Increase In DNA Content Research Articles

Somatic polyploidy supports biosynthesis and tissue function by increasing transcriptional output.

Cell size and biosynthetic capacity generally increase with increased DNA content. Somatic polyploidy has therefore been proposed to be an adaptive strategy to increase cell size in specialized tissues with high biosynthetic demands. However, if and how DNA concentration limits cellular biosynthesis in vivo is not well understood. Here, we show that polyploidy in the Caenorhabditis elegans intestine is critical for cell growth and yolk biosynthesis, a central role of this organ. Artificially lowering the DNA/cytoplasm ratio by reducing polyploidization in the intestine gave rise to smaller cells with dilute mRNA. Highly expressed transcripts were more sensitive to this mRNA dilution, whereas lowly expressed genes were partially compensated-in part by loading more RNA Polymerase II on the remaining genomes. Polyploidy-deficient animals produced fewer and slower-growing offspring, consistent with reduced synthesis of highly expressed yolk proteins. DNA-dilute cells had normal total protein concentration, which we propose is achieved by increasing the expression of translational machinery at the expense of specialized, cell-type-specific proteins.

TNK2 Inhibitor (R)-9bMS Causes Polyploidization Through Mitotic Failure by Targeting Aurora B.

TNK2 is a ubiquitously expressed nonreceptor-type tyrosine kinase. TNK2 participates in tumorigenesis, and TNK2 activation has been found in various cancers; therefore, TNK2 is a promising target for cancer chemotherapy. While the TNK2 inhibitor XMD16-5 is highly selective, it inhibits cytokinesis at higher concentrations by targeting Aurora B kinase, a key enzyme for cell division. Cytokinesis failure frequently generates polyploid cells, and the surviving polyploid cells risk leading to cancer development and malignant progression via chromosome instability. In this study, to investigate the possibility that (R)-9bMS, a TNK2 inhibitor structurally related to XMD16-5, drives malignant progression by inducing abnormal cell division, we examined its effects on cell division, Aurora B autophosphorylation, and colony formation. Cell count results showed a reduction in the number of A431, HeLa S3, HCT116, and MCF7 cells upon TNK2 inhibitor treatment. Microscopic observation indicated the formation of multinucleated and nucleus-enlarged cells. An increase in DNA content was confirmed with flow cytometry, which was underpinned by an increased number of centrosomes. Time-lapse imaging revealed mitotic failure, such as mitotic slippage and cytokinesis failure, as a cause of polyploidization. Of note, TNK2 knockdown significantly increased multinucleated cells, but the effect was quite weak, suggesting that TNK2 inhibition may only partially contribute to mitotic failure and polyploidization. Expectedly, Aurora B phosphorylation was reduced by (R)-9bMS like XMD16-5, but not by TNK2 knockdown. Collectively, TNK2 inhibitors (R)-9bMS and XMD16-5 induce polyploidization via mitotic failure caused by the inhibition of Aurora B kinase rather than TNK2. Notably, (R)-9bMS treatment promoted anchorage-independent colony formation, a hallmark of cancer. Our findings suggest that (R)-9bMS at a high concentration risks promoting cancer development or malignant progression. Therefore, caution should be used when using TNK2 inhibitors for cancers where TNK2 activation is not the transforming mutation and higher concentrations of TNK2 inhibitors are required to slow proliferation.

Postsynaptic BMP signaling regulates myonuclear properties in Drosophila larval muscles.

The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (size scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show that local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity.

High-Throughput Empirical and Virtual Screening to Discover Novel Inhibitors of Polyploid Giant Cancer Cells in Breast Cancer.

Therapy resistance in breast cancer is increasingly attributed to polyploid giant cancer cells (PGCCs), which arise through whole-genome doubling and exhibit heightened resilience to standard treatments. Characterized by enlarged nuclei and increased DNA content, these cells tend to be dormant under therapeutic stress, driving disease relapse. Despite their critical role in resistance, strategies to effectively target PGCCs are limited, largely due to the lack of high-throughput methods for assessing their viability. Traditional assays lack the sensitivity needed to detect PGCC-specific elimination, prompting the development of novel approaches. To address this challenge, we developed a high-throughput single-cell morphological analysis workflow designed to differentiate compounds that selectively inhibit non-PGCCs, PGCCs, or both. Using this method, we screened a library of 2,726 FDA Phase 1-approved drugs, identifying promising anti-PGCC candidates, including proteasome inhibitors, FOXM1, CHK, and macrocyclic lactones. Notably, RNA-Seq analysis of cells treated with the macrocyclic lactone Pyronaridine revealed AXL inhibition as a potential strategy for targeting PGCCs. Although our single-cell morphological analysis pipeline is powerful, empirically testing all existing compounds is impractical and inefficient. To overcome this limitation, we trained a machine learning model to predict anti-PGCC efficacy in silico, integrating chemical fingerprints and compound descriptions from prior publications and databases. The model demonstrated a high correlation with experimental outcomes and predicted efficacious compounds in an expanded library of over 6,000 drugs. Among the top-ranked predictions, we experimentally validated two compounds as potent PGCC inhibitors. These findings underscore the synergistic potential of integrating high-throughput empirical screening with machine learning-based virtual screening to accelerate the discovery of novel therapies, particularly for targeting therapy-resistant PGCCs in breast cancer.

Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry

Abstract Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions. Yet, we lack practical adaptations to fully harness the quantitative and high-throughput capabilities of most cytometers for many organisms. Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. In proof-of-principle experiments, we show that DNA content increase in S-phase correlates with de novo DNA synthesis and can be predicted by the presence of the replicative helicase MCM2-7 on genomic DNA.

#2592 Tubular cell polyploidy is involved in kidney compensatory hypertrophy

Abstract Background and Aims Chronic kidney disease (CKD) affects more than 10% of the population and it is associated with high risk of death for cardiovascular causes or progression towards end stage kidney disease (ESKD) requiring dialysis or kidney transplantation. CKD is characterized by progressive loss of nephrons, with consequent structural degeneration and fibrosis. Nephron number is established during prenatal kidney development and cannot increase. Consequently, following a reduction in kidney mass or in condition of altered metabolic demands, the remaining nephrons undergo compensatory hypertrophy. Compensatory hypertrophy results in increased functional output and has been observed in the absence of direct tissue injury after nephrectomy. Recently, we have identified a previously unknown mechanism of cellular adaptation: polyploidization of tubular epithelial cells (TC), and demonstrated that this is the essential mediator of kidney hypertrophy after acute kidney injury (AKI). Here, we hypothesize that TC polyploidy is involved in compensatory hypertrophy following nephrectomy. Method Polyploid TC are characterized by an increased DNA content in the absence of cell division. To discriminate polyploid cells from actively proliferating cells, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI2aR) technology. Nephrectomy was performed by removing the left kidney and mice were sacrificed at different days after uni- Nephrectomy (Nfx). Cytofluorimetric techniques were employed to characterize a subpopulation of polyploid TC and histology investigation was employed analyze fibrosis development. These results were further corroborated by single cell RNA-sequencing (scRNA-seq) analyses in vitro and in vivo. Results Employing mice that express the FUCCI2aR reported under the Pax8 promoter (which marks all TC), we found that TC polyploidy increases immediately after uni-Nfx. This was accompanied by an increased kidney to body mass ratio with a maximum peaks 3 days afterwards. Importantly, after uni-Nfx, YAP1 expression and nuclei localization (i.e., implying pathway activation) was exponentially increased, suggesting that the same mechanism that drives TC polyploidy after AKI controls TC polyploidization response after uni-Nfx. However, unlike in AKI, polyploidy after nephrectomy was not accompanied by structural alterations as shown by PAS and Masson's trichrome staining. Conversely, after uni-Nfx YAP1 knock-out mice, that are unable to undergo rapid polyploidization, displayed an impaired kidney function in the long term, characterized by extensive tubular dilations. Conclusion Polyploidization of TC mediates compensatory hypertrophy following nephrectomy via YAP1 activation

In Vitro Evaluation of Optimized PEEK Surfaces for Enhanced Osseointegration

The materials traditionally used for implant applications, such as titanium alloys, cobalt chromium, and zirconium, often require surface modifications to achieve the desired osseointegration. These materials still have the problematic stress-shielding effect. To limit stress shielding, PEEK is the superior alternative to fulfill implant needs. However, the traditional methods of modifying and functionalizing the surface of PEEK are often expensive, time consuming, and are not easily translated into commercialization. Sulfonation is a process, which is dependent on controllable factors. Thus far, no research has been performed to optimize the sulfonation process. Our data suggest that the process factors can be controlled and optimized. Cellular activity was examined on the optimized PEEK surfaces through testing with pre-osteoblast MC3T3-E1 cells through cell viability (MTT assay), cell proliferation (DNA assay), cell differentiation (ALP assay), and cell mineralization (Alizarin red assay). Overall, sulfonated and heat-treated PEEK exhibited a statistically significant increase in DNA content over the course of 21 days, indicating more cell proliferation and viability for that surface. In vitro testing results showed that the optimized sulfonated and heat-treated PEEK exhibited superior cell proliferation and mineralization performance over smooth PEEK and sulfonated-only PEEK.

Abstract 1991: Leiomyosarcoma poly-aneuploid cancer cells form in response to chemotherapy, contribute to chemoresistance, and lead to higher rates of metastatic recurrence

Abstract Poly-aneuploid cancer cells (PACCs) or as also known as “giant cells”, are a treatment resistant stem cell-like cancer phenotype. PACCs have been identified in numerous cancer types, and their presence appears to play a significant role in chemoresistance and poor patient outcomes. PACCs employ a multitude of polyploidization programs to enter this transient state in response to stress. After removal of stress, PACCs give rise to non-PACC progeny through potential mechanisms of neosis or depolyploidization that maintain chemotherapeutic resistance. Leiomyosarcoma (LMS) is a difficult to treat sarcoma that grows in smooth muscle that often develops chemoresistance. Here we report the first description of PACCs in LMS cell culture, LMS xenografts, and LMS patient tumor microarrays. LMS cells treated (doxorubicin, docetaxel, and gemcitabine) in vitro and in vivo lead to the development of mononucleated and multinucleated polypoid cells as the predominant phenotype via polyploidization. Polyploidization mechanisms identified included endocycling, endomitosis, and cell-cell fusion. In vitro, LMS PACCs had a significant increase in size and DNA content as well as an increase in resistance to chemotherapy as compared to parents. LMS PACCs also repopulated the culture with non-PACC LMS progeny cells. The PACC progeny cells maintained a significantly decreased (p<0.0001) response to chemotherapy compared to parent cells. PACCs were also identified in clinical samples from patients with LMS. Twenty-nine percent of samples (N=289) contained abnormally large tumor cells and fifty-eight percent contained abnormal nuclei. A positive correlation was observed in patient tumor samples between PACCs in metastatic tumors and recurrence (p=0.0277) and survival (p=0.0009) than those patient tumors without PACCs. The high rate of functional loss of TP53 in LMS (>90%) may contribute to the ability of PACCs to form abnormal, multiple nuclei. We therefore transfected LMS PACCs to express TP53 and observed an increased apoptotic response. Additionally, combining functional TP53 expression with low doses of doxorubicin increases the apoptotic response of LMS cells, potentially by targeting PACCs. LMS PACCs may contribute to the lack of response in patients with LMS. Identifying therapeutic approaches that prevent or target LMS PACCs, including TP53 replacement, may improve response to chemotherapy and improve outcomes for patients. Citation Format: Ryan Kennington, Gareth Mitchell, Emily Payne, Niraja Bhachech, Jonathan A. Fletcher, Ting Liu, Matt van de Rijn, Sarah Amend, Kenneth J. Pienta, Joshua D. Schiffman, Lisa M. Abegglen. Leiomyosarcoma poly-aneuploid cancer cells form in response to chemotherapy, contribute to chemoresistance, and lead to higher rates of metastatic recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1991.

Tissue-specific compensatory mechanisms maintain tissue architecture and body size independent of cell size in polyploid zebrafish

Genome duplications and ploidy transitions have occurred in nearly every major taxon of eukaryotes, but they are far more common in plants than in animals. Due to the conservation of the nuclear:cytoplasmic volume ratio increased DNA content results in larger cells. In plants, polyploid organisms are larger than diploids as cell number remains relatively constant. Conversely, vertebrate body size does not correlate with cell size and ploidy as vertebrates compensate for increased cell size to maintain tissue architecture and body size. This has historically been explained by a simple reduction in cell number that matches the increase in cell size maintaining body size as ploidy increases, but here we show that the compensatory mechanisms that maintain body size in triploid zebrafish are tissue-specific: A) erythrocytes respond in the classical pattern with a reduced number of larger erythrocytes in circulation, B) muscle, a tissue comprised of polynucleated muscle fibers, compensates by reducing the number of larger nuclei such that myofiber and myotome size in unaffected by ploidy, and C) vascular tissue compensates by thickening blood vessel walls, possibly at the expense of luminal diameter. Understanding the physiological implications of ploidy on tissue function requires a detailed description of the specific mechanisms of morphological compensation occurring in each tissue to understand how ploidy changes affect development and physiology.

On the distribution and diversity of tissue-specific somatic mutations in honey bee (Apis mellifera) drones

Somatic mutations originate from both exogenous (e.g. UV radiation, chemical agents) and endogenous (e.g., DNA replication, defective DNA repair) sources and can have significant impacts on an animal’s reproductive success. This may be especially true for haploid organisms that are susceptible to any deleterious alleles inherited from their parent and any that arise over their lifetime. Unfortunately, little is known about the rate of somatic mutation accumulation across individuals and tissues of haplodiploid animal populations, the functional processes through which they arise, and their distribution across tissues and the genome. Here, we generated short-read whole-genome sequencing data for four tissues of haploid honey bee males. We paired this with estimates of telomere length and tissue-specific DNA content to address three major questions: is there variance in somatic mutational load across haploid individuals and specific tissues therein, does increased DNA content in a tissue contribute to somatic mutational load, and does telomere length correlate with mutational load? Our results suggest that variance in somatic mutational load is better captured across individuals than across tissues, that tissue-specific DNA content is not associated with somatic mutation load, and that variance in telomere length does not correlate with somatic mutation loads across tissues. To our knowledge, this is the first observational study on somatic mutational load in Apoidea and likely Hymenoptera. It serves as a useful advent for additional studies understanding the processes through which haploids tolerate or repair somatic mutations.

Decellularisation and Characterisation of Porcine Pleura as Bioscaffolds in Tissue Engineering

Persistent air leaks caused by thoracic surgery, physical trauma, or spontaneous pneumothoraces are a cause of patient morbidity with need for extended chest tube durations and surgical interventions. Current treatment measures involve mechanical closure of air leaks in the compromised pleura. Organ and membrane decellularisation offers a broad range of biomimetic scaffolds of allogeneic and xenogeneic origins, exhibiting innate tissue‐specific characteristics. We explored a physicochemical method for decellularising porcine pleural membranes (PPM) as potential tissue‐engineered surrogates for lung tissue repair. Decellularised PPM (dPPM) was characterised with histology, quantitative assays, mechanical testing, and sterility evaluation. Cytotoxicity and recellularisation assays assessed biocompatibility of decellularised PPM (dPPM). Haematoxylin and Eosin (H&E) staining showed an evident reduction in stained nuclei in the dPPM, confirmed with nuclear staining and analysis ( ∗∗∗∗p < 0.0001). Sulphated glycosaminoglycans (sGAG) and collagen histology demonstrated minimal disruption to the gross structural assembly of core extracellular matrix (ECM) in dPPM. Confocal imaging demonstrated realignment of ECM fibres in dPPM against native control. Quantitative analysis defined a significant change in the angular distribution ( ∗∗∗∗p < 0.0001) and coherence ( ∗∗∗p < 0.001) of fibre orientations in dPPM versus native ECM. DNA quantification indicated ≥85% reduction in native nuclear dsDNA in dPPM ( ∗∗p < 0.01). Collagen and sGAG quantification indicated reductions of both ( ∗∗p < 0.01). dPPM displayed increased membrane thickness ( ∗∗∗p < 0.001). However, Young’s modulus (459.67 ± 10.36 kPa) and ultimate tensile strength (4036.22 ± 155.1 kPa) of dPPM were comparable with those of native controls at (465.82 ± 10.51 kPa) and (3912.9 ± 247.42 kPa), respectively. In vitro cytotoxicity and scaffold biocompatibility assays demonstrated robust human mesothelial cell line (MeT‐5A) attachment and viability. DNA quantification in reseeded dPPM with MeT‐5A cells exhibited significant increase in DNA content at day 7 ( ∗∗p < 0.01) and day 15 ( ∗∗∗∗p < 0.0001) against unseeded dPPM. Here, we define a decellularisation protocol for porcine pleura that represents a step forward in their potential tissue engineering applications as bioscaffolds.

Stress-granules, P-bodies, and cell aging: A bioinformatics study

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their physical state (or material properties). In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the physical state of these MLOs. Particular attention was paid to the proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs consists of intrinsically disordered proteins, 30–40% of which are potentially capable of liquid-liquid phase separation (LLPS). Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the SG dataset with senescence-related processes. Among SG proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. Proteins common to SGs and P-bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the physical state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.

Caspase-4 has a role in cell division in epithelial cells through actin depolymerization

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

Stomata variation in the process of polyploidization in Chinese chive (Allium tuberosum).

Stomatal variation, including guard cell (GC) density, size and chloroplast number, is often used to differentiate polyploids from diploids. However, few works have focused on stomatal variation with respect to polyploidization, especially for consecutively different ploidy levels within a plant species. For example, Allium tuberosum, which is mainly a tetraploid (2n = 4x = 32), is also found at other ploidy levels which have not been widely studied yet. We recently found cultivars with different ploidy levels, including those that are diploid (2n = 2x = 16), triploid (2n = 3x = 24), pseudopentaploid (2n = 34-42, mostly 40) and pseudohexaploid (2n = 44-50, mostly 48). GCs were evaluated for their density, size (length and width) and chloroplast number. There was no correspondence between ploidy level and stomatal density, in which anisopolyploids (approximately 57 and 53 stomata/mm2 in triploid and pseudopentaploid, respectively) had a higher stomatal density than isopolyploids (approximately 36, 43, and 44 stomata/mm2 in diploid, tetraploid and pseudohexaploid, respectively). There was a positive relationship between ploidy level and GC chloroplast number (approximately 44, 45, 51, 72 and 90 in diploid to pseudohexaploid, respectively). GC length and width also increased with ploidy level. However, the length increased approximately 1.22 times faster than the width during polyploidization. This study shows that GC size increased with increasing DNA content, but the rate of increase differed between length and width. In the process of polyploidization, plants evolved longer and narrower stomata with more chloroplasts in the GCs.

Seasonal dynamics of soil microbial growth, respiration, biomass, and carbon use efficiency in temperate soils

Soil microbial growth, respiration, and carbon (C) use efficiency (CUE) are essential parameters to understand, describe and model the soil carbon cycle. While seasonal dynamics of microbial respiration are well studied, little is known about how microbial growth and CUE change over the course of a year, especially outside the plant growing season. In this study, we measured soil microbial respiration, gross growth via 18O incorporation into DNA, and biomass in an agricultural field and a deciduous forest 16 times over the course of two years. We sampled soils to a depth of 5 cm from plots at which harvest residues or leaf litter remained on the plot or was removed. We observed strong seasonal variations of microbial respiration, growth, and biomass. All these microbial parameters were significantly higher at the forest site, which contained 4.3 % organic C compared to the agricultural site with 0.9 % organic C. CUE also varied strongly (0.1 to 0.7) but was overall significantly higher at the agricultural site compared to the forest site. We found that microbial respiration and to a lesser extent microbial growth followed the seasonal dynamics of soil temperature. Microbial growth was further affected by the presence of plants in the agricultural system or foliage in the forest. At low temperatures in winter, both microbial respiration and gross growth showed the lowest rates, whereas CUE (calculated from both respiration and growth) showed amongst the highest values determined during the two years, due to the higher temperature sensitivity of microbial respiration. Microbial biomass C strongly increased in winter. Surprisingly, this winter peak was not connected to high microbial growth or an increase in DNA content. This suggests that microorganisms accumulated C and N, potentially in the form of osmo- or cryoprotectants or increased in cell size but did not divide. This microbial winter bloom and following decline, where C is released from microbial biomass and freely available, might constitute a highly dynamic time in the annual C cycle in temperate soil systems. Highly variable CUE, which was observed in our study, and the fact that CUE is calculated from independently controlled microbial respiration and microbial growth, ask for great caution when CUE is used to describe soil microbial physiology, soil C dynamics or C sequestration. Instead, microbial respiration, microbial growth, and microbial biomass C should be investigated individually in combination to better understand the soil C cycle.

Cut homeodomain transcription factor is a novel regulator of growth and morphogenesis of cortex glia niche around neural cells.

Cortex glia in Drosophila central nervous system form a niche around neural cells for necessary signals to establish cross talk with their surroundings. These cells grow and expand their thin processes around neural cell bodies. Although essential for the development and function of the nervous system, how these cells make extensive and intricate connected networks remains largely unknown. In this study, we show that Cut, a homeodomain transcription factor, directly regulates the fate of the cortex glia, impacting neural stem cell (NSC) homeostasis. Focusing on the thoracic ventral nerve cord, we found that Cut is required for the normal growth and development of cortex glia and timely increase in DNA content through endocycle to later divide via acytokinetic mitosis. Knockdown of Cut in cortex glia significantly reduces the growth of cellular processes, the network around NSCs, and their progeny's cell bodies. Conversely, overexpression of Cut induces overall growth of the main processes at the expense of side ones. Whereas the Cut knockdown slows down the timely increase of DNA, the Cut overexpression results in a significant increase in nuclear size and volume and a 3-fold increase in DNA content of cortex glia. Further, we note that constitutively high Cut also interfered with nuclei separation during acytokinetic mitosis. Since the cortex glia form syncytial networks around neural cells, the finding identifies Cut as a novel regulator of glial growth and variant cell cycles to support a functional nervous system.

Stress induced by soil contamination with heavy metals and their effects on some biomarkers and DNA damage in maize plants at the vicinity of Ferronikel smelter in Drenas, Kosovo

The Ferronikel smelter in Drenas is one of the main industrial areas in the Kosovo and pollution by heavy metals causes serious threat for all living organisms on this area. The objective of this study was to determine the concentration of some heavy metals (Fe, Cu, Mn, Cr, Cd, Ni and Pb) in agricultural soils and in maize plants, and their potential toxic effects on this plant through some sensitive biochemical and molecular markers. Maize seedlings growth in nine soil samples from different locations of this area. The highest concentrations of heavy metals in soils and maize leaves were conducted close to the Ferronikel smelter, and in some locations, the nickel and chromium concertation in soils exceeded 800 mg kg−1. A significant effects of heavy metals induced toxicity resulted in the, build-up aminolevulinic acid and reduced activity of δ-aminolevulinic acid dehydratase, and chlorophyll content in the maize leaves. In general, maize seedlings growth in polluted locations showed an increase in nuclear DNA content and in G2M phase. We concluded that locations close to the smelter are affected by soil heavy metals pollution and these biochemical and molecular analysis would be a powerful ecotoxicological tool in biomonitoring of heavy metal pollution.

Establishment of evaluation system for biological remediation on organic pollution in groundwater using slow-release agents

In situ bioremediation through slow-release agents can continuously degrade organic pollutants for a long time and have high application potential in solving problems such as tailing and rebound. However, the existing evaluation system is difficult to reflect the performance of bioremediation through slow-release agents, which is not conducive to the promotion of technology. It is urgent to establish a targeted evaluation system. Therefore, based on the multi-criteria decision-making method (MCDA), a comprehensive evaluation model was established. The evaluation index system was constructed for bioremediation through slow-release agents consisting of 16 indicators including pollutant degradation rate, agent preparation cost, engineering operation and maintenance cost, secondary pollution, long-term degradation stability, slow release time, slow release stability, increase in functional microbial flora, increase in total DNA content, agent particle size, solid agent morphology, liquid agent viscosity, dispersibility in aqueous phase, zeta potential, operability of agent preparation, and engineering operation management difficulty. Then, the weight of the indicators was determined by using the best-worst method (BWM), and evaluation criteria was established based on relevant norms and literature. Both and the indicators aggregation simple additive weighting (SAW) method constitute a quantitative evaluation model. The above content together constitutes a new evaluation system for biological remediation on organic pollution in groundwater using slow-release agents, which was defined as AOBS evaluation system. In order to verify the rationality and scientificity of the evaluation system, a typical bioremediation slow-release agent was evaluated using the established AOBS evaluation system. The results showed that the evaluation system could reasonably and comprehensively evaluate bioremediation through slow-release agents and provide suggestions for agent improvement.

Cytotoxicity and genotoxicity of tributyltin in the early embryonic chick, Gallus gallus domesticus.

Tributyltin (TBT) is used in many commercial applications, including pesticides and antifouling paints, due to its biocidal properties. We examined the cytotoxicity and genotoxicity of TBT in the early chick embryo (Gallus gallus domesticus). Chick embryos (11 days) were treated with various doses of TBT to measure LD50 values for 24, 48, and 72h exposures, which were determined to be 110, 54, and 18μg/egg, respectively. The embryos were exposed to sub-lethal doses of TBT for evaluation of cytotoxicity and genotoxicity. An increase in the incidence of micronuclei (MN) was observed but it was not statistically significant. Induction of other nuclear abnormalities (ONA) after 72h TBT exposure was significant. A significant increase in comet assay tail DNA content was also detected in TBT-exposed embryos. Cytotoxicity was also evidenced by alteration in the polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratio and by an increase in the erythroblast population in treated organisms. The cytotoxicity and genotoxicity of TBT may have long-term complications in later stages of the life cycle.

#5741 POLYPLOID TUBULAR CELLS INITIATE A TGF-BETA CONTROLLED LOOP THAT SUSTAINS POLYPLOIDIZATION AND TUBULOINTERSTITIAL CROSSTALK DURING AKI-CKD TRANSITION

Abstract Background and Aims Acute Kidney Injury (AKI) is characterized by a sudden kidney failure accompanied by a transient decrease of kidney functionality. It is regarded as an important risk factor for chronic kidney disease (CKD) development, but the link is still elusive. Renal fibrosis, especially tubulointerstitial fibrosis, is the final manifestation of CKD and is characterized by an excessive synthesis and deposition of extracellular matrix associated with inflammatory infiltration, tubular epithelial (TC) cell damage and fibroblast activation. Although no targeted therapy yet exists to slow the progression of tubulointerstitial fibrosis, recent findings contributed to clarify the cellular and molecular mechanisms underlying its development and progression, posing TC at the center of this process. Accordingly, we have recently demonstrated that fibrosis and senescence are trade-offs of TC polyploidy occurring immediately after AKI to support fast kidney function recovery, but promoting consequent CKD. However, the mechanisms turning TC polyploidy to senescence and fibrosis still need to be elucidated. In this study, we propose that TC polyploidy is the primary driver of CKD progression after AKI. Methods Polyploid TC are characterized by an increased DNA content in the absence of cell division. To discriminate polyploid cells from actively proliferating cells, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology. AKI was triggered by unilateral ischemia reperfusion injury (IRI) or glycerol-induced rhabdomyolysis. This technology allows to follow the cell cycle phasing of living cells. Cell sorting and cytofluorimetric techniques were employed to isolate and characterize a subpopulation of polyploid TC that progressively accumulate DNA damage after AKI. These results were further corroborated by single cell RNA-sequencing (scRNA-seq) analyses in vitro and in vivo. Results In this study, we found that immediately after AKI, expression of cell cycle markers mostly identifies a population of DNA damaged polyploid TC. Employing transgenic mouse models and single cell RNA-sequencing we showed that after AKI, polyploid TC accumulate DNA damage and survive eventually resting in the G1 phase of cell cycle, while diploid cells do not survive DNA damage. This suggests that after AKI, polyploidization is a means to survive injury. Sorting of DNA-damaged polyploid TC showed that they express p21 and acquire a pro-fibrotic phenotype culminating in TGF-beta expression. in vitro analysis and single cell RNA-sequencing evidenced that TGF-beta directly promoted polyploidization, demonstrating that TC polyploidization is a self-sustained mechanism. Interactome analysis by single cell RNA-sequencing revealed that TGF-beta signaling fostered a reciprocal activation loop among polyploid TC, macrophages and fibroblasts to sustain kidney fibrosis and promote CKD progression. Conclusion Collectively, this study contributes to the ongoing revision of the paradigm of kidney tubule response to AKI supporting the existence of a tubulointerstitial crosstalk mediated by TGF-beta signaling produced by polyploid TC following DNA damage. Moreover, this study identifies polyploid TC as central actors during CKD progression with important implications for the development of novel targeted therapy to block CKD.