Outer Membrane Layer Research Articles

Label-Free LC-MS/MS Analysis Reveals Different Proteomic Profiles between Egg Yolks of Silky Fowl and Ordinary Chickens.

The proteomic profiles of Silky fowl egg yolk (SFEY) and Leghorn egg yolk (LEY) were analyzed by bottom-up label-free liquid chromatography–tandem mass spectrometry (LC-MS/MS). From a total of 186 identified proteins, 26 proteins were found significantly differentially abundant between two yolks, of which, 19 were up-regulated and 7 were down-regulated in SFEY, particularly, vitelline membrane outer layer protein 1, transthyretin and ovoinhibitor were up-regulated by 26, 25, and 16 times, respectively. In addition, there were 57 and 6 unique proteins in SFEY and LEY, respectively. Gene Ontology (GO) revealed SFEY contained relatively more abundant protease inhibitors and coagulation-related proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed differentially abundant proteins in SFEY may be actively involved in the regulation of the neuroactive ligand–receptor interaction pathway. This study provides a theoretical basis for the understanding of proteomic and biological differences between these two yolks and can guide for further exploration of nutritional and biomedical use of Silky fowl egg.

Dual-layer hollow fibre haemodialysis membrane for effective uremic toxins removal with minimal blood-bacteria contamination

Bacterial endotoxin contamination in dialysate may pass through haemodialysis membrane and cause a silent chronic microinflammation to kidney patients. Dual-layer hollow fibre (DLHF) membranes with dual function, biocompatible adsorptive and antibacterial effects were developed to solve the problem of incompatible membrane and endotoxin contamination. All membranes were fabricated via the co-extrusion dry-wet phase inversion technique. In this study, silica/α-mangostin nanoparticle was incorporated into the inner layer of membrane to enhance the biocompatibility of the membrane while maintaining its adsorption capacity. Activated carbon (AC) was incorporated in the outer layer of membrane for improved antibacterial property. The DLHF membranes were characterised based on its morphology and surface hydrophilicity. The performance of the DLHF membranes was evaluated in terms of permeability, urea, and creatinine removal capabilities, bovine serum albumin (BSA) rejection and antibacterial properties. The dense and small pore size on the outer layer of AC created a smoother surface for the DLHF membrane. The BSA rejection of the DLHF membranes was enhanced by 6–8% compared to that of unmodified single layer hollow fibre membrane. Silica/α-mangostin nanoparticle in the inner layer of membrane enhanced the removal of urea and creatinine by chemisorption. Result also showed that the incorporation of AC in the outer layer of DLHF membrane successfully filtered bacteria by bacteria entrapment. DLHF membrane with the combination of silica/α-mangostin nanoparticle in the inner layer and AC in the outer layer possessed the higher bacteria inhibition into blood compartment against Escherichia coli and Staphylococcus aureus, with removal rate of 68% and 75%, respectively, and better urea and creatinine removal by 60.57% and 75.18%, respectively, compared to single-layer PSf based membrane. The development of co-adsorptive biocompatible DLHF membrane can play an important role in improvement of kidney patient life.

Molecular markers for hemocyte subpopulations in crayfish Cherax quadricarinatus

Semigranular cells (SGCs) and granular cells (GCs) are two dominant groups of circulating hemocytes in crayfish Cherax quadricarinatus. Molecular markers are required for the clear classification of the hemocytes and the research of their function and differentiation. In this study, we compared the protein content of GCs and SGCs by using two workflows: one-dimensional gel electrophoresis followed by LC-MS/MS and in-solution digestion of cell lysate followed by LC-MS/MS. Cell type-specific proteins were identified, and their expression in SGCs and GCs was further investigated by RT-PCR, Western blotting, and immunofluorescence analysis. Three molecular markers for GCs (peroxinectin, a mannose-binding protein, and prophenoloxidase-activating enzyme 2a) and three molecular markers for SGCs (a vitelline membrane outer layer protein I-like protein, a C-type lectin, and a peptidase) were identified. The application of some of the markers in Eriocheir sinensis was also analyzed. These molecular markers are useful tools for the research of crustaceans hemocytes.

Mutually Exclusive Interactions of Rifabutin with Spatially Distinct Mycobacterial Cell Envelope Membrane Layers Offer Insights into Membrane-Centric Therapy of Infectious Diseases.

The mycobacterial cell envelope has spatially resolved inner and outer membrane layers with distinct compositions and membrane properties. However, the functional implication and relevance of this organization remain unknown. Using membrane biophysics and molecular simulations, we reveal a varied interaction profile of these layers with antibiotic Rifabutin, underlined by the structural and chemical makeup of the constituent lipids. The mycobacterial inner membrane displayed the highest partitioning of Rifabutin, which was located exclusively in the lipid head group/interfacial region. In contrast, the drug exhibited specific interaction sites in the head group/interfacial and hydrophobic acyl regions within the outer membrane. Altogether, we show that the design of membrane-active agents that selectively disrupt the mycobacterial outer membrane structure can increase drug uptake and enhance intracellular drug concentrations. Exploiting the mycobacterium-specific membrane–drug interaction profiles, chemotypes consisting of outer membrane-disruptive agents and antitubercular drugs can offer new opportunities for combinational tuberculosis (TB) therapy.

Stoichiometric analysis reveals a unique phosphatidylserine binding site in coagulation factor X

BackgroundCellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the plasma membrane, thereby allowing binding of many blood clotting proteins. We previously proposed the Anything But Choline (ABC) hypothesis to explain how PS and PE synergize to support binding of clotting proteins with gamma‐carboxyglutamate (Gla)‐rich domains, which posited that each Gla domain binds to a limited number of PS molecules and multiple PE molecules. However, the minimal number of PS molecules required to stably bind a Gla‐domain–containing blood clotting protein in the presence of excess PE was unknown. ObjectiveTo test the ABC hypothesis for factor X by determining the threshold binding requirement of PS molecules under conditions of PS–PE synergy. MethodsWe used surface plasmon resonance to investigate the stoichiometry of factor X binding to nanoscale membrane bilayers (Nanodiscs) of varying phospholipid composition. Results and ConclusionsWe quantified 1.05 ± 0.2 PS molecules per bound factor X molecule in Nanodiscs containing a mixture of 10% PS, 60% PE, and 30% phosphatidylcholine. Hence, there appears to be one truly PS‐specific binding site per Gla domain, while the remaining membrane binding interactions can be satisfied by PE.

Comparative transcriptome preliminary reveals the molecular mechanism of the growth rate of Procambarus clarkii

The red swamp crayfish (Procambarus clarkii) is a well-known invasive species. It has become one of the most important freshwater aquaculture resources in China. In this study, we reported the karyotype and whole mitochondrial genome of Procambarus clarkii. In addition, we performed transcriptome analysis of Procambarus clarkii from two different body size populations. The three large individuals, namely, the LS group, were randomly chosen, with body lengths and weights of 16.24–18.85 ​cm and 38.96–45.36 ​g, respectively. The three small individuals, namely, the SS group, were randomly chosen, with body lengths and weights of 10.50–12.70 ​cm and 19.56–25.95 ​g, respectively. Comparative transcriptomic analysis of Procambarus clarkii characterized 36 differentially expressed genes (DEGs), comprising 20 upregulated genes and 16 downregulated genes in the SS group. Functional analyses revealed that four genes (metalloreductase STEAP4-like (Steap4), myosin heavy chain fast skeletal muscle, muscle-like (MH), vitelline membrane outer layer protein I-like protein (VMO-I), and secreted protein acidic and rich in cysteine (SPARC)) were involved in the regulation of cell proliferation, muscle growth and energy metabolism, suggesting that four genes were related to growth in P. clarkii. We validated the expression levels of these four differentially expressed genes in LS and SS using Quantitative real-time PCR. These results provide insights into the regulatory mechanisms underlying the difference in P. clarkii growth.

DFF40 deficiency in cancerous T cells is implicated in chemotherapy drug sensitivity and resistance through the regulation of the apoptotic pathway

The regulation of the apoptotic pathway is one of the most studied mechanisms regarding cancer cell resistance. Many mutations have been linked to drug resistance. The DNA fragmentation factor 40 (DFF40) has been gaining interest regarding cancer cell response to chemotherapy and patient outcomes. Glioblastomas and uterine leiomyosarcomas have been shown to have a downregulation of DFF40 expression, conferring a poor patient prognosis. In concordance with these observations, in this study, we showed that DFF40 gene is also downregulated in breast, endocervical, ovarian, lung, pancreas and glioblastomas. DFF40 is the endonuclease responsible of DNA fragmentation during apoptosis. In this study, we sought to determine if a DFF40 deficiency in Jurkat T cells could impact the sensitivity to conventional chemotherapy drugs. CRISPR-cas9 generated DFF40 knockout (DFF40 KO) stable Jurkat cells and wild-type (DFF40 WT) cells were treated with different antimetabolites and topoisomerase II (TOP2) inhibitors, and cell viability was subsequently assessed. DFF40 deficient cells show chemoresistance to antimetabolites (e.g. methotrexate, 6-mercaptopurine and cytarabine) and surprisingly, they are more sensitive to TOP2 inhibitors (e.g. etoposide and teniposide). DFF40 deficient cells exposed to cytarabine present lower phosphatidylserine translocation levels to the outer cell membrane layer. Etoposide exposure in DFF40 deficient cells induces higher mortality levels and downregulation of Bcl-xL cells compared to DFF40 expressing T cells. The abolition of DFF40 expression in Jurkat cells significantly impairs histone H2AX phosphorylation following etoposide and cytarabine treatments. Our findings suggest that DFF40 is a novel key target in cancer cell resistance that potentially regulates genomic stability.

Daya Hambat Ekstrak Biji Kopi Robusta (Coffea Canephora) terhadap Bakteri Porphyromonas gingivalis (in vitro)

Periodontitis is mostly caused by plaque and Pophyromonas gingivalis bacteria as the main cause. The outer membrane layer of the Porphyromonas gingivalis wall produces pathogenic virulence factors, such as lipopolysaccharides which will activate inflammatory cells and cause phagocytosis of antigens thereby triggering free radicals. Robusta coffee beans naturally contain caffeine, phenolic compounds, trigonellin, and chlorogenic acids as antibacterial and anti-inflammatory. The purpose of this study was to determine the inhibition of Robusta (Coffea canephora) coffee bean extract 0.5%; 0.75%; 1%; 1.25%; 1.5% and 3% on the growth of Porphyromonas gingivalis in vitro and to find out the lowest concentration of Robusta (Coffea canephora) coffee bean extract which has inhibitory effect on the growth of Porphyromonas gingivalis. In this study were divided into 8 treatment groups namely positive control, negative control, 0.5% robusta coffee bean extract, 0.75%, 1%, 1.25%, 1.5% and 3%. Petridish dishes containing TSA media that have been sterilized, added P. gingivalis suspension with density according to Mc standard. Farland Then a sterile white test blank with a diameter of 6 mm that is still sterile is placed on top of the bacterial growth media in accordance with the placement of the treatment group and dropped with all 8 treatment materials. After 24 hours incubated in a desiccator, the inhibition of robusta coffee bean extracts against the growth of Porphyromonas gingivalis bacteria was observed and data collection was done by measuring the inhibition zone using calipers. The results obtained robusta coffee bean extract at concentrations of 3%, 1.5%, 1.25% and 1%, have an antibacterial power which is suspected because Robusta coffee beans naturally contain ingredients such as caffeine, polyphenols and chlorogenic acids which have antibacterial activity while the robusta coffee bean extract with a concentration of 0.5% and 0.75% does not have antibacterial power against Pophyromonas gingivalis. Robusta coffee bean extract with a concentration of 1% is the smallest concentration of Robusta (Coffea canephora) coffee bean extract which can inhibit the growth of Porphyromonas gingivalis.

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun’s lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

НЕПОЛНОЕ УДВОЕНИЕ ЧЕРВЕОБРАЗНОГО ОТРОСТКА КАК ПРИЧИНА ФОРМИРОВАНИЯ АППЕНДИКУЛЯРНОГО ИНФИЛЬТРАТА

The appendix anomalies are extremely rare malformations. Preoperative diagnosis of appendiceal duplication is often difficult and usually discovered incidentally during surgery forappendicitis. A clinical case of the development of chronic residual appendicitis in a 32-year-old woman with the abnormality of the vermiform appendix identified duplicity, type A according to the Cave-Wallbridge classification is described. During the first hospitalization, the patient was treated conservatively with a diagnosis of «a dense appendiceal infiltrate»; later, a planned appendectomy was performed. A duplication of the vermiform appendix type A was revealed during the examination of the removed appendix. Histopathological examination detected the underdevelopment of the wall of the accessory vermiform appendix - a complete absence of the outer longitudinal layer of the muscular membrane. The abnormally thin wall of the accessory appendix, as well as the related with it hypoperistalsis provoked the onset of chronic inflammation in the wall, which easily spread to the periappendiceal adipose tissue with the formation of a dense appendiceal infiltrate, which required surgical intervention. Thus, a rare clinical case of duplication of the vermiform appendix type A with forming of appendiceal infiltrate, associated with abnomality of muscular tissue, shown by the surgical findings and corroborated by pathology samples of intestinal tissue has been presented.

Interaction between polyene antifungal drug and saturated phospholipid monolayer regulated by calcium ions at the air-water interface

Amphotericin B (AmB) is a polyene antifungal drug, which could directly form pores on the sterol-free phospholipid monolayers. The interaction between AmB and phospholipid can be affected by calcium ions, but the mechanism is still unclear. DPPC is a saturated phospholipid with -PC group, which is often used to simulate the outer cell membrane leaflet. And DPPC is also the main constituent of pulmonary surfactants. In this work, the DPPC monolayer was used as a model membrane to study the effect of calcium ions on the interaction between AmB and phospholipid. The influence of different concentration of calcium ions on the elastic modulus, mean molecular area increment, excess Gibbs free energy and stability of the AmB/DPPC mixed monolayer has been researched at the surface pressure of 7.5 mN/m, 12.5 mN/m and 22.5 mN/m. The AmB/DPPC monolayers at the air-water interface have been observed in real-time by Brewster angle microscope and the microstructure of the Langmuir-Blodgett monolayer films transferred onto the mica have been researched by scanning electron microscope and atomic force microscope. The results showed that calcium ions had a significant influence on the elastic modulus, mean molecular area increment, excess Gibbs free energy, stability and microstructure of the AmB/DPPC monolayer. It has been indicated that the influence of calcium ions on the interaction between AmB and DPPC molecules mainly depended on the effect of calcium ions on the orientation of AmB molecules. The calcium ions could regulate the effect of AmB to the stability of the DPPC monolayer. This regulatory role changed with the different concentrations of calcium ions and the different phase states of the monolayer. This work provides useful information to further understand the influence mechanism of calcium ions on the interaction between AmB and saturated phospholipid with -PC group, which is helpful to find out the effect mechanism of calcium ion on the interaction between AmB and the outer layer of cell membrane or pulmonary surfactants in different phase states and to understand the toxicity mechanism of AmB on the cell membrane or lungs.

Prospects for the use of indicators of sialic acid metabolism in medicine (review of literature).

Sialic acids (SA) determine the degree of molecular hydrophilia, relieve binding together and their transportation, they increase mucin viscosity, stabilize the protein and membrane structure. Apart from that, SA are structural components of gangliosides participating in the formation of the outer layer of the plasma membrane. The degree of silyliation of glycoproteins and glycolipids is an important factor of molecular recognition in the cell, between the cells, between a cell and territorial matrix, as well as between a cell and some outer pathogenic factors. They can either mask the sites of recognition or be determinants of recognition. The most well-studied enzymes taking part in the SA metabolism and sialo-containing compounds are N-acetylneuraminate, cythydiltransferase, sialyltransferase, sialydase, aldolase SA and sialyl-O-acetylesterase. Numerous investigations have shown that aberrant sialylation is a specific feature of various changes and disorders of metabolism. Besides that, sialic acids are the first point of contact for different pathogenic microorganisms and the host's body due to their presence on the external surface of the cells and tissue of the mucous membrane. That is why the study of the above-mentioned various sialic acids fractions as well as of the activity of the enzymes participating in their metabolism in the blood plasma and tissues, and of the influence on the activity of these enzymes with the help of medicine can make an essential contribution to the diagnosis and treatment of many diseases.

Comparative proteomics elucidates the dynamics of ovarian development in the Chinese mitten crab Eriocheir sinensis

Ovarian development is a complex physiological process for crustacean reproduction that is divided into the oogonium proliferation stage, endogenous vitellogenic stage, exogenous vitellogenic stage, and oocyte maturation stage. Proteomics analysis offers a feasible approach to reveal the proteins involved in the complex physiological processes of any organism. Therefore, this study performed a comparative proteomics analysis of the ovary and hepatopancreas at three key ovarian stages, including stages I (oogonium proliferation), II (endogenous vitellogenesis) and IV (exogenous vitellogenesis), of the Chinese mitten crab Eriocheir sinensis using a label-free quantitative approach. The results showed that a total of 2,224 proteins were identified, and some key proteins related to ovarian development and nutrition metabolism were differentially expressed. The 26 key proteins were mainly involved in the ubiquitin/proteasome pathway (UPP), cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway during oogenesis. Fifteen differentially abundant proteins (DAPs) were found to participate in vitellogenesis and oocyte development, such as vitelline membrane outer layer protein 1 homolog, vitellogenin, vitellogenin receptor, heat shock 70 kDa protein cognate 3 and farnesyl pyrophosphate synthase. Forty-seven DAPs related to nutrition metabolism were identified, including the protein digestion, fatty acid metabolism, prostaglandin metabolism, lipid digestion and transportation, i.e. short-chain specific acyl-CoA dehydrogenase, acyl-CoA desaturase, fatty acid-binding protein, long-chain fatty acid CoA ligase 4, and hematopoietic prostaglandin D synthase. These results not only indicate proteins involved in ovarian development and nutrient deposition but also enhance the understanding of the regulatory pathways and physiological processes of crustacean ovarian development.

Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine.

T follicular helper (TFH) cells are crucial for Bcell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a(whichmediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on Bcells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.

Early Changes in Crayfish Hemocyte Proteins after Injection with a β-1,3-glucan, Compared to Saline Injected and Naive Animals.

Early changes in hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein β-1,3-glucan (laminarin) were investigated, as well as changes after saline (vehicle) injection and in naïve animals. Injection of saline resulted in rapid recruitment of granular hemocytes from surrounding tissues, whereas laminarin injection on the other hand induced an initial dramatic drop of hemocytes. At six hours after injection, the hemocyte populations therefore were of different composition. The results show that mature granular hemocytes increase in number after saline injection as indicated by the high abundance of proteins present in granular cell vesicles, such as a vitelline membrane outer layer protein 1 homolog, mannose-binding lectin, masquerade, crustin 1 and serine protease homolog 1. After injection with the β-1,3-glucan, only three proteins were enhanced in expression, in comparison with saline-injected animals and uninjected controls. All of them may be associated with immune responses, such as a new and previously undescribed Kazal proteinase inhibitor. One interesting observation was that the clotting protein was increased dramatically in most of the animals injected with laminarin. The number of significantly affected proteins was very few after a laminarin injection when compared to uninjected and saline-injected crayfish. This finding may demonstrate some problematic issues with gene and protein expression studies from other crustaceans receiving injections with pathogens or pattern recognition proteins. If no uninjected controls are included and no information about hemocyte count (total or differential) is given, expressions data for proteins or mRNAs are very difficult to properly interpret.

Comparative Yolk Proteomic Analysis of Fertilized Low and High Cholesterol Eggs during Embryonic Development.

Simple SummaryCholesterol exists in each cell of the living organism and plays a significant role in cellular functions. Several reports have demonstrated that high yolk cholesterol concentration affected embryo mortality, hatchability, and performance. The present results showed that high-cholesterol egg yolks had increased protein intensities and egg and yolk weight relative to low-cholesterol egg yolks. The differentially expressed yolk proteins were primarily involved in lipid transport, lipid localization, nutrient reservoir function, and embryo protection during embryonic development.The yolk is the principal part of the egg that contains vitamins, minerals, lipids, and proteins which are essential for embryo development and hatching. The egg yolk contains significant amounts of lipoproteins, triacylglycerides, and cholesterol, whose dynamics are indistinct during embryogenesis. The effects of cholesterol on the yolk protein abundance, intensity, and function are ill-defined during embryonic development. Using two-dimensional gel electrophoresis, eggs with respective high and low cholesterol protein abundance were investigated after 0, 2, 6, and 13 days of embryogenesis and further analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. The results revealed that the vitellogenin proteins are the most abundant egg yolk protein that showed proximity and a high degree of variation in isoelectric point and molecular weight. The results demonstrated increased expression of vitellogenin-1 and vitellogenin-3 at two days and vitellogenin-2 protein at 13 days of embryogenesis in both egg types. The ovoinhibitor, immunoglobulin lambda light chain precursor, Ig-gamma (clone-36 chicken), and beta-2-glycoprotein-1 precursor proteins were significantly expressed in high cholesterol eggs while haptoglobin protein PIT-54 and vitelline membrane outer layer proteins intensities were significant in low cholesterol eggs at two days of embryogenesis. The high cholesterol eggs showed a modest increase in egg weight, yolk weight, albumen height, yolk color, and egg strength relative to the low cholesterol eggs. The gene ontology enrichment analysis revealed that the differentially expressed proteins such as vitellogenin proteins were involved in lipid transport and lipid localization biological processes and showed nutrient reservoir activity function. The ovotransferrin regulated the biological processes of plasminogen activation and extracellular matrix disassembly and characterized the anchored component of the plasma membrane. The ovoinhibitor protein was involved in response to mineralocorticoid and corticosterone biological processes whereas the vitellin membrane outer layer protein constituted the extracellular exosome, extracellular organelle, and membrane-bounded vesicle cellular components. Collectively, our study revealed yolk protein abundance, molecular function, cellular components, and biological processes and concluded that yolk protein intensities were significantly altered by cholesterol concentration.

Molecular Simulations of Biogenesis Processes within the Bacterial Cell Envelope

The cell envelope serves as the front-line to both defence and pathogenicity in bacteria. Critical protein assemblies are required to mature, localise and assemble proteins, oligosaccharides and lipids around the cell. These enable protection against antibiotics, phages and toxins, and modulate cell structure and shape. Gram-negative bacteria contain two membrane layers. The outer membrane (OM) layer is comprised of an inner leaflet of phospholipids and an outer leaflet of a complex lipid-linked oligosaccharide, otherwise known as lipopolysaccharide (LPS).

Structure of the O-Antigen and the Lipid A from the Lipopolysaccharide of Fusobacterium nucleatum ATCC 51191.

Fusobacterium nucleatum is a common member of the oral microbiota. However, this symbiont has been found to play an active role in disease development. As a Gram‐negative bacterium, F. nucleatum has a protective outer membrane layer whose external leaflet is mainly composed of lipopolysaccharides (LPSs). LPSs play a crucial role in the interaction between bacteria and the host immune system. Here, we characterised the structure of the O‐antigen and lipid A from F. nucleatum ssp. animalis ATCC 51191 by using a combination of GC‐MS, MALDI and NMR techniques. The results revealed a novel repeat of the O‐antigen structure of the LPS, [→4)‐β‐d‐GlcpNAcA‐(1→4)‐β‐d‐GlcpNAc3NAlaA‐(1→3)‐α‐d‐FucpNAc4NR‐(1→], (R=acetylated 60 %), and a bis‐phosphorylated hexa‐acylated lipid A moiety. Taken together these data showed that F. nucleatum ATCC 51191 has a distinct LPS which might differentially influence recognition by immune cells.

Identification of Novel Proteins in Foam Nests of the Japanese Forest Green Tree Frog, Rhacophorus arboreus.

Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.

Do Multiple Drug Resistance Transporters Interfere with Cell Functioning under Normal Conditions?

Eukaryotic cells rely on multiple mechanisms to protect themselves from exogenous toxic compounds. For instance, cells can limit penetration of toxic molecules through the plasma membrane or sequester them within the specialized compartments. Plasma membrane transporters with broad substrate specificity confer multiple drug resistance (MDR) to cells. These transporters efflux toxic compounds at the cost of ATP hydrolysis (ABC-transporters) or proton influx (MFS-transporters). In our review, we discuss the possible costs of having an active drug-efflux system using yeast cells as an example. The pleiotropic drug resistance (PDR) subfamily ABC-transporters are known to constitutively hydrolyze ATP even without any substrate stimulation or transport across the membrane. Besides, some MDR-transporters have flippase activity allowing transport of lipids from inner to outer lipid layer of the plasma membrane. Thus, excessive activity of MDR-transporters can adversely affect plasma membrane properties. Moreover, broad substrate specificity of ABC-transporters also suggests the possibility of unintentional efflux of some natural metabolic intermediates from the cells. Furthermore, in some microorganisms, transport of quorum-sensing factors is mediated by MDR transporters; thus, overexpression of the transporters can also disturb cell-to-cell communications. As a result, under normal conditions, cells keep MDR-transporter genes repressed and activate them only upon exposure to stresses. We speculate that exploiting limitations of the drug-efflux system is a promising strategy to counteract MDR in pathogenic fungi.