Physalin A Induces Apoptosis and Autophagy in Hepatocellular Carcinoma via Inhibition of PI3K/Akt Signaling Pathway.

Physalin A (PA) is a bioactive withanolide with multiple pharmacological properties and has been indicated to be cytotoxic to hepatocellular carcinoma (HCC) cell line HepG2. This study aims to explore the mechanisms underlying PA antitumor activity in HCC. HepG2 cells were exposed to various concentrations of PA. Cell counting kit-8 assay and flow cytometry were implemented for evaluating cell viability and apoptosis, respectively. Immunofluorescence staining was utilized for detecting autophagic protein LC3. Western blotting was employed for measuring levels of autophagy-, apoptosis- and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling-related proteins. A xenograft mouse model was established to verify the antitumor activity of PA in vivo. PA impaired HepG2 cell viability, and triggered apoptosis as well as autophagy. Inhibiting autophagy augmented PA-evoked HepG2 cell apoptosis. PA repressed PI3K/Akt signaling in HCC cells and activating PI3K/Akt reversed PA-triggered apoptosis and autophagy. PA treatment inhibited tumor growth in tumor-bearing mice. PA triggers HCC cell apoptosis and autophagy by inactivating PI3K/Akt signaling.

Optimal therapeutic conditions for the neural stem cell-based management of ischemic stroke: a systematic review and network meta-analysis based on animal studies.

BACKGROUND: In order to promote the clinical translation of preclinical findings, it is imperative to identify the most optimal therapeutic conditions and adopt them for further animal and human studies. This study aimed to fully explore the optimal conditions for neural stem cell (NSC)-based ischemic stroke treatment based on animal studies. METHODS: The PubMed, Ovid-Embase, and Web of Science databases were searched in December 2021. The screening of search results, extraction of relevant data, and evaluation of study quality were performed independently by two reviewers. RESULTS: In total, 52 studies were included for data analysis. Traditional meta-analysis showed that NSCs significantly reduced the modified neurological severity score (mNSS) and volume of cerebral infarct in animal models of ischemic stroke. Network meta-analysis showed that allogeneic embryonic tissue was the best source of NSCs. Further, intracerebral transplantation was the most optimal route of NSC transplantation, and the acute phase was the most suitable stage for intervention. The optimal number of NSCs for transplantation was 1-5×105 in mouse models and 1×106 or 1.8×106 in rat models. CONCLUSIONS: We systematically explored the therapeutic strategy of NSCs in ischemic stroke, but additional research is required to develop optimal therapeutic strategies based on NSCs. Moreover, it is necessary to further improve and standardize the design, implementation, measuring standards, and reporting of animal-based studies to promote the development of better animal experiments and clinical research.

Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome.

Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex. Mutations in the Pyrin-encoding gene, MEFV, cause a human autoinflammatory disease known as familial Mediterranean fever. Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB, a major virulence factor of Clostridium difficile, which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn 41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.

Behavioural responses of zebrafish larvae to acute ethosuximide exposure.

The anticonvulsant drug ethosuximide has shown diverse anxiety-related activity in rodents, but research in zebrafish is limited. To evaluate the effects of acute ethosuximide exposure on locomotor activity and anxiety-related thigmotaxis behaviours of zebrafish larvae, the activity of AB strain zebrafish larvae at 5 and 7 days postfertilization (dpf) was analysed under normal constant illumination and stressful light-dark transitions. Under constant illumination, ethosuximide at concentrations of 2, 5 and 10 mmol/l increased the distances travelled and intensified locomotor responses to a novel environment. In addition, 40 mmol/l ethosuximide decreased the travel distance and attenuated the locomotor response to darkness. The effects were age related. Under constant illumination, ethosuximide at 40 mmol/l reduced thigmotaxis behaviours in larvae at both ages. Under the light-dark transition, 5 mmol/l ethosuximide reduced thigmotaxis behaviours in 7-dpf larvae. We concluded that under constant lighting, ethosuximide at low concentrations (2, 5 and 10 mmol/l) stimulated the locomotor activity of zebrafish larvae, whereas a high concentration (40 mmol/l) inhibited the activity. Ethosuximide at a low concentration (5 mmol/l) showed anxiolytic effects during the stressful light-dark transition in 7-dpf larvae. The effects of ethosuximide were age and concentration related.

Effects of picrotoxin on zebrafish larvae behaviors: A comparison study with PTZ.

Larval zebrafish (Danio rerio) have been suggested as a high-throughput experimental animal model for epilepsy-related genetic and developmental studies. The behavioral manifestations in response to the seizure-inducing drugs picrotoxin (PTX) (1, 5, 25, 125, or 625µM) or pentylenetetrazole (PTZ) (1, 2, 4, 8, or 16mM) under light-dark conditions were studied using zebrafish larvae at 5days post-fertilization (dpf). Two behavioral parameters, locomotor activity and thigmotaxis behavior, were analyzed. We conclude that high concentrations of PTX treatment increased locomotion and thigmotaxis in 5 dpf zebrafish larvae under continuous illumination and the locomotion of PTX-treated zebrafish was decreased under the dark condition. High concentrations of PTX treatment also increased thigmotaxis (an indicator of increased anxiety levels) in zebrafish larvae under both continuous illumination and dark condition. PTZ treatment increased the locomotion of 5 dpf zebrafish larvae under continuous illumination. However, 2mM PTZ decreased locomotion, and high concentrations of PTZ decreased the locomotion of larvae under dark conditions. High concentrations of PTZ treatment also increased thigmotaxis in the zebrafish larvae under both continuous illumination and dark condition. Compared with PTZ, PTX leads to higher levels of movement under light conditions and lower levels of movement under dark condition. However, the level of thigmotaxis in the zebrafish larvae was similar between the two drug treatments.

Developmental profiling of ASD-related shank3 transcripts and their differential regulation by valproic acid in zebrafish.

SHANK3 is a scaffolding protein that binds to various synaptic proteins at the postsynaptic density (PSD) of excitatory glutamatergic synapses. SHANK3 is not only strongly implicated in autism spectrum disorders (ASD) but also plays a critical role in human Phelan-McDermid syndrome (22q13.3 deletion syndrome). Accumulated experimental evidence demonstrates that the zebrafish model system is useful for studying the functions of ASD-related gene during early development. However, many basic features of shank3 transcript expression in zebrafish remain poorly understood. Here, we investigated temporal, spatial, and isoform-specific expression patterns of shank3 during zebrafish development on the basis of previous researches and the differential effects of each shank3 transcript expression after exposure to valproic acid (VPA), an ASD-associated drug. At first, we observed that both shank3a and shank3b were barely expressed at very early ages (before 24 h post-fertilization (hpf)), whereas their expression levels were increased and mainly enriched in the nervous system after 24 hpf. Secondly, all of the six shank3 transcripts gradually increased during the first 7 hpf and then decreased. Subsequently, they exhibited a second increasing peak between 1 month post-fertilization (mpf) and adulthood. Thirdly, VPA treatment affected the isoform-specific expression of zebrafish shank3. In particular, the mRNA expression levels of those isoforms that contain a SAM domain were significantly increased, whereas the mRNA expression level of those which contained an ANK domain but without a SAM domain was decreased. To conclude, our findings support the molecular diversity of shank3 in zebrafish and provide a molecular framework to understand the isoform-specific function of shank3 in zebrafish.

Phospholipase C gamma-1 is required for granulocyte maturation in zebrafish.

The regulation of hematopoiesis is generally evolutionarily conserved from zebrafish to mammals, including hematopoietic stem cell formation and blood cell lineage differentiation. In zebrafish, primitive granulocytes originate at two distinct regions, the anterior lateral plate mesoderm (A-LPM) and the intermediate cell mass (ICM). Few studies in the zebrafish have examined genes specifically required for the granulocytic lineage. In this study, we identified the responsible gene for a zebrafish mutant that has relatively normal hematopoiesis, except decreased expression of the granulocyte-specific gene mpx. Positional cloning revealed that phospholipase C gamma-1 (plcg1) was mutated. Deficiency of plcg1 function specifically affected development of granulocytes, especially the maturation process. These results suggested that plcg1 functioned specifically in zebrafish ICM granulopoiesis for the first time. Our studies suggest that specific pathways regulate the differentiation of the hematopoietic lineages.

Expression of histone deacetylase-1 and p300 in aristolochic acid nephropathy models.

Aristolochic acid nephropathy (AAN) is mainly caused by aristolochic acid I (AAI), but the actual mechanism is still uncertain. The current study explored the correlation among the expression of Smad7, p300, histone deacetylase-1 (HDAC1) and the development of AAN using transmission electron microscopy (TEM), RT-PCR, and western blotting in the AAN mouse model and in the AAN cell model. TEM revealed that the renal tubular epithelial cells from the AAI-treated mice presented organelle damages and nuclear deformation. We found that a certain dose of AAI caused renal fibrosis and induced renal tubular epithelial cells to differentiate into myofibroblasts. There was a gradual increase in the expression of HDAC1 mRNA and protein observed using RT-PCR and western blotting in the AAN cell model compared with the control group. Gradual decrease in the expression of Smad7 and p300 mRNA and protein was revealed in the AAN mouse and cell models compared with the control group. These results suggest that AAI dose dependently contributed to the development of AAN, and HDAC1 and p300 participate in the modulation of TGF-ß/Smad pathway-mediated renal interstitial fibrosis.

Piezo1-dependent regulation of pericyte proliferation by blood flow during brain vascular development.

Blood flow is known to regulate cerebrovascular development through acting on vascular endothelial cells (ECs). As an indispensable component of the neurovascular unit, brain pericytes physically couple with ECs and play vital roles in blood-brain barrier integrity maintenance and neurovascular coupling. However, it remains unclear whether blood flow affects brain pericyte development. Using in vivo time-lapse imaging of larval zebrafish, we monitored the developmental dynamics of brain pericytes and found that they proliferate to expand their population and increase their coverage to brain vessels. In combination with pharmacological and genetic approaches, we demonstrated that blood flow enhances brain pericyte proliferation through Piezo1 expressed in ECs. Moreover, we identified that EC-intrinsic Notch signaling is downstream of Piezo1 to promote the activation of Notch signaling in pericytes. Thus, our findings reveal a role of blood flow in pericyte proliferation, extending the functional spectrum of hemodynamics on cerebrovascular development.

Circadian regulation of developmental synaptogenesis via the hypocretinergic system.

The circadian clock orchestrates a wide variety of physiological and behavioral processes, enabling animals to adapt to daily environmental changes, particularly the day-night cycle. However, the circadian clock's role in the developmental processes remains unclear. Here, we employ the in vivo long-term time-lapse imaging of retinotectal synapses in the optic tectum of larval zebrafish and reveal that synaptogenesis, a fundamental developmental process for neural circuit formation, exhibits circadian rhythm. This rhythmicity arises primarily from the synapse formation rather than elimination and requires the hypocretinergic neural system. Disruption of this synaptogenic rhythm, by impairing either the circadian clock or the hypocretinergic system, affects the arrangement of the retinotectal synapses on axon arbors and the refinement of the postsynaptic tectal neuron's receptive field. Thus, our findings demonstrate that the developmental synaptogenesis is under hypocretin-dependent circadian regulation, suggesting an important role of the circadian clock in neural development.

Role of Olfactorily Responsive Neurons in the Right Dorsal Habenula-Ventral Interpeduncular Nucleus Pathway in Food-Seeking Behaviors of Larval Zebrafish.

The habenula (Hb) plays important roles in emotion-related behaviors. Besides receiving inputs from the limbic system and basal ganglia, Hb also gets inputs from multiple sensory modalities. Sensory responses of Hb neurons in zebrafish are asymmetrical: the left dorsal Hb and right dorsal Hb (dHb) preferentially respond to visual and olfactory stimuli, respectively, implying different functions of the left and right dHb. While visual responses of the left dHb (L-dHb) have been implicated in light-preference behavior, the significance of olfactory responses of the right dHb (R-dHb) remains under-examined. It was reported that the R-dHb can gate innate attraction to a bile salt. However, considering a broad range of odors that R-dHb respond to, it is of interest to examine the role of R-dHb in other olfactory behaviors, especially food seeking, which is essential for animals' survival. Here, using in vivo whole-cell recording and calcium imaging, we first characterized food extract-evoked responses of Hb neurons. Responsive neurons preferentially locate in the R- but not L-dHb and exhibit either ON- (~87%) or OFF-type responses (~13%). Interestingly, this right-to-left asymmetry of olfactory responses converts into a ventral-to-dorsal pattern in the interpeduncular nucleus (IPN), a main downstream target of Hb. Combining behavior assay, we further found that genetic dysfunction or lesion of the R-dHb and its corresponding downstream ventral IPN (V-IPN) impair the food seeking-associated increase of swimming activity. Thus, our study indicates that the asymmetrical olfactory response in the R-dHb to V-IPN pathway plays an important role in food-seeking behavior of zebrafish larvae.

The Locus Coeruleus Modulates Intravenous General Anesthesia of Zebrafish via a Cooperative Mechanism.

How general anesthesia causes loss of consciousness has been a mystery for decades. It is generally thought that arousal-related brain nuclei, including the locus coeruleus (LC), are involved. Here, by monitoring locomotion behaviors and neural activities, we developed a larval zebrafish model for studying general anesthesia induced by propofol and etomidate, two commonly used intravenous anesthetics. Local lesion of LC neurons via two-photon laser-based ablation or genetic depletion of norepinephrine (NE; a neuromodulator released by LC neurons) via CRISPR/Cas9-based mutation of dopamine-ß-hydroxylase (dbh) accelerates induction into and retards emergence from general anesthesia. Mechanistically, in vivo whole-cell recording revealed that both anesthetics suppress LC neurons' activity through a cooperative mechanism, inhibiting presynaptic excitatory inputs and inducing GABAA receptor-mediated hyperpolarization of these neurons. Thus, our study indicates that the LC-NE system plays a modulatory role in both induction of and emergence from intravenous general anesthesia.

Electrochemical oxidation of COD from real textile wastewaters: Kinetic study and energy consumption.

In the present study, the electrochemical oxidation of real wastewaters discharged by textile industry was carried out using a boron-doped diamond (BDD) anode. The effect of operational variables, such as applied current density (20-100 mA·cm-2), NaCl concentration added to the real wastewaters (0-3 g·L-1), and pH value (2.0-10.0), on the kinetics of COD oxidation and on the energy consumption was carefully investigated. The obtained experimental results could be well matched with a proposed kinetic model, in which the indirect oxidation mediated by electrogenerated strong oxidants would be described through a pseudo-first-order kinetic constant k. Values of k exhibited a linear increase with increasing applied current density and decreasing pH value, and an exponential increase with NaCl concentration. Furthermore, high oxidation kinetics resulted in low specific energy consumption, but this conclusion was not suitable to the results obtained under different applied current density. Under the optimum operational conditions, it only took 3 h to complete remove the COD in the real textile wastewaters and the specific energy consumption could be as low as 11.12 kWh·kg-1 COD. The obtained results, low energy consumption and short electrolysis time, allowed to conclude that the electrochemical oxidation based on BDD anodes would have practical industrial application for the treatment of real textile wastewater.

Effects of diphenylhydantoin on locomotion and thigmotaxis of larval zebrafish.

Antiepileptic effects of diphenylhydantoin (DPH) have been documented in animal studies and clinical research, while little is known about the effects of the drug on basic behaviors and anxiety-related behaviors. In order to understand neuroactivities of DPH deeply and administrate DPH in clinic rationally, it is necessary to study neurobehavioral effects of the drug. In the present study, the effects of DPH on the locomotor activity and thigmotaxis of zebrafish larvae at 5 days post fertilization (dpf) were explored under different illumination conditions. The influence of DPH on zebrafish larval responses to visual stimuli (sudden illumination transition from light to dark) was also investigated. Under light or dark condition, exposure to high concentrations of DPH resulted in decreased locomotor activity and thigmotaxis, whereas DPH treatment at low doses enhanced the locomotor activity. Additionally, sudden illumination transition induced robust increase in the locomotor activity and this phenomenon was not modified by DPH treatment. Our results suggest that DPH has potential stimulatory and inhibitory effects on the locomotor activity and possesses anxiolytic properties. In addition, responses of 5-dpf zebrafish larvae to visual stimuli were not modified by DPH treatment.

Anxiety-related behavioral responses of pentylenetetrazole-treated zebrafish larvae to light-dark transitions.

Pentylenetetrazole (PTZ), γ-aminobutyrate (GABA) antagonist, is a convulsant drug, known to induce anxiety and seizures in zebrafish. Changes in the mobility of zebrafish under light-dark transitions reflect anxiety level, serving as a useful behavioral readout. The effects of PTZ treatment have yet to be assayed in this manner. Zebrafish larvae (AB strain) at both 5dpf (days post-fertilization) and 7dpf were treated with different concentrations of PTZ. General locomotor activity and thigmotaxis were analyzed under continuous illumination (normal conditions) or alternating light-dark cycles (stressful conditions). Zebrafish larvae of 5dpf and 7dpf exhibited different sensitivities to PTZ. Anxiety level, measured in terms of response to illumination transitions under the influence of PTZ, demonstrated contrasting tendencies. Dark-light transitions dramatically increased the locomotor activity of zebrafish larvae receiving 8mM PTZ which was indicative of anxiety. This study suggests that PTZ increases the susceptibility by activating the neuron, which perhaps makes light change easier to influence the anxiety level of larvae. We provide useful evidence for putative anti-anxiety drug screening.

Influences of acute ethanol exposure on locomotor activities of zebrafish larvae under different illumination.

Larval zebrafish present unique opportunities to study the behavioral responses of a model organism to environmental challenges during early developmental stages. The purpose of the current study was to investigate the locomotor activities of AB strain zebrafish larvae at 5 and 7 days post-fertilization (dpf) in response to light changes under the influence of ethanol, and to explore potential neurological mechanisms that are involved in ethanol intoxication. AB strain zebrafish larvae at both 5 and 7 dpf were treated with ethanol at 0% (control), 0.1%, 0.25%, 0.5%, 1%, and 2% (v/v%). The locomotor activities of the larvae during alternating light-dark challenges, as well as the locomotor responses immediately following the light transitions, were investigated. The levels of various neurotransmitters were also measured in selected ethanol-treated groups. The larvae at 5 and 7 dpf demonstrated similar patterns of locomotor responses to ethanol treatment. Ethanol treatment at 1% increased the swimming distances of the zebrafish larvae in the dark periods, but had no effect on the swimming distances in the light periods. In contrast, ethanol treatment at 2% increased the swimming distances in the light periods, but did not potentiate the swimming activity in the dark periods, compared to controls. Differences in the levels of neurotransmitters that are involved in norepinephrine, dopamine, and serotonin pathways were also observed in groups with different ethanol treatments. These results indicated the behavioral studies concerning the ethanol effects on locomotor activities of zebrafish larvae could be carried out as early as 5 dpf. The 1% and 2% ethanol-treated zebrafish larvae modeled ethanol effects at different intoxication states, and the differences in neurotransmitter levels suggested the involvement of various neurotransmitter pathways in different ethanol intoxication states.

[Study on the nitrogen removal performance and the characteristics of denitrification bacterial community structure of biological aerated filter].

The nitrogen removal performance and the denitrification bacterial community structure of two pre-denitrification biological aeration filter (BAF) processes with different media combinations, the ceramic-zeolite BAF process (C-Z BAF) and the zeolite-ceramic BAF process (Z-C BAF), were compared at different C/N ratios. The results showed that the average TN removal efficiency of Z-C BAF was higher than that of C-Z BAF, and the advantage was more notable at low C/N ratio. When the C/N ratio decreased to 3.3, the average TN removal efficiency of Z-C BAF was 20% higher than that of C-Z BAF. Comparing the microbial community structure of the two combinations at different C/N ratios, it was found that the richness and diversity of denitrification bacterial community decreased significantly with the decrease of C/N ratio. At low C/N ratio, within the whole oxic filter of Z-C BAF, nosZ genetic T-RFs were detected with more than 20% relative abundance, whereas no nosZ genetic T-RFs were detected at the upper oxic filter of C-Z BAF by PCR amplification. The results indicated that the simultaneous nitrification and denitrification function of Z-C BAF was better than that of C-Z BAF. At low C/N ratio, the nitrogen removal efficiency of Z-C BAF was obviously higher than that of C-Z BAF. Thus, compared to C-Z BAF, Z-C BAF had greater potential on the treatment of wastewater with low organic concentration and low C/N ratio.