Immunofluorescence Research Articles

Protective Effect of Aloe-emodin on Cognitive Function in Copper-loaded Rats Based on The Inhibition of Hippocampal Neuron Ferroptosis.

Aloe-emodin (AE), a monomer derived from traditional Chinese medicine, has demonstrated remarkable efficacy in the clinical management of cognitive disorders. Ferroptosis (FPT), a specialized form of programmed cell death, plays a critical role in the pathological progression of various cognitive diseases. This study explored the therapeutic potential of AE in a rat model of Wilson's disease cognitive impairments (WDCI) and examined whether these effects are mediated through the silencing information regulator 1 (SIRT1)-regulated FPT signaling pathway. Employing techniques, such as the Morris water maze (MWM), Hematoxylin & eosin (H&E) staining, Transmission electron microscopy (TEM), Immunofluorescence (IF), assessments of oxidative stress markers, and measurements of FPT-related protein levels, we evaluated the extent of SIRT1-mediated FPT and the therapeutic efficacy of AE. The findings from the WD copper-loaded rat model experiments revealed that MWM, H&E, TEM, and IF outcomes indicated AE's potential to promote the restoration of learning and memory functions, ameliorate hippocampal neuronal morphological damage, and preserve cell membrane integrity. Results from western blot (WB) and ELISA analyses demonstrated that AE markedly upregulated the expression of SIRT1, nuclear factor erythroid-2-related factor 2 (Nrf2), solute carrier family 7 member 11 (SCL7A11), and glutathione peroxidase 4 (GPX4) proteins while simultaneously reversing the expression of oxidative stress markers such as malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD), and reactive oxygen species (ROS). Consequently, we posit that AE may attenuate WD copper-loaded rat model hippocampal neuronal FPT by activating the SIRT1-mediated signaling pathway. These findings suggested that AE mitigates WD copper-loaded rat model hippocampal neuronal damage through the activation of SIRT1-mediated FPT, thereby presenting a valuable candidate Chinese herbal monomer for the clinical treatment of WDCI.

Promotion of bone osteogenic and regeneration by resveratrol-derived carbon dots in inflammatory environments via a one-step hydrothermal method

In the context of bone fractures, local inflammation complicates the healing process, adversely affecting the recovery of bone defects. This inflammatory response impedes the regenerative potential of stem cells and induces the differentiation of macrophages into the M1 type, thereby inhibiting bone regeneration. To address this issue, we synthesized carbon dots (CDs) using resveratrol and citric acid. These CDs demonstrated excellent water solubility and retained the anti-inflammatory properties of resveratrol. In an inflammatory environment co-cultured with resveratrol carbon dots (RES-CDs), we observed a reduction in inflammatory factors such as iNOS and an increase in anti-inflammatory factors like CD206. Alkaline phosphatase (ALP) activity, Alizarin Red staining, and immunofluorescence assays indicated that MC3T3 cells produced more calcium nodules after co-culture with RES-CDs, along with enhanced expression of bone formation factors BMP-2 and ALP. Additionally, real-time polymerase chain reaction (qRT-PCR) and immunofluorescence analysis demonstrated that RES-CDs could induce bone tissue proliferation and differentiation by upregulating ALP, BMP-2, OCN, and OPN. In a rat cranial defect model, RES-CDs promoted tissue healing and bone proliferation at weeks 1 and 4 post-surgery. These findings underscore the potential of resveratrol carbon dots in promoting bone tissue healing while mitigating inflammation, thereby offering a promising approach for nanocomposite materials in tissue repair.

Gender-different effect of Src family kinases antagonism on photophobia and trigeminal ganglion activity

BackgroundSrc family kinases (SFKs) contribute to migraine pathogenesis, yet its role in regulating photophobia behaviour, one of the most common forms of migraine, remains unknown. Here, we addressed whether SFKs antagonism alleviates photophobia behavior and explored the underlying mechanism involving hypothalamus and trigeminal ganglion activity, as measured by the alteration of neuropeptide levels and transcriptome respectively.MethodsA rapid-onset and injury-free mouse model of photophobia was developed following intranasal injection of the TRPA1 activator, umbellulone. The role of SFKs antagonism on light aversion was assessed by the total time the mouse stays in the light and transition times between the dark and light compartments. To gain insight to the preventive mechanism of SFKs antagonism, hypothalamic neuropeptides levels were assessed using enzyme linked immunofluorescent assay and trigeminal ganglion activity were assessed using RNA-sequencing and qPCR analysis.ResultsSFKs antagonism by a clinically relevant SFKs inhibitor saracatinib reduced the total time in light and transition times in male mice, but not in females, suggesting SFKs play a crucial role in photophobia progressing and exhibit a male-only effect. SFKs antagonism had no effect on hypothalamic calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide levels of all mice investigated, suggesting the gender-different effect of saracatinib on light aversion appears to be independent of these hypothalamic neuropeptide levels. In trigeminal ganglion of male mice, photophobia is associated with profound alteration of differentially expressed genes, part of which were reversed by SFKs antagonism. Subsequent qPCR analysis showed SFKs antagonism displayed gender-different modulation of expression in some candidate genes, particularly noteworthy those encoding ion channels (trpm3, Scn8a), ATPase signaling (crebbp, Atp5α1) and kinase receptors (Zmynd8, Akt1).ConclusionsIn conclusion, our data revealed that SFKs antagonism reduced photophobia processing in male mice and exhibited gender-different modulation of trigeminal ganglion activity, primarily manifesting as alterations in the transcriptome profile. These findings underscore the potential of SFKs antagonism for allieving photophobia in males, highlighting its value in the emerging field of precision medicine.

Melanin concentrating hormone regulates the JNK/ERK signaling pathway to alleviate influenza A virus infection-induced neuroinflammation

Melanin concentrating hormone (MCH) controls many brain functions, such as sleep/wake cycle and memory, and modulates the inflammation response. Previous studies have shown that influenza A virus (IAV) infection-induced neuroinflammation leads to central nervous damage. This study investigated the potential effects of MCH against neuroinflammation induced by IAV infection and its mechanism. MCH (1 and 2 mg/ml) was administrated for 5 consecutive days before IAV infection. Pentobarbital-induced sleep tests, an open-field test, and a Morris water maze were performed to measure sleep quality, spatial learning and memory ability. Neuronal loss and microglial activation were observed with Nissl staining and immunofluorescence assay. The levels of inflammatory cytokines and the expression of the JNK/ERK signaling pathway were examined by ELISA and western blot. IAV infection led to poor sleep quality, impaired the ability of spatial learning and memory, caused neuronal loss and microglial activation in mice’s hippocampus and cortex. Meanwhile the level of inflammatory cytokines increased, and the JNK/ERK signaling pathway was activated after IAV infection. MCH administration significantly alleviated IAV-induced neuroinflammation, cognitive impairment, and sleep disorder, decreased the levels of inflammatory cytokines, and inhibited neuronal loss and microglial activation in the hippocampus and cortex by regulating the JNK/ERK signaling pathway. Therefore, MCH alleviated the neuroinflammation, spatial learning and memory impairment, and sleep disorder in IAV-infected mice by regulating the JNK/ERK signaling pathway.

Long non-coding RNA AC010457.1 promotes the growth and EMT of gastric cancer via the PI3K/AKT axis

Gastric cancer (GC) is a malignancy with a relatively high mortality rate. This study aimed to ascertain the prognostic significance of long non-coding RNA (lncRNA) AC010457.1 in GC and elucidate its role in disease progression. The Cancer Genome Atlas (TCGA) database was used to screen the prognosis-associated differentially expressed lncRNAs in GC patients. Kaplan-Meier curves, univariate and multivariate Cox regression analyses were applied to assess the prognostic significance of AC010457.1. In vitro and in vivo functional assays were performed to evaluate the effects of AC010457.1 on cellular proliferation and metastasis. Mechanistic investigations, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Western blotting (WB), Immunofluorescence (IF), and Immunohistochemistry (IHC), were used to explore the signaling pathways activated by AC010457.1. We demonstrated that AC010457.1 was abnormally upregulated in GC tissues, and that this aberrant upregulation was associated with a poor prognosis for GC patients. The functional experiments proved that the downregulated of AC010457.1 hindered GC cell proliferation, migration, and invasive potential. Furthermore, KEGG analysis revealed a significant association between AC010457.1 and the PI3K/AKT signaling pathway, which was further corroborated by WB analysis. Rescue experiments provided additional confirmation that AC010457.1 regulated PI3K/AKT promote GC proliferation, migration, and epithelial-mesenchymal transition (EMT). Collectively, our findings suggest that AC010457.1 overexpression serves as a distinct prognostic risk factor in GC and may represent a promising therapeutic target for the treatment of this malignancy.

Integrated bioinformatics analysis and experimental validation on malignant progression and immune cell infiltration of LTBP2 in gliomas

BackgroundGliomas are the highly aggressive brain tumor and also the most devastating human tumors. The latent TGF binding proteins (LTBP) had been found to be involved in malignant biological process and could be used as potent biomarkers in several solid tumors. While the role of LTBP family in human glioma remain to be elucidated.MethodsNormalized gene expression and corresponding clinical data of 2407 gliomas samples in public datasets were downloaded from Gliovis. Kaplan–Meier methods and Cox regression analysis was used for survival analyses.Western blot (WB) and Immunohistochemical (IHC) testing were employed to test LTBPs protein level in 154 gliomas samples. Correlation between LTBP2 expression and immune infiltration was evaluated by immunofluorescence (IF) and IHC in glioma tissues. CCK8 and flow cytometric analysis were used to detect the effect of LTBP2 on glioma cells. Orthotopic glioma- mouse models were utilized to evaluate effects in vivo.ResultsLTBP2 mRNA level was dramatically higher in glioma samples compared with non-tumor brain tissues in XENA-TCGA_GTEx, Gill and Gravendeel datasets (all P < 0.01), and its expression positively correlated with glioma WHO grade, IDH1/2 wildtype and mesenchymal subtypes. These results were confirmed by In-house cohort which was detected by WB and IHC. We found that gliomas patients with high LTBP2 level had shorter OS than those with low LTBP2 level. LTBP2 expression significantly associated with glioma immune score (Spearman r = 0.68, P < 0.01)) and strongly correlated with infiltration degreee of macrophages both in lower grade gliomas (LGG) and GBM. Knocking down LTBP2 obviously reduced proliferation and enhanced sensitivity to temozolomide in U87 and U251 cells. Nude mice with lower expression of LTBP2 had slower tumor growth, and accompanied by less tumor-associated macrophages (TAMs) infiltration detected by IHC staining in vivo. Finally, low LTBP2 expression glioma patients who received chemotherapy survived longer than patients with high LTBP2 expression.ConclusionLTBP2 could be used as a prognostic marker, and high LTBP2 expression related to abundant TAMs infiltration and with a worse response to chemotherapy.

Regulation of microglia inflammation and oligodendrocyte demyelination by Engeletin via the TLR4/RRP9/NF-κB pathway after spinal cord injury

Microglia polarization is crucial for neuroinflammatory response after spinal cord injury (SCI). Small molecule compounds and hub genes play an important role in regulating microglia polarization, reducing neuroinflammatory response and oligodendrocyte demyelination after SCI. In this study, suitable data sets were used to screen hub genes, and Western blot and Immunofluorescence (IF) experiments were used to confirm the expressions of proteins related to SDAD1, RRP9 and NF-κB pathways under LPS/SCI conditions. Engeletin (ENG) reduced microglia polarization and inflammation in vivo and in vitro via the SDAD1, RRP9 or NF-κB signaling pathways. In addition, ENG binds to the membrane receptor Toll-like receptor 4 (TLR4) through small molecule-protein docking. COIP experiment and protein-protein docking revealed protein-protein interaction (PPI) between RRP9 and SDAD1. By gene knock-down (KD) / overexpression (OE) and Western blot experiments, RRP9 and SDAD1 can regulate inflammatory response through NF-κB signaling and ribosome biogenesis pathway. Western blot analysis showed that CU increased the expression of SDAD1, RRP9 and NF-κB pathway related proteins through TLR1/2, while C34 decreased the expression of SDAD1 and RRP9 proteins through TLR4. These results suggest that ENG can reduce inflammation through TLR4/RRP9(SDAD1)/NF-κB signaling pathway. In addition, we demonstrated that oligodendrocyte apoptosis and demyelination could be influenced by the regulation of microglia and tissue inflammation. In conclusion, this study found the gene Rrp9/Sdad1 and the small molecule compound ENG, which control the inflammatory response of microglia, and further explored the related mechanism of oligodendrocyte demyelination, which has important theoretical significance.

High-throughput screening for small-molecule stabilizers of misfolded glucocerebrosidase in Gaucher disease and Parkinson’s disease

Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease, PD); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small proluminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and noninhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: The fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 directly visualized GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of small molecules targeting GCase, ultimately leading to a viable therapeutic for GD and PD.

Phylogenetic Analysis of Porcine Epidemic Diarrhea Virus (PEDV) during 2020-2022 and Isolation of a Variant Recombinant PEDV Strain.

Porcine epidemic diarrhea (PED) is an acute, highly contagious, and infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV can affect pigs of all ages, with 50~100% mortality in neonatal piglets and substantial economic losses in the swine industry. In the present study, 347 fecal and intestinal samples were collected from seven regions in China during 2020-2022. A comprehensive molecular investigation of the spike (S) gene of PEDV strains was carried out, which included phylogenetic analysis of the obtained PEDV sequences. Epidemiological surveillance data indicate that the GIIc subgroup strains are widely distributed among pigs. A PEDV strain was successfully isolated from positive small intestine samples and identified through RT-PCR detection using specific N gene primers of PEDV, indirect immunofluorescence assay (IFA), TEM analysis, genome sequencing, and full-length S gene analysis, named PEDV/SC/2022. RDP and SimPlot analysis showed that the isolate originated from the recombination of PEDV/AH2012 and PEDV/AJ1102. In conclusion, our findings contribute to the current understanding of PEDV epidemiology and provide valuable information for the control of PED outbreaks in China.

Induced tumor associated autoantibodies (iTAAs) by hapten plus drugs for targeting oncogenic nuclear antigens at sentinel lymph node of pancreatic cancer

Background: the abscopal is a hypothesis of effect on non-irradiated tumors after localized radiation therapy which associated with the products of tumor-associated gene as autoantibodies (aTAAs) in reaction to the tumor-associated antigens (TAAs). When TAAs interact with hapten within tumor after hapten plus chemotherapy drugs intratumoral injection to pancreatic cancer like tumor lysates vaccine, TAAs-hapten stimulates the immune system as a neu antigen and produces autologous tumor antibodies, it is called induced tumor-associated autoantibodies (iTAAs) which is lightly difference to aTAAs since epitope of antigen linked with hapten. Method: immunofluorescence (IF) was applied for detect the binding of the iTAAs in tumor cells at sentinel lymph node. Results: aTAAS naturally could not target and bind the TAAs in tumor since due to immune tolerance. The iTAAs can target and bind the TAAS, also the iTAAs targeted in the tumors of sentinel lymph node with complements C help, it was found Cmyc (P = 0.0017 at one week; P = 0.0001 at two weeks), p53 (P = 0.0373; 29.13 ± 6.91, and P = 0.0254), and Zeta (P = 0.1513, P = 0.0044) increased significantly in the tumor cells or perinuclear tumor cells in lymph node at one and two weeks after treatment. Conversely, IMP1 (P = 0.6154; P = 0.0138), Koc (P = 0.5684, P = 0.0103), Survivn (P = 0.1020; P = 0.0147) and Rala (P = 0.3226; P = 0.0249) increased significantly in the cell or perinuclear of tumor in lymph node at two weeks later following. Conclusions: it indicated all of iTAAs plays a function in the binding of original cellular tumor genes at sentinel lymph node while the aTAAs could not bind the cellular tumor genes at lymph node due to immune tolerance. We could make a hypothesis: TAAs inside the cancer cells’ nuclei can be targeted by the iTAAs which produced by hapten enhanced intratumoral chemotherapy. The iTAA may play a distinctive role in controlling tumor cell growth and resulting in an abscopal effect, which is one of hapten associated with TAA induced immune response.

Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

BackgroundIntervertebral disc degeneration(IVDD) is the primary etiology of low back pain and radicular pain. Recent studies have found that chemokines play a role in IVDD, but the underlying mechanism is largely unclear. MethodsBioinformatics analysis was employed to screen CXCL8 as the target gene. The expression levels of CXCL8 and CXCR2 were quantified using RT-qPCR, western blot(WB), immunohistochemistry(IHC), and enzyme-linked immuno-sorbent assay(ELISA). In the IVDD mouse model, X-ray images, Safranin O-fast green staining(SO-FG), IHC, and WB were conducted to assess the therapeutic effects of CXCL8 on IVDD. Reactive oxygen species (ROS) production, apoptosis of nucleus pulposus cells (NPCs), and the involvement of the NF-κB pathway were evaluated through WB, flow cytometry, immunofluorescence(IF), and Tunnel assay. ResultsIn our study, we observed that CXCL8 emerged as one of the chemokines that were up-regulated in IVDD. The mitigation of extracellular matrix degradation (ECM) and the severity of IVDD were significantly achieved by neutralizing CXCL8 or its receptor CXCR2(SB225002, CXCR2 antagonist). The release of CXCL8 from infiltrated macrophages within intervertebral discs (IVDs) was predominantly observed upon stimulation. CXCL8 exerted its effects on NPCs by inducing apoptosis and ECM degradation through the activation of CXCR2. Specifically, the formation of the CXCL8/CXCR2 complex triggered the NF-κB signaling pathway, resulting in an abnormal increase in intracellular ROS levels and ultimately contributing to the development of IVDD. ConclusionOur findings suggest that macrophage-derived CXCL8 and subsequent CXCR2 signaling play crucial roles in mediating inflammation, oxidative stress, and apoptosis in IVDD. Targeting the CXCL8/CXCR2 axis may offer promising therapeutic strategies to ameliorate IVDD. The translational potential of this articleThis study indicates that CXCL8 can effectively exacerbate the excessive apoptosis and oxidative stress of NPCs through activating the NF-κB pathway. This study may provide new potential targets for preventing and reversing IVDD.

Novel anti-CEA affibody for rapid tumor-targeting and molecular imaging diagnosis in mice bearing gastrointestinal cancer cell lines.

Gastrointestinal cancer is a common malignant tumor with a high incidence worldwide. Despite continuous improvements in diagnosis and treatment strategies, the overall prognosis of gastrointestinal tumors remains poor. Carcinoembryonic antigen (CEA) is highly expressed in various types of cancers, especially in gastrointestinal cancers, making it a potential target for therapeutic intervention. Therefore, the expression of CEA can be used as an indication of the existence of tumors, chosen as a target for molecular imaging diagnosis, and effectively utilized in the targeted therapy of gastrointestinal cancers. In this study, we report the selection and characterization of affibody molecules (ZCEA539, ZCEA546, and ZCEA919) specific to the CEA protein. Their ability to bind to recombinant and native CEA protein has been confirmed by surface plasmon resonance (SPR), immunofluorescence, and immunohistochemistry assays. Furthermore, Dylight755-labeled ZCEA affibody showed accumulation within the tumor site 1 h post injection and was continuously enhanced for 4 h. The Dylight755-labeled ZCEA affibody exhibited high tumor-targeting specificity in CEA+ xenograft-bearing mice and possesses promising characteristics for tumor-targeting imaging. Overall, our results suggest the potential use of ZCEA affibodies as fluorescent molecular imaging probes for detecting CEA expression in gastrointestinal cancer.

Thermosensitive hyaluronic acid-manganese-capsaicin complex nanogel improving NKG2D/CAR-T melanoma treatment through adjusting tumor microenvironment

Intratumorally injection of CAR-T cells to treat melanoma can reduce the incidence of systemic side effects and ensure an adequate concentration of CAR-T cells. However, few targeting ligands and hostile tumor microenvironment (TME) inhibit the CAR-T cells' survival and infiltration. An in situ hyaluronic acid‑manganese-capsaicin complex nanogel (HA-Mn-CAP Gel) was designed by forming complex nanogel based on the main skeleton material of hyaluronic acid (HA). It targeted CD44 and TRPV1 receptors through HA and CAP, concentrated and released Mn at melanoma, subsequently relieving the hypoxia in the tumor and increasing the expression of CAR-T cells' specific ligands. Cell experiments showed that HA-Mn-CAP Gel significantly enhanced the expression of representative NKG2D ligands (MICA/B and ULBP2/5/6) >2.7 times on A375 melanoma cells. Sequential administration of HA-Mn-CAP Gel and NKG2D/CAR-T increased the tumor inhibition rate about 1.5 times that of the NKG2D/CAR-T group in melanoma-bearing NSG mice. The results of immunohistochemistry and immunofluorescence assays showed that the combined HA-Mn-CAP Gel and NKG2D/CAR-T significantly increased the proliferation and infiltration of T cells, especially for CD8+ cells. Therefore, the new promising strategy of increasing the target ligand expression and adjusting TME before administering CAR-T cells is suitable for treating solid tumors.

Neutrophil chemotaxis score and chemotaxis-related genes have the potential for clinical application to prognosticate the survival of patients with tumours

As frontline cells, the precise recruitment of neutrophils is crucial for resolving inflammation and maintaining the homeostasis of the organism. Increasing evidence suggests the pivotal role of neutrophil chemotaxis in cancer progression and metastasis. Here, we collected clinical data and peripheral blood samples from patients with tumours to examine the alterations in the neutrophil quantity and chemotactic function using the Cell Chemotaxis Analysis Platform (CCAP). Transcriptome sequencing data of pan-cancer were obtained from The Cancer Genome Atlas (TCGA). Using the least absolute shrinkage and selection operator (LASSO) Cox regression model, we selected a total of 29 genes from 155 neutrophil- and chemotaxis-related genes to construct the ChemoScore model. Meanwhile, nomogram-based comprehensive model was established for clinical application. Furthermore, immunofluorescence (IF) staining was employed to assess the relationship between the neutrophils infiltrating and the survival outcomes of tumours. In this observational study, the chemotactic function of neutrophils was notably diminished in patients. The establishment and validation of ChemoScore suggested neutrophil chemotaxis to be a risk factor in most tumours, whereby higher scores were associated with poorer survival outcomes and were correlated with various immune cells and malignant biological processes. Moreover, IF staining of tumour tissue substantiated the adverse correlation between neutrophil infiltration and the survival of patients with lung adenocarcinoma (P = 0.0002) and colon adenocarcinoma (P = 0.0472). Taken together, patients with tumours demonstrated a decrease in chemotactic function. ChemoScore potentially prognosticates the survival of patients with tumours. Neutrophil chemotaxis provides novel directions and theoretical foundations for anti-tumour treatment.

A complex remodeling of cellular homeostasis distinguishes RSV/SARS-CoV-2 co-infected A549-hACE2 expressing cell lines.

Concurrent infections with two or more pathogens with analogous tropism, such as RSV and SARS-CoV-2, may antagonize or facilitate each other, modulating disease outcome. Clinically, discrepancies in the severity of symptoms have been reported in children with RSV/SARS-CoV-2 co-infection. Herein, we propose an in vitro co-infection model to assess how RSV/SARS-CoV-2 co-infection alters cellular homeostasis. To this end, A549-hACE2 expressing cells were either infected with RSV or SARS-CoV-2 alone or co-infected with both viruses. Viral replication was assessed at 72 hours post infection by droplet digital PCR, immunofluorescence, and transmission electron microscopy. Anti-viral/receptor/autophagy gene expression was evaluated by RT-qPCR and confirmed by secretome analyses and intracellular protein production. RSV/SARS-CoV-2 co-infection in A549-hACE2 cells was characterized by: 1) an increase in the replication rate of RSV compared to single infection; 2) an increase in one of the RSV host receptors, ICAM1; 3) an upregulation in the expression/secretion of pro-inflammatory genes; 4) a rise in the number and length of cellular conduits; and 5) augmented autophagosomes formation and/or alteration of the autophagy pathway. These findings suggest that RSV/SARS-CoV-2 co-infection model displays a unique and specific viral and molecular fingerprint and shed light on the viral dynamics during viral infection pathogenesis. This in vitro co-infection model may represent a potential attractive cost-effective approach to mimic both viral dynamics and host cellular responses, providing in future readily measurable targets predictive of co-infection progression.

Carbon monoxide-releasing molecule-3 ameliorates traumatic brain injury-induced cardiac dysfunctions via inhibition of pyroptosis and apoptosis.

Traumatic brain injury (TBI) frequently results in cardiac dysfunction and impacts the quality of survivors' life. It has been reported that carbon monoxide-releasing molecule-3 (CORM-3) administration immediately after hemorrhagic shock and resuscitation (HSR) ameliorated the HSR‑induced cardiac dysfunctions. The purpose of this study was to determine whether the application of CORM-3 on TBI exerted therapeutic effects against TBI-induced cardiac dysfunctions. Rats were randomly divided into four groups (n = 12) including Sham, TBI, TBI/CORM-3 and TBI/inactive CORM-3 (iCORM-3) groups. TBI was established by a weight-drop model. The rats in the TBI/CORM-3 group and TBI/iCORM-3 group were intravenously injected with CORM-3 and iCORM-3 (4mg/kg) following TBI, respectively. The time of death in the rats that did not survive within 24h was recorded. 24h post-trauma, the cardiac function, pathological change, serum troponin T and creatine kinase-MB (CK-MB) levels, pyroptosis, apoptosis and expressions of TUNEL staining, Gasdermin D (GSDMD), IL-1β, IL-18, ratio Bax/Bcl-2 were assessed by echocardiography, hematoxylin-eosin staining, chemiluminescence, immunofluorescence, and western blot assays, respectively. TBI-treated rats exhibited dramatically decreased ejection fraction and aggravated myocardial injury, increased mortality rate, elevated levels of serum troponin T and CK-MB, promoted cardiac pyroptosis and apoptosis, and upregulated expressions of cleaved caspase-3, GSDMD N-terminal fragments, IL-1β, IL-18, and ratio of Bax/Bcl-2, whereas CORM-3 partially reversed these changes. CORM-3 ameliorated TBI-induced cardiac injury and dysfunction. This mechanism may be responsible for the inhibition of pyroptosis and apoptosis in cardiomyocyte.

Performance Assessment of the New EUROLINE Neurologic Syndrome 15 Ag (IgG) for the Determination of Autoantibodies Associated with Neurological Disorders.

Here, we assess the performance of the new EUROLINE Neurologic Syndrome 15 Ag (IgG) which expands the EUROLINE Paraneoplastic Neurologic Syndrome 12 Ag by adding CDR2L (together with CDR2 targeted by anti-Yo), AK5, and Neurochondrin (NCDN). Many paraneoplastic as well as non-paraneoplastic autoantibodies (AAbs) have been described in neurological disorders in the last decade. By integrating the associated antigens into existing assays, the diagnostic work-up of patients is being improved and diagnostic gaps reduced. Sensitivity of each AAb was analyzed using a total of 194 clinically and diagnostically pre-characterized samples (Table 1). Specificity of each AAb was investigated using a minimum of 100 sera from healthy blood donors. Using the EUROLINE Neurologic Syndrome 15 Ag, autoantibody positivity was confirmed in 89-100% of samples. In particular, all samples for which clinical and tissue-based indirect immunofluorescence assay pre-characterization indicated anti-Yo positivity were anti-CDR2 and -CDR2L double positive. Anti-AK5 was determined in serum and cerebrospinal fluid (CSF) with a sensitivity of 90 and 100%, respectively, and anti-NCDN with a sensitivity of 100%. The individual specificities were ≥99%. This kit is a tool for the qualitative in vitro determination of AAbs against a large panel of 15 different neuronal autoantigens to support the diagnosis of neurologic syndromes. The parallel detection of anti-CDR2 and anti-CDR2L (both anti-Yo) increases the diagnostic significance, as double positivity is strongly related to paraneoplastic cerebellar degeneration.[Table: see text].

Effects of tenofovir disoproxil fumarate on intrahepatic viral burden and liver immune microenvironment in patients with chronic hepatitis B

BackgroundThe impact of nucleos(t)ide analogues on intrahepatic viral burden and immune microenvironment in patients with chronic hepatitis B (CHB) is not clear.ObjectiveWe aimed to characterise the effects of tenofovir disoproxil...

Whole-Tumor Clearing and Imaging of Intratumor Microbiota in Three Dimensions with miCDaL Strategy.

Acquiring detailed spatial information about intratumor microbiota in situ is challenging, which leaves 3D distributions of microbiota within entire tumors largely unexplored. Here, a modified iDISCO-CUBIC tissue clearing and D-amino acid microbiome labeling-based (miCDaL) strategy are proposed, that integrates microbiota in situ labeling, tissue clearing, and whole-mount tissue imaging to enable 3D visualization of indigenous intratumor microbiota. Leveraging whole-mount spatial resolution and centimeter-scale imaging depth, the 3D biogeography of microbiota is successfully charted across various tumors at different developmental stages, providing quantitative spatial insights in relation to host tumors. By incorporating an immunostaining protocol, 3D imaging of the immunologic microenvironment is achieved in both murine and human mammary tumors that is previously assumed to be bacteria-free. Notably, immune infiltrates, including T cells and NK cells, and tertiary lymphoid structures are conspicuously absent in bacteria-colonized regions. This 3D imaging strategy for mapping Indigenous intratumor microbiota offers valuable insights into host-microbiota interactions.

Occurrence of Encephalitozoon cuniculi and Encephalitozoon hellem in European Wild Rabbits (Oryctolagus cuniculus) in Southern Germany (Bavaria)

Encephalitozoon cuniculi and Encephalitozoon hellem are fungus-related, obligate intracellular pathogens belonging to the microsporidia. Both microorganisms occur in mammals, birds and even humans, thus revealing a zoonotic potential. Knowledge of their relevance in wild rabbits is very limited so far. Thus, the aim of the present study was to investigate the occurrence of E. cuniculi and E. hellem in wild rabbit populations in southern Germany (Bavaria). Therefore, blood and organ samples (brain/kidney) of 158 wild rabbits were investigated by immunofluorescence and PCR-based assays. Antibodies to E. cuniculi were detected in 24 of the 158 (15.2%) wild rabbits, while DNA of E. cuniculi was found in 7 (4.4%) and DNA of E. hellem was found in 3 (1.9%). Sequencing identified E. cuniculi genotype 1. This study provides the first E. cuniculi genotype determination in free-living wild rabbits worldwide and the first evidence of E. hellem in rabbits worldwide. Wild rabbits should, therefore, be regarded as a reservoir for both pathogens and, on the basis of molecular evidence from kidney tissue and presumed urine excretion, also as a source of infection for E. cuniculi for animals and humans.