ResearchPad - specimen-disruption https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[The role of the C<sub>2</sub>A domain of synaptotagmin 1 in asynchronous neurotransmitter release]]> https://www.researchpad.co/article/elastic_article_14623 Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.

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<![CDATA[TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in <i>Chlamydomonas reinhardtii</i>]]> https://www.researchpad.co/article/elastic_article_13864 Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.

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<![CDATA[Generation of targeted homozygosity in the genome of human induced pluripotent stem cells]]> https://www.researchpad.co/article/Nc0b5af8d-f419-410c-9036-89fcaed1eba6

When loss of heterozygosity (LOH) is correlated with loss or gain of a disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we developed a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor ML216 exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6, where human leukocyte antigen (HLA) loci are located. Genotyping and flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Although careful examination of HLA-homozygous hiPSCs generated from parental cells is needed for cancer predisposition and effectiveness of differentiation, they may help to mitigate the current shortcoming of hiPSC-based transplantation related to the immunological differences between the donor and host.

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<![CDATA[The genetic intractability of Symbiodinium microadriaticum to standard algal transformation methods]]> https://www.researchpad.co/article/5c75ac68d5eed0c484d08712

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, with the view to performing subsequent CRISPR-Cas9 mediated genome editing. Plasmid vectors designed for nuclear transformation containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation and agitation with silicon carbide whiskers. Chloroplast-targeted transformation was attempted using an engineered Symbiodinium chloroplast minicircle encoding a modified PsbA protein expected to confer atrazine resistance. We report that we have been unable to confer chloramphenicol or atrazine resistance on Symbiodinium microadriaticum strain CCMP2467.

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<![CDATA[Rapid and sensitive detection of NADPH via mBFP-mediated enhancement of its fluorescence]]> https://www.researchpad.co/article/5c6b2618d5eed0c4842892e3

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates.

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<![CDATA[Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation]]> https://www.researchpad.co/article/5c64490fd5eed0c484c2f524

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture.

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<![CDATA[Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi]]> https://www.researchpad.co/article/5c466535d5eed0c484517ffa

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.

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<![CDATA[CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells]]> https://www.researchpad.co/article/5c478caed5eed0c484bd3df4

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in 𝛾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of 𝛾-globin. These data suggest that the 1.7 kb region is not an autonomous 𝛾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.

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<![CDATA[A size-invariant bud-duration timer enables robustness in yeast cell size control]]> https://www.researchpad.co/article/5c269726d5eed0c48470ec25

Cell populations across nearly all forms of life generally maintain a characteristic cell type-dependent size, but how size control is achieved has been a long-standing question. The G1/S boundary of the cell cycle serves as a major point of size control, and mechanisms operating here restrict passage of cells to Start if they are too small. In contrast, it is less clear how size is regulated post-Start, during S/G2/M. To gain further insight into post-Start size control, we prepared budding yeast that can be reversibly blocked from bud initiation. While blocked, cells continue to grow isotropically, increasing their volume by more than an order of magnitude over unperturbed cells. Upon release from their block, giant mothers reenter the cell cycle and their progeny rapidly return to the original unperturbed size. We found this behavior to be consistent with a size-invariant ‘timer’ specifying the duration of S/G2/M. These results indicate that yeast use at least two distinct mechanisms at different cell cycle phases to ensure size homeostasis.

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<![CDATA[ALG-2 participates in recovery of cells after plasma membrane damage by electroporation and digitonin treatment]]> https://www.researchpad.co/article/5bae98e640307c0c23a1c14c

The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research suggests that ALG-2 is involved in membrane repair mechanisms, in line with several published studies linking ALG-2 to processes of membrane remodeling and transport, which may contribute to the fitness of cells or protect them from damage. To investigate the involvement of ALG-2 in cell recovery after membrane damage we disrupted the PDCD6 gene encoding the ALG-2 protein in DT-40 cells and exposed them to electroporation. ALG-2 knock-out cells were more sensitive to electroporation as compared to wild type cells. This phenotype could be reversed by reestablishing ALG-2 expression confirming that ALG-2 plays an important role in cell recovery after plasma membrane damage. We found that overexpression of wild type ALG-2 but not a mutated form unable to bind Ca2+ partially protected HeLa cells from digitonin-induced cell death. Further, we were able to inhibit the cell protective function of ALG-2 after digitonin treatment by adding a peptide with the ALG-2 binding sequence of ALIX, which has been proposed to serve as the ALG-2 downstream target in a number of processes including cell membrane repair. Our results suggest that ALG-2 may serve as a novel therapeutic target in combination with membrane damaging interventions.

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<![CDATA[From Spontaneous Motor Activity to Coordinated Behaviour: A Developmental Model]]> https://www.researchpad.co/article/5989dabeab0ee8fa60bafde8

In mammals, the developmental path that links the primary behaviours observed during foetal stages to the full fledged behaviours observed in adults is still beyond our understanding. Often theories of motor control try to deal with the process of incremental learning in an abstract and modular way without establishing any correspondence with the mammalian developmental stages. In this paper, we propose a computational model that links three distinct behaviours which appear at three different stages of development. In order of appearance, these behaviours are: spontaneous motor activity (SMA), reflexes, and coordinated behaviours, such as locomotion. The goal of our model is to address in silico four hypotheses that are currently hard to verify in vivo: First, the hypothesis that spinal reflex circuits can be self-organized from the sensor and motor activity induced by SMA. Second, the hypothesis that supraspinal systems can modulate reflex circuits to achieve coordinated behaviour. Third, the hypothesis that, since SMA is observed in an organism throughout its entire lifetime, it provides a mechanism suitable to maintain the reflex circuits aligned with the musculoskeletal system, and thus adapt to changes in body morphology. And fourth, the hypothesis that by changing the modulation of the reflex circuits over time, one can switch between different coordinated behaviours. Our model is tested in a simulated musculoskeletal leg actuated by six muscles arranged in a number of different ways. Hopping is used as a case study of coordinated behaviour. Our results show that reflex circuits can be self-organized from SMA, and that, once these circuits are in place, they can be modulated to achieve coordinated behaviour. In addition, our results show that our model can naturally adapt to different morphological changes and perform behavioural transitions.

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<![CDATA[Non-associative Potentiation of Perisomatic Inhibition Alters the Temporal Coding of Neocortical Layer 5 Pyramidal Neurons]]> https://www.researchpad.co/article/5989da26ab0ee8fa60b80c8b

Long-term potentiation of inhibitory GABAergic transmission controls synaptic integration and action potential generation of specific neocortical neurons.

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<![CDATA[Recurrent Inhibition to the Medial Nucleus of the Trapezoid Body in the Mongolian Gerbil (Meriones Unguiculatus)]]> https://www.researchpad.co/article/5989d9e4ab0ee8fa60b6abdc

Principal neurons in the medial nucleus of the trapezoid body (MNTB) receive strong and temporally precise excitatory input from globular bushy cells in the cochlear nucleus through the calyx of Held. The extremely large synaptic currents produced by the calyx have sometimes led to the view of the MNTB as a simple relay synapse which converts incoming excitation to outgoing inhibition. However, electrophysiological and anatomical studies have shown the additional presence of inhibitory glycinergic currents that are large enough to suppress action potentials in MNTB neurons at least in some cases. The source(s) of glycinergic inhibition to MNTB are not fully understood. One major extrinsic source of glycinergic inhibitory input to MNTB is the ventral nucleus of the trapezoid body. However, it has been suggested that MNTB neurons receive additional inhibitory inputs via intrinsic connections (collaterals of glycinergic projections of MNTB neurons). While several authors have postulated their presence, these collaterals have never been examined in detail. Here we test the hypothesis that collaterals of MNTB principal cells provide glycinergic inhibition to the MNTB. We injected dye into single principal neurons in the MNTB, traced their projections, and immunohistochemically identified their synapses. We found that collaterals terminate within the MNTB and provide an additional source of inhibition to other principal cells, creating an inhibitory microcircuit within the MNTB. Only about a quarter to a third of MNTB neurons receive such collateral inputs. This microcircuit could produce side band inhibition and enhance frequency tuning of MNTB neurons, consistent with physiological observations.

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<![CDATA[Functional Reorganization of the Default Mode Network across Chronic Pain Conditions]]> https://www.researchpad.co/article/5989dad3ab0ee8fa60bb707b

Chronic pain is associated with neuronal plasticity. Here we use resting-state functional magnetic resonance imaging to investigate functional changes in patients suffering from chronic back pain (CBP), complex regional pain syndrome (CRPS) and knee osteoarthritis (OA). We isolated five meaningful resting-state networks across the groups, of which only the default mode network (DMN) exhibited deviations from healthy controls. All patient groups showed decreased connectivity of medial prefrontal cortex (MPFC) to the posterior constituents of the DMN, and increased connectivity to the insular cortex in proportion to the intensity of pain. Multiple DMN regions, especially the MPFC, exhibited increased high frequency oscillations, conjoined with decreased phase locking with parietal regions involved in processing attention. Both phase and frequency changes correlated to pain duration in OA and CBP patients. Thus chronic pain seems to reorganize the dynamics of the DMN and as such reflect the maladaptive physiology of different types of chronic pain.

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<![CDATA[Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb77e

Background and Objectives

The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.

Methodology/Principal Findings

Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.

Conclusions/Significance

MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.

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<![CDATA[Kisspeptins Modulate the Biology of Multiple Populations of Gonadotropin-Releasing Hormone Neurons during Embryogenesis and Adulthood in Zebrafish (Danio rerio)]]> https://www.researchpad.co/article/5989da21ab0ee8fa60b7f1a7

Kisspeptin1 (product of the Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.

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<![CDATA[Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1) with DNA damage response genes]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be1128

Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

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<![CDATA[Neonatal NMDA Receptor Blockade Disrupts Spike Timing and Glutamatergic Synapses in Fast Spiking Interneurons in a NMDA Receptor Hypofunction Model of Schizophrenia]]> https://www.researchpad.co/article/5989daacab0ee8fa60ba9a85

The dysfunction of parvalbumin-positive, fast-spiking interneurons (FSI) is considered a primary contributor to the pathophysiology of schizophrenia (SZ), but deficits in FSI physiology have not been explicitly characterized. We show for the first time, that a widely-employed model of schizophrenia minimizes first spike latency and increases GluN2B-mediated current in neocortical FSIs. The reduction in FSI first-spike latency coincides with reduced expression of the Kv1.1 potassium channel subunit which provides a biophysical explanation for the abnormal spiking behavior. Similarly, the increase in NMDA current coincides with enhanced expression of the GluN2B NMDA receptor subunit, specifically in FSIs. In this study mice were treated with the NMDA receptor antagonist, MK-801, during the first week of life. During adolescence, we detected reduced spike latency and increased GluN2B-mediated NMDA current in FSIs, which suggests transient disruption of NMDA signaling during neonatal development exerts lasting changes in the cellular and synaptic physiology of neocortical FSIs. Overall, we propose these physiological disturbances represent a general impairment to the physiological maturation of FSIs which may contribute to schizophrenia-like behaviors produced by this model.

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<![CDATA[Waterborne exposure to BPS causes thyroid endocrine disruption in zebrafish larvae]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf502

Bisphenol S (BPS) is widely used as a raw material in industry, resulting in its ubiquitous distribution in natural environment, including the aqueous environment. However, the effect of BPS on the thyroid endocrine system is largely unknown. In this study, zebrafish (Danio rerio) embryos were exposed to BPS at 1, 3, 10, and 30 μg/L, from 2 h post-fertilization (hpf) to 168hpf. Bioconcentration of BPS and whole-body thyroid hormones (THs), thyroid-stimulating hormone (TSH) concentrations as well as transcriptional profiling of key genes related to the hypothalamic-pituitary-thyroid (HPT) axis were examined. Chemical analysis indicated that BPS was accumulated in zebrafish larvae. Thyroxine (T4) and triiodothyronine (T3) levels were significantly decreased at ≥ 10 and 30 μg/L of BPS, respectively. However, TSH concentration was significantly induced in the 10 and 30 μg/L BPS-treated groups. After exposure to BPS, the mRNA expression of corticotrophin releasing hormone (crh) and thyroglobulin (tg) genes were up-regulated at ≥10 μg/L of BPS, in a dose-response manner. The transcription of genes involved in thyroid development (pax8) and synthesis (sodium/iodide symporter, slc5a5) were also significantly increased in the 30 μg/L of BPS treatment group. Moreover, exposure to 10 μg/L or higher concentration of BPS significantly up-regulated genes related to thyroid hormone metabolism (deiodinases, dio1, dio2 and uridinediphosphate glucoronosyltransferases, ugt1ab), which might be responsible for the altered THs levels. However, the transcript of transthyretin (ttr) was significantly down-regulated at ≥ 3 μg/L of BPS, while the mRNA levels of thyroid hormone receptors (trα and trβ) and dio3 remained unchanged. All the results indicated that exposure to BPS altered the whole-body THs and TSH concentrations and changed the expression profiling of key genes related to HPT axis, thus triggering thyroid endocrine disruption.

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<![CDATA[The Effects of Disturbance on Hypothalamus-Pituitary-Thyroid (HPT) Axis in Zebrafish Larvae after Exposure to DEHP]]> https://www.researchpad.co/article/5989db17ab0ee8fa60bcd6f6

Di-(2-ethylhexyl) phthalate (DEHP) has the potential to disrupt the thyroid endocrine system, but the underlying mechanism is unknown. In this study, zebrafish (Danio rerio) embryos were exposed to different concentrations of DEHP (0, 40, 100, 200, 400 μg/L) from 2 to 168 hours post fertilization (hpf). Thyroid hormones (THs) levels and transcriptional profiling of key genes related to hypothalamus-pituitary-thyroid (HPT) axis were examined. The result of whole-body thyroxine (T4) and triiodothyronine (T3) indicated that the thyroid hormone homeostasis was disrupted by DEHP in the zebrafish larvae. After exposure to DEHP, the mRNA expressions of thyroid stimulating hormone (tshβ) and corticotrophin releasing hormone (crh) genes were increased in a concentration dependent manner, respectively. The expression level of genes involved in thyroid development (nkx2.1 and pax8) and thyroid synthesis (sodium/iodide symporter, nis, thyroglobulin, tg) were also measured. The transcripts of nkx2.1 and tg were significantly increased after DEHP exposure, while those of nis and pax8 had no significant change. Down-regulation of uridinediphosphate-glucuronosyl-transferase (ugt1ab) and up-regulation of thyronine deiodinase (dio2) might change the THs levels. In addition, the transcript of transthyretin (ttr) was up-regulated, while the mRNA levels of thyroid hormone receptors (trα and trβ) remained unchanged. All the results demonstrated that exposure to DEHP altered the whole-body thyroid hormones in the zebrafish larvae and changed the expression profiling of key genes related to HPT axis, proving that DEHP induced the thyroid endocrine toxicity and potentially affected the synthesis, regulation and action of thyroid hormones.

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