Interplay between Humoral and CLA+ T Cell Response against Candida albicans in Psoriasis

Candida albicans (CA) infections have been associated with psoriasis onset or disease flares. However, the integrated immune response against this fungus is still poorly characterized in psoriasis. We studied specific immunoglobulins in plasma and the CA response in cocultures of circulating memory CD45RA⁻ cutaneous lymphocyte antigen (CLA)^+/− T cell with autologous epidermal cells from plaque and guttate psoriasis patients (cohort 1, n = 52), and also healthy individuals (n = 17). A complete proteomic profile was also evaluated in plaque psoriasis patients (cohort 2, n = 114) regarding their anti-CA IgA levels. Increased anti-CA IgA and IgG levels are present in the plasma from plaque but not guttate psoriasis compared to healthy controls. CA cellular response is confined to CLA⁺ T cells and is primarily Th17. The levels of anti-CA IgA are directly associated with CLA⁺ Th17 response in plaque psoriasis. Proteomic analysis revealed distinct profiles in psoriasis patients with high anti-CA IgA. C-C motif chemokine ligand 18, chitinase-3-like protein 1 and azurocidin were significantly elevated in the plasma from plaque psoriasis patients with high anti-CA levels and severe disease. Our results indicate a mechanism by which Candida albicans exposure can trigger a clinically relevant IL-17 response in psoriasis. Assessing anti-CA IgA levels may be useful in order to evaluate chronic psoriasis patients.     

Application of the Mutant Libraries for Candida albicans Functional Genomics

Candida albicans is a typical opportunistic pathogen in humans that causes serious health risks in clinical fungal infections. The construction of mutant libraries has made remarkable developments in the study of C. albicans molecular and cellular biology with the ongoing advancements of gene editing, which include the application of CRISPR-Cas9 and novel high-efficient transposon. Large-scale genetic screens and genome-wide functional analysis accelerated the investigation of new genetic regulatory mechanisms associated with the pathogenicity and resistance to environmental stress in C. albicans. More importantly, sensitivity screening based on C. albicans mutant libraries is critical for the target identification of novel antifungal compounds, which leads to the discovery of Sec7p, Tfp1p, Gwt1p, Gln4p, and Erg11p. This review summarizes the main types of C. albicans mutant libraries and interprets their applications in morphogenesis, biofilm formation, fungus–host interactions, antifungal drug resistance, and target identification.     

Role of charge and hydrophobicity in translocation of cell-penetrating peptides into Candida albicans cells

Although the interactions of cell-penetrating peptides (CPPs) with mammalian cells have been widely studied, much less is known about their interactions with fungal cells. To study how the properties of CPPs affect translocation into fungal cells, we designed variants of the peptides pVEC and SynB with altered levels of charge and hydrophobicity and evaluated the translocation of the variants into the important human fungal pathogen Candida albicans. Charge played a greater role in translocation efficacy of the peptides than hydrophobicity, with a higher net positive charge leading to higher level of translocation into C. albicans and a higher level of cytosolic localization. Hydrophobicity had little effect on translocation efficacy, but a low level of hydrophobicity did lead to increased vacuolar localization and an energy-dependent translocation mechanism. Our results suggest that CPPs can be designed for desired levels of cargo delivery into fungal cells and for desired translocation mechanisms.     

mp65和sap2真核表达质粒对小鼠系统性白色念珠菌感染的免疫保护作用

目的研究白色念珠菌mp65和sap2基因真核表达质粒对小鼠系统性白色念珠菌感染的免疫保护作用。方法将pcDNA3.1-mp65和pcDNA3.1-sap2质粒分别或联合免疫BALB/c小鼠,ELISA法检测免疫小鼠外周血中抗MP65和抗Sap2蛋白特异性IgG抗体,流式细胞仪检测免疫小鼠脾脏中CD4+、CD8+T淋巴细胞百分含量的变化,并观察白色念珠菌攻击后免疫小鼠的存活率。结果免疫后小鼠血清中可检测出抗两种蛋白的特异性IgG抗体,两种质粒均能诱导脾脏中CD4+T淋巴细胞百分含量增高,并可提高白色念珠菌系统性感染小鼠的存活率。结论pcDNA3.1-mp65和pcDNA3.1-sap2质粒均能诱导小鼠的体液免疫和以CD4+T淋巴细胞为主的细胞免疫,且对白色念珠菌的系统性感染有一定的保护作用。     

GC-MS-Based Metabolomics Study of Single- and Dual-Species Biofilms of Candida albicans and Klebsiella pneumoniae

Candida albicans and Klebsiella pneumoniae frequently co-exist within the human host as a complex biofilm community. These pathogens are of interest because their association is also related to significantly increased morbidity and mortality in hospitalized patients. With the aim of highlighting metabolic shifts occurring in the dual-species biofilm, an untargeted GC-MS-based metabolomics approach was applied to single and mixed biofilms of C. albicans and K. pneumoniae. Metabolomic results showed that among the extracellular metabolites identified, approximately 40 compounds had significantly changed relative abundance, mainly involving central carbon, amino acid, vitamin, and secondary metabolisms, such as serine, leucine, arabitol, phosphate, vitamin B6, cyclo-(Phe-Pro), trehalose, and nicotinic acid. The results were related to the strict interactions between the two species and the different microbial composition in the early and mature biofilms.     

人乳头瘤病毒结构蛋白L1与白色念珠菌甘露糖蛋白CTL表位嵌合基因在昆虫细胞中的表达

目的构建携带人乳头瘤病毒(HPV)结构蛋白L1和白色念珠菌甘露糖蛋白(CaMp65)细胞毒性T细胞表位(CTL)基因的重组质粒,并在杆状病毒-昆虫细胞系统中进行表达。方法分别设计包含HPV6bL1和CaMp65CTL145-153的引物,经PCR扩增嵌合基因HPV6bL1/CaMp65CTL145-153,并进一步将其插入杆状病毒表达载体pFastBacTM1。在E.coliDH10Bac中组装成重组杆状病毒,在昆虫细胞sf-9中表达嵌合蛋白,并经SDS-PAGE和Westernblot分析鉴定。结果HPV6bL1/CaMp65CTL145-153嵌合蛋白在昆虫细胞sf-9中得到了表达,表达产物的相对分子质量为56000,与HPV6bL1单抗能产生特异性反应。结论HPV6bL1/CaMp65CTL145-153嵌合蛋白在杆状病毒-昆虫细胞系统中得到了成功表达,为防治HPV和白色念珠菌感染的基因工程疫苗的研究奠定了基础。     

Evaluation of Anti-Candida albicans Activity and Release of Ketoconazole in PMMA-G-PEG 4000 Films

Modified release systems depend on the selection of an appropriate agent capable of controlling the release of the drug, sustaining the therapeutic action over time, and/or releasing the drug at the level of a particular tissue or target organ. Polyethylene glycol 4000 (PEG 4000) is commonly employed in drug release formulations while polymethyl methacrylate (PMMA) is non-toxic and has a good solubility in organic solvents. This study aimed at the incorporation of ketoconazole in PMMA-g-PEG 4000 and its derivatives, thus evaluating its release profile and anti-Candida albicans and cytotoxic activities. Ketoconazole was characterized and incorporated into the copolymers. The ketoconazole incorporated in the copolymer and its derivatives showed an immediate release profile. All copolymers with ketoconazole showed activity against Candida albicans and were non-toxic to human cells in the entire concentration tested.     

Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.     

K143R Amino Acid Substitution in 14-α-Demethylase (Erg11p) Changes Plasma Membrane and Cell Wall Structure of Candida albicans

The opportunistic pathogen Candida albicans is responsible for life-threating infections in immunocompromised individuals. Azoles and polyenes are two of the most commonly used antifungals and target the ergosterol biosynthesis pathway or ergosterol itself. A limited number of clinically employed antifungals correspond to the development of resistance mechanisms. One resistance mechanism observed in clinical isolates of azole-resistant C. albicans is the introduction of point mutations in the ERG11 gene, which encodes a key enzyme (lanosterol 14-α-demethylase) on the ergosterol biosynthesis pathway. Here, we demonstrate that a point mutation K143R in ERG11 (C. albicans ERG11^K143R/K143R) contributes not only to azole resistance, but causes increased gene expression. Overexpression of ERG11 results in increased ergosterol content and a significant reduction in plasma membrane fluidity. Simultaneously, the same point mutation caused cell wall remodeling. This could be facilitated by the unmasking of chitin and β-glucan on the fungal cell surface, which can lead to recognition of the highly immunogenic β-glucan, triggering a stronger immunological reaction. For the first time, we report that a frequently occurring azole-resistance strategy makes C. albicans less susceptible to azole treatment while, at the same time, affects its cell wall architecture, potentially leading to exposure of the pathogen to a more effective host immune response.     

Single-Cell RNA Sequencing Analysis for Oncogenic Mechanisms Underlying Oral Squamous Cell Carcinoma Carcinogenesis with Candida albicans Infection

Oral squamous cell carcinoma (OSCC) carcinogenesis involves heterogeneous tumor cells, and the tumor microenvironment (TME) is highly complex with many different cell types. Cancer cell–TME interactions are crucial in OSCC progression. Candida albicans (C. albicans)—frequently pre-sent in the oral potentially malignant disorder (OPMD) lesions and OSCC tissues—promotes malignant transformation. The aim of the study is to verify the mechanisms underlying OSCC car-cinogenesis with C. albicans infection and identify the biomarker for the early detection of OSCC and as the treatment target. The single-cell RNA sequencing analysis (scRNA-seq) was performed to explore the cell subtypes in normal oral mucosa, OPMD, and OSCC tissues. The cell composi-tion changes and oncogenic mechanisms underlying OSCC carcinogenesis with C. albicans infec-tion were investigated. Gene Set Variation Analysis (GSVA) was used to survey the mechanisms underlying OSCC carcinogenesis with and without C. albicans infection. The results revealed spe-cific cell clusters contributing to OSCC carcinogenesis with and without C. albicans infection. The major mechanisms involved in OSCC carcinogenesis without C. albicans infection are the IL2/STAT5, TNFα/NFκB, and TGFβ signaling pathways, whereas those involved in OSCC carcinogenesis with C. albicans infection are the KRAS signaling pathway and E2F target down-stream genes. Finally, stratifin (SFN) was validated to be a specific biomarker of OSCC with C. albicans infection. Thus, the detailed mechanism underlying OSCC carcinogenesis with C. albicans infection was determined and identified the treatment biomarker with potential precision medicine applications.     

Clean Synthesis of Fatty Acids in an Intensive Lipase-Catalysed Bioreactor

The operation and performance of a laboratory-scale catalytic bioreactor used for the hydrolysis of mixed natural oils into free fatty acids in the presence of free lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) is described. The novelty of the reactor system is the intensification of the reaction kinetics by electrostatic spraying of the lipase as a dispersion of very fine droplets into the oil phase. The effect of phase ratio and field strength on the kinetics were measured for a simple batch type spray reactor. Coalescence rates within the electrostatic spray reactor were shown to be significantly higher than conventional stirred systems. The kinetic data were compared with a control experiment in which the free lipase suspension and oil were contacted by mechanical mixing in the absence of any externally applied electrical field. © 1997 SCI.     

水溶剂法合成葡萄糖脂肪酸酯

对水溶剂法合成葡萄糖脂肪酸酯的工艺作了正交设计实验,优选出水溶剂法合成葡萄糖脂肪酸酯的工艺参数为:n(葡萄糖)∶n(脂肪酸甲酯)=1.5∶1,乳化剂用量15%,催化剂用量2%,反应温度120℃,反应时间为2h。用此工艺参数可获得产率大于80%,单酯含量50%以上的葡萄糖脂肪酸酯的产品。     

Efficient Synthesis of α-Monoglycerides via Solventless Condensation of Fatty Acids with Glycerol Carbonate

Highly pure α-monoglycerides (5a–e) were successfully prepared in high yields by the condensation of fatty acids such as lauric, myristic, palmitic, stearic and oleic (2a–e) with glycerol carbonate (4-hydroxymethyl-1,3-dioxolan-2-one) (1) in the presence of triethylamine as catalyst. Reaction conditions and spectroscopic identification of products will be presented in this article.     

Peripheral Artery Disease Is Associated with a Deficiency of Erythrocyte Membrane n-3 Polyunsaturated Fatty Acids

Population-based data suggest that individuals who consume large dietary amounts of n-3 polyunsaturated fatty acids (PUFA) have lower odds of peripheral artery disease (PAD); however, clinical studies examining n-3 PUFA levels in patients with PAD are sparse. The objective of this study is to compare erythrocyte membrane fatty acid (FA) content between patients with PAD and controls. We conducted a cross-sectional study of 179 vascular surgery outpatients (controls, 34; PAD, 145). A blood sample was drawn and the erythrocyte FA content was assayed using capillary gas chromatography. We calculated the ratio of the n-3 PUFA eicosapentaenoic acid (EPA) to the n-6 PUFA arachidonic acid (ARA) as well as the omega-3 index (O3I), a measure of erythrocyte content of the n-3 PUFA, EPA, and docosahexaenoic acid (DHA), expressed as a percentage of total erythrocyte FA. Compared with controls, patients with PAD smoked more and were more likely to have hypertension and hyperlipidemia (p < 0.05). Patients with PAD had a lower mean O3I (5.0 ± 1.7% vs 6.0 ± 1.6%, p < 0.001) and EPA:ARA ratio (0.04 ± 0.02 vs 0.05 ± 0.05, p < 0.001), but greater mean total saturated fats (39.5 ± 2.5% vs 38.5 ± 2.6%, p = 0.01). After adjusting for several patient characteristics, comorbidities, and medications, an absolute decrease of 1% in the O3I was associated with 39% greater odds of PAD (odds ratio [OR] 1.39, 95% confidence interval [CI] 1.03–1.86, and p = 0.03). PAD was associated with a deficiency of erythrocyte n-3 PUFA, a lower EPA:ARA ratio, and greater mean total saturated fats. These alterations in FA content may be involved in the pathogenesis or development of poor outcomes in PAD.     

Reduction of Free Fatty Acids in Acidic Nonedible Oils by Modified K10 Clay

Biodiesel is a biofuel obtained from vegetable oils. The oils used as raw materials are usually refined edible vegetable oils. Nonedible acidic oils are unsuitable for biodiesel production unless reduction of the high content in free fatty acids (FFA) of these materials had been achieved. Obtaining a good raw material from unprofitable oils becomes an important research field. Additionally clays have a long history in industrial sorption and catalysis, some being commercially available and with properties that can be modified. In this work we present the results of the use of the montmorillonite clay K10 and two acid modified clays K10(I) and K10(II), in the esterification of stearic acid with methanol and 95 % of methyl stearate was obtained with K10(II). These clays were then used for the first time to reduce the acidity of enhanced FFA sunflower oil and they show to be very effective. Reduction of FFA from 11 to 4 % was obtained with K10(II) mainly due to 94 % conversion of FFA into fatty acid methyl esters (FAME). These clays were also tested with two waste oils, one from domestic use and the other gathered from different restaurants, and showed their ability to lower the acidity of these oils. Reactions were followed by ¹H NMR as well as quantitative determination of FFA and FAME. Clays were characterized by FTIR and XRD.     

Enzymatic Analysis of Positional Distribution of Fatty Acids in Solid Fat by 1,3-Selective Transesterification with Candida antarctica Lipase B

A protocol for the analysis of the positional distribution of fatty acids (FA) in solid triacylglycerols (TAG) was developed using sn-1(3) selective alcoholysis catalyzed by immobilized Candida antarctica lipase B (CALB). One part by weight of solid fat and ten parts by weight of ethanol (99.5 %) were warmed to liquefy the fat. After adding 0.44 parts by weight of CALB, the mixture was shaken at 50 °C for 10 min then at 30 °C for 2.8 h. The recovery of 2-MAG after the 3-h transesterification reaction was ca. 85 % of the maximum theoretical yield (33 mol%), with the loss of 15 % attributable to the acyl migration from sn-2 to sn-1(3). The recovery was similar to that of the solvent-free alcoholysis of structured lipids, 1,3-dipalmitoyl, 2-oleoyl glycerol and 1,3-dioleoyl, 2-palmitoyl glycerol, conducted at 30 °C for 3 h. In contrast, the acyl migration from sn-1(3) to sn-2 was hardly observed. Because the detected acyl migration was only in the direction of sn-2 to sn-1(3), and not vice versa, it is proposed to determine the FA composition of the sn-2 position of TAG by the gas chromatographic analysis of 2-MAG fraction recovered from the enzymatic reaction mixture, and the FA composition of sn-1(3) position by a mass balance using the FA composition of TAG and of the sn-2 position as inputs. The procedure was successfully applied to palm oil and shea butter, and docosahexaenoic acid (DHA)-rich single cell oil from Aurantiochytrium sp. KH105 for the first time.     

Synthesis of C20–38 Fatty Acids in Plant Tissues

Very-long-chain fatty acids (VLCFA) are involved in a number of important plant physiological functions. Disorders in the expression of genes involved in the synthesis of VLCFA lead to a number of phenotypic consequences, ranging from growth retardation to the death of embryos. The elongation of VLCFA in the endoplasmic reticulum (ER) is carried out by multiple elongase complexes with different substrate specificities and adapted to the synthesis of a number of products required for a number of metabolic pathways. The information about the enzymes involved in the synthesis of VLCFA with more than 26 atoms of Carbon is rather poor. Recently, genes encoding enzymes involved in the synthesis of both regular-length fatty acids and VLCFA have been discovered and investigated. Polyunsaturated VLCFA in plants are formed mainly by 20:1 elongation into new monounsaturated acids, which are then imported into chloroplasts, where they are further desaturated. The formation of saturated VLCFA and their further transformation into a number of aliphatic compounds included in cuticular waxes and suberin require the coordinated activity of a large number of different enzymes.     

Gb3 Glycosphingolipids with Fluorescent Oligoene Fatty Acids: Synthesis and Phase Behavior in Model Membranes

Glycosphingolipids are involved in a number of physiological and pathophysiological processes, and they serve as receptors for a variety of bacterial toxins and viruses. To investigate their function in lipid membranes, fluorescently labeled glycosphingolipids are highly desirable. Herein, a synthetic route to access Gb₃ glycosphingolipids with fluorescently labeled fatty acids, consisting of pentaene and hexaene moieties either at the terminus or in the middle of the acyl chain, has been developed. The fluorescent properties of the Gb₃ derivatives were investigated in small unilamellar vesicles composed of a raft-like mixture. Phase-separated giant unilamellar vesicles (GUVs) allowed the quantification of the apparent partitioning coefficients of the Gb₃ compounds by means of confocal fluorescence laser scanning microscopy. The determined partition coefficients demonstrate that the Gb₃ derivatives are preferentially localized in the liquid-disordered (l_d) phase. To analyze whether the compounds behave like their physiological counterparts, Cy3-labeled (Cy: cyanine) Shiga toxin B subunits (STxB) were specifically bound to Gb₃-doped GUVs. However, the protein was favorably localized in the l_d phase, in contrast to results reported for STxB bound to naturally occurring Gb₃, which is discussed in terms of the packing density of the lipids in the liquid-ordered (l_o) phase.