FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

Water- and nutrient-dependent effects of dietary restriction on Drosophila lifespan

Dietary restriction (DR) is a widely conserved intervention leading to lifespan extension. Despite considerable effort, the mechanisms underlying DR remain poorly understood. In particular, it remains unclear whether DR prolongs life through conserved mechanisms in different species. Here, we show that, in the most common experimental conditions, lifespan extension by DR is abolished by providing Drosophila with ad libitum water, without altering food intake, indicating that DR, as conventionally studied in flies, is fundamentally different from the phenomenon studied in mammals. We characterize an alternative dietary paradigm that elicits robust lifespan extension irrespective of water availability, and thus likely represents a more relevant model for mammalian DR. Our results support the view that protein:carbohydrate ratio is the main dietary determinant of fly lifespan. These findings have broad implications for the study of lifespan and nutrition

Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

Background: Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology: In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions: In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion

Pathological Endogenous α-Synuclein Accumulation in Oligodendrocyte Precursor Cells Potentially Induces Inclusions in Multiple System Atrophy.

Glial cytoplasmic inclusions (GCIs), commonly observed as α-synuclein (α-syn)-positive aggregates within oligodendrocytes, are the pathological hallmark of multiple system atrophy. The origin of α-syn in GCIs is uncertain; there is little evidence of endogenousα-syn expression in oligodendrocyte lineage cells, oligodendrocyte precursor cells (OPCs),and mature oligodendrocytes (OLGs). Here, based on in vitro analysis using primary rat cell cultures, we elucidated that preformed fibrils (PFFs) generated from recombinant human α-syn trigger multimerization and an upsurge of endogenous α-syn in OPCs, which is attributable to insufficient autophagic proteolysis. RNA-seq analysis of OPCs revealed that α-syn PFFs interfered with the expression of proteins associated with neuromodulation and myelination. Furthermore, we detected cytoplasmic α-syn inclusions in OLGs through differentiation of OPCs pre-incubated with PFFs. Overall, our findings suggest the possibility of endogenous α-syn accumulation in OPCs that contributes to GCI formation and perturbation of neuronal/glial support in multiple system atrophy brains

Electric dipole moments and the search for new physics

Static electric dipole moments of nondegenerate systems probe mass scales for physics beyond the Standard Model well beyond those reached directly at high energy colliders. Discrimination between different physics models, however, requires complementary searches in atomic-molecular-and-optical, nuclear and particle physics. In this report, we discuss the current status and prospects in the near future for a compelling suite of such experiments, along with developments needed in the encompassing theoretical framework.Comment: Contribution to Snowmass 2021; updated with community edits and endorsement

The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS

Comparative assessment of the cytotoxicity of six anti-inflammatory eyedrops in four cultured ocular surface cell lines, as determined by cell viability scores

Masahiko Ayaki,1 Atsuo Iwasawa,2 Yoshimi Niwano31Department of Ophthalmology, International University of Health and Welfare, Mita Hospital, Tokyo, 2Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 3Laboratory for Redox Regulation, Tohoku University Graduate School of Dentistry, Sendai, JapanPurpose: Anti-inflammatory eyedrops are often used in the treatment of corneal epithelial disorders. In the present study, we evaluated the cytotoxicity of six anti-inflammatory eyedrops in four ocular surface cell lines.Methods: The cytotoxicity of six commercially available anti-inflammatory ophthalmic solutions (ie, diclofenac, bromfenac, pranoprofen, betamethasone, and fluoromethorone) was assessed in three corneal cell lines and one conjunctival cell line. Cell viability was determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide and neutral red assays after exposing the cells to 10, 30, and 60 minutes of onefold, twofold, and tenfold dilutions of the drugs. Cytotoxicity was compared using the cell viability score (CVS), an integrated cytotoxic parameter that takes various factors into account, such as dilution by tear fluid or concentration by evaporation, drug exposure time, and ocular surface cell type.Results: Based on the CVS scores, the order of the anti-inflammatory eyedrops tested from least to most cytotoxic, with the active ingredient %CVS50, and %CVS40/80 for each solution given in parentheses, was as follows: Rinderon® (betamethasone, 100%, 100%) >0.02% Flumethoron® (fluoromethorone, 68%, 22%) = 0.1% Flumethoron® (fluoromethorone, 76%, 22%) >Bronuck® (0.1% bromfenac, 53%, −8%) = Diclod® (0.1% diclofenac, 44%, −15%) = Niflan® (pranoprofen, 50%, −19%). Rinderon® exhibited the least toxicity of all the anti-inflammatory eyedrops tested. Eyedrops containing non-steroidal anti-inflammatory drugs exhibited greater cytotoxicity than those containing steroids with benzalkonium at comparable concentrations. Concentration was the most significant factor affecting cell viability.Conclusion: The cytotoxicity of the anti-inflammatory eyedrops evaluated in the present study depended on both the pharmaceutical components and preservatives. The CVS is a concise indicator of drug cytotoxicity.Keywords: benzalkonium chloride, toxicity, cornea, cell viability score, anti-inflammatory dru