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ABSTRACTThe composition of the intestinal bacterial community is well described, but recent research suggests that the metabolism of these bacteria plays a larger role in health than which species are present. One fundamental aspect of gut bacterial metabolism that remains understudied is bacterial replication. Indeed, there exist few techniques which can identify actively replicating gut bacteria. In this study, we aimed to address this gap by adapting 5-ethynyl-2’-deoxyuridine (EdU) click chemistry (EdU-click), a metabolic labeling method, coupled with fluorescence-activated cell sorting and sequencing (FACS-Seq) to characterize replicating gut bacteria. We first used EdU-click with human gut bacterial isolates and show that many of them are amenable to this technique. We then optimized EdU-click and FACS-Seq for murine fecal bacteria and reveal that Prevotella UCG-001 and Ileibacterium are enriched in the replicating fraction. Finally, we labeled the actively replicating murine gut bacteria during exposure to cell wall-specific antibiotics in vitro. We show that regardless of the antibiotic used, the actively replicating bacteria largely consist of Ileibacterium, suggesting the resistance of this taxon to perturbations. Overall, we demonstrate how combining EdU-click and FACSeq can identify the actively replicating gut bacteria and their link with the composition of the whole community in both homeostatic and perturbed conditions. This technique will be instrumental in elucidating in situ bacterial replication dynamics in a variety of other ecological states, including colonization and species invasion, as well as for investigating the relationship between the replication and abundance of bacteria in complex communities.

Astrocytes and microglia play an orchestrated role following spinal cord injury; however, the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood. Herein, microglia were pharmacologically depleted and the effects on the astrocytic response were examined. We further explored the potential mechanisms involving the signal transducers and activators of transcription 3 (STAT3) pathway. For in vivo experiments, we constructed a contusion spinal cord injury model in C57BL/6 mice. To deplete microglia, all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397, starting 2 weeks prior to surgery until they were sacrificed. Cell proliferation was examined by 5-ethynyl-2-deoxyuridine (EdU) and three pivotal inflammatory cytokines were detected by a specific Bio-Plex ProTM Reagent Kit. Locomotor function, neuroinflammation, astrocyte activation and phosphorylated STAT3 (pSTAT3, a maker of activation of STAT3 signaling) levels were determined. For in vitro experiments, a microglia and astrocyte coculture system was established, and the small molecule STA21, which blocks STAT3 activation, was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia. PLX3397 administration disrupted glial scar formation, increased inflammatory spillover, induced diffuse tissue damage and impaired functional recovery after spinal cord injury. Microglial depletion markedly reduced EdU+ proliferating cells, especially proliferating astrocytes at 7 days after spinal cord injury. RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397. Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes. Importantly, in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration. These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation, and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.

Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods. [ABSTRACT FROM AUTHOR]

Background: This study aimed to explore the differential expression of peptides associated with ankylosing spondylitis (AS) patients, enabling identification of potential functional peptides to provide the basis for the novel intervention targets for AS. Material and Methods: 3 AS patients and 3 healthy volunteers were enrolled in this study. The expression profiles for peptides present in the plasma of AS patients and the healthy individual were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The physicochemical properties and biological functions of identified peptides were further analyzed by bioinformatics. The results of peptide identificationwere verified by cell viability analysis, usingCCK8 and Edu staining assay, and the differential peptides relevant to the disease were screened. Results: 52 differential peptides were successfully identified using mass spectrometry. 44 peptides were up-regulated, while eight were down-regulated. FGA-peptide (sequences: DSGEGDFLAEGGGVRGPR), C4A-peptide (sequences: NGFKSHAL), and TUBB-peptide (sequences: ISEQFTAMFR) were screened out that could significantly promote the proliferation of fibroblasts in AS patients. Bioinformatics analysis showed these differentially expressed peptides might be associated with "MHC class I protein binding" and "pathogenic Escherichia coli infection" pathways, which might further affect the progression of AS. Conclusion: This pilot study shows 3 differentially expressed peptides may have the potential function for the occurrence and development of AS, may provide novel insights into the underlying molecular mechanisms of AS based on peptide omics. [ABSTRACT FROM AUTHOR]

The paper explores the influence of discourse structure on text complexity. We assume that certain types of discourse units are easier to read than others, due to their explicit discourse structure, which makes their informational input more accessible. As a data source, we use the dataset from the MECO corpus, which contains eye movement data for 12 Russian texts read by 35 native speakers. We demonstrate that the approach relying on elementary discourse units (EDUs) can be felicitously used in the analysis of eye movement data, since fixation patterns on EDUs are similar to those on whole sentences. Our analysis has identified EDU outliers, which show shorter time of first fixation than estimated. We arranged these outliers into several groups associated with different discourse structures. First, these are statements with nominal predicates that set exposition of the text or macroproposition and, following those, EDUs that elaborate on the previous statement and signal the beginning of the narrative. Second, they are EDUs that serve as the middle component of a listing or a group of coordinated clauses or phrases. The final group represents EDUs that are part of an opposition, contrast or comparison. Discourse analysis based on EDUs has never been applied to eye movement data, so our project opens many avenues for further research of complexity of discourse structure.

Background & Aims: The enteric nervous system (ENS) is the largest part of the peripheral nervous system; moreover, abnormal ENS development and function are associated with multiple human pathologies. Data from several groups suggest that under normal physiological conditions in adult animals, enteric nerve cells do not replicate. A study by Kulkarni et al in 2017 challenged this view and proposed that nearly 70% of enteric neurons in the myenteric ganglia are born in 1 week. The authors of this study suggested that differences in DNA labelling times and DNA denaturation conditions might explain discrepancies with previous reports. Previous studies were carried out using different conditions and labelling techniques in various regions of the gastrointestinal tract; thus, conclusions have remained elusive. Methods: Here, we have eliminated those variables by analyzing the whole small intestine using the reagents and conditions that Kulkarni et al used. To exclude variables related to immunohistochemistry, we carried out parallel experiments with “click chemistry”-based detection of DNA replication. Results: Although proliferation was readily detected in the epithelium, we found no evidence of neuronal replication in the myenteric ganglia. Conclusions: We conclude that within 1 week under normal physiological conditions, myenteric neurons in the small intestine do not replicate.

Pancreatic beta cells are among the slowest replicating cells in the human body and have not been observed to increase in number except during the fetal and neonatal period, in cases of obesity, during puberty, as well as during pregnancy. Pregnancy is associated with increased beta cell mass to meet heightened insulin demands. This phenomenon raises the intriguing possibility that factors present in the serum of pregnant individuals may stimulate beta cell proliferation and offer insights into expansion of the beta cell mass for treatment and prevention of diabetes. The primary objective of this study was to test the hypothesis that serum from pregnant donors contains bioactive factors capable of inducing human beta cell proliferation. An immortalized human beta cell line with protracted replication (EndoC-βH1) was cultured in media supplemented with serum from pregnant and non-pregnant female and male donors and assessed for differences in proliferation. This experiment was followed by assessment of proliferation of primary human beta cells. Sera from five out of six pregnant donors induced a significant increase in the proliferation rate of EndoC-βH1 cells. Pooled serum from the cohort of pregnant donors also increased the rate of proliferation in primary human beta cells. This study demonstrates that serum from pregnant donors stimulates human beta cell proliferation. These findings suggest the existence of pregnancy-associated factors that can offer novel avenues for beta cell regeneration and diabetes prevention strategies. Further research is warranted to elucidate the specific factors responsible for this effect.

Since risk assessments of tropospheric ozone (O 3) are crucial for agricultural and forestry sectors, there is a growing body for realistic assessments by a stomatal flux-based approach in Free-Air Controlled Exposure (FACE) facilities. Ozone risks are normally described as relative risks (RRs), which are calculated by assuming the biomass or yield at zero O 3 dose as "reference". However, the estimation of the reference biomass or yield is challenging due to a lack of O 3 -clean-air treatment at the FACEs and the extrapolation without data in a low O 3 range increases the bias for estimating the reference values. Here, we reviewed a current methodology for the risk assessment at FACEs and presented a simple and effective way ("modified Paoletti's approach") of defining RRs just using biomass or yield data with a range of expected impacts under the FACE conditions hypothesizing three possible scenarios based on prediction limits using 95% credible intervals (CI) (1. Best fit using the intercept as reference, 2. Optimistic scenario using a lower CI and 3. Worst scenario using an upper CI). As a result, O 3 -sensitive species show a relatively narrow effect range (optimistic vs. worst scenario) whereas a wide range of response may be possibly taken in resistant species. Showing a possible effect range allows for a comprehensive understanding of the potential risks and its uncertainties related to a species sensitivity to O 3. As a supporting approach, we also recommend to use scientifically relevant tools (i.e., ethylenediurea treatments; mechanistic plant models) for strengthening the obtained results for the RRs against O 3. Interestingly, the moderately sensitive or resistant species showed non-linear rather than linear dose-response relationships, suggesting a need for the flexible functional form for the risk assessment to properly describe the complex plant response such as hormesis, which depends on their plasticity to O 3 stress. [ABSTRACT FROM AUTHOR]

Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains unresolved. Here, we address these concerns by examining the effects of BrdU and EdU on adult hippocampal neurogenesis and EdU on the perinatal somatic development of mice. We show that, in a wide range of doses, EdU and BrdU label similar numbers of cells in the dentate gyrus shortly after administration. Furthermore, whereas the administration of EdU does not affect the division and survival of neural progenitor within 48 h after injection, it does affect cell survival, as evaluated 6 weeks later. We also show that a single injection of various doses of EdU on the first postnatal day does not lead to noticeable changes in a panel of morphometric criteria within the first week; however, higher doses of EdU adversely affect the subsequent somatic maturation and brain growth of the mouse pups. Our results indicate the potential caveats in labeling the replicating DNA using thymidine analogs and suggest guidelines for applying this approach. [ABSTRACT FROM AUTHOR]

Stem cells hold promise for treating hair loss. Here an in vitro mouse model was developed using outer root sheaths (ORSs) isolated from hair follicles for studying stem cell-mediated dermal papillary regeneration.

Abstract Background Segmentation in arthropods typically occurs by sequential addition of segments from a posterior growth zone. However, the amount of tissue required for growth and the cell behaviors producing posterior elongation are sparsely documented. Results Using precisely staged larvae of the crustacean, Thamnocephalus platyurus, we systematically examine cell division patterns and morphometric changes associated with posterior elongation during segmentation. We show that cell division occurs during normal elongation but that cells in the growth zone need only divide ~ 1.5 times to meet growth estimates; correspondingly, direct measures of cell division in the growth zone are low. Morphometric measurements of the growth zone and of newly formed segments suggest tagma-specific features of segment generation. Using methods for detecting two different phases in the cell cycle, we show distinct domains of synchronized cells in the posterior trunk. Borders of cell cycle domains correlate with domains of segmental gene expression, suggesting an intimate link between segment generation and cell cycle regulation. Conclusions Emerging measures of cellular dynamics underlying posterior elongation already show a number of intriguing characteristics that may be widespread among sequentially segmenting arthropods and are likely a source of evolutionary variability. These characteristics include: the low rates of posterior mitosis, the apparently tight regulation of cell cycle at the growth zone/new segment border, and a correlation between changes in elongation and tagma boundaries.

The claustrum is an enigmatic brain structure thought to be important for conscious sensations. Recent studies have focused on gene expression patterns, connectivity, and function of the claustrum, but relatively little is known about its development. Interestingly, claustrum-enriched genes, including the previously identified marker Nurr1 , are not only expressed in the classical claustrum complex, but also embedded within lateral neocortical regions in rodents. Recent studies suggest that Nurr1 positive neurons in the lateral cortex share a highly conserved genetic expression pattern with claustrum neurons. Thus, we focus on the developmental progression and birth dating pattern of the claustrum and Nurr1 positive neurons in the lateral cortex. We comprehensively investigate the expression of Nurr1 at various stages of development in the rat and find that Nurr1 expression first appears as an elongated line along the anterior-posterior axis on embryonic day 13.5 (E13.5) and then gradually differentiates into multiple sub-regions during prenatal development. Previous birth dating studies of the claustrum have led to conflicting results, therefore, we combine 5-ethynyl-2′-deoxyuridine (EdU) labeling with in situ hybridization for Nurr1 to study birth dating patterns. We find that most dorsal endopiriform (DEn) neurons are born on E13.5 to E14.5. Ventral claustrum (vCL) and dorsal claustrum (dCL) are mainly born on E14.5 to E15.5. Nurr1 positive cortical deep layer neurons (dLn) and superficial layer neurons (sLn) are mainly born on E14.5 to E15.5 and E15.5 to E17.5, respectively. Finally, we identify ventral to dorsal and posterior to anterior neurogenetic gradients within vCL and DEn. Thus, our findings suggest that claustrum and Nurr1 positive neurons in the lateral cortex are born sequentially over several days of embryonic development and contribute toward charting the complex developmental pattern of the claustrum in rodents. [ABSTRACT FROM AUTHOR]

The claustrum is an enigmatic brain structure thought to be important for conscious sensations. Recent studies have focused on gene expression patterns, connectivity, and function of the claustrum, but relatively little is known about its development. Interestingly, claustrum-enriched genes, including the previously identified marker Nurr1, are not only expressed in the classical claustrum complex, but also embedded within lateral neocortical regions in rodents. Recent studies suggest that Nurr1 positive neurons in the lateral cortex share a highly conserved genetic expression pattern with claustrum neurons. Thus, we focus on the developmental progression and birth dating pattern of the claustrum and Nurr1 positive neurons in the lateral cortex. We comprehensively investigate the expression of Nurr1 at various stages of development in the rat and find that Nurr1 expression first appears as an elongated line along the anterior-posterior axis on embryonic day 13.5 (E13.5) and then gradually differentiates into multiple sub-regions during prenatal development. Previous birth dating studies of the claustrum have led to conflicting results, therefore, we combine 5-ethynyl-2′-deoxyuridine (EdU) labeling with in situ hybridization for Nurr1 to study birth dating patterns. We find that most dorsal endopiriform (DEn) neurons are born on E13.5 to E14.5. Ventral claustrum (vCL) and dorsal claustrum (dCL) are mainly born on E14.5 to E15.5. Nurr1 positive cortical deep layer neurons (dLn) and superficial layer neurons (sLn) are mainly born on E14.5 to E15.5 and E15.5 to E17.5, respectively. Finally, we identify ventral to dorsal and posterior to anterior neurogenetic gradients within vCL and DEn. Thus, our findings suggest that claustrum and Nurr1 positive neurons in the lateral cortex are born sequentially over several days of embryonic development and contribute toward charting the complex developmental pattern of the claustrum in rodents.

Neuronal loss is the most common and critical feature of a spectrum of brain traumas and neurodegenerative disorders such as Alzheimer's disease (AD). The capacity to generate new neurons in the central nervous system diminishes early during brain development and is restricted mainly to two brain areas in the mature brain: subventricular zone and subgranular zone. Extensive research on the impact of brain injury on endogenous neurogenesis and cognition has been conducted primarily using young animals, when neurogenesis is most active. However, a critical question remains to elucidate the effect of brain injury on endogenous neurogenesis and cognition in older animals, which is far more relevant for age-related neurodegenerative disorders such as AD. Therefore, we examined the impact of neuronal loss on endogenous neurogenesis in aged animals using CaM/Tet-DTA mice, a transgenic model of hippocampal cell loss. Additionally, we investigated whether the upregulation of adult neurogenesis could mitigate cognitive deficits following substantial hippocampal neuronal loss. Our findings demonstrate that aged CaM/Tet-DTA mice that sustain severe neuronal loss exhibit an upregulation of endogenous neurogenesis. However, despite this significant upregulation, neurogenesis alone is not able to mitigate the cognitive deficits observed. Our studies suggest that the aged brain has the capacity to stimulate neurogenesis post-injury; however, multiple therapeutic approaches, including upregulation of endogenous neurogenesis, will be necessary to recover brain function after severe neurodegeneration.

The relevance of the research resulting from the widespread introduction information space and computer technology into the educational process, which, along with indisputable advantages, is accompanied by risks, the least studied in terms of prediction. Due to the fact that by 2025, according to the project "Modern Digital Educational Environment in the Russian Federation", a digital educational space should be created, the aim of this research is to study the digital competence of future teachers and the attitude of parents to the implementation of this trend. During the research were used methods of surveys and interview: 258 first-year students (future teachers) and 365 parents of elementary school pupils were interviewed. The article substantiates necessity to study the behavior of the children using devices and gadgets, which should be taken into account designing the educational process of future teachers, as both teachers and students must have a reliable and objective notion about a degree of children immersion in the information space, digitalization risks and the degree of compliance of information and communication skills of pupils and future teachers by the time of their meeting at school. The article presents data on the degree of immersion in the Internet space of future teachers, children who have begun systematic training in rural and urban schools, as well as their parents. The article reflects the attitude of parents to online learning and the control of their children’ behavior in the Internet space. The results of the research can be used by teachers and researchers to design and monitor the digitalization of the educational process.

This brief editorial tries to make known the main research records and repositories, with the aim of being known and used early on by PhD students. It raises the importance of using the ORCID author’s ID and knowledge of other key records of the editorial process among which are the ISSN, the DOI and the ROR, referring to the journal, the document and the institution, respectively. Subsequently, the main databases whose primary function is as an academic search engine (Scopus, Web of Science, Dimensions and Google Scholar) are described. The main academic social networking sites are then described (ResearchGate and Academia.edu), finishing with the main characteristics of the Sci-Hub case. It concludes by mentioning the relevance of integrating all these digital platforms to open PhD students up to the digital world and with it a series of possibilities for exchanges.

Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains unresolved. Here, we address these concerns by examining the effects of BrdU and EdU on adult hippocampal neurogenesis and EdU on the perinatal somatic development of mice. We show that, in a wide range of doses, EdU and BrdU label similar numbers of cells in the dentate gyrus shortly after administration. Furthermore, whereas the administration of EdU does not affect the division and survival of neural progenitor within 48 h after injection, it does affect cell survival, as evaluated 6 weeks later. We also show that a single injection of various doses of EdU on the first postnatal day does not lead to noticeable changes in a panel of morphometric criteria within the first week; however, higher doses of EdU adversely affect the subsequent somatic maturation and brain growth of the mouse pups. Our results indicate the potential caveats in labeling the replicating DNA using thymidine analogs and suggest guidelines for applying this approach.

BackgroundThymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs.MethodsEdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10(-8) mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay.ResultsEdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express β-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1.DiscussionAt the recommended dosage of 10(-8) mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs.

Hemocytes, the cell of the hemolymph, play a key role in the immune response of pulmonate molluscs to various pathogens including trematode infection. The number of hemocytes is known to increase after immunization but the mechanism of their multiplication remains debatable, proliferative capacity being the stumbling block. Some scientists consider that hemocytes may proliferate, while others think that their multiplication is only possible in special hematopoietic centres. In this work we studied the proliferative activity of hemocytes in five species of pulmonate molluscs: Biomphalaria glabrata, Planorbarius corneus, Planorbis planorbis, Lymnaea stagnalis and Succinea putris. ImageStream technique was used for the study of the hemocyte populations of these molluscan species for the first time. The hemocytes of all the studied species were represented by two main types, granular cells and hyalinocytes. Microscopic and flow-cytometric study of the hemocytes with the use of EdU revealed some EdU-positive cells. However, the analysis of the cell cycle of the hemocytes showed that the amount of DNA in these cells was not increased. Thus, it remains unclear whether the cells of the hemolymph retain the capacity to multiplication.

Root stem cell niche functioning requires the formation and maintenance of the specific "auxin-rich domain" governed by directional auxin transport and local auxin production. Auxin maximum co-localizes with the WOX5 expression domain in the quiescent center that separates mitotically active proximal and distal root meristems. Here we unravel the interconnected processes happening under WOX5 overexpression by combining in vivo experiments and mathematical modeling. We showed that WOX5-induced TAA1-mediated auxin biosynthesis is the cause, whereas auxin accumulation, PIN transporters relocation, and auxin redistribution between proximal and distal root meristems are its subsequent effects that influence the formation of the well-described phenotype with an enlarged root cap. These findings helped us to clarify the role of WOX5, which serves as a local QC-specific regulator that activates biosynthesis of non-cell-autonomous signal auxin to regulate the distal meristem functioning. The mathematical model with WOX5-mediated auxin biosynthesis and auxin-regulated cell growth, division, and detachment reproduces the columella cells dynamics in both wild type and under WOX5 dysregulation. [ABSTRACT FROM AUTHOR]

Hemocytes, the cell of the hemolymph, play a key role in the immune response of pulmonate molluscs to various pathogens including trematode infection. The number of hemocytes is known to increase after immunization but the mechanism of their multiplication remains debatable, proliferative capacity being the stumbling block. Some scientists consider that hemocytes may proliferate, while others think that their multiplication is only possible in special hematopoietic centres. In this work we studied the proliferative activity of hemocytes in five species of pulmonate molluscs: Biomphalaria glabrata, Planorbarius corneus, Planorbis planorbis, Lymnaea stagnalis and Succinea putris. ImageStream technique was used for the study of the hemocyte populations of these molluscan species for the first time. The hemocytes of all the studied species were represented by two main types, granular cells and hyalinocytes. Microscopic and flow-cytometric study of the hemocytes with the use of EdU revealed some EdU-positive cells. However, the analysis of the cell cycle of the hemocytes showed that the amount of DNA in these cells was not increased. Thus, it remains unclear whether the cells of the hemolymph retain the capacity to multiplication. [ABSTRACT FROM AUTHOR]

The present study investigated growth, biochemical, physiological, yield and proteomic changes in 3 wheat varieties exposed to elevated CO2 (515 ppm) in a background of high ambient ozone in field. Ethylenediurea (EDU) was used as antiozonant. Average ozone concentration was 59 ppb and was sufficient enough to exert phytotoxic effects. Elevated carbon dioxide (eCO2) and EDU application individually or in combination negated the adverse effects of ozone by modulating antioxidants and antioxidative enzymes. Differential leaf proteomics revealed that at vegetative stage major changes in protein abundance were due to EDU treatment (47, 52 and 41 proteins in PBW-343, LOK1 and HD-2967, respectively). Combined treatment of eCO2 and EDU was more responsible for changes in 37 proteins during flowering stage of PBW-343 and LOK1. Functional categorization revealed more than 60% differentially abundant protein collectively belonging to carbon metabolism, protein synthesis assembly and degradation and photosynthesis. At both the growth stages, LOK1 was more responsive to eCO2 and combined treatment (eCO2 + EDU). HD-2967 was more positively responsive to EDU and combined treatment. eCO2 in combination of EDU protected these varieties against high ambient O3. [ABSTRACT FROM AUTHOR]

Background: Segmentation in arthropods typically occurs by sequential addition of segments from a posterior growth zone. However, the amount of tissue required for growth and the cell behaviors producing posterior elongation are sparsely documented. Results: Using precisely staged larvae of the crustacean, Thamnocephalus platyurus, we systematically examine cell division patterns and morphometric changes associated with posterior elongation during segmentation. We show that cell division occurs during normal elongation but that cells in the growth zone need only divide ~ 1.5 times to meet growth estimates; correspondingly, direct measures of cell division in the growth zone are low. Morphometric measurements of the growth zone and of newly formed segments suggest tagma-specific features of segment generation. Using methods for detecting two different phases in the cell cycle, we show distinct domains of synchronized cells in the posterior trunk. Borders of cell cycle domains correlate with domains of segmental gene expression, suggesting an intimate link between segment generation and cell cycle regulation. Conclusions: Emerging measures of cellular dynamics underlying posterior elongation already show a number of intriguing characteristics that may be widespread among sequentially segmenting arthropods and are likely a source of evolutionary variability. These characteristics include: the low rates of posterior mitosis, the apparently tight regulation of cell cycle at the growth zone/new segment border, and a correlation between changes in elongation and tagma boundaries. [ABSTRACT FROM AUTHOR]

To develop treatments for salivary gland dysfunction, it is important to understand how human salivary glands are maintained under normal homeostasis. Previous data from our lab demonstrated that murine salivary acinar cells maintain the acinar cell population through self‐duplication under conditions of homeostasis, as well as after injury. Early studies suggested that human acinar cells are mitotically active, but the identity of the resultant daughter cells was not clear. Using markers of cell cycle activity and mitosis, as well as an ex vivo 5‐Ethynyl‐2´‐deoxyuridine assay, we show that human salivary gland acinar cells divide to generate daughter acinar cells. As in mouse, our data indicate that human salivary gland homeostasis is supported by the intrinsic mitotic capacity of acinar cells. [ABSTRACT FROM AUTHOR]

Ozone (O 3) pollution is harmful to plants and ecosystems. Several chemicals have been evaluated to protect plants against O 3 deleterious effects. However, they are not adequately efficient and/or the environmental safety of their application is questioned. Hence, new chemicals that provide sufficient protection while being safer for environmental application are needed. This study investigates the response of two O 3 -sensitive plant species (Phaseolus vulgaris L. cv. Pinto and Nicotiana tabacum L. cv. Bel-W3) leaf-sprayed with deionized water (W, control), ethylenediurea (EDU, 1 mM) or melatonin at lower (1 mM) or higher (3 mM) concentrations (Mel_L and Mel_H, respectively), and then exposed to a square wave of 200 ppb O 3 , lasting 1 day (5 h day−1) for bean and 2 days (8 h day−1) for tobacco. In both species, the photosynthetic activity of O 3 -exposed plants was about halved. O 3 -induced membrane damage was also confirmed by increased malondialdehyde (MDA) byproducts compared to control (W). In EDU- and Mel-treated bean plants, the photosynthetic performance was not influenced by O 3 , leading to reduction of the incidence and severity of O 3 visible injury. In bean plants, Mel_L mitigated the detrimental effect of O 3 by boosting antioxidant enzyme activities or osmoprotectants (e.g. abscisic acid, proline, and glutathione transferase). In Mel_L-sprayed tobacco plants, O 3 negatively influenced the photosynthetic activity. Conversely, Mel_H ameliorated the O 3 -induced oxidative stress by preserving the photosynthetic performance, preventing membrane damage, and reducing the visible injuries extent. Although EDU performed better, melatonin protected plants against O 3 phytotoxicity, suggesting its potential application as a bio-safer and eco-friendlier phytoprotectant against O 3. It is worth noting that the content of melatonin in EDU-treated plants remained unchanged, indicating that the protectant mode of action of EDU is not Mel-related. [Display omitted] • Tobacco and bean plants treated with exogenous melatonin were subjected to ozone. • Melatonin mitigated the detrimental effect of ozone. • Melatonin preserved photosynthetic performance and reduced visible foliar injury. • Melatonin enhanced compatible solutes production and antioxidant enzyme activities. • An opportunity for safer and eco-friendlier phytoprotection against ozone emerges. [ABSTRACT FROM AUTHOR]

Achieving global food security and ensuring sustainable agriculture, the dual objectives of the second Sustainable Development Goal (SDG 2), necessitate immediate and collaborative efforts from developing and developed nations. The adverse effects of ozone on crop yields have the potential to significantly undermine the United Nations' ambitious target of attaining food security and ending hunger by 2030. This review examines the causes of growing tropospheric ozone, especially in India and China which lead to a substantial reduction in crop yield and forest biomass. The findings show that a nexus of high population, rapid urbanization and regional pollution sources aggravates the problem in these countries. It elucidates that when plants are exposed to ozone, specific cellular pathways are triggered, resulting in changes in the expression of genes related to hormone production, antioxidant metabolism, respiration, and photosynthesis. Assessing the risks associated with ozone exposure involves using response functions that link exposure-based and flux-based measurements to variables like crop yield. Precisely quantifying the losses in yield and economic value in food crops due to current ozone levels is of utmost importance in comprehending the risks ozone poses to global food security. We conclude that policymakers should focus on implementing measures to decrease the emissions of ozone precursors, such as enhancing vehicle fuel efficiency standards and promoting the use of cleaner energy sources. Additionally, efforts should be directed toward mapping or developing crop varieties that can tolerate ozone, applying protective measures at critical stages of plant growth and establishing ozone-related vegetation protection standards. [Display omitted] • Tropospheric ozone jeopardises food security. • Use of OTC and FACE systems to evaluate O 3 -crop response. • Exposure-based index AOT40 widely used in studies. • Use of emission control, chemical protectants, and bioengineering techniques to prevent yield losses. [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.

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.

Tardigrades, microscopic ecdysozoans known for extreme environment resilience, were traditionally believed to maintain a constant cell number after completing embryonic development, a phenomenon termed eutely. However, sporadic reports of dividing cells have raised questions about this assumption. In this study, we explored tardigrade post-embryonic cell proliferation using the model species Hypsibius exemplaris. Comparing hatchlings to adults, we observed an increase in the number of storage cells, responsible for nutrient storage. We monitored cell proliferation via 5-ethynyl-2'-deoxyuridine (EdU) incorporation, revealing large numbers of EdU+ storage cells during growth, which starvation halted. EdU incorporation associated with molting, a vital post-embryonic development process involving cuticle renewal for further growth. Notably, DNA replication inhibition strongly reduced EdU+ cell numbers and caused molting-related fatalities. Our study is the first to demonstrate using molecular approaches that storage cells actively proliferate during tardigrade post-embryonic development, providing a comprehensive insight into replication events throughout their somatic growth. Additionally, our data underscore the significance of proper DNA replication in tardigrade molting and survival. This work definitely establishes that tardigrades are not eutelic, and offers insights into cell cycle regulation, replication stress, and DNA damage management in these remarkable creatures as genetic manipulation techniques emerge within the field., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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 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.

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]

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]