Microtubule-associated Protein Research Articles

Promising and challenging phytochemicals targeting LC3 mediated autophagy signaling in cancer therapy.

Phytochemicals possess a wide range of anti-tumor properties, including the modulation of autophagy and regulation of programmed cell death. Autophagy is a critical process in cellular homeostasis and its dysregulation is associated with several pathological conditions, such as cancer, neurodegenerative diseases, and diabetes. In cancer, autophagy plays a dual role by either promoting tumor growth or suppressing it, depending on the cellular context. During autophagy, autophagosomes engulf cytoplasmic components such as proteins and organelles. LC3-II (microtubule-associated protein 1 light chain 3-II) is an established marker of autophagosome formation, making it central to autophagy monitoring in mammals. To explore the regulatory role of phytochemicals in LC3-mediated autophagy and their potential therapeutic impact on cancer. The review emphasizes the involvement of autophagy in tumor promotion and suppression, particularly focusing on autophagy-related signaling pathways like oxidative stress through the NRF2 pathway, and its implications for genomic stability in cancer development. The review focuses on a comprehensive analysis of bioactive compounds including Curcumin, Celastrol, Resveratrol, Kaempferol, Naringenin, Carvacrol, Farnesol, and Piperine. Literature on these compounds was examined to assess their influence on autophagy, LC3 expression, and tumor-related signaling pathways. A systematic literature search was conducted across databases including PubMed, Scopus, and Web of Science from inception to 2023. Studies were selected from prominent databases, focusing on their roles in cancer diagnosis and therapeutic interventions, particularly in relation to LC3-mediated mechanisms. Phytochemicals have been shown to modulate autophagy through the regulation of LC3-II levels and autophagic flux in cancer cells. The interaction between autophagy and other cellular pathways such as oxidative stress, inflammation, and epigenetic modulation highlights the complex role of autophagy in tumor biology. For instance, Curcumin and Resveratrol have been reported to either induce or inhibit autophagy depending on cancer type, influencing tumor progression and therapeutic responses. Targeting autophagy through LC3 modulation presents a promising strategy for cancer therapy. The dual role of autophagy in tumor suppression and promotion, however, necessitates careful consideration of the context in which autophagy is induced or inhibited. Future research should aim to delineate these context-specific roles and explore how phytochemicals can be optimized for therapeutic efficacy. Novel therapeutic strategies should focus on the use of bioactive compounds to fine-tune autophagy, thereby maximizing tumor suppression and inducing programmed cell death in cancer cells.

Caveolin 1 ameliorates nonesterified fatty acid-induced oxidative stress via the autophagy regulator Beclin 1 in bovine mammary gland epithelial cells

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis enhance uptake by the mammary gland and impair autophagy while causing oxidative stress. Caveolin 1 (CAV1) is closely related to autophagy and plays a role in regulating oxidative stress. The aim of this study was to explore the potential role of CAV1 on oxidative stress and autophagy during a high NEFA challenge in the immortalized bovine mammary epithelial cell line MAC-T. Mammary gland tissue biopsies and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) Holstein cows at 3 to 10 (average = 6) days in milk. Compared with healthy cows, ketotic cows had lower dry matter intake (DMI), daily milk yield, serum glucose and greater serum NEFA and BHBA, accompanied by greater milk fat and lower milk protein. Malondialdehyde (MDA) was greater but activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were lower in cows with clinical ketosis. A lower protein abundance of CAV1, Beclin 1, autophagy relative gene 5 (ATG5), and microtubule-associated protein 1 light chain 3 (LC3) as well as greater protein abundance of sequestosome-1 (SQSTM1, also called p62) were detected in mammary tissue of cows with clinical ketosis. In vitro, the MAC-T cells were treated with 0, 0.6 and 1.2 mM NEFA for 12 h or treated with 1.2 mM NEFA for various time points (0, 0.5, 1, 2, 4, 8, 12 and 24 h). Compared with 0 mM NEFA, protein abundance of CAV1, Beclin 1, ATG5 and LC3 was greater in the MAC-T challenged with 0.6 mM NEFA, but lower in the 1.2 mM NEFA group. Protein abundance of p62 was lower with 0.6 mM NEFA, but higher with 1.2 mM NEFA. In response to increasing doses of NEFA, mRNA abundance of CAV1, total antioxidant capacity (T-AOC) and SOD activity decreased while the level of reactive oxygen species (ROS) and content of MDA increased. The protein abundance of CAV1, Beclin 1, ATG5 and LC3 peaked at 0.5 h and 1 h, resulting in both linear and quadratic effects. The protein abundance of p62 decreased, reaching a nadir at 4 h in both a linear and quadratic manner. The silencing of CAV1 in MAC-T cells aggravated the 1.2 mM NEFA-induced decrease in Beclin 1 expression, impaired autophagy, and increase in oxidative stress, whereas the overexpression of CAV1 alleviated these effects. Pretreatment of MAC-T cells with Beclin 1 siRNA (si-Beclin 1) and overexpressing CAV1 followed by challenged with 1.2 mM NEFA reversed the CAV1 induced autophagy, thereby enhancing oxidative stress. Overall, these data suggest that CAV1 protects bovine mammary epithelial cells from NEFA-induced oxidative stress through enhancing the expression of Beclin 1 and activating autophagy.

Effect of phosphatase and tensin homolog-induced putative kinase 1/ E3 ubiquitin ligase Parkin mediated mitochondrial autophagy on chronic kidney disease myocardial injury and the intervention mechanism of Shenshuai recipe.

To study whether Shenshuai recipe (, SSR) can play a protective role on chronic kidney disease myocardial injury model through phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/E3 ubiquitin ligase Parkin (Parkin) mitochondrial autophagy pathway. Forty-eight nephrectomized rats were randomly divided into six groups: sham-operated group, model group, Benazepril group, low, medium and high-dose groups of SSR. The rats were given the cor-responding intervention for six weeks, then were sacrificed. Serum was examined by enzyme linked immunosorbent assay (ELISA). Cardiac ultrasound was used to detect cardiac function in 5/6 nephrectomized rats. Myocardial tissue was examined by light and electron microscopy; PINK1, Parkin, microtubule-associated protein1 light chain 3 II (LC3B), sequestosome 1 (P62), BECN1 (Beclin-1) and dynamin-related protein 1 (Drp-1) were measured by real time polymerase chain reaction (RT-PCR), Western blot (WB) and immunohistochemistry (IHC). The expression levels of blood urea nitrogen (BUN) and creatinine (SCr) in the model group were significantly higher than those in the sham-operated group, indicating that modeling was successful. SSR can protect myocardium by reducing the relative expression of creatine kinase myocardial isoenzyme and hypersensitivity cardiac troponin I (P<0.05). SSR can improve cardiac function in rats after ultrasound testing. SSR can improve the pathological manifestations of myocardial tissue after Masson staining. SSR can increase the number of autophagosomes and autophagiclysosomes in 5/6 nephrectomized rats (P<0.05). Determined by RT-PCR, WB and IHC, SSR can increase the relative expression of PINK1, Parkin, and LC3B (P<0.05), and decrease the relative expression of P62, Beclin-1 and Drp-1 (P<0.05). The PINK1/Parkin mitochondrial autophagy pathway in myocardial tissues in 5/6 nephrectomy CKD myocardial injury rats was inhibited. SSR can activate PINK1/Parkin mitochondrial autophagy to enhance mitochondrial autophagy, and play a protective role in myocardial tissues.

A herbal pair of Scutellaria barbata D. Don and Scleromitrion diffusum (Willd.) R.J. Wang induced ferroptosis in ovarian cancer A2780 cells via inducing heme catabolism and ferritinophagy

ObjectiveDespite the combination of Scutellaria barbata D. Don and Scleromitrion diffusum (Willd.) R.J. Wang (SB-SD) being a recognized Chinese medicinal herbal pair that is commonly used in the treatment of ovarian cancer, there is a poor understanding of their pharmacological mechanisms. This study examines the antitumor properties and potential mechanisms of SB-SD on human ovarian cancer A2780 cells through a multi-omics approach, establishing a pharmacological basis for clinical utilization. MethodsA range of mass ratios and reagents were used in the hot reflux extraction of SB-SD. The inhibitory effect of the SB-SD extracts on A2780 cell proliferation was assessed using the cell-counting kit 8 assay. A zebrafish tumor implantation model was used to evaluate the effects of SB-SD extracts on tumor growth and metastasis in vivo. Transcriptomics and proteomics were used to investigate alterations in biological pathways in A2780 cells after treatment with different concentrations of SB-SD extract. Cell cycle, cell apoptosis, intracellular free iron concentration, intracellular reactive oxygen species (ROS) concentration, malondialdehyde (MDA), and mitochondrial membrane potential were measured. Real-time quantitative reverse transcription polymerase chain reaction and Western blotting were utilized to investigate the effects of heme catabolism and ferritinophagy on ferroptosis induced by SB-SD extract in A2780 cells. ResultsThe 70% ethanol extract of SB-SD (a mass ratio of 4:1) inhibited A2780 cell proliferation significantly with a half maximal inhibitory concentration of 660 μg/mL in a concentration- and time-dependent manner. Moreover, it effectively suppressed tumor growth and metastasis in a zebrafish tumor implantation model. SB-SD extract induced the accumulation of free iron, ROS, MDA, and mitochondrial damage in A2780 cells. The mechanisms might involve the upregulated expression of ferritinophagy-related genes microtubule-associated protein 1 light chain 3, autophagy-related gene 5, and nuclear receptor coactivator 4. ConclusionsSB-SD extract effectively inhibited the development of ovarian cancer both in vitro and in vivo. Its mechanism of action involved inducing ferroptosis by facilitating heme catabolism and ferritinophagy. This herbal pair holds promise as a potential therapeutic option for ovarian cancer treatment and may be utilized in combination with routine treatment to improve the treatment outcomes of ovarian cancer patients.

The GhEB1C gene mediates resistance of cotton to Verticillium wilt.

The GhEB1C gene of the EB1 protein family functions as microtubule end-binding protein and may be involved in the regulation of microtubule-related pathways to enhance resistance to Verticillium wilt. The expression of GhEB1C is induced by SA, also contributing to Verticillium wilt resistance. Cotton, as a crucial cash and oil crop, faces a significant threat from Verticillium wilt, a soil-borne disease induced by Verticillium dahliae, severely impacting cotton growth and development. Investigating genes associated with resistance to Verticillium wilt is paramount. We identified and performed a phylogenetic analysis on members of the EB1 family associated with Verticillium wilt in this work. GhEB1C was discovered by transcriptome screening and was studied for its function in cotton defense against V. dahliae. The RT-qPCR analysis revealed significant expression of the GhEB1C gene in cotton leaves. Subsequent localization analysis using transient expression demonstrated cytoplasmic localization of GhEB1C. VIGS experiments indicated that silencing of the GhEB1C gene significantly increased susceptibility of cotton to V. dahliae. Comparative RNA-seq analysis showed that GhEB1C silenced plants exhibited altered microtubule-associated protein pathways and flavonogen-associated pathways, suggesting a role for GhEB1C in defense mechanisms. Overexpression of tobacco resulted in enhanced resistance to V. dahliae as compared to wild-type plants. Furthermore, our investigation into the relationship between the GhEB1C gene and plant disease resistance hormones salicylic axid (SA) and jasmonic acid (JA) revealed the involvement of GhEB1C in the regulation of the SA pathway. In conclusion, our findings demonstrate that GhEB1C plays a crucial role in conferring immunity to cotton against Verticillium wilt, providing valuable insights for further research on plant adaptability to pathogen invasion.

Genetically Encoded Microtubule Binders for Single-Cell Interrogation of Cytoskeleton Dynamics and Protein Activity.

Microtubule (MT) dynamics is tightly regulated by microtubule-associated proteins (MAPs) and various post-translational modifications (PTMs) of tubulin. Here, we introduce OligoMT and OligoTIP as genetically encoded oligomeric MT binders designed for real-time visualization and manipulation of MT behaviors within living cells. OligoMT acts as a reliable marker to label the MT cytoskeleton, while OligoTIP allows for live monitoring of the growing MT plus-ends. These engineered MT binders have been successfully utilized to label the MT network, monitor cell division, track MT plus-ends, and assess the effect of tubulin acetylation on the MT stability at the single-cell level. Moreover, OligoMT and OligoTIP can be repurposed as biosensors for quantitative assessment of drug actions and for reporting enzymatic activity. Overall, these engineered MT binders hold promise for advancing the mechanistic dissection of MT biology and have translational applications in cell-based high-throughput drug discovery efforts.

The role of PI3K signaling pathway in Alzheimer's disease.

Alzheimer's disease (AD) is a debilitating progressively neurodegenerative disease. The best-characterized hallmark of AD, which is marked by behavioral alterations and cognitive deficits, is the aggregation of deposition of amyloid-beta (Aβ) and hyper-phosphorylated microtubule-associated protein Tau. Despite decades of experimental progress, the control rate of AD remains poor, and more precise deciphering is needed for potential therapeutic targets and signaling pathways involved. In recent years, phosphoinositide 3-kinase (PI3K) and Akt have been recognized for their role in the neuroprotective effect of various agents, and glycogen synthase kinase 3 (GSK3), a downstream enzyme, is also crucial in the tau phosphorylation and Aβ deposition. An overview of the function of PI3K/Akt pathway in the pathophysiology of AD is provided in this review, along with a discussion of recent developments in the pharmaceuticals and herbal remedies that target the PI3K/Akt signaling pathway. In conclusion, despite the challenges and hurdles, cumulative findings of novel targets and agents in the PI3K/Akt signaling axis are expected to hold promise for advancing AD prevention and treatment.

Enhancement of cognitive function in mice with Alzheimer's disease through hyperbaric oxygen-induced activation of cellular autophagy.

In this study, we examined the effectiveness of hyperbaric oxygen (HBO) therapy in ameliorating cognitive deficits in mice with Alzheimer's disease (AD), while also assessing its impact on the autophagic pathway within the context of AD. 20 double-transgenic mice expressing the amyloid precursor protein and presenilin 1 (APP/PS1) were purposefully selected and randomly assigned to groups A and B. Concurrently, 20 C57BL/6 mice were chosen and randomly categorized into groups C and D, each consisting of 10 mice. Mice in groups B and D received HBO treatment. The Morris water maze assay was used to assess changes in mouse behavior. Immunohistochemistry techniques were used to quantify the expression levels of amyloid-beta 42 (Aβ42) and microtubule-associated protein 1A/1B-light chain 3 (LC3) in hippocampal tissues, while western blot analysis was used to investigate the levels of LC3-II, p62, phosphoinositide 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) proteins within hippocampal tissues. Mice allocated to group B exhibited reduced escape latency and prolonged dwell time in the target quadrant compared to other groups. Histological examination revealed conspicuous plaque-like deposits of Aβ42 in the hippocampal tissues of mice in groups A and B. Group B displayed diminished Aβ42-positive reactants and augmented microtubule-associated protein 1A/1B-LC3-positive reactants compared to group A. LC3-positive reactants were also detected in the hippocampal tissues of mice in groups C and D, surpassing the levels observed in groups A and B. Furthermore, group B demonstrated significantly lower expression of mTOR protein and markedly higher expression of LC3-II protein in mouse hippocampal tissues when compared to group A (P < 0.05). Conversely, there were no significant disparities noted in PI3K and p62 protein expression between groups B and A. Notably, no discernible discrepancies were observed in the expression levels of mTOR, PI3K, LC3-II, and p62 proteins between groups C and D within mouse hippocampal tissues. HBO treatment demonstrates efficacy in enhancing cognitive function in mice with AD and holds promise as a potential therapeutic intervention for AD by facilitating the activation of the mTOR pathway-mediated autophagy.

Further validation of the association between MAPT haplotype-tagging polymorphisms and Alzheimer's disease: neuropsychological tests, cerebrospinal fluid biomarkers, and APOE genotype.

Genetic studies have shown that variants in the microtubule-associated protein tau (MAPT) gene, which encodes tau protein, can increase the risk for Alzheimer's disease (AD). Additionally, two haplotypes of the MAPT gene (H1 and H2) are associated with various neurodegenerative disorders, including AD. This study aimed to test the association of MAPT haplotypes (H1 and H2) and MAPT haplotype-tagging polymorphisms (rs1467967, rs242557, rs3785883, rs2471738, del-In9, rs7521) with AD. The study included 964 individuals: 113 with AD, 53 with mild cognitive impairment (MCI), 54 with other dementias, and 744 healthy controls. The results showed that individuals carrying the A allele in the MAPT rs1467967 polymorphism, the GG genotype in the MAPT rs7521 polymorphism, and the G allele in the MAPT rs242557 polymorphism had worse performance on various neuropsychological tests. Carriers of the C allele in MAPT rs2471738 polymorphism and CC homozygotes also showed worse performance on neuropsychological tests and pathological levels of several cerebrospinal fluid (CSF) biomarkers. However, T allele carriers in the MAPT rs2471738 polymorphism were more represented among patients with dementia and apolipoprotein E (APOE) ɛ4 carriers. Carriers of the H2 MAPT haplotype had worse performance on various neuropsychological tests, consistent with our previous study, which associated the H2 MAPT haplotype with pathological levels of CSF AD biomarkers. Regarding the MAPT rs3785883 polymorphism, further research is needed since both the AA and GG genotypes were associated with pathological levels of CSF and plasma AD biomarkers. In conclusion, further genetic studies are needed to elucidate the role of MAPT haplotypes and MAPT haplotype-tagging polymorphisms in the development of AD.

Forebrain Eml1 depletion reveals early centrosomal dysfunction causing subcortical heterotopia.

Subcortical heterotopia is a cortical malformation associated with epilepsy, intellectual disability, and an excessive number of cortical neurons in the white matter. Echinoderm microtubule-associated protein like 1 (EML1) mutations lead to subcortical heterotopia, associated with abnormal radial glia positioning in the cortical wall, prior to malformation onset. This perturbed distribution of proliferative cells is likely to be a critical event for heterotopia formation; however, the underlying mechanisms remain unexplained. This study aimed to decipher the early cellular alterations leading to abnormal radial glia. In a forebrain conditional Eml1 mutant model and human patient cells, primary cilia and centrosomes are altered. Microtubule dynamics and cell cycle kinetics are also abnormal in mouse mutant radial glia. By rescuing microtubule formation in Eml1 mutant embryonic brains, abnormal radial glia delamination and heterotopia volume were significantly reduced. Thus, our new model of subcortical heterotopia reveals the causal link between Eml1's function in microtubule regulation and cell position, both critical for correct cortical development.

Synergistic Effect between the APOE ε4 Allele with Genetic Variants of GSK3B and MAPT: Differential Profile between Refractory Epilepsy and Alzheimer Disease.

Temporal Lobe Epilepsy (TLE) is a chronic neurological disorder characterized by recurrent focal seizures originating in the temporal lobe. Despite the variety of antiseizure drugs currently available to treat TLE, about 30% of cases continue to have seizures. The etiology of TLE is complex and multifactorial. Increasing evidence indicates that Alzheimer's disease (AD) and drug-resistant TLE present common pathological features that may induce hyperexcitability, especially aberrant hyperphosphorylation of tau protein. Genetic polymorphic variants located in genes of the microtubule-associated protein tau (MAPT) and glycogen synthase kinase-3β (GSK3B) have been associated with the risk of developing AD. The APOE ε4 allele is a major genetic risk factor for AD. Likewise, a gene-dose-dependent effect of ε4 seems to influence TLE. The present study aimed to investigate whether the APOE ɛ4 allele and genetic variants located in the MAPT and GSK3B genes are associated with the risk of developing AD and drug-resistant TLE in a cohort of the Mexican population. A significant association with the APOE ε4 allele was observed in patients with AD and TLE. Additional genetic interactions were identified between this allele and variants of the MAPT and GSK3B genes.

TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b−/− mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.

Macrophage-Activated Products of Lacticaseibacillus paracasei N1115 Promote the Development of Primary Hippocampal Neurons

To make a preliminary investigation of the effect of the immune pathway mediated by live Lacticaseibacillus paracasei N1115 on the development of primary hippocampal neurons cultured in vitro. Live Lacticaseibacillus paracasei N1115 suspension of an appropriate concentration was used as the experimental group. Peptidoglycan (PGN) and lipopolysaccharide (LPS) were used as positive controls, and RPMI1640 medium served as the blank control. These were co-cultured with RAW264.7 cells to obtain the co-culture mediums and the total cellular RNA, and to measure the expression and secretion of cytokines. After centrifugation, the supernatants were co-cultured with primary hippocampal neurons at appropriate ratios. The co-culture mediums were collected, and the total cellular RNA was extracted to measure the expression of genes related to synaptic development in neurons. Following immunofluorescence staining of the primary hippocampal neurons, the presynaptic and presynaptic membrane-associated proteins, including synaptophysin (SYP) and the postsynaptic density protein 95 (PSD95), and neuronal cell maturation markers, including microtubule-associated protein 2 (MAP-2), and doublecortin (DCX) were quantitatively analyzed. Additionally, the morphological development of the neurons were measured. Compared with the blank control, the mRNA expression levels of interleukin (IL)-6 and tumor necrosis factor- α (TNF-α) increased by 7471% and 926%, respectively, after the RAW264.7 cells were treated with live Lacticaseibacillus paracasei N1115, while their secretion levels increased by 184.16 pg/mL and 12320.76 pg/mL, respectively, all showing statistically significant differences (P<0.05). The activation ability of N1115 live bacteria was stronger than that of PGN but weaker than that of LPS, showing statistically significant differences (P<0.05). Compared with the blank control, following the intervention with the supernatant from the co-culture of N1115 live bacteria and RAW264.7 cells, the viability of primary hippocampal neurons in the 10% supernatant intervention group increased by 19.25%, showing statistically significant differences (P<0.05), and the mRNA expression of SYP and PSD95 increased by 137% and 159%, respectively, showing statistically significant differences (P<0.05). The total neurite length (489.88 μm) of the neurons of the group intervened with the supernatant of N1115 live bacteria was increased compared to that of the blank control group (381.51 μm), and the cell body area (2092.22 μm2), maximum neurite length (184.78 μm), total neurite length, average neurite length (108.38 μm), and branching points (4.84 s) were higher than those in the two positive control groups, showing statistically significant differences (P<0.05). Lacticaseibacillus paracasei N1115 can significantly activate the immune regulatory function of macrophages, thereby promoting the morphological development and synaptic function of nerve cells.

Study on the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells and the intervention effect of N-acetylcysteine

Objective: To investigate the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells (BEAS-2B cells), and to study the intervention effect and mechanism of N-acetylcysteine (NAC) on ultrafine carbon black-induced oxidative damage in BEAS-2B cells. Methods: In March 2023, BEAS-2B cells were used as research object, an in vitro airway model exposed to ultrafine carbon black was constructed. A control group and three carbon black exposure groups (50, 100, 200 μg/ml) were set up, and the cells were treated with corresponding concentrations of ultrafine carbon black for 24 hours. In addition, the experiment was divided into control group, NAC+ control group, 100 μg/ml carbon black exposure group and NAC+ exposure group. The corresponding groups were treated with 2 mmol/L NAC for 1 h and 100 μg/ml ultrafine carbon black for 24 h, respectively. Cell viability was measured by CCK-8 assay. Intracellular reactive oxygen species (ROS) level was detected by chemical fluorescence method. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected by colorimetry. The mRNA and protein expressions of autophagy-related genes[Atg5, Atg7, Beclin1, microtubule-associated protein light chain 3B (LC3B), p62 and lysosome-associated membrane protein 2 (LAMP2) ] and apoptosis-related genes [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and poly (ADP-ribose) polymerase 1 (PARP1) ] were determined by fluorescence quantitative PCR and Western blot. Cell apoptosis was determined by flow cytometry. Results: Compared with the control group, the relative survival rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly decreased, the levels of ROS and MDA were significantly increased, and the activities of SOD, GSH-Px and CAT were significantly decreased (P<0.05). The relative survival rate, ROS and MDA levels, SOD, GSH-Px and CAT activities were significantly correlated with the exposure dose of ultrafine carbon black (r(s)=-0.755, 0.826, 0.934, -0.810, -0.880, -0.840, P<0.05). Compared with the control group, the relative expression levels of Atg5, Atg7, Beclin1, LC3B, p62, LAMP2, Bax, Caspase3, Caspase9, PARP1 mRNA and Atg5, Atg7, Beclin1, LC3BⅡ, p62, LAMP2, Bax, cleaved Caspase3 (C-Caspase3), cleaved Caspase9 (C-Caspase9), cleaved PARP1 (C-PARP1) protein and the ratio of LC3BⅡ/LC3BⅠ in 50, 100 and 200 μg/ml carbon black exposure groups were significantly increased, while the relative expression levels of Bcl-2 mRNA and protein were significantly decreased (P<0.05). The changes of the above indexes were significantly correlated with the exposure dose of carbon black (r(s)=0.892, 0.879, 0.944, 0.892, 0.828, 0.880, 0.814, 0.794, 0.931, 0.918, 0.813, 0.866, 0.774, 0.695, 0.918, 0.761, 0.794, 0.944, 0.833, 0.866, 0.905, -0.886, -0.748, P<0.05). Compared with 100 μg/ml carbon black exposure group, the relative survival rate, the activities of SOD, GSH-Px and CAT in NAC+exposure group were significantly increased, while the levels of ROS and MDA were significantly decreased, and the relative expression levels of LC3B, p62 and Caspase3 mRNA and protein as well as the ratio of LC3BⅡ/LC3BⅠ were significantly decreased, and the differences were statistically significant (P<0.05). Compared with the control group, the apoptosis rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly increased (P<0.05), and there was a significant positive correlation between ultrafine carbon black exposure dose and cell apoptosis rate (r(s)=0.944, P<0.05). While compared with 100 μg/ml carbon black exposure group, the apoptosis rate of NAC+exposure group was significantly decreased, and the difference was statistically significant (P<0.05) . Conclusion: Cell autophagy and apoptosis may be important pathophysiological mechanisms of ultrafine carbon black-induced oxidative damage in BEAS-2B cells. NAC can alleviate the occurrence of BEAS-2B cell damage caused by ultrafine carbon black by regulating oxidative stress and the cascading autophagy and apoptosis pathways.

Investigation of Persistent Photoconductivity of Gallium Nitride Semiconductor and Differentiation of Primary Neural Stem Cells.

A gallium nitride (GaN) semiconductor is one of the most promising materials integrated into biomedical devices to play the roles of connecting, monitoring, and manipulating the activity of biological components, due to its excellent photoelectric properties, chemical stability, and biocompatibility. In this work, it was found that the photogenerated free charge carriers of the GaN substrate, as an exogenous stimulus, served to promote neural stem cells (NSCs) to differentiate into neurons. This was observed through the systematic investigation of the effect of the persistent photoconductivity (PPC) of GaN on the differentiation of primary NSCs from the embryonic rat cerebral cortex. NSCs were directly cultured on the GaN surface with and without ultraviolet (UV) irradiation, with a control sample consisting of tissue culture polystyrene (TCPS) in the presence of fetal bovine serum (FBS) medium. Through optical microscopy, the morphology showed a greater number of neurons with the branching structures of axons and dendrites on GaN with UV irradiation. The immunocytochemical results demonstrated that GaN with UV irradiation could promote the NSCs to differentiate into neurons. Western blot analysis showed that GaN with UV irradiation significantly upregulated the expression of two neuron-related markers, βIII-tubulin (Tuj-1) and microtubule-associated protein 2 (MAP-2), suggesting that neurite formation and the proliferation of NSCs during differentiation were enhanced by GaN with UV irradiation. Finally, the results of the Kelvin probe force microscope (KPFM) experiments showed that the NSCs cultured on GaN with UV irradiation displayed about 50 mV higher potential than those cultured on GaN without irradiation. The increase in cell membrane potential may have been due to the larger number of photogenerated free charges on the GaN surface with UV irradiation. These results could benefit topical research and the application of GaN as a biomedical material integrated into neural interface systems or other bioelectronic devices.

HN1 expression contributes to mitotic fidelity through Aurora A-PLK1-Eg5 axis.

Hematological and neurological expressed 1 (HN1) is homolog of Jupiter protein from Drosophila melanogaster where it functions as a microtubule-associated protein. However, in mammalian cells, HN1 is associated partially with y-tubulin in centrosomes, Stathmin for stabilizing microtubules, and Cdh1 for regulating Cyclin B1 for cell cycle regulation. Moreover, HN1 overexpression leads to early mitotic exit as well. Other molecular functions and interactions of HN1 are not clear yet. Here, based on our previous analysis where HN1 was shown to cluster supernumerary centrosomes and maintain mitotic spindle assembly, we further investigated the role of HN1 in centrosome maintenance and mitotic fidelity in PC-3 prostate and MDA-MB231 mammary cancer cell lines. The maturation-associated roles of HN1 during cell division by examining the AuroraA-PLK1 axis involving a plus end kinesin, Eg5 as well as pericentriolar matrix protein (PCM1) as components of centrosomes were established. We found that HN1 co-localized to centrioles with Eg5 and Aurora A to suppress aberrant spindle formation to ensure the fidelity of centriole/centrosome duplication when overexpressed. Consistently, depleting the HN1 expression using siRNA or shRNA resulted in an increased number of dysregulated mitotic spindle structures, where Aurora A as well as PLK1 co-localizations with Eg5 and PCM1 were disrupted. Further, the PLK1 and Aurora A kinase's phosphorylations also decreased, confirming the hypothesis that the cells struggle in mitotic progression, display nuclear and cytokinetic abnormalities with supernumerary but immature mononucleated centrosomes. In summary, we described the role of HN1 in centrosome nucleation/maturation in PLK1-Eg5 axis and concomitant mitotic spindle formation in human cells.

Etomidate protects retinal ganglion cells from hydrogen peroxide-induced injury via Nrf2/HO-1 pathway.

To determine whether etomidate (ET) has a protective effect on retinal ganglion cells (RGCs) injured with hydrogen peroxide (H2O2) and to explore the potential mechanism underlying the antioxidative stress effect of ET. Cultured RGCs were identified by double immunofluorescent labeling of microtubule-associated protein 2 and Thy1.1. An injury model of H2O2-induced RGCs oxidative stress was established in vitro. Cells were pretreated with different concentrations of ET (1, 5, and 10 µmol/L) for 4h, followed by further exposure to H2O2 at 1000 µmol/L. Cell counting kit 8 and Annexin V/propidium iodide assays were applied to detect the viabilities and apoptosis rates of the RGCs at 12, 24, and 48h after H2O2 stimulation. The levels of nitric oxide, malondialdehyde, and glutathione in culture media were measured at these time points. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to observe the effects of ET on the messenger RNA and protein expression of inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase 1 and the level of conjugated acrolein in RGCs at 12, 24, and 48h after H2O2 stimulation and in the retina at 12h after optic nerve transection (ONT). The applications of 5 and 10 µmol/L of ET significantly increased the viability of RGCs. Results from qRT-PCR indicated a decrease in the expression of iNOS and an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs at 12, 24 and 48h after H2O2 stimulation, as well as in ET-treated retinas at 12h after ONT. Western blot analysis revealed a decrease in the expression of iNOS and levels of conjugated acrolein, along with an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs in vitro and ET-treated retinas in vivo. ET is a neuroprotective agent in primary cultured RGCs injured by H2O2. The effect of ET is dose-dependent with the greatest effect being at 10 µmol/L. ET plays an antioxidant role by inhibiting iNOS, up-regulating Nrf2/HO-1, decreasing the production of acrolein, and increasing the scavenge of acrolein.

Resistin alleviates lipopolysaccharide-induced inflammation in bovine alveolar macrophages by activating the AMPK/mTOR signaling pathway and autophagy

ObjectiveResistin (RETN) is an adipocyte-specific hormone that participates in metabolism and modulates cellular inflammation. Our study aimed to assess the effects of RETN treatment on autophagy and the underlying molecular and biological mechanisms in bovine alveolar macrophages (BAMs). MethodsThe optimal concentration of RETN + lipopolysaccharide (LPS) on macrophages was screened and then used to co-culture with alveolar macrophages. Autophagosomes in BAMs were examined using a transmission electron microscope (TEM). Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of microtubule-associated protein light chain 3 (LC3) and p62. Western blot (WB) was used to detect the protein expressions of LC3 and p62. The distribution of LC3 and p62 proteins in the cells was observed by immunofluorescence (IF). The concentrations of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expression of adenosine-monophosphate-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), and p-mTOR was detected using WB. ResultsThe treatment of BAMs with RETN or LPS increased the number of autophagosomes and the ratio of LC3II/LC3I and decreased the expression level of p62 protein. RETN treatment significantly triggered autophagy compared to LPS treatment. Moreover, the ratios of p-AMPK/AMPK and p-mTOR/mTOR were upregulated and downregulated, respectively, after RETN treatment, suggesting that AMPK/mTOR signaling pathway activation is required for RETN-mediated autophagy in BAMs. Additionally, the ratio of LC3-II/LC3-I was lower, and the concentrations of IL-1β, IL-6, and TNF-α significantly decreased in the LPS and RETN co-treatment groups compared to the single LPS treatment group. However, both autophagy- and LPS-induced inflammation were partially alleviated by RETN treatment. ConclusionRETN can promote autophagy in BAMs by activating the AMPK/mTOR signaling pathway, it may help prevent LPS-induced inflammation.

Live-cell visualization of tau aggregation in human neurons

Alzheimer’s disease (AD) and more than twenty other dementias, termed tauopathies, are pathologically defined by insoluble aggregates of the microtubule-associated protein tau (MAPT). Although tau aggregation correlates with AD symptomology, the specific tau species, i.e., monomers, soluble oligomers, and insoluble aggregates that induce neurotoxicity are incompletely understood. We developed a light-responsive tau protein (optoTAU) and used viscosity-sensitive AggFluor probes to investigate the consequence(s) of tau aggregation in human neurons and identify modifiers of tau aggregation in AD and other tauopathies. We determined that optoTAU reproduces biological and structural properties of tau aggregation observed in human brains and the pathophysiological transition in tau solubility in live cells. We also provide proof-of-concept for the utilization of optoTAU as a pharmacological platform to identify modifiers of tau aggregation. These findings have broad implications for the characterization of aggregation-prone proteins and investigation of the complex relationship between protein solubility, cellular function, and disease progression.

Systems mapping of bidirectional endosomal transport through the crowded cell

Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under invitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors.