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Background: The location of coelomocyte proliferation in adult sea urchins is unknown and speculations since the early 1800s have been based on microanatomy and tracer uptake studies. In adult sea urchins (Strongylocentrotus purpuratus) with down-regulated immune systems, coelomocyte numbers increase in response to immune challenge, and whether some or all of these cells are newly proliferated is not known. The gene regulatory network that encodes transcription factors that control hematopoiesis in embryonic and larval sea urchins has not been investigated in adults. Hence, to identify the hematopoietic tissue in adult sea urchins, cell proliferation, expression of phagocyte specific genes, and expression of genes encoding transcription factors that function in the conserved regulatory network that controls hematopoiesis in embryonic and larval sea urchins were investigated for several tissues. Results: Cell proliferation was induced in adult sea urchins either by immune challenge through injection of heat-killed Vibrio diazotrophicus or by cell depletion through aspiration of coelomic fluid. In response to either of these stimuli, newly proliferated coelomocytes constitute only about 10% of the cells in the coelomic fluid. In tissues, newly proliferated cells and cells that express SpTransformer proteins (formerly Sp185/333) that are markers for phagocytes are present in the axial organ, gonad, pharynx, esophagus, and gut with no differences among tissues. The expression level of genes encoding transcription factors that regulate hematopoiesis show that both the axial organ and the pharynx have elevated expression compared to coelomocytes, esophagus, gut, and gonad. Similarly, an RNAseq dataset shows similar results for the axial organ and pharynx, but also suggests that the axial organ may be a site for removal and recycling of cells in the coelomic cavity. Conclusions: Results presented here are consistent with previous speculations that the axial organ may be a site of coelomocyte proliferation and that it may also be a center for cellular removal and recycling. A second site, the pharynx, may also have hematopoietic activity, a tissue that has been assumed to function only as part of the intestinal tract. [ABSTRACT FROM AUTHOR]

This study addresses the economic analysis and pattern of agrochemicals use among smallholder crop farmers in Edu Local Government Area of Kwara State, Nigeria. A multistage random sampling technique was used to select 144 small-scale crop farmers interviewed for the study. Descriptive statistics and farm budgeting techniques were used for data analysis. The result shows that a typical small-scale crop farmer in the study area is, on average, a 31-year-old male with 11 to 20 years of farming experience. These groups of farmers also cultivate 0.78 hectares and have a household size of 8. Herbicides are the predominant agrochemical used during the survey; it was mostly applied both before and after crop emergence. Usually, pesticides were applied without following safety instructions. Users of agrochemicals obtained a total gross margin of USD 1,469.95 per hectare with a profitability ratio of 1.22 which indicates that farming is a profitable business in the study area. The study recommends to increase awareness on farmer safety measures when handling agrochemicals; and to establish educational programs that will educate farmers on how to safely use agrochemicals. [ABSTRACT FROM AUTHOR]

Abstract Sea cucumber Apostichopus japonicus rely on the efficient innate immune mechanisms against invaders, in which the consumption and regeneration of coelomocytes take place at the same time. In the present study, histological features of putative hematopoietic tissues (HPTs) including the rete mirabile, the respiratory tree, the polian vesicle and the coelomic epithelium were characterized. The distribution of transcription factor GATA1 in coelomocytes and putative HPTs was examined by immunohistochemistry. In addition, cell proliferation using EdU labeling and coelomocyte distribution in different tissues using monoclonal antibody labeling were analyzed to further confirm the HPTs. The results showed that two homologs of GATA1 were detected with molecular weight of 43 and 90 kDa in coelomocytes, rete mirabile, respiratory tree and polian vesicle, whereas no signals were detected in the coelomic epithelium. A few cells were detected to be EdU-positive for coelomocytes, which accounted for approximately 9.5%. In the rete mirabile and the respiratory tree, the EdU signals were strong in cells of the tube wall. In the polian vesicle, numerous EdU-positive cells were detected in the cyst wall. In the coelomic epithelium, little EdU signaling was detected. Immunohistochemistry analysis by mAb 3F6 against A. japonicus coelomocytes showed that positive signals were observed in the tube wall of the rete mirabile, respiratory tree, cyst wall of the polian vesicle and in the coelomocyte antrum of coelomic epithelium. These results suggest that the rete mirabile, respiratory tree and polian vesicle are the HPTs of A. japonicus. Highlights • Rete mirabile, respiratory tree, and polian vesicle were suggested as HPTs. • Two homologs of transcription factor GATA1 were detected in the HPTs. • EdU signals were strong in coelomocytes and HPTs. [ABSTRACT FROM AUTHOR]

Abstract Castor bean (Ricinus communis L.) an important non-edible oilseed crop, is a prominent feed stock towards the generation of renewable materials for industrial production which has multiple applications ranging from cosmetics to biofuels industry. India accounts for 76% of the total world production of castor oil seed. However, major concern for developing countries like India where expanding economy led to rapid increases in gases like NOx, CO and VOCs photochemically form ozone. Ozone is strong oxidant that damages agriculture, ecosystems, and materials with considerable reduction in crop yields and crop quality. One way to reduce ozone induced loss is to focus on the adapting crops to ozone exposure by selecting cultivars with demonstrated ozone resistance. An experiment was conducted for ozone risk assessment of castor cultivars to select cultivar with demonstrated resistance against ozone pollution. This study comprise an open top chamber experiment with three treatments viz. (i) control (ambient ozone concentration), (ii) enhanced ozone (average 75 ppb for 4 h daily throughout the growing season), and (iii) EDU application. Results suggested that the ozone pollution substantially affected growth and physiology of castor cultivars. Crop biomass and yield was also negatively influenced by ozone pollution. Developed defence provided strength to withstand against ozone pollution to the experimental crop cultivars. However, developed defence is cultivar specific and positively correlated with the resistance against ozone pollution. Study concluded that the damage to ozone is directly dependent on the antioxidative potential of plant species. However, ozone adaptability is based on the genetic makeup of the cultivar and yield related loss to ozone can be minimizing by selecting ozone tolerant variety as seen in cultivar Nidhi-999. Graphical abstract Image 1 Highlights • Tropospheric ozone is detrimental for castor crop and reduces growth and yield. • ROS scavenging provided adaptability to castor cultivars for ozone pollution. • Developed defence for ozone is based on the genetic makeup of the cultivars. • Yield related loss to ozone can be minimize by selecting ozone tolerant variety as in cultivar Nidhi-999. Tropospheric ozone is detrimental for crop yield and yield related loss to ozone can be minimize by selecting ozone tolerant variety. [ABSTRACT FROM AUTHOR]

There are two most heavily used markers of cell proliferation, thymidine analogues 5-bromo-2′-deoxyuridine (BrdU) and 5-ethynyl-2′-deoxyuridine (EdU) that are incorporated into the DNA during its synthesis. In neurosciences, they are often used consecutively in the same animal to detect neuronal populations arising at multiple time points, their migration and incorporation. The effectivity of these markers, however, is not well established. Here, we studied the effectivity of equimolar doses of BrdU and EdU to label new cells and looked for the dose that will label the highest number of proliferating cells in the neurogenic ventricular zone (VZ) of adult songbirds. We found that, in male zebra finches (Taeniopygia guttata), the equimolar doses of BrdU and EdU did not label the same number of cells, with BrdU being more effective than EdU. Similarly, in liver, BrdU was more effective. The saturation of the detected brain cells occurred at 50 mg/kg BrdU and above 41 mg/kg EdU. Higher dose of 225 mg/kg BrdU or the equimolar dose of EdU did not result in any further significant increases. These results show that both markers are reliable for the detection of proliferating cells in birds, but the numbers obtained with BrdU and EdU should not be compared.

Measuring differences in cell cycle progression is often essential to understand cell behavior under different conditions, treatments and environmental changes. Cell synchronization is widely used for this purpose, but unfortunately, there are many cases where synchronization is not an option. Many cell lines, patient samples or primary cells cannot be synchronized, and most synchronization methods involve exposing the cells to stress, which makes the method incompatible with the study of stress responses such as DNA damage. The use of dual-pulse labelling using EdU and BrdU can potentially overcome these problems, but the need for individual sample processing may introduce a great variability in the results and their interpretation. Here, we describe a method to analyze cell proliferation and cell cycle progression by double staining with thymidine analogues in combination with fluorescent cell barcoding, which allows one to multiplex the study and reduces the variability due to individual sample staining, reducing also the cost of the experiment.

BACKGROUND: Skin-derived precursor cells play a regenerative role in wound healing, which can restore the physiological and aesthetic features of the skin by mitosis, proliferation and migration to the injury region. OBJECTIVE: To observe the structure of the mouse tail and the healing of the broken end, and then to investigate the correlation of these characteristics with the repair of skin trauma at the tail. METHODS: The tails of 2-day-old Kunming mice were cut off using ophthalmic microsurgical scissors to establish the mouse model of hair follicle regeneration in the tail. Regions in which the cells proliferated actively were determined. Wound healing samples were regularly taken, and embedded using OCT. Wound healing of the broken end was routinely observed by pathological staining. Cell proliferation was observed by immunofluorescence. Expression of AP-1 in the healing site was identified by immunofluorescent staining. RESULTS AND CONCLUSION: The structure of the mouse tail was complex, with rich blood supply. Good healing was observed in the broken end of the mouse tail. The cells in the hair follicles and dermal papillary layer showed active proliferation and mitosis, and thereby participated in wound repair and regeneration. Fluorescence staining results found that the positive expression of AP-1 was mainly concentrated in the epidermis and dermal papilla. These findings indicate that the epidermis and dermal papilla possess a strong ability to regenerate. [ABSTRACT FROM AUTHOR]

The study of organogenesis, tissue-homeostasis and regeneration requires the precise assessment of in vivo cell proliferation. To this end a host of methods have been developed to detect and quantify DNA synthesis in proliferating cells. These include cell labeling with various nucleotide analogues and fluorescence reporter-based animal models with each method presenting its idiosyncratic shortcomings. Quantitative assessment of epithelial cell turnover has been partly hampered due to their variable and limited in vivo accessibility and the requirement for harsher isolation procedures to procure single cells for FACS analysis. Here, we report a reliable protocol to study in vivo cell proliferation of epithelial cells in mice by repeatedly injecting EdU intravenously for an extended 12-day period. EdU incorporation was quantitated ex vivo by FACS after tissue dissociation in order to obtain single epithelial cell suspensions. As a lead population, we analyzed thymic epithelial cells (TECs), where we were able to label compartmentalized TEC subsets to saturation without apparent toxic effects on the thymus architecture or stress-sensitive TEC lineage differentiation. The data is in concordance with the prevailing model of medullary TEC terminal differentiation that includes the post-Aire stage. The same protocol was successfully applied to epithelial cells of various other organs – skin, lymph node, kidney and small intestine – tissues with widely varying frequencies and rates of proliferating epithelial cells. [ABSTRACT FROM AUTHOR]

Measuring the mean duration of synthesis-phase (Ts) and of the total cell-cycle (Tc) within progenitor cell populations can provide important insights into the biology governing these cells. Rather than a passive process that shows little variability across cellular contexts, the cell-cycle is instead highly regulated. For example, in the rodent forebrain, Ts is selectively lengthened in radial glial progenitor cells undergoing symmetric versus asymmetric division. This lengthening is thought to minimize the potential for copying errors that can occur during DNA replication. Manipulating cell-cycle duration can also affect cell fate, demonstrating that in certain circumstances cell-cycle duration is an instructive process. Currently, cell-cycle length is typically measured using either cumulative labeling with a single thymidine analogue, or via dual thymidine analogue labeling approaches. However, these methods are often time-consuming and inefficient. Here, using the embryonic mouse cerebral cortex as a model system, we describe a simplified dual thymidine analogue protocol using BrdU and EdU that can be used to measure Ts and Tc. The advantage of this protocol over cumulative labeling approaches is that only a single time-point is required for measurement. An additional benefit of this protocol over existing dual-analog approaches (CldU/IdU) is the antibody-free detection of EdU and the acid-free detection of BrdU, processes allowing for the parallel use of specific antibodies so as to measure the cell-cycle in immunologically defined cellular subpopulations. [ABSTRACT FROM AUTHOR]

Replication errors that are caused by mutagens are critical for living cells. The aim of the study was to analyze the distribution of a DNA replication pattern on chromosomes of the H. vulgare 'Start' variety using pulse 5-ethynyl-2'-deoxyuridine (EdU) labeling, as well as its relationship to the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and ray. To the best of our knowledge, this is the first example of a study of the effects of mutagens on the DNA replication pattern in chromosomes, as well as the first to use EdU labeling for these purposes. The duration of the cell cycle of the Hordeum vulgare 'Start' variety was estimated for the first time, as well as the influence of MH and γ ray on it. The distribution of the signals of DNA replication along the chromosomes revealed relationships between DNA replication, the chromatin structure, and DNA damage. MH has a stronger impact on replication than ray. Application of EdU seems to be promising for precise analyses of cell cycle disturbances in the future, especially in plant species with small genomes. [ABSTRACT FROM AUTHOR]

The present study evaluated the impact of high ambient O 3 on morphological, physiological and biochemical traits and leaf proteome in two high-yielding varieties of wheat using ethylene diurea (EDU) as foliar spray (200 and 300 ppm). Average ambient ozone concentration was 60 ppb which was more than sufficient to cause phytotoxic effects. EDU treatment resulted in less lipid peroxidation along with increased chlorophyll content, biomass and yield. EDU alleviated the negative effects of ozone by enhancing activities of antioxidants and antioxidative enzymes. Two dimensional electrophoresis (2DGE) analysis revealed massive changes in protein abundance in Kundan at vegetative stage (50% proteins were increased, 20% were decreased) and at flowering stage (25% increased, 18% decreased). In PBW 343 at both the developmental stages about 15% proteins were increased whereas 20% were decreased in abundance. Higher abundance of proteins related to carbon metabolism, defense and photorespiration conferred tolerance to EDU treated Kundan. In PBW343, EDU provided incomplete protection as evidenced by low abundance of many primary metabolism related proteins. Proteomic changes in response to EDU treatment in two varieties are discussed in relation to growth and yield. [ABSTRACT FROM AUTHOR]

The taxon Tardigrada, commonly called “water bears”, consists of microscopic, eight-legged invertebrates that are well known for their ability to tolerate extreme environmental conditions. Their miniscule body size means that tardigrades possess a small total number of cells, the number and arrangement of which may be highly conserved in some organs. Although mitoses have been observed in several organs, the rate and pattern of cell divisions in adult tardigrades has never been characterized. In this study, we incubated live tardigrades over a period of several days with a thymidine analog in order to visualize all cells that had divided during this time. We focus on the midgut, the largest part of the digestive system. Our results show that new cells in the midgut arise from the anterior and posterior ends of this organ and either migrate or divide toward its middle. These cells divide at a constant rate and all cells of the midgut epithelium are replaced in approximately one week. On the other hand, we found no cell divisions in the nervous system or any other major organs, suggesting that the cell turnover of these organs may be extremely slow or dependent on changing environmental conditions. [ABSTRACT FROM AUTHOR]

In this study, we present a systematic characterization of hair cell loss and regeneration in the chicken utricle in vivo. A single unilateral surgical delivery of streptomycin caused robust decline of hair cell numbers in striolar as well as extrastriolar regions, which in the striola was detected very early, 6 h post-insult. During the initial 12 h of damage response, we observed global repression of DNA replication, in contrast to the natural, mitotic hair cell production in undamaged control utricles. Regeneration of hair cells in striolar and extrastriolar regions occurred via high rates of asymmetric supporting cell divisions, accompanied by delayed replenishment by symmetric division. While asymmetric division of supporting cells is the main regenerative response to aminoglycoside damage, the detection of symmetric divisions supports the concept of direct transdifferentiation where supporting cells need to be replenished after their phenotypic conversion into new hair cells. Supporting cell divisions appear to be well coordinated because total supporting cell numbers throughout the regenerative process were invariant, despite the initial large-scale loss of hair cells. We conclude that a single ototoxic drug application provides an experimental framework to study the precise onset and timing of utricle hair cell regeneration in vivo. Our findings indicate that initial triggers and signaling events occur already within a few hours after aminoglycoside exposure. Direct transdifferentiation and asymmetric division of supporting cells to generate new hair cells subsequently happen largely in parallel and persist for several days. [ABSTRACT FROM AUTHOR]

Este artículo recoge la investigación realizada sobre la contribución de la lectura a la formaciónen género del alumnado del Grado de Educación Social. La investigación incluye las universidades españolas (29 públicas y 7 privadas). Se ha realizado una investigación descriptiva y censal. Se envió un cuestionario on-line al alumnado de 4º curso, que fue respondido por 213 personas, de las cuales un 86,4% han sido mujeres; pues es una titulación feminizada. El objetivo era conocer su opinión sobre las aportaciones de la lectura a su formación en género. Se ha calculado el alfa de Cronbach para las 45 variables ordinales. La validez cualitativa se ha realizado a través del juicio de 18 personas expertas en esta materia. Los datos muestran la importancia de las lecturas sobre género en su formación, por su contribución al desarrollo del pensamiento crítico y a su compromiso en la realización de actuaciones para conseguir la igualdad entre hombres y mujeres. Y, de este modo, luchar contra todo tipo de discriminación contra la mujer. This article describes a research on the contribution of reading to gender training of degree students in Social Education. The research includes the Spanish universities (29 public and 7 private). A descriptive and census research was carried out. An online questionnaire was sent to 4th year students, which was answered by 213 people, of which 86.4% were women; for it is a feminized degree. The aim was to know their opinion about the contribution of reading to their gender training. The qualitative validity has been obtained through the trial of 18 experts in this matter. The data show the importance of the readings on gender in their training due to its contribution to the development of critical thinking and its commitment to implementing actions aimed at achieving equality between men and women. Thus, it is a way to fight against all forms of discrimination against women. These data mostly indicate that it is at the University where they have been trained in equality between men and women.

Objectives: MiR-21 induces neoplastic transformation, cell proliferation, and metastasis and downregulates programmed cell death4 (PDCD4) in some cancers. The aim of this study was to investigate the roles and interactions of PDCD4 and miR-21 in human renal cell carcinoma (RCC). Materials and Methods: A total of 32 paired tumor and normal tissue specimens from RCC patients as well as three renal cancer cell lines (786-O, A498, caki-1) and one normal epithelial kidney cell line (HK-2) were studied. The expression levels of PDCD4 (protein and mRNA) and miR-21 were examined by Western blot analysis and by qRT-PCR and luciferase reporter assays. Furthermore, we transfected 786-O cells with pre-miR-21 (mimics) and anti-miR-21 (inhibitor) and then again analyzed the expression of PDCD4 protein and mRNA, and determined cell proliferation and transformation capabilities by EDU and soft agar colony formation assay. Results: MiR-21 expression was significantly upregulated in RCC, metastatic RCC specimens and renal cancer cell lines (A498, 786-O, caki-1) compared to normal non-metastatic RCC specimens and HK-2 cells (PPP>0.05). Moreover, we observed a significant reduction in PDCD4 protein levels in miR-21mimic-transfected cells, but a significant increase in miR-21inhibitor-transfected cells (PP>0.05). Furthermore, miR-21mimic-transfected cells exhibited increased cell proliferation and transformation capacity according to EDU analysis and soft agar formation assay, whereas miR-21inhibitor-transfected cells exhibited the opposite phenomenon(PConclusions: MiR-21 not only promoted cancer cell hyperplasia and contributed to tumor cell transformation and metastasis, but also post-transcriptionally downregulated PDCD4 protein expression. PDCD4 and miR-21 expression levels potentially play an important role in renal cell cancer.

The replication of a virus within its host cell involves numerous interactions between viral and cellular factors, which have to be tightly controlled in space and time. The intricate interplay between viral exploitation of cellular pathways and the intrinsic host defense mechanisms is difficult to unravel by traditional bulk approaches. In recent years, novel fluorescence microscopy techniques and single virus tracking have transformed the investigation of dynamic virus-host interactions. A prerequisite for the application of these imaging-based methods is the attachment of a fluorescent label to the structure of interest. However, their small size, limited coding capacity and multifunctional proteins render viruses particularly challenging targets for fluorescent labeling approaches. Click chemistry in conjunction with genetic code expansion provides virologists with a novel toolbox for site-specific, minimally invasive labeling of virion components, whose potential has just recently begun to be exploited. Here, we summarize recent achievements, current developments and future challenges for the labeling of viral nucleic acids, proteins, glycoproteins or lipids using click chemistry in order to study dynamic processes in virus-cell interactions.

The two neuronal populations in the cortex, pyramidal neurons and interneurons, can be separated based on neurotransmitter identity, however, within this segregation a large degree of diversity exists. Investigations into the molecular diversity of neurons are impeded by the inability to isolate cell populations born at different times for gene expression analysis. Developing interneurons may be distinguished by the expression of Glutamic Acid Decarboxylase-67 (GAD67). Neuronal birthdating using nucleoside analogs is an effective means of identifying coetaneous interneurons. Using these two features, neurotransmitter identity and birthdating, we have developed a method to isolate migrating interneurons using fluorescent-activated cell sorting (FACS) for RNA extraction and gene expression analysis. We utilized 5-ethynyl-2′-deoxyuridine (EdU) to birthdate interneuron cohorts and the GAD67 knock-in GFP transgenic mice to identify interneurons. In combination, we achieved simultaneous detection of GFP and EdU signals during FACS sorting of coetaneous interneurons with minimum loss of RNA integrity. RNA quality was deemed to be satisfactory by quantitative polymerase chain reaction (qPCR) for the interneuron-specific transcript Gad67 . [ABSTRACT FROM AUTHOR]

Ethylenediurea (EDU) is a synthetic chemical known to protect plants from the phytotoxic effects of tropospheric ozone (O3). Although many studies have proposed the use of EDU for studying the O3 effects under field conditions, its mechanism of action is not fully understood, and it is unclear whether it exerts a specific antiozonant action, or if it may also interact with other oxidative stresses. The aim of this work was to evaluate the effect of EDU on forest species in a Mediterranean environment where, during summer, vegetation is exposed to multiple oxidative stresses, such as O3 and drought. The experiment was conducted on Fraxinus ornus L. (Manna ash) plants growing in six mesocosms, three maintained under full irrigation, while the other three were subjected to drought for 84 days. In each mesocosm, three plants were sprayed every 15 days with 450 ppm EDU. Gas exchange and chlorophyll "a" fluorescence measurements carried out through the experimental period highlighted that EDU did not affect stomatal conductance and had an ameliorative effect on the functionality of drought-stressed plants, thus suggesting that it may act as a generic antioxidant. The implications of these findings for the applicability of EDU in field studies are discussed. [ABSTRACT FROM AUTHOR]

Objective: To explore a simple and effective method to track the proliferation and differentiation of hair follicle stem cells, and to lay a foundation for the study of the mechanism of proliferation and differentiation of hair follicle stem cells in the later stage. Vibrissa follicle outer root sheath of KM mouse was collected under sterile conditions 5% FCS+MEM was used as basic culture liquid with 4 different concentrations of EdU (5-Ethynyl-2'-deoxyuridine) as cells in the marker to the outer root sheath of the hair follicle regeneration culture and frozen section, after H.E. staining, the regenerative cells labeled with EdU were detected using KeyFluor488. Results: According to the results of H.E staining and the EdU labeling and staining, hair follicle outer root sheath regeneration morphological is worse when the cell marker concentration is high, and the marker effect of hair follicle outer root sheath cell proliferation is the best when the EdU concentration of 20 μmol/L. Conclusion: EdU cell markers of hair follicle outer root sheath stem cell proliferation activity has certain side effects affect on cell's normal physiological function, the concentration of 20 μmol/L EdU can be effectively labeled by the hair follicle stemcell proliferation, and on hair follicle regeneration structure effect is small. [ABSTRACT FROM AUTHOR]

Background: Injury to the white matter may lead to impaired neuronal signaling and is commonly observed following traumatic brain injury (TBI). Although endogenous repair of TBI-induced white matter pathology is limited, oligodendrocyte progenitor cells (OPCs) may be stimulated to proliferate and regenerate functionally myelinating oligodendrocytes. Even though OPCs are present throughout the adult brain, little is known about their proliferative activity following axonal injury caused by TBI. Objective: We hypothesized that central fluid percussion injury (cFPI) in mice, a TBI model causing wide-spread axonal injury, results in OPC proliferation. Methods: Proliferation of OPCs was evaluated in 27 cFPI mice using 5-ethynyl-2'-deoxyuridine (EdU) labeling and a cell proliferation assay at 2 (n=9), 7 (n = 8) and 21 (n = 10) days post injury (dpi). Sham-injured mice (n = 14) were used as controls. OPC proliferation was quantified by immunohistochemistry using the OPC markers NG2 and Olig2 in several white matter loci including the corpus callosum, external capsule, fimbriae, the internal capsule and cerebral peduncle. Results: The number of EdU/DAPI/Olig2-positive cells were increased in the cFPI group compared to sham-injured animals at 7 days post-injury (dpi; p≤0.05) in the majority of white matter regions. The OPC proliferation had subsided by 21 dpi. The number of EdU/DAPI/NG2 cells was also increase at 7 dpi in the external capsule and fimbriae. Conclusion: These results suggest that traumatic axonal injury in the mouse induces a transient proliferative response of residing OPCs. These proliferating OPCs may replace dead oligodendrocytes and contribute to remyelination, which needs evaluation in future studies. [ABSTRACT FROM AUTHOR]

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Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs. [ABSTRACT FROM AUTHOR]

Reactions of genetically identical cells to various exogenous and endogenous stimuli can vary significantly. One of the main factors of this non-genetic cellular heterogeneity is the cell cycle. The most convenient way to study the subcellular processes depending on the cell cycle stage is the synchronization of the cells. Toxic inhibitors of DNA replication and/or mitotic spindle assembly are typically used to synchronize cells. It is important to accurately select the synchronization method for a particular experiment. In this study, we performed a comparative analysis of the synchronization methods of normal and transformed human cells, paying special attention to the accuracy of synchronization and toxicity of the methods used. [ABSTRACT FROM AUTHOR]