Control Of Cell Number Research Articles

Protective Effect of Arzanol against H2O2-Induced Oxidative Stress Damage in Differentiated and Undifferentiated SH-SY5Y Cells.

Oxidative stress can damage neuronal cells, greatly contributing to neurodegenerative diseases (NDs). In this study, the protective activity of arzanol, a natural prenylated α-pyrone-phloroglucinol heterodimer, was evaluated against the H2O2-induced oxidative damage in trans-retinoic acid-differentiated (neuron-like) human SH-SY5Y cells, widely used as a neuronal cell model of neurological disorders. The pre-incubation (for 2 and 24 h) with arzanol (5, 10, and 25 μM) significantly preserved differentiated SH-SY5Y cells from cytotoxicity (MTT assay) and morphological changes induced by 0.25 and 0.5 mM H2O2. Arzanol reduced the generation of reactive oxygen species (ROS) induced by 2 h oxidation with H2O2 0.5 mM, established by 2',7'-dichlorodihydrofluorescein diacetate assay. The 2 h incubation of differentiated SH-SY5Y cells with H2O2 determined a significant increase in the number of apoptotic cells versus control cells, evaluated by propidium iodide fluorescence assay (red fluorescence) and NucView® 488 assay (green fluorescence). Arzanol pre-treatment (2 h) exerted a noteworthy significant protective effect against apoptosis. In addition, arzanol was tested, for comparison, in undifferentiated SH-SY5Y cells for cytotoxicity and its ability to protect against H2O2-induced oxidative stress. Furthermore, the PubChem database and freely accessible web tools SwissADME and pkCSM-pharmacokinetics were used to assess the physicochemical and pharmacokinetic properties of arzanol. Our results qualify arzanol as an antioxidant agent with potential neuroprotective effects against neuronal oxidative stress implicated in NDs.

In vitro and in vivo antimalarial activities of the ethanol extract of Erythrina sigmoidea stem bark used for the treatment of malaria in the Western Region of Cameroon

BackgroundMalaria is one of the leading causes of morbidity and/or mortality in tropical Africa. The spread and development of resistance to chemical antimalarial drugs and the relatively high cost of the latter are problems associated with malaria control and are reasons to promote the use of plants to meet healthcare needs to treat malaria. The aim of this study was to evaluate antiplasmodial activities of extracts of Erythrina sigmoidea (Mah quat), which is traditionally used for the treatment of malaria in the western region of Cameroon.Material and methodsThe ethanol extract of E. sigmoidea stem bark was obtained through the maceration process using 95% ethanol, while the aqueous extract was prepared by infusion. The in vitro antiplasmodial effect of extracts against P. falciparum chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains was determined using the Trager and Jensen method. On the other hand, the in vivo antimalarial activity of the extract was evaluated in mice infected with Plasmodium berghei strain NK65 using the Peters’ 4-day suppressive test and Ryley test (curative test). A total of 36 mice were used, subdivided into six groups of six mice each: one normal control, a negative control, a positive control, and three other groups for the tested product. Blood samples were collected on the 10th day of each test for hematological parameters.ResultsThe aqueous extract had an in vitro antiplasmodial activity against the chloroquine-sensitive strain with an IC50 of 29.51 ± 3.63 µg/mL and against the chloroquine-resistant strain with an IC50 of 35.23 ± 3.17 µg/mL. The highest in vitro antiplasmodial activity was observed with the ethanol extract against the chloroquine-sensitive strain with an IC50 of 6.44 ± 0.08 µg/mL and against the chloroquine-resistant strain with an IC50 of 7.53 ± 0.22 µg/mL. The ethanol extract demonstrated suppressive activity in vivo with reduction rates of 87.69%, 86.79%, and 81.08% at doses of 500 mg/kg, 250 mg/kg, and 125 mg/kg, respectively; and curative activity in vivo with reduction rates of 80%, 78.5%, and 77.5% at doses of 500 mg/kg, 250 mg/kg, and 125 mg/kg, respectively. The number of white blood cells in the negative control (44.55 ± 5.02 103/µL) was higher compared to the other groups. As for the red blood cells, we observed a massive destruction of the latter in the infected and untreated group (5.82 ± 1.50 106/µL) compared to the infected and ethanol extract-treated groups (8.74 ± 1.57 106/µL for 500 mg/kg, 7.54 ± 1.77 106/µL for 250 mg/kg, and 8.9 ± 1.50 106/µL for 125 mg/kg).ConclusionThis study provides scientific data on the use of E. sigmoidea by the local population for the treatment of malaria. It shows that E. sigmoidea has antiplasmodial activity, and we also see that there are differences between the parameters that we have in the treated groups and those of the untreated group. However, toxicity tests are necessary to assess its safety.

Angiogenesis and Apoptosis: Data Comparison of Similar Microenvironments in the Corpus Luteum and Tumors.

The corpus luteum is a temporary endocrine gland formed in the ovary after ovulation, and it plays a critical role in animal reproductive processes. Tumors rely on the development of an adequate blood supply to ensure the delivery of nutrients and oxygen and the removal of waste products. While angiogenesis occurs in various physiological and pathological contexts, the corpus luteum and tumors share similarities in terms of the signaling pathways that promote angiogenesis. In the corpus luteum and tumors, apoptosis plays a crucial role in controlling cell numbers and ensuring proper tissue development and function. Interestingly, there are similarities between the apoptotic-regulated signaling pathways involved in apoptosis in the corpus luteum and tumors. However, the regulation of apoptosis in both can differ due to their distinct physiological and pathological characteristics. Thus, we reviewed the biological events of the corpus luteum and tumors in similar microenvironments of angiogenesis and apoptosis.

The influence of Ras-associated binding protein 23 knockdown on the migration and invasion of esophageal squamous cell carcinoma cells and its mechanism

Objective: To investigate the role and the mechanism of Ras-associated binding protein23 (RAB23) in the migration and invasion of esophageal squamous cell carcinoma (ESCC) cells. Methods: RAB23 mRNA levels were measured in 16 pairs of ESCC and adjacent normal tissues via real-time polymerase chain reactions. RAB23 mRNA levels in the ESCC and adjacent normal tissues of dataset GSE20347 deposited in the Gene Expression Omnibus (GEO) database were also analyzed. Immunohistochemistry (IHC) was used to detect the RAB23 protein expressions in 106 pairs of ESCC and adjacent normal tissues, as well as in the lymph glands and primary tumor tissues of 33 patients with positive lymph nodes and 10 patients with negative lymph nodes. Endogenous RAB23 expression was transiently depleted using siRNAs (si-NC, si-RAB23-1, and si-RAB23-9) or stably reduced using shRNAs (sh-NC and sh-RAB23) in ESCC KYSE30 and KYSE150 cells, and the knockdown efficiency was tested using Western blot assays. Cell counting kit-8 assays and mouse xenograft models were used to test the proliferation of ESCC cells. Transwell assays and tail vein-pulmonary metastasis models in immunocompromised mice were used to examine the migration and invasion of ESCC cells. Cell adhesion assays were used to test the adhesion of ESCC cells. RNA-seq assays were used to analyze how RAB23 knockdown influenced the expression profile of ESCC cells and the implicated signal pathways were confirmed using Western blot assays. Results: The RAB23 mRNA expression in 16 cases of ESCC tissues was 0.009 7±0.008 9, which was markedly higher than that in adjacent normal tissues (0.003 2±0.003 7, P=0.006). GEO analysis on RAB23 expressions in ESCC and adjacent normal tissues showed that the RAB23 mRNA level in ESCC tissues (4.30±0.25) was remarkably increased compared with their normal counterparts (4.10±0.17, P=0.037). Among the 106 pairs of ESCC and tumor-adjacent normal tissues, 51 cases exhibited low expression of RAB23 and 55 cases showed high expression of RAB23, whereas in the paired tumor-adjacent normal tissues 82 cases were stained weakly and 24 strongly for RAB23 protein. These results indicated that RAB23 expression was markedly increased in ESCC tissues (P<0.001). Additionally, only 1 out of 33 primary ESCC tissues with positive lymph nodes showed low RAB23 protein expression. On the other hand, 7 samples of primary ESCC tissues with negative lymph nodes were stained strongly for RAB23 while its level in the other 3 samples was weak. These results showed that RAB23 expression was remarkably increased in primary ESCC tissues with positive lymph nodes compared with those with negative lymph nodes (P=0.024). Further tests showed that 32 out of 33 positive lymph nodes were stained strongly for RAB23, whereas no negative lymph nodes (n=10) exhibited high expression of RAB23 (P<0.001). Both transient and stable knockdown of endogenous RAB23 expression failed to cause detectable changes in the proliferation of KYSE30 cells in vitro and in vivo, but attenuated the migration and invasion of KYSE30 cells as well as the invasion of KYSE150 cells. RAB23 knockdown was found to significantly decrease the number of adhesive KYSE30 cells in the sh-RAB23 group (313.75±89.34) compared with control cells in the sh-NC group (1 030.75±134.29, P<0.001). RAB23 knockdown was also found to significantly decrease the number of adhesive KYSE150 cells in the sh-RAB23 group (710.5±31.74) compared with the number of control cells in the sh-NC group (1 005.75±61.09, P<0.001). RNA-seq assays demonstrated that RAB23 knockdown using two siRNAs targeting RAB23 mRNA markedly impaired focal adhesion-related signal pathways, and decreased the levels of phosphorylated FAK (p-FAK) and phosphorylated paxillin (p-paxillin) in KYSE30 and KYSE150 cells. Conclusions: Significantly increased RAB23 in ESCC tissues positively correlates with lymph node metastasis. Depleted RAB23 expression attenuates focal adhesion-related signal pathways, thus impairing the invasion, metastasis, and adhesion of ESCC cells.

Identification of key genes and validation of key gene aquaporin 1 on Wilms' tumor metastasis.

Wilms' tumor (WT) is one of the most common solid tumors in children with unsatisfactory prognosis, but few molecular prognostic markers have been discovered for it. Many genes are associated with the occurrence and prognosis of WT. This study aimed to explore the key genes and potential molecular mechanisms through bioinformatics and to verify the effects of aquaporin 1 (AQP1) on WT metastasis. Differentially expressed genes (DEGs) were generated from WT gene expression data sets from the Gene Expression Omnibus (GEO) database. Gene functional enrichment analysis was carried out with the Database for Annotation, Visualization and Integrated Discovery (DAVID). A protein-protein interaction network (PPI) was constructed and visualized by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and Cytoscape software. Minimal Common Oncology Data Elements (MCODE) was used to detect the important modules in the PPI network, and the important nodes (genes) in the PPI module were sorted by CytoHubba. RT-qPCR was performed to validate the expression of the key genes in WT. Wound healing and Transwell assays were used to detect the cell migration and invasion abilities of AQP1-overexpressing cells. Phalloidin-iFlour 488 was used to stain the cytoskeleton to observe how AQP1 overexpression affects cytoskeletal microfilament structure. A total of 73 co-expressed DEGs were chosen for further investigation. The importance of homeostasis and transmembrane transport of ions and water were highlighted by functional analysis. Gene regulatory network and PPI network were predicted. MCODE plug identified two important modules. Finally, top five key genes were identified using CytoHubba, including Renin (REN), nephrosis 2 (NPHS2), Solute Carrier Family 12 Member 3 (SLC12A3), Solute Carrier Family 12 Member 1 (SLC12A1) and AQP1. The five key genes were mainly enriched in cell volume and ion homeostasis. RT-qPCR confirmed the expression of the five key genes in WT. AQP1 was validated to be expressed at significantly lower levels in WT than in normal tissue. AQP1 overexpression significantly reduced the migratory and invasive capacity of Wit-49 cells, as evidenced by reducing the scratch healing rate and the number of perforated control cells by Wit-49 cells. AQP1 overexpression also reduced the expression of biomarkers of epithelial-mesenchymal transformation, decreased levels of vimentin and N-cadherin and increased expression of E-cadherin, resulting in decreased formation of conspicuous lamellipodial protrusions, characteristic of diminished WT cell invasion and migration. Our study reveals the key genes of WT. These key genes may provide novel insight for the mechanism and diagnosis of WT. AQP1 overexpression inhibited invasion, migration, EMT, and cytoskeletal rearrangement of WT cells, indicating that AQP1 plays a role in the pathogenesis of WT.

Epithelial apoptotic pattern emerges from global and local regulation by cell apical area

Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, we know little about whether and how cell geometry controls apoptosis commitment in developing tissues. Here, we combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. We found that early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. We showed that these global and local geometric characteristics are sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, we established that the coupling between these two geometric features and apoptotic cells is dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, our work provides important insights into the spatial regulation of cell death in tissues and improves our understanding of the mechanisms that control cell number and tissue size.

Demand-driven active droplet generation and sorting based on positive pressure-controlled fluid wall.

Droplet microfluidics is a rapidly advancing area of microfluidic technology, which offers numerous advantages for cell analysis, such as isolation and accumulation of signals, by confining cells within droplets. However, controlling cell numbers in droplets is challenging due to the uncertainty of random encapsulation which result in many empty droplets. Therefore, more precise control techniques are needed to achieve efficient encapsulation of cells within droplets. Here, an innovative microfluidic droplet manipulation platform had been developed, which employed positive pressure as a stable and controllable driving force for manipulating fluid within chips. The air cylinder, electro-pneumatics proportional valve, and the microfluidic chip were connected through a capillary, which enabled the formation of a fluid wall by creating a difference in hydrodynamic resistance between two fluid streams at the channel junction. Lowering the pressure of the driving oil phase eliminates hydrodynamic resistance and breaks the fluid wall. Regulating the duration of the fluid wall breakage controls the volume of the introduced fluid. Several important droplet microfluidic manipulations were demonstrated on this microfluidic platform, such as sorting of cells/droplets, sorting of droplets co-encapsulated cells and hydrogels, and active generation of droplets encapsulated with cells in a responsive manner. The simple, on-demand microfluidic platform was featured with high stability, good controllability, and compatibility with other droplet microfluidic technologies.

Examining the effect of Reelin on vascular endothelial cell polarity

Introduction: Vascular cells differentiate into motile leader cells and stalk cells, become polarized, and migrate toward growth factor signals during vessel formation. Our goal is to shed light on mechanisms contributing to coronary vessel formation. We showed that epicardial-specific deletion of the Tbx5 transcription factor in embryonic mouse hearts impaired coronary vessel formation and reduced cardiac expression of the RELN gene encoding Reelin extracellular matrix glycoprotein. Reelin is expressed in coronary vascular endothelial cells of embryonic mouse hearts. We silenced endogenous Reelin expression in human dermal microvascular endothelial cells (HDMECs) with small interfering RNAs (siRNA) designed to target human RELN and evaluated its role in these cells. Previously, we showed that RELN knockdown (KD) in HDMECs reduced cell migration. Cell polarity is a key to vascular endothelial cell migration, and polarization of the Golgi is vital for cell polarity. Reelin is known to modulate the Golgi in neuronal cells by affecting phosphorylation of the cytoplasmic linker associated protein CLASP2. Because CLASP2 is an upstream effector of microtubule reorganization in neuronal and endothelial cells, we hypothesize that RELN KD leads to CLASP2 phosphorylation and prevents polarization of vascular endothelial cells by reducing the association between Golgi-derived microtubules and actin filaments to inhibit cell migration. Study Objective: The purpose of this study was to examine the role of Reelin signaling in regulating vascular endothelial cell polarity to initiate migration during angiogenesis. Methods: We assayed the impact of Reelin on vascular endothelial cell polarity by conducting scratch wound assays on control or RELN KD HDMECs. Cells were allowed to migrate into a denuded region created by the scratch. After a 6-hour migration period, HDMECs were fixed and stained for immunofluorescence with the GM130 cis-Golgi marker and counterstained with DAPI to visualize the nucleus. Leader cells localized to the leading edge in the migration front of the scratch wound. We determined leader cell polarity by assessing the position of the Golgi relative to the nucleus. Also, we assessed CLASP2 in control and RELN KD cells. Results: We observed an equivalent number of leader cells in control and RELN KD HDMECs. However, 3-fold fewer RELN KD cells had the Golgi positioned in front of the nucleus versus control HDMECs. Thus, Reelin knockdown reduced the number of polarized Golgi in vascular endothelial cells. Conclusion: Our data indicates that Reelin may regulate cell migration and polarity through Golgi polarization. These data provide a plausible explanation for impairment of coronary vessel formation in Tbx5-deficient embryonic mouse hearts with reduced Reln cardiac expression. AHA 17AIREA33660773, PCOM CCDA This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Zic-r.b controls cell numbers in Ciona embryos by activating CDKN1B

The control of cell numbers and the establishment of cell types are two processes that are essential in early embryonic development. We have a reasonable understanding of how these processes occur individually, but we have considerably less sophisticated understanding of how these processes are linked. Tunicates have fixed cell lineages with predictable cell cycles, making them well suited to investigate these processes. In the ascidian Ciona, we show that the transcription factor Zic-r.b, known to be involved in establishing several cell types in early development also activates the expression of the cell cycle inhibitor CDKN1B. Zic-r.b is a major missing component of the cell division clock establishing specific cell numbers. We also show that a larvacean homolog of Zic-r.b is expressed one cell cycle earlier than its Ciona counterpart. The early expression in larvaceans may explain why they have half as many notochord cells as ascidians and may illustrate a general mechanism to evolve changes in morphology.

Growth, health status and gut microbiota of the scalloped spiny lobster (Panulirus homarus) at different salinities

The scalloped spiny lobster (Panulirus homarus) is a valuable species for aquaculture due to its high commercial value. However, limited information is available regarding the effect of environmental parameters on aquaculture of P. homarus. This study investigated the growth performance, health status and gut microbiota of P. homarus juveniles cultured at salinity 35 (control), 25, and 15 for 4 weeks. Compared to the control, the survival, weight gain, condition factor and haemolymph cell number were significantly reduced in P. homarus at salinity 15, while no significant differences were found in P. homarus at salinity 25. Compared with the control group, the antioxidant indices including total antioxidant capacity, superoxide dismutase and glutathione peroxidase in the 15 and 25 salinity groups were significantly increased, and malondialdehyde increased significantly only at salinity 15. Compared with salinity 35, salinity 15 significantly changed the structure of gut microbiota, increased within-habitat and between-habitat diversity, and increased the abundance of opportunistic pathogens Photobacterium and Vibrio. Unsurprisingly, the functions of the gut microbiota were mainly enriched in the metabolism pathway of the KEGG-level 1, and the KEGG-level 2 pathways with an enrichment abundance of >3000 were all in the metabolism pathway. Interestingly, compared with the salinity of 35, the antibiotic synthesis pathway was significantly inhibited, while the pathways related to bacterial life were significantly enhanced in the salinity 15. In conclusion, salinity changes affect growth, antioxidant capacity, and gut microbiota composition of P. homarus, and the effects of large-scale reductions in salinity (e.g., salinity 15) are generally detrimental.

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

Cell numbers, cell ratios, and developmental plasticity in the rod pathway of the mouse retina.

The precise specification of cellular fate is thought to ensure the production of the correct number of neurons within a population. Programmed cell death may be an additional mechanism controlling cell number, believed to refine the proper ratio of pre- to post-synaptic neurons for a given species. Here, we consider the size of three different neuronal populations in the rod pathway of the mouse retina: rod photoreceptors, rod bipolar cells, and AII amacrine cells. Across a collection of 28 different strains of mice, large variation in the numbers of all three cell types is present. The variation in their numbers is not correlated, so that the ratio of rods to rod bipolar cells, as well as rod bipolar cells to AII amacrine cells, varies as well. Establishing connectivity between such variable pre- and post-synaptic populations relies upon plasticity that modulates process outgrowth and morphological differentiation, which we explore experimentally for both rod bipolar and AII amacrine cells in a mouse retina with elevated numbers of each cell type. While both rod bipolar dendritic and axonal arbors, along with AII lobular arbors, modulate their areal size in relation to local homotypic cell densities, the dendritic appendages of the AII amacrine cells do not. Rather, these processes exhibit a different form of plasticity, regulating the branching density of their overlapping arbors. Each form of plasticity should ensure uniformity in retinal coverage in the presence of the independent specification of afferent and target cell number.

Effects of N‐oleoylethanolamide on Lymphoblasts Deficient in Tafazzin

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutations in the TAZ gene that encodes for the cardiolipin remodelling enzyme, Tafazzin. This syndrome is characterized by cardiac and skeletal myopathies, as well as immunological deficits that cause significant morbidity and mortality. Study of the molecular pathology of BTHS thus provides insight into the role of cardiolipin and mitochondria in cellular physiology, while addressing a clinically relevant disease. Current treatment options for BTHS are limited, but a recent trial suggests that PPARα activation may be therapeutic. The objective of this study was to quantitatively examine the growth properties and mitochondrial physiology of B-lymphoblasts derived from five donors with BTHS, as well as effects of treatment with oleoylethanolamide (OEA), a bioactive N-acylethanolamine. Cyclic neutropenia is a well-recognized aspect of the pathology of BTHS. We hypothesized that deficits in lymphoblast proliferation would also be evident, and that OEA, which activates receptors including PPARα, would demonstrate therapeutic benefit. One million B-lymphoblasts derived from BTHS donors and age- and sex-matched healthy controls were seeded on day 0 and counted daily for 4 days. On day 4, there were 27.7 ± 4.6% fewer BTHS B-lymphoblasts than control cells (P<0.05), indicating that lymphopenia is likely also a component of BTHS. Treatment of B-lymphoblasts with 1 μM OEA caused no significant effect on control cell numbers, but significantly improved deficits in each of the five BTHS lymphoblast lines, raising the mean cell number on day 4 from 3.74 × 106 to 4.25 × 106 cells, although a full rescue of growth was not achieved. Analysis of electron micrographs of samples from two distinct BTHS B-lymphoblast donors indicated that cells had larger mitochondria as compared with healthy controls (mean of 0.67 ± 0.07 µM2 versus 0.27 ± 0.16 µM2, respectively). OEA decreased mitochondrial size by 53.0 ± 10.9%, while increasing the total mitochondrial number per cell by 92.6 ± 58.6%. OEA reduced the elevated expression of the enzyme CDS1 in BTHS B-lymphoblasts to healthy control levels, suggesting improved cardiolipin levels may play a role. However, an initial donor pair comparison did not show an effect of OEA on cardiolipin content, suggesting other mechanisms should be explored. Higher immunodetectable levels of OPA1, a biomarker of mitochondrial fusion, were found in lymphoblasts from children with BTHS, and were attenuated by OEA. Results from this study newly identify lymphopenia as a probable immunological deficit in BTHS, and suggest that therapies targeting aberrant fission/fusion of the mitochondrial network, rather than cardiolipin content, per se, and may have clinical utility for treatment of this disease.

A non-canonical role for p27Kip1 in restricting proliferation of corneal endothelial cells during development.

The cell cycle regulator p27Kip1 is a critical factor controlling cell number in many lineages. While its anti-proliferative effects are well-established, the extent to which this is a result of its function as a cyclin-dependent kinase (CDK) inhibitor or through other known molecular interactions is not clear. To genetically dissect its role in the developing corneal endothelium, we examined mice harboring two loss-of-function alleles, a null allele (p27−) that abrogates all protein function and a knockin allele (p27CK−) that targets only its interaction with cyclins and CDKs. Whole-animal mutants, in which all cells are either homozygous knockout or knockin, exhibit identical proliferative increases (~0.6-fold) compared with wild-type tissues. On the other hand, use of mosaic analysis with double markers (MADM) to produce infrequently-occurring clones of wild-type and mutant cells within the same tissue environment uncovers a roughly three- and six-fold expansion of individual p27CK−/CK− and p27−/− cells, respectively. Mosaicism also reveals distinct migration phenotypes, with p27−/− cells being highly restricted to their site of production and p27CK−/CK− cells more widely scattered within the endothelium. Using a density-based clustering algorithm to quantify dispersal of MADM-generated clones, a four-fold difference in aggregation is seen between the two types of mutant cells. Overall, our analysis reveals that, in developing mouse corneal endothelium, p27 regulates cell number by acting cell autonomously, both through its interactions with cyclins and CDKs and through a cyclin-CDK-independent mechanism(s). Combined with its parallel influence on cell motility, it constitutes a potent multi-functional effector mechanism with major impact on tissue organization.

Statistical Complexity of Boolean Cellular Automata with Short-Term Reaction-Diffusion Memory on a Square Lattice

Memory is a ubiquitous phenomenon in biological systems, in which the present system state is not entirely determined by the current conditions but also depends on the time evolutionary path of the system. Specifically, many phenomena related to memory are characterized by chemical memory reactions that may fire under particular system conditions. These conditional chemical reactions contradict the extant approaches for modeling chemical kinetics and have increasingly posed significant challenges to mathematical modeling and computer simulation. Along these lines, we can imagine a memory module contributing to cell therapy or the synthetic differentiation of certain cells in a certain fashion after experiencing a brief stimulus. We demonstrate that information processing properties of cellular automata (CAs) can be controlled by a signal composed of excitation pulses. We discuss how cellular memory can be incorporated into more complex systems like CAs to understand the controlling of information processing performed by a medium with the use of a pulse signal propagated from a number of control cells. In this paper, we also investigate the potential application of cellular computation for constructing pseudorandom number generators (PRNGs). Furthermore, the PRNG scheme based on CAs with reaction-diffusion memory is proposed for its capability of generating ultrahigh-quality random numbers. However, the quality bottleneck of a practical PRNG lies in the limited cycle of the generator. To close the gap between the pure randomness generation and the short period, we propose and implement a memory algorithm based on a reaction-diffusion process in a chemical system for Boolean CAs. This scheme is characterized by a tradeoff between, on one hand, the rate of generation of random bits and, on the other hand, the degree of randomness that the series can deliver. These successful applications of the memory modeling framework suggest that this innovative theory is an effective and powerful tool for studying memory processes and conditional chemical reactions in a wide range of complex biological systems. This result also opens a new perspective to apply CAs as a computational engine for the robust generation of pure random numbers, which has important applications in cryptography and other related areas. © 2019, Complex Systems Publications, Inc. All rights reserved.

DLT/OsGRAS-32, regulating leaf width and thickness by controlling cell number in Oryza sativa

The improvement of rice leaf morphology is an important goal of rice plant breeding. Studying its molecular development mechanism will facilitate the breeding of a maximally efficient plant type. To further study the developmental mechanism of rice leaves, a dwarf broad-leaf mutant, g44, was obtained by ethyl methanesulfonate mutagenesis of the rice restorer line Jinhui10, and genetic analysis indicated that the trait was controlled by a pair of recessive nuclear gene. The locus was fine mapped on chromosome 6 with 16-kb physical distance. Sequencing revealed that an A-T base substitution occurred in the mutant, resulting in the early termination of LOC_Os06g03710/DLT translation in g44 and yielding only one residual peptide containing 558 amino acids. The phenotypic and sequencing results of the complementary transgenic plants and the allelic mutant g44-1 further confirmed this result. Mutant plants grown under field conditions showed dwarfing, a leaf colour of dark green, leaf widening, and grain enlargement. Paraffin section and cryosection analysis showed that the increase in the number of large vascular bundles and small vascular bundles and the increase in the spacing between adjacent small vascular bundles were the main reasons for the broadening of mutant leaves. In addition, the increase in thickness of the mesophyll cell layer may be the main reason for the increase in leaf colour darkness and net photosynthetic rate. The qPCR results preliminarily predicted that the increase in the number of mutant cells was caused by an enhanced cell division ability, and the DLT/G44 gene was proposed to participate in the regulation of cell division by suppressing the expression of cyclin-related genes.

Helicobacter pylori-Induced DNA Damage Is a Potential Driver for Human Gastric Cancer AGS Cells.

Helicobacter pylori is a major cause of gastric cancer. This study was aimed to explore the characteristic of DNA damage induced by H. pylori infection in gastric cancer AGS cells. After infection with H. pylori, the reactive oxygen species (ROS) levels in AGS cells were significantly higher than those in the uninfected cells. Cells with longer comet tails were detected after infection with H. pylori. The number of apurinic/apyrimidinic endonuclease 1- and phosphorylated H2AX-positive cells was significantly increased compared with the number of negative control cells. The expression of pChk1 and pChk2 was significantly upregulated by H. pylori infection. Cell growth was inhibited after H. pylori infection. All these results were dose dependent. The cell alterations were more significant upon infection with H. pylori at a multiplicity of infection (MOI) of 100:1 than at an MOI of 50:1. H. pylori infection can induce DNA single-strand breaks, DNA double-strand breaks, and cell cycle checkpoint activation after ROS generation in the gastric cancer cell line AGS, which is a potential driver for gastric cancer.

Abstract 4170: The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma

Abstract Introduction: Ewing sarcoma (ES) is a primary bone sarcoma occurring in adolescents and young adults, accounting for 15% of childhood/adolescent cancer. We previously reported that an RNA helicase, DDX3, regulates the translation of a variety of proteins in ES cells, including those modulating DNA damage repair. Therefore, we hypothesized that inhibiting DDX3, using a small molecule called RK-33, would radio-sensitize ES cells by preventing DNA damage repair. Methods: ES cell lines (MHH-ES and TC71) were transfected with DDX3- targeted shRNA or a control, scramble shRNA. RT-PCR and western blotting confirmed knockdown of DDX3. RK-33 cytotoxicity was evaluated using cell viability assay. DNA double strand breaks (DSB) were quantified by immunofluorescent staining for γH2AX, and subcellular localization of DDX3 was determined by immunofluorescence. ES patient derived xenografts (PDX) were subcutaneously implanted in 4 cohorts of NSG mice. Results: Expression of DDX3 mRNA and protein was decreased in DDX3-knockdown cells compared with controls. To validate that DDX3 is inhibited by RK-33, we compared the cytotoxicity of this agent in control cells and in the DDX3 knockdown cell lines. Parental ES and control cells (MHH-ES, TC71, MSD-10) were more sensitive to RK-33, with IC50 values that were significantly lower than their corresponding DDX3-knockdown lines (M2C7, M1F6; 4 μM, 2.7 μM and 2.6 μM vs 42 μM and 11.6 μM, respectively, p&amp;lt;0.05). To determine the effect on DNA damage repair, DSB were quantified in DDX3- knockdown cells and controls, treated with or without RK-33, at various time points after irradiation. At early time points, treatment with RK-33 had no impact on the number of ES and control cells (MHH-ES, TC71, MSD-10) with &amp;gt;10 γH2AX foci, but after 24 hours, the average number of irradiated cells with &amp;gt;10 foci was significantly higher in RK-33-treated cells than in cells treated with vehicle alone (p&amp;lt;0.01). The time needed for full recovery of radiation-induced DSB in untreated cells was less than 6 hr, while RK-33 pre-treated cells failed to recover even after 24 hr. Irradiated DDX3-knockdown cells demonstrated impaired DNA damage repair as well. To expand our investigation into the mechanism by which DDX3 regulates DNA damage repair, we studied the subcellular localization of the enzyme in response to radiation. We found that DDX3 translocates into the nucleus and colocalizes with γH2AX in irradiated cells, suggesting a dual role, direct and indirect, in DNA damage repair. In vivo, we found that animals treated with subtherapeutic external beam radiation concurrently with RK-33 had a substantial increase in tumor response. Our results suggest that DDX3 plays a crucial role in regulating DNA damage repair dynamics in ES, both indirectly by affecting the translation of involved proteins, and directly by localizing to sites of DSB. Therefore, inhibiting DDX3 using RK-33 may represent a novel approach to radiosensitization of cancer cells. Citation Format: Marwa Afifi, Breelyn A. Wilky, Catherine Kim, Venu Raman, David Loeb. The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4170.

Sodium Valproate Inhibits Small Cell Lung Cancer Tumor Growth on the Chicken Embryo Chorioallantoic Membrane and Reduces the p53 and EZH2 Expression.

The study aims to test the effect of different sodium valproate (NaVP) doses on small cell lung cancer NCI-H146 cells tumor in chicken embryo chorioallantoic membrane (CAM) model. Xenografts were investigated in the following groups: nontreated control and 5 groups treated with different NaVP doses (2, 3, 4, 6, and 8 mmol/L). Invasion of tumors into CAM in the nontreated group reached 76%. Tumors treated with 8 mmol/L NaVP doses significantly differed in tumor invasion frequency from the control and those treated with 2 mmol/L (P < .01). The calculated probability of 50% tumor noninvasion into CAM was when tumors were treated with 4 mmol/L of NaVP. Number of p53-positive cells in tumors was significantly reduced when treated with NaVP doses from 3 to 8 mmol/L as compared with control; number of EZH2-positive cells in control significantly differed from all NaVP-treated groups. No differences in p53- and EZH2-positive cell numbers were found among 4, 6, and 8 mmol/L NaVP-treated groups. Invaded tumors had an increased N-cadherin and reduced E-cadherin expression. The results indicate the increasing NaVP dose to be able to inhibit tumors progression. Expression of p53 and EZH2 may be promising target markers of therapeutic efficacy evaluation.

Optimizing critical source control of five priority-regulatory trace elements from industrial wastewater in China: Implications for health management.

Anthropogenic emissions of toxic trace elements (TEs) have caused worldwide concern due to their adverse effects on human health and ecosystems. Based on a stochastic simulation of factors' probability distribution, we established a bottom-up model to estimate the amounts of five priority-regulatory TEs released to aquatic environments from industrial processes in China. Total TE emissions in China in 2010 were estimated at approximately 2.27 t of Hg, 310.09 t of As, 318.17 t of Pb, 79.72 t of Cd, and 1040.32 t of Cr. Raw chemicals, smelting, and mining were the leading sources of TE emissions. There are apparent regional differences in TE pollution. TE emissions are much higher in eastern and central China than in the western provinces and are higher in the south than in the north. This spatial distribution was characterized in detail by allocating the emissions to 10 km × 10 km grid cells. Furthermore, the risk control for the overall emission grid was optimized according to each cell's emission and risk rank. The results show that to control 80% of TE emissions from major sources, the number of top-priority control cells would be between 200 and 400, and less than 10% of the total population would be positively affected. Based on TE risk rankings, decreasing the population weighted risk would increase the number of controlled cells by a factor of 0.3-0.5, but the affected population would increase by a factor of 0.8-1.5. In this case, the adverse effects on people's health would be reduced significantly. Finally, an optimized strategy to control TE emissions is proposed in terms of a cost-benefit trade-off. The estimates in this paper can be used to help establish a regional TE inventory and cyclic simulation, and it can also play supporting roles in minimizing TE health risks and maximizing resilience.