DNA Content Of Cells Research Articles

Bacterial filaments recover by successive and accelerated asymmetric divisions that allow rapid post-stress cell proliferation.

Filamentation is a reversible morphological change triggered in response to various stresses that bacteria might encounter in the environment, during host infection or antibiotic treatments. Here we re-visit the dynamics of filament formation and recovery using a consistent framework based on live-cells microscopy. We compare the fate of filamentous Escherichia coli induced by cephalexin that inhibits cell division or by UV-induced DNA-damage that additionally perturbs chromosome segregation. We show that both filament types recover by successive and accelerated rounds of divisions that preferentially occur at the filaments' tip, thus resulting in the rapid production of multiple daughter cells with tightly regulated size. The DNA content, viability and further division of the daughter cells essentially depends on the coordination between chromosome segregation and division within the mother filament. Septum positioning at the filaments' tip depends on the Min system, while the nucleoid occlusion protein SlmA regulates the timing of division to prevent septum closure on unsegregated chromosomes. Our results not only recapitulate earlier conclusions but provide a higher level of detail regarding filaments division and the fate of the daughter cells. Together with previous reports, this work uncovers how filamentation recovery allows for a rapid cell proliferation after stress treatment.

Biotechnology of the Tree Fern Cyathea smithii Hook. f. (Soft Tree Fern, Katote). I. Morphogenic Potential of Shoot Apical Dome, Plant Regeneration, and Nuclear DNA Content of Regenerants in the Presence of TDZ and NAA

Abstract In nature, the tree fern Cyathea smithii lacks meristem multiplication; therefore, a system of vegetative propagation in vitro is required to protect the species’ gene resources. Experiments were carried out on the apical dome of C. smithii J. D. Hooker (soft tree fern, Katote) to determine its morphogenic potential using 1/2 strength of Murashige and Skoog medium containing various concentration of thidiazuron [TDZ; 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea)] (0.01, 0.1, 1.0, and 5.0 µM) supplemented with naphthalene acetic acid (0.25 µM). The success of the treatments was determined by the number of regenerants per explant, level of regenerant development, and nuclear DNA content in the green mesophyll cells. The highest number of regenerants was achieved in the presence of 5.0 µM TDZ and the lowest at 0.01 µM TDZ. The quality of development of the regenerants was expressed as the number of formed leaves and their growth per individual plantlet. The highest growth and development of regenerants occurred at the lowest concentration of TDZ (0.01 µM), and the lowest growth and development of regenerants occurred at the highest TDZ concentration (5.0 µM); however, the number of regenerants showed the opposite. In gametophytes, the nuclear DNA content ranged from 4.74–4.78 pg representing haploid level, whereas in sporophytes it ranged from 8.45–9.13 pg and was diploid. These results prove the stability of the regenerant genome after TDZ treatment of meristematic cells.

NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing PINK1/Parkin-Mediated Mitophagy.

A long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) variant 1 (NEAT1v1), confers radioresistance to hepatocellular carcinoma (HCC) cells by inducing autophagy via γ-aminobutyric acid A receptor-associated protein (GABARAP). Radiation induces oxidative stress to damage cellular components and organelles, but it remains unclear how NEAT1v1 protects HCC cells from radiation-induced oxidative stress via autophagy. To address this, we precisely investigated NEAT1v1-induced autophagy in irradiated HCC cell lines. X-ray irradiation significantly increased cellular and mitochondrial oxidative stress and mitochondrial DNA content in HCC cells while NEAT1v1 suppressed them. NEAT1v1 concomitantly induced the phosphatase and tensin homolog-induced kinase 1 (PINK1)/parkin-mediated mitophagy. Interestingly, parkin expression was constitutively upregulated in NEAT1v1-overexpressing HCC cells, leading to increased mitochondrial parkin levels. Superoxide dismutase 2 (SOD2) was also upregulated by NEAT1v1, and GABARAP or SOD2 knockdown in NEAT1v1-overexpressing cells increased mitochondrial oxidative stress and mitochondrial DNA content after irradiation. Moreover, it was suggested that SOD2 was involved in NEAT1v1-induced parkin expression, and that GABARAP promoted parkin degradation via mitophagy. This study highlights the unprecedented roles of NEAT1v1 in connecting radioresistance and mitophagy in HCC.

Advances in Intraoperative Flow Cytometry.

Flow cytometry is the gold-standard laser-based technique to measure and analyze fluorescence levels of immunostaining and DNA content in individual cells. It provides a valuable tool to assess cells in the G0/G1, S, and G2/M phases, and those with polyploidy, which holds prognostic significance. Frozen section analysis is the standard intraoperative assessment for tumor margin evaluation and tumor resection. Here, we present flow cytometry as a promising technique for intraoperative tumor analysis in different pathologies, including brain tumors, leptomeningeal dissemination, breast cancer, head and neck cancer, pancreatic tumor, and hepatic cancer. Flow cytometry is a valuable tool that can provide substantial information on tumor analysis and, consequently, maximize cancer treatment and expedite patients' survival.

Modeling the Dynamics of Eukaryotic DNA Synthesis in Remembrance of Tunde Ogunnaike.

Two things Tunde loved were dynamics and probability. The work described herein combined them both, which explains why Tunde invariably asked me each time we talked how this work was proceeding. However, as I've come to appreciate as reminiscent of a surprisingly large amount of work in almost any researcher's career, I did not complete a peer-reviewed article on the matter while he could see it. We were broadly motivated by analysis of data for total DNA content in single cells, across thousands of cells. From such data one can estimate the proportions of cells in different phases of the cell cycle by fitting a mixture model for subpopulations of G0/G1 phase cells (1 relative copy of the genome), S phase cells (between 1 and 2 relative copies of the genome), and G2/M phase cells (2 relative copies of the genome). Given an asynchronously cycling population, Gaussian models are reasonable for the G0/G1 and G2/M subpopulations, but an appropriate functional form for the S-phase subpopulation was unclear. Since the probability of observing an S-phase cell is intimately related to the dynamics of DNA replication, we worked to derive a model for DNA replication dynamics from first principles, resulting in a closed-form, analytic expression for the dynamics of DNA synthesis. While quite arguably a somewhat superfluous effort, there is a certain satisfaction and academic beauty to modeling systems from a first-principles approach, and it can sometimes lead to unexpected scientific insights. Yet, while mathematically elegant, there was a fundamental issue with a key assumption that the so-called inter-origin distance distribution (distances between DNA replication initiation sites) was time-invariant. First, I present the model as developed previously. Then, to address the time-invariant inter-origin distance distribution issue, I provide a treatment of time-varying inter-origin distance distributions that, while mathematically simple, provides (i) mechanistic predictions for how all the DNA in a fertilized frog egg can be replicated "on time" despite some inter-origin distances initially exceeding the corresponding amount of allowable time and (ii) evidence that, based only on data from DNA content versus time and average inter-origin distances, somatic cell DNA is parsed into distinct regions whose replication is temporally separate.

DeepCAN: A Modular Deep Learning System for Automated Cell Counting and Viability Analysis.

Precise and quick monitoring of key cytometric features such as cell count, size, morphology, and DNA content is crucial in life science applications. Traditionally, image cytometry relies on visual inspection of hemocytometers. This approach is error-prone due to operator subjectivity. Recently, deep learning approaches have emerged as powerful tools enabling quick and accurate image cytometry applicable to different cell types. Leading to simpler, compact, and affordable solutions, these approaches revealed image cytometry as a viable alternative to flow cytometry or Coulter counting. In this study, we demonstrate a modular deep learning system, DeepCAN, providing a complete solution for automated cell counting and viability analysis. DeepCAN employs three different neural network blocks called Parallel Segmenter, Cluster CNN, and Viability CNN that are trained for initial segmentation, cluster separation, and viability analysis. Parallel Segmenter and Cluster CNN blocks achieve accurate segmentation of individual cells while Viability CNN block performs viability classification. A modified U-Net network, a well-known deep neural network model for bioimage analysis, is used in Parallel Segmenter while LeNet-5 architecture and its modified version Opto-Net are used for Cluster CNN and Viability CNN, respectively. We train the Parallel Segmenter using 15 images of A2780 cells and 5 images of yeasts cells, containing, in total, 14742 individual cell images. Similarly, 6101 and 5900 A2780 cell images are employed for training Cluster CNN and Viability CNN models, respectively. 2514 individual A2780 cell images are used to test the overall segmentation performance of Parallel Segmenter combined with Cluster CNN, revealing high Precision/Recall/F1-Score values of 96.52%/96.45%/98.06%, respectively. Cell counting/viability performance of DeepCAN is tested with A2780 (2514 cells), A549 (601 cells), Colo (356 cells), and MDA-MB-231 (887 cells) cell images revealing high analysis accuracies of 96.76%/99.02%, 93.82%/95.93%, and 92.18%/97.90%, 85.32%/97.40%, respectively.

RAD6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by aggravating mitotic spindle damage

BackgroundPaclitaxel (PTX), a first-line therapy for triple negative breast cancers (TNBC) induces anti-tumor activity by microtubule stabilization and inhibition of cell division. Its dose-limiting toxicity and short half-life, however, pose clinical challenges underscoring the need for strategies that increase its efficiency. RAD6, a E2 ubiquitin conjugating enzyme, is associated with centrosomes at all phases of cell cycle. Constitutive overexpression of the RAD6B homolog in normal breast cells induces centrosome amplification and multipolar spindle formation, indicating its importance in centrosome regulation.MethodsTNBC centrosome numbers were scored by pericentrin immunostaining. PTX sensitivities and interactions with SMI#9, a RAD6-selective small molecule inhibitor, on TNBC cell survival were analyzed by MTT and colony forming assays and an isogenic MDA-MB-468 TNBC model of PTX resistance. The molecular mechanisms underlying PTX and SMI#9 induced cytotoxicity were determined by flow cytometry, immunoblot analysis of cyclin B1 and microtubule associated protein TAU, and dual immunofluorescence staining of TAU and α-tubulin.ResultsOur data show aberrant centrosome numbers and that PTX sensitivities are not correlated with TNBC BRCA1 status. Combining PTX with SMI#9 synergistically enhances PTX sensitivities of BRCA1 wild-type and mutant TNBC cells. Whereas SMI#9/PTX combination treatment increased cyclin B1 levels in MDA-MB-468 cells, it induced cyclin B1 loss in HCC1937 cells with accumulation of reproductively dead giant cells, a characteristic of mitotic catastrophe. Cell cycle analysis revealed drug-induced accumulation of tetraploid cells in S and G2/M phases, and robust increases in cells with 4 N DNA content in HCC1937 cells. TAU overexpression is associated with reduced PTX efficacy. Among the six TAU isoforms, both SMI#9 and PTX downregulated 1N3R TAU in MDA-MB-468 and HCC1937 cells, suggesting a common mechanism of 1N3R regulation. Dual TAU and α-tubulin immunostaining showed that SMI#9 induces monopolar mitotic spindles. Using the isogenic model of PTX resistance, we show that SMI#9 treatment restores PTX sensitivity.ConclusionsThese data support a common mechanism of microtubule regulation by SMI#9 and PTX and suggest that combining PTX with RAD6 inhibitor may be beneficial for increasing TNBC sensitivities to PTX and alleviating toxicity. This study demonstrates a new role for RAD6 in regulating microtubule dynamics.

Two complementary approaches for efficient isolation of Sertoli cells for transcriptomic analysis.

Sertoli cells (SCs) are the only somatic cells that reside in seminiferous tubules of testis. They directly interact with and support the development of germ cells, thus have an indispensable role in the process of spermatogenesis. SCs first appear in a proliferative state and then, with the initiation of the first wave of spermatogenesis, progress to a mature “nurturing” state which supports lifelong continuous sperm production. During this development, the SC transcriptome must adapt rapidly as obstacles in SC maturation often result in deficiencies in male fertility. Due to its importance in spermatogenesis, a reliable, rapid, and precise method for the isolation of high purity, viable and unadulterated SC has been largely missing. We have developed an improved method for the preparation of a testicular single cell suspension comprised of two alternative protocols to separate SCs from the rest of the testicular cells by FACS. The first sorting scheme is based on their co-expression of surface specific markers, FSHr and Occludin-1, while the second focuses on the co-staining of SCs with FSHr-specific antibody and Hoechst 33342, which discriminates DNA content of testicular cells. The entire procedure can be completed in less than 3 h which permits the analysis of the development-related transcriptional profile of these cells. Notably, our comparative study showed that this method resulted in a SC transcriptome that is largely comparable to SCs which were briskly isolated due to their cell-specific expression of fluorescent protein. Interestingly, we also show that SCs sorted as FSHr+Occludin+ cells contained a tangible portion of transcripts from all types of testicular germ cells. Sorting of SCs according to their 2C DNA content significantly reduced the presence of these transcripts, thus seems to be the most suitable approach for accurate determination of the SC transcriptome. We believe that these novel approaches for the isolation of SCs will assist researchers in the elucidation of their function as well as their role in spermatogenesis and disorders related to male infertility.

Protective effect of 5-heptadecylresorcinol against obesity-associated skeletal muscle dysfunction by modulating mitochondrial biogenesis via the activation of SIRT3/PGC-1α signaling pathway

Obesity-associated skeletal muscle mitochondrial dysfunction contributed to exercise capacity decline. 5-heptadecylresorcinol (AR-C17) has been proven effective against obesity and insulin resistance. However, its effects on skeletal muscle performance remains unclear. In this study, we investigate whether AR-C17 could protect against high-fat diet induced C57 BL/6J mice skeletal muscle dysfunction. The results indicated that AR-C17 intervention significantly improved skeletal muscle exercise capacity according to the strength and endurance test. AR-C17 showed no effect on skeletal muscle myofiber type conversion and myogenesis, but could significantly enhance PGC-1α mediated mitochondrial biogenesis. Moreover, AR-C17 significantly attenuated PA-induced reduction of mitochondrial DNA content in C2C12 cells and promoted mitochondrial biogenesis regulator gene PGC-1α and its downstream signaling pathway protein expression. In addition, AR-C17 increased the protein levels of SIRT3, while the protective effect was abolished by SIRT3 inhibitor (3-TYP), indicating AR-C17 ameliorated obesity induced skeletal muscle dysfunction might through SIRT3/PGC-1α signaling pathway.

A Feulgen glimpse into genome evolution during range expansion: a case study of the subterranean amphipod Niphargus schellenbergi

In 1923, Robert Feulgen designed a quantitative approach to measure the relative DNA content of a species (its so-called “C-value”, which is defined as half of the DNA content of a somatic cell and is expressed in pg). The Feulgen approach has several advantages: it can be used on ethanol-preserved samples, it is relatively inexpensive, and it does not require sophisticated equipment (Hardie et al. 2002, Jeffery and Gregory 2014, Kasten 2003). Gathering DNA content information is a key preliminary step for whole-genome sequencing of non-model species and also yield important information for ecological and evolutionary studies as well as for conservation (Jeffery et al. 2013), since genome size has been shown to be strongly correlated to extinction risk in animals and plants (Vinogradov 2004, Vinogradov 2003). Among invertebrates, Niphargidae is among the largest families of freshwater subterranean amphipods in the world, and more than 450 niphargid species have been described (Horton et al. 2021). Thus, it is considered an ideal model for evolutionary and ecological studies. Using a new, improved Feulgen protocol, we generated novel genome size estimates for 26 samples of Niphargus schellenbergi collected from central and western European niphargids. The obtained C-values ranged from 2.75 to 5.25 pg. Such a two-fold intraspecific variation is unusual but not unheard of: it occurs also in the monogonont rotifer Brachionus asplanchnoidis (Stelzer et al. 2021). Further research will be required to find out the mechanisms responsible for this variation, which may result from copy-number variation, polyploidy and/or various amounts of transposable elements.

Role of ERK signaling in bladder urothelium in response to cyclophosphamide injury.

Mice with inducible urothelial deletion of fibroblast growth factor receptor 2 (ShhCreERT2;Fgfr2 Fl/Fl ) injured with cyclophosphamide had aberrant basal cell endoreplication and poor regeneration. The endoreplication correlated with an absence of phosphorylated (activated) ERK expression in urothelium. We assessed whether inhibiting ERK activity phenocopied the urothelial defects in injured Fgfr2 mutant mice. We co‐administered cyclophosphamide and an ERK inhibitor (ERKi) systemically in mice and assessed general histology and immunofluorescence for various markers post injury. Since AKT also signals downstream of FGFR2, we assessed effects of an AKT inhibitor (AKTi) on cyclophosphamide injury. ERK knockdown did not affect urothelial injury or proliferation 24 h after cyclophosphamide. Conversely, ERK inhibition led to larger basal cell nuclei, more submucosal hemorrhage and attenuated uroplakin staining 3 days after injury versus vehicle‐treated mice. Compared to vehicle‐treated mice, ERKi‐treated mice had a trend for more Ki67+ urothelial cells and had statistically fewer phospho‐Histone H3+ cells normalized to Ki67 and higher basal cell DNA content, consistent with endoreplication 3 days after injury. Ten days after injury, ERKi‐treated mice still had signs of poor urothelial regeneration with absent or aberrant expression of differentiation markers and ectopic lumenal expression of keratin 14 (basal progenitor marker). Co‐administration of the AKTi led to no apparent urothelial defects 3 days after cyclophosphamide. Thus, ERK knockdown (but not AKT knockdown) leads to urothelial regenerative responses after cyclophosphamide reminiscent of Fgfr2 mutant mice. Together, it appears that FGFR2 acts through ERK to prevent aberrant urothelial basal cell endoreplication and ensure normal regeneration after cyclophosphamide.

Impact of Sr2+ and hypoxia on 3D triple cultures of primary human osteoblasts, osteocytes and osteoclasts

An in vitro bone triple culture involving human primary osteoblasts, osteocytes and osteoclasts enables the investigation of bone healing factors, drugs or biomaterials in a model system for native bone tissue. The present study analyses the impact of Sr2+ as well as hypoxic cultivation (5% O2 content or chemically induced by Co2+) on bone cells. The three cell types were cultivated together in the presence of 100 µM Sr2+, hypoxic conditions or in the presence of 75 µM Co2+. After cultivation the cell types were separated and analysed on mRNA and protein level individually. In response to Sr2+ osteoblasts showed a downregulation of IBSP expression and a stimulation of ALP activity. Osteocyte gene marker expression of PDPN, MEPE, RANKL, OPG, osteocalcin and likewise the amount of secreted osteocalcin was reduced in the presence of Sr2+. Activity of osteoclast-specific enzymes TRAP and CAII was enhanced compared to the Sr2+ free control. Hypoxic conditions induced by both 5% O2 or a Co2+ treatment led to decreased DNA content of all bone cells and downregulated expression of osteoblast markers ALPL and IBSP as well as osteocyte markers PDPN, RANKL and OPG. In addition, Co2+ induced hypoxia decreased gene and protein expression of osteocalcin in osteocytes. In response to the Co2+ treatment, the TRAP gene expression and activity was increased. This study is the first to analyse the effects of Sr2+ or hypoxia on triple cultures with primary human bone cells. The investigated in vitro bone model might be suitable to reduce animal experiments in early stages of biomaterial and drug development.

No evidence for temporally balanced selection on larval Pacific oysters Crassostrea gigas: a comment on Durland et al. (2021).

No evidence for temporally balanced selection on larval Pacific oysters Crassostrea gigas: a comment on Durland et al. (2021).

MtDNA content in cumulus cells does not predict development to blastocyst or implantation.

STUDY QUESTIONIs relative mitochondrial DNA (mtDNA) content in cumulus cells (CCs) related to embryo developmental competence in humans and/or the bovine model?SUMMARY ANSWERmtDNA content in CCs provides a poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer.WHAT IS KNOWN ALREADYCCs are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in CCs could be used as a proxy to infer oocyte developmental potentialSTUDY DESIGN, SIZE, DURATIONmtDNA content was analyzed in CCs obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage or oocytes developing to blastocysts.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman CCs samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis.MAIN RESULTS AND THE ROLE OF CHANCEmtDNA content in human and bovine CCs did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding CCs.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThe lack of correlation found between mtDNA content in human CCs and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, mono-ovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable.WIDER IMPLICATIONS OF THE FINDINGSThe use of molecular markers for oocyte developmental potential in CCs could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in CCs is not a good proxy for oocyte quality.STUDY FUNDING/COMPETING INTEREST(S)This research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by the Madrid Region Government. The authors declare no competing interests.

A chromosome-level, haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate whole-genome assembly

Vanilla planifolia, the species cultivated to produce one of the world’s most popular flavors, is highly prone to partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report here the first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that the diploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequently observed. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gb with a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequencies and the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linking gene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complex molecular regulation of endoreplication along the chromosomes. This high-quality, anchored assembly represents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidation of this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a user-friendly integrated web portal that enables centralized access to high-throughput genomic and other omics data and interoperable use of bioinformatics tools.

Effects of Cartilage Progenitor Cells, Bone Marrow Mesenchymal Stem Cells and Chondrocytes on Cartilage Repair as Seed Cells: An in vitro Study

PurposeTo determine the effects of cartilage progenitor cells, bone marrow mesenchymal stem cells and chondrocytes on cartilage repair as seed cells.MethodsPorcine cartilage progenitor cells (CPCs), bone marrow mesenchymal stem cells (BMSCs) and chondrocytes (CCs) were obtained from the femoropatellar joints of young pigs, and seeded in agarose gel as a graft. During the 28-day culture, proliferation ability was measured by MTT assay, and gene expression of Collagen I, Collagen II, Aggrecan and SOX 9 were measured by qPCR. Qualitative and quantitative analysis of collagen, glycosaminoglycan and DNA were appraised by immunohistochemical staining and biochemical assay, and integration strength was analyzed by push-out tests.ResultsAfter 28-day culture, proliferation ability of CPCs and BMSCs was higher than CCs. Collagen, glycosaminoglycan, DNA content and chondrocyte-related genes expression in the cartilage progenitor cells seeded gel were significantly higher than the other two gels. Integration strength in the cartilage progenitor cells seeded gel was also higher compared with the other two gels.ConclusionCompared with CCs and BMSCs, CPCs in vitro have dominance in the ability of cell proliferation and differentiation as seed cells in tissue engineering.

Targeting IKKε in Androgen-Independent Prostate Cancer Causes Phenotypic Senescence and Genomic Instability.

Advanced prostate cancer will often progress to a lethal, castration-resistant state. We previously demonstrated that IKKε expression correlated with the aggressiveness of prostate cancer disease. Here, we address the potential of IKKε as a therapeutic target in prostate cancer. We examined cell fate decisions (proliferation, cell death, and senescence) in IKKε-depleted PC-3 cells, which exhibited delayed cell proliferation and a senescent phenotype, but did not undergo cell death. Using IKKε/TBK1 inhibitors, BX795 and Amlexanox, we measured their effects on cell fate decisions in androgen-sensitive prostate cancer and androgen-independent prostate cancer cell lines. Cell-cycle analyses revealed a G2-M cell-cycle arrest and a higher proportion of cells with 8N DNA content in androgen-independent prostate cancer cells only. Androgen-independent prostate cancer cells also displayed increased senescence-associated (SA)-β-galactosidase activity; increased γH2AX foci; genomic instability; and altered p15, p16, and p21 expression. In our mouse model, IKKε inhibitors also decreased tumor growth of androgen-independent prostate cancer xenografts but not 22Rv1 androgen-sensitive prostate cancer xenografts. Our study suggests that targeting IKKε with BX795 or Amlexanox in androgen-independent prostate cancer cells induces a senescence phenotype and demonstrates in vivo antitumor activity. These results strengthen the potential of exploiting IKKε as a therapeutic target.

Dysregulated HIC1 and RassF1A expression in vitro alters the cell cytoskeleton and exosomal Piwi-interacting RNA

HIC1 and RassF1A methylation, which cause loss of gene function, are found in various cancers, including renal cell carcinoma (RCC), and could alter cell stiffness and the content of extracellular vesicles (EVs). These physiological changes may provide a tumoral survival advantage and thus could serve as cellular biomarkers for monitoring cell transformation, although direct associations between these changes and cell transformation remain to be established. As we found HIC1 and RassF1A methylation and expression changes in RCC samples, we examined the effects of gain and loss of HIC1 and RassF1A expression on cell DNA content, cytoskeletal structure, and Piwi-interacting RNA (piRNA) expression in EVs. We found HIC1 and RassF1A hypermethylation and abnormal expression in RCC patient samples was independent of the somatic mutations found in publicly available data. Cell stiffness was reduced in accordance with disrupted cytoskeleton conformation after knockdown of HIC1 or RassF1A. Gain or loss of HIC1 expression induced instability in genomic content, abnormal RassF1A expression disturbed cytoskeletal structure, and the abnormal expression of either gene altered piRNA content in EVs. These results suggest a causal relationship between abnormal tumor suppressor gene expression, cell stiffness, and piRNA expression.

Reduced Maternal Circulating Cell-Free Mitochondrial DNA Is Associated With the Development of Preeclampsia.

BackgroundCirculating cell‐free mitochondrial DNA (ccf‐mtDNA) is a damage‐associated molecular pattern that reflects cell stress responses and tissue damage, but little is known about ccf‐mtDNA in preeclampsia. The main objectives of this study were to determine (1) absolute concentrations of ccf‐mtDNA in plasma and mitochondrial DNA content in peripheral blood mononuclear cells and (2) forms of ccf‐mtDNA transport in blood from women with preeclampsia and healthy controls. In addition, we sought to establish the association between aberrance in circulating DNA‐related metrics, including ccf‐mtDNA and DNA clearance mechanisms, and the clinical diagnosis of preeclampsia using bootstrapped penalized logistic regression.Methods and ResultsAbsolute concentrations of ccf‐mtDNA were reduced in plasma from women with preeclampsia compared with healthy controls (P≤0.02), while mtDNA copy number in peripheral blood mononuclear cells did not differ between groups (P>0.05). While the pattern of reduced ccf‐mtDNA in patients with preeclampsia remained, DNA isolation from plasma using membrane lysis buffer resulted in 1000‐fold higher ccf‐mtDNA concentrations in the preeclampsia group (P=0.0014) and 430‐fold higher ccf‐mtDNA concentrations in the control group (P<0.0001). Plasma from women with preeclampsia did not induce greater Toll‐like receptor‐9–induced nuclear factor kappa‐light‐chain enhancer of activated B cells‐dependent responses in human embryonic kidney 293 cells overexpressing the human TLR‐9 gene (P>0.05). Penalized regression analysis showed that women with preeclampsia were more likely to have lower concentrations of ccf‐mtDNA as well as higher concentrations of nuclear DNA and DNase I compared with their matched controls.ConclusionsWomen with preeclampsia have aberrant circulating DNA dynamics, including reduced ccf‐mtDNA concentrations and DNA clearance mechanisms, compared with gestational age–matched healthy pregnant women.

Detoxification of aflatoxin B1 by H2SO3 during maize wet processing, and toxicity assessment of the transformation product of aflatoxin B1

Maize is susceptible to contamination with aflatoxins (AFs). Consumption of AFs contaminated maize can cause poisoning and even death. The goals of this study were to determine the detoxification efficacy of sulfurous acid (H2SO3) on aflatoxin B1 (AFB1) within the range of soaking parameters used in industrial maize production, to identify the transformation product of AFB1 and analyze the cytotoxicity of transformation product. AFB1 contaminated maize was soaked in different concentrations of H2SO3 and the results indicated that soaking time and temperature could significantly influence the detoxification efficiency; 25–31 μg/kg of AFB1 in maize could be detoxified by 0.2–0.3 % of H2SO3. The transformation product was obtained using mass spectrometry and molecular formula analysis. It was identified as C17H14O9S, named as AFB1-HSO3. The toxicity of AFB1-HSO3 was evaluated. Cell morphology showed that the damage caused by AFB1 was significantly greater than that caused by AFB1-HSO3. It also found that the effects of AFB1-HSO3 on the activity, ATP and DNA content of Hep G2 cells were significantly less than that of AFB1. It was concluded that AFB1-HSO3 was less toxic than AFB1 in this system. These results suggest that H2SO3 can be used as a potential detoxification agent in industrial maize production.