Stress granule and P-body clearance: Seeking coherence in acts of disappearance.

Stress granules and P-bodies are conserved cytoplasmic biomolecular condensates whose assembly and composition are well documented, but whose clearance mechanisms remain controversial or poorly described. Such understanding could provide new insight into how cells regulate biomolecular condensate formation and function, and identify therapeutic strategies in disease states where aberrant persistence of stress granules in particular is implicated. Here, I review and compare the contributions of chaperones, the cytoskeleton, post-translational modifications, RNA helicases, granulophagy and the proteasome to stress granule and P-body clearance. Additionally, I highlight the potentially vital role of RNA regulation, cellular energy, and changes in the interaction networks of stress granules and P-bodies as means of eliciting clearance. Finally, I discuss evidence for interplay of distinct clearance mechanisms, suggest future experimental directions, and suggest a simple working model of stress granule clearance.

Self-efficacy to overcome exercise barriers in individuals with cancer-related lymphedema.

Although cancer survivors are recommended to exercise, they may lack confidence (self-efficacy) to be active. This research aimed to measure exercise barriers and related selfefficacy in individuals with cancer-related lymphedema as well as examine relationships between self-efficacy and participant characteristics. A cross-sectional survey was undertaken in individuals with cancer-related lymphedema using a validated 14-item Likert scale assessing self-efficacy to overcome general and lymphedema-specific exercise barriers (0%=not at all confident, 100%=extremely confident). Demographic, medical and lymphedema data were also collected. Of 109 participants (52% response), 79% (n=86) had breast cancer-related lymphedema. Participants were found to be moderately confident to exercise when facing general (48% [95% CI: 44, 52]) and lymphedema- specific exercise barriers (51% [95% CI: 47, 55]). Participants who were female, sedentary (p<0.05), had lymphedema for ≥2 years, and reported greater symptom burden (p<0.05) recorded lower general exercise barriers selfefficacy. Lower lymphedema-specific exercise barriers self-efficacy was reported by individuals who were sedentary, had cancers other than breast, and higher symptom burden. These findings suggest general and lymphedema- specific barriers challenge exercise confidence in those with cancer-related lymphedema, and strategies tailored to improve confidence in overcoming exercise barriers are warranted. Supporting individuals to be sufficiently active during and following cancer treatment should consider behavior change strategies tailored to the unique needs faced by individuals with lymphedema.

HARLEY mitigates user bias and facilitates efficient quantification and co-localization analyses of foci in yeast fluorescence images.

Quantification of cellular structures in fluorescence microscopy data is a key means of understanding cellular function. Unfortunately, numerous cellular structures present unique challenges in their ability to be unbiasedly and accurately detected and quantified. In our studies on stress granules in yeast, users displayed a striking variation of up to 3.7-fold in foci calls and were only able to replicate their results with 62-78% accuracy, when re-quantifying the same images. To facilitate consistent results we developed HARLEY (Human Augmented Recognition of LLPS Ensembles in Yeast), a customizable software for detection and quantification of stress granules in S. cerevisiae. After a brief model training on ~ 20 cells the detection and quantification of foci is fully automated and based on closed loops in intensity contours, constrained only by the a priori known size of the features of interest. Since no shape is implied, this method is not limited to round features, as is often the case with other algorithms. Candidate features are annotated with a set of geometrical and intensity-based properties to train a kernel Support Vector Machine to recognize features of interest. The trained classifier is then used to create consistent results across datasets. For less ambiguous foci datasets, a parametric selection is available. HARLEY is an intuitive tool aimed at yeast microscopy users without much technical expertise. It allows batch processing of foci detection and quantification, and the ability to run various geometry-based and pixel-based colocalization analyses to uncover trends or correlations in foci-related data. HARLEY is open source and can be downloaded from https://github.com/lnilya/harley .

Correction: Fernandes et al. Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation. Biomolecules 2020, 10, 1367.

In the original article [...].

Visual outcome following posterior capsule rupture during manual small incision cataract surgery.

BACKGROUND: The quality of cataract surgery can be measured by visual outcome, which is sometimes limited by intraoperative complications, most commonly posterior capsular rupture. AIMS: The aim of the study was to assess visual outcome at the last visit (≥8 weeks) following posterior capsule rupture (PCR) in patients who had manual small incision cataract surgery (MSICS) managed without access to an automated vitrector. METHODS: A review of medical records of all manual small incision cataract surgeries performed between January 2013 and December 2016 at the National Eye Centre, Kaduna, Nigeria was conducted. Descriptive statistics and logistic regression analysis were performed using STATA 14.0 to examine risk factors for the development of a poor visual outcome and to assess the impact of PCR on development of poor visual outcome. RESULTS: In total, 405 patients were operated on with MSICS (50.6% males). Mean age was 62.4 (SD 12.6) years. PCR was the most common complication (n = 19 (4.7%)). The proportion of good outcomes (≥6/18) rose from 12.4% non-PCR and 0.0% for those with PCR at day 1 postoperative review, to 71.5 and 26.3%, respectively, by final follow up (P = 0.001). Patients with PCR were 7.0 (P = 0.0001) times more likely to have borderline/poor visual outcome (<6/18) compared to those without PCR. Age >60 years increased the odds of borderline/poor by 1.4 times (P = 0.002). CONCLUSION: PCR significantly affects the visual outcome of cataract patients in settings with no facilities for automated vitrectomy. Minimizing complications will improve visual outcome of cataract patients and increase uptake of cataract surgical services.

RNAs as Regulators of Cellular Matchmaking.

RNA molecules are increasingly being identified as facilitating or impeding the interaction of proteins and nucleic acids, serving as so-called scaffolds or decoys. Long non-coding RNAs have been commonly implicated in such roles, particularly in the regulation of nuclear processes including chromosome topology, regulation of chromatin state and gene transcription, and assembly of nuclear biomolecular condensates such as paraspeckles. Recently, an increased awareness of cytoplasmic RNA scaffolds and decoys has begun to emerge, including the identification of non-coding regions of mRNAs that can also function in a scaffold-like manner to regulate interactions of nascently translated proteins. Collectively, cytoplasmic RNA scaffolds and decoys are now implicated in processes such as mRNA translation, decay, protein localization, protein degradation and assembly of cytoplasmic biomolecular condensates such as P-bodies. Here, we review examples of RNA scaffolds and decoys in both the nucleus and cytoplasm, illustrating common themes, the suitability of RNA to such roles, and future challenges in identifying and better understanding RNA scaffolding and decoy functions.

EDC3 phosphorylation regulates growth and invasion through controlling P-body formation and dynamics.

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P-bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P-body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P-body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P-bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin ß1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer-relevant functions and suggest that modulation of P-body activity may represent a new paradigm for cancer treatment.

Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation.

Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in G3BP1/2ΔΔ cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events.

RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly.

P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3'UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3'UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3'UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA 'scaffolds'. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3'UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures.

Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast.

Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA-protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls "Tet On/Tet Off" regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies.

Cdc48/VCP and Endocytosis Regulate TDP-43 and FUS Toxicity and Turnover.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mislocalize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mislocalization and aggregation are implicated in ALS pathology, though the mechanism(s) of TDP-43 and FUS cytoplasmic toxicity remains unclear. Recently, we determined that the endocytic function aids the turnover (i.e., protein degradation) of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 cofactor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in Saccharomyces cerevisiae Cdc48 physically interacts and colocalizes with TDP-43, as does VCP, in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function but not on autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identify a role for Cdc48/VCP and endocytic function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting the cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise.

Canine MAS1: monoallelic expression is suggestive of an imprinted gene.

Imprinted genes are epigenetically modified in a parent-of-origin dependent manner and as a consequence are differentially expressed, with one allele typically expressed while the other is repressed. In canine, the insulin like growth factor 2 receptor gene (IGF2R) is imprinted with predominant expression of the maternally inherited allele. Because imprinted genes usually occur in clusters, we examined the allelic expression pattern of the gene encoding the canine Mas receptor (MAS1), which is located upstream of IGF2R on canine chromosome 1 and is highly conserved in mammals. In this report we describe monoallelic expression of canine MAS1 in the neonatal umbilical cord of several individuals and we identify the expressed allele as maternally inherited. These data suggest that canine MAS1 is an imprinted gene.

Stress Granules and ALS: A Case of Causation or Correlation?

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by cytoplasmic protein aggregates within motor neurons. These aggregates are linked to ALS pathogenesis. Recent evidence has suggested that stress granules may aid the formation of ALS protein aggregates. Here, we summarize current understanding of stress granules, focusing on assembly and clearance. We also assess the evidence linking alterations in stress granule formation and dynamics to ALS protein aggregates and disease pathology.

What are the priorities for improving cataract surgical outcomes in Africa? Results of a Delphi exercise.

PURPOSE: The quality of cataract surgery delivered in sub-Saharan Africa (SSA) is a significant constraint to achieving the elimination of avoidable blindness. No published reports from routine SSA cataract services attain the WHO benchmarks for visual outcomes; poor outcomes (<6/60) often comprise 20% in published case series. This Delphi exercise aimed to identify and prioritise potential interventions for improving the quality of cataract surgery in SSA to guide research and eye health programme development. METHODS: An initial email open-question survey created a ranked list of priorities for improving quality of surgical services. A second-round face-to-face discussion facilitated at a Vision 2020 Research Mentorship Workshop in Tanzania created a refined list for repeated ranking. RESULTS: Seventeen factors were agreed that might form target interventions to promote quality of cataract services. Improved training of surgeons was the top-ranked item, followed by utilisation of biometry, surgical equipment availability, effective monitoring of outcomes of cataract surgery by the surgeon, and well-trained support staff for the cataract pathway (including nurses seeing post-operative cases). CONCLUSION: Improving the quality of cataract surgery in SSA is a clinical, programmatic and public health priority. In the absence of other evidence, the collective expert opinion of those involved in ophthalmic services regarding the ranking of factors to promote quality improvement, refined through this Delphi exercise, provides us with candidate intervention areas to be evaluated.

Endocytosis regulates TDP-43 toxicity and turnover.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. ALS-affected motor neurons exhibit aberrant localization of a nuclear RNA binding protein, TDP-43, into cytoplasmic aggregates, which contributes to pathology via unclear mechanisms. Here, we demonstrate that TDP-43 turnover and toxicity depend in part upon the endocytosis pathway. TDP-43 inhibits endocytosis, and co-localizes strongly with endocytic proteins, including in ALS patient tissue. Impairing endocytosis increases TDP-43 toxicity, aggregation, and protein levels, whereas enhancing endocytosis reverses these phenotypes. Locomotor dysfunction in a TDP-43 ALS fly model is also exacerbated and suppressed by impairment and enhancement of endocytic function, respectively. Thus, endocytosis dysfunction may be an underlying cause of ALS pathology.

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

Role of Autophagy and Apoptosis in Non-Small-Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC) constitutes 85% of all lung cancers, and is the leading cause of cancer-related death worldwide. The poor prognosis and resistance to both radiation and chemotherapy warrant further investigation into the molecular mechanisms of NSCLC and the development of new, more efficacious therapeutics. The processes of autophagy and apoptosis, which induce degradation of proteins and organelles or cell death upon cellular stress, are crucial in the pathophysiology of NSCLC. The close interplay between autophagy and apoptosis through shared signaling pathways complicates our understanding of how NSCLC pathophysiology is regulated. The apoptotic effect of autophagy is controversial as both inhibitory and stimulatory effects have been reported in NSCLC. In addition, crosstalk of proteins regulating both autophagy and apoptosis exists. Here, we review the recent advances of the relationship between autophagy and apoptosis in NSCLC, aiming to provide few insights into the discovery of novel pathogenic factors and the development of new cancer therapeutics.