The most commonly farmed fish species in Bosnia and Herzegovina’s aquaculture are from the family Salmonidae, including brook trout
which is reared both for consumption and stocking purposes. A ...number of farmers complained about the elevated frequency of anatomical deformities in the smolts and fingerlings of brook trout, decreasing their fitness rate and causing significant financial loss. Since it has been shown that occurrence of deformities is correlated with the low genetic diversity and high inbreeding, this study aimed to assess intra- and interpopulation diversity of
from different freshwater fish farms in Bosnia and Herzegovina by observing variation in mitochondrial and nuclear genome. Total of 109 samples of brook trout from three hatcheries located at the Neretva River were analyzed for the mitochondrial control region and seven nuclear microsatellite loci. Both PCR-RFLP and sequencing revealed only one haplotype of the control region in all investigated trout. Overall, a low number of genotypes was evident across all the observed loci. Values of genetic diversity and polymorphic information content followed the increase in the number of alleles per locus. In general, values of inbreeding coefficient were generally very high, while the genetic diversity and observed heterozygosity had low rates. The results of our study are congruent with the findings of previous studies in which developmental deformities were concomitant with the low genetic diversity and inbreeding depression. It is, therefore, strongly advised to regularly supplement the broodstock with new, unrelated individuals, as it is of vital importance for sustaining a satisfying level of genetic diversity and preventing inbreeding depression. Additionally, maintaining good management practices regarding the fluctuation of water temperature, exposure to pollution, nutrition, etc., will further contribute to the prevention of this detrimental condition.
Topoisomerase-I is required for DNA replication. It acts by preventing torsional stress caused by DNA winding during replication fork progression. Topoisomerase-I inhibitors are widely used in many ...cancer therapies, in light of their anti-proliferative activity. However, their use as chemotherapeutics is associated with significant toxicity due to the off-target effects on healthy cells. We analyzed the dose-time-toxicity profile of a clinically employed topoisomerase-I inhibitor, i.e. topotecan, on primary CD4+T cells. This cell type was chosen to model a typical in-vivo interaction, due to the wide use of topotecan in the treatment of T-cell lymphomas. Our results show that a clinically achievable concentration of topotecan can induce toxic effects in healthy CD4+ T cells as early as 7 hours of the in vitro treatment. Toxicity of the drug was markedly increased by prolonging the post-treatment follow-up, but not by increasing concentrations, suggesting that clinical doses of topotecan can induce cell death and DNA damage in non-cancerous activated CD4+ T lymphocytes.
HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization and mechanism of uncoating are under debate. We adapted a strategy to visualize individual ...reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.
Hormones and growth factors (GFs) are signaling molecules implicated in the regulation of a variety of cellular processes. They play important roles in both healthy and tumor cells, where they ...function by binding to specific receptors on target cells and activating downstream signaling cascades. The stages of tumor progression are influenced by hormones and GF signaling. Hypoxia, a hallmark of cancer progression, contributes to tumor plasticity and heterogeneity. Most solid tumors contain a hypoxic core due to rapid cellular proliferation that outgrows the blood supply. In these circumstances, hypoxia-inducible factors (HIFs) play a central role in the adaptation of tumor cells to their new environment, dramatically reshaping their transcriptional profile. HIF signaling is modulated by a variety of factors including hormones and GFs, which activate signaling pathways that enhance tumor growth and metastatic potential and impair responses to therapy. In this review, we summarize the role of hormones and GFs during cancer onset and progression with a particular focus on hypoxia and the interplay with HIF proteins. We also discuss how hypoxia influences the efficacy of cancer immunotherapy, considering that a hypoxic environment may act as a determinant of the immune-excluded phenotype and a major hindrance to the success of adoptive cell therapies.
The maintenance of genome stability and cellular homeostasis depends on the temporal and spatial coordination of successive events constituting the classical DNA damage response (DDR). Recent ...findings suggest close integration and coordination of DDR signaling with specific cellular processes. The mechanisms underlying such coordination remain unclear. We review emerging crosstalk between DNA repair factors, chromatin remodeling, replication, transcription, spatial genome organization, cytoskeletal forces, and liquid–liquid phase separation (LLPS) in mediating DNA repair. We present an overarching DNA repair framework within which these dynamic processes intersect in nuclear space over time. Collectively, this interplay ensures the efficient assembly of DNA repair proteins onto shifting genome structures to preserve genome stability and cell survival.
Chromatin remodeling, replication, and transcription facilitate classical DDR signaling cascades.3D genome reorganization, LLPS of DNA repair factors, and cytoskeletal forces cooperate to promote DDR signaling as well as damaged DNA mobility and repair.Coordination of the sequential recruitment of dynamic DDR factors onto mobile chromosome structures supports DNA repair.The development of methods to study DNA dynamics at a genome-wide level in living cells will be necessary to fully understand the dynamic nature of DNA repair.
Topoisomerase-I is required for DNA replication. It acts by preventing torsional stress caused by DNA winding during replication fork progression. Topoisomerase-I inhibitors are widely used in many ...cancer therapies, in light of their anti-proliferative activity. However, their use as chemotherapeutics is associated with significant toxicity due to the off-target effects on healthy cells. We analyzed the dose-time-toxicity profile of a clinically employed topoisomerase-I inhibitor, i.e. topotecan, on primary CD4+T cells. This cell type was chosen to model a typical in-vivo interaction, due to the wide use of topotecan in the treatment of T-cell lymphomas. Our results show that a clinically achievable concentration of topotecan can induce toxic effects in healthy CD4+ T cells as early as 7 hours of the in vitro treatment. Toxicity of the drug was markedly increased by prolonging the post-treatment follow-up, but not by increasing concentrations, suggesting that clinical doses of topotecan can induce cell death and DNA damage in non-cancerous activated CD4+ T lymphocytes.
The nuclear envelope can form complex structures in physiological and pathological contexts. Current approaches to quantify nuclear envelope structures can be time-consuming or inaccurate. Here, we ...present a protocol to measure nuclear envelope tubules induced by DNA double-strand breaks using a mid-throughput approach. We describe steps for the induction of these nuclear envelope structures and 3D image analysis using machine-learning-based image segmentation. This protocol can be applied to analyze various nuclear envelope structures in contexts beyond DNA repair.
For complete details on the use and execution of this protocol, please refer to Shokrollahi et al.1
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•Instructions for preparing nuclear envelope tubules•Guidance on 3D imaging enables visualization of nuclear envelope tubules•Steps for quantifying nuclear surfaces using machine-learning-based segmentation•This protocol enables mid-throughput quantification of nuclear envelope tubules
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
The nuclear envelope can form complex structures in physiological and pathological contexts. Current approaches to quantify nuclear envelope structures can be time-consuming or inaccurate. Here, we present a protocol to measure nuclear envelope tubules induced by DNA double-strand breaks using a mid-throughput approach. We describe steps for the induction of these nuclear envelope structures and 3D image analysis using machine-learning-based image segmentation. This protocol can be applied to analyze various nuclear envelope structures in contexts beyond DNA repair.
Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside ...metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4
T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.
HIV‐1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV‐1 infection is accompanied by metabolic alterations, such as oxidative ...stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac‐infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV‐1 latency. We found that PML NBs are hyper‐SUMOylated and that PML protein is degraded via the ubiquitin–proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV‐1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.
Synopsis
HIV‐1 infection leads to alterations in redox and iron metabolism of the host cell. Here, HIV‐1‐induced reshaping of cellular metabolism is shown to regulate HIV‐1 latency establishment and reactivation via regulation of PML nuclear body dynamics.
HIV‐1 replication hijacks cellular metabolism by inducing oxidative stress and iron import.
Redox and iron imbalance leads to degradation of the HIV‐1 latency marker PML via the ubiquitin/proteasome pathway.
Upregulation of antioxidant responses facilitates cell survival and replenishment of PML, but can lead to HIV‐1 latency establishment.
Latency reversal recapitulates the metabolic dysregulation induced during productive infection.
Activation of cellular oxidative stress responses and iron import upon HIV‐1 infection triggers latency establishment via remodeling of PML nuclear bodies.
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