Dynamics of actively dividing prokaryotes in the western Mediterranean Sea.

Microbial community metabolism and functionality play a key role modulating global biogeochemical processes. However, the metabolic activities and contribution of actively growing prokaryotes to ecosystem energy fluxes remain underexplored. Here we describe the temporal and spatial dynamics of active prokaryotes in the different water masses of the Mediterranean Sea using a combination of bromodeoxyuridine labelling and 16S rRNA gene Illumina sequencing. Bulk and actively dividing prokaryotic communities were drastically different and depth stratified. Alteromonadales were rare in bulk communities (contributing 0.1% on average) but dominated the actively dividing community throughout the overall water column (28% on average). Moreover, temporal variability of actively dividing Alteromonadales oligotypes was evinced. SAR86, Actinomarinales and Rhodobacterales contributed on average 3-3.4% each to the bulk and 11, 8.4 and 8.5% to the actively dividing communities in the epipelagic zone, respectively. SAR11 and Nitrosopumilales contributed less to the actively dividing than to the bulk communities during all the study period. Noticeably, the large contribution of these two taxa to the total prokaryotic communities (23% SAR11 and 26% Nitrosopumilales), especially in the meso- and bathypelagic zones, results in important contributions to actively dividing communities (11% SAR11 and 12% Nitrosopumilales). The intense temporal and spatial variability of actively dividing communities revealed in this study strengthen the view of a highly dynamic deep ocean. Our results suggest that some rare or low abundant phylotypes from surface layers down to the deep sea can disproportionally contribute to the activity of the prokaryotic communities, exhibiting a more dynamic response to environmental changes than other abundant phylotypes, emphasizing the role they might have in community metabolism and biogeochemical processes.

Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells.

DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3'-single-stranded DNA (3'-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2'-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2'-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

Assaying Cell Cycle Status Using Flow Cytometry.

In this chapter, four methods are described that can be used to assess cell cycle status in flow cytometry. The first method is based on the simultaneous analysis of cellular DNA content using a fluorescent DNA dye (propidium iodide) and of a nuclear proliferation marker (Ki-67). The second is based on the differential staining of DNA and RNA using Hoechst 33342 and Pyronin Y: this method is particularly useful to distinguish quiescent cells in G0 phase from G1 cells. Finally, two methods are described based on DNA incorporation of the synthetic nucleosides BrdU and EdU.

Detection of base analogs incorporated during DNA replication by nanopore sequencing.

DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.

Applicability of the proposed GHS subcategorization criterion for LLNA:BrdU-ELISA (OECD TG442B) to the CBA/J strain mouse.

The Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is a hazard classification and communication system for providing information on the safe handling of chemicals worldwide. In this study, we evaluated the applicability of the newly proposed GHS subcategorization criterion for murine local lymph node assay:2-bromodeoxyuridine enzyme-linked immunosorbent assay (LLNA:BrdU-ELISA), Category 1A:EC1.6 ≤6%, Category 1B:EC1.6 >6%, to data derived from LLNA:BrdU-ELISA performed in the CBA/J strain mouse. Fifteen chemicals categorized in GHS hazard Category 1 sensitizers listed in the LLNA performance standard were tested by LLNA:BrdU-ELISA in the CBA/J strain mouse and were classified according to the new criterion. The results revealed that all of the GHS 1A or 1B category chemicals classified according to the EC3 values derived from radioisotopic LLNA (LLNA-RI) could be correctly assigned into the respective 1A and 1B categories using the newly proposed GHS subclassification criterion. In addition, analysis of the correlation between the reported EC3 values and EC1.6 values derived from the LLNA:BrdU-ELISA performed in the CBA/J strain mouse confirmed the existence of a strong correlation (r = 0.9076, P < .0001). These findings suggest that the newly proposed GHS subcategorization criterion for LLNA:BrdU-ELISA is potentially applicable for practical use in GHS subcategorization.

Stochastic modeling reveals kinetic heterogeneity in post-replication DNA methylation.

DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes.

We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2'-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL-G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.

Effects of lecithin-based nanoemulsions on skin: Short-time cytotoxicity MTT and BrdU studies, skin penetration of surfactants and additives and the delivery of curcumin.

Surfactants are important ingredients in pharmaceutical and cosmetic formulations, as in creams, shampoos or shower gels. As conventional emulsifiers such as sodium dodecyl sulfate (SDS) have fallen into disrepute due to their skin irritation potential, the naturally occurring lecithins are being investigated as a potential alternative. Thus, lecithin-based nanoemulsions with and without the drug curcumin, known for its wound healing properties, were produced and characterised in terms of their particle size, polydispersity index (PDI) and zeta potential and compared to SDS-based formulations. In vitro toxicity of the produced blank nanoemulsions was assessed with primary human keratinocytes and fibroblasts using two different cell viability assays (BrdU and EZ4U). Further, we investigated the penetration profiles of the deployed surfactants and oil components using combined ATR-FTIR/tape stripping experiments and confirmed the ability of the lecithin-based nanoemulsions to deliver curcumin into the stratum corneum in tape stripping-UV/Vis experiments. All manufactured nanoemulsions showed droplet sizes under 250 nm with satisfying PDI and zeta potential values. Viability assays with human skin cells clearly indicated that lecithin-based nanoemulsions were superior to SDS-based formulations. ATR-FTIR tests showed that lecithin and oil components remained in the superficial layers of the stratum corneum, suggesting a low risk for skin irritation. Ex vivo tape stripping experiments revealed that the kind of oil used in the nanoemulsion seemed to influence the depth of curcumin penetration into the stratum corneum.

Symposium review: Determinants of milk production: Understanding population dynamics in the bovine mammary epithelium.

The mammary gland undergoes distinct periods of growth, development, and secretory activity. During bovine lactation, a gradual decrease in the number of mammary epithelial cells largely accounts for the decline in milk production with advancing lactation. The net decline in cell number (approx. 50%) is due to cell death but is simultaneously accompanied by cell renewal. Although the rate of cell proliferation is slow, by the end of lactation most cells in the gland were formed after calving. Typically milking is terminated when cows are in the final 2 mo of pregnancy. This causes regenerative involution, wherein extensive cell replacement and mammary growth occurs. We hypothesized that replacement of senescent secretory cells and progenitor cells during the dry period increases milk yield in the next lactation. Analysis of global gene expression revealed networks and canonical pathways during regenerative involution that support cell turnover and mammary growth, and reflect oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Immune responses consistent with influx of neutrophils, macrophages, and lymphocytes, and processes that support mammary differentiation and lactogenesis were also evident. Data also suggest that replication of stem and progenitor cells occurs during the dry period. Relying on long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine mammary stem cells. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%), predominantly estrogen receptor-negative, and localized in a basal or suprabasal layer of the epithelium. Analyses of gene expression in laser-microdissected LREC are consistent with the concept that LREC represent stem cells and progenitor cells, which differ in properties and location within the epithelial layer. We identified potential markers for these cells and have increased their number by infusing xanthosine through the teat canal of prepubertal heifers. Altering population dynamics of mammary stem and progenitor cells during the mammary cycle may be a means to increase efficiency of milk production.

Cell Proliferation High-Content Screening on Adherent Cell Cultures.

Pulse-chase experiments using 5-bromo-2'-deoxyuridine (BrdU), or the more recent EdU (5-etynil-2'-deoxyuridine), enable the identification of cells going through S phase. This chapter describes a high-content proliferation assay pipeline for adherent cell cultures. High-throughput imaging is followed by high-content data analysis using a non-supervised ImageJ macroinstruction that segments the individual nuclei, determines the nucleoside analogue absence/presence, and measures the signal of up to two additional nuclear markers. Based upon the specific combination with proliferation-specific protein immunostaining, the percentage of cells undergoing different phases of the cell cycle (G0, G1, S, G2, and M) might be established. The method can be also used to estimate the proliferation (S phase) rate of particular cell subpopulations identified through labelling with specific nuclear markers.

A Proximity Ligation-Based Method to Detect RNA-DNA Association.

We recently developed a method for assessing RNA-DNA interactions using proximity ligation assays (PLA). This technique, termed the "RNA-DNA interaction assay" (RDIA), involves differentially labeling DNA and RNA with EdU and BrU, respectively. Once labeled, PLA is performed to assess if the labeled molecules are in close proximity. Here we provide a detailed description of the modified RDIA protocol utilizing currently commercially available BrdU antibodies. As an example, we show its ability to detect nascent transcripts on recently synthesized DNA in both cultured H1299 cells and mouse embryonic stem cells.

DNA Damage Does Not Cause BrdU Labeling of Mouse or Human ß-Cells.

Pancreatic ß-cell regeneration, the therapeutic expansion of ß-cell number to reverse diabetes, is an important goal. Replication of differentiated insulin-producing cells is the major source of new ß-cells in adult mice and juvenile humans. Nucleoside analogs such as BrdU, which are incorporated into DNA during S-phase, have been widely used to quantify ß-cell proliferation. However, reports of ß-cell nuclei labeling with both BrdU and γ-phosphorylated H2A histone family member X (γH2AX), a DNA damage marker, have raised questions about the fidelity of BrdU to label S-phase, especially during conditions when DNA damage is present. We performed experiments to clarify the causes of BrdU-γH2AX double labeling in mouse and human ß-cells. BrdU-γH2AX colabeling is neither an age-related phenomenon nor limited to human ß-cells. DNA damage suppressed BrdU labeling and BrdU-γH2AX colabeling. In dispersed islet cells, but not in intact islets or in vivo, pro-proliferative conditions promoted both BrdU and γH2AX labeling, which could indicate DNA damage, DNA replication stress, or cell cycle-related intrinsic H2AX phosphorylation. Strategies to increase ß-cell number must not only tackle the difficult challenge of enticing a quiescent cell to enter the cell cycle, but also achieve safe completion of the cell division process.

BrdU incorporation in multiparameter flow cytometry: A new cell cycle assessment approach in multiple myeloma.

BACKGROUND: Mutiple myeloma (MM) is a neoplasia characterized by the accumulation of malignant plasma cells (PC) in the bone marrow. Although proliferation markers have been studied in MM, none of the current staging systems include them. Moreover, approaches used to analyze proliferation do not separate MM cells (MMCs) from normal PC. METHODS: In this study, we combined multiparameter flow cytometry and BrdU incorporation or Ki67 staining to analyze MM cell proliferation in 44 monoclonal gammopathy of undetermined significance (MGUS), 153 newly diagnosed MM patients and 69 MM patients at relapse. The prognostic value of proliferation assessment was analyzed in 60 newly diagnosed patients treated with high-dose chemotherapy supported by autologous hematopoietic stem cell transplantation. RESULTS: The median number of proliferating malignant PC significantly increases during MM disease progression. MM patients with a percentage of proliferating MMCs greater than 1.42% using BrdU/DAPI or greater than 1.1% using ki67/DAPI, are associated with a significantly shorter event free survival compared with patients with a lower percentage of proliferating MMCs. CONCLUSIONS: Combination of flow cytometry with BrdU or ki67/DAPI staining could become a standard for the determination of MM cell proliferation. Furthermore, in the context of new effective myeloma treatment options, assessment of MM cell proliferation may be valuable, in clinical trials, to identify novel agents that could significantly affect the small proliferative compartment of MM cells. © 2018 International Clinical Cytometry Society.

Whole-Mount BrdU Staining with Fluorescence In Situ Hybridization in Planarians.

Detection of cell proliferation based on the incorporation of 5'-bromo-2'-deoxyuridine (BrdU) has become a standard approach for studying stem cell and progenitor cell populations in developing and adult tissue. In this chapter, we describe three BrdU administration methods for planarians and a staining protocol combining BrdU detection with whole-mount fluorescence in situ hybridization (FISH). Collectively, these protocols enable the combined analysis of BrdU-incorporation and endogenous gene expression, as for example during lineage tracing applications.

A Suzuki-Miyaura method for labelling proliferating cells containing incorporated BrdU.

The 5-bromo-2'-deoxyuridine (BrdU) incorporation cell proliferation assay is the most commonly used method for assessing DNA replication. The current detection of BrdU in cells relies on antibody immunostaining, but has various limitations including low antibody specificity and poor tissue penetration. In this study, we utilised a Suzuki-Miyaura reaction to develop a chemical method to label cellular BrdU with fluorescent boronic acid probes. The coupling conditions were optimised for complex cellular environments, and the key observation was the need to use oxygen scavengers and zerovalent palladium to prevent side reactions and increase the rate of coupling. The reliability and specificity of the BrdU Suzuki-Miyaura labelling method were verified under various biological conditions. The applicability of the BrdU Suzuki-Miyaura labelling methodology was also investigated, and we show that labelling cellular BrdU is highly sensitive and reliable, which is comparable to the ideal performance of BrdU immunostaining. Moreover, the Suzuki-Miyaura reaction protocol provides high BrdU recognition specificity. Taken together, the BrdU Suzuki-Miyaura labelling protocol provides an attractive alternative to the more traditional cell proliferation assay.

A Mild Aqueous Sonogashira Reaction as a Fluorescent Labeling Strategy for 5-Bromide-2'-Deoxyuridine.

C5-modified uridines are a valuable class of nucleoside analogues, both as potent chemotherapy agents and through their use as the conjunction site in DNA labeling strategies. As an important C5-modified uridine, BrdU has been used in cell proliferation assays since the 1980s. Currently, the detection of BrdU relies on traditional immunostaining; however, this approach has its limitations. Thus, it is desirable, albeit difficult, to develop chemistry methods to fluorescently label BrdU in a cellular context. In the present study, we report our efforts toward developing a robust chemistry methodology for BrdU fluorescent labeling. The Sonogashira reaction was chosen as the key reaction, and various alkynyl groups (aliphatic or aryl) containing fluorescent dyes were synthesized to cross-couple with BrdU. Various bases and catalyst systems were screened to evaluate the optimum conditions. A mild aqueous Sonogashira reaction (K2PdCl4, S-Phos, n-Bu4N⁺OH-, Sodium d-isoascorbate, EtOH/H2O = 1:1, 37 °C, Ar) was obtained to enable high-yielding BrdU fluorescent labeling.

HepG2 and human healthy hepatocyte in vitro culture and co-culture in PCL electrospun platforms.

The discovery of new drugs to treat pathological cells in the case of aggressive liver primary cancer is imposing the identification of high-throughput screening systems to predict the in vivo response of new therapeutic molecules, in order to reduce current use of animals and drug testing costs. Recently, micro/nanostructured scaffolds have been adopted to reproduce the hepatic microenvironment due to their higher similarity to the biological niche with respect to the traditional two-dimensional culture plate, so providing novel in vitro models for reliably understanding molecular mechanisms related to cancer cells activity. Herein, we propose the study of electrospun scaffolds made of polycaprolactone as in vitro model that can mimic the morphological organization of native extracellular matrix and the co-culture of hepatic cell lines-i.e., HepG2, human healthy hepatocytes (HHH). The micro- and nano-scale morphological features of fibers with diameter equal to (3.22 ± 0.42) µm and surface roughness of (17.84 ± 4.43) nm-allow the reproduction of the in vivo scenario influencing the adhesion and proliferation rate of the cultured cells. A much lower proliferation rate is observed for the HepG2 cells compared to the HHH cells, when cultured on the fibrous scaffolds over a time course of 4 weeks. Moreover, results on oxidative stress mechanisms indicate an antioxidant effect of fibers mainly in the case of co-colture, thus suggesting a promising use as new in vitro models to explore alternative therapeutic strategies in hepatocarcinoma treatment.

CyTOF Mass Cytometry for Click Proliferation Assays.

Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single-cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S-phase determination in cell cycle applications, combining well-established click chemistry with a novel iodo-deoxyuridine (IdU) azide derivative and a cross-reactive anti-IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU-based assays for complex and multiparametric experiments. It provides a feasible cost-effective approach for detecting ethynyl-labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.