BrdU Labeling Research Articles

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

JAK2V617F Mutant Megakaryocytes Contribute to Hematopoietic Aging in a Murine Model of Myeloproliferative Neoplasm

IntroductionRecent studies implicated megakaryocytes (MKs) in regulating hematopoietic stem cell (HSC) function. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). In the present study, we investigated the effects of JAK2V617F-bearing MKs on HSC aging in a murine model of MPN during a 2-yr follow up.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) and Pf4-Cre mice (which express Cre under the promoter of platelet factor 4, a MK-specific gene) were crossed to generate MK lineage-specific human JAK2V617F transgenic mouse line (Pf4 +FF1 +).ResultsDuring aging, the Pf4 +FF1 +mice maintained an essential thrombocythemia phenotype with no evidence of transformation to leukemia or myelofibrosis (Figure 1).Myeloid-biased HSCs (Lin -cKit +Sca1 +CD150 +CD48 -CD41 +) were significantly expanded in 1yr old and 2yr old Pf4 +FF1 +mice compared to age-matched control mice, while no difference in myeloid-biased HSC numbers was detected between young (6mo) Pf4 +FF1 +mice and control mice. Competitive repopulation assays and serial transplantation assays showed that HSCs from old Pf4 +FF1 +mice had a reduced engraftment and self-renewal capacity with a skewed differentiation towards the myeloid lineage. Results from the serial transplantation experiments also indicated that the functional decline of HSCs in aged Pf4 +FF1 +mice were HSC-intrinsic and was not reversible. Both cell cycle analysis by Hoechst33342 and Pyronin Y staining and cell proliferation analysis by in vivo BrdU labeling revealed that the JAK2V617F mutant MK niche can promote hematopoietic aging in old Pf4 +FF1 +mice by increasing HSC proliferation/cycling. Taken together, the Pf4 +FF1 +mice demonstrated several hallmarks of accelerated HSC aging.(Figure 2)Next, we examined the hematopoietic microenvironment in the old Pf4 +FF1 + mice. Using whole-mount immunofluorescent staining and confocal imaging, we found that the cKit +HSPCs were located further from MKs in old Pf4 +FF1 +mice compared to old control mice. We also found that CD45 -CD31 +Sca1 - sinusoidal marrow endothelial cell (EC) number (by flow cytometry analysis) and marrow vascular area (by in vivo VE-cadherin staining) were significantly decreased in aged Pf4 +FF1 +mice compared to control mice. Tube formation assays confirmed that conditioned medium from old Pf4 +FF1 +MK culture significantly inhibited EC angiogenesis in vitro. These findings suggest that the JAK2V617F mutant MK niche not only altered its own interaction with HSCs during aging, but also suppressed the vascular niche function to promote HSC aging. (Figure 3)To further understand the mechanisms by which JAK2V617F mutant MKs promote HSC aging, we performed both RNA sequencing and targeted cytokine arrays of wild-type and JAK2V617F mutant MKs from both young (6mo) and old (2yr) Pf4-cre control and Pf4 +FF1 +mice. We found that HSC aging had a profound effect on MK transcriptomic profiles and dysregulated pathways in cell adhesion molecules, MAPK signaling, NF-kappa B signaling, hematopoietic cell lineage, and cytokine-cytokine receptor interaction were highly upregulated in old JAK2V617F mutant MKs compared to young JAK2V617F mutant MKs. FAS ligand, IL-12, tissue inhibitor of metalloproteinases-1, IL-10, IL-6, MIG, macrophage inflammatory protein 1a, GCSF, IL-5, MIP-1g were produced in increased amounts in old mutant MKs compared to old wild-type MKs. Many of these factors are involved in inflammation, angiogenesis, extracellular matrix remodeling, and hematopoiesis regulation.Sensitive PCR assays confirmed that human JAK2 gene was expressed in MKs but not in HSCs in 2yr old Pf4 +FF1 +mice. In vitro co-culture and in vivo co-transplantation assays provided further evidence that the JAK2V617F mutant MKs affected wild-type HSC function directly. Therefore, the hematopoietic aging phenotype we have observed was not caused by any direct effect of the JAK2V617F mutation on HSC function because the Pf4 promoter was ‘leaky’.ConclusionsResults from this study support that, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging in MPNs – the JAK2V617F-bearing MKs can alter the hematopoietic niche to accelerate HSC aging, and HSC aging in turn can profoundly remodel the niche e.g. by affecting MK transcriptomics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ascorbic acid ameliorates corneal endothelial dysfunction and enhances cell proliferation via the noncanonical GLUT1-ERK axis

BackgroundThe pumping function of corneal endothelial cells (CECs) plays a pivotal role in the maintenance of corneal water homeostasis. Corneal endothelial dysfunction (CED) leads to corneal edema and opacity, but with the exception of keratoplasty, no optimal therapeutic strategies have been established for CED. In this study, we aimed to investigate the ameliorative effect of ascorbic acid (AA) on CED and the underlying mechanism of action in the corneal endothelium. MethodsRabbit corneal endothelial damage was induced by anterior chamber injection of benzalkonium chloride (BAK). AA was topically administered to the corneal surface, and the transparency and thickness of the cornea were assessed by external eye photography, slit-lamp photography, and ultrasonic pachymetry. To further analyze the mechanism, rabbit CECs and immortalized human CECs (B4G12 cells) were cultured. A ferric reducing/antioxidant and AA (FRASC) assay was performed to measure the AA concentration. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (BrdU) labeling assays, and protein expression was examined by liquid chromatography–mass spectrometry (LC/MS) and immunoblotting. The involvement of glucose transporter 1 (GLUT1) and phospho-ERK was evaluated via GLUT1-siRNA and phospho-ERK inhibitor (PD98059) treatment. InterpretationWe observed that topical AA ameliorates BAK-induced rabbit corneal endothelial damage. Furthermore, we demonstrated that AA is transported into B4G12 cells via GLUT1, and afterward, AA increases ERK phosphorylation and promotes cell proliferation. Our findings indicate that CEC proliferation stimulated via the noncanonical AA-GLUT1-ERK axis contributes to AA-enhanced healing of CED.

Matricellular Protein Cilp1 Promotes Myocardial Fibrosis in Response to Myocardial Infarction.

[Figure: see text].

The Proliferation of Dentate Gyrus Progenitors in the Ferret Hippocampus by Neonatal Exposure to Valproic Acid.

Prenatal and neonatal exposure to valproic acid (VPA) is associated with human autism spectrum disorder (ASD) and can alter the development of several brain regions, such as the cerebral cortex, cerebellum, and amygdala. Neonatal VPA exposure induces ASD-like behavioral abnormalities in a gyrencephalic mammal, ferret, but it has not been evaluated in brain regions other than the cerebral cortex in this animal. This study aimed to facilitate a comprehensive understanding of brain abnormalities induced by developmental VPA exposure in ferrets. We examined gross structural changes in the hippocampus and tracked proliferative cells by 5-bromo-2-deoxyuridine (BrdU) labeling following VPA administration to ferret infants on postnatal days (PDs) 6 and 7 at 200 μg/g of body weight. Ex vivo short repetition time/time to echo magnetic resonance imaging (MRI) with high spatial resolution at 7-T was obtained from the fixed brain of PD 20 ferrets. The hippocampal volume estimated using MRI-based volumetry was not significantly different between the two groups of ferrets, and optical comparisons on coronal magnetic resonance images revealed no differences in gross structures of the hippocampus between VPA-treated and control ferrets. BrdU-labeled cells were observed throughout the hippocampus of both two groups at PD 20. BrdU-labeled cells were immunopositive for Sox2 (>70%) and almost immunonegative for NeuN, S100 protein, and glial fibrillary acidic protein. BrdU-labeled Sox2-positive progenitors were abundant, particularly in the subgranular layer of the dentate gyrus (DG), and were denser in VPA-treated ferrets. When BrdU-labeled Sox2-positive progenitors were examined at 2 h after the second VPA administration on PD 7, their density in the granular/subgranular layer and hilus of the DG was significantly greater in VPA-treated ferrets compared to controls. The findings suggest that VPA exposure to ferret infants facilitates the proliferation of DG progenitors, supplying excessive progenitors for hippocampal adult neurogenesis to the subgranular layer.

Bromodeoxyuridine Labelling to Determine Viral DNA Localization in Fluorescence and Electron Microscopy: The Case of Adenovirus.

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), which are incorporated into the nucleic acids during replication or transcription. In adenovirus infections, BrdU has been used to localize newly synthesized viral genomes in the nucleus, where it is key to distinguish between host and viral DNA. Here, we describe our experience with methodological variations of BrdU labeling to localize adenovirus genomes in fluorescence and electron microscopy. We illustrate the need to define conditions in which most of the newly synthesized DNA corresponds to the virus and not the host, and the amount of BrdU provided is enough to incorporate to the new DNA molecules without hampering the cell metabolism. We hope that our discussion of problems encountered and solutions implemented will help other researches interested in viral genome localization in infected cells.

Inflammation- and Gut-Homing Macrophages, Engineered to De Novo Overexpress Active Vitamin D, Promoted the Regenerative Function of Intestinal Stem Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gut. Available drugs aim to suppress gut inflammation. These drugs have significantly delayed disease progression and improved patients’ quality of life. However, the disease continues to progress, underscoring the need to develop novel therapies. Aside from chronic gut inflammation, IBD patients also experience a leaky gut problem due to damage to the intestinal epithelial layer. In this regard, epithelial regeneration and repair are mediated by intestinal stem cells. However, no therapies are available to directly enhance the intestinal stem cells’ regenerative and repair function. Recently, it was shown that active vitamin D, i.e., 1,25-dihydroxyvitamin D or 1,25(OH)2D, was necessary to maintain Lgr5+ intestinal stem cells, actively cycling under physiological conditions. In this study, we used two strategies to investigate the role of 1,25(OH)2D in intestinal stem cells’ regenerative function. First, to avoid the side effects of systemic high 1,25(OH)2D conditions, we used our recently developed novel strategy to deliver locally high 1,25(OH)2D concentrations specifically to inflamed intestines. Second, because of the Lgr5+ intestinal stem cells’ active cycling status, we used a pulse-and-chase strategy via 5-bromo-2′-deoxyuridine (BrdU) labeling to trace the Lgr5+ stem cells through the whole epithelial regeneration process. Our data showed that locally high 1,25(OH)2D concentrations enhanced intestinal stem cell migration. Additionally, the migrated cells differentiated into mature epithelial cells. Our data, therefore, suggest that local delivery of high 1,25(OH)2D concentrations is a promising strategy to augment intestinal epithelial repair in IBD patients.

Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines. Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway. We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes. Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.

Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.

Crohn’s Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 μM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.

Dynamics of Spermatogenesis and Change in Testicular Morphology under 'Mating' and 'Non-Mating' Conditions in Medaka (Oryzias latipes).

Here, we report that the gross morphology of the testes changes under 'non-mating' or 'mating' conditions in medaka (Oryzias latipes). During these conditions, an efferent duct expands and a histological unit of spermatogenesis, the lobule, increases its number under 'non-mating' conditions. Based on BrdU labeling experiments, lower mitotic activity occurs in gonial cells under 'non-mating' conditions, which is consistent with the reduced number of germ cell cysts. Interestingly, the total number of type A spermatogonia was maintained, regardless of the mating conditions. In addition, the transition from mitosis to meiosis may have been retarded under the 'non-mating' conditions. The minimum time required for germ cells to become sperm, from the onset of commitment to spermatogenesis, was approximately 14 days in vivo. The time was not found to significantly differ between 'non-mating' and 'mating' conditions. The collective data suggest the presence of a mechanism wherein the homeostasis of spermatogenesis is altered in response to the mating conditions.

Ddx20, DEAD box helicase 20, is essential for the differentiation of oligodendrocyte and maintenance of myelin gene expression.

Oligodendrocytes form myelin sheaths that surround axons, contributing to saltatory conduction and proper central nervous system (CNS) function. Oligodendrocyte progenitor cells (OPCs) are generated during the embryonic stage and differentiate into myelinating oligodendrocytes postnatally. Ddx20 is a multifunctional, DEAD-box helicase involved in multiple cellular processes, including transcription, splicing, microRNA biogenesis, and translation. Although defects in each of these processes result in abnormal oligodendrocyte differentiation and myelination, the involvement of Ddx20 in oligodendrocyte terminal differentiation remains unknown. To address this question, we used Mbp-Cre mice to generate Ddx20 conditional knockout (cKO) mice to allow for the deletion of Ddx20 from mature oligodendrocytes. Mbp-Cre;Ddx20 cKO mice demonstrated small body sizes, behavioral abnormalities, muscle weakness, and short lifespans, with mortality by the age of 2months old. Histological analyses demonstrated significant reductions in the number of mature oligodendrocytes and drastic reductions in the expression levels of myelin-associated mRNAs, such as Mbp and Plp at postnatal day 42. The number of OPCs did not change. A thin myelin layer was observed for large-diameter axons in Ddx20 cKO mice, based on electron microscopic analysis. A bromodeoxyuridine (BrdU) labeling experiment demonstrated that terminal differentiation was perturbed from ages 2 weeks to 7 weeks in the CNS of Mbp-Cre;Ddx20 cKO mice. The activation of mitogen-activated protein (MAP) kinase, which promotes myelination, was downregulated in the Ddx20 cKO mice based on immunohistochemical detection. These results indicate that Ddx20 is an essential factor for terminal differentiation of oligodendrocytes and maintenance of myelin gene expression.

Abstract 3040: Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma

Abstract Background: In many adult cancers, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Our aims are to identify patients most likely to benefit from selinexor combination therapy and define the cellular mechanism responsible this effect. We hypothesize that selinexor and the proteasome inhibitor, bortezomib, act synergistically to decrease NF-kB-mediated proliferation by halting excessive exportation and proteasome degradation of IkB, an XPO1 cargo protein. Methods: We performed ion intensity-based label-free semiquantitative proteomic profiling on primary tumor material from fifty patients with uniformly treated high-risk NB, half of whom were long term survivors and half of whom died <18 months from diagnosis from disease progression. We cross-referenced this dataset with gene expression profiling data available through the R2 database. A panel of neuroblastoma cell lines was screened for XPO1 expression using Western blotting, sensitivity to selinexor and bortezomib as single agents, and in combination using cellular viability assays. Localization and quantification of IkB after vehicle or selinexor treatment was done by confocal imaging, using immunofluorescent labeling and Volocity software. Flow cytometry was used to define treatment effect on cell cycle arrest and apoptosis using BrdU and Annexin-V labeling, respectively. siRNA-mediated knockdown of IkB was used to investigate the relationship between presence of IkB and drug effectiveness. NF-kB activity was measured with a luciferase assay. Results: High tumor protein and gene expression of XPO1 correlates with poor outcome. Neuroblastoma cell lines demonstrate variably high XPO1 expression compared to normal tissue, as well as sensitivity to selinexor (IC50 range 4-300nM). IkB is retained in the nuclear compartment after treatment with selinexor. When selinexor is used in combination with bortezomib the effect on cell proliferation is synergistic. Flow cytometry demonstrates that selinexor alone contributes to G1-0 arrest while combination treatment results in significantly enhanced apoptosis. Measurement of NF-kB transcriptional activity shows decrease in activity with single agents and the least amount of activity in combination and siRNA against IkB diminishes the effect of single and combination treatment. Conclusions: This work demonstrates that XPO1 expression is a biomarker of highly aggressive neuroblastoma and may define patients most likely to be responsive to treatment with selinexor-based combinatorial therapies. Synergistic treatment effect with selinexor and bortezomib is mediated, in part, through the presence of IkB to regulate NF-kB signaling. Citation Format: Basia Galinski, Yossef Landesman, Daniel Weiser. Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3040.

Effect of Tongfu Xingshen capsule on the endogenous neural stem cells of experimental rats with intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) can stimulate neural regeneration, promoting tissue repair and recovery of nerve function. Tongfu Xingshen capsule (TXC) is a Chinese medicinal formula used to treat ICH and has been shown to protect brain tissue and improve nerve function in clinical studies. However, the effect of TXC on endogenous neural stem cells (NSCs) remains elusive. To explore the mechanisms underlying TXC action, a rat model of ICH was established. The effects of TXC on the proliferation and differentiation of NSCs were assessed in the subventricular zone (SVZ). TXC significantly improved nerve function defects, decreased brain water content and restored blood-brain barrier integrity. Additionally, BrdU labeling showed that both high and low doses of TXC significantly increased the proportion of actively cycling NSCs positive for Nestin and glial fibrillary acidic protein, but did not affect the proliferation rates of NeuN-positive neurons. Finally, TXC also upregulated the mRNA levels of brain-derived neurotrophic factor and its receptor, TrκB, in affected brain tissues. Taken together, TXC accelerated neural repair and functional recovery after brain injury by potentially enhancing the proliferation and differentiation of endogenous NSCs into astroglial cells in the SVZ area.

MCM2-7 and FANCD2 Are Required for RAD51 Function in Homologous Recombination and ICL Repair

MCM2-7 helicase is required for DNA replication and has been implicated in the maintenance of genomic instability, as more than 90% of MCM complexes are in dormant origins. Decreased levels of MCM2-7 complex on chromatin lead to genomic instability. The Fanconi anemia (FA) pathway, whose proteins are defective in the genetic disorder that entails bone marrow failure, congenital defects, and acute myeloid leukemia, is also important in maintaining genomic instability through the regulation of homologous recombinatorial repair (HRR) of interstrand cross links (ICL). FANCD2 is the central protein in the FA pathway, exhibiting monoubiquitination in response to DNA damage, and has been shown to interact with MCM2-7. Here we show that monoubiquitinated FANCD2 interacts with MCM2-7 and is required for maintenance of MCM2-7 on chromatin upon DNA interstrand crosslinking (ICL) drug mitomycin C treatment. FANCD2-MCM2-7 also interacts with RAD51, an interaction which is decreased in the absence of FANCD2 or with the expression of the ubiquitination mutant FANCD2(K561R). Also, RAD51 chromatin localization and homologous recombination (HR) function is impaired in MCM7 knockdown cells. MCM2, MCM5, or MCM7 knock down cells exhibit hypersensitive to ICL and G2/M accumulation as well, and the hypersensitivity of MCM7 knock down cells can be rescued by re-expressing MCM7. Importantly FANCD2 and MCM7 are epistatic for MMC hypersensitivity. At the levels of MCM7 knock down, we observe that cells are not defective for replication as measured by BrdU labeling and are not hypersensitive to replication inhibitor hydroxyurea (HU) but remain ICL sensitive. Using recombinant protein reconstitution in vitro, we observe MCM2-7 and FANCI-FANCD2 dependent RAD51 filament stabilization and strand exchange, which are key steps in HRR. MCM2-7 proteins defective for helicase activity and thus replication defective remain functional in these in vitro biochemical assays. On the other hand, a FANCI DNA binding mutant completely abrogates these activities, suggesting that the FANCD2-FANCI-MCM2-7-RAD51 complex requires a regulated interaction with DNA and is separable from MCM2-7 replication function. Taken together, the FA pathway, MCM2-7, and RAD51 work cooperatively in the biochemical and cell response to DNA damage ICL. DisclosuresNo relevant conflicts of interest to declare.

HDAC8 Regulates Long-Term Hematopoietic Stem Cell Maintenance Under Stress By Modulating p53 Activity

The functional integrity and maintenance of long-term hematopoietic stem cells (LT-HSC) is dependent on a critical balance of quiescence, apoptosis and proliferation states. As a member of the class I histone deacetylase (HDAC), HDAC8 modulates acetylation of lysine residues, a protein modification important for regulation of numerous biological processes. In this study, we show that Hdac8 is highly expressed in the phenotypic LT-HSC (Lin-cKit+Sca1+CD150+CD48-) population in the bone marrow (BM) of adult mice. To determine the function of HDAC8 in adult hematopoiesis, we generated conditional Hdac8 deficient mice using the Mx1-Cre and a floxed Hdac8 allele (Mx1-Cre/Hdac8f/f(y)) andconfirmed that Hdac8 is successfully deleted (Hdac8 △/△ ) after induction with polyinosinic polycytidylic acid (14 mg/kg, 7 doses). We observed largely normal hematopoietic differentiation capacity of Hdac8 △/△ cells. However, the frequency of phenotypic LT-HSC population (0.04968% vs. 0.03575%; n=10-13; p=0.0038) was significantly higher 6 weeks after Hdac8 deletion and the expansion shifted to the phenotypic multipotent progenitor population by one year (0.1571% vs. 0.06683%; n=5-8; p=0.0025). In vitro colony-forming cell (CFC) assay demonstrated that while Hdac8 △/△ and control progenitorsgenerated similar numbers of total CFC in the first plating, significantly reduced Hdac8 △/△ CFC were seen in secondary and tertiary platings. Using Mx1-Cre/Hdac8f/f(y) mice with a dual fluorescence Rosa26mT/mG (mT/mG) Cre reporter allele (GFP+ after deletion) in hematopoietic repopulation assays, we found similar frequencies and numbers of GFP+ donor-derived lineage populations and phenotypic stem and progenitor cells. However, Hdac8 △/△ GFP+ donor-derived multi-lineage repopulation was significantly compromised upon secondary (p=0.0227, n=3) and tertiary (p=0.0109, n=5-8) transplantation. Cell cycle analysis by in vivo BrdU labeling revealed that Hdac8 deficient LT-HSCs display reduced quiescence (35.36% vs. 56.15% G0; n=5-6; p=0.01918) and increased cycling (45.36% vs. 28.03% S phase; n=5-6; p=0.0077), consistent with significantly reduced expression of Cdkn1a (p=0.0025) and Necdin (p=0.019). These results indicate that Hdac8 deletion perturbs homeostasis of LT-HSC and hematopoietic output over time.We previously showed that Hdac8 functions to deacetylate p53 protein in inv(16) acute myeloid leukemia. Thus, we hypothesize that Hdac8 could perhaps modulate p53 activity in LT-HSC. Co-immunoprecipitation assay revealed that Hdac8 binds to the p53 protein, which is strongly enhanced upon irradiation (IR; 3Gy). Enhanced p53 acetylation and increased p53 target gene expression including Bax, Noxa, Puma and Cdkn1a were seen after IR in Hdac8 △/△ BM cells. Specifically, Hdac8 △/△ LT-HSCs are significantly more sensitive to apoptosis after IR. We crossed Mx1-Cre/Hdac8f/f(y) mice with the p53ERTAM knock-in (KI) mice carrying a p53 fusion protein completely dependent on ectopic 4-hydroytamoxifen. Inactivation of p53 in Hdac8 △/△ /p53ERKI/KI LT-HSCs reversed the increased IR-induced apoptosis and elevated expression of Noxa and Puma seen in Hdac8 △/△ LT-HSCs. To test whether Hdac8 modulates p53 activation via its deacetylase activity, we treated control BM cells with an HDAC8-selective inhibitor (HDAC8i) targeting HDAC8 catalytic site before exposure to IR (3Gy). Treatment with HDAC8i significantly enhanced the responsiveness of p53 targets to IR, supporting that these effects are dependent on HDAC8 deacetylase activity. Loss of quiescence and hyperactivation of p53 could render LT-HSC higher sensitivity to hematopoietic stressors such as ablation with 5-fluorouracil (5-FU). We tested this by treating control, Hdac8 △/△, or Hdac8 △/△ /p53ERKI/KI mice with serial 5-FU (100 mg/kg) every 7 days. Dramatically impaired hematopoietic recovery (white blood cell, red blood cell and platelet counts) and increased lethality was seen in Hdac8 △/△ mice challenged with serial 5-FU treatment (75% Hdac8 △/△ , n=30 vs. 5.9% control n=34). This hypersensitivity to hematopoietic ablation is completely rescued in Hdac8Δ/Δ/p53ERKI/KI mice (no lethality seen,n=7), indicating that this effect is indeed mediated by p53. Altogether, this study highlights HDAC8 as a novel regulator of LT-HSC in that Hdac8 functions to modulate p53 activity in LT-HSCs to ensure cell survival and maintenance under stress. DisclosuresNo relevant conflicts of interest to declare.

Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed

Nontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including Vibrio spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2′-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active Vibrio spp. in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active Vibrio spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of V. cholerae, V. vulnificus, and V. parahaemolyticus using both methods, while V. aesturianus and V. shilonii were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including P. shigelloides, B. cereus and E. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in Vibrio spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active Vibrio spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.

Characterization of substantia nigra neurogenesis in homeostasis and dopaminergic degeneration: beneficial effects of the microneurotrophin BNN-20

BackgroundLoss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) underlines much of the pathology of Parkinson’s disease (PD), but the existence of an endogenous neurogenic system that could be targeted as a therapeutic strategy has been controversial. BNN-20 is a synthetic, BDNF-mimicking, microneurotrophin that we previously showed to exhibit a pleiotropic neuroprotective effect on the dopaminergic neurons of the SNpc in the “weaver” mouse model of PD. Here, we assessed its potential effects on neurogenesis.MethodsWe quantified total numbers of dopaminergic neurons in the SNpc of wild-type and “weaver” mice, with or without administration of BNN-20, and we employed BrdU labelling and intracerebroventricular injections of DiI to evaluate the existence of dopaminergic neurogenesis in the SNpc and to assess the origin of newborn dopaminergic neurons. The in vivo experiments were complemented by in vitro proliferation/differentiation assays of adult neural stem cells (NSCs) isolated from the substantia nigra and the subependymal zone (SEZ) stem cell niche to further characterize the effects of BNN-20.ResultsOur analysis revealed the existence of a low-rate turnover of dopaminergic neurons in the normal SNpc and showed, using three independent lines of experiments (stereologic cell counts, BrdU and DiI tracing), that the administration of BNN-20 leads to increased neurogenesis in the SNpc and to partial reversal of dopaminergic cell loss. The newly born dopaminergic neurons, that are partially originated from the SEZ, follow the typical nigral maturation pathway, expressing the transcription factor FoxA2. Importantly, the pro-cytogenic effects of BNN-20 were very strong in the SNpc, but were absent in other brain areas such as the cortex or the stem cell niche of the hippocampus. Moreover, although the in vitro assays showed that BNN-20 enhances the differentiation of NSCs towards glia and neurons, its in vivo administration stimulated only neurogenesis.ConclusionsOur results demonstrate the existence of a neurogenic system in the SNpc that can be manipulated in order to regenerate the depleted dopaminergic cell population in the “weaver” PD mouse model. Microneurotrophin BNN-20 emerges as an excellent candidate for future PD cell replacement therapies, due to its area-specific, pro-neurogenic effects.

Studies on the role of alpha 7 nicotinic acetylcholine receptors in K562 cell proliferation and signaling.

The results we obtained from this study gave information about the determination of alpha 7 nicotinic acetylcholine receptor (α7-nACh) expression in human erythroleukemia cells, as well as whether it has a role in calcium release and cell proliferation in the presence of nicotinic agonist, antagonists. Determining the roles of α7 nicotinic receptors in erythroleukemia cells will also contribute to leukemia-related signal transduction studies. This study is primarily to determine the role of nicotinic agonists and antagonists in cell proliferation, α7 nicotinic acetylcholine receptor expression, and calcium release. The aim of this study, which is a continuation and an important part of our previous studies on the cholinergic system, has contributed to the literature on the human erythroleukemia cell signaling mechanism. Cell viability was evaluated by the trypan blue exclusion test and Bromodeoxyuridine/5-Bromo-2'-deoxyuridine (BrdU) labeling. Acetylcholine, nicotinic alpha 7 receptor antagonist methyllycaconitine citrate, and cholinergic antagonist atropine were used to determine the role of α7-nACh in K562 cell proliferation. In our experiments, the fluorescence spectrophotometer was used in Ca2+ measurements. The expression of nicotinic alpha 7 receptor was evaluated by western blot. The stimulating effect of acetylcholine in K562 cell proliferation was reversed by both the α7 nicotinic antagonist methyllycaconitine citrate and the cholinergic antagonist, atropine. Methyllycaconitine citrate inhibited K562 cell proliferation partially explained the roles of nicotinic receptors in signal transduction. While ACh caused an increase in intracellular Ca2+, methyllycaconitine citrate decreased intracellular Ca2+ level in K562 cell. The effects of nicotinic agonists and/or antagonists on erythroleukemic cells on proliferation, calcium level contributed to the interaction of nicotinic receptors with different signaling pathways. Proliferation mechanisms in erythroleukemic cells are under the control of the α7 nicotinic acetylcholine receptor via calcium influx and different signalling pathway.

A Matrix Metalloproteinase Mediates Tracheal Development in Bombyx mori.

The trachea of insects is a tubular epithelia tissue that transports oxygen and other gases. It serves as a useful model for the studying of the cellular and molecular events involved in epithelial tube formation. Almost all of the extracellular matrix can be degraded by Matrix metalloproteinases (MMPs), which is closely related to the processes of development and regeneration. The regulation of trachea by MMPs is roughly known in previous studies, but the detailed regulation mechanism and involved gene function are not fully explored. In this article, we found MMP1 expressed highly during tracheal remodeling, and knocked out it makes the tracheal branch number reduced in Bombyx mori. In trachea of transgenic BmMMP1-KO silkworm, the space expanding of taenidium and epidermal cells and the structure of apical membrane were abnormal. To explore the underlying mechanism, we detected that DE-cadherin and Integrin β1 were accumulated in trachea of transgenic BmMMP1-KO silkworm by immunohistochemistry. Moreover, 5-Bromo-2′-Deoxyuridine (BrdU) labeling showed that knockout of BmMMP1 in silkworm inhibited tracheal cell proliferation, and BmMMP1 also regulated the proliferation and migration of BmNS cells. All of the results demonstrated that BmMMP1 regulates the development of the tracheal tissue by expanding the space of tracheal cuticles and increases the number of tracheal branches by degrading DE-cadherin and Integrin β1.

Mild therapeutic hypothermia improves neurological outcomes in a rat model of cardiac arrest

Cardiac arrest (CA) is the leading cause of death in humans. Research has shown that mild therapeutic hypothermia (MTH) can reduce neurological sequelae and mortality after CA. Nevertheless, the mechanism remains unclear. This study aimed to determine whether MTH promotes neurogenesis, attenuates neuronal damage, and inhibits apoptosis of neurons in rats after CA. Sprague–Dawley rats were divided into the normothermia and mild hypothermia groups. The rats in the normothermia and hypothermia groups were exposed to 2 h of normothermia (36–37℃) and hypothermia (32–33℃), respectively, immediately after resuscitation from 5 min of asphyxial CA. Corresponding control groups not subjected to CA were included. On days 1–6, 5-bromodeoxyuridine (BrdU) 100 mg/kg/day was administered intraperitoneally. The animals were euthanized 1 week after CA. Compared with the normothermia group, the hypothermia group showed a significant increase in the number of doublecortin (DCX) immune-positive cells in the subgranular zone of the hippocampus 1 week after CA. Neurogenesis was assessed using double immunofluorescent labeling of BrdU with neuronal-specific nuclear protein (NeuN)/DCX. There was no marked change in the number of newborn mature (BrdU+-NeuN+) neurons, though there was a significant increase in the number of newborn immature (BrdU+-DCX+) neurons in the hypothermia than in the normothermia group 1 week after CA. Neuronal injury and apoptosis in the CA1 region of the hippocampus, assessed using NeuN immunofluorescence and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays, were significantly reduced in the hypothermia group 1 week after CA. Moreover, mild hypothermia increased the expression of cold-shock protein RNA-binding motif protein 3 (RBM3) in the early stage (24 h/48 h) after CA. These results suggested that mild hypothermia promotes generation of neuronal cells, reduces neuronal injury, and inhibits apoptosis of neurons, which may be related to RBM3 expression.