Repli-seq Sample Preparation using Cell Sorting with Cell-Permeant Dyes.

Replication timing is significantly correlated with gene expression and chromatin organization, changes dynamically during cell differentiation, and is altered in diseased states. Genome-wide analysis of replication timing is performed in actively replicating cells by Repli-seq. Current methods for Repli-seq require cells to be fixed in large numbers. This is a barrier for sample types that are sensitive to fixation or are in very limited numbers. In this article, we outline optimized methods to process live cells and intact nuclei for Repli-seq. Our protocol enables the processing of a smaller number of cells per sample and reduces processing time and sample loss while obtaining high-quality data. Further, for samples that tend to form clumps and are difficult to dissociate into a single-cell suspension, we also outline methods for isolation, staining, and processing of nuclei for Repli-seq. The Repli-seq data obtained from live cells and intact nuclei are comparable to those obtained from the standard protocols. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Live cell isolation and staining Alternate Protocol: Nuclei isolation and staining.

Epigenomic signatures associated with spontaneous and replication stress-induced DNA double strand breaks.

Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced in vitro by mild level of DNA replication stress, such as DNA polymerase inhibition or nucleotide pool disturbance. The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300 kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.

You shall not pass! Unveiling the barriers for cohesin-mediated loop extrusion.

Dequeker and colleagues performed elegant in vivo, in silico, and in vitro experiments to demonstrate that the MCM complex, an essential DNA replication factor, is an obstacle for the DNA loop formation by cohesin.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands.

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Towards a model for aerosol removal by rain scavenging: The role of physical-chemical characteristics of raindrops.

A one-year study was carried out in León, Spain, in order to characterize physically and chemically the precipitation. With the aim of studying the scavenging process of atmospheric pollutants, scavenging ratio and removal coefficients were calculated through physical parameters of raindrops (obtained by disdrometer data) and through chemical properties of aerosols. Finally, linear models for the prediction of the chemical composition of rainwater and the efficiency of the removal effect were established. In general, the rainwater was dominated by NH4+ > SO42- > NO3- in all seasons. Higher ion concentrations and conductivity and lowest pH were observed in summer, due to the low volume of rain. In winter, the high values of Na+ and Cl- in the rainwater showed the contribution from marine sources, while in summer the high concentrations of Ca2+, Mg2+, SO42-, NH4+ and NO3- reflected the contribution from both crustal and anthropogenic sources. The linear models revealed that the amount of dissolved organic carbon and of the water-soluble ions in rain samples, Ca2+, SO42-, NO3-, increases with the volume swept by the falling drops. Insoluble carbon fraction has a negative dependence with the volume swept and positive with the diameter of the raindrop. Removal coefficients are affected by the concentration in the air of each species before precipitation, the duration of the event and the time elapsed between two precipitation events.

Characterization of aerosol sources in León (Spain) using Positive Matrix Factorization and weather types.

A one-year aerosol sampling campaign, between 2016 and 2017, was conducted in a suburban area of León city, Spain. An association between the Positive Matrix Factorization (PMF) results and air masses through circulation weather types was carried out, through the construction of linear models from the PM10 concentrations and its chemical composition. The aerosol sources, identified by PMF six-factor solution, were: traffic (29%), aged sea salt (26%), secondary aerosols (16%), dust (13%), marine aerosol (7%) and biomass burning (3%). Traffic and secondary factors showed the highest PM10 contribution in the hybrid cyclonic types with wind component from the first and second quadrant. Anticyclonic types with wind component from the first quadrant exhibited high values of secondary, aged sea salt and dust factors. The highest contributions of the dust factor were also associated with northerly types. The linear models built for estimating the source apportionment of PM10, from aerosol chemical composition and geostrophic flow, showed positive coefficients for: westerly flows (WF) in marine factor, southerly flows (SF) in secondary and traffic factors, and shear southerly vorticities (ZS) in dust factor. Negative dependences were observed for ZS in aged sea salt factor and for SF in dust factor. The PM10 mass concentration calculated by the linear models and by the PMF model were strongly correlated. This can be very useful to determine the contribution of a specific source to PM10 in León, only by knowing some meteorological and chemical variables.

Insights into HP1a-Chromatin Interactions.

Understanding the packaging of DNA into chromatin has become a crucial aspect in the study of gene regulatory mechanisms. Heterochromatin establishment and maintenance dynamics have emerged as some of the main features involved in genome stability, cellular development, and diseases. The most extensively studied heterochromatin protein is HP1a. This protein has two main domains, namely the chromoshadow and the chromodomain, separated by a hinge region. Over the years, several works have taken on the task of identifying HP1a partners using different strategies. In this review, we focus on describing these interactions and the possible complexes and subcomplexes associated with this critical protein. Characterization of these complexes will help us to clearly understand the implications of the interactions of HP1a in heterochromatin maintenance, heterochromatin dynamics, and heterochromatin's direct relationship to gene regulation and chromatin organization.

Molecular effects of dADD1 misexpression in chromatin organization and transcription.

BACKGROUND: dADD1 and dXNP proteins are the orthologs in Drosophila melanogaster of the ADD and SNF2 domains, respectively, of the ATRX vertebrate's chromatin remodeler, they suppress position effect variegation phenotypes and participate in heterochromatin maintenance. RESULTS: We performed a search in human cancer databases and found that ATRX protein levels were elevated in more than 4.4% of the samples analyzed. Using the Drosophila model, we addressed the effects of over and under-expression of dADD1 proteins in polytene cells. Elevated levels of dADD1 in fly tissues caused different phenotypes, such as chromocenter disruption and loss of banding pattern at the chromosome arms. Analyses of the heterochromatin maintenance protein HP1a, the dXNP ATPase and the histone post-translational modification H3K9me3 revealed changes in their chromatin localization accompanied by mild transcriptional defects of genes embedded in heterochromatic regions. Furthermore, the expression of heterochromatin embedded genes in null dadd1 organisms is lower than in the wild-type conditions. CONCLUSION: These data indicate that dADD1 overexpression induces chromatin changes, probably affecting the stoichiometry of HP1a containing complexes that lead to transcriptional and architectural changes. Our results place dADD1 proteins as important players in the maintenance of chromatin architecture and heterochromatic gene expression.

Immune profiling and identification of prognostic immune-related risk factors in human ovarian cancer.

Suppression of immune reactivity by increased expression of co-inhibitory receptors has been discussed as a major reason as to why the immune system fails to control tumor development. Elucidating the co-inhibitory expression pattern of tumor-infiltrating lymphocytes in different cancer types will help to develop future treatment strategies. We characterized markers reflecting and affecting T-cell functionality by flow cytometry on lymphocytes isolated from blood, ascites and tumor from advanced ovarian cancer patients (n = 35). Significantly higher proportions of CD4+ and CD8+ T-cells expressed co-inhibitory receptors LAG-3, PD-1 and TIM-3 in tumor and ascites compared to blood. Co-expression was predominantly observed among intratumoral CD8+ T-cells and the most common combination was PD-1 and TIM-3. Analysis of 26 soluble factors revealed highest concentrations of IP-10 and MCP-1 in both ascites and tumor. Correlating these results with clinical outcome revealed the proportion of CD8+ T-cells without expression of LAG-3, PD-1 and TIM-3 to be beneficial for overall survival. In total we identified eight immune-related risk factors associated with reduced survival. Ex vivo activation showed tumor-derived CD4+ and CD8+ T-cells to be functionally active, assessed by the production of IFN-γ, IL-2, TNF-α, IL-17 and CD107a. Blocking the PD-1 receptor resulted in significantly increased release of IFN-γ suggesting potential reinvigoration. The ovarian tumor environment exhibits an inflammatory milieu with abundant presence of infiltrating immune cells expressing inhibitory checkpoints. Importantly, we found subsets of CD8+ T-cells with double and triple expression of co-inhibitory receptors, supporting the need for multiple checkpoint-targeting agents to overcome T-cell dysfunction in ovarian cancer.

Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma.

Glioblastoma multiforme (GBM) presents the most malignant form of glioma, with a 5-year survival rate below 3% despite standard therapy. Novel immune-based therapies in improving treatment outcomes in GBM are therefore warranted. Several molecularly defined targets have been identified mediating anti-GBM cellular immune responses. Mesothelin is a tumor-associated antigen (TAA) which is expressed in several solid tumors with different histology. Here, we report the immunological significance of mesothelin in human malignant glioma. Expression of mature, surface-bound mesothelin protein was found to bein human GBM defined by immunofluorescence microscopy, and on freshly isolated, single cell suspension of GBM tumor cells and GBM tumor cell lines, determined by based on flow cytometric analysis. Peripheral blood (PB) from patients with GBM, stimulated with mesothelin peptides and IL-2, IL-15 and IL-21, exhibited increased antigen-specific IFN-γ and TNF-α production. Anti-mesothelin directed T-cell responses could also be detected in tumor - infiltrating lymphocytes (TIL) isolated from GBM speciments. Furthermore, T cells cultured in the presence of IL-2, IL-15 and IL-21 displayed enhanced mesothelin-specific CD4+ and CD8+ subset proliferation, based on ELISA and flow cytometric readouts. Mesothelin-specific IgG antibodies as well as (shed) mature mesothelin protein were detected in plasma samples from patients with GBM by indirect ELISA. Finally yet importantly, we identified distinct immune recognition hotspots within the mature mesothelin component, defined by peptide-specific IFN-γ responses from peripheral T-cells from patients with GBM. Mesothelin may therefore qualify as a viable target for immunotherapeutic approaches for patients with GBM.

Characterization of infiltrating lymphocytes in human benign and malignant prostate tissue.

Immune checkpoint blockade has shown promising results in numerous cancer types. However, in prostate cancer (PC), absent or limited responses have been reported. To investigate further, we compared the phenotype of infiltrating T-cells isolated from prostate tissue from patients with PC (n = 5), benign prostatic hyperplasia (BPH) (n = 27), BPH with concurrent PC (n = 4) and controls (n = 7). The majority of T-cells were CD8+ and had a CCR7-CD45RO+ effector memory phenotype. However, the yield of T-cells isolated from PC lesions was on average 20-fold higher than that obtained from control prostates. Furthermore, there were differences between the prostate conditions regarding the percentage of T-cells expressing several activation markers and co-inhibitory receptors. In conclusion, many prostate-infiltrating T-cells express co-inhibitory receptors PD-1 and LAG-3, regardless of prostate condition. Despite the observed increase in counts and percentages of PD-1+ T-cells in PC, the concomitant demonstration of high percentage of PD-1+ T-cells in control prostates suggests that PD-1 may play a role in controlling the homeostasis of the prostate rather than in contributing to PC-associated immune-suppression. Thus, PD-1 may not be a good candidate for checkpoint blockade in PC and these data are relevant for evaluation of clinical trials and in designing future immunotherapeutic approaches of PC.

MAIT cells accumulate in placental intervillous space and display a highly cytotoxic phenotype upon bacterial stimulation.

During pregnancy, the maternal immune system must tolerate the developing foetus, and yet retain a potent antimicrobial response to prevent infections. Mucosal associated invariant T (MAIT) cells recognize microbial-derived vitamin B metabolites presented on the MR1 molecule, but their presence and function at the foetal-maternal interface is not known. We here isolated mononuclear cells from paired samples of peripheral blood (PB), intervillous blood (IVB), and decidua parietalis (DP) following uncomplicated term pregnancies. Interestingly, MAIT cells were highly enriched in IVB compared to PB and DP. The activation status of IVB MAIT cells was similar to that of PB MAIT cells, except for a lower expression of PD-1. Both IVB MAIT cells and conventional T cells were more dominated by an effector memory phenotype compared to PB MAIT cells and T cells. IVB MAIT cells also responded more vigorously with expression of IFN-γ, granzyme B, and perforin in response to Escherichia coli stimulation compared to PB. MR1 was not expressed in syncytiotrophoblasts, but in placental villous and decidual macrophages. These data indicate that maternal MAIT cells accumulate in the intervillous space of the placenta and that they are highly armed to quickly respond if bacteria are encountered at the foetal-maternal interface.

Polycyclic aromatic hydrocarbons and their derivatives (nitro-PAHs, oxygenated PAHs, and azaarenes) in PM2.5 from Southern European cities.

Atmospheric particulate matter (PM2.5) samples were collected over two one month periods during winter and summer in three Southern European cities (Oporto - traffic site, Florence - urban background, Athens - suburban). Concentrations of 27 polycyclic aromatic hydrocarbons (PAHs), 15 nitro-PAHs (NPAHs), 15 oxygenated-PAHs (OPAHs) and 4 azaarenes (AZAs) were determined. On average, the winter-summer concentrations of ΣPAHs were 16.3-5.60, 7.75-3.02 and 3.44-0.658ngm-3 in Oporto, Florence and Athens, respectively. The corresponding concentrations of ΣNPAHs were 15.8-9.15, 10.9-3.36 and 15.9-2.73ngm-3, whilst ΣOPAHs varied in the ranges 41.8-19.0, 11.3-3.10 and 12.6-0.704ngm-3. Concentrations of ΣAZAs were always below 0.5ngm-3. Irrespective of the city, the dominant PAHs were benzo[b+j+k]fluoranthene, retene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene. The most abundant OPAH in all cities was 1,8-naphthalic anhydride, whereas 5-nitroacenaphthene was the prevailing NPAH. The ΣOPAHs/ΣPAHs and ΣNPAHs/ΣPAHs were higher in summer than in winter, suggesting increasing formation of derivatives by photochemical degradation of PAHs. Molecular diagnostic ratios suggested that, after traffic, biomass burning was the dominant emission source. Apart from being influenced by seasonal sources, the marked differences between winter and summer may indicate that these diagnostic ratios are particularly sensitive to photodegradation, and thus should be applied and interpreted cautiously. The lifetime excess cancer risk from inhalation was, in part, attributable to PAH derivatives, acclaiming the need to include these compounds in regular monitoring programmes. On average, 206, 88 and 26 cancer cases per million people were estimated, by the World Health Organisation method, for the traffic-impacted, urban background and suburban atmospheres of Oporto, Florence and Athens, respectively.

IFN type I and II induce BAFF secretion from human decidual stromal cells.

B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers' children exhibit higher BAFF levels in cord blood than non-farmers' children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy.

Frontline Science: Placenta-derived decidual stromal cells alter IL-2R expression and signaling in alloantigen-activated T cells.

This study investigated how stromal cells affect the IL-2 pathway in alloantigen-activated T cells. We found that decidual stromal cells (DSCs) from term placentas promoted a high production of IL-2 in cultures with alloantigen-activated T cells. The intensity of expression of cluster of differentiation 25 (CD25; IL-2Rα) on T cells was increased by DSCs, whereas the frequency and intensity of expression of the signaling subunits CD122 (IL-2Rß) and CD132 (IL-2Rγc) were reduced. Consequently, uptake of IL-2 and STAT5 phosphorylation (pSTAT5) was abrogated. DSCs also decreased the proportion of pSTAT5+ T cells in response to IL-15, which also use CD122 for signaling. Addition of DSCs to the allogeneic cultures did not increase the expression of programmed death 1 (PD-1) or CD95, indicating that they did not promote T cell exhaustion. However, exogenous recombinant (r)IL-2 in similar concentrations in the same setting increased the expression of CD95 and down-regulated CD122 in T cells. The antiproliferative effect of sirolimus (SRL) and cyclosporine A (CsA), which target the IL-2 signaling pathway, was diminished by DSCs in vitro. To conclude, DSCs affect IL-2 production and IL-2R expression and signaling, which may contribute to the stromal cell-mediated immune modulation and phenotype shift seen in activated T cells. Altered proliferation in cultures when combining DSCs and SRL or CsA may be of clinical importance, as stromal cells are used in trials for acute inflammation and are often used in combination with conventional immunosuppressive therapies.

Source apportionment of fine and coarse particles at a roadside and urban background site in London during the 2012 summer ClearfLo campaign.

London, like many major cities, has a noted air pollution problem, and a better understanding of the sources of airborne particles in the different size fractions will facilitate the implementation and effectiveness of control strategies to reduce air pollution. Thus, the trace elemental composition of the fine and coarse fraction were analysed at hourly time resolution at urban background (North Kensington, NK) and roadside (Marylebone Road, MR) sites within central London. Unlike previous work, the current study focuses on measurements during the summer providing a snapshot of contributing sources, utilising the high time resolution to improve source identification. Roadside enrichment was observed for a large number of elements associated with traffic emissions (Al, S, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Rb and Zr), while those elements that are typically from more regional sources (e.g. Na, Cl, S and K) were not found to have an appreciable increment. Positive Matrix Factorization (PMF) was applied for the source apportionment of the particle mass at both sites with similar sources being identified, including sea salt, airborne soil, traffic emissions, secondary inorganic aerosols and a Zn-Pb source. In the fine fraction, traffic emissions was the largest contributing source at MR (31.9%), whereas it was incorporated within an "urban background" source at NK, which had contributions from wood smoke, vehicle emissions and secondary particles. Regional sources were the major contributors to the coarse fraction at both sites. Secondary inorganic aerosols (which contained influences from shipping emissions and coal combustion) source factors accounted for around 33% of the PM10 at NK and were found to have the highest contributions from regional sources, including from the European mainland. Exhaust and non-exhaust sources both contribute appreciably to PM10 levels at the MR site, highlighting the continuing importance of vehicle-related air pollutants at roadside.

A comparison between thermal-optical transmittance elemental carbon measured by different protocols in PM2.5 samples.

Although controlled procedures for the determination of carbonaceous fractions are of importance for any air quality measurements, currently no reference method for elemental carbon (EC) and organic carbon (OC) analysis is established yet in Europe. The implementation of the different thermal evolution protocols available in the literature, differing in temperature and duration of the heating ramps, affects the results and can result in a wide variation of EC and OC values. In this study three different protocols for thermal-optical-transmittance analysis of EC and OC were compared, namely He-870 (a variation of the NIOSH protocol), He-550 (a proxy of the IMPROVE protocol), and EUSAAR_2. Measurements were carried out on PM2.5 samples collected on Quartz fibre filters in three sites of different typology: urban background and urban traffic in Florence (Italy) and regional background in Livorno (Italy). The samples were analysed before and after a washing procedure to remove possible water-soluble organic compounds (WSOC), which may enhance the charring process, complicating the EC quantification. This study evidenced a very good agreement for TC measurement (at 2-3% level) and some discrepancies in EC measurement (up to 40%), as expected. WSOC and Pyrolitic Carbon (PyC) present a good correlation, independently of site typology, demonstrating that water soluble compound can be responsible of charring mechanism during the He phase.

Intra-arterial Administration of Placenta-Derived Decidual Stromal Cells to the Superior Mesenteric Artery in the Rabbit: Distribution of Cells, Feasibility, and Safety.

Selective administration of mesenchymal stromal cells to the mesenteric arteries is a potential technique to overcome pulmonary trapping and increase the density of transplanted cells in extensive mural inflammation of the intestine, such as in inflammatory bowel disease and graft-versus-host disease. We injected 5 × 10(6) (111)In-oxine-labeled human decidual stromal cells (DSCs) to the rabbit superior mesenteric artery (SMA) using clinical routine catheters guided by an angiographical system under sterile conditions. We used longitudinal single-photon emission tomography at 6 h and at 1, 2, and 5 days to assess trafficking and distribution of DSCs. We used digital subtraction angiography, computed tomography, and hematoxylin and eosin stainings to determine biodistribution of cells and to assess safety end points. We found that selective injection of human DSCs to the rabbit SMA does not result in acute embolic complications. Furthermore, we found that IV administration resulted in extensive retention of the radiolabeled DSCs in the lungs, corroborating previous studies on pulmonary trapping. In sharp contrast, selective injections to the SMA resulted in uptake distributed in the intestine supplied by the SMA and in the liver, indicating that this approach could significantly increase the fraction of injected DSCs reaching the target tissue.