Clock knockout in inhibitory neurons reduces predisposition to epilepsy and influences anxiety-like behaviors in mice.

Epilepsy is a brain disorder affecting up to 1 in 26 individuals. Despite its clinical importance, the molecular mechanisms of epileptogenesis are still far from clarified. Our previous study showed that disruption of Clock in excitatory neurons alters cortical circuits and leads to generation of focal epilepsy. In this study, a GAD-Cre;Clockflox/flox mouse line with conditional Clock gene knockout in inhibitory neurons was established. We observed that seizure latency was prolonged, the severity and mortality of pilocarpine-induced seizure were significantly reduced, and memory was improved in GAD-Cre;Clockflox/flox mice. We hypothesize that mice with CLOCK knockout in inhibitory neurons have increased threshold for seizure, opposite from mice with CLOCK knockout in excitatory neurons. Further investigation showed Clock knockout in inhibitory neurons upregulated the basal protein level of ARC, a synaptic plasticity-associated immediate-early gene product, likely through the BDNF-ERK pathway. Altered basal levels of ARC may play an important role in epileptogenesis after Clock deletion in inhibitory neurons. Although sEPSCs and intrinsic properties of layer 5 pyramidal neurons in the somatosensory cortex exhibit no changes, the spine density increased in apical dendrite of pyramidal neurons in CLOCK knockout group. Our results suggest an underlying mechanism by which the circadian protein CLOCK in inhibitory neurons participates in neuronal activity and regulates the predisposition to epilepsy.

microRNA profilings identify plasma biomarkers and targets associated with pediatric epilepsy patients.

BACKGROUND: Although previous studies show that microRNAs (miRNAs) can potentially be used as diagnostic markers for epilepsy, there are very few analyses of pediatric epilepsy patients. METHODS: miRNA profiles using miRNA-seq was performed on plasma samples from 14 pediatric epileptic patients and 14 healthy children. miRNA miR-27a-3p that were significantly changed between two groups were further evaluated. The potential target genes of miR-27a-3p were screened through unbiased mRNA-seq and further validated using Western blot and immunohistochemistry in HEK-293T cells and in the brains of mice with epilepsy induced by lithium chloride-pilocarpine. RESULTS: We found 82 upregulated and 76 downregulated miRNAs in the plasma from pediatric patients compared with controls (p < 0.01), of which miR-27a-3p exhibited a very low p value (p < 0.0001) and validated in additional plasma samples. Two genes, GOLM1 and LIMK1, whose mRNA levels were decreased (p < 0.001) with the increase of miR-27a-3p were further validated in both HEK-293T cells and in epileptic mice. CONCLUSIONS: MiR-27a-3p exhibits potential as a diagnostic and therapeutic marker for epilepsy. We postulate that additional studies on the downstream targets of miR-27a-3p will unravel its roles in epileptogenesis or disease progression. IMPACT: A total of 158 differentially expressed miRNAs were detected in plasma between epileptic and control children. Plasma miR-27a-3p was one of the miRNAs with a low p value. GOLM1 and LIMK1 were validated as downstream target genes of miR-27a-3p. miR-27a-3p has potential as a diagnostic and therapeutic marker for epilepsy.

Chlorogenic acid exerts neuroprotective effect against hypoxia-ischemia brain injury in neonatal rats by activating Sirt1 to regulate the Nrf2-NF-κB signaling pathway.

BACKGROUND: Neonatal hypoxic-ischemic brain injury (HIE) is caused by perinatal asphyxia, which is associated with various confounding factors. Although studies on the pathogenesis and treatment of HIE have matured, sub-hypothermia is the only clinical treatment available for HIE. Previous evidence indicates that chlorogenic acid (CGA) exerts a potential neuroprotective effect on brain injury. However, the role of CGA on neonatal HI brain damage and the exact mechanism remains elusive. Here, we investigate the effects of CGA on HI models in vivo and in vitro and explore the underlying mechanism. METHODS: In the in vivo experiment, we ligated the left common carotid artery of 7-day-old rats and placed the rats in a hypoxic box for 2 h. We did not ligate the common carotid artery of the pups in the sham group since they did not have hypoxia. Brain atrophy and infarct size were evaluated by Nissl staining, HE staining and 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining. Morris Water Maze test (MWM) was used to evaluate neurobehavioral disorders. Western-blotting and immunofluorescence were used to detect the cell signaling pathway. Malondialdehyde (MDA) content test, catalase (CAT) activity detection and Elisa Assay was used to detect levels of inflammation and oxidative stress. in vitro experiments were performed on isolated primary neurons. RESULT: In our study, pretreatment with CGA significantly decreased the infarct volume of neonatal rats after HI, alleviated brain edema, and improved tissue structure in vivo. Moreover, we used the Morris water maze to verify CGA's effects on enhancing the learning and cognitive ability and helping to maintain the long-term spatial memory after HI injury. However, Sirt1 inhibitor EX-527 partially reversed these therapeutic effects. CGA pretreatment inhibited neuronal apoptosis induced by HI by reducing inflammation and oxidative stress. The findings suggest that CGA potentially activates Sirt1 to regulate the Nrf2-NF-κB signaling pathway by forming complexes thereby protecting primary neurons from oxygen-glucose deprivation (OGD) damage. Also, CGA treatment significantly suppresses HI-induced proliferation of glial. CONCLUSION: Collectively, this study uncovered the underlying mechanism of CGA on neonatal HI brain damage. CGA holds promise as an effective neuroprotective agent to promote neonatal brain recovery from HI-induced injury. Video Abstract.

The efficacy of high-flow oxygen versus conventional oxygen for asthma control: a meta-analysis of randomized controlled studies.

Introduction: The efficacy of high-flow oxygen versus conventional oxygen therapy for asthma control remains controversial. Aim: This meta-analysis aims to explore the influence of high-flow oxygen versus conventional oxygen therapy on asthma control. Material and methods: We have searched PubMed, Embase, Web of Science, EBSCO, and Cochrane library databases, and included randomized controlled trials (RCTs) assessing the efficacy of high-flow oxygen versus conventional oxygen therapy for asthma control. Results: Four RCTs are included in this meta-analysis. Overall, compared with conventional oxygen therapy for asthma, high-flow oxygen is associated with a significantly lower dyspnoea score (standard mean difference (SMD) = -0.63; 95% confidence interval (CI): -1.08 to -0.17; p = 0.008), but reveals no remarkable influence on PaCO2 (SMD = 0.28; 95% CI: -0.22 to 0.77; p = 0.28), PaO2 (SMD = 0.44; 95% CI: -1.34 to 2.22; p = 0.63), intubation rate (OR = 1.09; 95% CI: 0.15 to 8.21; p = 0.93) or hospital length of stay (SMD = -0.07; 95% CI: -0.41 to 0.27; p = 0.67). Conclusions: High-flow oxygen may benefit to reduce/may be more beneficial in reducing the dyspnoea score than conventional oxygen therapy for asthma, but shows no improvement in PaCO2, PaO2, intubation or hospital length of stay.

Molecular basis for specific regulation of neuronal kinesin-3 motors by doublecortin family proteins.

Doublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx's linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals.

A dominant dendrite phenotype caused by the disease-associated G253D mutation in doublecortin (DCX) is not due to its endocytosis defect.

Doublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans. The microtubule-binding activity of DCX is well-described and is important for its function, such as supporting neuronal migration and dendrite growth during development. Previous work showed that microtubule binding is not sufficient for DCX-mediated promotion of dendrite growth and that domains in DCX's C terminus are also required. The more C-terminal regions of DCX bind several other proteins, including the adhesion receptor neurofascin and clathrin adaptors. We recently identified a role for DCX in endocytosis of neurofascin. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we now asked if disruption of neurofascin endocytosis underlies the DCX-G253D-associated pathology. We first demonstrated that DCX functions in endocytosis as a complex with both the clathrin adaptor AP-2 and neurofascin: disrupting either clathrin adaptor binding (DCX-ALPA) or neurofascin binding (DCX-G253D) decreased neurofascin endocytosis in primary neurons. We then investigated a known function for DCX, namely, increasing dendrite growth in cultured neurons. Surprisingly, we found that the DCX-ALPA and DCX-G253D mutants yield distinct dendrite phenotypes. Unlike DCX-ALPA, DCX-G253D caused a dominant-negative dendrite growth phenotype. The endocytosis defect of DCX-G253D thus was separable from its detrimental effects on dendrite growth. We recently identified Dcx-R59H as a dominant allele and can now classify Dcx-G253D as a second Dcx allele that acts dominantly to cause pathology, but does so via a different mechanism.

TUBB4A mutations result in specific neuronal and oligodendrocytic defects that closely match clinically distinct phenotypes.

Hypomyelinating leukodystrophies are heritable disorders defined by lack of development of brain myelin, but the cellular mechanisms of hypomyelination are often poorly understood. Mutations in TUBB4A, encoding the tubulin isoform tubulin beta class IVA (Tubb4a), result in the symptom complex of hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC). Additionally, TUBB4A mutations are known to result in a broad phenotypic spectrum, ranging from primary dystonia (DYT4), isolated hypomyelination with spastic quadriplegia, and an infantile onset encephalopathy, suggesting multiple cell types may be involved. We present a study of the cellular effects of TUBB4A mutations responsible for H-ABC (p.Asp249Asn), DYT4 (p.Arg2Gly), a severe combined phenotype with hypomyelination and encephalopathy (p.Asn414Lys), as well as milder phenotypes causing isolated hypomyelination (p.Val255Ile and p.Arg282Pro). We used a combination of histopathological, biochemical and cellular approaches to determine how these different mutations may have variable cellular effects in neurons and/or oligodendrocytes. Our results demonstrate that specific mutations lead to either purely neuronal, combined neuronal and oligodendrocytic or purely oligodendrocytic defects that closely match their respective clinical phenotypes. Thus, the DYT4 mutation that leads to phenotypes attributable to neuronal dysfunction results in altered neuronal morphology, but with unchanged tubulin quantity and polymerization, with normal oligodendrocyte morphology and myelin gene expression. Conversely, mutations associated with isolated hypomyelination (p.Val255Ile and p.Arg282Pro) and the severe combined phenotype (p.Asn414Lys) resulted in normal neuronal morphology but were associated with altered oligodendrocyte morphology, myelin gene expression, and microtubule dysfunction. The H-ABC mutation (p.Asp249Asn) that exhibits a combined neuronal and myelin phenotype had overlapping cellular defects involving both neuronal and oligodendrocyte cell types in vitro. Only mutations causing hypomyelination phenotypes showed altered microtubule dynamics and acted through a dominant toxic gain of function mechanism. The DYT4 mutation had no impact on microtubule dynamics suggesting a distinct mechanism of action. In summary, the different clinical phenotypes associated with TUBB4A reflect the selective and specific cellular effects of the causative mutations. Cellular specificity of disease pathogenesis is relevant to developing targeted treatments for this disabling condition.

Characterization, promoter analysis and expression of the interleukin-6 gene in blunt snout bream, Megalobrama amblycephala.

Interleukin-6 (IL-6) is one of the most important multifunctional cytokines, playing essential roles in mediating the innate and adaptive immune responses. In this study, il-6 gene and its promoter from blunt snout bream, Megalobrama amblycephala, were characterized, and its expression at the transcript level in healthy fish and after bacterial infection was determined by quantitative real-time PCR. The results showed that the M. amblycephala il-6 (Mamil-6) cDNA had an ORF of 699 bp, encoding 232 amino acids, and contained 9 instable motifs in the 3' UTR. The deduced MamIL-6 possessed a 24-amino acid signal peptide and was located in the cytoplasm. Although sequence alignment and phylogenetic analysis revealed that IL-6 is poorly conserved in vertebrates, the protein and genomic structure of il-6 gene was well conserved. Analysis of the Mamil-6 promoter revealed the presence of a conserved TATA box and six major cis-regulatory elements, including C/EBPß (NF-IL6), AP-1, CRE, GRE, GATA and NF-κB binding sites. In healthy fish, Mamil-6 was the most abundant in the spleen. After Aeromonas hydrophila infection, Mamil-6 was significantly up-regulated in all 6 immune-related tissues examined, suggesting that Mamil-6 plays an important role in the blunt snout bream immune system.

Doublecortin (Dcx) family proteins regulate filamentous actin structure in developing neurons.

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule-binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semiquantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, widespread differences are found in actin-associated proteins as compared with wild-type axons. Decreases in actin-binding proteins α-actinin-1 and α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued by full-length Dcx and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C-terminal S/P-rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin-like kinase 1 deficiency is consistent with actin defect, as these axons are selectively deficient in axon guidance, but not elongation.

Dietary Chito-oligosaccharide attenuates LPS-challenged intestinal inflammation via regulating mitochondrial apoptotic and MAPK signaling pathway.

To investigate the regulatory effects of Chito-oligosaccharide (COS) on the anti-oxidative, anti-inflammatory, and MAPK signaling pathways. A total of 40 28-day-old weaned piglets were randomly allotted to 4 equal groups [including the control group, lipopolysaccharide (LPS) group, COS group, and COS*LPS group]. On the morning of d 14 and 21, piglets were injected with saline or LPS. At 2 h post-injection, whole blood samples were collected on d 14 and 21, and small intestine and liver samples were collected and analyzed on d 21. The results showed that COS inhibited the LPS-induced increase of malondialdehyde (MDA) concentration and hepatic TNF-α cytokines. COS significantly increased the serum total antioxidant capability (T-AOC) value on d 14, and total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) activities in both serum and liver on d 21. Furthermore, it increased hepatic catalase (CAT) activity. COS also increased the LPS-induced decrease in serum IgG concentrations. Immunohistochemical analysis results showed that COS significantly increased the jejunal and ileal Caspase 3, and ileal CD4+ values challenged by LPS. Dietary COS decreased the LPS-induced jejunal and ileal BAX and CCL2 mRNA levels, markedly decreased ileal COX2 and SOD1 mRNA levels, while increasing ileal iNOS. Furthermore, COS significantly increased the LPS-induced jejunal and ileal p-P38 and MyD88, as well as jejunal P38, while it effectively suppressed jejunal JNK1, and jejunal and ileal JNK2, p-JNK1, and p-JNK2 protein expressions. These results demonstrated that COS could be beneficial by attenuating LPS-challenged intestinal inflammation via regulating mitochondrial apoptotic and MAPK signaling pathways.

Dietary supplementation with mulberry leaf flavonoids and carnosic acid complex enhances the growth performance and antioxidant capacity via regulating the p38 MAPK/Nrf2 pathway.

Introduction: This study aimed to investigate the regulatory effects of mulberry leaf flavonoids and carnosic acid complex (MCC) on the growth performance, intestinal morphology, antioxidant, and p38 MAPK/Nrf2 pathway in broilers. Methods: A total of 256 healthy 8-day-old female yellow-feathered broilers were randomly divided into 4 equal groups: a control group (CON) fed a basal diet, an antibiotic group (CTC) supplemented with 50 mg/kg chlortetracycline, and two experimental groups (MCC75, MCC150) fed basal diets with 75 mg/kg and 150 mg/kg of MCC, respectively. The experiment lasted for 56 days, with days 1-28 designated as the initial phase and days 29-56 as the growth phase. Results: The results on the growth performance showed that diets supplemented with MCC and CTC decreased the feed-to-gain ratio (F/G), diarrhea rate, and death rate, while significantly increasing the average daily weight gain (ADG) (p < 0.05). Specifically, the MCC150 group enhanced intestinal health, indicated by reduced crypt depth and increased villus height-to-crypt depth ratio (V/C) as well as amylase activity in the jejunum. Both the MCC and CTC groups exhibited increased villus height and V/C ratio in the ileal (p < 0.05). Additionally, all treated groups showed elevated serum total antioxidant capacity (T-AOC), and significant increases in catalase (CAT) and glutathione peroxidase (GSH-Px) activities were observed in both the MCC150 and CTC groups. Molecular analysis revealed an upregulation of the jejunal mRNA expression levels of PGC-1α, Nrf2, and Keap1 in the MCC and CTC groups, as well as an upregulation of ileum mRNA expression levels of P38, PGC-1α, Nrf2, and Keap1 in the MCC150 group, suggesting activation of the p38-MAPK/Nrf2 pathway. Discussion: These findings indicate that dietary supplementation with MCC, particularly at a dosage of 150 mg/kg, may serve as a viable antibiotic alternative, enhancing growth performance, intestinal health, and antioxidant capacity in broilers by regulating the p38-MAPK/Nrf2 pathway.

Genomic evidence for climate-linked diversity loss and increased vulnerability of wild barley spanning 28 years of climate warming.

The information on how plant populations respond genetically to climate warming is scarce. Here, landscape genomic and machine learning approaches were integrated to assess genetic response of 10 wild barley (Hordeum vulgare ssp. spontaneum; WB) populations in the past and future, using whole genomic sequencing (WGS) data. The WB populations were sampled in 1980 and again in 2008. Phylogeny of accessions was roughly in conformity with sampling sites, which accompanied by admixture/introgressions. The 28-y climate warming resulted in decreased genetic diversity, increased selection pressure, and an increase in deleterious single nucleotide polymorphism (dSNP) numbers, heterozygous deleterious and total deleterious burdens for WB. Genome-environment associations identified some candidate genes belonging to peroxidase family (HORVU2Hr1G057450, HORVU4Hr1G052060 and HORVU4Hr1G057210) and heat shock protein 70 family (HORVU2Hr1G112630). The gene HORVU2Hr1G120170 identified by selective sweep analysis was under strong selection during the climate warming of the 28-y, and its derived haplotypes were fixed by WB when faced with the 28-y increasingly severe environment. Temperature variables were found to be more important than precipitation variables in influencing genomic variation, with an eco-physiological index gdd5 (growing degree-days at the baseline threshold temperature of 5 °C) being the most important determinant. Gradient forest modelling revealed higher predicted genomic vulnerability in Sede Boqer under future climate scenarios at 2041-2070 and 2071-2100. Additionally, estimates of effective population size (Ne) tracing back to 250 years indicated a forward decline in all populations over time. Our assessment about past genetic response and future vulnerability of WB under climate warming is crucial for informing conservation efforts for wild cereals and rational use strategies.

The de novo missense mutation F224S in GABRB2, identified in epileptic encephalopathy and developmental delay, impairs GABAAR function.

Genetic variants in genes encoding subunits of the γ-aminobutyric acid-A receptor (GABAAR) have been found to cause neurodevelopmental disorders and epileptic encephalopathy. In a patient with epilepsy and developmental delay, a de novo heterozygous missense mutation c.671 T > C (p.F224S) was discovered in the GABRB2 gene, which encodes the ß2 subunit of GABAAR. Based on previous studies on GABRB2 variants, this new GABRB2 variant (F224S) would be pathogenic. To confirm and investigate the effects of this GABRB2 mutation on GABAAR channel function, we conducted transient expression experiments using GABAAR subunits in HEK293T cells. The GABAARs containing mutant ß2 (F224S) subunit showed poor trafficking to the cell membrane, while the expression and distribution of the normal α1 and γ2 subunits were unaffected. Furthermore, the peak current amplitude of the GABAAR containing the ß2 (F224S) subunit was significantly smaller compared to the wild type GABAAR. We propose that GABRB2 variant F224S is pathogenic and GABAARs containing this ß2 mutant reduce response to GABA under physiological conditions, which could potentially disrupt the excitation/inhibition balance in the brain, leading to epilepsy.

Effect of 25 hydroxyvitamin D on attention deficit and hyperactivity in school-age children with ADHD.

BACKGROUND: To observe the serum levels of 25 hydroxyvitamin D [25 (OH) D] in healthy school-age children and children with attention deficit hyperactivity disorder (ADHD) and to analyze the effects of serum 25 (OH) D on the symptoms of attention deficit and hyperactivity in school-age children with ADHD. METHODS: According to the Diagnostic and Statistical Manual of Mental Disorders DSM-IV diagnostic criteria for ADHD in children, 80 healthy children aged 6 years or less than 10 years old and children diagnosed with ADHD in the Department of Rehabilitation Medicine, Department of Pediatrics and Department of Physical Examination of our hospital were randomly selected as research subjects. The serum 25 (OH) D level, attention deficit hyperactivity (Swanson, Nolan, and Pelham, version IV [SNAP-IV] parental version) score and Conners child behavior (PSQ) index were observed and compared between the 2 groups. In addition, the children with ADHD whose serum 25 (OH) D was lower than normal were treated with supplemental VitD3, and the changes in serum 25 (OH) D, SNAP-IV parental score and PSQ index of ADHD children were observed and compared. RESULTS: Serum 25(OH)D was insufficient or deficient in 26 healthy children, but the SNAP-IV score and PSQ index were normal. Serum 25(OH)D was lower than normal in 69 patients in the ADHD group, which was negatively correlated with SNAP-IV score (r = -0.3479, P = .0034) and negatively correlated with PSQ index (r = -0.3566, P = .0026). After vitamin D3 (VitD3) supplementation in 69 children with serum 25(OH)D levels lower than the normal ADHD group, it was found that the SNAP-IV score (r = -0.4654, P = .0037) and PSQ index (r = -0.5680, P = .0002) of 34 children with ADHD were negatively correlated with the increase in serum 25(OH)D. The SNAP-IV score and PSQ index of the other 35 children with ADHD showed no correlation with the increase in serum 25 (OH) D (P > .05). CONCLUSION SUBSECTIONS: Serum 25(OH)D levels lower than normal are more common in school-age children, and levels lower than normal are not the key pathogenic factor of ADHD in school-age children, but serum 25(OH)D levels lower than normal may be the upregulation factor of attention deficit and hyperactivity disorder expression in some school-age children with ADHD. The lower level of serum 25(OH)D may be closely related to the severity of ADHD symptoms in some children.

An ice- air-water-NAPL multiphase model for simulating NAPL migration in subsurface system under freeze-thaw condition.

Non-aqueous phase liquid (NAPL) leakage poses serious threats to human health and the environment. Understanding NAPL migration and distribution in subsurface systems is crucial for developing effective remediation strategies. Multiphase flow modeling is an important tool to quantitatively describe the NAPL migration process in the subsurface. However, most multiphase flow models are built for temperatures typical of warmer climates and above freezing conditions, only considering two phases (water-NAPL) or three phases (air-water-NAPL). To date, few studies simulate NAPL migration in a four-phase system (ice-air-water-NAPL), which would be more appropriate for cold regions. In this study, we developed a coupled non-isothermal multiphase transport model to quantitatively describe NAPL migration in a four-phase (ice, gas, water, NAPL) system. The ice phase was added in the continuity equations and the constitutive relationship between unfrozen water content and temperature was applied to solve the energy and flow equations. The developed mathematical model was evaluated using a two-dimensional experiment under freeze-thaw cycles (FTCs) with an R2 = 0.8803 between the simulated and observed NAPL saturation. Next, we evaluated the effect of freezing-induced changes in pressure and density between LNAPL and DNAPL on NAPL distribution under freeze-thaw condition. Simulation results show that ignoring the impact of ice formation and thawing during freeze-thaw cycles for LNAPL and DNAPL transport simulations can result in up to a 48% and 13% difference in model predictions of local NAPL saturations respectively, affecting model predictions of overall NAPL spatial distributions and potentially predicted remediation effectiveness.

Chlorogenic acid alleviates hypoxic-ischemic brain injury in neonatal mice.

Recent studies have shown that chlorogenic acid (CGA), which is present in coffee, has protective effects on the nervous system. However, its role in neonatal hypoxic-ischemic brain injury remains unclear. In this study, we established a newborn mouse model of hypoxic-ischemic brain injury using a modified Rice-Vannucci method and performed intraperitoneal injection of CGA. We found that CGA intervention effectively reduced the volume of cerebral infarct, alleviated cerebral edema, restored brain tissue structure after injury, and promoted axon growth in injured brain tissue. Moreover, CGA pretreatment alleviated oxygen-glucose deprivation damage of primary neurons and promoted neuron survival. In addition, changes in ferroptosis-related proteins caused by hypoxic-ischemic brain injury were partially reversed by CGA. Furthermore, CGA intervention upregulated the expression of the key ferroptosis factor glutathione peroxidase 4 and its upstream glutamate/cystine antiporter related factors SLC7A11 and SLC3A2. In summary, our findings reveal that CGA alleviates hypoxic-ischemic brain injury in neonatal mice by reducing ferroptosis, providing new ideas for the treatment of neonatal hypoxic-ischemic brain injury.

Ferroptosis is Involved in Hypoxic-ischemic Brain Damage in Neonatal Rats.

Ferroptosis is an iron-dependent form of regulated cell death, which is driven by loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and subsequent accumulation of lipid peroxidation. Ferroptosis is implicated in various diseases involving neuronal injury. However, the role of ferroptosis in hypoxic-ischemic brain damage (HIBD) has not been elucidated. The objectives of this study were to evaluate whether ferroptosis is involved in hypoxic-ischemic brain damage and its mechanisms through the HIBD model. A 7-day-old male Sprague-Dawley neonatal rat HIBD model was established by blocking the left common carotid artery. Laser speckle contrast imaging, immunohistochemical staining, transmission electron microscopy were used to measure the effects of ferroptosis on HIBD. Brain tissue on the damaged side in the HIBD group showed atrophied, even liquefied, glial cells increased, and blood perfusion was significantly reduced. The HIBD group insult significantly increased reactive oxygen species levels, as well as the protein levels of iron metabolism-related proteins transferrin receptor (TFRC), ferritin heavy chain (FHC), and ferritin light chain (FLC), while reducing the levels of Solute Carrier Family 7 Member 11 (SLC7A11), glutathione (GSH), and GPX4. These changes resulted in diminished cellular antioxidant capacity and mitochondrial damage, causing neuronal ferroptosis in the cerebral cortex. We conclude that ferroptosis plays a role in HIBD in neonatal rats. Ferroptosis-related mechanisms such as abnormalities in iron metabolism, amino acid metabolism, and lipid peroxidation regulation play important roles in HIBD.

Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking.

Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT-based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport in neurons from Dcx-/y or Dcx-/y;Dclk1-/- mice by reducing dynein's association with MTs and disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and regulating the composition of the dynein motor complex.

RNA-seq and qRT-PCR analyses reveal the physiological response to acute hypoxia and reoxygenation in Epinephelus coioides.

Hypoxia is a critical problem in intensive Epinephelus coioides aquaculture systems. In the present study, the physiological responses of E. coioides muscle to acute hypoxic stress (DO = 0.6 ± 0.1 mg/L) and reoxygenation (DO = 6.0 ± 0.1 mg/L) were analyzed by transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR). RNA-seq was conducted on the muscle tissues of E. coioides in the hypoxia-tolerant (EMS), hypoxia-sensitive (EMW), and normoxic (CM) groups. Among the three groups, a total of 277 differentially expressed genes (DEGs) were identified. KEGG analysis revealed that the pathways significantly enriched after hypoxic stress are involved in the immune response, glycolysis/gluconeogenesis, energy metabolism, vasodilation and proliferation, cell proliferation, and apoptosis. qRT‒PCR verified that the differentially expressed genes FIH-1, PHD-2, PPARα, BCL-XL, LDH-A, and Flt-1 were significantly upregulated after hypoxic stress and returned to normal levels after reoxygenation, suggesting that these DEGs play important roles in responding to hypoxia treatment. In addition, the HIF-1 signaling pathway was also activated under hypoxic stress, and qRT‒PCR confirmed that the expression level of HIF-1α was significantly elevated under acute hypoxic stress, indicating that the HIF-1 signaling pathway is the central pathway in the E. coioides hypoxic response mechanism and activates other related pathways to adapt to hypoxic stress. These pathways jointly regulate energy metabolism, substance synthesis, blood vessel proliferation, cell proliferation, and differentiation and prolong survival time. These results provide ideas for understanding physiological regulation after hypoxic stress and reoxygenation and provide basic insights for the future breeding of hypoxia-tolerant E. coioides.

Simvastatin Inhibits NLRP3 Inflammasome Activation and Ameliorates Lung Injury in Hyperoxia-Induced Bronchopulmonary Dysplasia via the KLF2-Mediated Mechanism.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly found in premature infants. Excessive inflammation and oxidative stress contribute to BPD occurrence and development. Simvastatin, as an inhibitor of HMG-CoA reductase, has been reported to have antioxidative and anti-inflammatory effects. However, its effect and possible mechanisms in hyperoxia-induced lung injury are rarely reported. In this study, in vivo and in vitro experiments were conducted to investigate whether simvastatin could ameliorate hyperoxia-induced lung injury and explore its potential mechanism. For the in vivo study, simvastatin could improve alveolar development after hyperoxic lung injury and reduce hyperoxic stress and inflammation. The in vitro study revealed that simvastatin can reduce inflammation in A549 cells after high-oxygen exposure. Simvastatin suppressed NLRP3 inflammasome activation and played anti-inflammatory and antioxidant roles by increasing KLF2 (Krüppel-like factor 2) expression. In vitro experiments also revealed that these effects of simvastatin were partially reversed by KLF2 shRNA, indicating that KLF2 was involved in simvastatin effects. In summary, our findings indicate that simvastatin could downregulate NLRP3 inflammasome activation and attenuate lung injury in hyperoxia-induced bronchopulmonary dysplasia via KLF2-mediated mechanism.