Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay.

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion.

Generation of recombinant bioluminescent Escherichia coli for quantitative determination of bacterial adhesion.

Bacterial adhesion to urinary catheter was evaluated by measuring the light emitted from a recombinant bioluminescent glycocalyx producer Escherichia coli strain. Generation of the bioluminescent strain was carried out by transforming the bacterial cells with pUCP18-GFP plasmid that contains a green fluorescence gene. Light emission measurement was closely correlated with the number of the adherent cells, giving a detectable signal from 1.2 X 10² cells. The efficiency of this assay was confirmed by testing the antiadherent effect of subinhibitory concentrations of ciprofloxacin with the aid of a model for in-vitro catheter colonization. There was no significant difference in the percentage reduction of adherent cells obtained by both light emission measurement and viable cell count techniques.

The role of miR-765 in human cancers.

MicroRNAs, a collection of short noncoding RNAs, are promising biomarkers for identifying cancer in its early stages and tracking the effectiveness of treatment. This is due to their critical role in regulating gene expression and other vital biological functions via cell-level epigenetic mechanisms. This review brings together data on the molecular and clinical effects of miR-765 on different types of cancer. Significant variation in miR-765 levels has been observed in a variety of cancer types, suggesting that it could have an oncogene or tumor suppressor role. A number of pathways, including PLP2/Notch, VEGFA/Akt1, PDX1, KLK4, RUNX2, DPF3, EMP3, APE1, ERK/EMT axis, and others, are impacted by the inclusion of miR-765 in their analysis. MiR-765 is an essential biomarker that shows promise as a diagnostic tool for various types of cancer. The latest research has identified them as reliable predictive markers for detecting tumor development at an early stage. Based on our study, miR-765 shows promising potential as a biomarker for prognosis in multiple types of cancer. Specifically, we suggest that miR-765 could be an early detection marker for tumor development, progression, and metastasis.

Polystyrene-based magnetic hydrogels for elimination of some toxic metal cations from aqueous solutions.

Three smart novel polystyrene-based hydrogel nanocomposites were introduced for recovery of some divalent heavy metals (Cu2+ ad Pb2+) from simulated solutions for industrial wastewater remediation. In this regard, magnetic nanoparticles were prepared then fabricated with polystyrene waste and a copolymer of acrylic acid and acrylamide in presence of an initiator/crosslinking system. The chemical amendment process was established by the IR spectroscopy and the prepared hydrogels were characterized by the AFM microscopy and XRD analysis. Moreover, the elimination efficacy of the prepared hydrogels was monitored for Cu2+ and Pb2+versus different temperatures and pH values in simulated solution. The data showed a direct relation between the contact time and the amount of the metal removed. Moreover, it was found that the removal efficiency decreases within the region between LCST of polyacrylamide and HCST of polystyrene. It was also proved that maximum metal removal was attained at neutral pH. The maximum removal was achieved by the hydrogel with minimum magnetite content with removal capacity of 128 mg/g of Cu2+ and 122 mg/g of Pb2+ in single removal experiments at optimum test conditions. In double removal experiments, the prepared hydrogels showed obvious selectivity towards Cu2+ ions. The reusability of the investigated hydrogels was examined at three swelling-deswelling cycles.

A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion.

Endocytosis is a fundamental process underlying diverse eukaryotic physiology. The terminal stage of this process is membrane fusion between the perimeter membrane of a late endosome filled with intraluminal vesicles, or multivesicular body (MVB), and the lysosome membrane to facilitate catabolism of internalized biomaterials or surface polytopic proteins. To comprehensively understand the mechanisms underlying MVB-lysosome membrane fusion, we developed a quantitative, cell-free assay to study this SNARE-mediated event in molecular detail using Saccharomyces cerevisiae and its vacuolar lysosome, or vacuole, as models. This involves separately isolating organelles from two yeast strains each expressing a different complementary fusion probe targeted to the lumen of either MVBs or vacuoles. Isolated organelles are mixed in vitro under fusogenic conditions. Upon MVB-vacuole membrane fusion, luminal contents mix to facilitate probe interaction, reconstituting ß-lactamase activity recorded by a colorimetric enzyme activity assay. This method accommodates a multitude of approaches (e.g., genetics, addition of purified protein reagents) to study this process in isolation, and in theory could be repurposed to study other SNARE-mediated fusion events within cells.

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion.

Loss-of-function mutations in human endosomal Na+(K+)/H+ exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function.

Rab-Effector-Kinase Interplay Modulates Intralumenal Fragment Formation during Vacuole Fusion.

Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity.

Selective Lysosomal Transporter Degradation by Organelle Membrane Fusion.

Lysosomes rely on their resident transporter proteins to return products of catabolism to the cell for reuse and for cellular signaling, metal storage, and maintaining the lumenal environment. Despite their importance, little is known about the lifetime of these transporters or how they are regulated. Using Saccharomyces cerevisiae as a model, we discovered a new pathway intrinsic to homotypic lysosome membrane fusion that is responsible for their degradation. Transporter proteins are selectively sorted by the docking machinery into an area between apposing lysosome membranes, which is internalized and degraded by lumenal hydrolases upon organelle fusion. These proteins have diverse lifetimes that are regulated in response to protein misfolding, changing substrate levels, or TOR activation. Analogous to endocytosis for controlling surface protein levels, the "intralumenal fragment pathway" is critical for lysosome membrane remodeling required for organelle function in the context of cellular protein quality control, ion homeostasis, and metabolism.

Homo-Tandem Polymer Solar Cells with VOC >1.8 V for Efficient PV-Driven Water Splitting.

Efficient homo-tandem and triple-junction polymer solar cells are constructed by stacking identical subcells composed of the wide-bandgap polymer PBDTTPD, achieving power conversion efficiencies >8% paralleled by open-circuit voltages >1.8 V. The high-voltage homo-tandem is used to demonstrate PV-driven electrochemical water splitting with an estimated solar-to-hydrogen conversion efficiency of ≈6%.

Concordant proficiency in measurement of T-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents.

The gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-gamma ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-gamma ELISPOT responses. These findings also illustrate the ability to standardize the IFN-gamma ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.

Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes.

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.