Approaches to Visualising Endocytosis of LDL-Related Lipoproteins.

Endocytosis is the process by which molecules are actively transported into cells. It can take on a variety of forms depending on the cellular machinery involved ranging from specific receptor-mediated endocytosis to the less selective and actin-driven macropinocytosis. The plasma lipoproteins, which deliver lipids and other cargo to cells, have been intensely studied with respect to their endocytic uptake. One of the first molecules to be visualised undergoing endocytosis via a receptor-mediated, clathrin-dependent pathway was low-density lipoprotein (LDL). The LDL molecule has subsequently been shown to be internalised through multiple endocytic pathways. Dissecting the pathways of lipoprotein endocytosis has been crucial to understanding the regulation of plasma lipid levels and how lipids enter cells in the arterial wall to promote atherosclerosis. It has also aided understanding of the dysregulation that occurs in plasma lipid levels when molecules involved in uptake are defective, as is the case in familial hypercholesterolemia (FH). The aim of this review is to outline the many endocytic pathways utilised for lipoprotein uptake. It explores the various experimental approaches that have been applied to visualise lipoprotein endocytosis with an emphasis on LDL and its more complex counterpart, lipoprotein(a) [Lp(a)]. Finally, we look at new developments in lipoprotein visualisation that hold promise for scrutinising endocytic pathways to finer detail in the future.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis.

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae ADΔΔ is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae ADΔΔ, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Engineering a Cysteine-Deficient Functional Candida albicans Cdr1 Molecule Reveals a Conserved Region at the Cytosolic Apex of ABCG Transporters Important for Correct Folding and Trafficking of Cdr1.

Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is a major cause of azole antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR transporter has been solved. The objective of this project was to investigate the role of the 23 Cdr1 cysteine residues in the stability, trafficking, and function of the protein when expressed in the eukaryotic model organism, Saccharomyces cerevisiae The biochemical characterization of 18 partially cysteine-deficient Cdr1 variants revealed that the six conserved extracellular cysteines were critical for proper expression, localization, and function of Cdr1. They are predicted to form three covalent disulfide bonds that stabilize the large extracellular domains of fungal PDR transporters. Our investigations also revealed a novel nucleotide-binding domain motif, GX2[3]CPX3NPAD/E, at the peripheral cytosolic apex of ABCG transporters that possibly contributes to the unique ABCG transport cycle. With this knowledge, we engineered an "almost cysteine-less," yet fully functional, Cdr1 variant, Cdr1P-CID, that had all but the six extracellular cysteines replaced with serine, alanine, or isoleucine (C1106I of the new motif). It is now possible to perform cysteine-cross-linking studies that will enable more detailed biochemical investigations of fungal PDR transporters and confirm any future structure(s) solved for this important protein family.IMPORTANCE Overexpression of the fungal pleiotropic drug resistance (PDR) transporter Cdr1 is a major cause of antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR ABC transporter has been solved. Cdr1 contains 23 cysteines; 10 are cytosolic and 13 are predicted to be in the transmembrane or the extracellular domains. The objective of this project was to create, and biochemically characterize, CDR1 mutants to reveal which cysteines are most important for Cdr1 stability, trafficking, and function. During this process we discovered a novel motif at the cytosolic apex of PDR transporters that ensures the structural and functional integrity of the ABCG transporter family. The creation of a functional Cys-deficient Cdr1 molecule opens new avenues for cysteine-cross-linking studies that will facilitate the detailed characterization of an important ABCG transporter family member.

The influence of impact delivery mode, lactation time, infant gender, maternal age and rural or urban life on total number of Lactobacillus in breast milk Isfahan - Iran.

BACKGROUND: Breast milk is known as the most crucial postpartum issue in metabolic and immunologic programming of neonatal health. Human milk microbial changes over Lactation. The factors influencing the milk microbiome as well as potential impact of microbes on infant health have not yet been discovered. The objective was to identify pre- and post-natal factors that can potentially influence the bacterial communities inhabiting human milk. MATERIALS AND METHODS: Breast milk samples (n = 40) with all full-term breastfed infants were collected from lactating randomized. Information on personal characteristics, dietary habits, information about infants were collected after birth. The samples were plated with serial dilutions on three selective culture media man rogosa sharp and then colonies were counted. Colonies tested for catalase reaction, Gram-staining and microscopic examination. RESULTS: The result of this study showed that the overall incidence of positive Lactobacillus in mother's milk was 87.5%. The results based on (infant gender, mode of delivery, rural or urban and lactation time) rural or urban and lactation time were significant (P < 0.05). The results showed that all of the variables were significant in this regression model (P < 0.001). The median of log10 Lactobacillus counts in rural mothers, vaginal delivery, infant male gender and Lactation time for first 3-month were meaningfully high. CONCLUSIONS: The findings of this study about the breast milk Lactobacillus potential probiotic bacteria of healthy Iranian mothers, suggested that the breast milk microbiome is significantly influenced by several factors, mode of delivery, rural or urban and lactation time.

A novel method of anal fissure laser surgery: a pilot study.

Anal fissure is a common painful problem, affecting all age groups. Its pathophysiology is based on high sphincter pressures and reduced blood supplying and treatments which means that it generally reduces anal pressures and increases anodermal blood flow. Since each of the anal fissure's routine therapies has some limitations such as definite risk of permanent fecal incontinence and high recurrence rate, we tried to find a more effective and less invasive procedure. In this pilot study which was implemented on 25 male and female patients aged 20-75 years, diagnosed clinically with chronic anal fissure, the Carbon Dioxide Laser Fractional was used to treat patients. In order to first remove fibrotic and granulation tissues, the base and the edges of the fissure were laser beamed. Eight spots were made on the sphincter by the laser on its continuous mode; somehow, they were passed through the full thickness of sphincter without interrupting its continuance. Afterwards, the area around the fissure ulcer was irradiated by deep fractional mode of the laser to stimulate the submucosa to regenerate and rejuvenate. After going through this procedure, patients were followed up within 6 months to 1 year. Pain, bleeding, and constipation were significantly improved. None of the patients had recurrence after a 1-year follow-up, and none of them had fecal incontinence and/or inability to control the passage of gas too. This study revealed that this new laser-based surgery is a simple, safe, and effective procedure to treat the anal fissure that can be performed with local anesthesia in an outpatient clinic with minimal postoperative morbidity.

How much in vitro cholesterol reducing activity of lactobacilli predicts their in vivo cholesterol function?

BACKGROUND: Based on literature, in vitro cholesterol removal of lactic acid bacteria has been accounted for their in vivo cholesterol reduction. But recently it has been proposed that such in vitro characteristic may not be directly relevant to their in vivo activity. The objective of this study was to find how much in vitro cholesterol reducing potential of Lactobacillus plantarum A7 (LA7), a native strain isolated from an infant fecal flora, reflects its in vivo efficiency. LA7 previously showed serum cholesterol reducing capability in mice subjected to fatty diet. Here, we investigate whether the given strain is capable of in vitro cholesterol assimilation or consumption. METHOD: LA7 was cultured in whole milk and de-Man-Rogosa-Sharpe (MRS) added with water-soluble cholesterol. Colorimetric method was adopted for cholesterol determination in both cultured media during incubation period. RESULTS: No cholesterol assimilation was detected by growth and incubation of the active culture in either of the medium. Thus, in vivo cholesterol function of LA7 was not caused by cholesterol consumption. A comprehensive review of literature on the related studies also showed that there are other documented studies which evidenced the uncertainty of the direct relation between in vitro and in vivo studies. CONCLUSION: Cholesterol removal from the cultured media may not be considered as an appropriate integral index for selection of Lactobacillus strains with cholesterol-lowering activity.