Electron Tomography of Organelles and Vesicles in the Investigation of SNARE Function and Localization.

Electron tomography can provide additional morphological information not easily obtained by conventional transmission electron microscopy of thin sections. It uses a goniometer stage in the electron microscope to tilt the specimen and collect a series of 2D images from different orientations, which are combined to provide a 3D volume tomogram and a colored reconstruction of the morphological feature(s) of interest. Here we describe the protocols for its use in visualizing changes in organelle morphology after depletion of the SNARE proteins VAMP7 and VAMP8 and to study VAMP7 localization on endolysosomes/lysosomes.

Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase.

Lived Experience-Led Research Agenda to Address Early Death in People With a Diagnosis of a Serious Mental Illness: A Consensus Statement.

Importance: People with serious mental illness (SMI), defined as a diagnosis of schizophrenia spectrum disorder, bipolar disorder, or disabling major depressive disorder) die approximately 10 to 25 years earlier than the general population.

Objective: To develop the first-ever lived experience-led research agenda to address early mortality in people with SMI.

Evidence Review: A virtual 2-day roundtable comprising 40 individuals convened on May 24 and May 26, 2022, and used a virtual Delphi method to arrive at expert group consensus.

Juvenile mucopolysaccharidosis plus disease caused by a missense mutation in VPS33A.

A rare and fatal disease resembling mucopolysaccharidosis in infants, is caused by impaired intracellular endocytic trafficking due to deficiency of core components of the intracellular membrane-tethering protein complexes, HOPS, and CORVET. Whole exome sequencing identified a novel VPS33A mutation in a patient suffering from a variant form of mucopolysaccharidosis. Electron and confocal microscopy, immunoblotting, and glycosphingolipid trafficking experiments were undertaken to investigate the effects of the mutant VPS33A in patient-derived skin fibroblasts.

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway.

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered lysosome-associated membrane protein (LAMP)-carrier vesicles around multivesicular bodies, as well as the appearance of 'hourglass' profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8.

Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29.

VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface.

Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples.

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples.

Antibiotic Review Kit for Hospitals (ARK-Hospital): study protocol for a stepped-wedge cluster-randomised controlled trial.

Background: To ensure patients continue to get early access to antibiotics at admission, while also safely reducing antibiotic use in hospitals, one needs to target the continued need for antibiotics as more diagnostic information becomes available. UK Department of Health guidance promotes an initiative called 'Start Smart then Focus': early effective antibiotics followed by active 'review and revision' 24-72 h later. However in 2017, < 10% of antibiotic prescriptions were discontinued at review, despite studies suggesting that 20-30% of prescriptions could be stopped safely.

The lysosomal disease caused by mutant VPS33A.

A rare lysosomal disease resembling a mucopolysaccharidosis with unusual systemic features, including renal disease and platelet dysfunction, caused by the defect in a conserved region of the VPS33A gene on human chromosome 12q24.31, occurs in Yakuts-a nomadic Turkic ethnic group of Southern Siberia. VPS33A is a core component of the class C core vacuole/endosome tethering (CORVET) and the homotypic fusion and protein sorting (HOPS) complexes, which have essential functions in the endocytic pathway.

The Lysosome and Intracellular Signalling.

In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis.

Mammalian γ2 AMPK regulates intrinsic heart rate.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia).

Families' experiences of raising concerns in health care services: An interpretative phenomenological analysis.

Background: This exploratory study aimed to increase understanding of the experiences of families of people with intellectual disabilities when noticing and raising concerns in services. A qualitative design was employed.

Methods: Seven participants (all female) were recruited through local and national voluntary agencies; five were mothers of people with intellectual disabilities, one was the aunt and one the sister.

Relationship of low doses of alcohol voluntarily consumed during adolescence and early adulthood with subsequent behavioral flexibility.

Previous alcohol use is associated with impaired decision-making and impulsivity in humans, but the relationship between alcohol use and decision-making/impulsivity is unclear. In two experiments, we determined whether chronic intermittent access to alcohol during adolescence and early adulthood would alter or be correlated with performance in a go/no-go reversal task, a devaluation task, or operant extinction. Rats received 6 weeks of chronic intermittent access to 20% alcohol or water from postnatal day 26 to 66 and then behavioral testing was initiated 1.

Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria.

Background: Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit.

Exosomes bind to autotaxin and act as a physiological delivery mechanism to stimulate LPA receptor signalling in cells.

Autotaxin (ATX; also known as ENPP2), the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA), is a secreted enzyme. Here we show that, once secreted, ATX can bind to the surface of cell-secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA.

Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active.

High autistic trait individuals do not modulate gaze behaviour in response to social presence but look away more when actively engaged in an interaction.

Autism is characterised by difficulties in social functioning, notably in interactions with other people. Yet, most studies addressing social difficulties have used static images or, at best, videos of social stimuli, with no scope for real interaction. Here, we study one crucial aspect of social interactions-gaze behaviour-in an interactive setting.

Prior alcohol consumption does not impair go/no-go discrimination learning, but causes over-responding on go trials, in rats.

Prior alcohol use is associated with impaired response inhibition in humans, including in laboratory go/no-go discrimination tasks. In two experiments, we determined whether chronic intermittent access to alcohol would alter go/no-go discrimination learning. Rats received 4-6 weeks of chronic intermittent access to 20% alcohol (alone or accompanied by saline or 1.

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I.

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes.

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects.

Lysosome fusion in cultured mammalian cells.

In mammalian cells, lysosomes fuse with late endosomes to form endolysosomes from which lysosomes are reformed. Lysosomal fusion events were initially inferred from light and electron microscopy studies, demonstrated in cell-free content mixing assays and, more recently, shown directly with live cell microscopy. Currently, there is a focus on studying lysosome fusion in cultured cells using various forms of microscopy, especially under conditions in which the use of overexpression of dominant-negative protein constructs or the use of RNA interference to deplete individual proteins allows the investigation of the molecular machinery of fusion.

Repetition Suppression in Ventral Visual Cortex Is Diminished as a Function of Increasing Autistic Traits.

Repeated viewing of a stimulus causes a change in perceptual sensitivity, known as a visual aftereffect. Similarly, in neuroimaging, repetitions of the same stimulus result in a reduction in the neural response, known as repetition suppression (RS). Previous research shows that aftereffects for faces are reduced in both children with autism and in first-degree relatives.