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Exposure to radiation causes DNA damage and cell death in enterocytes and inhibition of ISC proliferation. (A) Midguts were stained with anti-γ-H2Av antibody and DAPI. Guts from esg-Gal4, UAS-GFP flies were dissected 30 min after irradiation with (bottom panels) or without (top panels) 100 Gy. Right panels are magnified images of the white square in the left side panels. Yellow asterisks indicate the both γ-H2Av and GFP positive cells. White asterisks indicate the γ-H2Av negative GFP positive cells. Scale bar indicates 40 µm. (B) Midguts were stained with anti-GFP antibody and DAPI. Guts from upd3-Gal4, UAS-GFP flies were dissected 30 min and 3 days after irradiation with (bottom panels) or without (top panels) 100 Gy. Scale bar indicates 40 µm. (C) Midguts were stained with anti-cPARP antibody, anti-LacZ antibody and DAPI. Guts from esg-Gal4, UAS-GFP, Dl-LacZ, UAS-CD8-PARP-Venus flies were dissected 1 day after irradiation with (bottom panels) or without (top panels) 100 Gy. Yellow asterisk indicates the both cPARP and LacZ positive cell. Scale bar indicates 20 µm. (D) Ethidium bromide-Acridine Orange staining was performed in guts of w¹¹¹⁸ female flies after irradiation (100 Gy) at indicated time points. The results are plotted as the mean number of apoptotic cells per gut and presented and mean apoptotic cells and error bars indicate S.D. of 2 independent experiments with at least 10 guts each. (E) Relative fold change in the expression of Hid, Rpr and Puc in the dissected gut 24 h after irradiation. The error bars indicate S.D. of 4 replicates. (***p < 0.001, **p < 0.01, nsp > 0.05 by t-test).
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Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. We have developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, we performed an unbiased GWAS sc...
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Citations
... However, Sdc did not contribute to fGSC protection ( Figure S4), suggesting tissuespecific roles. In ISCs, the proximal cause of death upon Sdc knockdown remains to be determined, although the DNA damage acquired by these cells seems a likely driving force, with physiological DNA damage and genotoxic stress known to contribute to ISC elimination (Nagy et al., 2018;Sharma et al., 2020). ...
Tissue maintenance is underpinned by resident stem cells whose activity is modulated by microenvironmental cues. Using Drosophila as a simple model to identify regulators of stem cell behaviour and survival in vivo , we have identified novel connections between the conserved transmembrane proteoglycan Syndecan, nuclear properties and stem cell function. In the Drosophila midgut, Syndecan depletion in intestinal stem cells results in their loss from the tissue, impairing tissue renewal. At the cellular level, Syndecan depletion alters cell and nuclear shape, and causes nuclear lamina invaginations and DNA damage. In a second tissue, the developing Drosophila brain, live imaging revealed that Syndecan depletion in neural stem cells results in nuclear envelope remodelling defects which arise upon cell division. Our findings reveal a new role for Syndecan in the maintenance of nuclear properties in diverse stem cell types.
Graphical Abstract
... We previously demonstrated an intrinsic requirement for the RBP Musashi (Msi) in GSC maintenance in the Drosophila testis [14]. In the Drosophila midgut, overexpression of Msi promotes stem cell proliferation after radiation-induced damage [18]. Its vertebrate orthologues, Msi-1 and Msi-2, function in the regulation, proliferation and maintenance of stem cells in multiple tissues [19][20][21][22][23][24][25][26] and play key roles in vertebrate spermatogenesis [27,28] and folliculogenesis [29]. ...
... Drosophila Msi has known roles in determining fate outcome of ectodermal sensory organ precursors [36], photoreceptor [32] and crystal cell determination [44]. Msi is required for maintenance of spermatogonial GSCs [14] and modulates intestinal stem cell proliferation after radiation induced damage [18]. These studies Sex-specific differences in Drosophila RBP function for maintenance of GSC identity have been described Our analysis has revealed that Msi isoforms exhibit divergent expression in the germline and soma of both the Drosophila testis and ovary. ...
The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. We have identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, we show that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.
In insects, melanism, a fundamental pigmentation process, is of significant importance in evolutionary biology due to its complex genetic foundation. We investigated the role of the RNA-binding gene Musashi (msi) in melanism in Laodelphax striatellus, a Hemiptera species. We identified a single L. striatellus msi homolog, Lsmsi, encoding a 357 amino acid protein with 2 RNA recognition motifs. RNA interference-mediated knockdown of LsMsi resulted in complete body melanism and increased cuticular permeability. Additionally, we found the involvement of G protein-coupled receptor A42 and tyrosine hydroxylase (Th) in L. striatellus melanism. Knockdown of LsTh lightened the epidermis, showing dehydration signs, while LsA42 knockdown enhanced LsTh expression, leading to melanism. Surprisingly, Lsmsi knockdown decreased both LsA42 and LsTh expression, which was expected to cause whitening but resulted in melanism. Further, we found that Lsmsi influenced downstream genes like phenoloxidase homolog LsPo and dopa decarboxylase (Ddc) homolog LsDdc in the tyrosine-mediated melanism pathway. Extending to Nilaparvata lugens and Sogatella furcifera, we demonstrated the conserved role of msi in melanism among Delphacidae. Given MSI proteins' roles in cancer and tumors in vertebrates, our study is the first to link msi in insects to Delphacidae body color melanization via the tyrosine-mediated pathway, offering fresh perspectives on the genetic basis of insect melanism and msi functions.
Lung cancer is one of the most common types of cancer worldwide. Non-small cell lung cancer (NSCLC), typically caused by KRAS and TP53 driver mutations, represents the majority of all new lung cancer diagnoses. Overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) has been associated with NSCLC progression. To investigate the role of MSI2 in NSCLC development, we compared the tumorigenesis in mice with lung-specific Kras -activating mutation and Trp53 deletion, with and without Msi2 deletion (KPM2 versus KP mice). KPM2 mice showed decreased lung tumorigenesis in comparison with KP mice. In addition, KPM2 lung tumors showed evidence of decreased proliferation, but increased DNA damage, marked by increased levels of phH2AX (S139) and phCHK1 (S345), but decreased total and activated ATM. Using cell lines from KP and KPM2 tumors, and human NSCLC cell lines, we found that MSI2 directly binds ATM mRNA and regulates its translation. MSI2 depletion impaired DNA damage response (DDR) signaling and sensitized human and murine NSCLC cells to treatment with PARP inhibitors in vitro and in vivo. Taken together, we conclude that MSI2 supports NSCLC tumorigenesis, in part, by supporting repair of DNA damage by controlling expression of DDR proteins. These results suggest that targeting MSI2 may be a promising strategy for lung cancers treated with DNA-damaging agents.
The numerous beneficial probiotic properties of Lactobacillus reuteri (L. reuteri) include decreasing metabolic syndrome, preventing disorders linked to oxidative stress, improving gut flora imbalances, controlling immunological function, and extending life span. Exposure to ionizing radiation is closely associated with several disorders. We examined the protective and salvaging effects of L. reuteri on ionizing radiation-induced injury to the intestinal tract, reproductive system, and nervous system of Drosophila melanogaster. We also examined its effects on lifespan, antioxidant capacity, progeny development, and behavioral aspects to assess the interaction between L. reuteri and ionizing radiation-induced injury. The findings demonstrated that L. reuteri improved the median survival time following irradiation and greatly extended its lifespan. In addition, it raised SOD activity, reduced ROS levels in intestinal epithelial cells, and increased the quantity of intestinal stem cells. Furthermore, L. reuteri enhanced the adult male flies' capacity to move. It also successfully safeguarded the generations' growth and development. L. reuteri dramatically enhanced expression of the AMPKα gene and regulated expression of its pathway-related gene, mTOR, as well as the autophagy-related genes Atg1 and Atg5 in female Drosophila exposed to irradiation. Notably, no prior reports have been made on the possible effects of L. reuteri on injuries caused by irradiation. As a result, our research offers important new information regarding L. reuteri's possible role as a shield against ionizing radiation-induced injury.
Background and aim:
While preliminary evidence points to pro-tumorigenic roles for the Musashi (MSI) RNA-binding proteins Musashi-1 (MSI1) and Musashi-2 (MSI2) in some breast cancer subtypes, no data exist for inflammatory breast cancer (IBC).
Methods:
MSI gene expression was quantified in IBC SUM149PT cells. We then used small interfering RNA-based MSI1 and MSI2 double knockdown (DKD) to understand gene expression and functional changes upon MSI depletion. We characterized cancer stem cell characteristics, cell apoptosis and cell cycle progression via flow cytometry, mammospheres via spheroid assays, migration and proliferation via digital holographic microscopy, and cell viability using BrdU assays. Chemoresistance was determined for paclitaxel and cisplatin with MTT assays and radioresistance was assessed with clonogenic analyses. In parallel, we supported our in vitro data by analyzing publicly available patient IBC gene expression datasets.
Results:
MSI1 and MSI2 are upregulated in breast cancer generally and IBC specifically. MSI2 is more commonly expressed compared to MSI1. MSI DKD attenuated proliferation, cell cycle progression, migration, and cell viability while increasing apoptosis. Stem cell characteristics CD44(+)/CD24(-), TERT and Oct4 were associated with MSI expression in vivo and were decreased in vitro after MSI DKD as was ALDH expression and mammosphere formation. In vivo, chemoresistant tumors were characterized by MSI upregulation upon chemotherapy application. In vitro, MSI DKD was able to alleviate chemo- and radioresistance.
Conclusions:
The Musashi RNA binding proteins are dysregulated in IBC and associated with tumor proliferation, cancer stem cell phenotype, chemo- and radioresistance. MSI downregulation alleviates therapy resistance and attenuates tumor proliferation in vitro.
Environmental and occupational low-dose radiation (LDR) exposure may be harmful for health but the previous reports regarding effect of LDR on cognition are contradictory. Here we investigated the effect of long-term LDR exposure on cognition. In this study, male Balb/c mice’ cognitive functions were tested at 15 weeks after being exposed to 0.5 Gy LDR in 10 fractions at each dose of 0.05 Gy. The results demonstrated that long-term LDR exposure increases escape latency and the time spent in finding exits in mice compared with non LDR exposure. Meanwhile, the inflammation-related proteins including NFκB and p38 also increased. Lipopolysaccharide (LPS) increased and short-chain fatty acid (SCFA) levels decreased following long term LDR exposure. Treatment with microbiota-derived LPS and SCFAs reversed these effects in mice. Furthermore, the gut barrier integrity was damaged in a time-dependent manner with the decreased expression of intestinal epithelial-related biomarkers such as ZO-1 and occludin. Mechanistically, long after exposure to LDR, increased LPS levels may cause cognitive impairment through the regulation of Akt/mTOR signaling in the mouse hippocampus. These findings provide new insight into the clinical applications of LDR and suggest that the gut microbiota–plasma LPS and SCFAs–brain axis may underlie long-term LDR-induced cognition effects.
As pectin is widely used as a food and feed additive due to its tremendous prebiotic potentials for gut health. Yet, the underlying mechanisms associated with its protective effect remain unclear. Twenty-four piglets (Yorkshire × Landrace, 6.77 ± 0.92 kg) were randomly divided into three groups with eight replicates per treatment: (1) Control group (CON), (2) Lipopolysaccharide-challenged group (LPS), (3) Pectin-LPS group (PECL). Piglets were administrated with LPS or saline on d14 and 21 of the experiment. Piglets in each group were fed with corn-soybean meal diets containing 5% citrus pectin or 5% microcrystalline cellulose. Our result showed that pectin alleviated the morphological damage features by restoring the goblet numbers which the pig induced by LPS in the cecum. Besides, compared with the LPS group, pectin supplementation elevated the mRNA expression of tight junction protein [Claudin-1, Claudin-4, and zonula occludens-1 (ZO-1)], mucin (Muc-2), and anti-inflammatory cytokines [interleukin 10 (IL-10), and IL-22]. Whereas pectin downregulated the expression of proinflammatory cytokines (IL-1β, IL-6, IL-18), tumor necrosis factor-&alpha (TNF-α), and NF-κB. What is more, pectin supplementation also significantly increased the abundance of beneficial bacteria (Lactobacillus, Clostridium_sensu_stricto_1, Blautia, and Subdoligranulum), and significantly reduced the abundance of harmful bacteria, such as Streptococcus. Additionally, pectin restored the amount of short-chain fatty acids (SCFAs) after being decreased by LPS (mainly Acetic acid, Propionic acid, and Butyric acid) to alleviate gut injury and improve gut immunity via activating relative receptors (GPR43, GPR109, AhR). Mantel test and correlation analysis also revealed associations between intestinal microbiota and intestinal morphology, and intestinal inflammation in piglets. Taken together, dietary pectin supplementation enhances the gut barrier and improves immunity to ameliorate LPS-induced injury by optimizing gut microbiota and their metabolites.
Cells have evolved mechanisms that allow them to respond to DNA damage to preserve genomic integrity and maintain tissue homeostasis. These responses include the activation of the cell cycle checkpoints and the repair mechanisms or the induction of apoptosis that eventually will eliminate damaged cells. These “life” vs. “death” decisions differ depending on the cell type, stages of development, and the proliferation status of the cell. The apoptotic response after DNA damage is of special interest as defects in its induction could contribute to tumorigenesis or the resistance of cancer cells to therapeutic agents such as radiotherapy. Multiples studies have elucidated the molecular mechanisms that mediate the activation of the DNA damage response pathway (DDR) and specifically the role of p53. However, much less is known about how the different cellular responses such as cell proliferation control and apoptosis are coordinated to maintain tissue homeostasis. Another interesting question is how the differential apoptotic response to DNA damage is regulated in distinct cell types. The use of Drosophila melanogaster as a model organism has been fundamental to understand the molecular and cellular mechanisms triggered by genotoxic stress. Here, we review the current knowledge regarding the cellular responses to ionizing radiation as the cause of DNA damage with special attention to apoptosis in Drosophila: how these responses are regulated and coordinated in different cellular contexts and in different tissues. The existence of intrinsic mechanisms that might attenuate the apoptotic pathway in response to this sort of DNA damage may well be informative for the differences in the clinical responsiveness of tumor cells after radiation therapy.
Elevated serum urate (hyperuricemia) promotes crystalline monosodium urate tissue deposits and gout, with associated inflammation and increased mortality. To identify modifiers of uric acid pathologies, we performed a fly Genome-Wide Association Study (GWAS) on purine metabolites using the Drosophila Genetic Reference Panel strains. We tested the candidate genes using the Drosophila melanogaster model of hyperuricemia and uric acid crystallization ("concretion formation") in the kidney-like Malpighian tubule. Medusa (mda) activity increased urate levels and inflammatory response programming. Conversely, whole-body mda knockdown decreased purine synthesis precursor phosphoribosyl pyrophosphate, uric acid, and guanosine levels; limited formation of aggregated uric acid concretions; and was sufficient to rescue lifespan reduction in the fly hyperuricemia and gout model. Levels of mda homolog FAM214A were elevated in inflammatory M1- and reduced in anti-inflammatory M2-differentiated mouse bone marrow macrophages, and influenced intracellular uric acid levels in human HepG2 transformed hepatocytes. In conclusion, mda/FAM214A acts in a conserved manner to regulate purine metabolism, promotes disease driven by hyperuricemia and associated tissue inflammation, and provides a potential novel target for uric acid-driven pathologies.
