Retrospective Investigation and Research on Fall Events Among Hospitalized Patients in the Rehabilitation Department.

Objective: We investigated the clinical characteristics, fall outcomes, and related factors of falls in patients who were hospitalized in the rehabilitation department, and explored strategies to reduce the incidence of falls and prevent falls in patients. Methods: Data from 60 patients who fell in the rehabilitation department between 2016 and 2021 were analyzed for clinical characteristics, associated factors, incidence of falls, injuries, and patient demographics. Under the random stratified sampling method, 60 patients who did not fall during the same period were selected as the control group, and relevant data was collected. Measurement data were compared using an independent sample t-test. Enumeration data were compared using chi-squared (χ2) test was employed to compare these data between the two groups. Non-parametric data were analyzed using the Mann-Whitney U-test. Factors potentially influencing falls were scrutinized through both univariate and binary logistic regression analyses. Results: The median annual incidence of falls among patients who were hospitalized in the rehabilitation department was 0.04%, while the overall fall injury rate was 60%. Falls were most prevalent within 30 days of hospitalization (71.67%). The most common fall-related condition was craniocerebral disease (83.33%). The incidents of falls location of fall were mainly reported in nearby areas of rehabilitation ward (70%). Most accidents occurred between 7:00 a.m.-12:00 p.m. and 3:01 p.m.-6:00 p.m. (63.33%), and dyskinesia was the most common cause of falls (71.67%). There were 39 patients (65.00%) with Barthel Index (BI) scores ranging between 40-60. Conclusion: Patients in the rehabilitation department had a greater incidence of falls and fall injuries. Within 30 days of admission, patients with moderately dependent craniocerebral disorders and dyskinesia frequently experienced falls during typical daytime shifts in areas characterized by endemic conditions.

Extracellular release in the quality control of the mammalian mitochondria.

Mammalian cells release a wealth of materials to their surroundings. Emerging data suggest these materials can even be mitochondria with perturbed morphology and aberrant function. These dysfunctional mitochondria are removed by migrating cells through membrane shedding. Neuronal cells, cardiomyocytes, and adipocytes send dysfunctional mitochondria into the extracellular space for nearby cells to degrade. Various studies also indicate that there is an interplay between intracellular mitochondrial degradation pathways and mitochondrial release in handling dysfunctional mitochondria. These observations, in aggregate, suggest that extracellular release plays a role in quality-controlling mammalian mitochondria. Future studies will help delineate the various types of molecular machinery mammalian cells use to release dysfunctional mitochondria. Through the studies, we will better understand how mammalian cells choose between intracellular degradation and extracellular release for the quality control of mitochondria.

Nanoscale details of mitochondrial constriction revealed by cryoelectron tomography.

Mitochondria adapt to changing cellular environments, stress stimuli, and metabolic demands through dramatic morphological remodeling of their shape, and thus function. Such mitochondrial dynamics is often dependent on cytoskeletal filament interactions. However, the precise organization of these filamentous assemblies remains speculative. Here, we apply cryogenic electron tomography to directly image the nanoscale architecture of the cytoskeletal-membrane interactions involved in mitochondrial dynamics in response to damage. We induced mitochondrial damage via membrane depolarization, a cellular stress associated with mitochondrial fragmentation and mitophagy. We find that, in response to acute membrane depolarization, mammalian mitochondria predominantly organize into tubular morphology that abundantly displays constrictions. We observe long bundles of both unbranched actin and septin filaments enriched at these constrictions. We also observed septin-microtubule interactions at these sites and elsewhere, suggesting that these two filaments guide each other in the cytosolic space. Together, our results provide empirical parameters for the architecture of mitochondrial constriction factors to validate/refine existing models and inform the development of new ones.

Monitoring Stub1-Mediated Pexophagy.

Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality of peroxisomes. During this process, heat shock protein 70 and the ubiquitin E3 ligase, Stub1, translocate onto peroxisomes to be turned over to initiate pexophagy. The Stub1 ligase activity allows the accumulation of ubiquitin and other autophagy-related modules on targeted peroxisomes. Elevating reactive oxygen species (ROS) levels within the peroxisomal lumen can activate Stub1-mediated pexophagy. One can, therefore, use dye-assisted ROS generation to trigger and monitor this pathway. This article outlines the procedures for using two classes of dyes, fluorescent proteins and synthetic fluorophores, to initiate pexophagy within mammalian cell cultures. These dye-assisted ROS generation-based protocols can not only be used to target all the peroxisomes within a cell population globally but can also permit the manipulation of individual peroxisomes within single cells. We also describe how Stub1-mediated pexophagy can be followed using live-cell microscopy.

Parkin coordinates mitochondrial lipid remodeling to execute mitophagy.

Autophagy has emerged as the prime machinery for implementing organelle quality control. In the context of mitophagy, the ubiquitin E3 ligase Parkin tags impaired mitochondria with ubiquitin to activate autophagic degradation. Although ubiquitination is essential for mitophagy, it is unclear how ubiquitinated mitochondria activate autophagosome assembly locally to ensure efficient destruction. Here, we report that Parkin activates lipid remodeling on mitochondria targeted for autophagic destruction. Mitochondrial Parkin induces the production of phosphatidic acid (PA) and its subsequent conversion to diacylglycerol (DAG) by recruiting phospholipase D2 and activating the PA phosphatase, Lipin-1. The production of DAG requires mitochondrial ubiquitination and ubiquitin-binding autophagy receptors, NDP52 and optineurin (OPTN). Autophagic receptors, via Golgi-derived vesicles, deliver an autophagic activator, EndoB1, to ubiquitinated mitochondria. Inhibition of Lipin-1, NDP52/OPTN, or EndoB1 results in a failure to produce mitochondrial DAG, autophagosomes, and mitochondrial clearance, while exogenous cell-permeable DAG can induce autophagosome production. Thus, mitochondrial DAG production acts downstream of Parkin to enable the local assembly of autophagosomes for the efficient disposal of ubiquitinated mitochondria.

Golgi quality control and autophagy.

Organelles can easily be disrupted by intracellular and extracellular factors. Studies on ER and mitochondria indicate that a wide range of responses are elicited upon organelle disruption. One response thought to be of particular importance is autophagy. Cells can target entire organelles into autophagosomes for removal. This wholesale nature makes autophagy a robust means for eliminating compromised organelles. Recently, it was demonstrated that the Golgi apparatus is a substrate of autophagy. On the other hand, various reports have shown that components traffic away from the Golgi for elimination in an autophagosome-independent manner when the Golgi apparatus is stressed. Future studies will reveal how these different pieces of machinery coordinate to drive Golgi degradation. Quantitative measurements will be needed to determine how much autophagy contributes to the maintenance of the Golgi apparatus.

Enhanced enzymatic production of cholesteryl 6'-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori.

BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

A cryo-electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane.

Cryo-electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the 'endosomal sorting complex required for transport' machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

Hsc70/Stub1 promotes the removal of individual oxidatively stressed peroxisomes.

Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. Here, using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we find that cells individually remove ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70 s mediates the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology and U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to promote their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes is sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. We further determine that Stub1 mutants found in Ataxia patients are defective in pexophagy induction. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.

Correction: Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection.

[This corrects the article DOI: 10.1371/journal.pgen.1003007.].

Vesicular transport mediates the uptake of cytoplasmic proteins into mitochondria in Drosophila melanogaster.

Mitochondrial aging, which results in mitochondrial dysfunction, is strongly linked to many age-related diseases. Aging is associated with mitochondrial enlargement and transport of cytosolic proteins into mitochondria. The underlying homeostatic mechanisms that regulate mitochondrial morphology and function, and their breakdown during aging, remain unclear. Here, we identify a mitochondrial protein trafficking pathway in Drosophila melanogaster involving the mitochondria-associated protein Dosmit. Dosmit induces mitochondrial enlargement and the formation of double-membraned vesicles containing cytosolic protein within mitochondria. The rate of vesicle formation increases with age. Vesicles originate from the outer mitochondrial membrane as observed by tracking Tom20 localization, and the process is mediated by the mitochondria-associated Rab32 protein. Dosmit expression level is closely linked to the rate of ubiquitinated protein aggregation, which are themselves associated with age-related diseases. The mitochondrial protein trafficking route mediated by Dosmit offers a promising target for future age-related mitochondrial disease therapies.

Intracellular galectins control cellular responses commensurate with cell surface carbohydrate composition.

Galectins are ß-galactoside-binding animal lectins primarily found in the cytosol, while their carbohydrate ligands are mainly distributed in the extracellular space. Cytosolic galectins are anticipated to accumulate on damaged endocytic vesicles through binding to glycans initially displayed on the cell surface and subsequently located in the lumen of the vesicles, and this can be followed by cellular responses. To facilitate elucidation of the mechanism underlying this process, we adopted a model system involving induction of endocytic vesicle damage with light that targets the endocytosed amphiphilic photosensitizer disulfonated aluminum phthalocyanine. We demonstrate that the levels of galectins around damaged endosomes are dependent on the composition of carbohydrates recognized by the proteins. By super resolution imaging, galectin-3 and galectin-8 aggregates were found to be distributed in distinct microcompartments. Importantly, galectin accumulation is significantly affected when cell surface glycans are altered. Furthermore, accumulated galectins can direct autophagy adaptor proteins toward damaged endocytic vesicles, which are also significantly affected following alteration of cell surface glycans. We conclude that cytosolic galectins control cellular responses reflect dynamic modifications of cell surface glycans.

Omegasome-proximal PtdIns(4,5)P2 couples F-actin mediated mitoaggregate disassembly with autophagosome formation during mitophagy.

Cells govern their homeostasis through autophagy by sequestering substrates, ranging from proteins to aggregates and organelles, into autophagosomes for lysosomal degradation. In these processes cells need to coordinate between substrate remodeling and autophagosome formation for efficient engulfment. We found that in Parkin-mediated mitophagy, mitochondria to be turned over first become grape-like mitoaggregates, followed by their disassembly into smaller pieces via the actinomyosin system. At the disassembly step, we observed spatially-associated, synchronous formation of circular F-actin and BATS-labeled autophagy initiation sites near mitochondria, suggesting coordination between substrate downsizing and autophagosome formation during mitophagy. Interestingly, PtdIns(4,5)P2, instead of PtdIns(3)P, regulates this mitophagy-associated formation of circular F-actin and BATS-sites. Selective depletion of PtdIns(4,5)P2 near omegasomes, the endoplasmic reticulum (ER) subdomains involved in autophagosome formation, impaired mitoaggregate disassembly. Our findings demonstrate the presence of a pool of PtdIns(4,5)P2 adjacent to omegasomes, and that they coordinate mitoaggregate disassembly with autophagy initiation during Parkin-mediated mitophagy.

Srv2 Is a Pro-fission Factor that Modulates Yeast Mitochondrial Morphology and Respiration by Regulating Actin Assembly.

Dynamic processes such as fusion, fission, and trafficking are important in the regulation of cellular organelles, with an abundant literature focused on mitochondria. Mitochondrial dynamics not only help shape its network within cells but also are involved in the modulation of respiration and integrity. Disruptions of mitochondrial dynamics are associated with neurodegenerative disorders. Although proteins that directly bind mitochondria to promote membrane fusion/fission have been studied intensively, machineries that regulate dynamic mitochondrial processes remain to be explored. We have identified an interaction between the mitochondrial fission GTPase Dnm1/DRP1 and the actin-regulatory protein Srv2/CAP at mitochondria. Deletion of Srv2 causes elongated-hyperfused mitochondria and reduces the reserved respiration capacity in yeast cells. Our results further demonstrate that the irregular network morphology in Δsrv2 cells derives from disrupted actin assembly at mitochondria. We suggest that Srv2 functions as a pro-fission factor in shaping mitochondrial dynamics and regulating activity through its actin-regulatory effects.

Triggering Mitophagy with Photosensitizers.

One can utilize light illumination to stimulate mitochondrial reactive oxygen species production through the use of mitochondria-specific photosensitizers. By proper tuning of the light dosage, the methodology permits probing of a multitude of mitochondrial damage responses, including mitophagy. This light-controllable trick offers unique opportunities for the investigation of mitophagy-one can spatiotemporally define mitochondrial damage, alter the number of impaired mitochondria, as well as modulate the severity of the mitochondrial injury. This light-activated mitophagy can be adapted not only to single-cell imaging techniques but also to cell population-based biochemical assays.

Staining of Lipid Droplets with Monodansylpentane.

Monodansylpentane (MDH) is a fluorophore that displays selective labeling of lipid droplets (LDs). The dye preferentially segregates into the neutral lipid cores of LDs and emits blue fluorescence, compatible with the simultaneous use of green and red fluorescent reporters in multi-color live-cell imaging. MDH can be used for visualizing LDs not only in cell cultures, but also in fixed tissues such as the fat body and ovaries from Drosophila. MDH is therefore a versatile marker for LDs in fluorescence microscopy.

Optogenetic probing of mitochondrial damage responses.

It is now possible to functionally impair mitochondria through light illumination with high specificity. These optogenetic tools permit precise control on the timing, location, and extent of mitochondrial damage within a cell population with subcellular resolution, allowing quantitative probing of the various types of mitochondrial damage responses within cells. This approach can generally be extended toward the probing of other organelle damage responses.

Bit-by-bit autophagic removal of parkin-labelled mitochondria.

Eukaryotic cells maintain mitochondrial integrity through mitophagy, an autophagic process by which dysfunctional mitochondria are selectively sequestered into double-layered membrane structures, termed phagophores, and delivered to lysosomes for degradation. Here we show that small fragments of parkin-labelled mitochondria at omegasome-marked sites are engulfed by autophagic membranes one at a time. Using a light-activation scheme to impair long mitochondrial tubules, we demonstrate that sites undergoing bit-by-bit mitophagy display preferential ubiquitination, and are situated where parkin-labelled mitochondrial tubules and endoplasmic reticulum intersect. Our observations suggest contact regions between the endoplasmic reticulum and impaired mitochondria are initiation sites for local LC3 recruitment and mitochondrial remodelling that support bit-by-bit, parkin-mediated mitophagy. These results help in understanding how cells manage to fit large and morphologically heterogeneous mitochondria into micron-sized autophagic membranes during mitophagy.

Spatiotemporally controlled induction of autophagy-mediated lysosome turnover.

Lysosomes are the major degradative compartments within cells, harbouring a wide variety of hydrolytic enzymes within their lumen. Release of lysosomal hydrolases from lysosomes into the cell cytoplasm results in cell death. Here we report that damaged lysosomes undergo autophagic turnover. Using a light-induced lysosome impairing scheme that can be controlled spatially and temporally within a cell, we show that damaged lysosomes are selectively ubiquitinated, recruit autophagic proteins and are eventually incorporated into autolysosomes for degradation. We propose that autophagic removal of lysosomes, which we term lysophagy, is a surveillance mechanism that alleviates cells from the adverse effects of lysosomal damage. We envision our method to induce lysosomal damage will enable detailed molecular studies of the lysophagy pathway in the future.