Immunofluorescence Staining Method Research Articles

Electroacupuncture alleviates myocardial ischemia-reperfusion injury by inhibiting hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.

Accumulating evidence suggests that electroacupuncture (EA) has obvious therapeutic effects and unique advantages in alleviating myocardial ischemia-reperfusion injury (MIRI), while the underlying neuromolecular mechanisms of EA intervention for MIRI have not been fully elucidated. The aim of the study is to investigate the role of the neural pathway of hypothalamic paraventricular nucleus (PVN) neurons projecting to the rostral ventrolateral medulla (RVLM) in the alleviation of MIRI rats by EA preconditioning. MIRI models were established by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for 2h. Electrocardiogram recording, chemogenetics, enzyme-linked immunosorbent assay, multichannel physiology recording and haematoxylin-eosin and immunofluorescence staining methods were conducted to demonstrate that the firing frequencies of neurons in the PVN and the expression of c-Fos decreased by EA pretreatment. Meanwhile, EA preconditioning significantly reduced the levels of creatine kinase isoenzymes (CK-MB), cardiac troponin I (cTnI) and lactic dehydrogenase (LDH). Virus tracing showed a projection connection between PVN and RVLM. The inhibition of the PVN-RVLM neural pathway could replicate the protective effect of EA pretreatment on MIRI rats. However, the activation of the pathway weakened the effect of EA preconditioning. EA pretreatment alleviated MIRI by regulating PVN neurons projecting to RVLM. This work provides novel evidence of EA pretreatment for alleviating MIRI.

Britanin alleviates chondrocyte ferroptosis in osteoarthritis by regulating the Nrf2-GPX4 axis

BackgroundOsteoarthritis (OA) is a chronic joint disease characterized by degeneration of the articular cartilage or inflamed joints. Britanin is a guaiacyl-type sesquiterpene lactone possessing anti-inflammatory and antioxidant activities. MethodsIn this study, western blotting, Reverse-transcription PCR (RT-PCR), and immunofluorescent staining method were used to clarifying the molecular mechanisms of britanin in alleviating chondrocyte ferroptosis. The effect of britanin on reactive oxygen species (ROS) and lipid peroxidation levels in chondrocytes was examined by Dihydroethidium (DHE) stain and fluorescent dye BODIPY581 / 591 C11. The destabilized medial meniscus (DMM) model was used to mimic OA, then britanin was injected into the knee articular cavity. The morphological analysis was performed by hematoxylin and eosin (H&E) staining, Safranin O-Fast green staining and Micro-computed tomography (Micro-CT) analysis. Immunohistochemical analysis and Immunofluorescence assay was conducted to verify the effect of britanin in vivo. ResultsThe results revealed that britanin treatment regulates the expression of chondrocyte extracellular matrix–related factors, such as ADAMTS5, MMP13, SOX9, aggrecan, and COL2A1. Britanin also significantly reversed the influence of interleukin (IL)-1β on the levels of reactive oxygen species and lipid peroxidation and upregulated glutathione peroxidase 4 (GPX4) expression in IL-1β-induced chondrocytes. Erastin could alleviate the effects of britanin on chondrocytes. Moreover, britanin could prevent IL-1β-induced oxidative stress and ferroptosis in chondrocytes. Britanin also upregulated the expression of nuclear factor E2–related factor 2 (Nrf2) in IL-1β-induced chondrocytes, and Nrf2 activator played a similar role as that of britanin in IL-1β-induced chondrocytes. Furthermore, injecting britanin into the knee joints of OA rats alleviated cartilage destruction caused by surgical resection. ConclusionsThese findings confirmed that britanin can inhibit ferroptosis in OA chondrocytes through the Nrf2-GPX4 pathway. Thus, britanin has the potential to be developed as a drug for OA therapy.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

3'-Daidzein Sulfonate Sodium Protects against Glutamate-induced Neuronal Injuries by Regulating NMDA Receptors.

It was previously found that 3'-Daidzein Sulfonate Sodium (DSS) exhibits protective effects on cerebral ischemia/reperfusion injury (CI/RI). This study aimed to explore the underlying molecular mechanisms involved in the neuroprotective effects of DSS against ischemic stroke. In this study, rats with transient middle cerebral artery occlusion (tMCAO) were used as an in vivo model, whereas PC12 cells treated with glutamate alone and rat primary cortical neurons treated with the combination of glutamate and glycine were used as in vitro models. Cell viability and lactate dehydrogenase (LDH) release were used to evaluate cell injury. Cell apoptosis was determined by flow cytometry. Quantitative polymerase chain reaction (qPCR), Western blotting, and immunofluorescent staining methods were used to determine the mRNA expressions and protein levels and location. It was found that DSS significantly suppressed the impaired viability of PC12 cells induced by glutamate. DSS also increased cell viability while reducing the LDH release and apoptosis in primary cortical neurons injured by glutamate and glycine. In addition, DSS decreased GluN2B subunit expression while enhancing the expressions of GluN2A subunit and PSD95 in tMCAO rats' brains. This study demonstrated that DSS protects against excitotoxic damage in neurons induced by CI/RI through regulating the expression of NMDA receptors and PSD95. Our findings provide experimental evidence for the potential clinical administration of DSS in ischemic stroke.

SGLT2i Alleviates Atherosclerosis by Inhibiting NHE1 Activation to Protect against Macrophage Senescence Induced by Angiotensin II.

Sodium-dependent glucose transporter (SGLT2) inhibitors (SGLT2i) have been found to have anti-atherosclerotic effects in clinical treatment. The aim of this study was to explore whether angiotensin II (Ang II) induces changes in the expression of Na+/H+ exchanger of cytoplasmic membrane channel proteins (NHE1) and SGLT2 in macrophages and whether dapagliflozin (DAPA), an SGLT2i, protects against Ang II induced macrophage senescence by inhibiting NHE1 activation to alleviate Atherosclerosis (AS). After intervention with DAPA plus gavage or feeding them a high-fat diet, the mice's aortas were dissected, and oil red O staining was performed. Cell proliferation and toxicity detection, western blot, immunofluorescence, and β-galactosidase staining methods were adopted to detect cell activity, expressions of senescence-related genes, and number of senescent cells after different concentrations of Ang II or DAPA or plasmid NHE1 were treated with RAW264.7 cells. (1) The formation of AS plaques in ApoE -/- mice showed a downward trend under DAPA. (2) After the intervention of Ang II, the cell activity of RAW264.7 decreased, and the expression of senescent cells and related genes increased. (3) Under the Ang II condition, the expression of SGLT2 and NHE1 increased, and SGLT2, NHE1, and senescence-related genes decreased with the addition of DAPA. (4) The expression of NHE1, senescent cells and related genes decreased in RAW264.7 cells after DAPA treatment with plasmid NHE1 intervention. SGLT2i alleviates atherosclerosis by inhibiting NHE1 activation to protect against macrophage senescence induced by Ang II.

Abstract 2865: Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma

Abstract Pediatric Neuroblastoma (NB) is one of the most prevalent solid tumors in children that arise from embryonic neural crest cells and poses a significant challenge in the field of oncology. There are about 700 to 800 new cases of neuroblastoma diagnosed each year in the United States, and accounts for approximately 15% of all childhood cancer mortalities. Since this malignancy predominantly affects infants and young children, it is imperative to decipher the underlying molecular factors driving its pathogenesis. The novel molecular therapeutic approach includes the use of epigenetic modifiers as drugs. This approach aims to reverse significant epigenetic events underlying cancer pathogenesis, particularly abnormalities in histone modifications and DNA methylation. The reduction of both DNA and histone methylation levels leads to the reactivation of tumor suppressor genes and inhibits cancer cell proliferation and cell growth. In this study, we used selective and reversible small molecule agents such as MDM2 inhibitor (RG7388) and epigenetic modifiers (CM272 and SAHA) to block cell proliferation by inducing cell cycle arrest via enforcing Epigenetic alterations. Our initial experiments were designed to understand the effects of RG7388, CM272, and SAHA treatments on NB and their impact on cell cycle progression. It was hypothesized that the decrease in DNA methylation and increase in histone modification- augment p21 expression via the use of MDM2 inhibitors and epigenetic modifiers. To test this hypothesis, the NB cells (SK-N-SK and IMR-32) were treated with RG7388, CM272, and SAHA for 24 hours. The extent of Cell death was determined for NB cells by using the Trypan Blue Dye Exclusion (TBDE) method, and CellTiter-Glo® Luminescent Cell Viability Assay. Induction of apoptosis was determined by using the Immunofluorescence staining method with DEVD-amc substrate. The results of our study suggested that RG7388, CM272, and SAHA were able to induce cell death in the SK-N-SH cells following 24-hour treatment. Furthermore, we analyzed the levels of Tumor suppressor (p53), cell cycle arrest (p21, p27, AurkB, CDC25A, CyclinD) cell death (Bax, Bcl2, LC3, Beclin, PARP, Cleaved PARP, RIP1, RIP3, MLKL, p-AKT), and Epigenetic biomarker (DNMT1, DNMT3A, DNMT3B, acetyl histoneH3, H4, and H2b) protein expression levels using western blot analysis. The results showed significant elevation of p53, p21, p27, and acetylation of histone family biomarkers after treatment when compared with the control group. In summary, our results indicate that RG7388, CM272, and SAHA treatments can effectively induce cellular responses and prevent cell growth and progression by enforcing epigenetic changes in the NB cells. (This research was supported by the National Pediatric Cancer Foundation, Tampa, Florida, and the Royal Dames of Cancer Research Inc. of Ft. Lauderdale, Florida) Citation Format: Tahani Momenah, Umamaheswari Natarajan, Shyam Jaganathan, Appu Rathinavelu. Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2865.

Abstract 5891: An image-based phenotypic screen identified CBP/p300 as new cancer drug resistance target and enabled the development of the clinical candidate TT125-802

Abstract Resistance to targeted therapies is a major challenge in oncology. Genetic mechanisms of resistance such as gatekeeper mutations in the primary drug target can be readily identified. However, drug targets responsible for non-genetic resistance mechanisms such as transcriptional reprogramming remain elusive. Here we describe an innovative phenotypic screening approach that led to the development of the clinical candidate TT125-802 and identification of its target CBP/p300 which we discovered as a novel regulator of transcriptional resistance to a range of targeted therapies. Targeted cancer therapies have been reported to induce profound transcriptional reprogramming leading to drug resistance. For instance, inhibition of oncogenic MAPK signaling using a BRAF/MEK inhibitor combination in BRAF-V600E mutated melanoma, triggers expression of the pluripotency transcription factor SOX2 along with broader expression of associated stemness and EMT genes. We used this system as a model to screen for small molecules that prevent transcriptional escape mechanisms to targeted cancer therapies. For this, we developed an automated high-throughput immunofluorescence staining and analysis method of SOX2 as a surrogate marker for resistance-conferring transcriptional reprogramming. A hit-like high diversity library of 16’000 small molecules was screened using an image-based multi-parameter readout with an integrated counter-screen for general cell toxicity. A battery of downstream assessments including patentability, synthetic tractability, physico-chemical properties, and dose-dependent modulation of transcriptional resistance signatures was used to select the chemical scaffold TT125 for further hit to lead optimization. Hit to lead optimization was initially target agnostic. To de-orphan the scaffold, a chemical probe of TT125 was developed in house and used in a chemoproteomics assay. The transcriptional co-activators CBP/p300 were identified as the target of TT125. Biochemical assays revealed that the TT125 scaffold binds specifically to the bromodomain of CBP/p300 but not the histone acetyl transferase domain. Moreover, TT125 was confirmed to bind highly selectively to the bromodomain of CBP/p300 but not other bromodomain-containing proteins such as BRD2 or BRD4. Structure-based drug design along with in vivo PD models using transcriptional short-term readouts were used for lead optimization that resulted in the development of the potent and highly selective clinical candidate TT125-802. Validating our phenotypic approach to drug discovery and development, TT125-802 is currently being evaluated in a phase I oncology trial. The modular setup of our phenotypic screening system has the potential to power the discovery of additional novel modulators of transcriptional drug resistance. Citation Format: Dorothea Gruber, Charles-Henry Fabritius, Thomas Bohnacker, Martin Schwill, Sara Laudato, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Stefanie Flückiger-Mangual. An image-based phenotypic screen identified CBP/p300 as new cancer drug resistance target and enabled the development of the clinical candidate TT125-802 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5891.

In vitro anti-Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou.

Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism. Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein. Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 μg/mL, and minimum bactericidal concentration (MBC) values were 160-320 μg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity. Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.

Inhibition of Autophagy on Melatonin‑Induced Apoptosis in MCF-7 and MDA-MB-231 Cell Lines

Aim: We looked at the connection between autophagy and apoptosis after our prior research indicated that melatonin could cause MCF-7 and MDA-MB-231 cells in the present study.
 Material and Method: In order to investigate the autophagy inhibition’s effect on the melatonin-induced BC cells’ apoptosis, melatonin and/or 3-methyladenine (3-MA, autophagy inhibitor) have been utilized. Melatonin was applied to the cells following a 5-mM 3-MA pre-cultivation. Then, apoptosis was detected by the TUNEL method. The technique for double immunofluorescence labeling was used to identify the molecular alterations in Bax/Bcl-2 expression. To evaluate the cell viability, the MTT test was used.
 Results: When an autophagy inhibitor, 3-MA, and melatonin treatment were co-administered in MCF-7 cells, apoptosis was decreased, compared to melatonin treatment alone, but it was not significant. In addition, 3-MA application downregulated Bax expression compared with melatonin alone treatment. Combined therapy markedly elevated apoptosis and significantly up-regulated Bax protein in MDA-MB-231 cells. 
 Conclusion: Taken together, in MCF-7 cells, autophagy’s inhibition contributes to the downregulation of apoptosis, whereas increased apoptosis is seen in MDA-MB-231 cells. Inhibiting autophagy in these cells treated with melatonin could serve as a self-defense mechanism, and This might be a good strategy for breast cancer adjuvant treatment.

Neutrophil extracellular traps (NETs) and Th-2 dominant immune responses in chronic granulomatous chromobalstomycosis.

Chromoblastomycosis (CBM), a chronic, granulomatous, suppurative mycosis of the skin and subcutaneous tissue, is caused by several dematiaceous fungi. The formation of granulomas, tissue proliferation, and fibrosis in response to these pathogenic fungi is believed to be intricately linked to host immunity. To understand this complex interaction, we conducted a comprehensive analysis of immune cell infiltrates, neutrophil extracellular traps (NETs) formation, and the fibrosis mechanism in 20 CBM lesion biopsies using immunohistochemical and immunofluorescence staining methods. The results revealed a significant infiltration of mixed inflammatory cells in CBM granulomas, prominently featuring a substantial presence of Th2 cells and M2 macrophages. These cells appeared to contribute to the production of collagen I and III in the late fibrosis mechanism, as well as NETs formation. The abundance of Th2 cytokines may act as a factor promoting the bias of macrophage differentiation toward M2, which hinders efficient fungal clearance while accelerates the proliferation of fibrous tissue. Furthermore, the expression of IL-17 was noted to recruit neutrophils, facilitating subsequent NETs formation within CBM granulomas to impede the spread of sclerotic cells. Understanding of these immune mechanisms holds promise for identifying therapeutic targets for managing chronic granulomatous CBM.

Visualizing Lipophagy as a New Mechanism of the Synthesis of Sex Steroids in Human Ovary and Testis Using Immunofluorescence Staining Method.

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.

Role of vascular endothelial growth factor D in lung adenocarcinoma immunotherapy response.

To identify key genes associated with tumor-associated macrophages (TAMs), tumor immunotherapy, in the prognosis of lung adenocarcinoma (LUAD). The mRNA expression profiles of LUAD samples were obtained from The Cancer Genome Atlas (TCGA) database. The "CIBERSORT" R package was employed to calculate the proportion of innate immune cell infiltration in both tumor and adjacent normal tissues. TAM-associated genes in LUAD were identified to construct a prognostic risk model using weighted gene correlation network analysis (WGCNA), Least Absolute Shrinkage and Selection Operator (LASSO), and multivariate Cox regression analyses (COX). The IMvigor210 cohort was utilized to validate the roles of these genes as predictors of immunotherapy response. Tissue microarrays, immunofluorescence staining, and mRNA level detection methods were used to determine the correlation of risk factors in LUAD tissues. CIBERSORT analysis revealed significant differences in innate immune cells between tumor and adjacent tissues. Seventy-four differential genes linked to these cells were identified from WGCNA. Four hub genes (endothelin receptor type B, vascular endothelial growth factor D (VEGFD), latent transforming growth factor beta binding protein 4 (LTBP4), and fibroblast growth factor receptor 4 (FGFR4)) in the TAM prognostic model were identified as independent prognostic risk factors (P < 0.05). VEGFD expression was identified as a low-risk factor for LUAD prognosis prediction (P < 0.05). Moreover, low-risk patients exhibited higher sensitivity to anti-PD-L1 therapy compared to high-risk patients (P < 0.05). VEGFD levels were negatively correlated with programmed cell death 1 (PD-1) levels (r = -0.363; P < 0.05), suggesting that VEGFD may serve as a predictor for anti-PD-1 treatment. VEGFD is associated with innate immunity in LUAD, it can predict LUAD prognosis, and therefor may be a potential predictor for anti-PD-1 treatment in patients with LUAD.

Pilot testing of a simple alternative tissue processing method for immunofluorescence staining

Background: The image quality of tissues obtained with high-resolution imaging systems depends on several factors including the quality of sections prepared by different tissue processing methods. Fresh frozen sections and formalin-fixed paraffinembedded (FFPE) sections are the two routinely employed methods in histopathological examination having their own merits and demerits. Aims and Objectives: We tested the feasibility of a hybrid tissue processing method that could reduce the processing time without compromising the quality of processed and stained tissues. Materials and Methods: Bovine pancreatic tissue was utilized to standardize the process. The steps included fixation of tissue in formalin, followed by embedding in optimal cutting temperature compound and then cryo-sectioning to obtain 50 μm slices for immunostaining. The prepared thick slices were immunostained with islet markers and assessed by confocal imaging. Results: The images revealed that the staining quality was comparable with FFPE sections, though the tissue processing time was significantly curtailed. The modified Leica microsystems scoring system revealed a nearly 80% overall total score for the processed tissue specimens, indicating acceptable levels of quality of the tissues. We also observed that the staining quality of these formalin-fixed cryo-slices was preserved without tissue deterioration for up to 1 month when stored in formalin, indicating its storage potential for future applications. Conclusion: The proof of concept of the hybrid tissue processing method tested in this study will encourage researchers to extrapolate its use to the human pancreas, other solid organs, and other staining techniques for evaluating its general utility.

Neurotropin Alleviates Bortezomib-Induced Neurotoxicity Via Microglial Neuroinflammation

Objective: To investigate the effect of neurotropin (NTP) on the prevention and amelioration of neurotoxicity induced by bortezomib (BTZ)-based chemotherapy regimens and the possible mechanism of action, and to provide new ideas for the prevention mechanism and treatment of bortezomib-induced peripheral neuropathy (BIPN). Methods: The BIPN model was constructed by injecting bortezomib （ BTZ group ）or bortezomib+saline ( BTZ+NS group)into the tail vein of C57BL/6 mice. After successful modeling, NTP was added to take intraperitoneal injections for 4 weeks, which were defined to be NTP group. In order to assess the severity of BIPN, systemic toxicity, pain behavioral and sensorimotor kinetic changes in mice of different group were observed while transmission electron microscopy examination was used to observe the damage of peripheral nerve ultrastructure and myelin thickness. Immunofluorescence staining method was used to detected the activation of spinal cord microglial and their expression level of inflammatory factors such as IL-1β, IL-6 and TNF-α. ELISA was used to detected the expression of IL-1β, IL-6 and TNF-α in the dorsal horn homogenate of the spinal cord; Western blot to detect the protein expression of pathway-associated proteins JAK/STAT. Results: The Compared with the Control group, the BTZ and BTZ+NS groups showed significant decreases in mechanical, thermal pain thresholds and motor coordination compared with the Control group (P&amp;lt;0.05), and ultrastructural damage to the peripheral nerves, with demyelinating lesions and a significant reduction in the overall thickness of myelin sheaths in myelinated fibers (0.62±0.08 Vs 0.56±0.12, P＜0.01). NTP administration significantly improved pain behavioral changes, reduced ultrastructural damage, reversed demyelinating lesions and significantly increased myelin thickness (0.56±0.06 Vs 0.62±0.08, P＜0.01) in model mice compared with BTZ group. Immunofluorescence staining suggested that microglia in the dorsal horn of the spinal cord of mice in the BTZ group and the BTZ+NS group were significantly activated, which were shown the fluorescence ratio of IBA-1 + was significantly increased (2.55±0.61 Vs 0.55±0.04，2.42±0.89 Vs 0.55±0.04，P＜0.0001), while NTP significantly improved the activation of microglia in the model mice (0.71±0.13 Vs 2.55±0.61, 0.71±0.13 Vs 2.42±0.89, P&amp;lt;0.0001). The co-staining results suggest that the increase in inflammatory factors may originate from microglia. ELISA results that the expression level of IL-6 and TNF-α in NTP administration group significantly decreased compared with the group of BTZ group (IL-6: 37.87±23.2 Vs 111.8±6.0, P＜0.05; TNF-α: 547.9±17.11 Vs 998.8±333.4, P＜0.05) and the BTZ+NS group (IL-6: 37.87±23.2 Vs 88.3±15.58, P&amp;lt;0.05; TNF-α: 547.9±17.11 Vs 768.3±37.45, P&amp;lt;0.05) in the dorsal horn homogenate of the spinal cord. The results of spinal cord proteomics showed that neurotropin could induce the changes of differential proteins, such as PI3K-AKT, MAPK, JAK/STAT, which were related to inflammation. Moreover, these differential proteins can be significantly enriched in the JAK/STAT KEGG Pathway. The KEGG pathway of differential protein enrichment in the spinal cord of differential BIPN mice was detected by proteomics chip after NTP treatment, and the JAK/STAT pathway was significantly down-regulated after NTP treatment. Western blot showed that the protein expression of JAK1/STAT3 decreased after NTP administration. Conclusion: Neurotropin alleviates bortezomib induced neurotoxicity through microglial neuroinflammation, and may reduce the release of glial inflammation by regulating JAK1/STAT3 signaling pathway, thus playing a therapeutic role in BIPN. Repeated experiments are needed for further research. Keywords: Neurotropin, Bortezomib, Peripheral neuropathy, Neuroinflammation

Effect of Shao's five-needle therapy pretreatment on airway inflammatory response in asthmatic rats based on ROS/TXNIP/NLRP3 pathway.

To explore the possible mechanism of Shao's five-needle therapy pretreatment on relieving airway inflammatory response in asthmatic rats. Forty SPF-grade SD rats were randomly divided into a blank group, a model group, an acupuncture group, and a medication group, with 10 rats in each group. Except the blank group, asthma model was established by aerosol inhalation of ovalbumin in the other 3 groups. The rats in the acupuncture group were treated with acupuncture at "Dazhui" (GV 14) and bilateral "Feishu" (BL 13) and "Fengmen" (BL 12), with each session lasting for 20 min. Acupuncture was given before each motivating, once daily for 7 consecutive days. The rats in the medication group were treated with intraperitoneal injection of dexamethasone sodium phosphate solution before each motivating, once daily for 7 days. General situation of the rats was observed in each group; ELISA method was used to detect the levels of inflammatory cytokines interleukin (IL)-1β and IL-18 in serum; immunofluorescence staining method was performed to assess the expression of reactive oxygen species (ROS) in lung tissues; Western blot method was used to measure the protein expression of thioredoxin interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in lung tissues. The rats in the blank group exhibited normal behavior, while those in the model group showed signs of respiratory distress, ear scratching, cheek rubbing, and dysphoria. Compared with the model group, the rats in the acupuncture group and the medication group showed stable respiration and relatively agile responses. Compared with those in the blank group, the serum levels of IL-18 and IL-1β were elevated (P<0.01), the expression intensity of ROS was increased, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were increased (P<0.01) in the model group. Compared with those in the model group, the serum levels of IL-18 and IL-1β were reduced (P<0.01), the expression intensity of ROS was lowered, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were reduced (P<0.01) in the acupuncture group and the medication group. Compared with the medication group, the protein expression of ASC in lung tissue was reduced in the acupuncture group (P<0.05). Pretreatment of Shao's five-needle therapy could alleviate airway inflammatory response in asthmatic rats by reducing ROS levels and decreasing the aggregation and activation of pathway-related proteins in the ROS/TXNIP/NLRP3 pathway, ultimately leading to decreased secretion of IL-1β and IL-18. This mechanism may contribute to the effectiveness of Shao's five-needle therapy in preventing and treating asthma.

A New Target of Electroacupuncture Pretreatment Mediated Sympathetic Nervous to Improve MIRI: Glutamatergic Neurons in Fastigial Nucleus of the Cerebellum

Ischemic heart disease is a fatal cardiovascular disease that irreversibly impairs the function of the heart, followed by reperfusion leading to a further increase in infarct size. Clinically, we call it myocardial ischemia–reperfusion injury (MIRI). A growing number of clinical observations and experimental studies have found electroacupuncture (EA) to be effective in alleviating MIRI. This study attempts to investigate whether glutamatergic neurons in fastigial nucleus (FN) of the cerebellum are involved in EA pretreatment to alleviate MIRI via sympathetic nerves, and the potential mechanisms of EA pretreatment process. A MIRI model was established by ligating the coronary artery of the left anterior descending branch of the heart for 30 minutes, followed by 2 hours of reperfusion. Multichannel physiological recordings, electrocardiogram, cardiac ultrasound, chemical genetics, enzyme-linked immunosorbent assay and immunofluorescence staining methods were combined to demonstrate that EA pretreatment inhibited neuronal firing and c-Fos expression in FN of the cerebellum and reduced cardiac sympathetic firing. Meanwhile, EA pretreatment significantly reduced cardiac ejection fraction (EF), shortening fraction (SF), percentage infarct area, decreased myocardial norepinephrine (NE), creatine kinase isoenzyme MB (CK-MB) concentrations, and improved MIRI-induced myocardial tissue morphology. The results were similar to the inhibition of glutamatergic neurons in FN. However, the activation of glutamatergic neurons in FN diminished the aforementioned effects of EA pretreatment. This study revealed that glutamatergic neurons in FN of the cerebellum is involved in EA pretreatment mediated sympathetic nervous and may be a potential mediator for improving MIRI.

Focal ischemic stroke modifies microglia-derived exosomal miRNAs: potential role of mir-212-5p in neuronal protection and functional recovery

BackgroundIschemic stroke is a severe type of stroke with high disability and mortality rates. In recent years, microglial exosome-derived miRNAs have been shown to be promising candidates for the treatment of ischemic brain injury and exert neuroprotective effects. Mechanisms underlying miRNA dysregulation in ischemic stroke are still being explored. Here, we aimed to verify whether miRNAs derived from exosomes exert effects on functional recovery.MethodsMiR-212-5p agomir was employed to upregulate miR-212-5p expression in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Western blot analysis, qRT–PCR and immunofluorescence staining and other methods were applied to explore the underlying mechanisms of action of miR-212-5p.ResultsThe results of our study found that intervention with miR-212-5p agomir effectively decreased infarct volume and restored motor function in MCAO/R rats. Mechanistically, miR-212-5p agomir significantly reduced the expression of PlexinA2 (PLXNA2). Additionally, the results obtained in vitro were similar to those achieved in vivo.ConclusionIn conclusion, the present study indicated that PLXNA2 may be a target gene of miR-212-5p, and miR-212-5p has great potential as a target for the treatment and diagnosis of ischemic stroke.

A single-center experience of using immunofluorescence staining of blood smears for the diagnosis of hereditary thrombocytopathies

The method of immunofluorescence staining of blood smears is a recently developed approach to the remote diagnosis of various platelet pathologies including MYH9 disorders/MYH9-related disease, biallelic Bernard–Soulier syndrome, Glanzmann thrombasthenia, gray platelet syndrome, and others. We report here the experience of introducing this method at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology (Moscow, Russia), which is the main national pediatric hematology hospital that provides diagnosis and therapy to children with blood disorders throughout the country. Our study aimed to transfer this relatively labor-intensive and skill-sensitive method and introduce it into routine laboratory practice, and to perform its validation. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology.

Giardiasis in the Southwest Region of the United States: Causes and Preventive Measures

Giardia is a protozoan parasite of the small intestine, and a leading cause of diarrheal diseases worldwide in a variety of animals, as well as humans. Despite the decrease, Giardiasis remains the most commonly reported intestinal parasitic infection principally in the Southwestern region of the United States. The parasite is typically transmitted by fecal-oral route and may be transmitted by direct intake or indirectly from contaminated food or water. Symptomatic Giardiasis is often treated to reduce the duration of symptoms, to prevent future complications, and to minimize transmission of the parasite to other hosts. The clinical image of Giardiasis is mixed, with high variability in severity of clinical disease which can become chronic or be followed by post-infectious sequelae. At present, treatment options include nitroimidazoles derivatives; especially metronidazole, which has been the mainstay of treatment for decades and is still widely used. The Navajo Nation located in the Southwestern region of the United States has suffered from the scarcity of water expediting Navajo populations to use water resources such as underground water, river streams, lakes and surface water as their main source of accessibility. The lack of adequate domestic and municipal water has led several Navajo families to become ill with waterborne infectious diseases like Giardiasis. More recently, molecular methods have become amateur in diagnosis for testing patient samples as well as water samples for Giardiasis. Through direct immunofluorescent staining and other methods that detect intact organisms have improved the detection of Giardia trophozoites or cysts. An understanding of clinical and molecular variability of organisms is important for effective treatment and the response of remedies that are commonly used. The molecular classification of bacteria and viruses isolated from Indigenous patients might have their own specification that can be correlated with isolates from water samples collected from the same localities. This correlation could help us in source tracking and finding solutions for the communities who are already at risk due to the lack of infrastructure and resources. In this review of literature, the causes and preventive measures of Giardiasis are covered to potentially help understand the importance behind this parasitic disease, more specifically on the Navajo Nation of the Southwest of the US.

Collection, Fixation, and Antibody Staining of Drosophila Embryos.

The Drosophila embryonic central nervous system has been used for decades as a model for understanding the genetic regulation of axon guidance and other aspects of neural development. Foundational studies using antibody staining to examine the embryonic ventral nerve cord in wild-type and mutant animals led to the discovery of evolutionarily conserved genes that regulate fundamental aspects of axon guidance, including midline crossing of axons. The development of the regular, segmentally repeating structure of axon pathways in the ventral nerve cord can illustrate basic principles of axon guidance to beginning students and can also be used by expert researchers to characterize new mutants, detect genetic interactions between known genes, and precisely quantify variations in gene function in engineered mutant lines. Here, we describe a protocol for collecting and fixing Drosophila embryos and visualizing axon pathways in the embryonic ventral nerve cord using immunofluorescence or immunohistochemical staining methods. As embryogenesis in Drosophila takes ∼24 h to complete, a 1-d collection yields embryos representing all stages of development from newly fertilized through ready-to-hatch larvae, allowing investigation of multiple developmental events within a single batch of collected embryos. The methods described in this protocol should be accessible to introductory laboratory courses as well as seasoned investigators in established research laboratories.