Cell surface associated protein mucin 15 (MUC15) is elevated in preeclampsia.

BACKGROUND: Mucins are a family of proteins that protect the epithelium. A particular type of mucin, MUC15 is highly expressed in the placenta. This study aimed to characterise MUC15 in preeclampsia and investigate its role in placental stem cell biology. METHODS: MUC15 mRNA and protein were measured in placentas from patients with early onset (<34 weeks' gestation) preeclampsia. Circulating serum MUC15 was measured via ELISA. MUC15 was localised in the placenta using in situ hybridisation. MUC15 mRNA expression was measured across differentiation of human trophoblast stem cells (hTSCs) to syncytiotrophoblast and extravillous trophoblasts. MUC15 was measured after syncytialised hTSCs were cultured in hypoxic (1% O2) and proinflammatory (TNF α, IL-6) conditions. MUC15 secretion was assessed when syncytialised hTSCs were treated with brefeldin A (impairs protein trafficking) and batimastat (inhibits matrix metalloproteinases). RESULTS: MUC15 protein was significantly increased in the placenta (P = 0.0003, n = 32 vs n = 20 controls) and serum (P = 0.016, n = 32 vs n = 22 controls) of patients with preeclampsia, whilst MUC15 mRNA remained unchanged (n = 61 vs n = 18 controls). MUC15 mRNA (P = 0.005) and protein secretion (P = 0.006) increased following differentiation to syncytiotrophoblast cells. In situ hybridisation confirmed MUC15 localised to the syncytiotrophoblast cell within the placenta. Neither hypoxic or inflammatory conditions changed MUC15 mRNA expression or secretion. Brefeldin A treated hTSCs did not alter MUC15 secretion, whilst batimastat reduced MUC15 secretion (P = 0.044). CONCLUSIONS: MUC15 is increased in early onset preeclampsia and is cleaved by matrix metalloproteinases. Increased MUC15 may reflect a protective mechanism associated with placental dysfunction. Further research will aid in confirming this.

Absorption and Transport Mechanism of Red Meat-Derived N-glycolylneuraminic Acid and Its Damage to Intestinal Barrier Function through the NF-κB Signaling Pathway.

N-glycolylneuraminic acid (Neu5Gc) is a specific factor in red meat that induces intestinal disease. Our aim was to investigate the effect of Neu5Gc on the intestinal barrier as well as its mechanism of endocytosis and exocytosis. Ten specific inhibitors were used to explore the mechanism of Neu5Gc endocytosis and exocytosis by Caco-2 cells. Amiloride hydrochloride and cytochalasin D had the strongest inhibitory effect on the endocytosis of Neu5Gc. Sodium azide, dynasore, chlorpromazine hydrochloride, and nystatin also inhibited Neu5Gc endocytosis. Dynasore exhibited a stronger inhibitory effect than that of chlorpromazine hydrochloride or nystatin alone. Exocytosis inhibitors, including nocodazole, brefeldin A, monensin, and bafilomycin A, inhibited the transmembrane transport of Neu5Gc. Monensin promoted the exocytosis of Neu5Gc from Caco-2 cells. In another experiment, we observed no significant inhibitory effects of monensin and brefeldin A. Dietary concentrations of Neu5Gc induced prominent damage to intestinal tight junction proteins zonula occludens-1 (ZO-1), occludin, and claudin-1 and promoted the phosphorylation of IκB-α and P65 to activate the canonical Nuclear Factor kappa-B (NF-κB) pathway. Neu5Gc increased the RNA levels of pro-inflammatory factors IL-1ß, IL-6, and TNF-α and inhibited those of anti-inflammatory factors TGF-ß and IL-10. BAY, an NF-κB signaling pathway inhibitor, attenuated these changes. Reductions in the levels of ZO-1, occludin, and claudin-1 were recovered in response to BAY. Our data reveal the endocytosis and exocytosis mechanism of Neu5Gc and prove that Neu5Gc can activate the canonical NF-κB signaling pathway, regulate the transcription of inflammatory factors, thereby damaging intestinal barrier function.

Microscopy and Immunocytochemistry-Based Methods to Study Cell Wall Biosynthetic Enzymes in the Golgi.

The Golgi apparatus has essential roles in all eukaryotic cells, and its importance in plants is further exemplified by a critical role in building a cellulosic cell wall. The Golgi apparatus houses numerous cell wall-synthesizing or cell wall-modifying enzymes to generate the complex cell wall structure. However, several putative cell wall biosynthetic candidates await characterization, which requires verification of the subcellular localization and enzymatic products. Here, we describe detailed methods to analyze the localization of proteins that are transiently produced in tobacco leaves or stably produced in transgenic plants, by confocal microscopy using fluorescent-tagged proteins along with known Golgi markers or the trafficking inhibitor brefeldin A. We also detail a procedure to analyze the enzymatic products through antibody-based immunoblotting after cell wall enrichment.

The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis.

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin. This response depends on the concerted activities of the auxin transporters such as PIN-FORMED (PIN) proteins for auxin efflux and AUXIN RESISTANT 1 (AUX1) for auxin influx. However, how the auxin gradient is established remains elusive. Here we identified a new mutant with a short root, strong auxin distribution in the lateral root cap and an impaired gravitropic response. The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor (URI). AtURI interacted with prefoldin 2 (PFD2) and PFD6, two ß-type PFD members that modulate actin and tubulin patterning in roots. The auxin reporter DR5rev :GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips. Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter PIN2 is disrupted in aturi-1 roots as well as in pfd mutants. We propose that AtURI cooperates with PFDs to recycle PIN2 and modulate auxin distribution.

ER-stress-induced secretion of circulating glucose-regulated protein 78kDa (GRP78) ameliorates pulmonary artery smooth muscle cell remodelling.

Pulmonary arterial hypertension (PAH) is driven by vascular remodelling due to inflammation and cellular stress, including endoplasmic reticulum stress (ER stress). The main ER-stress chaperone, glucose-regulated protein 78 kDa (GRP78), is known to have protective effects in inflammatory diseases through extracellular signalling. The aim of this study is to investigate its significance in PAH. Human pulmonary arterial smooth muscle cells (PASMC) were stimulated with compounds that induce ER stress, after which the secretion of GRP78 into the cell medium was analysed by western blot. We found that when ER stress was induced in PASMC, there was also a time-dependent secretion of GRP78. Next, naïve PASMC were treated with conditioned medium (CM) from the ER-stressed donor PASMC. Incubation with CM from ER-stressed PASMC reduced the viability, oxidative stress, and expression of inflammatory and ER-stress markers in target cells. These effects were abrogated when the donor cells were co-treated with Brefeldin A to inhibit active secretion of GRP78. Direct treatment of PASMC with recombinant GRP78 modulated the expression of key inflammatory markers. Additionally, we measured GRP78 plasma levels in 19 PAH patients (Nice Group I) and correlated the levels to risk stratification according to ESC guidelines. Here, elevated plasma levels of GRP78 were associated with a favourable risk stratification. In conclusion, GRP78 is secreted by PASMC under ER stress and exhibits protective effects from the hallmarks of PAH in vitro. Circulating GRP78 may serve as biomarker for risk adjudication of patients with PAH. Proposed mechanism of ER-stress-induced GRP78 secretion by PASMC. Extracellular GRP78 can be measured as a circulating biomarker and is correlated with favourable clinical characteristics. Conditioned medium from ER-stressed PASMC reduces extensive viability, ROS formation, inflammation, and ER stress in target cells. These effects can be abolished by blocking protein secretion in donor cells by using Brefeldin A.

Identification of secreted proteins by comparison of protein abundance in conditioned media and cell lysates.

Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach. The subset of proteins demonstrating increased conditioned media to cell lysate ratio in the presence of Brefeldin A identified candidates for unconventional secretion via a pathway independent of ER to Golgi trafficking.

Ca2+ transport via TRPV6 is regulated by rapid internalization of the channel.

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca2+-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca2+]i with a half time in the range of 0.5-1 h. Upon wash-out of the toxin, the [Ca2+]i rose to a steady-state level within 2-3 h. Consistently, the synchronized forward trafficking of TRPV6VL-eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6VL-bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca2+ transport.

Brefeldin A and kifunensine modulate LPS-induced lung endothelial hyperpermeability in human and bovine cells.

Endothelial hyperpermeability is the hallmark of acute respiratory distress syndrome (ARDS). Laborious efforts in the investigation of the molecular pathways involved in the regulation of the vascular barrier shall reveal novel therapeutic targets toward that respiratory disorder. Herein, we investigate in vitro the effects of the α-1,2-mannosidase 1 inhibitor kifunensine (KIF) and brefeldin A (BFA) in the lipopolysaccharides (LPS)-induced endothelial breakdown. Our results suggest that BFA opposes the deteriorating effects of KIF [unfolded protein response (UPR) suppressor] toward the lung microvasculature. Since KIF is a UPR suppressor, and brefeldin A is a UPR inducer, we suggest that a carefully devised UPR manipulation may deliver novel therapeutic avenues in diseases related to endothelial barrier dysfunction (e.g., ARDS and sepsis).

GNOM-dependent endocytosis maintains polar localisation of the borate exporter BOR1 in Arabidopsis.

BACKGROUND INFORMATION: Plants use transporters polarly localised in the plasma membrane for the directional transport of nutrients. The boric acid/borate (B) exporter BOR1 is localised polarly in the inner lateral domain of the plasma membrane in various root cells for efficient translocation of B under B limitation. With a high B supply, BOR1 is ubiquitinated and transported to vacuoles for degradation. The polar localisation and vacuolar targeting of BOR1 are maintained by different endocytosis mechanisms. RESULTS: We demonstrated that one of the most utilised inhibitors in endosomal recycling, brefeldin A (BFA), inhibits the polar localisation of BOR1. BFA inhibits a subset of guanine-nucleotide exchange factors (ARF-GEFs), regulators of vesicle formation. Using a transgenic line expressing BFA-resistant engineered GNOM, we identified GNOM as the key ARF-GEF in endocytosis and maintenance of the polar localisation of BOR1. CONCLUSIONS AND SIGNIFICANCE: We found that BFA inhibits the polar localisation of BOR1 by inhibiting GNOM activity. Our results suggest that GNOM-dependent endocytosis contributes to the maintenance of the polar localisation of BOR1 under B limitation. We propose a model of BOR1 transcytosis initiated from GNOM-dependent endocytosis.

Class II Arfs require a brefeldin-A-sensitive factor for Golgi association.

Arf proteins are small Ras-family GTPases which recruit clathrin and COPI coats to Golgi membranes and regulate components of the membrane trafficking machinery. It is believed membrane association and activity of Arfs is coupled to GTP binding, with GTP hydrolysis required for vesicle uncoating. In humans, four Arf proteins (Arf1, Arf3, Arf4 and Arf5) are Golgi-associated. Conflicting reports have suggested that HA-GFP-tagged Class II ARFs (Arf4 and Arf5) are recruited to membrane independently of the brefeldin A sensitive exchange factor GBF1, suggesting regulation fundamentally different from the Class I Arfs (Arf1, Arf3), or alternately that the GTPase cycle of GFP-tagged Class II Arfs is similar to other Arfs. We show that these results depend on the fluorescent tag, with Arf4-HA-GFP tag resistant to brefeldin, but Arf4-GFP acting similarly to Arf1-GFP in brefeldin-sensitivity and photobleach assays. Arf4-HA-GFP could be partially reverted to the behavior of Arf4-GFP by mutation of two aspartic acids in the HA tag to alanine. Our results, which indicate a high sensitivity of Arf4 to tagging, can explain the discrepancies between previous studies. We discuss the implications of this study for future work with tagged Arfs.

Secretion of Phospholipase Dδ Functions as a Regulatory Mechanism in Plant Innate Immunity.

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.

The Phytophthora infestans Haustorium Is a Site for Secretion of Diverse Classes of Infection-Associated Proteins.

The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection.IMPORTANCE There are many different classes of proteins secreted from Phytophthora infestans that may influence or facilitate infection. Elucidating where and how they are secreted during infection is an important step toward developing methods to control their delivery processes. We used an inhibitor of conventional secretion to identify the following different classes of infection-associated extracellular proteins: cell wall-degrading and cell wall-modifying enzymes, microbe-associated molecular pattern-like proteins that may elicit immune responses, and apoplastic effectors that are predicted to suppress immunity. In contrast, secretion of a cytoplasmic effector that is translocated into host cells is nonconventional, as it is insensitive to inhibitor treatment. This evidence further supports the finding that proteins that are active in the apoplast and effector proteins that are active in the host cytoplasm are differentially secreted by P. infestans Critically, it demonstrates that a disease-specific developmental structure, the haustorium, is a major secretion site for diverse protein classes during infection.

Use of Brefeldin A and Wortmannin to Dissect Post-Golgi Organelles Related to Vacuolar Transport in Arabidopsis thaliana.

Eukaryotic cells comprise various organelles surrounded by the membrane. Each organelle is characterized by unique proteins and lipids and has its own specific functions. Single membrane-bounded organelles, including the Golgi apparatus, endosomes, and vacuoles are connected by membrane trafficking. Identifying the organelle localization of a protein of interest is essential for determining the proteins physiological functions. Here, we describe methods for determining protein subcellular localization using the inhibitors brefeldin A and wortmannin in Arabidopsis thaliana.

Therapeutic Delivery Specifications Identified Through Compartmental Analysis of a Mesenchymal Stromal Cell-Immune Reaction.

Despite widespread preclinical success, mesenchymal stromal cell (MSC) therapy has not reached consistent pivotal clinical endpoints in primary indications of autoinflammatory diseases. Numerous studies aim to uncover specific mechanisms of action towards better control of therapy using in vitro immunomodulation assays. However, many of these immunomodulation assays are imperfectly designed to accurately recapitulate microenvironment conditions where MSCs act. To increase our understanding of MSC efficacy, we herein conduct a systems level microenvironment approach to define compartmental features that can influence the delivery of MSCs' immunomodulatory effect in vitro in a more quantitative manner than ever before. Using this approach, we notably uncover an improved MSC quantification method with predictive cross-study applicability and unveil the key importance of system volume, time exposure to MSCs, and cross-communication between MSC and T cell populations to realize full therapeutic effect. The application of these compartmental analysis can improve our understanding of MSC mechanism(s) of action and further lead to administration methods that deliver MSCs within a compartment for predictable potency.

Manganese transport and toxicity in polarized WIF-B hepatocytes.

Manganese (Mn) toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains whereas ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to >250 µM MnCl2. However, the hepatocytes were resistant to 4-h exposures of up to 500 µM MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor, brefeldin A, did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile. NEW & NOTEWORTHY Polarized WIF-B hepatocytes express manganese (Mn) transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14. Fpn and ZIP14 localize to basolateral domains. ZnT10-positive vesicles were adjacent to apical bile compartments, and ZIP8-positive vesicles were distributed uniformly throughout the cytoplasm. WIF-B hepatocyte Mn export was resistant to hepcidin but inhibited by brefeldin A, pointing to an efflux mechanism involving ZnT10-mediated uptake of Mn into vesicles that subsequently fuse with and empty their contents across the apical bile canalicular membrane.

The Golgi entry core compartment functions as a COPII-independent scaffold for ER-to-Golgi transport in plant cells.

Many questions remain about how the stacked structure of the Golgi is formed and maintained. In our previous study, we challenged this question using tobacco BY-2 cells and revealed that, upon Brefeldin A (BFA) treatment, previously undescribed small punctate structures containing a particular subset of cis-Golgi proteins are formed adjacent to the ER-exit sites and act as scaffolds for Golgi regeneration after BFA removal. In this study, we analyzed these structures further. The proteins that localize to these punctate structures originate from the cis-most cisternae. 3D time-lapse observations show that the trans-Golgi marker is transported through these structures during Golgi regeneration. These data indicate that the cis-most cisternae have a specialized region that receives cargo from the ER, which becomes obvious upon BFA treatment. Expression of a dominant mutant form of SAR1 does not affect the formation of the punctate structures. We propose to call these punctate structures the 'Golgi entry core compartment' (GECCO). They act as receivers for the rest of the Golgi materials and are formed independently of the COPII machinery.This article has an associated First Person interview with the first author of the paper.

Agrobacterium-delivered virulence protein VirE2 is trafficked inside host cells via a myosin XI-K-powered ER/actin network.

Agrobacterium tumefaciens causes crown gall tumors on various plants by delivering transferred DNA (T-DNA) and virulence proteins into host plant cells. Under laboratory conditions, the bacterium is widely used as a vector to genetically modify a wide range of organisms, including plants, yeasts, fungi, and algae. Various studies suggest that T-DNA is protected inside host cells by VirE2, one of the virulence proteins. However, it is not clear how Agrobacterium-delivered factors are trafficked through the cytoplasm. In this study, we monitored the movement of Agrobacterium-delivered VirE2 inside plant cells by using a split-GFP approach in real time. Agrobacterium-delivered VirE2 trafficked via the endoplasmic reticulum (ER) and F-actin network inside plant cells. During this process, VirE2 was aggregated as filamentous structures and was present on the cytosolic side of the ER. VirE2 movement was powered by myosin XI-K. Thus, exogenously produced and delivered VirE2 protein can use the endogenous host ER/actin network for movement inside host cells. The A. tumefaciens pathogen hijacks the conserved host infrastructure for virulence trafficking. Well-conserved infrastructure may be useful for Agrobacterium to target a wide range of recipient cells and achieve a high efficiency of transformation.

An Insight into the Secondary Metabolism of Muscodor yucatanensis: Small-Molecule Epigenetic Modifiers Induce Expression of Secondary Metabolism-Related Genes and Production of New Metabolites in the Endophyte.

Muscodor spp. are proficient producers of bioactive volatile organic compounds (VOCs) with many potential applications. However, all members of this genus produce varying amounts and types of VOCs which suggests the involvement of epigenetics as a possible explanation. The members of this genus are poorly explored for the production of soluble compounds (extrolites). In this study, the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes from an endophyte, Muscodor yucatanensis Ni30, were cloned and sequenced. The PKS genes belonged to reduced, partially reduced, non-reduced, and highly reduced subtypes. Strains over-expressing PKS genes were developed through the use of small-molecule epigenetic modifiers (suberoylanilide hydroxamic acid (SAHA) and 5-azacytidine). The putative epigenetic variants of this organism differed considerably from the wild type in morphological features and cultural characteristics as well as metabolites that were produced. Each variant produced a different set of VOCs distinct from the wild type, and several VOCs including methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)hexane-2,4-diol and 2-carboxymethyl-3-n-hexylmaleic appeared in the variant strains, the production of which could be attributed to the activity of otherwise silent PKS genes. The bioactive extrolite brefeldin A was isolated and characterized from the wild type. However, this metabolite was not detected in EV-1, but instead, two other products were isolated and characterized as ergosterol and xylaguaianol C. Hence, M. yucatanensis has the genetic potential to produce several previously undetectable VOCs and organic solvent soluble products. It is also the case that small-molecule epigenetic modifiers can be used to produce stable variant strains of fungi with the potential to produce new molecules. Finally, this work hints to the prospect that the epigenetics of an endophytic microorganism can be influenced by any number of environmental and chemical factors associated with its host plant which may help to explain the enormous chemical diversity of secondary metabolic products found in Muscodor spp.

Alternative autophagy, brefeldin A and viral trafficking pathways.

Two topics that have attracted recent attention in the field of autophagy concern the source of the membrane that is used to form the autophagosome during macroautophagy and the role of noncanonical autophagic pathways. The 2 topics may converge when considering the intersection of autophagy with viral infection. We suggest that noncanonical autophagy, which is sensitive to treatment with brefeldin A, may converge with the infectious cycles of certain DNA and RNA viruses that utilize membrane from the ER and cis-Golgi.

The border-to-border distribution method for analysis of cytoplasmic particles and organelles.

Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.