Mouse Rad1 deletion enhances susceptibility for skin tumor development

Cells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown. To address this question, we constructed mice with a deletion of Mrad1. Matings between heterozygous Mrad1 mutant mice produced Mrad1+/+ and Mrad1+/- but no Mrad1-/- progeny, suggesting the Mrad1 null is embryonic lethal. Mrad1+/- mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals. Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells. These data suggest that Mrad1 is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells

A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage

The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity

Effects of Simulated Microgravity on Embryonic Stem Cells

There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG

Environmental filtering controls soil biodiversity in wet tropical ecosystems

9 páginas..- 4 figuras.- referencias.- Supplementary data to this article can be found online at https://doi. org/10.1016/j.soilbio.2022.108571The environmental factors controlling soil biodiversity along resource gradients remain poorly understood in wet tropical ecosystems. Aboveground biodiversity is expected to be driven by changes in nutrient availability in these ecosystems, however, much less is known about the importance of nutrient availability in driving soil biodiversity. Here, we combined a cross-continental soil survey across tropical regions with a three decades' field experiment adding nitrogen (N) and phosphorus (P) (100 kg N ha(-1)y(-1) and 100 kg P ha(-1)y(-1)) to Hawai'ian tropical forests with contrasting substrate ages (300 and 4,100,000 years) to investigate the influence of nutrient availability to explain the biodiversity of soil bacteria, fungi, protists, invertebrates and key functional genes. We found that soil biodiversity was driven by soil acidification during long-term pedogenesis and across environmental gradients, rather than by nutrient limitations. In fact, our results showed that experimental N additions caused substantial acidification in soils from Hawai'i. These declines in pH were related to large decreases in soil biodiversity from tropical ecosystems in four continents. Moreover, the microbial activity did not change in response to long-term N and P additions. We concluded that environmental filtering drives the biodiversity of multiple soil organisms, and that the acidification effects associated with N additions can further create substantial undesired net negative effects on overall soil biodiversity in naturally tropical acid soils. This knowledge is integral for the understanding and management of soil biodiversity in tropical ecosystems globally.Supported by a Ramón y Cajal grant (RYC2018-025483-I), a “Ayuda P.P. 2020. Desarrollo Lineas Investigación Propias (UPO), a project from the Spanish Ministry of Science and Innovation (PID2020-115813RA-I00), and a project PAIDI 2020 from the Junta de Andalucía (P20_00879). H.Y.C. is supported by National Natural Science Foundation of China (32101335), China Postdoctoral Science Foundation (2021M690589), Innovation Project of Young Technological Talents in Changchun City (21QC07), and Fundamental Research Funds for the Central Universities (2412021QD014). J.P.V. is thankful to DST and SERB (Science and Engineering Research Board), India for financial support for plant-microbe interaction research. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.Peer reviewe

The global contribution of soil mosses to ecosystem services

Soil mosses are among the most widely distributed organisms on land. Experiments and observations suggest that they contribute to terrestrial soil biodiversity and function, yet their ecological contribution to soil has never been assessed globally under natural conditions. Here we conducted the most comprehensive global standardized field study to quantify how soil mosses influence 8 ecosystem services associated with 24 soil biodiversity and functional attributes across wide environmental gradients from all continents. We found that soil mosses are associated with greater carbon sequestration, pool sizes for key nutrients and organic matter decomposition rates but a lower proportion of soil-borne plant pathogens than unvegetated soils. Mosses are especially important for supporting multiple ecosystem services where vascular-plant cover is low. Globally, soil mosses potentially support 6.43 Gt more carbon in the soil layer than do bare soils. The amount of soil carbon associated with mosses is up to six times the annual global carbon emissions from any altered land use globally. The largest positive contribution of mosses to soils occurs under a high cover of mat and turf mosses, in less-productive ecosystems and on sandy and salty soils. Our results highlight the contribution of mosses to soil life and functions and the need to conserve these important organisms to support healthy soils.The study work associated with this paper was funded by a Large Research Grant from the British Ecological Society (no. LRB17\1019; MUSGONET). D.J.E. is supported by the Hermon Slade Foundation. M.D.-B. was supported by a Ramón y Cajal grant from the Spanish Ministry of Science and Innovation (RYC2018-025483-I), a project from the Spanish Ministry of Science and Innovation for the I + D + i (PID2020-115813RA-I00 funded by MCIN/AEI/10.13039/501100011033a) and a project PAIDI 2020 from the Junta de Andalucía (P20_00879). E.G. is supported by the European Research Council grant agreement 647038 (BIODESERT). M.B. is supported by a Ramón y Cajal grant from Spanish Ministry of Science (RYC2021-031797-I). A.d.l.R is supported by the AEI project PID2019-105469RB-C22. L.W. and Jianyong Wang are supported by the Program for Introducing Talents to Universities (B16011) and the Ministry of Education Innovation Team Development Plan (2013-373). The contributions of T.G. and T.U.N. were supported by the Research Program in Forest Biology, Ecology and Technology (P4-0107) and the research projects J4-3098 and J4-4547 of the Slovenian Research Agency. The contribution of P.B.R. was supported by the NSF Biological Integration Institutes grant DBI-2021898. J. Durán and A. Rodríguez acknowledge support from the FCT (2020.03670.CEECIND and SFRH/BDP/108913/2015, respectively), as well as from the MCTES, FSE, UE and the CFE (UIDB/04004/2021) research unit financed by FCT/MCTES through national funds (PIDDAC)

Data from: Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions

Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects

Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects

Measurement of intracellular pH using flow cytometry with carboxy‐SNARF‐1

The new intracellular pH (pHi) dye carboxy‐seminaphthorhodafluor (SNARF‐1) was compared to the established dye 2,3‐dicyanohydroquinone (DCH) using flow cytometry. Both dyes give high‐resolution pHi measurements. SNARF‐1 remains trapped within cells much longer than DCH, so that pHi can be monitored during and after treatments with chemicals or hyperthermia. The toxicity of the dyes is similar, and both dyes can be used at concentrations that result in low toxicity to cells. Adequate staining of cells with SNARF‐1 is dependent on the cell concentration. The absolute pHi values indicated by SNARF‐1 are higher than values measured with DCH. However, the trends measured by both dyes are consistent, and both are useful for making pHi measurements. © 1993 Wiley‐Liss, Inc

Effects of SMG on NADPH oxidase2 (Nox2) and NADPH oxidase4 (Nox4) expression in <i>Rad9</i>^<i>+/+</i> and <i>Rad9</i>^<i>-/-</i> MES cells.

<p>(A) Quantitative real-time PCR analysis of Nox2 mRNA expression in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells exposed to 1G or SMG condition for 1 or 5 days. The expression levels of Nox2 were normalized to the endogenous control GAPDH expression. (B) Quantitative real-time PCR analysis of Nox4 mRNA expression in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells exposed to 1G or SMG condition for 1 or 5 days. The expression levels of Nox4 were normalized to the endogenous control GAPDH expression. (C) Western blot analysis of Nox2 protein expression in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells exposed to 1G or SMG condition for 1 or 5 days. GAPDH was used as an internal control. The representative results of three independent experiments were shown. (D) Quantitative comparison of Nox2 expression. Data were derived from three independent experiments. The expression levels of Nox2 protein were normalized to the endogenous control GAPDH protein expression. The data represent mean ± SD of three independent experiments. Student’s t test, *P<0.05.</p

Effects of SMG on antioxidant enzyme activity in MES cells.

<p><i>Rad9</i>^{<b><i>+/+</i></b>} MES cells and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells were cultured under 1G and SMG for 1 and 5 days, respectively. The activities of the antioxidant enzymes in MES cell lysates were determined. (A) Histograms of superoxide dismutase enzyme activity. (B) Histograms of catalase enzyme activity. (C) Histogram of Glutahione peroxidase. The data represent mean ± SD of three independent experiments.</p