Effect of Aphidicolin,a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication,on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.     

Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex

The developing prenatal brain is particularly susceptible to environmental disturbances. During prenatal brain development, synapses form between neurons, resulting in neural circuits that support complex cognitive functions. In utero exposure to environmental factors such as pharmaceuticals that alter the process of synapse formation increases the risk of neurodevelopmental abnormalities. However, there is a lack of research into how specific environmental factors directly impact the developing neural circuitry of the human brain. For example, selective serotonin reuptake inhibitors are commonly used throughout pregnancy to treat depression, yet their impact on the developing fetal brain remains unclear. Recently, human brain models have provided unprecedented access to the critical window of prenatal brain development. In the present study, we used human neurons and cortical spheroids to determine whether the selective serotonin reuptake inhibitor fluoxetine alters neurite and synapse formation and the development of spontaneous activity within neural circuits. We demonstrate that cortical spheroids express serotonin transporter, thus recapitulating the early developmental expression of serotonin transporter associated with cortical pyramidal neurons. Cortical spheroids also appropriately express serotonin receptors, such as synaptic 5-HT2A and glial 5-HT5A. To determine whether fluoxetine can affect developing neural circuits independent of serotonergic innervation from the dorsal and medial raphe nuclei, we treated cortical neurons and spheroids with fluoxetine. Fluoxetine alters neurite formation in a dose-dependent fashion. Intriguingly, in cortical spheroids, neither acute nor chronic fluoxetine significantly altered excitatory synapse formation. However, only acute, but not chronic fluoxetine exposure altered inhibitory synaptogenesis. Finally, fluoxetine reversibly suppresses neuronal activity in a dose-dependent manner. These results demonstrate that fluoxetine can acutely alter synaptic function in developing neural circuits, but the effects were not long-lasting. This work provides a foundation for future studies to combine serotonergic innervation with cortical spheroids and assess the contributions of fluoxetine-induced alterations in serotonin levels to brain development.     

Concurrent Physiological and Pathological Angiogenesis in Retinopathy of Prematurity and Emerging Therapies

Retinopathy of prematurity (ROP) is an ocular vascular disease affecting premature infants, characterized by pathological retinal neovascularization (RNV), dilated and tortuous retinal blood vessels, and retinal or vitreous hemorrhages that may lead to retinal detachment, vision impairment and blindness. Compared with other neovascular diseases, ROP is unique because of ongoing and concurrent physiological and pathological angiogenesis in the developing retina. While the disease is currently treated by laser or cryotherapy, anti-vascular endothelial growth factor (VEGF) agents have been extensively investigated but are not approved in the U.S. because of safety concerns that they negatively interfere with physiological angiogenesis of the developing retina. An ideal therapeutic strategy would selectively inhibit pathological but not physiological angiogenesis. Our group recently described a novel strategy that selectively and safely alleviates pathological RNV in animal models of ROP by targeting secretogranin III (Scg3), a disease-restricted angiogenic factor. The preclinical profile of anti-Scg3 therapy presents a high potential for next-generation disease-targeted anti-angiogenic therapy for the ROP indication. This review focuses on retinal vessel development in neonates, the pathogenesis of ROP and its underlying molecular mechanisms, including different animal models, and provides a summary of current and emerging therapies.     

Wogonin induces reactive oxygen species production and cell apoptosis in human glioma cancer cells

Glioma is the most common primary adult brain tumor with poor prognosis because of the ease of spreading tumor cells to other regions of the brain. Cell apoptosis is frequently targeted for developing anti-cancer drugs. In the present study, we have assessed wogonin, a flavonoid compound isolated from Scutellaria baicalensis Georgi, induced ROS generation, endoplasmic reticulum (ER) stress and cell apoptosis. Wogonin induced cell death in two different human glioma cells, such as U251 and U87 cells but not in human primary astrocytes (IC 50 > 100 μM). Wogonin-induced apoptotic cell death in glioma cells was measured by propidine iodine (PI) analysis, Tunnel assay and Annexin V staining methods. Furthermore, wogonin also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Moreover, treatment of wogonin also increased a number of signature ER stress markers glucose-regulated protein (GRP)-78, GRP-94, Calpain I, and phosphorylation of eukaryotic initiation factor-2α (eIF2α). Treatment of human glioma cells with wogonin was found to induce reactive oxygen species (ROS) generation. Wogonin induced ER stress-related protein expression and cell apoptosis was reduced by the ROS inhibitors apocynin and NAC (N-acetylcysteine). The present study provides evidence to support the fact that wogonin induces human glioma cell apoptosis mediated ROS generation, ER stress activation and cell apoptosis.     

Carotenoids from Marine Sources as a New Approach in Neuroplasticity Enhancement

An increasing number of people experience disorders related to the central nervous system (CNS). Thus, new forms of therapy, which may be helpful in repairing processes’ enhancement and restoring declined brain functions, are constantly being sought. One of the most relevant physiological processes occurring in the brain for its entire life is neuroplasticity. It has tremendous significance concerning CNS disorders since neurological recovery mainly depends on restoring its structural and functional organization. The main factors contributing to nerve tissue damage are oxidative stress and inflammation. Hence, marine carotenoids, abundantly occurring in the aquatic environment, being potent antioxidant compounds, may play a pivotal role in nerve cell protection. Furthermore, recent results revealed another valuable characteristic of these compounds in CNS therapy. By inhibiting oxidative stress and neuroinflammation, carotenoids promote synaptogenesis and neurogenesis, consequently presenting neuroprotective activity. Therefore, this paper focuses on the carotenoids obtained from marine sources and their impact on neuroplasticity enhancement.     

Role of the Novel Peptide Phoenixin in Stress Response and Possible Interactions with Nesfatin-1

The novel peptide phoenixin was shown to be involved in several physiological processes ranging from reproduction to food intake. Interest in this protein has steadily increased over the last few years and its known implications have become much broader, playing a role in glucose homeostasis, anxiety, nociception, and pruritus. Phoenixin is expressed in a multitude of organs such as the small intestine, pancreas, and in the hypothalamus, as well as several other brain nuclei influencing numerous physiological functions. Its highly conserved amino-acid sequence amongst species leads to the assumption, that phoenixin might be involved in essential physiological functions. Its co-expression and opposing functionality to the extensively studied peptide nesfatin-1 has given rise to the idea of a possible counterbalancing role. Several recent publications focused on phoenixin’s role in stress reactions, namely restraint stress and lipopolysaccharide-induced inflammation response, in which also nesfatin-1 is known to be altered. This review provides an overview on the phoenixins and nesfatin-1 properties and putative effects, and especially highlights the recent developments on their role and interaction in the response to response.     

空气负离子与健康

作为衡量和评价空气质量高低的重要指标之一,空气负离子(negative air ion,NAI)浓度对生活区和旅游区环境质量的改善有重大意义。阐述了NAI的产生机理及其对人体内外环境的保健作用,讨论了城市环境、海滨环境和森林环境对空气NAI和人体健康的影响,为改善生活质量提供了科学参考,同时,为城市中人们的日常活动或休假给出了健康效益更高的建议。     

Assessment of Normal Tissue Radiosensitivity by Evaluating DNA Damage and Repair Kinetics in Human Brain Organoids

DNA-double strand break (DSB), detected by immunostaining of key proteins orchestrating repair, like γH2AX and 53BP1, is well established as a surrogate for tissue radiosensitivity. We hypothesized that the generation of normal brain 3D organoids (“mini-brains”) from human induced pluripotent stem cells (hiPSC) combined with detection of DNA damage repair (DDR) may hold the promise towards developing personalized models for the determination of normal tissue radiosensitivity. In this study, cerebral organoids, an in vitro model that stands in its complexity between 2D cellular system and an organ, have been used. To quantify radiation-induced response, immunofluorescent staining with γH2AX and 53BP1 were applied at early (30 min, initial damage), and late time points (18 and 72 h, residual damage), following clinical standard 2 Gy irradiation. Based on our findings, assessment of DDR kinetics as a surrogate for radiosensitivity in hiPSC derived cerebral organoids is feasible. Further development of mini-brains recapitulating mature adult neuronal tissue and implementation of additional signaling and toxicity surrogates may pave the way towards development of next-generation personalized assessment of radiosensitivity in healthy neuronal tissue.     

DNA Homeostasis and Senescence: Lessons from the Naked Mole Rat

As we age, our bodies accrue damage in the form of DNA mutations. These mutations lead to the generation of sub-optimal proteins, resulting in inadequate cellular homeostasis and senescence. The build-up of senescent cells negatively affects the local cellular micro-environment and drives ageing associated disease, including neurodegeneration. Therefore, limiting the accumulation of DNA damage is essential for healthy neuronal populations. The naked mole rats (NMR) are from eastern Africa and can live for over three decades in chronically hypoxic environments. Despite their long lifespan, NMRs show little to no biological decline, neurodegeneration, or senescence. Here, we discuss molecular pathways and adaptations that NMRs employ to maintain genome integrity and combat the physiological and pathological decline in organismal function.     

Dysregulation of Astrocyte Ion Homeostasis and Its Relevance for Stroke-Induced Brain Damage

Ischemic stroke is a leading cause of mortality and chronic disability. Either recovery or progression towards irreversible failure of neurons and astrocytes occurs within minutes to days, depending on remaining perfusion levels. Initial damage arises from energy depletion resulting in a failure to maintain homeostasis and ion gradients between extra- and intracellular spaces. Astrocytes play a key role in these processes and are thus central players in the dynamics towards recovery or progression of stroke-induced brain damage. Here, we present a synopsis of the pivotal functions of astrocytes at the tripartite synapse, which form the basis of physiological brain functioning. We summarize the evidence of astrocytic failure and its consequences under ischemic conditions. Special emphasis is put on the homeostasis and stroke-induced dysregulation of the major monovalent ions, namely Na⁺, K⁺, H⁺, and Cl⁻, and their involvement in maintenance of cellular volume and generation of cerebral edema.     

Flexible Assembly of an Enzyme Cascade on a DNA Triangle Prism Nanostructure for the Controlled Biomimetic Generation of Nitric Oxide

Spatial organization of multiple enzymes at specific positions for a controlled reaction cascade has attracted wide attention in recent years. Here, we report the construction of a biomimetic enzyme cascade organized on DNA triangle prism (TP) nanostructures to enable the efficient catalytic production of nitric oxide (NO) on a single microbead. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), were assembled at adjacent locations on a DNA TP nanostructure by using DNA‐binding protein adaptors with small interenzyme distances. In the cascade, the first enzyme, GOx, converts glucose into gluconic acid in the presence of oxygen. The produced H₂O₂ intermediate is rapidly transported to the second enzyme, HRP, which oxides hydroxyurea into NO and other nitroxyl species. The pH near the surface of the negatively charged DNA nanostructures is believed to be lower than that in the bulk solution; this creates an optimal pH environment for the anchored enzymes, which results in higher yields of the NO product. Furthermore, the multienzyme system was immobilized on a microbead mediated by a DNA adaptor, and this enabled the efficient catalytic generation of gas molecules in the microreactor. Therefore, this work provides an alternative route for the biomimetic generation of NO through enzyme cascades. In particular, the dynamic binding capability of the DNA sequence enabled the positions of the protein enzyme and the DNA nanostructure to be reversed, which allowed the cascade catalysis to be modulated.     

Non-Invasive Preimplantation Genetic Testing for Aneuploidy and the Mystery of Genetic Material: A Review Article

This review focuses on recent findings in the preimplantation genetic testing (PGT) of embryos. Different preimplantation genetic tests are presented along with different genetic materials and their analysis. Original material concerning preimplantation genetic testing for aneuploidy (PGT-A) was sourced by searching the PubMed and ScienceDirect databases in October and November 2021. The searches comprised keywords such as ‘preimplantation’, ‘cfDNA’; ‘miRNA’, ‘PGT-A’, ‘niPGT-A’, ‘aneuploidy’, ‘mosaicism’, ‘blastocyst biopsy’, ‘blastocentesis’, ‘blastocoel fluid’, ‘NGS’, ‘FISH’, and ‘aCGH’. Non-invasive PGT-A (niPGT-A) is a novel approach to the genetic analysis of embryos. The premise is that the genetic material in the spent embryo culture media (SECM) corresponds to the genetic material in the embryo cells. The limitations of niPGT-A are a lower quantity and lesser quality of the cell-free genetic material, and its unknown origin. The concordance rate varies when compared to invasive PGT-A. Some authors have also hypothesized that mosaicism and aneuploid cells are preferentially excluded from the embryo during early development. Cell-free genetic material is readily available in the spent embryo culture media, which provides an easier, more economic, and safer extraction of genetic material for analysis. The sampling of the SECM and DNA extraction and amplification must be optimized. The origin of the cell-free media, the percentage of apoptotic events, and the levels of DNA contamination are currently unknown; these topics need to be further investigated.     

Long-range charge transfer in DNA: transient structural distortions control the distance dependence

Damage to DNA is often caused by oxidative reactions. In one such process, an electron is lost from a base, forming its radical cation. Further reaction of the radical cation can lead to permanent change, which results in mutation. This Account is a report on oxidative damage to DNA caused by irradiation of anthraquinone derivatives, which are either randomly bound to the DNA or attached to it covalently at specific locations. Radical cations introduced in the DNA by the excited quinone cause damage both near to it and far away. We describe a mechanism for long-range charge transport in DNA that depends on its spontaneous structural distortion, which we call phonon-assisted polaron hopping. This mechanism, and its extension, provides a framework for understanding the reactions and charge-transport properties of DNA.     

Neuroinflammation in Post-Ischemic Neurodegeneration of the Brain: Friend,Foe, or Both?

One of the leading causes of neurological mortality, disability, and dementia worldwide is cerebral ischemia. Among the many pathological phenomena, the immune system plays an important role in the development of post-ischemic degeneration of the brain, leading to the development of neuroinflammatory changes in the brain. After cerebral ischemia, the developing neuroinflammation causes additional damage to the brain cells, but on the other hand it also plays a beneficial role in repair activities. Inflammatory mediators are sources of signals that stimulate cells in the brain and promote penetration, e.g., T lymphocytes, monocytes, platelets, macrophages, leukocytes, and neutrophils from systemic circulation to the brain ischemic area, and this phenomenon contributes to further irreversible ischemic brain damage. In this review, we focus on the issues related to the neuroinflammation that occurs in the brain tissue after ischemia, with particular emphasis on ischemic stroke and its potential treatment strategies.     

Cardiovascular and Cerebrovascular Implications of Growth Restriction: Mechanisms and Potential Treatments

Fetal growth restriction (FGR) is a common complication of pregnancy, resulting in a fetus that fails to reach its genetically determined growth potential. Whilst the fetal cardiovascular response to acute hypoxia is well established, the fetal defence to chronic hypoxia is not well understood due to experiment constraints. Growth restriction results primarily from reduced oxygen and nutrient supply to the developing fetus, resulting in chronic hypoxia. The fetus adapts to chronic hypoxia by redistributing cardiac output via brain sparing in an attempt to preserve function in the developing brain. This review highlights the impact of brain sparing on the developing fetal cardiovascular and cerebrovascular systems, as well as emerging long-term effects in offspring that were growth restricted at birth. Here, we explore the pathogenesis associated with brain sparing within the cerebrovascular system. An increased understanding of the mechanistic pathways will be critical to preventing neuropathological outcomes, including motor dysfunction such as cerebral palsy, or behaviour dysfunctions including autism and attention-deficit/hyperactivity disorder (ADHD).     

DNA nanotechnology: a future perspective

In addition to its genetic function, DNA is one of the most distinct and smart self-assembling nanomaterials. DNA nanotechnology exploits the predictable self-assembly of DNA oligonucleotides to design and assemble innovative and highly discrete nanostructures. Highly ordered DNA motifs are capable of providing an ultra-fine framework for the next generation of nanofabrications. The majority of these applications are based upon the complementarity of DNA base pairing: adenine with thymine, and guanine with cytosine. DNA provides an intelligent route for the creation of nanoarchitectures with programmable and predictable patterns. DNA strands twist along one helix for a number of bases before switching to the other helix by passing through a crossover junction. The association of two crossovers keeps the helices parallel and holds them tightly together, allowing the assembly of bigger structures. Because of the DNA molecule''s unique and novel characteristics, it can easily be applied in a vast variety of multidisciplinary research areas like biomedicine, computer science, nano/optoelectronics, and bionanotechnology.     

Cortical Dysplasia and the mTOR Pathway: How the Study of Human Brain Tissue Has Led to Insights into Epileptogenesis

Type II focal cortical dysplasia (FCD) is a neuropathological entity characterised by cortical dyslamination with the presence of dysmorphic neurons only (FCDIIA) or the presence of both dysmorphic neurons and balloon cells (FCDIIB). The year 2021 marks the 50th anniversary of the recognition of FCD as a cause of drug resistant epilepsy, and it is now the most common reason for epilepsy surgery. The causes of FCD remained unknown until relatively recently. The study of resected human FCD tissue using novel genomic technologies has led to remarkable advances in understanding the genetic basis of FCD. Mechanistic parallels have emerged between these non-neoplastic lesions and neoplastic disorders of cell growth and differentiation, especially through perturbations of the mammalian target of rapamycin (mTOR) signalling pathway. This narrative review presents the advances through which the aetiology of FCDII has been elucidated in chronological order, from recognition of an association between FCD and the mTOR pathway to the identification of somatic mosaicism within FCD tissue. We discuss the role of a two-hit mechanism, highlight current challenges and future directions in detecting somatic mosaicism in brain and discuss how knowledge of FCD may inform novel precision treatments of these focal epileptogenic malformations of human cortical development.     

Neuroplasticity and Multilevel System of Connections Determine the Integrative Role of Nucleus Accumbens in the Brain Reward System

A growing body of evidence suggests that nucleus accumbens (NAc) plays a significant role not only in the physiological processes associated with reward and satisfaction but also in many diseases of the central nervous system. Summary of the current state of knowledge on the morphological and functional basis of such a diverse function of this structure may be a good starting point for further basic and clinical research. The NAc is a part of the brain reward system (BRS) characterized by multilevel organization, extensive connections, and several neurotransmitter systems. The unique role of NAc in the BRS is a result of: (1) hierarchical connections with the other brain areas, (2) a well-developed morphological and functional plasticity regulating short- and long-term synaptic potentiation and signalling pathways, (3) cooperation among several neurotransmitter systems, and (4) a supportive role of neuroglia involved in both physiological and pathological processes. Understanding the complex function of NAc is possible by combining the results of morphological studies with molecular, genetic, and behavioral data. In this review, we present the current views on the NAc function in physiological conditions, emphasizing the role of its connections, neuroplasticity processes, and neurotransmitter systems.