Experimental Biology and Medicine最新文献

Although it is well documented in animal research that an early exposure to general anesthetics during critical stages of synaptogenesis disturbs normal brain development ultimately leading to cognitive and affective impairments, it is less clear whether and how surgical interventions and/or underlying systemic inflammation impact the detrimental effects of general anesthetics. Some emerging evidence suggests that aseptic systemic inflammation preceding exposure to the commonly used general anesthetics worsens anesthesia-induced neuroapoptosis and activates inflammasome pathways while resulting in impaired cognitive-affective behaviors. To improve our understanding of the underlying mechanisms, here we focused on multicellular interactions between damaged neurons and microglia since microglia is the resident macrophages within the brain that respond to stress. Using infant rats (post-natal day 7) and most commonly used inhaled anesthetic, sevoflurane, we examine microglia role in sevoflurane-induced inflammation-propagated developmental neurotoxicity. We show that sevoflurane exposure leads to a significant neuroapoptosis in young rat pup hippocampal subiculum, a neuroapoptosis that is worsened in the setting of systemic inflammation caused by either lipopolysaccharide (LPS) injection or trauma (tibial fracture). The worsening is not only shown in terms of the intensity of neuroapoptosis but in its duration and onset. We further report that sevoflurane-induced neuroapoptosis triggers activation of microglia, which in turn releases proinflammatory cytokine MCP-1 and upregulates endothelial cell adhesion molecule, ICAM-1. This leads to T-lymphocyte infiltration in the hippocampal subiculum, an event that further perpetuates microglia activation in an attempt to control neuroapoptosis which is suggested by the fact that microglia depletion leads to a significant worsening of sevoflurane-induced developmental neuroapoptosis. Our work gets us a step closer to making our animal work more relevant to the clinical setting and hence more translational. This is vitally important considering that exposure to anesthesia is exceedingly rare in the absence of any kind of a pathological process.

Although substantial progress has been made in the last three decades towards our understanding of how general anesthetics (GAs) act at the molecular level, much less is known about how GAs cause loss of consciousness at the level of neuronal networks. The role of thalamus as an important brain region in anesthetic-induced hypnosis is relatively well established, but the specific roles of voltage-gated ion channels in different functional regions of the thalamus in anesthetic mechanisms are not well studied. To address this gap in knowledge, we selectively silenced the Cacna1g gene that encodes the low-threshold-activated Ca_V3.1 T-type voltage-gated calcium channel subunit by injecting short-hairpin RNA (shRNA) into midline and intralaminar - nonspecific thalamus (MIT) and sensory - specific ventrobasal (VB) thalamic nuclei in wild-type (WT) mice. Control animals were injected with scrambled shRNA. To validate our silencing approach, we performed patch-clamp experiments in acute thalamic slices ex vivo. In injected animals we determined anesthetic endpoints such as hypnosis measured with loss of righting reflex (LORR) and immobilization measured with loss of withdrawal reflex (LOWR) in vivo after administration of a traditional volatile GA isoflurane. Effective Ca_V3.1 channel knock-down was documented by greatly diminished amplitudes of T-currents and absence of rebound burst firing in our patch-clamp recordings from thalamic slices. We found that knocking down Ca_V3.1 channels in MIT significantly decreased inhaled isoflurane concentration that is required to induce LORR, but it did not affect speed of anesthetic induction and the immobilizing effect of isoflurane. In contrast, knocking down the Ca_V3.1 channel in the VB thalamus did not affect any of the measured anesthetic endpoints. Hence, we concluded that Ca_V3.1 channels in nonspecific MIT thalamus have a preferential role in anesthetic hypnosis when compared to the sensory VB thalamus.

Intracerebral hemorrhage (ICH), as bleeding from ruptured vessels within the brain, is the second leading neuropathological problem following ischemic stroke. In the present study, the involvement of epithelial growth factor receptor (EGFR)-tyrosine kinase (TK) signaling underlying ICH-related neurodegeneration was investigated using afatinib, a clinically available EGFR-tyrosine kinase inhibitor (EGFR-TKI). We employed hemin (a breakdown product of hemoglobin) to mimic the pathophysiology of ICH in primary cultured cortical neurons. Using a lactate dehydrogenase (LDH) assay, incubation of hemin concentration- and time-dependently induced neuronal death. Simultaneous incubation of afatinib (10 nM) significantly inhibited hemin (30 μM)-induced neuronal death. Immunofluorescent data demonstrated that co-treatment of afatinib for 1 h attenuated hemin (30 μM)-induced elevation in phosphorylated-EGFR (p-EGFR) immunoreactivity and neurite impairment. Western blot assay demonstrated that co-incubation of afatinib for 16 h diminished hemin-induced elevation in p-EGFR and p-AKT, tumor necrosis factor-α and cyclooxygenase 2 (two proinflammatory biomarkers) as well as heme oxygenase-1 (HO-1, an enzyme catalyzing heme/hemin), glutathione hydroperoxidase 4 and receptor-interacting protein 3 (two biomarkers of ferroptosis and necroptosis). In addition, co-treatment of afatinib for 24 h inhibited hemin-induced NO production in the culture medium. In conclusion, our study shows that afatinib via blocking EGFR-AKT signaling inhibits hemin-induced EGFR-AKT activation, neuroinflammation, HO-1 expression and programed cell death, suggesting that EGFR-AKT signaling is involved in hemin-induced neurotoxicity and may be a druggable target for ICH.

Epilepsy affects more than 70 million people worldwide. A seizure focus that develops in different cortical brain regions can present as either focal or generalized seizures. Temporal lobe epilepsy is a highly pharmacoresistant form of epilepsy that involves the amygdala, hippocampus with or without hippocampal sclerosis as well as other limbic structures. Loss and/or dysfunction of GABAergic inhibitory neurons play a critical role in tipping the balance toward excitation. Synchronous burst firing is a feature of inhibitory neurons that is thought to regulate and rectify large excitatory neuronal networks in the BLA and is thought to underlie higher cognitive function. Acid sensing ion channels (ASIC) activated by decreases in pH, the presence of ammonium ion or a slight lowering of temperature are present on excitatory and inhibitory neurons and can alter excitability. The net effect of the activation of ASIC1a channels in the BLA is inhibition. ASIC1a channels are active in the basal state, enhancing primarily GABAergic inhibition by direct depolarization of interneurons but also by indirect excitation of interneurons via ASIC1a-mediated depolarization of pyramidal neurons. In this study, we examine the contribution of ASIC1a channel activation on alpha-linolenic acid (ALA)-induced GABAergic inhibitory synchronous burst firing in the BLA. Our results show that ALA initiates inhibitory bursts that are dependent, in part, on the activation of ASIC1a channels that may in turn be mediated by mature brain-derived neurotrophic factor.

Cancer/testis antigen HCA587, also known as MAGE-C2, highly expressed in a wide range of malignant tumors with unique immunological characteristics, serves as a potential target for tumor immunotherapy. Synthetic long peptides from HCA587 (HCA587 SLP) were proved to be highly immunogenic and promising in the application of cancer vaccine composed of Freud's adjuvant (FA) and CpG 1826, whereas, scarce CD8⁺ T cell response may limit their anti-tumor effects during previous research. In this study, notwithstanding the multiple potential of IFN-α in immune modulation, there was no evidence of IFN-α in enhancing the immune response elicited by HCA587 SLP vaccine (HCA587 SLP + FA + CpG 1826). Given the unpleasant side-effects of Freud's adjuvant, then we applied AddaVax as a substitute for Freud's adjuvant, and we demonstrated that HCA587 SLP, formulated with AddaVax and CpG 2395, could induce more robust immune response in comparison with combined use of AddaVax and CpG 1826 through ELISpot assay. Furthermore, both IFN-γ-secreting CD4⁺ T cell and CD8⁺ T cell responses could be elicited by HCA587 SLP in combination with AddaVax and CpG 2395, and CD8⁺ T cell response was most obviously observed under the condition of 10-h inhibition of cytokine secretion by brefeldin A post 10-h stimulation with HCA587 SLP, suggesting that cross presentation of exogenous long peptides to CD8⁺ T cells may require more time than direct presentation to CD4⁺ T cells. This vaccine formulation also conferred protection against challenge with HCA587-expressing B16 melanoma presented by delayed tumor growth and prolonged survival compared. This formulation of HCA587 SLP vaccine holds promise for the treatment of patients with cancer in future clinical trials.

Adverse drug events are harms associated with drug use, whether the drug is used correctly or incorrectly. Identifying adverse drug events is vital in pharmacovigilance to safeguard public health. Drug safety surveillance can be performed using unstructured data. A comprehensive and accurate list of drug names is essential for effective identification of adverse drug events. While there are numerous sources for drug names, RxNorm is widely recognized as a leading resource. However, its effectiveness for unstructured data analysis in drug safety surveillance has not been thoroughly assessed. To address this, we evaluated the drug names in RxNorm for their suitability in unstructured data analysis and developed a refined set of drug names. Initially, we removed duplicates, the names exceeding 199 characters, and those that only describe administrative details. Drug names with four or fewer characters were analyzed using 18,000 drug-related PubMed abstracts to remove names which rarely appear in unstructured data. The remaining names, which ranged from five to 199 characters, were further refined to exclude those that could lead to inaccurate drug counts in unstructured data analysis. We compared the efficiency and accuracy of the refined set with the original RxNorm set by testing both on the 18,000 drug-related PubMed abstracts. The results showed a decrease in both computational cost and the number of false drug names identified. Further analysis of the removed names revealed that most originated from only one of the 14 sources. Our findings suggest that the refined set can enhance drug identification in unstructured data analysis, thereby improving pharmacovigilance.

Human respiratory syncytial virus (hRSV) is one of the most prevalent viruses infecting children globally. In this study, we employed the RT-RPA with CRISPR/Cas12a detection methodology to detect and differentiate RSV-A and RSV-B, particularly in resource-limited settings. The detection limit for RSV-A and RSV-B was approximately 10² and 10³ copies/reaction, respectively. The assay revealed 100% specificity in detecting both RSV-A and RSV-B. Diagnostic accuracy was 90.32 and 93.55% for RSV-A and RSV-B, respectively, compared to RT-qPCR. These data indicate a proficient strategy for RSV screening, demonstrating promise for prospective applications in detecting diverse viral infections.

Inflammatory breast cancer (IBC) is a rare and aggressive breast cancer subtype with poor survival. Identifying novel biomarkers is needed to predict survival for this highly progressive form of breast cancer. In this retrospective study, we investigated pan-immune-inflammation value (PIV), a novel immune-inflammation-based biomarker which combined the peripheral blood parameters (lymphocytes, monocytes, neutrophils, and platelets) in a retrospective cohort of 143 IBC patients. Then we explored the difference of PIV levels in IBC and non-IBC cohorts and the relationship between PIV and clinical characteristics in IBC patients. The survival rates of disease-free survival (DFS) and overall survival (OS) in IBC patients were analyzed and univariate and multivariate statistics were used to evaluate the prognostic value. PIV had the most significantly predictive value in IBC patients compared with other peripheral blood parameters. The mean PIV value in IBC patients was significantly higher than non-IBC patients, and the significant difference between the IBC and non-IBC was also observed in subgroups with different clinical stages and pathologic types. Furthermore, PIV performed an extensive systemic immune prognostic factor on both DFS and OS in IBC patients, and PIV was identified an independent prognostic indicator for survival outcome in IBC patients in univariate and multivariate models. Our retrospective study demonstrated the prognostic value of PIV in IBC patients, suggesting the potential application of PIV in IBC treatment outcomes. PIV would also provide some insights into the mechanisms underlying the role of immune and inflammation in IBC development and progression.

Nonerythroid spectrins are proteins important in maintaining the structural integrity and flexibility of the cell and nuclear membranes and are essential for a number of functionally important cellular processes. One of these proteins, nonerythroid α spectrin (αSpII), plays a critical role in DNA repair, specifically repair of DNA interstrand crosslinks (ICLs), where it acts as a scaffold, recruiting repair proteins to sites of damage. Loss or breakdown of αSpII is an important factor in a number of disorders. One of these is Fanconi anemia (FA), a genetic disorder characterized by bone marrow failure, chromosome instability, cancer predisposition, congenital abnormalities and a defect in DNA ICL repair. Significantly, breakdown of αSpII occurs in cells from a number of FA complementation groups, due to excessive cleavage by the protease, μ-calpain, leading to defective repair of DNA ICLs in telomeric and non-telomeric DNA. Knockdown of μ-calpain in FA cells by μ-calpain siRNA results in restoration of αSpII levels to normal and repair of DNA ICLs in telomeric and non-telomeric DNA, demonstrating the importance of αSpII stability in the repair process. It is hypothesized that there is a mechanistic link between excessive cleavage of αSpII by μ-calpain and defective DNA ICL repair in FA and that FA proteins, which are deficient in FA, play a key role in maintaining the stability of αSpII and preventing its cleavage by μ-calpain. All of these events are proposed to be important key factors involved in the pathophysiology of FA and suggest new avenues for potential therapeutic intervention.

Colorectal cancer (CRC) remains a major contributor to cancer-related morbidity and mortality. While Dachshund homolog 1 (DACH1) was recognized as a critical regulator in cancer progression, its role in promoting or suppressing tumor development remains a subject of ongoing debate. This study aimed to elucidate the role of DACH1 in CRC progression and its underlying regulation mechanisms. The expression levels of Methyltransferase 1 (DNMT1) and DACH1, as well as its methylation status were assessed through a combination of TCGA data analysis and experimental validation using immunohistochemistry, PCR, methylation-specific PCR, and bisulfite sequencing RCR on 120 clinical samples, comprising normal mucosa, adenomas, and adenocarcinomas. The relationships among them were evaluated using Pearson or Spearman correlation analysis. The associations between the DACH1 and DNMT1 levels and clinicopathological parameters were examined to determine their clinical relevance. A progressive decrease in DACH1 expression and a concomitant increase in DACH1 promoter methylation and DNMT1 expression were observed from normal mucosa to adenoma and adenocarcinoma tissues. Higher DNMT1 expression and lower DACH1 expression were associated with poorer clinical outcomes, including worse tumor differentiation, lymphatic metastasis, and advanced tumor stages. Paired analysis of tissues from the same patient further validated their inverse expression patterns during CRC progression. DNMT1-mediated DACH1 epigenetic silencing plays a critical role in CRC progression, suggesting that the DNMT1-DACH1 regulatory axis may serve as a potential biomarker and therapeutic target in CRC.