A Comprehensive Study on Nanoparticle Drug Delivery to the Brain: Application of Machine Learning Techniques.

The delivery of drugs to specific target tissues and cells in the brain poses a significant challenge in brain therapeutics, primarily due to limited understanding of how nanoparticle (NP) properties influence drug biodistribution and off-target organ accumulation. This study addresses the limitations of previous research by using various predictive models based on collection of large data sets of 403 data points incorporating both numerical and categorical features. Machine learning techniques and comprehensive literature data analysis were used to develop models for predicting NP delivery to the brain. Furthermore, the physicochemical properties of loaded drugs and NPs were analyzed through a systematic analysis of pharmacodynamic parameters such as plasma area under the curve. The analysis employed various linear models, with a particular emphasis on linear mixed-effect models (LMEMs) that demonstrated exceptional accuracy. The model was validated via the preparation and administration of two distinct NP formulations via the intranasal and intravenous routes. Among the various modeling approaches, LMEMs exhibited superior performance in capturing underlying patterns. Factors such as the release rate and molecular weight had a negative impact on brain targeting. The model also suggests a slightly positive impact on brain targeting when the drug is a P-glycoprotein substrate.

2,3,7,8-tetrachlorodibenzo-p-dioxin induces multigenerational testicular toxicity and biosynthetic disorder of testosterone in BALB/C mice: Transcriptional, histopathological and hormonal determinants.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent environmental contaminant, is an endocrine disrupter with a proven reproductive toxicity in mammals. However, its effects on male fertility across generations are still elusive. The current work evaluates the toxicity of dioxin on male reproductive system in two separate groups of BALB/C mice; a group of pubertal males directly exposed to TCDD (referred to as DEmG), and a group of indirectly exposed males (referred to as IDEmG) comprises of F1, F2 and F3 males born from TCDD-exposed pregnant females. Both groups were exposed to 25 µg TCDD/kg body weight for a week. Our data show that males of TCDD-DEmG exhibited significant alterations in the expression of certain genes involved in the detoxification of TCDD and the biosynthesis of testosterone. This was accompanied with testicular pathological symptoms, including a sloughing in the germinal epithelium and a congestion of blood vessels in interstitial tissue with the presence of multinuclear cells into seminiferous tubule, with a 4-fold decline in the level of serum testosterone and reduced sperm count. Otherwise, the male reproductive toxicity across F1, F2 and F3 generations from TCDD-IDEmG was mainly characterized by: i) a reduce in body and testis weight. ii) a decrease in gene expression of steriodogenesis enzyme, e.g., AhR, CYP1A1, CYP11A1, COX1, COX2, LOX5 and LOX12. iii) a remarked and similar testicular histopathology that found for DEmG, iv) a serious decline in serum testosterone. v) a decreased male-to-female ratio. vi) a low sperm count with increasing abnormalities. Thus, pubertal or maternal exposure to TCDD provokes multigenerational male reproductive toxicity in mice, ultimately affecting the spermatogenesis and suggesting that the hormonal alternation and sperm abnormality are the most marked effects of the indirect exposure of mammalian male to TCDD.

Female-to-male differential transcription patterns of miRNA-mRNA networks in the livers of dioxin-exposed mice.

Non-coding microRNAs (miRNAs) have important roles in regulating the expression of liver mRNAs in response to xenobiotic-exposure, but their roles concerning dioxins such as TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) are less clear. This report concerns the potential implication of liver (class I) and circulating (class II) miRNAs in hepatotoxicity of female and male mice after acute exposure to TCDD. The data show that, of a total of 38 types of miRNAs, the expression of eight miRNAs were upregulated in both female and male mice exposed to TCDD. Inversely, the expression of nine miRNAs were significantly downregulated in both animal genders. Moreover, certain miRNAs were preferentially induced in either females or males. The potential downstream regulatory effects of miRNAs on their target genes was evaluated by determining the expression of three group of genes that are potentially involved in cancer biogenesis, other diseases and in hepatotoxicity. It was found that certain cancer-related genes were more highly expressed females rather than males after exposure to TCDD. Furthermore, a paradoxical female-to-male transcriptional pattern was found for several disease-related and hepatotoxicity-related genes. These results suggest the possibility of developing of new miRNA-specific interfering molecules to address their dysfunctions as caused by TCDD.

Transcriptional, hormonal and histological alterations in the ovaries of BALB/c mice exposed to TCDD in connection with multigenerational female infertility.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins, has a proven reproductive toxicity. Due to the lack of evidence on the multigenerational female reproductive toxicity of TCDD through the maternal exposure, the current study aims to evaluate, on the one hand, the acute reproductive toxicity of TCDD on adult female pre-gestational exposed to a critical single dose of TCDD (25 µg/kg) for a week (group referred to as AFnG; adult female/non-gestation). On the other hand, the transcription, hormonal and histological effects of TCDD on the females of two generations F1 and F2, were also investigated after the exposure of pregnant females to TCDD on gestational day 13 (GD13) (group referred to as AFG; adult female/gestation). First, our data showed alternations in the ovarian expressional pattern of certain key genes involved in the detoxification of TCDD as well as in the biosynthesis of steroidal hormones. The expression of Cyp1a1 was highly induced in TCDD-AFnG group, but reduced in both F1 and F2. While the transcripts levels of Cyp11a1 and 3ßhsd2 were decreased, Cyp19a1 transcripts were increased as a function of TCDD exposure. This was synchronized with a dramatic increase in the level of estradiol hormone in the females of both experimental groups. Beside a significant reduce in their size and weight, ovaries of TCDD-exposed females showed serious histological alterations marked by atrophy of the ovary, congestion in the blood vessels, necrosis in the layer of granular cells, dissolution of the oocyte and nucleus of ovarian follicles. Finally, the female fertility was dramatically affected across generations with a reduced male\female ratio. Our data indicate that the exposure of pregnant female to TCDD has serious negative effects in the female productive system across generations and suggest the use of hormonal alternation as biomarker to monitor and assess the indirect exposure of these generations to TCDD.

Mammalian lipid droplets: structural, pathological, immunological and anti-toxicological roles.

Mammalian lipid droplets (LDs) are specialized cytosolic organelles consisting of a neutral lipid core surrounded by a membrane made up of a phospholipid monolayer and a specific population of proteins that varies according to the location and function of each LD. Over the past decade, there have been significant advances in the understanding of LD biogenesis and functions. LDs are now recognized as dynamic organelles that participate in many aspects of cellular homeostasis plus other vital functions. LD biogenesis is a complex, highly-regulated process with assembly occurring on the endoplasmic reticulum although aspects of the underpinning molecular mechanisms remain elusive. For example, it is unclear how many enzymes participate in the biosynthesis of the neutral lipid components of LDs and how this process is coordinated in response to different metabolic cues to promote or suppress LD formation and turnover. In addition to enzymes involved in the biosynthesis of neutral lipids, various scaffolding proteins play roles in coordinating LD formation. Despite their lack of ultrastructural diversity, LDs in different mammalian cell types are involved in a wide range of biological functions. These include roles in membrane homeostasis, regulation of hypoxia, neoplastic inflammatory responses, cellular oxidative status, lipid peroxidation, and protection against potentially toxic intracellular fatty acids and lipophilic xenobiotics. Herein, the roles of mammalian LDs and their associated proteins are reviewed with a particular focus on their roles in pathological, immunological and anti-toxicological processes.

Caleosin/peroxygenases: multifunctional proteins in plants.

BACKGROUND: Caleosin/peroxygenases (CLO/PXGs) are a family of multifunctional proteins that are ubiquitous in land plants and are also found in some fungi and green algae. CLO/PXGs were initially described as a class of plant lipid-associated proteins with some similarities to the oleosins that stabilize lipid droplets (LDs) in storage tissues, such as seeds. However, we now know that CLO/PXGs have more complex structures, distributions and functions than oleosins. Structurally, CLO/PXGs share conserved domains that confer specific biochemical features, and they have diverse localizations and functions. SCOPE: This review surveys the structural properties of CLO/PXGs and their biochemical roles. In addition to their highly conserved structures, CLO/PXGs have peroxygenase activities and are involved in several aspects of oxylipin metabolism in plants. The enzymatic activities and the spatiotemporal expression of CLO/PXGs are described and linked with their wider involvement in plant physiology. Plant CLO/PXGs have many roles in both biotic and abiotic stress responses in plants and in their responses to environmental toxins. Finally, some intriguing developments in the biotechnological uses of CLO/PXGs are addressed. CONCLUSIONS: It is now two decades since CLO/PXGs were first recognized as a new class of lipid-associated proteins and only 15 years since their additional enzymatic functions as a new class of peroxygenases were discovered. There are many interesting research questions that remain to be addressed in future physiological studies of plant CLO/PXGs and in their recently discovered roles in the sequestration and, possibly, detoxification of a wide variety of lipidic xenobiotics that can challenge plant welfare.

Effects of dioxins on animal spermatogenesis: A state-of-the-art review.

The male reproductive system is especially affected by dioxins, a group of persistent environmental pollutants, resulting in irreversible abnormalities including effects on sexual function and fertility in adult males and possibly on the development of male offspring. The reproductive toxicity caused by dioxins is mostly mediated by an aryl hydrocarbon receptor (AhR). In animals, spermatogenesis is a highly sensitive and dynamic process that includes proliferation and maturation of germ cells. Spermatogenesis is subject to multiple endogenous and exogenous regulatory factors, including a wide range of environmental toxicants such as dioxins. This review discusses the toxicological effects of dioxins on spermatogenesis and their relevance to male infertility. After a detailed categorization of the environmental contaminants affecting the spermatogenesis, the exposure pathways and bioavailability of dioxins in animals was briefly reviewed. The effects of dioxins on spermatogenesis are then outlined in detail. The endocrine-disrupting effects of dioxins in animals and humans are discussed with a particular focus on their effects on the expression of spermatogenesis-related genes. Finally, the impacts of dioxins on the ratio of X and Y chromosomes, the status of serum sex hormones, the quality and fertility of sperm, and the transgenerational effects of dioxins on male reproduction are reviewed.

Insights into the control of taxane metabolism: Molecular, cellular, and metabolic changes induced by elicitation in Taxus baccata cell suspensions.

More knowledge is needed about the molecular/cellular control of paclitaxel (PTX) production in Taxus spp. cell cultures. In this study, the yield of this anticancer agent in Taxus baccata cell suspensions was improved 11-fold after elicitation with coronatine (COR) compared to the untreated cells, and 18-fold when co-supplemented with methyl-ß-cyclodextrins (ß-CDs). In the dual treatment, the release of taxanes from the producer cells was greatly enhanced, with 81.6% of the total taxane content being found in the medium at the end of the experiment. The experimental conditions that caused the highest PTX production also induced its maximum excretion, and increased the expression of taxane biosynthetic genes, especially the flux-limiting BAPT and DBTNBT. The application of COR, which activates PTX biosynthesis, together with ß - CDs, which form inclusion complexes with PTX and related taxanes, is evidently an efficient strategy for enhancing PTX production and release to the culture medium. Due to the recently described role of lipid droplets (LDs) in the trafficking and accumulation of hydrophobic taxanes in Taxus spp. cell cultures, the structure, number and taxane storage capacity of these organelles was also studied. In elicited cultures, the number of LDs increased and they mainly accumulated taxanes with a side chain, especially PTX. Thus, PTX constituted up to 50-70% of the total taxanes found in LDs throughout the experiment in the COR + ß - CD-treated cultures. These results confirm that LDs can store taxanes and distribute them inside and outside cells.

Characterization of lipid droplets from a Taxus media cell suspension and their potential involvement in trafficking and secretion of paclitaxel.

KEY MESSAGE: Our paper describes the potential roles of lipid droplets of Taxus media cell suspension in the biosynthesis and secretion of paclitaxel and, therefore, highlights their involvement in improving its production. Paclitaxel (PTX) is a highly potent anticancer drug that is mainly produced using Taxus sp. cell suspension cultures. The main purpose of the current study is to characterize cellular LDs from T. media cell suspension with a particular focus on the biological connection of their associated proteins, the caleosins (CLOs), with the biosynthesis and secretion of PTX. A pure LD fraction obtained from T. media cells and characterized in terms of their proteome. Interestingly, the cellular LD in T. media sequester the PTX. This was confirmed in vitro, where about 96% of PTX (C0PTX,aq [mg L-1]) in the aqueous solution was partitioned into the isolated LDs. Furthermore, silencing of CLO-encoding genes in the T. media cells led to a net decrease in the number and size of LDs. This coincided with a significant reduction in expression levels of TXS, DBAT and DBTNBT, key genes in the PTX biosynthesis pathway. Subsequently, the biosynthesis of PTX was declined in cell culture. In contrast, treatment of cells with 13-hydroperoxide C18:3, a substrate of the peroxygenase activity, induced the expression of CLOs, and, therefore, the accumulation of cellular LDs in the T. media cells cultures, thus increasing the PTX secretion. The accumulation of stable LDs is critically important for effective secretion of PTX. This is modulated by the expression of caleosins, a class of LD-associated proteins with a dual role conferring the structural stability of LDs as well as regulating lipidic bioactive metabolites via their enzymatic activity, thus enhancing the biosynthesis of PTX.

Functional involvement of caleosin/peroxygenase PdPXG4 in the accumulation of date palm leaf lipid droplets after exposure to dioxins.

Dioxins are highly injurious environmental pollutants with proven toxicological effects on both animals and humans, but to date their effects on plants still need to be studied in detail. We identified a dioxin-inducible caleosin/peroxygenase isoform, PdPXG4, that is mostly expressed in leaves of date palm seedlings and exhibits a specific reductase activity towards the 13-hydroperoxide of C18:2 and C18:3 (HpODE and HpOTrE, respectively). After exposure to TCDD, lipid droplets (LDs) isolated from TCDD-exposed leaves were about 6.5-15.7-fold more active in metabolizing 13-HpOTrE compared with those isolated from non-exposed leaves. A characteristic spectrum of leaf dioxin-responsive oxylipins (LDROXYL) was detected in dioxin-exposed seedlings. Of particular importance, a group of these oxylipins, referred to as Class I, comprising six congeners of hydroxides fatty acids derived from C18:2 and C18:3, was exclusively found in leaves after exposure to TCDD. The TCDD-induced oxylipin pattern was confirmed in vitro using terbufos, a typical inhibitor towards the PdPXG4 peroxygenase activity. Of particular interest, the response of terbufos-pretreated protoplasts to TCDD was drastically reduced. Together, these findings suggest that PdPXG4 is implicated in the establishment of a dioxin-specific oxylipin signature in date palm leaves soon after their exposure to these pollutants.

Involvement of hepatic lipid droplets and their associated proteins in the detoxification of aflatoxin B1 in aflatoxin-resistance BALB/C mouse.

The highly potent carcinogen, Aflatoxin B1, induces liver cancer in many animals including humans but some mice strains are highly resistant. This murine resistance is due to a rapid detoxification of AFB1. Hepatic lipid droplets (LDs) ultimately impact the liver functions but their potential role in AFB1 detoxification has not been addressed. This study describes the structural and functional impacts on hepatic LDs in BALB/C mice after exposure to 44 (low dose) or 663 (high dose) µg AFB1/kg of body weight. After 7 days, the liver of AFB1-dosed mice did not accumulate any detectable AFB1 or its metabolites and this was associated with a net increase in gene transcripts of the AhR-mediating pathway. Of particular interest, the livers of high-dose mice accumulated many more LDs than those of low-dose mice. This was accompanied with a net increase in transcript levels of LD-associated protein-encoding genes including Plin2, Plin3 and Cideb and an alteration in the LDs lipid profiles that could be likely due to the induction of lipoxygenase and cyclooxygenase genes. Interestingly, our data suggest that hepatic LDs catalyze the in vitro activation of AFB1 into AFB1-exo-8,9-epoxide and subsequent hydrolysis of this epoxide into its corresponding dihydrodiol. Finally, transcript levels of CYP1A2, CYP1B1, GSTA3 and EH1 genes were elevated in livers of high-dose mice. These data suggest new roles for hepatic LDs in the trapping and detoxifying of aflatoxins.

Exposure of Aspergillus flavus NRRL 3357 to the Environmental Toxin, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin, Results in a Hyper Aflatoxicogenic Phenotype: A Possible Role for Caleosin/Peroxygenase (AfPXG).

Aflatoxins (AFs) as potent food contaminants are highly detrimental to human and animal health. The production of such biological toxins is influenced by environmental factors including pollutants, such as dioxins. Here, we report the biological feedback of an active AF-producer strain of A. flavus upon in vitro exposure to the most toxic congener of dioxins, the 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD). The phenotype of TCDD-exposed A. flavus was typified by a severe limitation in vegetative growth, activation of conidia formation and a significant boost in AF production. Furthermore, the level of reactive oxygen species (ROS) in fungal protoplast was increased (3.1- to 3.8-fold) in response to TCDD exposure at 10 and 50 ng mL-1, respectively. In parallel, superoxide dismutase (SOD) and catalase (CAT) activities were, respectively, increased by a factor of 2 and 3. In contrast to controls, transcript, protein and enzymatic activity of caleosin/peroxygenase (AfPXG) was also significantly induced in TCDD-exposed fungi. Subsequently, fungal cells accumulated fivefold more lipid droplets (LDs) than controls. Moreover, the TCDD-exposed fungi exhibited twofold higher levels of AFB1. Interestingly, TCDD-induced hyperaflatoxicogenicity was drastically abolished in the AfPXG-silencing strain of A. flavus, suggesting a role for AfPXG in fungal response to TCDD. Finally, TCDD-exposed fungi showed an increased in vitro virulence in terms of sporulation and AF production. The data highlight the possible effects of dioxin on aflatoxicogenicity of A. flavus and suggest therefore that attention should be paid in particular to the potential consequences of climate change on global food safety.

The cytochrome P450BM-1 of Bacillus megaterium A14K is induced by 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin: Biophysical, molecular and biochemical determinants.

The current study describes biological changes in Bacillus megaterium A14K cells growing in the presence of 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin (TCDD), the most potent congener of dioxins. The results indicate that the metabolizing of 2,3,7,8-TCDD by BmA14K was accompanied with a novel morphological and biophysical profile typified by the growth of single cells with high levels of biosurfactant production, surface hydrophobicity and cell membrane permeability. Moreover, the TCDD-grown bacteria exhibited a specific fatty acid profile characterized by low ratios of branched/straight chain fatty acids (BCFAs/SCFAs) and saturated/unsaturated fatty acids (SFAs/USFAs) with a specific "signature" due to the presence of branched chain unsaturated fatty acids (BCUFAs). This was synchronized with a significant induction of P450BM-1, an unsaturated fatty acid-metabolizing enzyme in B. megaterium. Subsequently, the profile of oxygenated fatty acids in the TCDD-grown bacteria was typified by the presence of 5,6-epoxy derived from unsaturated C15, C16 and C17 fatty acids, that were absent in control bacteria. A net increase was also detected in both hydroxylated and epoxidized fatty acids, especially those derived from C15:0 and C16:1, respectively, suggesting a specific TCDD-induced "signature" of oxygenated fatty acids in BmA14K. Overall, this study sheds light on the use of B. megaterium A14K as a promising bioindicator/biodegrader of dioxins.

Evolutionary, structural and functional analysis of the caleosin/peroxygenase gene family in the Fungi.

BACKGROUND: Caleosin/peroxygenases, CLO/PXG, (designated PF05042 in Pfam) are a group of genes/proteins with anomalous distributions in eukaryotic taxa. We have previously characterised CLO/PXGs in the Viridiplantae. The aim of this study was to investigate the evolution and functions of the CLO/PXGs in the Fungi and other non-plant clades and to elucidate the overall origin of this gene family. RESULTS: CLO/PXG-like genes are distributed across the full range of fungal groups from the basal clades, Cryptomycota and Microsporidia, to the largest and most complex Dikarya species. However, the genes were only present in 243 out of 844 analysed fungal genomes. CLO/PXG-like genes have been retained in many pathogenic or parasitic fungi that have undergone considerable genomic and structural simplification, indicating that they have important functions in these species. Structural and functional analyses demonstrate that CLO/PXGs are multifunctional proteins closely related to similar proteins found in all major taxa of the Chlorophyte Division of the Viridiplantae. Transcriptome and physiological data show that fungal CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression and are upregulated in response to a range of biotic and abiotic stresses as well as participating in key metabolic and developmental processes such as lipid metabolism, signalling, reproduction and pathogenesis. Biochemical data also reveal that the Aspergillus flavus CLO/PXG has specific functions in sporulation and aflatoxin production as well as playing roles in lipid droplet function. CONCLUSIONS: In contrast to plants, CLO/PXGs only occur in about 30% of sequenced fungal genomes but are present in all major taxa. Fungal CLO/PXGs have similar but not identical roles to those in plants, including stress-related oxylipin signalling, lipid metabolism, reproduction and pathogenesis. While the presence of CLO/PXG orthologs in all plant genomes sequenced to date would suggest that they have core housekeeping functions in plants, the selective loss of CLO/PXGs in many fungal genomes suggests more restricted functions in fungi as accessory genes useful in particular environments or niches. We suggest an ancient origin of CLO/PXG-like genes in the 'last eukaryotic common ancestor' (LECA) and their subsequent loss in ancestors of the Metazoa, after the latter had diverged from the ancestral fungal lineage.

Identification of a dioxin-responsive oxylipin signature in roots of date palm: involvement of a 9-hydroperoxide fatty acid reductase, caleosin/peroxygenase PdPXG2.

Dioxins are highly hazardous pollutants that have well characterized impacts on both animal and human health. However, the biological effects of dioxins on plants have yet to be described in detail. Here we describe a dioxin-inducible caleosin/peroxygenase isoform, PdPXG2, that is mainly expressed in the apical zone of date palm roots and specifically reduces 9-hydroperoxide fatty acids. A characteristic spectrum of 18 dioxin-responsive oxylipin (DROXYL) congeners was also detected in date palm roots after exposure to dioxin. Of particular interest, six oxylipins, mostly hydroxy fatty acids, were exclusively formed in response to TCDD. The DROXYL signature was evaluated in planta and validated in vitro using a specific inhibitor of PdPXG2 in a root-protoplast system. Comparative analysis of root suberin showed that levels of certain monomers, especially the mono-epoxides and tri-hydroxides of C16:3 and C18:3, were significantly increased after exposure to TCDD. Specific inhibition of PdPXG2 activity revealed a positive linear relationship between deposition of suberin in roots and their permeability to TCDD. The results highlight the involvement of this peroxygenase in the plant response to dioxin and suggest the use of dioxin-responsive oxylipin signatures as biomarkers for plant exposure to this important class of xenobiotic contaminants.

Evolutionary and genomic analysis of the caleosin/peroxygenase (CLO/PXG) gene/protein families in the Viridiplantae.

Bioinformatics analyses of caleosin/peroxygenases (CLO/PXG) demonstrated that these genes are present in the vast majority of Viridiplantae taxa for which sequence data are available. Functionally active CLO/PXG proteins with roles in abiotic stress tolerance and lipid droplet storage are present in some Trebouxiophycean and Chlorophycean green algae but are absent from the small number of sequenced Prasinophyceaen genomes. CLO/PXG-like genes are expressed during dehydration stress in Charophyte algae, a sister clade of the land plants (Embryophyta). CLO/PXG-like sequences are also present in all of the >300 sequenced Embryophyte genomes, where some species contain as many as 10-12 genes that have arisen via selective gene duplication. Angiosperm genomes harbour at least one copy each of two distinct CLO/PX isoforms, termed H (high) and L (low), where H-forms contain an additional C-terminal motif of about 30-50 residues that is absent from L-forms. In contrast, species in other Viridiplantae taxa, including green algae, non-vascular plants, ferns and gymnosperms, contain only one (or occasionally both) of these isoforms per genome. Transcriptome and biochemical data show that CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression. CLO/PXG proteins can associate with cytosolic lipid droplets and/or bilayer membranes. Many of the analysed isoforms also have peroxygenase activity and are involved in oxylipin metabolism. The distribution of CLO/PXG-like genes is consistent with an origin >1 billion years ago in at least two of the earliest diverging groups of the Viridiplantae, namely the Chlorophyta and the Streptophyta, after the Viridiplantae had already diverged from other Archaeplastidal groups such as the Rhodophyta and Glaucophyta. While algal CLO/PXGs have roles in lipid packaging and stress responses, the Embryophyte proteins have a much wider spectrum of roles and may have been instrumental in the colonisation of terrestrial habitats and the subsequent diversification as the major land flora.

Arabidopsis plants exposed to dioxin result in a WRINKLED seed phenotype due to 20S proteasomal degradation of WRI1.

Dioxins are highly toxic persistent organic pollutants bioaccumulated by both plants and animals that cause severe developmental abnormalities in humans. We investigated the effects of dioxins on seed development in Arabidopsis. Plants were exposed to various concentrations of the most toxic congener of dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the effects on seed development were analysed in-depth at transcriptome, proteome and metabolome levels. Exposure to dioxin led to generalized effects on vegetative tissues plus a specific set of perturbations to seed development. Mature seeds from TCDD-treated plants had a characteristic 'wrinkled' phenotype, due to a two-thirds reduction in storage oil content. Transcriptional analysis of a panel of genes related to lipid and carbohydrate metabolism was consistent with the observed biochemical phenotypes. There were increases in WRI1 and LEC1 expression but decreases in ABI3 and FUS3 expression, which is puzzling in view of the low seed oil phenotype. This anomaly was explained by increased expression of 20S proteasome components that resulted in a substantial degradation of WRI1 protein, despite the up-regulation of the WRI1 gene. Our findings reveal novel effects of dioxins that lead to altered gene regulation patterns that profoundly affect seed development in Arabidopsis.

The Peroxygenase Activity of the Aspergillus flavus Caleosin, AfPXG, Modulates the Biosynthesis of Aflatoxins and Their Trafficking and Extracellular Secretion via Lipid Droplets.

Aflatoxins (AF) are highly detrimental to human and animal health. We recently demonstrated that the Aspergillus flavus caleosin, AfPXG, had peroxygenase activity and mediated fungal development and AF accumulation. We now report the characterization of an AfPXG-deficient line using reference strain NRRL3357. The resulting fungal phenotype included a severe decrease in mycelium growth, failure to sporulate, and reduced AF production. Increasing cellular oxidative status by administration of hydrogen peroxide and cumene hydroperoxide did not restore the AfPXG-deficient phenotype, which suggests that AfPXG-deficiency is not directly related to oxidative stress. To investigate possible alternative roles of AfPXG, a gain of function approach was used to overexpress AfPXG, with the reporter gene Gfp, in an AfPXG-deficient line, termed AfPXG+ . The resulting phenotype included elevated numbers of stable lipid droplets (LDs) plus enhanced AF production. Highly purified LDs from AfPXG+ cultures sequestered AF and this ability was positively correlated with overall LD number. Site-specific mutagenesis of AfPXG to delete Histidine 85 (AfPXGHis85), a residue essential for its catalytic activity, or deletion of the putative LD targeting domain (AfPXGD126-140), showed that AfPXG-peroxygenase activity was required for AF biosynthesis and that integration of AF into LDs was required for their export via a LD-dependent pathway. Ectopic expression in fungal cells of the plant LD-associated protein, oleosin, also resulted in both additional LD accumulation and enhanced AF secretion. These results suggest that both fungal LDs and their associated caleosin proteins are intimately involved in the biosynthesis, trafficking, and secretion of AF.

Biochemical, Molecular, and Transcriptional Highlights of the Biosynthesis of an Effective Biosurfactant Produced by Bacillus safensis PHA3, a Petroleum-Dwelling Bacteria.

Petroleum crude oil (PCO)-dwelling microorganisms have exceptional biological capabilities to tolerate the toxicity of petroleum contaminants and are therefore promising emulsifier and/or degraders of PCO. This study describes a set of PCO-inhabiting bacterial species, one of which, identified as Bacillus safensis PHA3, produces an efficient biosurfactant which was characterized as a glycolipid. Fourier transform infrared spectrometer, nuclear magnetic resonance, Thin layer chromatography, HPLC, and GC-MS analysis of the purified biosurfactant revealed that the extracted molecule under investigation is likely a mannolipid molecule with a hydrophilic part as mannose and a hydrophobic part as hexadecanoic acid (C16:0). The data reveal that: (i) PHA3 is a potential producer of biosurfactant (9.8 ± 0.5 mg mL-1); (ii) pre-adding 0.15% of the purified glycolipid enhanced the degradation of PCO by approximately 2.5-fold; (iii) the highest emulsifying activity of biosurfactant was found against the PCO and the lowest was against the naphthalene; (iv) the optimal PCO-emulsifying activity was found at 30-60°C, pH 8 and a high salinity. An orthologous gene encodes a putative ß-diglucosyldiacylglycerol synthase (ß-DGS) was identified in PHA3 and its transcripts were significantly up-regulated by exogenous PAHs, i.e., pyrene and benzo(e)pyrene but much less by mid-chain n-alkanes (ALKs) and fatty acids. Subsequently, the accumulation of ß-DGS transcripts coincided with an optimal growth of bacteria and a maximal accumulation of the biosurfactant. Of particular interest, we found that PHA3 actively catalyzed the degradation of PAHs notably the pyrene and benzo(e)pyrene but was much less effective in the mono-terminal oxidation of ALKs. Such characteristics make Bacillus safensis PHA3 a promising model for enhanced microbial oil recovery and environmental remediation.

Biochemical, Transcriptional, and Bioinformatic Analysis of Lipid Droplets from Seeds of Date Palm (Phoenix dactylifera L.) and Their Use as Potent Sequestration Agents against the Toxic Pollutant, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin.

Contamination of aquatic environments with dioxins, the most toxic group of persistent organic pollutants (POPs), is a major ecological issue. Dioxins are highly lipophilic and bioaccumulate in fatty tissues of marine organisms used for seafood where they constitute a potential risk for human health. Lipid droplets (LDs) purified from date palm, Phoenix dactylifera, seeds were characterized and their capacity to extract dioxins from aquatic systems was assessed. The bioaffinity of date palm LDs toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins was determined. Fractioned LDs were spheroidal with mean diameters of 2.5 µm, enclosing an oil-rich core of 392.5 mg mL(-1). Isolated LDs did not aggregate and/or coalesce unless placed in acidic media and were strongly associated with three major groups of polypeptides of relative mass 32-37, 20-24, and 16-18 kDa. These masses correspond to the LD-associated proteins, oleosins, caleosins, and steroleosins, respectively. Efficient partitioning of TCDD into LDs occurred with a coefficient of log K LB/w,TCDD = 7.528 ± 0.024; it was optimal at neutral pH and was dependent on the presence of the oil-rich core, but was independent of the presence of LD-associated proteins. Bioinformatic analysis of the date palm genome revealed nine oleosin-like, five caleosin-like, and five steroleosin-like sequences, with predicted structures having putative lipid-binding domains that match their LD stabilizing roles and use as bio-based encapsulation systems. Transcriptomic analysis of date palm seedlings exposed to TCDD showed strong up-regulation of several caleosin and steroleosin genes, consistent with increased LD formation. The results suggest that the plant LDs could be used in ecological remediation strategies to remove POPs from aquatic environments. Recent reports suggest that several fungal and algal species also use LDs to sequester both external and internally derived hydrophobic toxins, which indicates that our approach could be used as a broader biomimetic strategy for toxin removal.