager Upright Microscope equipped with fluorescence at 4x magnification. DAPI stained sections were used to define thalamic nuclei boundary. Thalamic nuclear (dLGN) size was quantified in serial sections using ImageJ, with borders determined as described above. Experimenters were blind to conditions. Data is displayed as a percentage change in baseline (control) measure (n = 6 control and n = 6 enucleates). Localization and comparisons of somatosensory INCs at the S-V boundary across conditions were analyzed using an electronic micrometer measuring the distance from the cortical midline to the most medial labeled cell observed from a somatosensory cortex dye placement as was done in previous studies , 23 . The observed reduction in dLGN size was more pronounced in enucleated tissue as compared to controls when assessed 4 days following early enucleation, and 8 days prior to eye opening .Although brain plasticity associated with sensory deprivation has been documented in several sensory systems, the precise timing of early effects of visual deprivation through bilateral enucleation has not been well described. In this study, the development of the visual sensory thalamic relay, the dLGN, was examined at two time points after early newborn bilateral enucleation: postnatal day (P) 1, and P4 . The area of ephrin A5 expression remained within boundaries of the reduced nucleus at both P1 and P4. Despite a reduction in dLGN size, enucleation at birth did not appear to impact the development of thalamic afferents, observed by making dye placements in somatosensory and visual cortices . Decreased ephrin A5 expression was also detected in medial somatosensory cortical areas of P1 enucleated brains when compared to controls (compare P < 0.01). Ephrin A5 percentage of area above threshold in P1 visual cortex ROI (compare P < 0.05). amalgam  . Transcripts in control somatosensory cortex were detected in middle layers. Transcripts were also detected in middle layers of enucleated somatosensory cortex, although the expression border was lateral to that of control cases (compare arrows in P < 0.001). A significant difference in ephrin A5 area above threshold between enucleated and control visual cortices was detected (compare P < 0.0001).At P4, expression of l brains , with vel brains . Furtherl brains  in both  compare , with en compare  when comIn order to determine the effects of bilateral enucleation at birth on INCs early in development, DiI and DiA were placed in postmortem brains of P1 and P4 control and enucleated mice. Cortical area boundaries have been previously determined using specific arrangement of INCs within different sensory areas ,18–19. IP = 0.50; P = 0.20), as present in P10 blind mice [In P4 control and enucleated brains, DiA placement in the parietal somatosensory cortex retrogradely labeled cells in locations rostral and caudal ; green tind mice . This suephrin A5. This reduced Ephrin A5 expression in the dLGN occurs alongside reduced cortical gene expression in enucleated brains when compared to controls. These changes in gene expression occur before shifts in intraneocortical connectivity are observed, as we did not observe alterations in dye tracing at P1 or P4.In summary, by removing both eyes at birth, we observed immediate changes in subcortical and cortical regions. Correlated with a reduction in dLGN size, we demonstrate the reduction of a well-characterized guidance molecule, and the very important role of sensory input in the formation of sensory thalamic nuclei and neocortical areas [ephrin A5 expression [first, followed by a re-routing of cortical connections or vice versa. The study described here analyzes the impact of newborn bilateral enucleation, or visual sensory deprivation, on the brain within the first postnatal week in order to determine which feature of cortical organization was altered first as a result of the deprivation. Based on previous work from our laboratory, we hypothesized that an abrupt change in sensory input via bilateral enucleation would induce a rapid change in gene expression leading to a later alteration of intraneocortical connectivity.There is ample data to support the role that genes play in early brain patterning al areas ,18–19,21al areas , howeverephrin A5 expression in cortex as early as 24 hours post-blindness. Specifically, DAPI staining and in situ RNA hybridization revealed a dorsomedial reduction in dLGN size and ephrin A5 expression in both P1 and P4 enucleates  is knocked out in the mouse, the expression gradient of Pax6 shifts caudally [Effects of short-term enucleation on neocortical gene expression In addition to changes in LGN size and gene expression, early bilateral enucleation altered caudally . Thus, wephrin A5 gene expression, emphasize the contribution of retinal waves to both cellular and molecular mechanisms of the developing cortex and provide grounds to speculate the role of ephrin A5 in mediating cross-modal neocortical plasticity as a result of modifying patterns of peripheral activity.It is not surprising that we see a shift in gene expression levels in our early enucleation experiments, as the developing mammalian brain is subject to anatomical and functional reorganization following peripheral alternations during development –41. Addiephrin A5 expression in P1 and P4 neocortex, including the lateral positioning of expression in P4, observed in enucleated mice may not be a ‘shift’ in expression, or a shift in area position, but rather a delay in developmental processes. This notion stems from an observation that cortical expression in P1 and P4 enucleated brains is similar to pre- and perinatal expression in the same regions of neocortex in wild-type mice [ephrin A5 expression in P1 sensory-motor cortex at P4 seen here, strongly supports the idea of delayed expression of key developmental genes following peripheral modifications (Figs ephrin A5 expression observed in our P1 and P4 enucleated brains, may illustrate a potential mechanism underlying prolonged critical periods in neocortical development. In order to determine if the short-term plasticity actually caused a shift in borders or area position within sensory and motor cortex, multi-unit electrophysiology would be needed to determine functionality of the region. Unfortunately, this is not feasible at these early ages. However, tracing data presented here linking regions of interest within the cortex to appropriate thalamic nuclei suggest that the areas have not changed their position within cortex.The alterations in ype mice –19. MoreEffects of short-term bilateral enucleation on ipsilateral INCs Ipsilateral somatosensory and visual cortex INC development is well characterized in the first week of life in CD-1 mice ,19. In tEarly developmental processes have been shown to involve and rely heavily on endogenous spontaneous activity from the retina and sensory input transmitted through the eyelid ,57–59. Ovice versa. Previous data led us to hypothesize that acute visual deprivation achieved through bilateral enucleation would first generate a change in cortical gene expression followed by remodeling of sensory INCs in cortex. Our data supports this hypothesis as a rapid change in cortical ephrin A5 expression is detected just one day after bilateral enucleation in the newborn mouse neocortex with normal connections prevailing until P4. Our initial study demonstrated that by P10, cortical ephrin A5 expression and somatosensory INCs were altered, leading us to conclude that the alteration of sensory INCs in our blindness model occurs between P5 and P10, after a change in gene expression. A rapid effect of bilateral enucleation was also seen in the thalamus. Just 24 hours after enucleation, we observed that the dLGN was significantly reduced in size following removal of all spontaneous retinal activity. By studying the effects of significant perturbations of sensory system development on the anatomy of sensory nuclei, important developmental genes and INCs, we have begun to characterize the immediate effects of enucleation and the relationship between vision loss, dLGN anatomy and cortical ephrin A5 expression.In this study we used a sensory deprivation model to determine whether deprivation leads to a change in gene expression followed by a remodeling of intraneocortical connections, or ephrin A5 expression in these regions only 1 day after sensory deprivation. These rapid effects observed both in the thalamus and cortex within the first postnatal week offer novel insights into potential interactions of molecular processes and peripheral sensory input, highlighting the importance of retinal activity in the first few days of life for proper establishment of precise temporal gene expression in sensory areas. These data suggest that cortical gene expression plays a role in the development of proper intraneocortical connectivity, providing further evidence for the interaction of the two features of brain development first discussed by Huffman [ephrin A5 mutant mice would need to be conducted to better interpret the role of this gene and activity-dependent mechanisms in the formation and maintenance of INCs in the first few days and weeks of post-natal development.We suggest that input from the retina is critical for precise gene expression in and development of thalamic nuclei and cortical sensory areas, as we observed modifications in  Huffman . Further


Ki67 labeling index (LI) is used as a predictive marker and is associated with prognosis in breast cancer. However, standardised methodologies for measurement are lacking which has limited its application in clinical practice. In this study, we evaluated the interobserver concordance of visual assessment of Ki67 LI in breast cancer.Ki67- immunostained slides of 160 cases of primary invasive breast cancer were visual assessed by five breast pathologists with two different methods to choose the scoring fields: (1) hot-spot score, (2) average score. Proportions of positive invasive tumor cells at 10 % intervals were scored. The intra-class correlation coefficient (ICC) was used to assess the interobserver reproducibility.P<0.0001). Average score method  demonstrated a better correlation than hot-spot score method . (2) By respective means according to two score methods, all cases were classified into three groups . The concordance was relatively low in intermediate Ki67 LI group compared with low and high Ki67 LI groups. (3) All cases were classified into three groups by paired-difference (d) between means of hot-spot score and average score . The consistency was observed to decrease with increasing paired-difference according to both methods.(1) A perfect concordance of Ki67 LI was demonstrated according to both score methods (Visual assessment of Ki67 LI at 10 % intervals is a candidate for a standard method in breast cancer clinical practice. Average score and hot-spot score of visual assessment both demonstrated a perfect concordance, and an overall average assessment across the whole section including hot spots may be a better method. Interobserver concordance of intermediate Ki67 LI in which most cutoffs are located for making clinical decisions was relatively low. Uncontrolled proliferation is an important feature of malignant tumors. The nuclear proliferation marker Ki67 is used as a predictive marker for response to chemotherapy and is associated with patient prognosis in breast cancer –7. Assesth St. Gallen Consensus Meeting suggested that the Ki67 LI is important for distinguishing between ‘‘luminal A” and ‘‘luminal B (HER2-negative)” breast cancer subtypes and advised adjuvant chemotherapy for luminal B but not for luminal A [In 2011, the 12uminal A . Therefouminal A –13. CurrManual counting of as many as 1000 cancer cells is often recommended to evaluate Ki-67 LI , but thiOne hundred and sixty cases of primary invasive breast cancer were extracted from the pathology database of Fudan University Shanghai Cancer Center. An important object of this study was to evaluate the interobserver concordance of visual assessment in intermediate Ki67 LI group in which most cutoffs are located for making clinical decisions. Since most of grade 1 breast cancers are ER/PR strongly positive cases with low Ki67 LI, Ki67 LI is not crucial in decision-making of the treatment in these patients. So we included less grade 1, but more grade 2 and grade 3 cases of invasive breast cancer in this study. All patients underwent surgery at the Cancer Center in 2012 without neoadjuvant chemotherapy. Clinicopathological features of all cases were reviewed. All specimens were fixed with 10% neutral phosphate-buffered formalin and paraffin-embedded. 4μm-thick slices of representative tumor blocks were stained with hematoxylin and eosin (H&E) and stained on Benchmark XT system . MIB-1 clone antibody was used  for Ki67 staining.Ki67 assessment was performed by five breast pathologists independently, in a blinded manner. Our pathology department has been a highly specialized center and the five observers included in this study were all experienced breast pathologists. Specific instructions were given to the five pathologists for Ki67 assessment. For each case, an H&E slide from the same paraffin block was used for morphology observation. Nuclear staining of any intensity was defined as Ki67 positive. The Ki67 LI was visually scored for the percentage of nuclei positive tumor cells among all tumor cells in invasive tumor area. The whole slide was scanned under low-power microscopy first. At least three high-power (×40 objective) fields should be selected to represent the spectrum of staining seen on initial overview of the whole slide. In heterogeneously stained samples , each paP value less than 0.05 was considered to be statistical significant. The computations were done with the statistical software package SPSS 19.0 for Windows .To assess the interobserver reproducibility of Ki67 LI according to two score methods, the intra-class correlation coefficient (ICC) was estimated with a 95% confidence interval (CI) using two-way random models. Higher ICC usually indicates better consistency. There is no universally accepted standard criteria for the ICC, the following criteria similar to the kappa coefficient were used in our study : 0.00–0.Our study was approved by Ethics Institutional Review Board of Fudan University Shanghai Cancer Center. The patient records/information was anonymized and de-identified prior to analysis.Clinicopathological features of 160 cases of primary invasive breast carcinoma were summarized in P<0.0001) (The detailed data of scores of each pathologist was provided in <0.0001) . AverageMost of the proposed cutoff values of Ki67 for making clinical decisions are between 10%-30%. By respective means according to two score methods, all cases were classified into three groups  to allow categorical data analyses. The ICC was relatively low in intermediate Ki67 LI group (11%-30%) compared with low (≤10%) and high (>30%) Ki67 LI groups according to both methods ; Fig 3. To evaluate the correlation between Ki67 staining distribution (heterogeneous or homogenous) and reproducibility of assessment, all cases were classified into three groups by paired-difference (d) between means of hot-spot score and average score . The smaller the paired-difference between hot-spot score and average score, the more homogenous staining of Ki67 was indicated. ICC of three groups was evaluated. The consistency among five pathologists was observed to decrease with increasing paired-difference according to both methods ; Fig 4. The nuclear protein Ki67 is often used as a marker of cellular proliferation, and is a good predictive and prognostic marker widely used in clinical practice of breast cancer –7. StudiThere is no standard method yet to assess Ki67 in breast cancer, and there are still many technical issues to be resolved, such as how to count positive cells, how to select scoring areas. In 2011, the International Ki67 in Breast Cancer working group published recommendations for Ki67 assessment in breast cancer. The guideline aimed for better analysis, reporting, and use of Ki67 that should minimize interlaboratory variability and improve inter-study comparability of Ki67 results. This includes using an overall average score and scoring 1000 cancer cells, with 500 cells as the absolute minimum . HoweverBiological heterogeneity of Ki67 staining can occur across breast cancer specimens, and the location and extent of the area of the cancer that should be scored is controversial, which has been an important reason of the low interobserver reproducibility. Excellent concordance has been reported when the same area is counted using printed photographs . The appOur study also showed that the staining distribution of Ki67 was associated with the reproducibility of assessment. Ki67 staining was homogenous in some slides and heterogeneous in others. According to paired-difference between means of hot-spot score and average score of each case, all cases were divided into three groups . Smaller paired-difference meant more homogenous in staining. The consistency among five pathologists was observed to decrease with increasing paired- difference according to both methods. A best agreement was demonstrated in d<5 group compared with 5≤d<10 and d≥10 groups among five pathologists. Our study indicated that biological heterogeneity of Ki67 staining is an important reason of the low interobserver reproducibility, and observers may reach better Ki67 LI agreement in homogenous staining slides than in heterogeneous staining ones.Clinical decision-making in breast cancer treatment often relies on a Ki67 cutoff to classify patients into “Ki67 high” or “Ki67 low” risk groups. The choice of the cutoff has a major impact in practice, as it may determine which patients receive more aggressive therapy. However, discrete cutoff values to distinguish high from low Ki67 expression have not been clearly established and remain controversial ,18–20. MIn conclusion, our study suggested that visual assessment of Ki67 LI on IHC staining at 10% intervals is a candidate for a standard method in breast cancer clinical practice. Average score and hot-spot score of visual assessment both demonstrated a perfect concordance of Ki67 LI, and an overall average assessment across the whole slide including hot spots may be a better visual assessment method in routine practice. Interobserver concordance of intermediate Ki67 LI in which most cutoffs are located for making clinical decisions was relatively low. Well-validated methodologies to evaluate the “gray zone” around the cutoff points of Ki67 may allow more accurate risk estimation and therefore better clinical management.S1 Dataset(XLS)Click here for additional data file.


Dendroctonus ponderosae Hopkins) is an important disturbance agent of western North American forests and recent outbreaks have affected tens of millions of hectares of trees. Most western North American pines (Pinus spp.) are hosts and are successfully attacked by mountain pine beetles whereas a handful of pine species are not suitable hosts and are rarely attacked. How pioneering females locate host trees is not well understood, with prevailing theory involving random landings and/or visual cues. Here we show that female mountain pine beetles orient toward volatile organic compounds (VOCs) from host limber pine (Pinus flexilis James) and away from VOCs of non-host Great Basin bristlecone pine (Pinus longaeva Bailey) in a Y-tube olfactometer. When presented with VOCs of both trees, females overwhelmingly choose limber pine over Great Basin bristlecone pine. Analysis of VOCs collected from co-occurring limber and Great Basin bristlecone pine trees revealed only a few quantitative differences. Noticeable differences included the monoterpenes 3-carene and D-limonene which were produced in greater amounts by host limber pine. We found no evidence that 3-carene is important for beetles when selecting trees, it was not attractive alone and its addition to Great Basin bristlecone pine VOCs did not alter female selection. However, addition of D-limonene to Great Basin bristlecone pine VOCs disrupted the ability of beetles to distinguish between tree species. When presented alone, D-limonene did not affect behavior, suggesting that the response is mediated by multiple compounds. A better understanding of host selection by mountain pine beetles could improve strategies for managing this important forest insect. Moreover, elucidating how Great Basin bristlecone pine escapes attack by mountain pine beetles could provide insight into mechanisms underlying the incredible longevity of this tree species.The tree-killing mountain pine beetle ( Dendroctonus ponderosae Hopkins) is one of the most ecologically and socioeconomically important forest insects in North America. Outbreaks of this native insect during the early 21st century have been extensive, with over 3.5 million hectares of tree mortality in 2009 alone [The mountain pine beetle  and the non-host Great Basin bristlecone pine (Pinus longaeva Bailey). Limber pine is a favored and highly-productive host of MPBs [P. flexilis/P. longaeva Series” [In this study, we investigated whether pioneering female MPBs use VOCs to choose between the host limber pine ( of MPBs  and limb of MPBs . In cont of MPBs  and conc Series” ). These  Series” , includi Series” . This stThe objectives of this work were to 1) collect and analyze VOCs of co-occurring limber and bristlecone pines as potential foraging cues for the MPB, and 2) assess the behavioral responses of female MPBs to limber and bristlecone pine VOCs in a Y-tube olfactometer. We also explored the role of candidate individual volatile compounds in the behavioral response of MPBs. We hypothesized that VOCs differ between tree species and serve as a readily available cue that foraging MPBs can use in host finding.Pinus contorta Dougl.) infested with MPB larvae from the Bear River Range of Northern Utah  and from the Caribou-Targhee National Forest in Southern Idaho . Sections from the bole of the trees (~60 cm long) were transported to the US Forest Service’s Rocky Mountain Research Station laboratory in Logan, UT and ends sealed with paraffin wax to reduce desiccation. The sections were placed in Percival incubator cabinets (12 hours of light per 24-hour cycle) at room temperature (ca. 21°C) to facilitate larval development to the adult stage (approximately 70 to 80 days). Emerging adults were collected daily and placed in petri dishes with moistened filter paper and stored in a refrigerator at approximately 3°C until use. Gender was determined using characters on the seventh abdominal tergite [Adult mountain pine beetles were obtained from two locations infested with MPB (separated by about 90 km) by felling two mature lodgepole pines  and Wheeler Peak, NV  by cutting branches approximately 50 cm in length from a randomly selected bristlecone pine and limber pine from each site, and refrigerating them in sealed plastic bags at approximately 3°C until use. VOCs from these samples were collected and analyzed at time of use as described below. The USDA Forest Service, Humboldt-Toiyabe and Caribou-Targhee National Forests, and the Utah Division of Forestry, Fire, and State Lands granted permission for use of all field sites.VOCs were collected from co-occurring limber pine and bristlecone pine trees of similar size in the Spring Mountains near Las Vegas, Nevada  and on Cave Mountain near Ely, Nevada . Trees of similar size were selected for sampling and VOCs were collected from lower branches (≤ 3 m above ground) which correspond to the heights at which dispersing MPBs fly ,27. ApprVOCs were eluted from traps using 200 μl of dichloromethane and 1,000 ng of n-nonyl-acetate added as an internal standard. Samples were analyzed using an Agilent 7890A gas chromatograph (GC) coupled with a 5975C mass spectrometer and separated on a HP-1ms  column; helium was used as the carrier gas. The GC oven was maintained at 35°C for 3 minutes and then increased by 5°C per minute to 125°C, then 25°C per minute to 250°C. Quantifications were made relative to internal standards using ChemStation software , and identifications of compounds confirmed by comparing retention times and mass spectra to commercial standards. Measurements of VOC emissions (ng per hour per gram) were on a fresh weight basis.Statistical analyses for VOCs were performed using the non-parametric Kruskal-Wallis one-way analysis of variance  using R We used a Y-tube olfactometer to investigate the response of adult female MPBs to airborne cues, following the methodology of others . Y-tubeTrials were conducted in a greenhouse at temperatures between 20–27°C. Mountain pine beetles are positively phototactic , so to an-hexane ; 500 μl of 200 ng/μl hexane solution added to each septum. Control septa were treated with 500 μl of n-hexane only. Treated and control septa were left in a fume hood at room temperature and release rates checked each day as described above. Release rates of both compounds responded similarly: amounts released mimicked that of limber pine foliage used in Y-tube trials on day 4 after treatment for D-limonene (approximately 80 ng per hr) and day 5 after treatment for 3-carene (approximately 50 ng per hr) after treatment. We collected and analyzed a small number of foliage samples with the commercial compound added to verify that the target compound was present and in greater abundance.We used rubber septa treated with synthetic VOCs to test how individual compounds affect beetle behavior following methods outlined by Runyon et al. . We chosStatistical analyses for Y-tube trials were performed using chi-square tests with the Yate’s continuity correction for small sample sizes : for eacP < 0.05; The VOCs emitted by Great Basin bristlecone pine and limber pine at Cave Mountain were similar. Both species emitted the same 28 VOCs  and diffAdult female MPBs overwhelmingly chose the Y-tube arm with limber pine VOCs over the arm with clean air Click here for additional data file.S2 AppendixVOCs reported in ng per hr per gram.(XLSX)Click here for additional data file.S1 FigNote different scale for α-pinene.(EPS)Click here for additional data file.


The data suggest that when used quantitatively the TRFLP protocol tested is a powerful, repeatable method for AM fungal community analysis. However, we suggest some limitations to its use for population-level analyses. We found no evidence of PCR bias, supporting the quantitative use of other PCR-based methods for the study of AM fungi such as next generation amplicon sequencing. This finding greatly improves our confidence in methods that quantitatively examine AM fungal communities, providing a greater understanding of the ecology of these important fungi.Arbuscular mycorrhizal (AM) fungi are widely distributed microbes that form obligate symbioses with the majority of terrestrial plants, altering nutrient transfers between soils and plants, thereby profoundly affecting plant growth and ecosystem properties. Molecular methods are commonly used in the study of AM fungal communities. However, the biases associated with PCR amplification of these organisms and their ability to be utilized quantitatively has never been fully tested. We used Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to characterise artificial community templates containing known quantities of defined AM fungal genotypes. This was compared to a parallel  Arbuscular mycorrhizal (AM) fungi form the most abundant symbiosis on earth, colonising the roots of approximately two thirds of terrestrial plants One of the most widely used DNA-based methods is the analysis of Terminal Restriction Fragment Length Polymorphisms  to study AM fungal communities  were amplified with the universal eukaryotic primer NS31 Escherichia coli cells by incubation with 25µl competent E. coli cells  at 4°C for 30 minutes, followed by a heatshock of 42°C for 45 seconds and rotary incubation with 475µl Super Optimal broth with Catabolite repression (SOC) at 37°C for 1 hour. The resulting bacteria were plated onto LB-agar amended with X-gal and ampicillin. These were incubated at 37°C overnight before storing at 4°C. Putative positive transformants were amplified using primers SP6 and T7  using primer SP6 and BigDyeTM terminator cycling conditions on an ABI 3130 capillary sequencer . Sequences were aligned using ClustalX in silico suggested they would each produce uniquely sized TRFs with each of two enzymes commonly used in TRFLP analyses: Alu-I and Hinf-I; 2) the AM1/NS31 insert had the same orientation within the plasmid; 3) each genotype showed ≤97% pairwise sequence similarity to all other genotypes used, reflecting commonly used cut-offs for defining Operational Taxonomic Units (OTUs). These sequences have been submitted to the European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database (accession numbers KJ749695-KJ749700).Artificial community templates were produced by mixing PCR products from 18S rDNA clone libraries produced for a study of AM fungi in soybean roots To generate sufficient genetic material to produce the templates, PCR products derived from the chosen genotypes were amplified again using the same protocol as before (with primers SP6 and T7), but with only 15 cycles to reduce the likelihood of errors. The resulting PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and divided into three separate aliquots and the concentration of DNA in each was measured in triplicate using a nanodrop-1000 spectrophotometer .Six different artificial community templates were produced by combining four, five or six of the genotypes in different proportions . These dAll artificial communities were characterised using TRFLPs. This was performed three times on each of the 27 templates to provide technical replicates to examine the repeatability of the protocol. To confirm that each genotype produced the predicted TRFs, the three aliquots of each genotype were also analysed. All templates were analysed by amplifying a 550 bp partial fragment of SSU rDNA by PCR using HotstarTaq Plus DNA polymerase (Qiagen) with primers AM1 and NS31 labelled on the 5′ end with the fluorescent markers HEX and FAM respectively . The 25µl reactions contained 0.64 mM dNTPs, 0.2µM of each primer, 0.5 mg dried skimmed milk powder  and the manufacturer's reaction buffer . PCR products were cleaned using a QIAquick PCR Purification Kit (Qiagen) and the concentrations of DNA they contained were measured using a nanodrop-1000 spectrophotometer.Hinf-I and Alu-I (Promega) in a reaction volume of 15µl, with manufacturer's buffer and 2µg BSA for 15 hours. Restriction products were cleaned using a QIAquick PCR Purification Kit (Qiagen). A size standard (GS600-LIZ) was added and samples were run on an ABI 3130 genetic analyzer (Applied Biosystems Inc.). GeneMapper v4.0 software (Applied Biosystems Inc.) was used to determine peak size and quantity. Peaks greater than 100 fluorescent units in height representing TRFs longer than 100 base pairs (bp) were analysed using a bin width of 2 bp and the local southern size calling method. Peak area was used to measure TRF abundance rather than peak height, as using peak height can down-weight longer fragments as they produce wider peaks due to their longer retention time during electrophoresis Alu-I digestion and FAM labelled TRFs from Hinf-I digestion were further analysed, as other TRFs were predicted to be undetectable and uninformative as they were identical for all genotypes and <50 bp.Eight microlitres of each cleaned PCR product were separately digested using five units of in silico digestion (using Hinf-I and Alu-I cutting sites) of the six distinct AM fungal genotype sequences. The resulting virtual TRFs were then combined in the same ratios as the artificial community templates. This produced nine predicted theoretical AM fungal communities with which to compare the observed artificial communities empirically analysed using TRFLPs. In addition, the size of possible under-digested terminal fragments, formed where the enzymes did not cleave the DNA at all possible cutting sites . ANOVAs and Tukey's honestly significant difference tests were used to compare the templates' observed PC1 and PC2 scores to determine if they could be differentiated from each other using these community characterisation measures. Identical analyses were also performed on the predicted data.Principal component analysis (PCA) was used to examine whether the TRFLP protocol used could differentiate between the nine artificial communities, when analysed using ordination based on raw species abundance matrices. Covariance PCA was separately performed on the observed data from the empirically tested templates and the predicted data based on All possible Euclidean distances between the PC scores of all possible pairs of templates from the observed data were calculated and identical calculations were also performed on the predicted data. To determine the relationship between observed and predicted TRFLP data, the two sets of distances were then compared with a Mantel test . Bray Curtis dissimilarity coefficients between pairs of templates were also calculated for all predicted and observed results and similarly compared with a Mantel test, to evaluate TRFLP data as a quantitative measure of community differences.Margalef's and Simpson's diversity indices were calculated from both the predicted and observed TRFs to determine how accurately the TRFLP protocol predicted the communities' α diversity levels. These indices were chosen as Margalef's index is primarily influenced by the richness of a community whereas Simpson's is primarily influenced by evenness. The relationship between predicted and observed indices were tested using linear regression.Unless stated otherwise, all analyses were conducted using the R statistical language with the ‘vegan’ and ‘ade4’ libraries Alu-I and unique FAM labelled fragments with enzyme Hinf-I (There was little variation in length (527 bp–548 bp) of the AM1/NS31 amplicons generated and all sequences had low GC content (39.7%–41.7%), typical of AM fungal AM1/NS31 amplicons. Genotype 2 was similar to sequences from voucher specimens of Gigasporaceae whereas the remaining five genotypes were from the Glomeraceae. The AM1/NS31 amplicon of sequences differed from each other by an average of 9.7% . Genotypes were predicted to produce unique HEX labelled TRFs when digested with enzyme e Hinf-I .Hinf-I digested amplicons derived from single genotypes showed that an average of 99.2% of the total fragments produced were detected as a single peak, representing the same size fragment as that predicted by the theoretical digests. The sizes of the remaining TRFs corresponded to predicted pseudo-TRFs, which the in silico analysis suggested would be produced if the sequences had not fully digested  which corresponded to the sizes of predicted pseudo-TRFs . Tukey's honestly significant difference tests showed all templates could be differentiated from each other using either or both their PC1 or PC2 scores . Comparisons of predicted and observed Bray Curtis dissimilarity coefficients across templates also produced a significant positive relationship .Comparison of predicted and observed Euclidean distances between PC scores across templates produced a significant positive relationship  may over inflate richness estimates in silico digestions (The observed pseudo-TRFs show that even when using TRFLPs qualitatively, with ‘diagnostic peaks' being used to indicate the presence of a particular genotype in a sample (e.g. as used in ref. gestions , making Finally, when applying the conclusions of this experiment to future studies, standard methodological caveats on the biological interpretation of molecular analyses must also be applied. All TRFLP and NGS analyses measure the relative abundances of fungal taxa within samples, and should therefore not be used to infer differences in absolute abundances of taxa across samples. Furthermore, AM1 and NS31 are thought to amplify the vast majority of AM fungi, but exclude the Paraglomeraceae This investigation has shown that TRFLPs can be used quantitatively to discriminate between different AM fungal communities containing the same genotypes in different relative abundances, and accurately measure the differences between them. In addition we have shown that the method is robust and reliable with little error in repeatability. The protocol described therefore has great potential to enhance our understanding of a wide variety of aspects of AM fungal ecology. The results also suggest there was no PCR bias in the protocol, supporting the use of AM fungal relative abundance data from other PCR based methods such as NGS.