Prostate carcinoma (PRAD) is one of the most frequently diagnosed malignancies amongst men worldwide. It is well-known that androgen receptor (AR) plays a pivotal role in a vast majority of prostate tumors. However, recent evidence emerged stating that estrogen receptors (ERs) may also contribute to prostate tumor development. Moreover, progression and aggressiveness of prostate cancer may be associated with differential expression genes of epithelial-to-mesenchymal transition (EMT). Therefore we aimed to assess the significance of receptors status as well as EMT marker genes expression among PRAD patients in accordance to their age and Gleason score.We analyzed TCGA gene expression profiles of 497 prostate tumor samples according to 43 genes involved in EMT and 3 hormone receptor genes  as well as clinical characteristic of cancer patients. Then patients were divided into four groups according to their age and 5 groups according to Gleason score. Next, we evaluated PRAD samples according to relationship between the set of variables in different combinations and compared differential expression in subsequent groups of patients. The analysis was applied using R packages: FactoMineR, gplots, RColorBrewer and NMF.MFA analysis resulted in distinct grouping of PRAD patients into four age categories according to expression level of AR, ESR1 and ESR2 with the most distinct group of age less than 50 years old. Further investigations indicated opposite expression profiles of EMT markers between different age groups as well as strong association of EMT gene expression with Gleason score. We found that depending on age of prostate cancer patients and Gleason score EMT genes with distinctly altered expression are: KRT18, KRT19, MUC1 and COL4A1, CTNNB1, SNAI2, ZEB1 and MMP3.Our major observation is that prostate cancer from patients under 50 years old compared to older ones has entirely different EMT gene expression profiles showing potentially more aggressive invasive phenotype, despite Gleason score classification. Prostate adenocarcinoma (PRAD) is second the most common solid neoplasm worldwide  .The current PRAD grading system based on histological expansion rate was developed between 1966 and 1974 by Donald Gleason et al. . Despitein vivo significance of EMT with respect to clinical course of the disease, however there are still some discrepancies, which should be investigated [Epithelial-to-mesenchymal transition (EMT) is a basic mechanism that plays a central role in development, tissue regeneration, architecture and remodeling in physiology as well as pathological migratory properties of cancer cells. In greater detail, EMT describes a process of loss of epithelial character towards gain of mesenchymal properties. This is done mainly due to loss of cell-cell communication, adhesion and reorganization of cytoskeleton leading to a switch from apical-basal to front-rear polarity of cells. Therefore, the EMT may be considered as molecular mechanism of acquisition of invasive properties as well as enhancing tumor aggressiveness and migratory potential through involvement of regulatory pathways (mainly transforming growth factor β (TGF-β), phospahtydylinositol-3-kinase (PI3K) and mitogen-associated protein kinase (MAPK)) and particular genes  . In factstigated .The association between patient age and aggressiveness (and hence Gleason score) of prostate tumor has not been well investigated so far. There are many reports suggesting that younger men experience PRAD of completely different biology compared to those diagnosed at higher age . This raSignaling via androgen receptor (AR) plays a pivotal role in both, the development and function of normal prostate gland as well as tumorigenesis of the prostate. Canonical signaling through AR comprises modulation of transcriptional activity of particular genes through AR nuclear translocation and binding to androgen response elements (AREs) on its targets followed by recruitment or crosstalk with various transcription factors (Tfs). SurprisTo date, several studies indicated that sex hormones such as testosterone and estradiol are declining with age in males. More particularly, androgen levels and estrogen levels are unbalanced in men of higher age, it has been showed that androgen loses its significance in favor of estrogen followed by altered body composition and increased BMI in olders .Considering the fact that the levels of androgens and estrogens in men change with age and taking into account the impact of estrogen and androgen hormones in PRAD progression and development, we have studied biological differences of PRAD between younger and older patients. Moreover, we also examined the significance of EMT and signaling via AR and both ERs status in prostate according to patient age and Gleason score.AR, ESR1, ESR2 in the biology of prostate cancer regarding the effect of their expression on tumor recurrence according to patient’s age  through disease-free survival (DFS) analysis. We found a shift in the significance of the receptors for tumor recurrence; higher rates of AR expression are unfavorable for prostate tumor in younger patients, however it loses its significance in the oldest group, in contrast to ESR1, where higher expression is generally unfavorable. In greater detail, we identified lowered expression of AR as favorable in groups of ≤50 and 51–60 years old , whereas in groups of older patients (61–70 and 70> years old) AR lost its significance. In contrast, ESR1 did not show any importance in groups of ≤50 and 70> years old in tumor recurrence, although its lowered expression was associated with better prognosis in remaining groups of 51–60 and 61–70 years old . Finally, lowered expression of ESR2 correlated with good prognosis in group of 51–60 years old , whereas relatively higher expression was favorable in group of 61–70 years old  while not relevant in the youngest and oldest patients. Summarized results are presented in Firstly, we examined the significance of three hormone receptors: Subsequently, we focused on biological differences between prostate tumors regarding sets of variables such as patient age, Gleason score as well as hormone receptors expression. Patients were dimensionally partitioned by applying the Multiple Factor Analysis (MFA).AR, ESR1 and ESR2, patient age and Gleason score as supplementary variable. The analysis resulted in plots of individuals (patients) shown as ESR1 was unfavorable for tumor recurrence, thus it may indicate that the shift from a high AR expression in patients aged ≤50 years old towards high expression of ESR1 in patients aged 70> years old is caused by the occurrence of andropause in the oldest group  through the insight into expression of EMT marker genes in correlation with patients age and Gleason score. Primarily, we selected from the literature 43 well-known EMT markers . Subsequst males .Furthermore, from the initial 43 EMT markers we subsequently focused on the 31 genes , which were mostly differentiating patients ≤50 vs 70> years old. As can be seen in the Subsequently we evaluated epithelial apart from mesenchymal state markers and their separate contribution to biology of the tumors in respect to patients age as well as dimensional partitioning of PRAD cases. The variant considering all divided EMT markers enabled us to identify two phenomena: first, reversed profiles between epithelial and mesenchymal states especially in groups of ≤50 and 70> years old  and secoa priori should be true). As expected, we found that expression of selected EMT markers pretty well differentiates Gleason scores into separate partitions along with the dimensions with 29.03% of total variability, thus bearing out the strong relationship between tumor aggressiveness characterized on the one hand by expression of EMT markers and on the other, by clinical parameter—Gleason score  cohort from cBioportal due to the sufficient number of patients and available information of Gleason score. Those gene expression data comes from different cohort of patients and were obtained using microarray analysis. Although patients with Gleason 10 are missing, this analysis confirmed general tendency that profiles of EMT markers are distinct among Gleason score especially in patient with high Gleason score . SeveralCTNNB1, CDH1, SMAD2, SMAD3, TCF3, LEF1 in younger patients and overexpression of SNAI1 and underexpression of KRT5, KRT19, OCLN, CDH2 and MUC1 which clearly indicates different characteristics of prostate tumor in younger vs older patients  according to patients age. We observed that the same Gleason score differs in the EMT signature according to patient age. In particular, we found primarily overexpression of patients .TP53 gene, as we should suspect. Second in mutation rate is androgen receptor, the castrate resistant cancer, thought there is less than 10% mutations found in COSMIC reported prostate cancer. Therefore, as in the most sporadic cancers there is no specific highly represented mutations in PRAD. Nevertheless, in the era of personalized medicine, even relatively rare mutations can be important in treatment profiling.Cancer has been recently considered as genetic disorder, which results from many alterations at molecular level with mutations among others. To complete the insight into prostate tumorigenesis biology we additionally examined the significance of the mutations borne by patients, although their role seems marginal . Tables In our study we aimed to evaluate DFS prognostic effect of androgen and estrogen receptors gene expression according to patient age in prostate cancer. Multifaceted association of receptors gene expression with DFS inclined us to explore biological and clinical differences between age groups of patients with prostate adenocarcinoma. For this purpose we analyzed epithelial to mesenchymal transition marker genes which are responsible for cancer cell aggressiveness, metastasis and poor prognosis in various tumors. We performed multiple factor analysis to integrate the relationship between the groups of variables  describing the individuals (patients). Our results showed that accordingly to patient age, gene expression and Gleason score, the most diverse group form patients below 50 years old.AR is critical for development and proliferation of androgen-refractory prostate tumor cell lines LNCaP-Rf and LNCaP-C4 [AR overexpression and amplification in prostate cancer cells has been shown to correlate with lower recurrence-free survival rates [AR in malignant epithelial cells and a reduction of AR positive nuclear in peritumoral stromal cells had increased risk of relapse following radical prostatectomy [AR decreased expression is significantly associated with better DFS prognosis but only for relatively young patients below 50 years old and for the age group 51–60. Moreover, for patients under 50 years old, AR expression profile points to unfavorable prognosis due to the fact that in this group AR expression is upregulated in comparison to normal adjacent tissue –26. In pnd LNCaP  but alsond LNCaP . Moreoveer cells . Gene pond stage . Studiesnd stage . Furthergression . Our anaor genes . Moreoveor genes , howeverpatients .We also investigated the mechanism of epithelial-to-mesenchymal transition and have looked into differences in the expression of EMT genes according to patient age and Gleason score. Since it is well known that Gleason score is a clinical representation of aggressiveness, it should be a direct association between Gleason grade and EM transition. And it is in fact, that that Gleason grades are clearly separated from the other according to the EMT genes, with Gleason 10 as the most distant phenotype  but at the same time much more mesenchymal markers which are targets of SNAI1 are increased   .ZEB1 and SNAI1 as well as many mesenchymal markers like VIM, CDH2, MMP2, MMP3, MMP9 are downregulated. But at the same time these patients are characterized by overexpression of the adhesion proteins CDH1 and CTNNB1 which according to the literature could be advantageous for invasiveness and colonization. On the other hand, reversed profiles of epithelial and mesenchymal markers could be seen in older prostate cancer patients. In cancers from patients older than 70 years more significant seems to be the upregulation of key transcription factor SNAI1 and decreased expression of genes responsible for epithelial phenotype KRT19, CDH1, OCLN and MUC1. Summarizing, our analysis shows distinct and biologically significant differences in expression profile of sex hormone receptors as well as epithelial to mesenchymal transition genes that are associated with disease progression depicted as Gleason score. However, despite of Gleason score, distinct profiles of EMT genes expression may explain more aggressive phenotype of prostate cancer in younger patients.Metalloproteases are important factors involved in degradation of extracellular matrix and basement membrane thus contribute to the alteration of cell-matrix adhesion ,59. Comphttp://cancergenome.nih.gov/, data status of Jan 28, 2016). The methods of biospecimen procurement, RNA isolation and RNA sequencing were previously described by The Cancer Genome Atlas Research Network [We performed The Cancer Genome Atlas (TCGA) RNA-Seq expression profiling  and clinical characteristics of 499 PRAD patients  regarding recurrence of the disease, we divided patients into four groups in accordance to their age: ≤50 y.o. (35 patients), 51–60 y.o. (188 patients), 61–70 y.o. (237 patients) and 70> y.o. (37 patients). Clinical characteristics of cohort patients are shown in To determine the relevance of patient age and its associations with hormone receptors  by applying the DFS analysis. According to the calculated cutoff points we were able to split patients into subgroups of favorable/unfavorable recurrence prognosis with regards to the expression level of particular hormone receptor. The analysis was performed separately for each age subset using freely available Cutoff Finder R script [We examined the significance of hormone receptors , and EMT marker genes. Primarily, 43 genes known as markers of epithelial-to-mesenchymal transition have been selected from the literature  as well as Gleason score (supplementary variable); 2) partitioning regarding the age groups and combined effect of 43 EMT marker genes as well as Gleason score (supplementary variable); 3) partitioning regarding the age groups and combined effect of epithelial/mesenchymal state markers; 4) partitioning regarding the Gleason score and EMT marker genes. The MFA was applied using packages: FactoMineR and factoextra [R environment [The MFA was conducted in four variants according to the following parameters: 1) partitioning regarding the age groups and combined effect of expression of hormone receptors  study which is available in cBioPortal. The clinical parameter not include age information, nevertheless we could perform hierarchical clustering analysis using Gleason score information. To determine the relevance of patients Gleason score and its association with EMT marker genes we divided patients into four groups according to their Gleason score: Gleason 6 (41 patients), Gleason 7 (76 patients), Gleason 8 (11 patients), Gleason 9 (11 patients).http://www.cbioportal.org/) tools. The results have been validated with COSMIC database .Additionally, we analyzed PRAD patients regarding mutations using cBioPortal Click here for additional data file.S2 FigCDH1, CTNNB1, MMP3, KRT5, SMAD3 with simultaneous lowered expression of KRT18, KRT19, VIM, MMP2, COL4A1, CDH11, CDH2, ITGA5 (D).The expression of EMT markers indicates partition of PRAD cases into patients age (A), however no associations in Gleason score (B). The projections along Dim2 show significant contribution of EMT in distinct partitioning of patients of age ≤50 years old (C). The grouping results from opposite profiles of expression of particular EMT markers in the youngest vs the oldest patients: heightened expression of (TIFF)Click here for additional data file.S3 FigThe expression of epithelial (A) vs mesenchymal (B) state markers in separate partitions PRAD patients oppositely. The projections representing contribution of epithelial (C) and mesenchymal (D) state markers along the dimensions indicate significant shit in the EMT model in the age groups, especially in the youngest and the oldest men. In addition, the shift towards more aggressive mesenchymal character of the tumor is observable in the contrasting expression of particular genes involved in the EMT (E) epithelial state markers, F) mesenchymal state markers).(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.


Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on “Unconventional Protein and Membrane Traffic” (UPMT) during 4–7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes. The definition of the conventional secretory pathway arose from studies realized during the 1960s and 1970s  pathways where these proteins bypass intermediate compartments involved in secretion or exocytosis, such as the Golgi apparatus . CurrentDuring the four days, the meeting was characterized by presentations grouped in seven sessions to cover topics related to the definition of UPS in a broad sense, and these presentations are summarized in this report organized into five sections from . The speMost eukaryotic leaderless cytoplasmic proteins reach the cell surface by UPS. Such proteins include members of the Annexin family, cytoskeletal proteins, Heat Shock Proteins, members of the interleukin family, fibroblast growth factors, and others . They discussed unpublished data on the direct transport of the cytoplasmic protein fibroblast growth factor 2 across the PM. This transport, as reported by Nickel , requireAnother likely example of direct translocation by pore formation was showed by Kevin Moreau  regarding the transport to the cell surface of Annexin A2 and Galectin-3. Cell surface Annexin A2 acts as a plasminogen receptor able to stimulate fibrinolysis and cell migration through the extracellular matrix proteolysis while cell surface Galectin-3 serves as a cell-to-cell and cell-to-matrix adhesion/interaction molecule . He presMembers of the interleukin family are a case study for this kind of UPS. Interleukin-1β is an essential mediator of inflammation after infection and injury. This cytokine is produced as an inactive precursor, which must be cleaved to its active form via a molecular complex called the inflammasome. Interleukin-1β is released from the cell via an unknown process. The group of David Brough  discussed its recent publication about the release of Interleukin-1β via a mechanism that requires membrane permeabilization . The mecInterleukin-1β can be also be secreted by a vesicular mean of transport. In human monocytes, Interleukin-1β secretion is mediated by secretory lysosomes, induced by exogenous ATP, once more underlining the complex crosstalk among different secretory mechanisms . HoweverAutophagic vesicles could be also used for unconventional protein secretion from the cell. For example, William Jackson  described how Poliovirus 1, a Picornavirus responsible for human disease, uses autophagic vesicles to promote virus replication and exit from the cell . In partA novel compartment, formed by a tubulovesicular structure surrounded by a cup-shaped membrane, named compartment for unconventional protein secretion (CUPS), is induced in yeast by nutrient starvation triggering secretion of the signal sequence-lacking Acb1 protein. The biogenesis of the compartment for unconventional protein secretion requires several proteins, including an ER exit site/Golgi resident protein, Grh1. Amy Curwin  from the Malhotra lab reported that the major endosomal sorting complex required for transport-III subunit Snf7 localizes transiently to this novel compartment, and promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature compartment for unconventional protein secretion containing Acb1 ,37. The Ustilago maydis that lacks a classical N-terminal secretion signal but is secreted in the fragmentation zone between mother and daughter cells during cytokinesis [N-glycosylation signal, but—crucially—glycosylation largely inactivates the enzyme. Stock et al. realized that this feature could be harnessed to prove that a secreted protein fused to GUS does indeed bypass the ER [U. maydis for protein production. Indeed, inappropriate N-glycosylation can be a problem in the heterologous production of pharmaceutical proteins. Cts1 can act as a carrier to avoid unwanted ER processing whilst retaining the advantages of protein secretion for facilitated downstream processing. Another leaderless mammalian protein, indoleamine 2,3-dioxygenase 1, seems to be secreted by UPS, even if the exact unconventional mechanism is still unknown. Maria Teresa Pallotta  presented unpublished data regarding this protein, an enzyme that exerts regulatory functions in autoimmune and inflammatory settings [Kerstin Schipper  presented new data on the chitinase Cts1 from the fungus okinesis . Her teaokinesis . The bacs the ER . Schippesettings . She demIt is clear from the above paragraphs that the UPS pathways of leaderless proteins to the extra cellular space are important for the crucial roles of these proteins in normal physiology as well as in human diseases, but also in innate immune response. The majority of examples come from mammalian and yeast biology; therefore, to further characterize these pathways, more research efforts are needed in other disciplines like for example plant biology.UPS can take also place by a transportation mode in which both soluble and membrane bound proteins initially enter the conventional secretory pathway whether through ER translocation as they possess ER-targeting signals or through ER association because they are associated with the cytosolic side of ER membranes. Then, these proteins could be subsequently transported to the cell surface or to the vacuole bypassing the Golgi apparatus. There are some examples of animal proteins transported through this kind of pathway , but mucMature autophagosomes, the autophagy organelle, are double-membrane vesicles decorated with the autophagy-related (ATG) protein ATG8 on both surfaces. Autophagosomes, moving along the cytoskeletal network, are transported to the tonoplast with which they fuse to deliver autophagic bodies for enzymatic recycling, or to the PM for secretion. These autophagy pathways can be also used in the cell for other purposes. For example, Alexandra Boeske, from Dieter Willbold’s lab , discussed the function of ATG8 homologs GABARAP proteins in HIV-1 Nef protein pathogenesis and its unconventional secretion as a follow-up inquiry into previously identified HIV-1 Nef interactors. To get the signal peptide lacking HIV-1 Nef protein out of the cell it needs at least one of GABARAP paralogs to be active, forming a complex with HIV-1 Nef in perinuclear region from where it is secreted to the extracellular space via PM localized patches. Interestingly, also GABARAP non-binding mutant HIV-1 Nef was detected within extracellular vesicles indicating a parallel mechanism of HIV-1 Nef GABARAP dependent export, however not dependent on the direct GABARP–HIV-1 Nef interaction. Interestingly, it was recently shown that, in transfected cells, a proportion of Nef localized to ER membranes where Nef physically interacted with the ER chaperone calnexin .N-glycoproteome of the rat lysosomal and plasma membranes is much higher than that of the plant tonoplast, and is very similar to that of the Arabidopsis thaliana PM [Hordeum vulgare Vacuolar Protein Sorting 24 (HvVPS24) has a putative protein sorting role in seed storage protein trafficking in barley endosperm [A new perspective about direct ER to vacuole import  was openliana PM . Emanuelliana PM ,47,48,49liana PM . For exaliana PM . Interesndosperm . HvVPS24A. thaliana. In post-invasive immunity, moreover, VPS9a organizes and mediates cellular endomembrane trafficking promoting the correct delivery of membrane material, likely also from the ER membrane, to the encasement structure.Studies on plant-pathogens interactions are producing very interesting results on UPS occurrence in plants mediated by exosome release. Many filamentous pathogens enter plant cells by penetrating the host cell wall. Then they initiate intracellular feeding structures, called haustoria, by invagination of the plant’s PM. Haustorial penetration generates structural and biochemical cascade responses in the plant cells, as a part of the plant innate immune system, which leads to the formation of plants defense structures. During the resistance response a dome-shaped cell wall apposition, named “papilla”, is formed in the outer cell wall of epidermal cells at the site of penetration, which successively evolves to encase the entire haustorial body. Exosome-mediated secretion contributes to papilla formation and multivesicular bodies are often abundant in the plant cytoplasm around papilla ,54. MadsIn both animals and plants, increasing numbers of possible cargoes for the pathways discussed in this section have been discovered. These are often related to the defense against pathogens and, in plants, to transport of seed storage proteins in vacuoles. In all reports unpublished data were in the center of presentations and indicated increasing awareness of cell biology community both working on animals as well as on plant models to the central importance of the endomembrane system and autophagy related processes not only in cytoplasm/proteins degradation, but especially in the formation of endomembrane containers involved also in the UPS.Intercellular channels represent a pathway for the transport of proteins, RNA and other macromolecules, largely independent of conventional secretory pathway. These have been identified in both plants and animals where they function in development, response to abiotic stress conditions and diseases. In plants, intercellular channels known as plasmodesmata are inserted in cell wall domains enriched in the polysaccharide callose providing membrane and cytoplasmic continuity for symplastic molecular transport ,56. An aAn entire focus session, chaired by Yoselin Benitez-Alfonso , was dedicated to this topic during the meeting. Emmanuelle Bayer  focused on plasmodesmata specialized membrane organization. Electron tomography micrographs revealed details on plasmodesmata ultrastructure as never seen before. Their work shows that within the pores, ER-PM junctions undergo substantial remodeling during cell differentiation and tissue growth that vary from direct membrane contact to intermembrane gap of about 10 nm spanned by spokes. They showed that in newly divided cells, plasmodesmata displayed almost non-existent space between the desmotubule and the plasmodesmata suggesting the absence of cytoplasmic sleeve. Intriguingly, transport of macromolecules was still effective across this type of connections suggesting that there is no simple correlation between ER-PM spacing and the extent of cell-to-cell connectivity. Their work questioned the function of membrane contacts within plasmodesmata.Chris Hawes  presented data on the role of reticulons in cell plates and plasmodesmata formation, suggesting that specific members of the reticulon family (RTN3 and RTN6) target mature plasmodesmata and developing cell plate . StudiesThe role of the ER-actin transport network in virus movement through plasmodesmata was presented by Manfred Heinlein . Tobamoviruses, such as the Tobacco mosaic virus, are transported in the form of viral replication complexes that target plasmodesmata by means of its movement protein . The forYka Helariutta  presented new results on his research on symplastic interfaces and their role in phloem transport. Sieve pores connect the enucleated sieve elements and together with plasmodesmata connections in companion cells they form the route for vascular phloem communication. The work in Helariutta lab carefully dissects the mechanisms underlying the formation of sieve pores and the regulation of plasmodesmata aiming to identify opportunities to engineer long distance signaling. Characterization of proteins, such as the putative choline transporter CHER1 , involveMedicago truncatula. As a result, root infection and nodulation, after inoculation with the symbiotic nitrogen-fixing bacteria rhizobia, were significantly improved. Her research indicates that callose deposition at plasmodesmata cell walls is significantly downregulated soon after inoculation, concomitant with enhanced symplastic communication. Inducing callose degradation prior inoculation facilitates the establishment of the symbiotic interaction. The identity of the symplastic factors involved and how they regulate root development and response to pathogenic and non-pathogenic microbes are still unknown.Yoselin Benitez-Alfonso elaborated on plasmodesmata role in development and disease response and presented new evidence that point to plasmodesmata as regulator of beneficial symbiotic interactions. Using proteins involved in the degradation of callose, symplastic transport between epidermal and cortical tissues was enhanced in the model legume Chiara Zurzolo  talked about the formation, regulation and function of very different type of intercellular channels: tunneling nanotubes in human cells. Different from plasmodesmata in plants, tunneling nanotubes can connect very distant cells but they still transport big macromolecular proteins including GFP. Similarities with filopodia structures, precursors for dendritic spines in neurons , suggestIn summary, talks on this focus session contributed to the understanding of the processes underlying the formation, regulation and function of intercellular channels in plants and animals. Moreover, a platform for communication between researchers in tunneling nanotubes and plasmodesmata was established aiming to create common knowledge on these intriguing structures and their roles in UPS pathways.During the meeting, it became apparent that proteins involved in conventional protein and membrane secretion can also play additional unconventional roles, and it was reported in several talks that uncharacterized processes, from exocyst tethering complex formation to the role of interfering SNARE or that of the RAB proteins, can lead to the formation of membranous structures involved in UPS. In the following subsections, are all the different cases belonging to The exocyst tethering complex is very recently and surprisingly implicated, not only in exocytosis, but also autophagy in both animals and plants ,66,67,68A. thaliana, whereas there are two or more genes for all the other exocyst components and up to 23 gene members for the EXO70 subunit. Therefore, different exocyst complexes could correspond to the great diversification of endomembrane structure and function in plants. The work of Pečenková and colleagues is focalized on the EXO70B1 and B2, which are highly homologous. Both subunits are involved in defense against pathogens and the EXO70B1 is also implicated in autophagy-related transport to vacuole. Based on preliminary results from microscopic observations they concluded that the role for EXO70Bs could be to support the unconventional cargo transportation and secretion. Rosana Sanchez-Lopez  provided another example of the importance of endomembrane dynamics in biotic interactions by documenting the importance of GNOM related ARF guanine-nucleotide exchange factor (GEF) in nodulation induced in Vicia faba by Rhizobacteria.Indeed, Tamara Pečenková  discussed the possible role of the plant exocyst complex on transport in defense against pathogens attacks. In plants, the exocyst tethering complex has been shown to participate in numerous events as cell wall differentiation and maturation, cell plate formation, plasma membrane recycling, autophagy and plant defense ,69. ThisA. thaliana, where a wide variety of reporter lines and characterized cargo proteins are available [It would be interesting to test the role of each single component of the exocyst complex with the suite of chemical inhibitors of membrane trafficking discussed by Glenn Hicks . Chemical genomics—the use of small molecule inhibitors combined with genetic screening or reporter systems—is well-established as a tool for investigating endomembrane trafficking in plants. This is particularly true for vailable . He presvailable .To explain homo- and heterotypic fusions among large compartments, it will probably be necessary to investigate in more detail the unconventional function of important regulatory proteins such as the soluble NSF attachment protein receptor (SNARE) proteins. These tail anchored proteins are essential for the specificity of vesicular targeting but can also contribute to the definition of membrane identity by assuming alternative roles . An updaA. thaliana. One of the puzzles in vesicle fusion is the apparent over-abundance of SNARE proteins relative to their requirement for vesicle fusion. He has proposed a new class of SNARE, the iSNARE of interfering SNARE that can become non-fusogenic when over-expressed [A. thaliana protoplasts SYP51 and SYP52 typically function as t-SNARES, however when localized to the tonoplast these proteins take on inhibitory or non-fusogenic roles. Different roles for these two highly similar SNARE proteins were also presented, identifying SYP51 as essential for trafficking to the central vacuole [Another example of an unconventional role for a conventional trafficking protein was provided by Gian Pietro di Sansebastiano , who at short notice described their work on the syntaxin 5 proteins in xpressed . In A. t vacuole .Several talks focused on a family of small GTPases, known as RAB proteins, which are frequently described as “master regulators” of membrane trafficking. RABs are highly conserved molecular switches that help to specify a unique identity for membranes to which they are transiently attached. Activated, GTP-bound RABs interact with a wide variety of other proteins known as “effectors”. Although their structure and core mechanisms are highly conserved, RAB proteins have diversified extensively between different organisms. Over 60 RAB genes have been identified in humans and their roles in regulating intracellular traffic throughout the endomembrane system is well known. However, less well established roles for RAB proteins include signaling, cell cycle control and cellular migration.The group of Cinzia Progida  used human cells to develop cell migration and proliferation assays that could be used as the basis for an RNAi screen for RAB proteins. The RAB7 group is known to regulate transport between the late endosomes and the Golgi apparatus, and previous work by the Progida group had identified an additional role for RAB7b as a coordinator of cytoskeleton organization through direct interactions with myosin II and by activating RhoA and therefore actin remodeling . As remoAn alternative way to gain insight into the function of proteins is to physically isolate them and identify interacting partners. One classical method for establishing interactions is to use co-immunoprecipitation, where one protein is isolated by affinity purification using antibodies raised to either an epitope on the bait protein itself or—more commonly—to an established fusion protein such as GFP. Alex Jones  presented data from a large proteomics dataset that used fluorescent proteins fused to seven target proteins and gentle protein extraction conditions to isolate not just direct protein interactions but large sections of the endomembrane system to which they are attached . Heard eA. thaliana was then presented by Grégory Mouille . Well-defined polymeric xyloglucan structures are present in the plant cell wall. While core xyloglucan biosynthesis is located in the Golgi key maturation steps occur in the apoplast. Several cell wall residing enzymes that carry out the trimming are known and mutants are available. Grégory Mouille used sophisticated mass spectrometric technologies combined with chemical genomics (5000 compounds tested) to characterize the cell wall structure and phenotype of these mutants. This approach reveals uncharacterized subcellular compartments involved in the dynamic and polarized trimming of the cell wall polysaccharide [In plants, cell wall material and enzymes, such as xyloglucan, are cargos of RAB-regulated routes. A chemical genomic approach to study xyloglucan metabolism and trafficking in the model plant ccharide ,80.Aspergillus. In higher fungi, the AP-2 complex appears to have lost the clathrin binding domain (in β2 subunit). Nevertheless, it does interact with endocytosis markers, lipid flippases, sphingolipid and sterols biosynthetic machineries and contributes to proper apical membrane lipid composition. The question arisen during the meeting was if this imply a possibility of similar features in some other adaptor complexes.George Diallinas  focused on the unexpected function of the AP-2 adaptor complex outside of clathrin dependent endocytosis in hyphal tip growth of To mount adaptive immune responses requires the cell surface expression of Major Histocompatibility Class II molecules loaded with antigenic peptide. Oddmund Bakke  reported on his previous studies focused on Major Histocompatibility Class II—associated Invariant chain , which serves as a chaperone for Major Histocompatibility Class II molecules and mediates trafficking to the endosomal pathway in humans . In factThis section is a miniature showcase of the diversity of topics that UPMT meeting attendees work on. With such diversity in proteins of the endomembrane system and organisms presented in this section, it can be hard to find a point of commonality. However, the molecular mechanisms of secretion and membrane trafficking, conventional or not, provided a thread of continuity between the subjects. The same concept can be applied to the next Intra- and intercellular communication is a fundamental mechanism for the coordination of organs during development and growth, stress adaptation, immunity and host-pathogen interactions in different biological kingdoms. Traffic and exchange of proteins, hormonal peptides and nucleic acids among cells play an essential role. A. thaliana plants [A. thaliana, indicate a putative role of UPS in pollen tube guidance towards the ovules for fertilization.Daphne Goring  focused on the exocyst, exosomes and autophagy in the regulation of plant sexual reproduction . Normal a plants , suggestLiwen Jiang  debated the crosstalk existing between UPS and autophagy, describing multiple transport pathways and showing how distinct mechanisms regulate the conventional secretory pathway and UPS in plant cells . Plant c2 consumption.Cristiano Simone  discussed the unconventional transport of FoxO3A, a transcription factor working in normal conditions on genomic DNA in the nucleus, to mitochondria during nutrient deprivation in a process dependent of MEK/ERK and the AMPK pathways. These kinases provide the final signature by phosphorylating serines 12 and 30 on FoxO3A N-terminal domain, which is required for the recruitment to the mitochondrial membrane. After that, FoxO3A is imported and processed by the mitochondrial processing peptidase system. Inside mitochondria, FoxO3A activates the transcription of the mitochondrial genome, thus leading to increased OJürgen Soll and his coworkers  use biochemical and electron microscopic techniques to uncover transport and signal transduction processes in green plants . One focDaniel Garcia Cabanillas  covered the replication of the Turnip mosaic virus, a positive-sense (+) RNA plant virus, which induces substantial endomembrane system remodeling during infection. The virus, indeed, is known to induce ER-derived vesicles, commonly known as “viral replication factories”, which house viral RNA as well as viral and host proteins required for its replication. These vesicles are also involved in the intercellular trafficking of the Turnip mosaic virus and are released at the ER in a COPII-dependent transport. The viral membrane associated protein 6K2 has an essential role in vesicle formation . The worDrosophila model was also observed [+ channel TM9SF4 is especially abundant in kidneys and its knockdown inhibits autophagy normally activated via mTOR signaling pathway.Valeria Crippa from the Angelo Poletti’s lab  presented results devoted to the function of small heat shock protein chaperon B8 in autophagic disposal of misfolded proteins implied in motor neuron diseases. The B8 protein, upregulated in motor neuron diseases patients, was shown to increase an autophagic/lysosomal degradation of misfolded proteins in a complex with co-chaperons (including E3 ligase CHIP) in cellular models and a clear protective function in observed . Moving Andrea Pompa  presented unpublished data on the maturation and unconventional trafficking of the CLV3 protein in transgenic tobacco plants. The CLV3 protein is the precursor of a small 12 amino acid active peptide recognized at the cell surface by CLV1/2 receptor complexes  and coopT. kok-saghyz. Natural rubber is synthetized and compartmentalized in particles of some plants and fungi, but the ontogeny and development of these particles is not well understood [Hevea brasiliensis or its alternative Parthenium argentatum growing in arid regions, the plant Taraxacum would be of interest for rubber production in temperate areas like parts of the US or Europe. Muhammad Ghaffar presented first insights into rubber ontogeny in secretory laticifer cells of Taraxacum. He observed rubber being initially formed in the ER-Golgi membrane system. Rubber particles were also found in vesicles located in the cytoplasm and in plastids of laticifer cells. The findings contribute to understanding rubber ontogeny and hence to establishing T. kok-saghyz as a novel rubber producer.Muhammad Akbar Abdul Ghaffar  described another process of intracellular trafficking. He applied transmission electron microscopy to study rubber ontogeny in the alternative rubber crop derstood . In contA. thaliana to the fungus U. maydis, was a good idea because the audience might struggle to follow the talks if the speaker’s research background is too distant from their own research field. Occasionally this might have been the case, but in general the meeting was very successful and on the last day, during an open final discussion, there was unanimity among the participants that the second UPMT meeting should be held in 2018.The first “Unconventional Protein and Membrane Traffic” meeting was something of a gamble with a happy conclusion. The UPS topic is intrinsically very diverse and heterogeneous because it involves many pathways, cargoes, cell types, and triggering conditions. Moreover, the organizers had not been sure that bringing together scientists working on different model systems, from primary human monocytes to yeast cells, or from the plant


The prevalence of dry eye following cataract surgery was reported as high as 55.7%, this acute and iatrogenic disorder urgently required appropriate clinical management. The purpose of this study is to compare the efficacy of diquafosol sodium ophthalmic solution (DQS) and conventional artificial tears (AT) for the treatment of dry eye following cataract surgery.The PubMed, Embase, and the Cochrane Central Register of Controlled Trials were searched from their earliest entries through June 2017 to obtain the studies, which evaluated the efficacy of DQS for patients with dry eye after cataract surgery. The relevant data were analyzed using StataSE 12.0 software. The PRISMA checklist was used as protocol of the meta-analysis and the guideline was followed. The weighted mean difference, relative risk, and their 95% confidence interval were used to assess the strength of the association.P < .05). Although the scores of symptom questionnaire could not be pooled, the results of each study also proved that DQS could significantly better relieve the symptoms of postoperative dry eye.The authors identified 21 references of which 4 studies evaluating the efficacy of DQS for patients with dry eye after cataract surgery were included. The dataset consisted of 291 patients of dry eye following cataract surgery (371 postoperative eyes). The pooling result of our study suggested that the DQS could significantly better improve the indices like corneal and conjunctival fluorescein staining scores, tear breakup time, and Schirmer I test than AT (Based on the available evidence, topical DQS has a superior efficacy than AT in the management of dry eye after cataract surgery; however, further researches with larger sample sizes and focus on indicators such as higher-order aberrations, symptom questionnaire scores, and cost-effective ratio are required to reach a firmer conclusion. According to the results of recent studies, such subjective symptoms are caused by the surgery-induced dry eye.–5With the significant process in development and refining of the surgical techniques and apparatus, today, cataract surgery has become one of the safest and most effective ophthalmic surgical procedures, which are performed on numerous patients. However, many patients still complain of some postoperative symptoms, such as ocular soreness, pain, burning sensation, foreign-body sensation, and poor vision. Until now, effective interventions of patients with those symptoms have not been established. Several risk factors for postoperative dry eye were reported by previous studies, including forceful opening of the eye lids, thermal damage from the operative microscope, sterilizing conjunctival sac and lids with povidone–iodine, topical anesthetics, transection of the corneal nerves by clear corneal incision, preservatives in eye drops, and topical nonsteroidal anti-inflammatory drugs.–12 These factors cause the malfunction of ocular surface epithelium and tear film, then induce dry eye via the vicious cycle between unstable tear film and damaged epithelium.,13,14 According to the Japanese criteria of dry eye, the prevalence of postoperative dry eye was reported to be as high as 55.7%, this acute and iatrogenic disorder urgently required appropriate clinical management.,13–15As a multifactorial disease of the tears and ocular surface, dry eye is always manifested as ocular discomfort, visual disturbance, and tear film instability, which could cause potential damage to the ocular surface.,17 Previous studies reported that DQS could effectively relieved various symptoms of dry eye and improve the visual function of the dry eye patients.–19With deeper understanding of the pathophysiology of dry eye, the optimal intervention has shifted from simply lubricating the ocular surface by artificial tears (AT) or sodium hyaluronate to applying medications to increase the secretion of water and mucin. Recently, 3% diquafosol sodium ophthalmic solution (DQS) was approved for the treatment of dry eye. DQS is a purinergic P2Y2 receptor agonist, which may stimulate the secretion of water and mucin from conjunctival epithelial cells and goblet cells.,20–22 In addition, some studies suggested that the addition of DQS or AT may equally relieve dry eye after cataract surgery. As DQS is significantly more expensive than common AT, we need more solid evidence to conclude whether the application of DQS in our routine regimen of postoperative therapy for dry eye following cataract surgery is appropriate.However, the effect of DQS on dry eye after cataract surgery was scarcely studied and relatively unclear, the sample sizes of previous studies were relatively small and may not detect the difference as statistically significant.Until now, no meta-analysis in this field has focused on this problem. Thus, we undertook a meta-analysis to evaluate the efficacy of DQS for the treatment of dry eye following cataract surgery, in order to provide a reference for the decision making of ophthalmologists.2 Since this is a systematic review, ethical approval is not required.This meta-analysis was performed strictly according to the guidelines, the “preferred reporting items for systematic reviews and meta-analysis (the PRISMA statement).”2.1The PubMed, Embase, and the Cochrane Central Register of Controlled Trials were searched from their earliest entries through June 2017. The following keywords or corresponding Medical Subject Headings (Mesh) were used: “cataract surgery”, “diquafosol”, and “Randomized Controlled Trial”. The detailed electronic search strategy of PubMed was  OR random ∗ [Title/Abstract])) AND ((diquafosol [Title/Abstract]) OR “diquafosol” [Supplementary Concept])) AND (((“Cataract Extraction” [Mesh] OR “Cataract” [Mesh])) OR Cataract [Title/Abstract]). The searches started at May 25, 2017 and ended at June 1, 2017. The reference lists of the relevant articles were also manually reviewed to further identify potentially related studies. No language restriction was imposed.2.2Inclusion criteria were participants: patients with visually significant cataract; intervention: cataract surgery; comparison: postoperative dry eye managed with the use of diquafosol versus AT; outcomes: at least one of the followings: tear breakup time (BUT), corneal and conjunctival fluorescein staining scores, subjective symptom questionnaire, and Schirmer I test; and methodological criterion: randomized controlled trial.Exclusion criteria were other differences between the experimental group and control group beside the administration of diquafosol and AT; relative risk (RR) or weighted mean difference (WMD) could not be estimated as insufficient data; animal studies or cadaver subjects; and redundant publications.2.3: a trial with a score of 7 or more was considered high quality, more than 4 but no more than 7 was considered moderate quality, and no more than 4 was considered low quality. Disagreements were evaluated by kappa text and were resolved by discussing with the corresponding author (Y-XC).After obtaining the list of potential relevant articles, the Endnote software was used to remove the duplicates. Then the titles and abstracts of the remaining studies were reviewed to filter out the unrelated articles. The next procedure was achieving the full text of each article and reviewing them, the studies that met the eligibility criteria and fail the exclusion criteria were included. Two authors (XZ and SX) extracted relevant data independently, including the first author's name, publication year, design, sample size (patients and eyes), group size, average age, gender ratio, application method, other intervention protocol, and outcome. The corresponding authors of the included articles would be contacted if the requisite data were unavailable. The data of updated publications involving same cohort of cases would be extracted synthetically. Two authors (YZ and SX) independently assessed the methodological quality of each included randomized controlled trial by 12-item scale2.42 were used to evaluate the statistical heterogeneity. If heterogeneity was low , a fixed-effect model would be used. If substantial heterogeneity existed , both sensitivity analysis and subgroup analyses would be performed to identify the source of the heterogeneity. If the heterogeneity could not be eliminated, a random-effect model would be used when the result of meta-analysis had clinical homogeneity, or a descriptive analysis would be used.Statistical analyses were performed with StataSE 12.0 software . The WMD and 95% confidence interval (CI) were calculated for continuous data, and the RR and 95% CI were calculated for dichotomous data. Chi-squared test and IP < .05 was considered to indicate statistical significance.Publication bias was evaluated by Begg funnel plot and the Egger linear regression test, a 33.1Initially, 21 potentially relevant studies were identified in our search. We firstly removed 14 duplicate studies by Endnote software, then screened off 1 unrelated article after reviewing titles and abstracts, and excluded 2 studies for their unrelated data after reviewing the full texts. Eventually, 4 studies were included in our meta-analysis. The dataset consisted of 291 patients of dry eye following cataract surgery, including 371 postoperative eyes. Among them, 178 eyes were in the DQS group and 193 eyes in the AT group. The sample sizes varied from 32 to 154 patients. The demographic characteristics of the case group and control group were similar in every study. Table 3.2,20,21 included 133 eyes in the DQS group and 144 eyes in the AT group described the BUT before the treatment of postoperative dry eye. When pooling the data of pretherapeutic BUT, fixed-effect models were used as Chi-squared test manifested no heterogeneity. The results of meta-analysis showed no statistical difference of BUT between the 2 groups before the treatment . The random-effect model was used for the reason that regardless of the exclusion or inclusion of every study, the pooling results were all same and all studies were of high quality. The forest plot indicated that the post-therapeutic BUT of DQS group was significantly increased than that of AT group  and Egger test (P = .338) indicated that publication bias did not affect our results.Begg test . Unstable tear film might increase HOAs and contribute to glare, halo, decreased night vision, and impaired contrast sensitivity.As far as we know, this is the first meta-analysis evaluating the efficacy of DQS and AT for treatment of dry eye following cataract surgery that includes all the available evidence of high quality. The satisfactory heterogeneity and the insignificant publication bias made our study highly reliable and might provide valuable instructions for ophthalmologist. However, the following limitations still exist: although all the available data had been pooled together by the most reliable way, the final sample size in our meta-analysis was still relatively small, more researches of high quality were need to get a more solid conclusion; a descriptive analysis was used for symptom questionnaire scores; and there were insufficient data to analyze the HOAs. Further researches should focus on these 2 points as they are the most direct indices for evaluating the efficacy of 2 drugs; currently, DQS is only approved in Japan and South Korea, the possible effect of ethnic differences should be further evaluated; as DQS is significantly more expensive than common AT, cost-effective ratio should also be evaluated to get a more solid evidence to conclude whether the application of DQS in our routine regimen of postoperative therapy for cataract surgery is appropriate.5Based on the available evidence, DQS has a superior efficacy than AT in the management of dry eye after cataract surgery, supported by better corneal and conjunctival fluorescein staining scores, BUT, and Schirmer I test. Given the limitations in our study, further researches with larger sample sizes and focus on indicators such as HOAs, symptom questionnaire scores, and cost-effective ratio are required to reach a firmer conclusion.The authors would like to thank the editor and anonymous reviewers for the valuable comments and suggestions.


Metastatic melanoma is the most deadly type of skin cancer. Despite the success of immunotherapy and targeted agents, the majority of patients experience disease recurrence upon treatment and die due to their disease. Long non-coding RNAs (lncRNAs) are a new subclass of non-protein coding RNAs involved in (epigenetic) regulation of cell growth, invasion, and other important cellular functions. Consequently, recent research activities focused on the discovery of these lncRNAs in a broad spectrum of human diseases, especially cancer. Additional efforts have been undertaken to dissect the underlying molecular mechanisms employed by lncRNAs. In this review, we will summarize the growing evidence of deregulated lncRNA expression in melanoma, which is linked to tumor growth and progression. Moreover, we will highlight specific molecular pathways and modes of action for some well-studied lncRNAs and discuss their potential clinical implications. BRAF gene in approximately 95% of all cases, the most common exchange being a valine to glutamic acid referred as V600EBRAF mutation ..HES1 locmtBRAF melanoma compared to triplewt (RAS/BRAF/NF1) melanoma and expression levels were negatively correlated with melanoma progression [RMEL3 was first described together with RMEL1 and RMEL2 by Sousa et al. who revealed that all RMEL lncRNAs were almost exclusively expressed in melanocytes and melanoma . Interesgression .V600EBRAF melanoma cell lines. Additionally, MAPK and PI3K pathway activators and effectors were also impacted negatively. Multiple genes for these activators and effectors were found to be correlated with RMEL3, thus indicating the requirement of RMEL3 for MAPK and PI3K signaling  [Survival associated mitochondrial melanoma-specific oncogenic non-coding RNA (SAMMSON) is located 30 kb downstream of the melanoma-specific oncogene melanogenesis associated transcription factor (MITF) and is co-amplified in around 10% of all melanoma cases, although studies demonstrated that SAMMSON acts in o-factor C. ExpresS, TP53) . By targS, TP53) .SnoRNA host gene 5 (SNHG5) is part of the non-coding multiple small nucleolar RNA host gene family and encompasses 524 base pairs . The sitSPRY4 gene and extends to exon 3 [SPRY4, both genes were found to be functionally and transcriptionally independent [SPRY4 intronic transcript 1  is initiated in intron 1 of the o exon 3 . Even thependent .SPRY4-IT1 was shown to be over-proportionally represented in melanoma cell lines as well as in human melanoma samples compared to melanocytes . It is mUCA1 was first discovered in bladder transitional cell carcinoma and so far two isoforms have been identified .A study comparing the expression of six cancer-implicated lncRNAs in melanoma to their respective expression in paired adjacent healthy tissue, found elevated expression of UCA1 in melanoma, especially at advanced stages ,125. KnoLong noncoding RNAs are now widely recognized as contributing factors which play diverse and complex roles in cancer. Moreover, they are gaining increasing attention as potential biomarkers and represent a novel class of target molecules. However, we are only beginning to understand the complexity of tumorigenic processes and the role of lncRNAs in melanoma as well as in other cancer types. The clinical integration of lncRNAs as prognostic and predictive biomarkers in conjunction with additional cancer targets, could provide a chance to increase the therapeutic benefit.Nevertheless, several challenges lie ahead of us. First of all, we have to learn more about the molecular mechanisms and processes employed and controlled by lncRNAs. This task is not trivial and requires multiple techniques as well as the development of novel methods that allow us to observe and capture the lncRNAs and their respective interaction partners and sites in a highly specific manner. For example, in addition to our current understanding that lncRNAs exert their biological effect through interaction with DNA/RNA or proteins, it might be possible that lncRNAs directly interact with other metabolites, such as lipids or sugars . IdentifSecondly, the in vivo function of lncRNAs is difficult to study, due to the low conservation of most lncRNAs . AdditioFinally, these novel animal models could represent valuable tools to develop effective therapeutic reagents against lncRNAs, which are currently considered difficult to target. Reducing the expression or blocking the function of oncogenic lncRNAs with, e.g., small molecules, might pave the way to novel treatment strategies and their clinical application in the future.


KAT) and lysine deacetylase (KDAC) complexes in brain development and the different neurodevelopmental disorders that are associated with dysfunctional lysine (de)acetylation machineries.Embryonic development is critical for the final functionality and maintenance of the adult brain. Brain development is tightly regulated by intracellular and extracellular signaling. Lysine acetylation and deacetylation are posttranslational modifications that are able to link extracellular signals to intracellular responses. A wealth of evidence indicates that lysine acetylation and deacetylation are critical for brain development and functionality. Indeed, mutations of the enzymes and cofactors responsible for these processes are often associated with neurodevelopmental and psychiatric disorders. Lysine acetylation and deacetylation are involved in all levels of brain development, starting from neuroprogenitor survival and proliferation, cell fate decisions, neuronal maturation, migration, and synaptogenesis, as well as differentiation and maturation of astrocytes and oligodendrocytes, to the establishment of neuronal circuits. Hence, fluctuations in the balance between lysine acetylation and deacetylation contribute to the final shape and performance of the brain. In this review, we summarize the current basic knowledge on the specific roles of lysine acetyltransferase ( Adaptability and plasticity are the key features of the brain, which grant rapid and coordinated responses to environmental changes. In order to insure the complex function of the brain, a series of mechanisms must work in a coordinated manner starting early on during embryonic development and continuing during postnatal and adult life. Embryonic development defines the size and functionality of the adult brain since the bulk of neurogenesis occurs prenatally while only few neurons are produced after birth At the molecular level, brain development and maintenance are controlled both by cell-intrinsic mechanisms, such as transcriptional programs controlling proliferation and differentiation of NSCs, and extrinsic mechanisms including extracellular cues influencing neuronal migration, synapse formation, and neuronal activity Acetylation of histones is a reversible epigenetic mechanism that can be influenced by external factors The mammalian CNS arises from the ectoderm germ layer of the early embryo after a series of complex morphological changes. During early development, the neural tube is formed to subsequently generate the early CNS and peripheral nervous system The paradigms on how the brain is formed are mainly based on studies conducted on the cerebral cortex, hippocampus, or cerebellum. With the onset of neurogenesis, starting around embryonic day 10.5 (E10.5), the proliferative area within the cerebral cortex is subdivided into the VZ, which contains APs, and the subventricular zone (SVZ), which contains intermediate or basal progenitors (BPs) Indeed, to achieve a fully functional brain, it is essential to control gene expression profiles for the generation of appropriate cell fates. The production of neurons and glial cells, as well as the maintenance of reduced stem cell populations in the adult brain, is accomplished by the complex interplay of intrinsic and extrinsic signals. Therefore, cell autonomous mechanisms including intrinsic transcriptional programs that control cell cycle progression, centrosome activity, or the polar distribution of cell fate determinants, are of great importance Lysine acetylation and deacetylation represent prominent posttranslational modifications of histone tails that influence chromatin structure and epigenetic states. Lysine acetylation is mediated by lysine or histone acetyltransferases (KATs or HATs), whereas lysine deacetylation is catalyzed by lysine or histone deacetylases (KDACs or HDACs) N-acetyltransferases (GNATs) include different families of KATs that share similar structural features and functional roles. The GNAT superfamily in humans includes HAT1 (KAT1), GCN5 (KAT2A), p300/CREB-binding protein (CBP)-associated protein , elongator acetyltransferase complex subunit 3 , cysteine rich protein 2 binding protein , activating transcription factor 2 (ATF-2), and HAT4 KATs, which are evolutionarily conserved from yeast to humans, catalyze the transfer of acetyl groups from acetyl-CoA onto lysine residues of acceptor proteins . There a++ levels and play important roles in the regulation of cellular homeostasis, metabolism, and lifespan KDACs are enzymes responsible for the removal of acetyl groups from lysine residues . In cont+, whereas Class I, II, and IV KDACs need zinc in order to function Contrary to what was initially thought, lysine acetylation also occurs in a vast number of proteins besides histones. Protein acetylation has emerged as an important posttranslational modification regulating many cellular processes Abnormal lysine acetylation activity or histone acetylation levels have been linked to different pathological conditions or alterations in brain development both in humans and in mouse models . MutatioNumerous studies using cellular and animal models have been performed to unravel the function of different KAT and KDAC complexes during brain development. Generally, these studies have focused on the effects of histone acetylation on important developmental transcriptional programs. Moreover, the acetylation of proteins other than histones has also been related to different neurodevelopmental processes such as NP fate determination and neuronal migration p300 and Cbp null mouse embryos, as well as double heterozygous Cbp/p300 embryos, exhibit defects in neural tube closure and die before E12 p300-knockout or Cbp-knockout heterozygous mice are viable, and have the normal general brain morphology and long-term memory despite exhibiting impaired behavior at birth Cbp knockout or Cbp siRNA knockdown results in decreased differentiation of cortical NPs and neural precursors into neurons and glial cells within the ganglionic eminence, without affecting their survival or proliferation α1-tubulin), and glial differentiation genes . These analyses are suggestive of an involvement of CBP in the transcriptional activation of NP differentiation genes p300/CBP family members are required for proper brain development and mutations in the genes encoding these proteins are associated with the human Rubinstein-Taybi syndrome (RSTS) Pcaf does not seem to show noticeable effects in mice during embryonic development Gcn5 leads to embryonic lethality in mice around E10.5. These mice exhibit severe developmental defects, such as growth retardation, increased apoptosis, as well as failure to generate the neural tube and dorsal mesoderm-derived lineages Pcaf and Gcn5 leads to more severe defects and lethality around E7 Gcn5 mutations, including a hypomorphic Gcn5 mutation, a catalytically-dead Gcn5 point mutation, and a double knockout p53/Gcn5 mutation, exhibit exencephaly and neural tube closure defects and die pre- or peri-natally Gcn5 in the CNS by Nestin-Cre expression results in microcephaly and a decrease in the NSC mass due to decreased cell proliferation that is associated with lower expression of N-Myc target genes Pcaf knockout mice exhibit an impaired short-term memory two months after birth, accompanied by subtle differences in hippocampal morphology. Progressively, these mice exhibit an age-related decline in short- and long-term memories Elp1 and Elp3 causes decreased neuronal migration and reduced levels of α-tubulin acetylation, thus identifying a key role of these molecules in regulating the migration and maturation of projection neurons GCN5 and PCAF show 89.5% amino acid sequence homology, whereas other GNAT family members are more diversified Although mutations in the MYST family members often lead to neurodevelopmental defects in humans, not many studies have been conducted to address their specific roles during brain development. This may be due to the fact that while heterozygous mutations of these factors exhibit no phenotypes, homozygous mutations of most of these members, including TIP60, HBO1, and MOF, as well as cofactors like transformation/transcription domain-associated protein (TRRAP) or DNA methyltransferase 1 associated protein 1 (DMAP1), lead to early embryonic lethality around the blastocyst stage or post gastrulation Morf was first identified in a mouse gene-trap screen for genes important in neural development MORF cause different neurodevelopmental disorders in humans, the mechanisms through which MORF regulates brain development are not fully understood Moz, coding for a closely-related KAT with MORF, have been generated by two different laboratories. These mice exhibit embryonic or perinatal lethality, craniofacial abnormalities, and defects in the hematopoietic system INK4a in NSCs in order to avoid replicative senescence Trrap leads to early embryonic lethality around the blastocyst stage Trrap in the embryonic brain by using Nestin-Cre causes cell proliferation defects accompanied by premature AP differentiation, leading to severe brain atrophy. Strikingly, unscheduled differentiation of Trrap-deleted NPs is attributed to cell cycle defects TRRAP is an essential cofactor of different GNAT and MYST family KAT complexes The balance between lysine acetylation and deacetylation is as much dependent on KATs as it is on KDACs. In fact, it is the kinetics of KAT versus KDAC activity that dictates the final acetylation status of histones and other proteins, thus determining subsequently their functionality Hdac1 is expressed in NPs, whereas Hdac2 is expressed in post-mitotic neurons in the CNS of mice at E13.5 Hdac1 or Hdac2 alone causes lethality, although at different developmental stages Hdac1 or Hdac2 in the mouse CNS using Nestin-Cre does not have any obvious consequences on brain development, combined deletion of Hdac1 and Hdac2 results in embryonic or perinatal lethality, due to cell death and failure of NPs to differentiate into mature neurons Hdac1+/-Hdac2-/- mice display impaired brain development and perinatal lethality, due to reduced NP proliferation and premature differentiation mediated by overexpression of protein kinase C γ (PKCγ) Although sharing 86% amino acid sequence homology and binding to the same transcription repression complexes, HDAC1 and HDAC2 display different expression patterns during brain development, thus indicating their specific functions in this process. Strikingly, Hdac3 in the CNS using Nestin-Cre causes perinatal lethality, which is accompanied by major abnormalities in the cytoarchitecture of the neocortex and cerebellum Hdac8 knockout mice exhibit perinatal lethality due to massive ossification defects in the skull, which lead to brain tissue herniation and brain hemorrhage, suggesting a specific role of HDAC8 in regulating skull morphogenesis rather than brain development The deletion of Hdac4-null mice die perinatally due to severe bone malformations and chondrocyte hypertrophy Hdac4 in the brain using Thy1- or Nestin-Cre does not result in any obvious defects. These in vivo studies suggest that HDAC4 does not play important roles during brain development or its function is compensated for by other KDACs Hdac9 and Hdac6 knockout mice show no obvious neural phenotypes Class II KDACs exhibit specific expression patterns during embryonic development and adulthood. Nonetheless, their roles during brain development remain mostly unknown. Many studies have linked HDAC4 with neuronal survival in cell cultures in utero electroporation, and knockout models to investigate their functions. It has been shown that SIRT1 regulates the cell fate of NPs and is required for neurite outgrowth, axonogenesis, and dendritic branching through multiple mechanisms including the activation of AKT/glycogen synthase kinase 3 (GSK3) signaling and inhibition of pro-neural genes from the Notch-hairy/enhancer of split (Hes) axis Sirt6-null mice, display profound abnormalities, including low insulin, hypoglycemia, and premature aging, and die within four weeks after birth Sirt6 in the brain using Nestin-Cre leads to postnatal growth retardation in mice due to low levels of growth hormone and insulin-like growth factor 1 (IGF-1) through yet unknown mechanisms Among the sirtuin family members, SIRT1 and SIRT2 seem to play opposite roles during brain development. Different studies have been performed using cellular systems, in vivo and in vitro in mice and humans. All have shown that inhibition of KDACs causes dramatic effects during brain development. In humans, treatment with valproic acid (VPA), an inhibitor for Class I and II HDACs, during pregnancy is associated with the occurrence of autism in the progeny Sox2There are a remarkable number of studies investigating the effects of HDACi on neurodevelopmental processes Given the important role of KATs and KDACs in controlling transcription and other cellular processes, most of the mutations in the genes encoding these enzymes are likely to be incompatible with life. However, owing to a high degree of substrate redundancy and the presence of hypomorphic mutations, a number of human syndromes have been mapped to mutations on both KAT and KDAC coding genes. Common features of these syndromes are brain abnormalities, usually including primary microcephaly, mental retardation, global developmental delay, and craniofacial dimorphism 180849 and 613684) CBP gene have been reported in approximately half of the RSTS patients p300 gene are estimated to represent 5%–8% of all cases CBP gene duplications cause a disorder called 16p13.3 duplication syndrome (OMIM 613458), which is characterized by a mild to moderate intellectual disability (ID) and abnormal facial and skeletal morphology CBP cause similar brain manifestations shows that a tight control of CBP level and function is essential for efficient brain development. Moreover, de Vries et al. CBP mutations cause overlapping features of RSTS and Filippi syndrome (OMIM 272440). Filippi syndrome is usually caused by mutations in the mitotic spindle protein cytoskeleton associated protein 2-like (CKAP2L) and is characterized by microcephaly, short stature, syndactyly, intellectual disability, and facial dysmorphism (OMIM 272440).Mutations in genes encoding CBP/p300 family members lead to RSTS, a congenital autosomal dominant disorder also known as broad thumb-hallux syndrome , characterized by severe mental retardation, distinctive facial appearance, and other skeletal problems MORF haploinsufficiency has been proposed as a rare cause of Noonan syndrome-like disorder that is characterized by short stature, retarded bone age, attention deficit hyperactivity with learning disability, and distinct facial features BRD1 that encodes bromodomain-containing protein 1, an essential cofactor of MOZ/MORF/HBO1 complexes, has been repetitively identified as a susceptibility gene for schizophrenia and bipolar disorder Different gene mutations leading to C-terminal truncations of MORF cause the rare genitopatellar syndrome (OMIM 606170), a condition characterized by microcephaly, severe psychomotor retardation, ID, genital abnormalities, missing or underdeveloped kneecaps, and other abnormalities HDAC8, which encodes the class I KDAC enzyme, have been identified in Wilson-Turner X-linked mental retardation syndrome (OMIM 309585), a neurological disorder characterized by ID, dysmorphic facial features, hypogonadism, short stature, and truncal obesity HDAC8 mutations are also found in Cornelia de Lange-like syndrome, which is characterized by, among other features, distinct facial features, growth failure, and ID HDAC4, which encodes the class IIA KDAC enzyme, is located on the chromosome 2q37 whose heterozygous loss is associated with chromosome 2q37 deletion syndrome, also called brachydactyly-mental retardation syndrome (OMIM 600430). This syndrome shows mild to moderate mental disabilities, behavioral abnormalities, dysmorphic facial features, brachydactyly type E, and short stature HDAC9 has been identified in a small proportion of schizophrenia patients HDAC6, which encodes the class IIB KDAC enzyme, suppress miR433-mediated posttranscriptional regulation and cause overexpression of HDAC6, resulting in chondrodysplasia with platyspondyly, distinctive brachydactyly, hydrocephaly, and microphtalmia syndrome (OMIM 300863), which exhibits, among other symptoms, hydrocephaly and macrocephaly Mutations in HDAC9 copy number has been correlated with an increased risk of autism in utero exposure to VPA correlates with ASD occurrence in humans and rodents Autism spectrum disorder (ASD) includes different disorders characterized by persistent deficits in social communication, interaction, and repetitive patterns of behavior during early childhood with significant functional impairments later in life 312750) is an X-linked neurodevelopmental disorder caused by mutations in MEPC2, which encodes methyl CpG binding protein 2 that is able to regulate transcription 300624), which is the second most common cause of mental impairment after trisomy 21 and characterized by moderate to severe mental retardation, macroorchidism, and distinct facial features FMR1, resulting in extensive local methylation, silencing of FMR1, and loss of fragile X mental retardation protein (FMRP). Given the extensive chromatin rearrangements caused by the CGG-triplet repeat, HDAC inhibitors have been used in different models to reactivate the transcription of FMR1The Rett syndrome (RTT; OMIM Several studies have highlighted the importance of maternal behavior both prenatally and perinatally on lysine acetylation and its consequences on brain development. Guo et al. Clinical research as well as cellular and mouse models have revealed the critical role of a delicate control of the balance between lysine acetylation and deacetylation in brain development. Lysine acetylation and deacetylation are involved in all levels of brain development starting from NP survival and proliferation, cell fate decisions, neuronal maturation and migration, differentiation and maturation of astrocyte and oligodendrocyte lineages, and finally synapse formation and establishment of neuronal circuits . The comHDACi has been used to treat different human diseases, including cancer The authors declare no competing financial interest.


Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer. Initially described by Ehrlich in 1879 , neutropThroughout time, neutrophils have been considered the “kamikaze” cells that arrive first at the site of injury and immolate themselves while killing the invading pathogens with a variety of mechanisms that include phagocytosis, NETosis (active extrusion of DNA to generate Neutrophils Extracellular Traps (NETs)), secretion of reactive oxygen species (ROS), hyperchlorous acid, and antimicrobial proteins  . HoweverIn light of this new understanding of neutrophil biology, it is not surprising that their role in tumor progression has finally started to be evaluated.Only recently have neutrophils and their role in cancer received the attention they deserved. Their homeostatic alteration as part of tumor induced Sweet’s syndrome (acute febrile neutrophilic dermatosis) , in leuk+ neutrophil to CD8 lymphocytes ratio is predictive of worse outcome , it , it 6], Given the fact that most neutrophils in advanced stage tumors are pro-tumoral, strategies are being tested to limit PMN trafficking at the tumor site. The redundancy of chemotactic signals can raise concerns on the efficacy of these strategies however, interesting results were obtained with these approaches. In patients with hepatocellular carcinoma the presence of high expression of CXCR2 and its ligands CXCL5 and CXCL8 (IL-8) on tumor cells are associated with neutrophil recruitment and neoplastic cell spreading .The administration of antibodies against IL-8 (ABX-IL-8) attenuated the growth of bladder cancer xenograft models , and decAs mentioned above, chemotactic factors are usually redundant. Indeed, other ligands such as CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, oxysterol, and migration inhibitory factor (MIF) are able to induce neutrophil recruitment and activation via CXCR1 and CXCR2 ,125,126.AhCreER; fl/+Apc; fl/flPten mice), CXCR2 deficiency was shown to suppress inflammation-driven tumorigenesis in the skin and intestine, as well as spontaneous adenocarcinoma formation [Targeting receptors instead of ligands offers the advantage of making it easier to overcome the overproduction of chemokines secreted by tumors and resolve some of the chemokine redundancy. Thus, strategies have been developed to inhibit CXCR2 and have shown promising results . In the ormation . In AB1 ormation . Howeverormation .CXCR6 and its ligand CXCL6 are involved in neutrophil recruitment in both a direct and indirect manner. Tumor cells and lymphocytes express CXCR6 on their surface and evidence of CXCR6 expression in tumor infiltrating neutrophils was found in patients with pancreatic carcinoma . CXCR6 en-telopeptide (a bone turnover rates marker) in 14% of the patients suggesting at least some therapeutic efficacy. A phase 1 trial for the CCR5 antagonist Maraviroc in colorectal liver metastasis is currently being performed, but no results are yet posted (NCT01736813).It is important to note that even when chemokine receptors are targeted, the redundancy and pleiotropism of these pathways may dramatically reduce therapeutic efficacy. This possibility is exemplified by the disappointing results of clinical trials using CCR1 antagonists for the treatment of rheumatoid arthritis, multiple sclerosis, and COPD . DespiteNeutrophil elastase (NE), a serine proteinase characterized by broad substrate specificity, is particularly important in neutrophil function and can mediate the cleavage of nearly all components of the extracellular matrix including cytokines, cytokine receptors, integrins, and inert elastic fibers . ConsideIn accordance with these NE pro-tumoral roles, the usage of the specific NE inhibitor in the treatment of pancreatic cancer reduced tumor proliferation and migratory capacity . SimilarSince inhibition of neutrophil elastase is considered an important step in the treatment of pulmonary diseases like chronic obstructive pulmonary disease (COPD) and in alleviating the symptoms of cystic fibrosis; many inhibitors have been designed and are currently being tested in clinical trials  which taMetalloproteinases (MMPs) are involved in many steps of cancer progression. Their presence and relative abundance is often related to cancer stage and patient prognosis . Tumor-a165-CTGF complex restores the angiogenic function of VEGF165 isoform, that is inhibited in the complexed form. [Pro-MMP9 facilitates VEGF bioavailability , and itsed form. ,150. Mored form.  facilitaed form. ,150.Inhibition of MMP9 by the endogenous inhibitor TIMP-1 reduces angiogenesis in chorioallantoic membrane assays  and promThe selective interference with prometastatic neutrophil functions represents an attractive new strategy for cancer treatment. A recent study has demonstrated that in the MMTV-PyMT breast cancer model, neutrophils express high levels of the lipids leukotriene B4 (LTB4) and cysteinyl leukotrienes C4, D4 and E4 (LTC/D/E4), products of the arachidonate 5-lipoxygenase (Alox5) enzyme . These mNeutrophils are finally being recognized for the important role they play in tumor progression. Depending on their polarization, these cells can promote tumor growth by favoring tumor angiogenesis, metastasis, and immune suppression; or they can promote tumor eradication by a direct tumor cytotoxicity and by orchestrating the adaptive immune response against the tumor.Still, the differentiation and/or polarization pathways, the role and cooperation of different tumor derived factors on neutrophil chemotaxis and polarization, the influence of sexual hormones on the biology of these cells, and the mechanisms that regulate their pro-tumoral and anti-tumoral action needs to be defined. Additionally, there is the critical need for the identification of functional markers able to discriminate between the different PMN subsets and polarization states. Nevertheless, promising therapeutic strategies aimed at eliminating or inhibiting pro-tumorigenic neutrophils, while increasing the frequency and or activity of anti-tumoral neutrophils, are being developed.In summary, in the 100 years since Metchnikoff’s death, many studies are still required to dissect the biology of neutrophils that may lead to important therapeutic applications for the immunological treatment of tumors.


It is hypothesized that the rise of early non-vascular land plants, and the later evolution of roots and vasculature, drove the long-term shift towards a high-oxygen, low CO2 climate that eventually permitted the evolution of mammals and, ultimately, humans. However, very little is known about the productivity of the early terrestrial biosphere, which depended on the acquisition of the limiting nutrient phosphorus via fungal symbiosis. Recent laboratory experiments have shown that plant–fungal symbiotic function is specific to fungal identity, with carbon-for-phosphorus exchange being either enhanced or suppressed under superambient CO2. By incorporating these experimental findings into a biogeochemical model, we show that the differences in these symbiotic nutrient acquisition strategies could greatly alter the plant-driven changes to climate, allowing drawdown of CO2 to glacial levels, and altering the nature of the rise of oxygen. We conclude that an accurate depiction of plant–fungal symbiotic systems, informed by high-CO2 experiments, is key to resolving the question of how the first terrestrial ecosystems altered our planet.Fossil evidence from the Rhynie chert indicates that early land plants, which evolved in a high-COThis article is part of a discussion meeting issue ‘The Rhynie cherts: our earliest terrestrial ecosystem revisited’. To determine the movement of phosphorus from fungus to plant, 33P-orthophosphate solution was supplied to fungus-only compartments in pot-based microcosms and incubated for 21 days. After harvest, 33P activity was measured in plant tissues using acid digestion and liquid scintillation. In the same experimental microcosms, 14CO2 was supplied to the liverworts for one complete photoperiod before being measured in the fungus-only compartments within the pots through sample oxidation and liquid scintillation . Totn et al. ,46, resp33P per unit of plant-fixed carbon transferred to the fungus under a Palaeozoic atmospheric CO2 concentration than when compared with a modern ambient CO2 atmosphere, but liverworts with Mucoromycotina fungal symbionts display the opposite trend .2.2 by incorporating phosphorus effects on global plant biomass and the ability of fungi to affect plant phosphorus uptake into the COPSE biogeochemical model (Carbon Oxygen Phosphorus Sulphur Evolution) [We examined the implications of contrasting plant–fungal relationships  in respoolution) .Mbio, which comprises constraints based on the expansion of the terrestrial biosphere onto the land surface (E), limitation by surface temperature (VT), prevalence of wildfires (Vfire) and the atmospheric concentrations of CO2  to explicitly affect the mass of the biosphere, in order to represent the effects of phosphorus limitation on both carbon burial and chemical weathering:VP) is calculated from the available weathered phosphorus (phosw) multiplied by an uptake efficiency parameter (kuptake), which allows for possible fungal-driven changes to plant phosphorus acquisition:0phosw is the present-day rate of phosphorus weathering used to normalize the expression. The climatic limiting factors are defined to constrain biomass in the following ways, and are unaltered from the original COPSE model [Pland), which is amended to ensure that it cannot exceed phosphorus supply from weathering:kland is the present-day fraction of weathered P buried on land. Land-derived organic carbon burial (locb) is calculated from the relative value of Pland and a present-day biomass burial rate (klocb) :Psea), where it stimulates marine productivity.The COPSE model ,3 reconsa) wherein terrestrial productivity increase phosphorus weathering and uptake, which in turn further increases productivity.Our modified Fungal COPSE (Fun-COPSE) model allows us to observe the possible long-term climate effects associated with changes in plant phosphorus uptake efficiency, and how this may influence our reconstructions of Paleozoic climate. In particular, our introduction of a direct phosphorus limitation of the terrestrial vegetation introduces a positive feedback loop to the model a whereinkuptake defined following the relationships between CO2 concentration and phosphorus-for-carbon exchange efficiency observed in experiments with Mucoromycotina and Glomeromycotina fungi. Figure 3c shows representative curves that are fitted to the experimental data [RCO2 is the relative atmospheric CO2 concentration. For Glomeromycotina, we require We experiment by running the model with tal data , which tkuptake). The assumed relationship for Glomeromycotina is particularly strong, based on the observed order-of-magnitude increase in phosphorus-for-carbon exchange at the elevated CO2 concentration. The strength of the relationship may be overestimated in the experimental systems. In nature, the fungus forms symbioses and provides phosphorus to multiple plant partners. In addition, the fungus likely competes for phosphorus resources with diverse soil microbial communities. As a result of this, and together with the observation that such a high phosphorus uptake efficiency results in numerical instability in the model due to positive feedbacks, we reduce the maximum value of More experimental data are needed to further constrain the relationships between fungal identity, phosphorus acquisition and climate, but the present formulation allows us to assess the potential climate impacts of a variable terrestrial phosphorus uptake efficiency  influences modelled NPP, atmospheric CO2 and O2 concentrations, and global temperature in response to land colonization during the Palaeozoic Era.We use our model to examine how the nature of plant phosphorus uptake via fungal partners . Relative terrestrial NPP scales with the supply of the limiting nutrient phosphate, and therefore is also significantly different between steady states (b). Efficient phosphorus uptake at superambient CO2 results in enhanced organic carbon sequestration, which contributes to a reduction in CO2 (c) and drives a rise in atmospheric O2 (d). Assuming less efficient phosphorus uptake at high CO2, as observed in liverwort–Mucoromycotina symbioses [2 and lower O2 concentrations in the atmosphere .Broadly, Fun-COPSE shows that changes to plant–fungal phosphorus uptake, such as those observed in laboratory conditions under high atmospheric COing rate a. Relatiy states b. Efficin in CO2 c and driheric O2 d. Assumiymbioses , resultsmosphere c,d.FiguThe time-integrated model predictions for the Early Palaeozoic are similarly affected by phosphorus uptake efficiency . As in tFurther uncertainty remains regarding the productivity of the early terrestrial biosphere, and the effects of early plants on the global carbon cycle and climate. This topic has been the focus of considerable debate in the literature, which has recently turned to the question of the ability of early terrestrial plants to enhance silicate weathering rates and liberate phosphorus ,22,23. O2 to such a degree that global average surface temperature may be sufficiently low to trigger the Late Ordovician glaciation, without requiring additional tectonic considerations. In this case, atmospheric oxygen concentration rises to modern levels by approximately 420 Ma, coincident with the first evidence for wildfires [2 concentration and temperature remains high throughout the early colonization phase, and O2 remains less than 10% atm.Assuming a phosphorus uptake efficiency greater than that observed today, as might be expected if Glomeromycotina were the dominant fungal symbionts of early land plants , thus if plant phosphorus uptake was suppressed globally under high CO2, then one must appeal either to an increased efficiency of phosphorus weathering or to currently unknown tectonic or palaeogeographic factors. Nevertheless, such extremes in model output show that the ability of the terrestrial biosphere to acquire phosphorus, rather than simply its ability to affect continental weathering, must be taken into account in future investigations.These scenarios show entirely different drivers for Palaeozoic environmental change, with the latter implying that the early terrestrial biosphere may not have been responsible for either glaciation or substantial Oevidence b,c, thusIt is likely that the earliest land plants were cosmopolitan in their choice of fungal symbionts , because2 concentrations reflective of conditions at various points in the Earth's history. These experiments should be designed to allow quantification of the relative costs and benefits to plants and fungi of symbiosis, with the results being used to construct models of the terrestrial biosphere that include phosphorus-for-carbon exchange and phosphorus recycling.A greater understanding of the magnitude and balance of fungal partnering requires further experiments conducted on extant examples of early diverging plants with a suite of fungal symbionts at a range of atmospheric COOur findings show significant Earth system sensitivity to phosphorus uptake from mycorrhizal fungi. Current dynamic global vegetation models (e.g. SDGVM ) linked 2 concentrations. Uncovering the nature of the Palaeozoic climate transition, and therefore the emergence of the human-habitable world, relies heavily on understanding the ability of the early terrestrial biosphere to acquire and use phosphorus. Such advances must come from a linked campaign of experiments and trait-based modelling, informed by palaeobotanical studies on preserved ancient ecosystems, such as the Rhynie chert. Efforts must also be made to integrate aspects of the geochemical literature, such as the relationships between erosion, soil shielding and global chemical weathering fluxes [Fossil evidence from the Rhynie chert has provided unique insights into the plant–fungal relationships during the early period of plant evolution and has allowed for the focused experimental study of how these partnerships may have functioned in response to changing atmospheric COg fluxes .4.2 depends greatly on the functioning of plant–fungal symbioses and phosphorus cycling. The experiments of Field et al. [The global terrestrial biosphere response to high COd et al. ,43 show


The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom, which began in 1986 and has affected nearly 200,000 cattle, is waning to a conclusion, but leaves in its wake an outbreak of human Creutzfeldt-Jakob disease, most probably resulting from the consumption of beef products contaminated by central nervous system tissue. Although averaging only 10-15 cases a year since its first appearance in 1994, its future magnitude and geographic distribution  cannot yet be predicted. The possibility that large numbers of apparently healthy persons might be incubating the disease raises concerns about iatrogenic transmissions through instrumentation  and blood and organ donations. Government agencies in many countries continue to implement new measures to minimize this risk.


Bryostatin 1, an anti-neoplastic agent and protein kinase C activator, has dose-limiting toxicity manifesting as myalgia. Studies in vivo have suggested that this myalgia may be caused by impairment of oxidative metabolism as mitochondrial capacity, muscle reoxygenation and proton washout from muscle are reduced by bryostatin, possibly as a result of vasoconstriction. To investigate these mechanisms further, and to enable use of bryostatin for prolonged periods, the effect of a vasodilator on the established effects of bryostatin on calf metabolism was studied using 31P magnetic resonance spectroscopy and near infrared spectroscopy. Six patients with disseminated melanoma were examined on four occasions: before and 1 week after initiation of long-term nifedipine (10 mg twice daily) treatment and then 4 and 48 h after bryostatin infusion (25 micrograms m(-2)). Nifedipine impaired muscle oxidative metabolism but had no effect on proton efflux or muscle reoxygenation rate. In the presence of nifedipine, two of the effects of bryostatin, impaired reoxygenation rate and reduced proton efflux, were abolished, but the impaired mitochondrial activity remained. These results show that nifedipine counteracted the vasoconstrictive effect of bryostatin 1. However, because nifedipine itself had an unexpected effect on mitochondrial metabolism, it was not possible to assess whether nifedipine modified bryostatin's effect on this variable. There was no additive detrimental effect of bryostatin on mitochondrial metabolism and nifedipine did not reduce the clinical toxicity of bryostatin 1, which cannot therefore be due to vasoconstriction.