Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells.

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

Lysophosphatidylglycerol (LPG) phospholipase D maintains membrane homeostasis in Staphylococcus aureus by converting LPG to lysophosphatidic acid.

Lysophospholipids are deacylated derivatives of their bilayer forming phospholipid counterparts that are present at low concentrations in cells. Phosphatidylglycerol (PG) is the principal membrane phospholipid in Staphylococcus aureus and lysophosphatidylglycerol (LPG) is detected in low abundance. Here, we used a mass spectrometry screen to identify locus SAUSA300_1020 as the gene responsible for maintaining low concentrations of 1-acyl-LPG in S. aureus. The SAUSA300_1020 gene encodes a protein with a predicted amino terminal transmembrane α-helix attached to a globular glycerophosphodiester phosphodiesterase (GDPD) domain. We determined that the purified protein lacking the hydrophobic helix (LpgDΔN) possesses cation-dependent lysophosphatidylglycerol phospholipase D activity that generates both lysophosphatidic acid (LPA) and cyclic-LPA products and hydrolyzes cyclic-LPA to LPA. Mn2+ was the highest affinity cation and stabilized LpgDΔN to thermal denaturation. LpgDΔN was not specific for the phospholipid headgroup and degraded 1-acyl-LPG, but not 2-acyl-LPG. Furthermore, a 2.1 Å crystal structure shows that LpgDΔN adopts the GDPD variation of the TIM barrel architecture except for the length and positioning of helix α6 and sheet ß7. These alterations create a hydrophobic diffusion path for LPG to access the active site. The LpgD active site has the canonical GDPD metal binding and catalytic residues, and our biochemical characterization of site-directed mutants support a two-step mechanism involving a cyclic-LPA intermediate. Thus, the physiological function of LpgD in S. aureus is to convert LPG to LPA, which is re-cycled into the PG biosynthetic pathway at the LPA acyltransferase step to maintain membrane PG molecular species homeostasis.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

Cytidine diphosphate diacylglycerol synthase is essential for mitochondrial structure and energy production in Arabidopsis thaliana.

Cytidine diphosphate diacylglycerol (CDP-DAG), an important intermediate for glycerolipid biosynthesis, is synthesized under the catalytic activity of CDP-DAG synthase (CDS) to produce anionic phosphoglycerolipids such as phosphatidylglycerol (PG) and cardiolipin (CL). Previous studies showed that Arabidopsis CDSs are encoded by a small gene family, termed CDS1-CDS5, the members of which are integral membrane proteins in endoplasmic reticulum (ER) and in plastids. However, the details on how CDP-DAG is provided for mitochondrial membrane-specific phosphoglycerolipids are missing. Here we present the identification of a mitochondrion-specific CDS, designated CDS6. Enzymatic activity of CDS6 was demonstrated by the complementation of CL synthesis in the yeast CDS-deficient tam41Δ mutant. The Arabidopsis cds6 mutant lacking CDS6 activity showed decreased mitochondrial PG and CL biosynthesis capacity, a severe growth deficiency finally leading to plant death. These defects were rescued partly by complementation with CDS6 or supplementation with PG and CL. The ultrastructure of mitochondria in cds6 was abnormal, missing the structures of cristae. The degradation of triacylglycerol (TAG) in lipid droplets and starch in chloroplasts in the cds6 mutant was impaired. The expression of most differentially expressed genes involved in the mitochondrial electron transport chain was upregulated, suggesting an energy-demanding stage in cds6. Furthermore, the contents of polar glycerolipids in cds6 were dramatically altered. In addition, cds6 seedlings lost the capacity for cell proliferation and showed a higher oxidase activity. Thus, CDS6 is indispensable for the biosynthesis of PG and CL in mitochondria, which is critical for establishing mitochondrial structure, TAG degradation, energy production and seedling development.

Exploring Skin Wound Healing Models and the Impact of Natural Lipids on the Healing Process.

Cutaneous wound healing is a complex biological process involving a series of well-coordinated events aimed at restoring skin integrity and function. Various experimental models have been developed to study the mechanisms underlying skin wound repair and to evaluate potential therapeutic interventions. This review explores the diverse array of skin wound healing models utilized in research, ranging from rodent excisional wounds to advanced tissue engineering constructs and microfluidic platforms. More importantly, the influence of lipids on the wound healing process is examined, emphasizing their role in enhancing barrier function restoration, modulating inflammation, promoting cell proliferation, and promoting remodeling. Lipids, such as phospholipids, sphingolipids, and ceramides, play crucial roles in membrane structure, cell signaling, and tissue repair. Understanding the interplay between lipids and the wound microenvironment provides valuable insights into the development of novel therapeutic strategies for promoting efficient wound healing and tissue regeneration. This review highlights the significance of investigating skin wound healing models and elucidating the intricate involvement of lipids in the healing process, offering potential avenues for improving clinical outcomes in wound management.

Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype.

Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.

Ruscogenin interacts with DPPC and DPPG model membranes and increases the membrane fluidity: FTIR and DSC studies.

Ruscogenin, a kind of steroid saponin, has been shown to have significant anti-oxidant, anti-inflammatory, and anti-thrombotic characteristics. Furthermore, it has the potential to be employed as a medicinal medication to treat a variety of acute and chronic disorders. The interaction of a drug molecule with cell membranes can help to elucidate its system-wide protective and therapeutic effects, and it's also important for its pharmacological activity. The molecular mechanism by which ruscogenin affects membrane architecture is still a mystery. Ruscogenin's interaction with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) and anionic dipalmitoyl phosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) was studied utilizing two non-invasive approaches, including: Fourier Transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry. Ruscogenin caused considerable alterations in the phase transition profile, order, dynamics and hydration state of head groups and glycerol backbone of DPPC and DPPG MLVs at all concentrations. The DSC results indicated that the presence of ruscogenin decreased the main phase transition temperature (Tm) and enthalpy (ΔH) values of both membranes and increased half height width of the main transition (ΔT1/2). The FTIR results demonstrated that all concentrations (1, 3, 6, 9, 15, 24 and 30 mol percent) of ruscogenin disordered the DPPC MLVs both in the gel and liquid crystalline phases while it increased the order of DPPG MLVs in the liquid crystalline phase. Moreover, ruscogenin caused an increase in the dynamics of DPPC and DPPG MLVs in both phases. Additionally, it enhanced the hydration of the head groups of lipids and the surrounding water molecules implying ruscogenin to interact strongly with both zwitterionic and charged model membranes.

Phosphatidylglycerol-specific phospholipase C from Amycolatopsis sp. NT115 strain: purification, characterization, and gene cloning.

Recently, phosphatidylglycerol (PG) focused on its important role in chloroplast photosynthesis, mitochondrial function of the sperm, an inhibitory effect on SARS-CoV-2 ability to infect naïve cells, and reducing lung inflammation caused by coronavirus disease 2019. To develop an enzymatic PG determination method as the high-throughput analysis of PG, a PG-specific phospholipase C (PG-PLC) was found in the culture supernatant of Amycolatopsis sp. NT115. PG-PLC (54 kDa by SDS-PAGE) achieved the maximal activity at pH 6.0 and 55 °C and was inhibited by detergents, such as Briji35, Tween 80, and sodium cholate, but not by EDTA and metal ions, except for Zn2+. The open reading frame of the PG-PLC gene consisted of 1620 bp encoding 515-amino-acid residues containing the preceding 25-amino-acid residues (Tat signal peptide sequence). The putative amino acid sequence of PG-PLC was highly similar to those of metallophosphoesterases; however, its substrate specificity was completely different from those of known PLCs.

Analysis of serum metabolome of laborers exposure to welding fume.

OBJECTIVE: Welding fume exposure is inevitable of welding workers and poses a severe hazard to their health since welding is a necessary industrial process. Thus, preclinical diagnostic symptoms of worker exposure are of great importance. The aim of this study was to screen serum differential metabolites of welding fume exposure based on UPLC-QTOF-MS/MS. METHODS: In 2019, 49 participants were recruited at a machinery manufacturing factory. The non-target metabolomics technique was used to clarify serum metabolic signatures in people exposed to welding fume. Differential metabolites were screened by OPLS-DA analysis and Student's t-test. The receiver operating characteristic curve evaluated the discriminatory power of differential metabolites. And the correlations between differential metabolites and metal concentrations in urine and whole blood were analyzed utilizing Pearson correlation analysis. RESULTS: Thirty metabolites were increased significantly, and 5 metabolites were decreased. The differential metabolites are mainly enriched in the metabolism of arachidonic acid, glycero phospholipid, linoleic acid, and thiamine. These results observed that lysophosphatidylcholine (20:1/0:0) and phosphatidylglycerol(PGF1α/16:0) had a tremendous anticipating power with relatively increased AUC values (AUC > 0.9), and they also presented a significant correlation of Mo concentrations in whole blood and Cu concentrations in urine, respectively. CONCLUSION: The serum metabolism was changed significantly after exposure to welding fume. Lysophosphatidylcholine (20:1/0:0) and phosphatidylglycerol (PGF1α/16:0) may be a potential biological mediator and biomarker for laborers exposure to welding fume.

Dioleoylphosphatidylglycerol Inhibits Heat Shock Protein B4 (HSPB4)-Induced Inflammatory Pathways In Vitro.

Our previous work shows that dioleoylphosphatidylglycerol (DOPG) accelerates corneal epithelial healing in vitro and in vivo by unknown mechanisms. Prior data demonstrate that DOPG inhibits toll-like receptor (TLR) activation and inflammation induced by microbial components (pathogen-associated molecular patterns, PAMPs) and by endogenous molecules upregulated in psoriatic skin, which act as danger-associated molecular patterns (DAMPs) to activate TLRs and promote inflammation. In the injured cornea, sterile inflammation can result from the release of the DAMP molecule, heat shock protein B4 (HSPB4), to contribute to delayed wound healing. Here, we show in vitro that DOPG inhibits TLR2 activation induced in response to HSPB4, as well as DAMPs that are elevated in diabetes, a disease that also slows corneal wound healing. Further, we show that the co-receptor, cluster of differentiation-14 (CD14), is necessary for PAMP/DAMP-induced activation of TLR2, as well as of TLR4. Finally, we simulated the high-glucose environment of diabetes to show that elevated glucose levels enhance TLR4 activation by a DAMP known to be upregulated in diabetes. Together, our results demonstrate the anti-inflammatory actions of DOPG and support further investigation into its development as a possible therapy for corneal injury, especially in diabetic patients at high risk of vision-threatening complications.

Insights into the Formation of Intermolecular Complexes of Fluorescent Probe 10-N-Nonyl Acridine Orange with Cardiolipin and Phosphatidylglycerol in Bacterial Plasma Membrane by Molecular Modeling.

In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic biological probe 10-N-nonyl acridine orange (NAO) with the bilayer, mimicking a plasma membrane of Gram-negative bacteria. Fluorescent probes serve as an effective tool to study the localization of different components in biological membranes. Revealing the molecular details of their interaction with membrane phospholipids is important both for the interpretation of experimental results and future design of lipid-specific stains. By means of coarse-grained (CG) MD, we studied the interactions of NAO with a model membrane, imitating the plasma membrane of Gram-negative bacteria. In our simulations, we detected different NAO forms: monomers, dimers, and stacks. NAO dimers had the central cardiolipin (CL) molecule in a sandwich-like structure. The stacks were formed by NAO molecules interlayered with anionic lipids, predominantly CL. Use of the CG approach allowed to confirm the ability of NAO to bind to both major negatively charged phospholipids, phosphatidylglycerol (PG) and CL, and to shed light on the exact structure of previously proposed NAO-lipid complexes. Thus, CG modeling can be useful for the development of new effective and highly specific molecular probes.

Membrane lipid remodeling is required for photosystem II function under low CO2.

Membrane lipid remodeling in plants and microalgae has a crucial role in their survival under nutrient-deficient conditions. Aquatic microalgae have low access to CO2 , an essential carbon source for photosynthetic assimilates; however, 70-90 mol% of their membrane lipids are sugar-derived lipids (glycolipids) such as monogalactosyldiacylglycerol (MGDG). In this study, we discovered a new system of membrane lipid remodeling responding to CO2 in Synechocystis sp. PCC 6803, a unicellular, freshwater cyanobacterium. As compared with higher CO2 (HC; 1% CO2 ), under ambient air (lower CO2 : LC), phosphatidylglycerol (PG) content was increased at the expense of MGDG content. To explore the biological significance of this alteration in content, we generated a transformant of Synechocystis sp. PCC 6803 overexpressing sll0545 gene encoding a putative phosphatidic acid phosphate (oxPAP), which produces diacylglycerol that is used for the synthesis of glycolipids, and examined the effect on membrane lipid remodeling and phototrophic growth responding to LC. Photosystem II (PSII) activity and growth rate were inhibited under LC in oxPAP cells. PG content was substantially reduced, and MGDG and sulfoquinovosyldiacylglycerol contents were increased in oxPAP cells as compared with control cells. These phenotypes in oxPAP cells were recovered under the HC condition or PG supplementation. Increased PG content may be required for proper functioning of PSII under LC conditions.

LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain.

BACKGROUND: Aminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region. RESULTS: Using genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human ß-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria. CONCLUSIONS: Overall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

Genetically-determined variations in photosynthesis indicate roles for specific fatty acid species in chilling responses.

Using a population of recombinant inbred lines (RILs) cowpea (Vigna unguiculata. L. Walp), we tested for co-linkages between lipid contents and chilling responses of photosynthesis. Under low-temperature conditions (19°C/13°C, day/night), we observed co-linkages between quantitative trait loci intervals for photosynthetic light reactions and specific fatty acids, most strikingly, the thylakoid-specific fatty acid 16:1Δ3trans found exclusively in phosphatidylglycerol (PG 16:1t). By contrast, we did not observe co-associations with bulk polyunsaturated fatty acids or high-melting-point-PG (sum of PG 16:0, PG 18:0 and PG 16:1t) previously thought to be involved in chilling sensitivity. These results suggest that in cowpea, chilling sensitivity is modulated by specific lipid interactions rather than bulk properties. We were able to recapitulate the predicted impact of PG 16:1t levels on photosynthetic responses at low temperature using mutants and transgenic Arabidopsis lines. Because PG 16:1t synthesis requires the activity of peroxiredoxin-Q, which is activated by H2 O2 and known to be involved in redox signalling, we hypothesise that the accumulation of PG 16:1t occurs as a result of upstream effects on photosynthesis that alter redox status and production of reactive oxygen species.

Origin of cyanobacterial thylakoids via a non-vesicular glycolipid phase transition and their impact on the Great Oxygenation Event.

The appearance of oxygenic photosynthesis in cyanobacteria is a major event in evolution. It had an irreversible impact on the Earth, promoting the Great Oxygenation Event (GOE) ~2.4 billion years ago. Ancient cyanobacteria predating the GOE were Gloeobacter-type cells lacking thylakoids, which hosted photosystems in their cytoplasmic membrane. The driver of the GOE was proposed to be the transition from unicellular to filamentous cyanobacteria. However, the appearance of thylakoids expanded the photosynthetic surface to such an extent that it introduced a multiplier effect, which would be more coherent with an impact on the atmosphere. Primitive thylakoids self-organize as concentric parietal uninterrupted multilayers. There is no robust evidence for an origin of thylakoids via a vesicular-based scenario. This review reports studies supporting that hexagonal II-forming glucolipids and galactolipids at the periphery of the cytosolic membrane could be turned, within nanoseconds and without any external source of energy, into membrane multilayers. Comparison of lipid biosynthetic pathways shows that ancient cyanobacteria contained only one anionic lamellar-forming lipid, phosphatidylglycerol. The acquisition of sulfoquinovosyldiacylglycerol biosynthesis correlates with thylakoid emergence, possibly enabling sufficient provision of anionic lipids to trigger a hexagonal II-to-lamellar phase transition. With this non-vesicular lipid-phase transition, a framework is also available to re-examine the role of companion proteins in thylakoid biogenesis.

Lipids in photosynthetic protein complexes in the thylakoid membrane of plants, algae, and cyanobacteria.

In the thylakoid membrane of cyanobacteria and chloroplasts, many proteins involved in photosynthesis are associated with or integrated into the fluid bilayer matrix formed by four unique glycerolipid classes, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol. Biochemical and molecular genetic studies have revealed that these glycerolipids play essential roles not only in the formation of thylakoid lipid bilayers but also in the assembly and functions of photosynthetic complexes. Moreover, considerable advances in structural biology have identified a number of lipid molecules within the photosynthetic complexes such as PSI and PSII. These data have provided important insights into the association of lipids with protein subunits in photosynthetic complexes and the distribution of lipids in the thylakoid membrane. Here, we summarize recent high-resolution observations of lipid molecules in the structures of photosynthetic complexes from plants, algae, and cyanobacteria, and evaluate the distribution of lipids among photosynthetic protein complexes and thylakoid lipid bilayers. By integrating the structural information into the findings from biochemical and molecular genetic studies, we highlight the conserved and differentiated roles of lipids in the assembly and functions of photosynthetic complexes among plants, algae, and cyanobacteria.

Impacts of phosphatidylglycerol on plastid gene expression and light induction of nuclear photosynthetic genes.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis, and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development, with decreased chlorophyll accumulation, impaired thylakoid formation, and down-regulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species (ROS)-responsive genes were up-regulated in pgp1-2. However, ROS production was not enhanced in the mutant even under strong light, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered down-regulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor gene regulating chloroplast development, in pgp1-2 up-regulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in plastid gene expression and plant development.

Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation.

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.

Innate Immune System Activation, Inflammation and Corneal Wound Healing.

Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.

Cutaneous Permeation of a Percutaneously Applied Glucocorticoid Using Plant-Based Anionic Phospholipids in Hydrogenated Vegetable Oil: A Preliminary Study.

Purpose: It is important that, when corticosteroids are used therapeutically, concentrations be reduced as much as possible to mitigate potential adverse events and side effects. This preliminary study compares the permeation for the delivery of a corticosteroid in a 1% hydrocortisone-supplemented topical cream containing anionic polar phospholipids (APP) in hydrogenated vegetable oil (triglyceride) versus a market-leading 1% hydrocortisone in a mineral hydrocarbon-based skin cream. Methods: Using the Franz diffusion cell method with cadaveric skin, the permeation of a 1% hydrocortisone-supplemented cream containing APP (test preparation) was compared with a commercially available 1% hydrocortisone cream (control preparation). The principal APP in the test preparation were phosphatidylinositol, phosphatidylserine and phosphatidylglycerol. Permeation was determined at 4 and 8 h time intervals. Results: The permeation values for the 1% hydrocortisone supplemental APP cream (test preparation) were comparatively very high 1180 ng/cm2 at 4 h and 2173 ng/cm2 at 8 h, in contrast to the 1% hydrocortisone cream (control preparation) values of 13 ng/cm2 at 4 h and 98 ng/cm2 at 8 h. Permeation of skin cream increased significantly from 0 to 4 and 8 h, when comparing the APP test preparation with the control preparation (p < 0.001). This translates, respectively, into the 90-fold greater and a 20-fold greater penetration of the test preparation APP cream over the 1% hydrocortisone cream at 4 h and 8 h time points. Conclusions: This preliminary study demonstrates the enhanced permeation of 1% hydrocortisone when applied topically to the skin in an APP skin cream vehicle. This enhanced permeation suggests the potential use of APP technology to deliver therapeutically effective hydrocortisone treatment to the skin at markedly reduced concentrations of steroid. As such, APP technology may offer an improved approach to the treatment of dermatoses associated with inflammatory diseases and conditions requiring prolonged topical corticosteroid therapy.