Candida albicans stimulates formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection.

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 are required for C. albicans to stimulate c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorates OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans.

Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells.

During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays and have low virulence in immunocompetent mouse models of disseminated infection. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to αv integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells but can be restored when mouse endothelial cells are engineered to express human gC1qR or αv integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells.

Life Cycle of Cryptococcus neoformans.

Cryptococcus neoformans is a ubiquitous environmental fungus and an opportunistic pathogen that causes fatal cryptococcal meningitis. Advances in genomics, genetics, and cellular and molecular biology of C. neoformans have dramatically improved our understanding of this important pathogen, rendering it a model organism to study eukaryotic biology and microbial pathogenesis. In light of recent progress, we describe in this review the life cycle of C. neoformans with a special emphasis on the regulation of the yeast-to-hypha transition and different modes of sexual reproduction, in addition to the impacts of the life cycle on cryptococcal populations and pathogenesis.

Potential drug targets for multiple sclerosis identified through Mendelian randomization analysis.

Multiple sclerosis is a complex autoimmune disease, and several therapies for multiple sclerosis have been developed and widely used. However, existing medications for multiple sclerosis were far from satisfactory due to their failure to suppress relapses and alleviate disease progression. Novel drug targets for multiple sclerosis prevention are still needed. We performed Mendelian randomization to explore potential drug targets for multiple sclerosis using summary statistics from the International Multiple Sclerosis Genetics Consortium (nCase = 47 429, nControl = 68 374) and further replicated in UK Biobank (nCase = 1356, nControl = 395 209) and FinnGen cohorts (nCase = 1326, nControl = 359 815). Genetic instruments for 734 plasma and 154 CSF proteins were obtained from recently published genome-wide association studies. The reverse causality detection using bidirectional Mendelian randomization analysis and Steiger filtering, Bayesian co-localization, and phenotype scanning that searched previously reported genetic variant-trait associations were implemented to consolidate the Mendelian randomization findings further. In addition, the protein-protein interaction network was performed to reveal potential associations among proteins and/or present multiple sclerosis medications. At Bonferroni significance (P < 5.63 × 10-5), Mendelian randomization analysis revealed six protein-multiple sclerosis pairs. In plasma, per standard deviation increase in FCRL3, TYMP and AHSG had a protective effect. Odds ratios for the proteins above were 0.83 (95% CI, 0.79-0.89), 0.59 (95% CI, 0.48-0.71) and 0.88 (95% CI, 0.83-0.94), respectively. In CSF, per 10-fold increase in MMEL1 (OR, 5.03; 95% CI, 3.42-7.41) increased the risk of multiple sclerosis, while SLAMF7 (OR, 0.42; 95% CI, 0.29-0.60) and CD5L (OR, 0.30; 95%CI, 0.18-0.52) decreased the risk. None of the six proteins had reverse causality. Bayesian co-localization suggested that FCRL3 [coloc.abf-posterior probability of hypothesis 4 (PPH4) = 0.889], TYMP (coloc.susie-PPH4 = 0.896), AHSG (coloc.abf-PPH4 = 0.957, coloc.susie-PPH4 = 0.973), MMEL1 (coloc.abf-PPH4 = 0.930) and SLAMF7 (coloc.abf-PPH4 = 0.947) shared the same variant with multiple sclerosis. FCRL3, TYMP and SLAMF7 interacted with target proteins of current multiple sclerosis medications. MMEL1 was replicated in both UK Biobank and FinnGen cohorts. Our integrative analysis suggested that genetically determined levels of circulating FCRL3, TYMP, AHSG, CSF MMEL1 and SLAMF7 had causal effects on multiple sclerosis risk. These findings suggested those five proteins might be promising drug targets for multiple sclerosis and warrant further clinical investigation, especially FCRL3 and SLAMF7.

Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects ß-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.

Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung.

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.

Genome-wide identification and expression analysis of the R2R3-MYB gene family in tobacco (Nicotiana tabacum L.).

BACKGROUND: The R2R3-MYB transcription factor is one of the largest gene families in plants and involved in the regulation of plant development, hormone signal transduction, biotic and abiotic stresses. Tobacco is one of the most important model plants. Therefore, it will be of great significance to investigate the R2R3-MYB gene family and their expression patterns under abiotic stress and senescence in tobacco. RESULTS: A total of 174 R2R3-MYB genes were identified from tobacco (Nicotiana tabacum L.) genome and were divided into 24 subgroups based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were especially conserved among the NtR2R3-MYB genes, especially members within the same subgroup. The NtR2R3-MYB genes were distributed on 24 tobacco chromosomes. Analysis of gene duplication events obtained 3 pairs of tandem duplication genes and 62 pairs of segmental duplication genes, suggesting that segmental duplications is the major pattern for R2R3-MYB gene family expansion in tobacco. Cis-regulatory elements of the NtR2R3-MYB promoters were involved in cellular development, phytohormones, environmental stress and photoresponsive. Expression profile analysis showed that NtR2R3-MYB genes were widely expressed in different maturity tobacco leaves, and however, the expression patterns of different members appeared to be diverse. The qRT-PCR analysis of 15 NtR2R3-MYBs confirmed their differential expression under different abiotic stresses (cold, salt and drought), and notably, NtMYB46 was significantly up-regulated under three treatments. CONCLUSIONS: In summary, a genome-wide identification, evolutionary and expression analysis of R2R3-MYB gene family in tobacco were conducted. Our results provided a solid foundation for further biological functional study of NtR2R3-MYB genes in tobacco.

Identification of biomarkers, immune infiltration landscape, and treatment targets of ischemia-reperfusion acute kidney injury at an early stage by bioinformatics methods.

BACKGROUND: Mechanisms underlying ischemia/reperfusion injury-acute kidney injury (IRI-AKI) are not fully elucidated. We conducted an integrative analysis of IRI-AKI by bioinformatics methods. METHODS: We screened gene expression profiles of the IRI-AKI at early phase from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified and enrichment pathways were conducted based on gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and Gene set enrichment analysis (GSEA). Immune cell infiltration analysis was performed to reveal the change of the microenvironment cell types. We constructed protein-protein interaction (PPI), and Cytoscape with plug-ins to find hub genes and modules. We performed robust rank aggregation (RRA) to combine DEGs and analyzed the target genes for miRNA/transcription factor (TF) and drug-gene interaction networks. RESULTS: A total of 239 and 384 DEGs were identified in GSE87024 and GSE34351 separately, with the 73 common DEGs. Enrichment analysis revealed that the significant pathways involve mitogen-activated protein kinase (MAPK) signaling, interleukin-17, and tumor necrosis factor (TNF) signaling pathway, etc. RRA analysis detected a total of 27 common DEGs. Immune cell infiltration analysis showed the plasma cells reduced and T cells increased in IRI-AKI. We identified JUN, ATF3, FOS, EGR1, HMOX1, DDIT3, JUNB, NFKBIZ, PPP1R15A, CXCL1, ATF4, and HSPA1B as hub genes. The target genes interacted with 23 miRNAs and 116 drugs or molecular compounds such as curcumin, staurosporine, and deferoxamine. CONCLUSION: Our study first focused on the early IRI-AKI adopting RRA analysis to combine DEGs in different datasets. We identified significant biomarkers and crucial pathways involved in IRI-AKI and first construct the immune landscape and detected the potential therapeutic targets of the IRI-AKI by drug-gene network.

Determination of 241Am in Environmental Samples: A Review.

The determination of 241Am in the environment is of importance in monitoring its release and assessing its environmental impact and radiological risk. This paper aims to give an overview about the recent developments and the state-of-art analytical methods for 241Am determination in environmental samples. Thorough discussions are given in this paper covering a wide range of aspects, including sample pre-treatment and pre-concentration methods, chemical separation techniques, source preparation, radiometric and mass spectrometric measurement techniques, speciation analyses, and tracer applications. The paper focuses on some hyphenated separation methods based on different chromatographic resins, which have been developed to achieve high analytical efficiency and sample throughput for the determination of 241Am. The performances of different radiometric and mass spectrometric measurement techniques for 241Am are evaluated and compared. Tracer applications of 241Am in the environment, including speciation analyses of 241Am, and applications in nuclear forensics are also discussed.

Long-term ambient PM2.5 exposure associated with cardiovascular risk factors in Chinese less educated population.

INTRODUCTION: Long-term exposure to ambient air pollution is related to major cardiovascular risk factors including diabetes, hypertension, hyperlipidemia and overweight, but with few studies in high-concentration nations like China so far. We aimed to investigate the association between long-term exposure to ambient fine particulate matter (particles with an aerodynamic diameter ≤ 2.5 µm, PM2.5) and major cardiovascular risk factors in China. METHODS: Adult participants with selected biochemical tests were recruited from the Chinese Physiological Constant and Health Condition (CPCHC) survey conducted from 2007 to 2011. Gridded PM2.5 data used were derived from satellite-observed data with adjustment of ground-observed data. District-level PM2.5 data were generated to estimate the association using multivariate logistic regression model and generalized additive model. RESULTS: A total of 19,236 participants from the CPCHC survey were included with an average age of 42.8 ± 16.1 years, of which nearly half were male (47.0%). The annual average PM2.5 exposure before the CPCHC survey was 33.4 (14.8-53.4) µg/m3, ranging from 8.0 µg/m3 (Xiwuqi) to 94.7 µg/m3 (Chengdu). Elevated PM2.5 was associated with increased prevalence of hypertension (odds ratio (OR) =1.022, 95% confidence interval (95%CI): 1.001, 1.043) and decreased prevalence of overweight (OR = 0.926, 95%CI: 0.910, 0.942). Education significantly interacted with PM2.5 in association with all the interesting risk factors. Each 10 µg/m3 increment of PM2.5 was associated with increased prevalence of diabetes (OR = 1.118, 95%CI: 1.037, 1.206), hypertension (OR = 1.101, 95%CI: 1.056, 1.147), overweight (OR = 1.071, 95%CI: 1.030, 1.114) in participants with poor education, but not in well-educated population. PM2.5 exposure was negatively associated with hyperlipidemia in all participants (OR = 0.939, 95%CI: 0.921, 0.957). The results were robust in all the sensitivity analyses. CONCLUSION: Association between long-term PM2.5 exposure and cardiovascular risk factors might be modified by education. PM2.5 was associated with a higher prevalence of diabetes, hypertension, and overweight in a less-educated population with time-expose dependency. Long-term exposure to PM2.5 might be associated with a lower prevalence of hyperlipidemia.

Transformation of Cryptococcus neoformans by electroporation using a transient CRISPR-Cas9 expression (TRACE) system.

The basidiomycete Cryptococcus neoformans is not only a clinically important pathogen, but also a model organism for studying microbial pathogenesis and eukaryotic biology. One key factor behind its rise as a model organism is its genetic amenability. The widely used methods for transforming the C. neoformans species complex are Agrobacterium-mediated transformation (AMT) for random insertional mutagenesis and biolistic transformation for targeted mutagenesis. Electroporation was introduced to C. neoformans in early 1990s. Although electroporation is economic and yields a large number of transformants, introduced DNA rarely integrates into cryptococcal genome, which limits its use. Biolistic transformation, although costly and inefficient, has been the only method used in targeted mutagenesis in the past two decades. Several modifications, including the use of a donor DNA with split markers, a drug-resistant selection marker, and a recipient strain deficient in non-homologous end joining (NHEJ), have since modestly increased the frequency of genome integration and the rate of homologous replacement of the DNA introduced by electroporation. However, electroporation was not the method of choice for transformation until the recent adoption of CRISPR-Cas9 systems. We have developed a Transient CRISPR-Cas9 coupled with Electroporation System (TRACE), which dramatically facilitates targeted mutagenesis in the Cryptococcus species complex. TRACE combines the high transformation efficiency of electroporation with the high rates of DNA integration due to the transiently expressed CRISPR-Cas9. Here, we briefly discussed the history of electroporation for Cryptococcus transformation and provided detailed procedures for electroporation and the cassettes construction of the TRACE system for various genetic manipulations.

Glucosamine stimulates pheromone-independent dimorphic transition in Cryptococcus neoformans by promoting Crz1 nuclear translocation.

Morphotype switch is a cellular response to external and internal cues. The Cryptococcus neoformans species complex can undergo morphological transitions between the yeast and the hypha form, and such morphological changes profoundly affect cryptococcal interaction with various hosts. Filamentation in Cryptococcus was historically considered a mating response towards pheromone. Recent studies indicate the existence of pheromone-independent signaling pathways but their identity or the effectors remain unknown. Here, we demonstrated that glucosamine stimulated the C. neoformans species complex to undergo self-filamentation. Glucosamine-stimulated filamentation was independent of the key components of the pheromone pathway, which is distinct from pheromone-elicited filamentation. Glucosamine stimulated self-filamentation in H99, a highly virulent serotype A clinical isolate and a widely used reference strain. Through a genetic screen of the deletion sets made in the H99 background, we found that Crz1, a transcription factor downstream of calcineurin, was essential for glucosamine-stimulated filamentation despite its dispensability for pheromone-mediated filamentation. Glucosamine promoted Crz1 translocation from the cytoplasm to the nucleus. Interestingly, multiple components of the high osmolality glycerol response (HOG) pathway, consisting of the phosphorelay system and some of the Hog1 MAPK module, acted as repressors of glucosamine-elicited filamentation through their calcineurin-opposing effect on Crz1's nuclear translocation. Surprisingly, glucosamine-stimulated filamentation did not require Hog1 itself and was distinct from the conventional general stress response. The results demonstrate that Cryptococcus can resort to multiple genetic pathways for morphological transition in response to different stimuli. Given that the filamentous form attenuates cryptococcal virulence and is immune-stimulatory in mammalian models, the findings suggest that morphogenesis is a fertile ground for future investigation into novel means to compromise cryptococcal pathogenesis.

Pheromone independent unisexual development in Cryptococcus neoformans.

The fungus Cryptococcus neoformans can undergo a-α bisexual and unisexual reproduction. Completion of both sexual reproduction modes requires similar cellular differentiation processes and meiosis. Although bisexual reproduction generates equal number of a and α progeny and is far more efficient than unisexual reproduction under mating-inducing laboratory conditions, the α mating type dominates in nature. Population genetic studies suggest that unisexual reproduction by α isolates might have contributed to this sharply skewed distribution of the mating types. However, the predominance of the α mating type and the seemingly inefficient unisexual reproduction observed under laboratory conditions present a conundrum. Here, we discovered a previously unrecognized condition that promotes unisexual reproduction while suppressing bisexual reproduction. Pheromone is the principal stimulus for bisexual development in Cryptococcus. Interestingly, pheromone and other components of the pheromone pathway, including the key transcription factor Mat2, are not necessary but rather inhibitory for Cryptococcus to complete its unisexual cycle under this condition. The inactivation of the pheromone pathway promotes unisexual reproduction despite the essential role of this pathway in non-self-recognition during bisexual reproduction. Nonetheless, the requirement for the known filamentation regulator Znf2 and the expression of hyphal or basidium specific proteins remain the same for pheromone-dependent or independent sexual reproduction. Transcriptome analyses and an insertional mutagenesis screen in mat2Δ identified calcineurin being essential for this process. We further found that Znf2 and calcineurin work cooperatively in controlling unisexual development in this fungus. These findings indicate that Mat2 acts as a repressor of pheromone-independent unisexual development while serving as an activator for a-α bisexual development. The bi-functionality of Mat2 might have allowed it to act as a toggle switch for the mode of sexual development in this ubiquitous eukaryotic microbe.

Synthesis and Characterization of Photothermal Osmium Carbolong Complexes.

Metallacycles with chelating polydentate conjugated-carbon chain ligands are called carbolong complexes, which are expected to have interesting properties. In this work, the preparation of 12-carbon carbolong complexes in which all of the coordinated atoms in the equatorial plane are carbon atoms was studied. With the help of the well-established mechanism, a new approach to prepare coplanar carbolong complexes bearing different organic functional groups was developed by adding different terminal alkynes in sequence. In the presence of HBF4 , these coplanar carbolong complexes were converted to η3 -allyl osmapentalene derivatives, which can be produced directly from the reaction of cyclopropaosmapentalene 1 with terminal alkynes in the presence of AgBF4 under anhydrous conditions. This study offers a new route for the preparation of functional osmium carbolong complexes with excellent photothermal properties, which can be used to prepare photothermal materials.

Metallapentalenofuran: Shifting Metallafuran Rings Promoted by Substituent Effects.

Bulky substituents play important roles in controlling the reaction pathways or producing selected products. This work reports that the shift of metallafuran rings in a metallapentalenofuran complex can be promoted by the substituent effect via a reversible C-H bond reductive elimination and oxidative addition. The starting osmapentalyne, a so-called 7-carbon carbolong complex, was produced by the oxidation of a metallapentalenofuran with FeCl3 . It was then allowed to react with nucleophiles, followed by a C-H activation, to give the aforementioned metallapentalenofuran complex. This work enriches the family of carbolong complexes and reveals a new strategy to promote, but not prevent reactions by the bulky substituents.

Reference Equations for the Six-Minute Walk Distance in the Healthy Chinese Han Population, Aged 18-30 Years.

BACKGROUND: The six-minute walk test (6MWT) is a safe, simple, inexpensive tool for evaluating the functional exercise capacity. However, there is a lack of standard reference equations for the six-minute walk distance (6MWD) in the healthy Chinese Han population with an age of 18-30 years. The aims of the present study were as follows: 1) to measure the anthropometric data and the walking distance in a sample of healthy Chinese Han population, aged 18-30 years; 2) to construct reference equations for the 6MWD; 3) to compare the measured 6MWD of our cohort with previously published equations. METHODS: The anthropometric data, demographic, lung function and the walking distance of Chinese Han population, aged 18-30 years, were prospectively measured using a standardized protocol. Informed consent was obtained from each participant and the approval was obtained from the ethics committee of Wenzhou People's Hospital. The 6MWT was performed twice and the longer 6MWD was used for further analysis. RESULTS: A total of 355 subjects (176 female and 179 male) completed the 6MWT, and the average walking distance was 627.3 ± 52.88 m. The walking distance was achieved by females compared with males (607.4 ± 51.00 m vs. 646.9 ± 47.15 m; p < 0.0001) and active subjects compared with non-active subjects (646.1 ± 48.27 m vs. 611.6 ± 51.52 m; p < 0.0001). Univariate analysis showed age, height, body mass index, resting blood pressure, heart rate and blood pressure after the walk test and difference in heart rate before and after the walk test were significantly correlated with the 6MWD. Stepwise multiple regression analysis showed that height and difference in heart rate before and after the walk test were independent predictors associated with the 6MWD. The reference equations from Caucasian, Canadian and Chilean populations tend to overestimate the walking distance in our subjects, while Brazilian and Arabian equations tend to underestimate the walking distance. There was no significant difference in the walking distance between Korean equations and the current study. CONCLUSION: In summary, height and difference in heart rate before and after the walk test were the most significant predictors of the 6MWD, and the regression equations could explain approximately 38% and 31% of the distance variance in the female and male groups, respectively.

Allicin prevents oxidized low-density lipoprotein-induced endothelial cell injury by inhibiting apoptosis and oxidative stress pathway.

BACKGROUND: Vascular endothelial apoptosis is significantly associated with atherosclerosis and cardiovascular diseases, for which oxidized low-density lipoprotein (ox-LDL) is a major risk factor. Allicin, the primary active ingredient of garlic, has been found to have cardiovascular protective effect by changing the fatty-acid composition, but its effect on ox-LDL-induced vascular endothelial injury remains unclear. We investigated the protective effect of allicin on cell viability, LDH release, apoptosis and apoptotic signaling in human umbilical vein endothelial cells (HUVECs). METHODS: In cultured HUVEC cell line, ox-LDL induced injury was investigated. The cell viability and injury were evaluated by using cell proliferation Assay kit and LDH release assay. The apoptosis was evaluated by the Annexin V-FITC kit. The activity of caspase-3 was assessed using a colorimetric caspase-3 assay kit. The ROS production was evaluated by fluorometric assay and NADPH oxidase activity was assessed with a GENMED kit. RESULTS: Exposure of HUVECs to ox-LDL (150 µg/ml) reduced cell viability, induced apoptosis and increased activity of caspase-3, NADPH oxidase, and reactive oxygen species (ROS) production. The pretreatment with allicin (30 and 100 µM) significantly rescued the cell viability, inhibited ox-LDL-induced apoptosis and activity of caspase-3, NADPH oxidase and ROS production in HUVECs, and the protective effect is concentration-dependent. The allicin (100 µM) alone did not show significant difference from control. Our study demonstrated that allicin protected HUVECs from ox-LDL-induced endothelial injury by reducing the apoptosis, mediated by inhibition of caspase-3 and NADPH oxidase related apoptotic signaling. CONCLUSIONS: Allicin prevents ox-LDL-induced endothelial cell injury by inhibiting apoptosis and oxidative stress pathway.

Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.

Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, ß-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.

Morphology and its underlying genetic regulation impact the interaction between Cryptococcus neoformans and its hosts.

Cryptococcus neoformans is a fungus that causes the majority of fatal cryptococcal meningitis cases worldwide. This pathogen is capable of assuming different morphotypes: yeast, pseudohypha, and hypha. The yeast form is the most common cell type observed clinically. The hyphal and pseudohyphal forms are rarely observed in the clinical setting and are considered attenuated in virulence. However, as a ubiquitous environmental pathogen, Cryptococcus interacts with various organisms, and it is known to be parasitic to different hosts. Capitalizing on recent discoveries, morphogenesis regulators were manipulated to examine the impact of cell shape on the cryptococcal interaction with three different host systems: the soil amoeba Acanthamoeba castellanii (a protist), the greater wax moth Galleria mellonella (an insect), and the murine macrophage cell line J774A.1 (mammalian cells). The regulation of Ace2 and morphogenesis (RAM) pathway is a highly conserved pathway among eukaryotes that regulates cytokinesis. Disruption of any of five RAM components in Cryptococcus renders cells constitutively in the pseudohyphal form. The transcription factor Znf2 is the master activator of the yeast to hyphal transition. Deletion of ZNF2 locks cells in the yeast form, while overexpression of this regulator drives hyphal growth. Genetic epistasis analyses indicate that the RAM and the Znf2 pathways regulate distinct aspects of cryptococcal morphogenesis and independently of each other. These investigations using the Cryptococcus RAM and ZNF2 mutants indicate that cell shape, cell size, and likely cell surface properties weigh differently on the outcome of cryptococcal interactions with different hosts. Thus, certain traits evolved in Cryptococcus that are beneficial within one host might be detrimental when a different host is encountered.

Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein.

Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility.