During prolonged nutrient restriction, developing animals redistribute vital nutrients to favor brain growth at the expense of other organs. In
, such brain sparing relies on a glia-derived growth ...factor to sustain proliferation of neural stem cells. However, whether other aspects of neural development are also spared under nutrient restriction is unknown. Here we show that dynamically growing somatosensory neurons in the
peripheral nervous system exhibit organ sparing at the level of arbor growth: Under nutrient stress, sensory dendrites preferentially grow as compared to neighboring non-neural tissues, resulting in dendrite overgrowth. These neurons express lower levels of the stress sensor FoxO than neighboring epidermal cells, and hence exhibit no marked induction of autophagy and a milder suppression of Tor signaling under nutrient stress. Preferential dendrite growth allows for heightened animal responses to sensory stimuli, indicative of a potential survival advantage under environmental challenges.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 provides a highly efficient and flexible genome editing technology with numerous potential applications ranging from gene ...therapy to population control. Some proposed applications involve the integration of CRISPR/Cas9 endonucleases into an organism's genome, which raises questions about potentially harmful effects to the transgenic individuals. One example for which this is particularly relevant are CRISPR-based gene drives conceived for the genetic alteration of entire populations. The performance of such drives can strongly depend on fitness costs experienced by drive carriers, yet relatively little is known about the magnitude and causes of these costs. Here, we assess the fitness effects of genomic CRISPR/Cas9 expression in
cage populations by tracking allele frequencies of four different transgenic constructs that allow us to disentangle 'direct' fitness costs due to the integration, expression, and target-site activity of Cas9, from fitness costs due to potential off-target cleavage. Using a maximum likelihood framework, we find that a model with no direct fitness costs but moderate costs due to off-target effects fits our cage data best. Consistent with this, we do not observe fitness costs for a construct with Cas9HF1, a high-fidelity version of Cas9. We further demonstrate that using Cas9HF1 instead of standard Cas9 in a homing drive achieves similar drive conversion efficiency. These results suggest that gene drives should be designed with high-fidelity endonucleases and may have implications for other applications that involve genomic integration of CRISPR endonucleases.
Endothelial dysfunction, which leads to ischemic events under atherosclerotic conditions, can be attenuated by antagonizing the thromboxane-prostanoid receptor (TP) that mediates the vasoconstrictor ...effect of prostanoids including prostacyclin (PGI
2
). This study aimed to determine whether antagonizing the E prostanoid receptor-3 (EP3; which can also be activated by PGI
2
) adds to the above effect of TP deficiency (TP
–/–
) under atherosclerotic conditions and if so, the underlying mechanism(s). Atherosclerosis was induced in ApoE
–/–
mice and those with ApoE
–/–
and TP
–/–
. Here, we show that in phenylephrine pre-contracted abdominal aortic rings with atherosclerotic lesions of ApoE
–/–
/TP
–/–
mice, although an increase of force (which was larger than that of non-atherosclerotic controls) evoked by the endothelial muscarinic agonist acetylcholine to blunt the concurrently activated relaxation in ApoE
–/–
counterparts was largely removed, the relaxation evoked by the agonist was still smaller than that of non-atherosclerotic TP
–/–
mice. EP3 antagonism not only increased the above relaxation, but also reversed the contractile response evoked by acetylcholine in NO synthase-inhibited atherosclerotic ApoE
–/–
/TP
–/–
rings into a relaxation sensitive to I prostanoid receptor antagonism. In ApoE
–/–
atherosclerotic vessels the expression of endothelial NO synthase was decreased, yet the production of PGI
2
(which evokes contraction via both TP and EP3) evoked by acetylcholine was unaltered compared to non-atherosclerotic conditions. These results demonstrate that EP3 blockade adds to the effect of TP
–/–
in uncovering the dilator action of natively produced PGI
2
to alleviate endothelial dysfunction in atherosclerotic conditions.
Using scallop as raw material and sodium stearate as modifier, the modified scallop powder was prepared by wet modification. Using single factor and response surface experiments to explore the ...effects of modification temperature, modifier dosage, and modification time on the modification effect of scallop shell powder. The scallop shell powder was characterized by Fourier infrared spectrometer, scanning electron microscope and X-ray diffractometer. The modified scallop shell powder and chitosan were made into a composite film, and the influence of the mass fraction of the modified scallop shell powder on the properties of the composite film was investigated. The experimental results showed that the best modification conditions of scallop shell powder were: Sodium stearate addition amount 2.53%, modification temperature 80.70 ℃, modification time 43.19 min. The activation of the fan shellshell powder produced under these conditions could reach up to 76.41%. Combined with characterization mapping analysis, sodi
Neuron development is a highly orchestrated process that involves the precise coordination of cellular events, molecular signals, and environmental cues. This intricate process lays the foundation ...for the formation of functional neural circuits, allowing for proper information processing and communication within the nervous system. Understanding the mechanisms underlying neuron development is crucial for unraveling the complexities of brain function and for advancing our knowledge of neurological disorders and potential therapeutic interventions. To dissect the process of neuron development, optogenetics, with its ability to provide precise spatiotemporal manipulation of protein activity, offers a tremendous opportunity to investigate the complex signaling networks in developmental processes. In my dissertation research, I conducted a comprehensive investigation into both extrinsic and intrinsic factors during neuron development. The study focused on three main areas: understanding the response of neurons to nutrient stress, elucidating the role of terminal selectors in determining neuron identity post-mitotically, and developing optogenetics and CRISPR tools for studying neuron development. In the first phase of my research, my colleagues and I demonstrated that somatosensory neurons in the Drosophila peripheral nervous system exhibit organ sparing during nutrient stress. Sensory dendrites preferentially grow compared to non-neural tissues, resulting in dendrite overgrowth. These neurons exhibit lower levels of the stress sensor FoxO, leading to a milder suppression of Tor signaling and no marked induction of autophagy. Preferential dendrite growth enhances animal responses to sensory stimuli, potentially providing a survival advantage under environmental challenges.Next, I identified H6-like-homeobox (Hmx) and Cut as terminal selectors for class II (C2) and III (C3) da neurons, respectively, in Drosophila dendritic arborization (da) sensory neurons. Cut is constantly expressed in C3da neurons to inhibit Hmx expression and maintain C3da identity, while Hmx is required in C2da neurons to suppress Cut expression. Loss of each transcription factor results in upregulation of the other, leading to neuron type conversion.To further advance my research, I developed an innovative optogenetics system, OptoTrap, to manipulate endogenous proteins. I demonstrated that OptoTrap effectively traps GFP-tagged endogenous proteins of diverse sizes, subcellular locations, and functions, allowing for the manipulation of protein function in neurons and epithelial cells. Illumination conditions can be fine-tuned to achieve graded phenotypes and dissect the roles of proteins such as kinesin heavy chain (Khc) and αTubulin84B in dendrite morphogenesis.Besides, together with colleagues, I improved the CRISPR-TRiM tool by optimizing multiplexed gRNA design, enhancing gRNA efficiency in somatic tissues and the germline. I also developed methods to label mutant cells in tissue-specific mutagenesis using co-CRISPR reporters. Additionally, I created genetic reagents for converting Gal4 drivers into tissue-specific Cas9 lines through HACK.In summary, this dissertation underscored the delicate and intricate processes involved in the development of neurons and represented my efforts in advancing the tools to study neuronal development in Drosophila.
The F prostanoid receptor (FP), which accounts for the therapeutic effect of PGF
in uterine atony that leads to postpartum hemorrhage and maternal morbidity, could possibly mediate vasoconstrictor ...effect in small or resistance arteries to elevate blood pressure that limits the clinical use of the agent in patients with cardiovascular disorders. This study aimed to test the above hypothesis with genetically altered mice. Ex vivo and in vivo experiments were performed on control wild-type (WT) mice and mice with deficiencies in FP (FP
) or thromboxane (Tx)-prostanoid receptor (the original receptor of TxA
; TP
), and/or those with an additional deficiency in E prostanoid receptor-3 (one of the vasoconstrictor receptors of PGE
; EP3
). Here, we show that PGF
indeed evoked vasoconstrictor responses in the above-mentioned tissues of WT mice, which were however unaltered by FP
. Interestingly, such contractile responses were reversed into dilations by TP
/EP3
. A similar pattern of results was observed with the pressor effect of PGF
under in vivo conditions. However, TP
alone (which could largely remove the contractile responses) did not result in relaxation to PGF
. Also, either the ex vivo vasodilator effect or the in vivo depressor response of PGF
obtained after the removal of TP and EP3-mediated actions was unaltered by FP
. Therefore, both the ex vivo vasoconstrictor action in small or resistance arteries and the systemic pressor effect of PGF
can reflect vasoconstrictor activities derived from the non-FP receptors TP and EP3 outweighing a concurrently activated dilator effect, which is again independent of FP.
Necroinflammation plays an important role in disease settings such as acute kidney injury (AKI). We and others have elucidated that prostaglandins, which are critically involved in inflammation, may ...activate E-prostanoid 3 receptor (EP3) at low concentrations. However, how EP3 blockade interacts with regulated cell death and affects AKI remains unknown. In this study, AKI was induced by ischemia-reperfusion (30 minutes/24 hours) in Ep3 knockout (Ep3-/-), bone marrow chimeric, myeloid conditional EP3 knockout and corresponding control mice. The production of prostaglandins E2 and I2 was markedly increased after ischemia-reperfusion, and either abrogation or antagonism of EP3 ameliorated the injury. EP3 deficiency curbed inflammatory cytokine release, neutrophil infiltration and serum high-mobility group box 1 levels, but additional TLR4 inhibition with TAK-242 did not offer further protection against the injury and inflammation. The protection of Ep3-/- was predominantly mediated by suppressing Mixed Lineage Kinase domain-Like-dependent necroptosis, resulting from the inhibition of cytokine generation and the switching of cell death modality from necroptosis to apoptosis through caspase-8 up-regulation, in part due to the restraint of IL-6/JAK2/STAT3 signaling. EP3 deficiency failed to further alleviate the injury when necroptosis was inhibited. Ep3-/- in bone marrow-derived cells, particularly that in myeloid cells, protected kidneys to the same extent as that of global EP3 deletion. Thus, our results demonstrate that EP3 deficiency especially that on myeloid cells, ameliorates ischemic AKI via curbing inflammation and breaking the auto-amplification loop of necroinflammation. Hence, EP3 may be a promising target for the prevention and/or treatment of AKI.
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The F prostanoid receptor (FP), which accounts for the therapeutic effect of PGF2α in uterine atony that leads to postpartum hemorrhage and maternal morbidity, could possibly mediate vasoconstrictor ...effect in small or resistance arteries to elevate blood pressure that limits the clinical use of the agent in patients with cardiovascular disorders. This study aimed to test the above hypothesis with genetically altered mice. Ex vivo and in vivo experiments were performed on control wild‐type (WT) mice and mice with deficiencies in FP (FP−/−) or thromboxane (Tx)‐prostanoid receptor (the original receptor of TxA2; TP−/−), and/or those with an additional deficiency in E prostanoid receptor‐3 (one of the vasoconstrictor receptors of PGE2; EP3−/−). Here, we show that PGF2α indeed evoked vasoconstrictor responses in the above‐mentioned tissues of WT mice, which were however unaltered by FP−/−. Interestingly, such contractile responses were reversed into dilations by TP−/−/EP3−/−. A similar pattern of results was observed with the pressor effect of PGF2α under in vivo conditions. However, TP−/− alone (which could largely remove the contractile responses) did not result in relaxation to PGF2α. Also, either the ex vivo vasodilator effect or the in vivo depressor response of PGF2α obtained after the removal of TP and EP3‐mediated actions was unaltered by FP−/−. Therefore, both the ex vivo vasoconstrictor action in small or resistance arteries and the systemic pressor effect of PGF2α can reflect vasoconstrictor activities derived from the non‐FP receptors TP and EP3 outweighing a concurrently activated dilator effect, which is again independent of FP.
Vasomotor reactions of prostacyclin (prostaglandin I2; PGI2) can be collectively modulated by thromboxane prostanoid receptor (TP), E‐prostanoid receptor‐3 (EP3), and the vasodilator I prostanoid ...receptor (IP). This study aimed to determine the direct effect of PGI2 on renal arteries and/or the whole renal vasculature and how each of these receptors is involved. Experiments were performed on vessels or perfused kidneys of wild‐type mice and/or mice with deficiency in TP (TP−/−) and/or EP3. Here we show that PGI2 did not evoke relaxation, but instead resulted in contraction of main renal arteries (from ~0.001‐0.01 µM) or reduction of flow in perfused kidneys (from ~1 µM); either of them was reversed into a dilator response in TP−/−/EP3−/− counterparts. Also, we found that in renal arteries although it has a lesser effect than TP−/− on the maximal contraction to PGI2 (10 µM), EP3−/− but not TP−/− resulted in relaxation to the prostanoid at 0.01‐1 µM. Meanwhile, TP−/− only significantly reduced the contractile activity evoked by PGI2 at ≥0.1 µM. These results demonstrate that PGI2 may evoke an overall vasoconstrictor response in the mouse renal vasculature, reflecting activities of TP and EP3 outweighing that of the vasodilator IP. Also, our results suggest that EP3, on which PGI2 can have a potency similar to that on IP, plays a major role in the vasoconstrictor effect of the prostanoid of low concentrations (≤1 µM), while TP, on which PGI2 has a lower potency but higher efficacy, accounts for a larger part of its maximal contractile activity.
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