Immunohistochemical Experiments Research Articles

Interaction of Polyamine-modified Marine Peptide Aculeine A with Cell Membranes: Disruption or Entry

Aculeine A (ACU-A, 1), a marine sponge-derived peptide toxin with a novel posttranslational modification with long-chain polyamine (LCPA), exerts potent hemolytic and cytotoxic activity against mammalian erythrocytes and cultured cells, respectively. Here we found that toxic actions of 1 were attenuated by negatively charged phospholipids and glycosaminoglycans (GAGs) suggesting that 1 interacts primarily with cell surfaces through ionic interactions. In HeLa cells, 1 induced cytotoxicity with characteristic membrane blebbing at 37 °C, while the treatment at 4 °C did not show such adverse phenotype. The immunohistochemical and mass spectral experiments together showed that 1 penetrated cell membrane and localized in the cytosol efficiently at 37 °C but not at 4 °C, suggesting that the intracellular translocation of 1 requires some energy-dependent cellular process. These results together suggested that 1 could act either as a membrane disrupting or a cell penetrating agent depending on cell types.

Analysis of ferroptosis-associated genes in Crohn's disease based on bioinformatics.

Ferroptosis, a novel mode of apoptosis has recently been shown to be associated with fibrosis, tumor, cardiovascular, and other diseases. In this study, using bioinformatic analysis, we identified ferroptosis genes associated with Crohn's disease (CD) and performed biological function analysis, identified potential drug targets, and provided new directions for the future treatment of CD. Differential expression analysis was performed using the GSE186582 dataset from the Gene Expression Omnibus (GEO) database. Ferroptosis-associated genes were downloaded from the FerrDB database, and overlapping genes associated with CD and ferroptosis were extracted. Then, we performed functional enrichment analysis, constructed a protein-protein interaction network (PPI), identified the correlation between hub genes and immune infiltration, performed external validation using a second and third dataset (GSE102133, GSE95095), and identified potential therapeutic agents. Finally, we validated the protein expression levels of the identified hub genes by immunohistochemical staining in the colon tissues from CD and healthy participants. A total of 28 ferroptosis-associated genes associated with CD were identified in our analysis, which included 22 up-regulated and 6 down-regulated genes. Gene Ontology (GO) analysis showed that these genes are essential for the apical plasma membrane and amide transport, and Metascape analysis showed that these genes mainly act on IL-4 and IL-13 signaling pathways. Five hub genes, PTGS2, IL6, IL1B, NOS2, and IDO1, were identified by a protein interaction network, and external validation of these hub genes showed statistically significant differences in expression between the CD patients and normal participants (p < 0.05), and all AUC values were greater than 0.8. Further, we predicted the top 10 drugs used to treat CD. Immune infiltration results suggest that Hub gene is related to T cells, macrophages, dendritic cells, and other immune cells. Finally, the results of immunohistochemical experiments showed that the protein expression of the hub gene was higher in CD colon tissue than in normal subjects (p < 0.05). Bioinformatics analysis showed that ferroptosis is closely related to the development of CD, and the prediction of potential drugs provides new targets for the treatment of CD. Moreover, five hub genes identified are potentially new and effective markers for the diagnosis of CD.

Comprehensive analysis of alfa defensin expression and prognosis in human colorectal cancer.

Colorectal cancer (CRC) is a serious threat to human health. Screening new biomarkers can provide basis for improving the prognosis and individualized treatment of CRC. Although some members of the defensin family were found increased in pancreatic cancer and CRC, their exact function and clinical significance remain unclear. In this study, the expression, correlation, mutation, and functional enrichment of several defensin family members in pancreatic cancer and CRC were analyzed using tumor public databases and verified in several patients. Results showed no significant correlation between the expression levels of DEFA1-4 and CRC. The expression levels of DEFA5 and DEFA6 significantly increased in CRC tissues compared with those in normal tissues. DEFA5 may be associated with better prognosis of CRC, while DEFA6 may be associated with poor prognosis. Immunohistochemistry (IHC) experiments showed that the expression of DEFA6 was significantly higher in adenoma than in normal mucosa and slightly higher in carcinoma than in normal mucosa. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that DEFAs were closely related to hsa05202: transcriptional misregulation in cancer and Hsa04015: Rap1 signaling pathway. DEFA5 may be a stable and good prognostic marker, and DEFA6 may be a poor prognostic marker in CRC of metastasis. Overall, DEFA5 and DEFA6 have a certain degree of sensitivity and specificity in predicting CRC.

Perinatal Western-style diet alters serotonergic neurons in the macaque raphe nuclei.

The neurotransmitter serotonin is a key regulator of neurotransmission, mood, and behavior and is essential in neurodevelopment. Dysfunction in this important neurotransmitter system is connected to behavioral disorders such as depression and anxiety. We have previously shown that the developing serotonin system is sensitive to perinatal exposure to Western-style diet (WSD). To advance our hypothesis that perinatal WSD has a long-term impact on the serotonergic system, we designed a fluorescent immunohistochemistry experiment using antibodies against tryptophan hydroxylase 2 (TPH2) and vesicular glutamate transporter 3 (VGLUT3) to probe protein expression in the raphe subnuclei in 13-month-old Japanese macaques (Macaca fuscata; n = 22). VGLUT3 has been shown to be coexpressed in TPH2+ cells in the dorsal raphe (DR) and median raphe nucleus (MnR) of rodent raphe nuclei and may provide information about the projection site of serotonergic fibers into the forebrain. We also sought to improve scientific understanding of the heterogeneity of the serotonin production center for the central nervous system, the midbrain raphe nuclei. In this immunohistochemical study, we provide the most detailed characterization of the developing primate raphe to date. We utilize multi-level modeling (MLM) to simultaneously probe the contribution of WSD, offspring sex, and raphe anatomical location, to raphe neuronal measurements. Our molecular and morphological characterization revealed that the 13-month-old macaque DR is remarkably similar to that of adult macaques and humans. We demonstrate that vesicular glutamate transporter 3 (VGLUT3), which rodent studies have recently shown can distinguish raphe populations with distinct projection targets and behavioral functions, likewise contributes to the heterogeneity of the primate raphe. This study provides evidence that perinatal WSD has a long-term impact on the density of serotonin-producing neurons, potentially limiting serotonin availability throughout the brain. Due to the critical involvement of serotonin in development and behavior, these findings provide important insight into the mechanisms by which maternal nutrition and metabolic state influence offspring behavioral outcomes. Finally, these findings could inform future research focused on designing therapeutic interventions to optimize neural development and decrease a child's risk of developing a mental health disorder.

Humulus lupulus L. Extract Protects against Senior Osteoporosis through Inhibiting Amyloid β Deposition and Oxidative Stress in APP/PS1 Mutated Transgenic Mice and Osteoblasts

As aging progresses, β-amyloid (Aβ) deposition and the resulting oxidative damage are key causes of aging diseases such as senior osteoporosis (SOP). Humulus lupulus L. (hops) is an important medicinal plant widely used in the food, beverage and pharmaceutical industries due to its strong antioxidant ability. In this study, APP/PS1 mutated transgenic mice and Aβ-injured osteoblasts were used to evaluate the protective effects of hops extracts (HLE) on SOP. Mice learning and memory levels were assessed by the Morris water maze. Mice femurs were prepared for bone micro-structures and immunohistochemistry experiments. The deposition of Aβ in the hippocampus, cortex and femurs were determined by Congo red staining. Moreover, protein expressions related to antioxidant pathways were evaluated by Western blotting. It was found that HLE markedly improved learning abilities and ameliorated memory impairment of APP/PS1 mice, as well as regulated antioxidant enzymes and bone metabolism proteins in mice serum. Micro-CT tests indicated that HLE enhanced BMD and improved micro-architectural parameters of mice femur. More importantly, it was discovered that HLE significantly reduced Aβ deposition both in the brain and femur. Further in vitro results showed HLE increased the bone mineralization nodule and reduced the ROS level of Aβ-injured osteoblasts. Additionally, HLE increased the expression of antioxidant related proteins Nrf2, HO-1, NQO1, FoxO1 and SOD-2. These results indicated that Humulus lupulus L. extract could protect against senior osteoporosis through inhibiting Aβ deposition and oxidative stress, which provides a reference for the clinical application of hops in the prevention and treatment of SOP.

Latilactobacillus sakei Furu2019 and stachyose as probiotics, prebiotics, and synbiotics alleviate constipation in mice.

Slow transit constipation (STC) is a common disorder in the digestive system. This study aimed to evaluate the effects of stachyose (ST) and Latilactobacillus sakei Furu 2019 (L. sakei) alone or combined on diphenoxylate-induced constipation and explore the underlying mechanisms using a mouse model. ICR mice were randomly divided into five groups. The normal and constipation model groups were intragastrically administrated with PBS. The ST, L. sakei, and synbiotic groups were intragastrically administrated with ST (1.5 g/kg body weight), alive L. sakei (3 × 109 CFU/mouse), or ST + L. sakei (1.5 g/kg plus 3 × 109 CFU/mouse), respectively. After 21 days of intervention, all mice except the normal mice were intragastrically administrated with diphenoxylate (10 mg/kg body weight). Defecation indexes, constipation-related intestinal factors, serum neurotransmitters, hormone levels, short-chain fatty acids (SCFAs), and intestinal microbiota were measured. Our results showed that three interventions with ST, L. sakei, and synbiotic combination (ST + L. sakei) all alleviated constipation, and synbiotic intervention was superior to ST or L. sakei alone in some defecation indicators. The RT-PCR and immunohistochemical experiment showed that all three interventions relieved constipation by affecting aquaporins (AQP4 and AQP8), interstitial cells of Cajal (SCF and c-Kit), glial cell-derived neurotrophic factor (GDNF), and Nitric Oxide Synthase (NOS). The three interventions exhibited a different ability to increase the serum excitatory neurotransmitters and hormones (5-hydroxytryptamine, substance P, motilin), and reduce the serum inhibitory neurotransmitters (vasoactive intestinal peptide, endothelin). The result of 16S rDNA sequencing of feces showed that synbiotic intervention significantly increased the relative abundance of beneficial bacteria such as Akkermansia, and regulated the gut microbes of STC mice. In conclusion, oral administration of ST or L. sakei alone or combined are all effective to relieve constipation and the symbiotic use may have a promising preventive effect on STC.

Total flavonoids of Litchi seed attenuate stem cell-like properties in breast cancer by regulating Notch3 signaling pathway

Ethnopharmacological relevanceBreast cancer has been the most commonly-diagnosed cancer worldwide, and the treatment and prognosis of which are often limited by breast cancer stem cells (BCSCs). Litchi seeds have shown good anti-cancer activity in various cancers including prostate cancer, lung cancer and breast cancer. However, the activity and underlying mechanism of Litchi seeds against BCSCs remain unknown. Aim of the studyTo investigate the activity and mechanism of total flavonoids of litchi seed (TFLS) against BCSCs in vitro and in vivo. Materials and methodsTwo orthotopic xenograft mouse models were established using HCC1806 cells pretreated or untreated with TFLS to determine whether TFLS could target BCSCs in vivo. Mammosphere formation and flow cytometry assays were employed to evaluate the effect of TFLS on BCSCs in vitro. The underlying mechanism was investigated using RT-qPCR, Western blot, immunohistochemistry and immunofluorescence experiments. ResultsTFLS could significantly inhibit the viability of HCC1806, MCF-7 and HCC1937 cells in vitro and suppress the growth of HCC1806 cells in vivo. TFLS attenuated stem cell-like properties of breast cancer through reducing the percentage of CD44+CD24-/low cells, inhibiting the mammospheres formation and down-regulating the mRNA and protein levels of cancer stem cells related markers (Oct4, Nanog, Sox2) in MCF-7 and HCC1806 cells. Meanwhile, TFLS suppressed the tumor-initiating ability of BCSCs via reducing the percentage of CD44+CD24-/low cells in tumor and lowering tumor incidence rate in orthotopic xenograft mice. In addition, TFLS treatments restricted the expression and nuclear translocation of Notch3, subsequently down-regulated Hes1 and Runx2 expressions. ConclusionsTFLS could suppress the growth of breast cancer and eliminate breast cancer stem cells by inhibiting the Notch3 signaling pathway.

Artesunate Alleviates Hyperoxia-Induced Lung Injury in Neonatal Mice by Inhibiting NLRP3 Inflammasome Activation.

Bronchopulmonary dysplasia (BPD) is a chronic respiratory disease in preterm infants that may cause persistent lung injury. Artesunate exhibits excellent anti-inflammatory in lung injury caused by various factors. This study aimed to investigate the effect of the artesunate on hyperoxia-induced lung injury in neonatal mice and its mechanism. A BPD model of hyperoxic lung injury in neonatal mice was established after hyperoxia (75% oxygen) exposure for 14 days, and part of the mice received intraperitoneal injections of the artesunate. H&E staining was used to observe the pathology of lung tissue, and the degree of oxidative stress in the lung tissue was determined by commercial kits. The levels of inflammatory cytokines in the serum and lung tissues of neonatal mice were detected by an enzyme-linked immunosorbent assay. Immunohistochemical experiments were performed to further evaluate the expression of IL-1β. The real-time quantitative polymerase chain reaction was used to determine the mRNA level of the NLRP3 inflammasome. The western blot assay was used to measure the levels of NLRP3 inflammasome and NF-κB pathway-related proteins. Artesunate ameliorated weight loss and lung tissue injury in neonatal mice induced by hyperoxia. The level of malondialdehyde was decreased, while the activity of superoxide dismutase and the level of glutathione increased after artesunate treatment. Artesunate reduced the level of inflammation cytokines TNF-α, IL-6, and IL-1β in the serum and lung. Moreover, artesunate inhibited the mRNA expression and protein levels of NLRP3, ASC, and caspase-1, as well as the phosphorylation of the NF-κB and IκBα. Our findings suggest that artesunate treatment can attenuate hyperoxia-induced lung injury in BPD neonatal mice by inhibiting the activation of NLRP3 inflammasome and the phosphorylation of the NF-κB pathway.

The prognostic effect of immunohistochemical staining rates in patients with non-muscle-invasive bladder cancer.

Despite the follow-up protocols developed in non-muscle-invasive bladder cancer patients, progression and recurrence could not be prevented. We aimed to investigate whether proteins such as OCT-4, CD47, p53, Ki-67, and Survivin, which increase in bladder cancer cells, can be used as prognostic markers for patients with non-muscle-invasive bladder cancer. The study included a total of 89 patients with newly diagnosed non-muscle-invasive bladder cancer between January 2015 and December 2020. Levels of OCT-4, CD47, p53, Kİ-67, and Survivin proteins in cancer cells were determined with a semi-quantitative immunohistochemical experiment. Pathological data and survival rates were compared according to the staining rates. Data obtained in the study were analyzed statistically with SPSS 22.0 (SPSS, Chicago, IL, USA). The mean age of the patients was 64.25 ± 9.91 years, and the median follow-up period was 55 months. Recurrence rate was determined to be 36% (n = 32), and the rate of progression at 40.4% (n = 36). The staining rates were stronger for each marker in the progression group and advanced-stage tumors (p < 0.001). The findings of the multivariate analysis carried out as part of the study showed that older age and higher tumor stage were independent risk factors for recurrence-free survival (HR = 1.048 and 7.074, respectively; P = 0.02). Also, higher tumor stages, diameters, and grades were associated with reduced progression-free survival (HR = 0.105, 0.395, 0.225, respectively; P < 0.05). Although immunohistochemical staining rates are promising, it is more appropriate to use tumor characteristics when assessing survival rate in patients with non-muscle-invasive bladder cancer.

Downregulation of HS6ST2 Inhibits Cervical Cancer Cell Migration and Invasion in Vivo and in Vitro.

Emerging evidence indicates the importance of heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) in a number of developmental processes. Little is known regarding its biological function in regulating cervical cancer (CC) progression. In this study, we aim to explore the role of HS6ST2 in CC progression. The transcriptome sequencing data of CC tissues from three databases, GSE64217, GSE138080, and GSE63514, was examined for genes with significant changes. The expression profile for HS6ST2 within CC tissue was then assessed through fluorescence quantitative PCR and immunohistochemistry and compared to data from patients with clinicopathological features. A multivariate survival analysis was performed using the COX regression. The real-time quantitative PCR assessed the HS6ST2 expression profile within CC cellular cultures. The results of knocking down HS6ST2, considering the proliferative activity and invasiveness of CC cultures in vitro, were detected through cell viability assay, clonogenic assessment, tumorsphere formation analysis, 3D invasion experiment and transwell assay. The impact of HS6ST2 knockdown in CC proliferation was also evaluated in vivo using a nude mice model. HS6ST2 was severely upregulated within CC tissues across the three explored databases (GSE64217, GSE138080, and GSE63514). Fluorescent quantitative PCR and immunohistochemistry experiments identified HS6ST2 as highly upregulated within patients CC tissues. Survival analysis taking into account the parameters of lymph node metastasis, Federation of Gynecology and Obstetrics (FIGO) stage, depth of invasion, pathological grade, and HS6ST2 expression level demonstrated that individuals with downregulated HS6ST2 exhibited considerably extended progression-free survival (PFS) and overall survival (OS) in comparison to upregulated HS6ST2 cases. According to the findings of COX univariate analysis, the parameters lymph node metastasis, FIGO stage, depth of invasion, pathological grade, and HS6ST2 expression level, all showed a statistically significant correlation with effect upon prognosis of CC patients. The FIGO stage, depth of invasion and expression level of HS6ST2 were identified as independent risk variables influencing CC case prognosis within subsequent COX multivariate analysis. Cell function experiments proved that HS6ST2 knockdown can considerably diminish the proliferative potential, stemness and invasive traits of CC cells. Tumor formation experiments in nude mice in vivo demonstrated that knocking down HS6ST2 can significantly thwart CC cellular proliferative properties within animal models. The clinicopathological features and the survival time of the patients significantly correlate with the level of HS6ST2 expression in CC tissue samples.

Activation of P2X4 receptors in midbrain cerebrospinal fluid-contacting nucleus leads to mechanical hyperalgesia in chronic constriction injury rats.

Neuropathic pain is a refractory pain state, and its mechanism is still not clear. Previous studies have shown that the purine receptor P2X4R expressed on hyperactive microglia in the spinal cord is essential for the occurrence and development of neuropathic pain. The cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) in the midbrain has been found to play an important role in the descending inhibition system of modulation. However, there have been no studies on P2X4R in the CSF-contacting nucleus involved in neuropathic pain. To investigate whether P2X4R is expressed in the CSF-contacting nucleus and whether its expression in the CSF-contacting nucleus is involved in the regulation of neuropathic pain, we used a model of chronic sciatic nerve ligation injury (CCI) to simulate neuropathic pain conditions. Immunohistochemistry experiments were conducted to identify the expression of P2X4R in the CSF-contacting nuclei in CCI rats, and western blot analysis showed a significant increase in P2X4R levels 7days after modeling. Then, we packaged a P2rx4 gene-targeting shRNA in scAAV9 to knock down the P2X4R level in the CSF-contacting nucleus, and we found that CCI-induced mechanical hyperalgesia was reversed. In conclusion, P2X4R expressed in the CSF-contacting nucleus is involved in the process of neuropathic pain, and downregulating P2X4R protein in the CSF-contacting nucleus can reverse the occurrence and development of hyperalgesia, which could represent a potent therapeutic strategy for neuropathic pain.

Paiteling induces apoptosis of cervical cancer cells by down-regulation of the E6/E7-Pi3k/Akt pathway: A network pharmacology

Ethnopharmacological relevanceHuman papillomavirus (HPV) infection is considered to be the main pathogen causing intraepithelial neoplasia. Paiteling (PTL) has been used to treat intraepithelial neoplasia caused by human papillomavirus (HPV) infection for more than 20 years in China, but its specific mechanism of action is not very clear, and further research is still needed. ObjectiveThis study designed a comprehensive strategy to study the pharmacological mechanism of paiteling in regulating cervical cancer cell apoptosis by integrating LC-MS/MS, network pharmacology and pharmacological experiments. MethodsWe used liquid chromatography–tandem mass spectrometry to detect the active substances in PTL and performed protein–protein interaction analysis on the intersection of the targets of these key compounds and the targets of intraepithelial neoplasia. Additionally, by using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG), the potential pathway of PTL against HPV-induced intraepithelial neoplasia was predicted. Finally, we used HeLa and Ect1/E6E7 cells for experimental verification. ResultsThe protein–protein interaction network predicted that AKT1, TP53, MYC, STAT3, MTOR, and MAPK were pivotal targets for PTL to inhibit epithelial neoplasia. KEGG enrichment analysis showed that the Pi3k/Akt pathway and HPV infection had scientific significance. Compared to the control group, after PTL diluent stimulated HeLa and Ect1/E6E7 cells for 24 h, cell viability, migration, and invasion capabilities were significantly reduced, and cell apoptosis was significantly increased, conforming to a dose-effect relationship and time-effect relationship. PCR, cellular immunohistochemistry, and western blot experiments showed that PTL reduced the expression of E6, Pi3k, E7, Akt, Bcl-xl, while increasing the expression of Bad in HeLa and Ect1/E6E7 cells. ConclusionPTL can induce cervical cancer cell apoptosis by inhibiting the E6/E7-Pi3k/Akt signaling pathway. It may provide an effective alternative strategy of traditional Chinese medicine for the treatment of epithelial neoplasia caused by HPV infection.

Cell senescence-associated genes predict the malignant characteristics of glioblastoma

BackgroundGlioblastoma (GBM) is the most malignant, aggressive and recurrent primary brain tumor. Cell senescence can cause irreversible cessation of cell division in normally proliferating cells. According to studies, senescence is a primary anti-tumor mechanism that may be seen in a variety of tumor types. It halts the growth and spread of tumors. Tumor suppressive functions held by cellular senescence provide new directions and pathways to promote cancer therapy.MethodsWe comprehensively analyzed the cell senescence-associated genes expression patterns. The potential molecular subtypes were acquired based on unsupervised cluster analysis. The tumor immune microenvironment (TME) variations, immune cell infiltration, and stemness index between 3 subtypes were analyzed. To identify genes linked with GBM prognosis and build a risk score model, we used weighted gene co-expression network analysis (WGCNA), univariate Cox regression, Least absolute shrinkage and selection operator regression (LASSO), and multivariate Cox regression analysis. And the correlation between risk scores and clinical traits, TME, GBM subtypes, as well as immunotherapy responses were estimated. Immunohistochemistry (IHC) and cellular experiments were performed to evaluate the expression and function of representative genes. Then the 2 risk scoring models were constructed based on the same method of calculation whose samples were acquired from the CGGA dataset and TCGA datasets to verify the rationality and the reliability of the risk scoring model. Finally, we conducted a pan-cancer analysis of the risk score, assessed drug sensitivity based on risk scores, and analyzed the pathways of sensitive drug action.ResultsThe 3 potential molecular subtypes were acquired based on cell senescence-associated genes expression. The Log-rank test showed the difference in GBM patient survival between 3 potential molecular subtypes (P = 0.0027). Then, 11 cell senescence-associated genes were obtained to construct a risk-scoring model, which was systematically randomized to distinguish the train set (n = 293) and the test set (n = 292). The Kaplan-Meier (K-M) analyses indicated that the high-risk score in the train set (P < 0.0001), as well as the test set (P = 0.0053), corresponded with poorer survival. In addition, the high-risk score group showed a poor response to immunotherapy. The reliability and credibility of the risk scoring model were confirmed according to the CGGA dataset, TCGA datasets, and Pan-cancer analysis. According to drug sensitivity analysis, it was discovered that LJI308, a potent selective inhibitor of RSK pathways, has the highest drug sensitivity. Moreover, the GBM patients with higher risk scores may potentially be more beneficial from drugs that target cell cycle, mitosis, microtubule, DNA replication and apoptosis regulation signaling.ConclusionWe identified potential associations between clinical characteristics, TME, stemness, subtypes, and immunotherapy, and we clarified the therapeutic usefulness of cell senescence-associated genes.

The Persistence of HIV Diversity, Transcription, and Nef Protein in Kaposi's Sarcoma Tumors during Antiretroviral Therapy.

Epidemic Kaposi's sarcoma (KS), defined by co-infection with Human Herpes Virus 8 (HHV-8) and the Human Immunodeficiency Virus (HIV), is a major cause of mortality in sub-Saharan Africa. Antiretroviral therapy (ART) significantly reduces the risk of developing KS, and for those with KS, tumors frequently resolve with ART alone. However, for unknown reasons, a significant number of KS cases do not resolve and can progress to death. To explore how HIV responds to ART in the KS tumor microenvironment, we sequenced HIV env-nef found in DNA and RNA isolated from plasma, peripheral blood mononuclear cells, and tumor biopsies, before and after ART, in four Ugandan study participants who had unresponsive or progressive KS after 180-250 days of ART. We performed immunohistochemistry experiments to detect viral proteins in matched formalin-fixed tumor biopsies. Our sequencing results showed that HIV diversity and RNA expression in KS tumors are maintained after ART, despite undetectable plasma viral loads. The presence of spliced HIV transcripts in KS tumors after ART was consistent with a transcriptionally active viral reservoir. Immunohistochemistry staining found colocalization of HIV Nef protein and tissue-resident macrophages in the KS tumors. Overall, our results demonstrated that even after ART reduced plasma HIV viral load to undetectable levels and restored immune function, HIV in KS tumors continues to be transcriptionally and translationally active, which could influence tumor maintenance and progression.

FAM3D as a Prognostic Indicator of Head and Neck Squamous Cell Carcinoma Is Associated with Immune Infiltration.

Globally, head and neck squamous cell carcinoma (HNSCC) is a common malignant tumor with high morbidity and mortality. Hence, it is important to find effective biomarkers for the diagnosis and prediction of the prognosis of patients with HNSCC. FAM3D had been proven to be vital in other cancers. However, its predictive and therapeutic value in HNSCC is unclear. Therefore, it is valuable to explore the association between the expression level of FAM3D and its impacts on the prognosis and tumor microenvironment in HNSCC. The Cancer Genome Atlas (TCGA) dataset, Genotype-Tissue Expression (GTEx) dataset, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset, and The Human Protein Atlas (THPA) website were used to assess HNSCC expressions in tumor and nontumor tissues. Then, we further conducted immunohistochemistry experiment as internal cohort to validate the same results. The Cox regression analysis, Kaplan-Meier analysis, and nomograms were performed to find the predictive prognostic value of FAM3D in HNSCC patients and its relationship with the clinicopathological features in HNSCC. The Gene Expression Omnibus (GEO) dataset was utilized to externally verify the prognosis value of FAM3D in HNSCC. Gene Set Enrichment Analysis (GESA) was applied to search the molecular and biological functions of FAM3D. The association between FAM3D and immune cell infiltration was investigated with the Tumor Immune Estimating Resource, version 2 (TIMER2). The relationships between FAM3D expression and tumor microenvironment (TME) scores, immune checkpoints, and antitumor compound half-maximal inhibitory concentration predictions were also explored. In different datasets, FAM3D mRNA and protein levels were all significantly lower in HNSCC tissues than in normal tissues, and they were strongly inversely associated with tumor grade, stage, lymph node metastasis, and T stage. Patients with high-FAM3D-expression displayed better prognosis than those with low-FAM3D-expression. FAM3D was also determined to be a suitable biomarker for predicting the prognosis of patients with HNSCC. This was externally validated in the GEO dataset. As for gene and protein level, the functional and pathway research results of FAM3D indicated that it was enriched in alteration of immune-related pathways in HNSCC. The low-expression group had higher stromal and ESTIMATE scores by convention than the high-expression group. FAM3D expression were found to be positively correlated with immune infiltrating cells, such as cancer-associated fibroblasts, myeloid-derived suppressor cells, macrophage cells, T cell CD8+ cells, regulatory T cells, and T cell follicular helper cells. FAM3D's relationships with immune checkpoints and sensitivity to antitumor drugs were also investigated. Our study explored the impact of FAM3D as a favorable prognostic marker for HNSCC on the tumor immune microenvironment from multiple perspectives. The results may provide new insights into HNSCC-targeted immunotherapy.

Single-cell discovery of the scene and potential immunotherapeutic target in hypopharyngeal tumor environment

Hypopharyngeal carcinoma is a cancer with the worst prognosis. We constructed the first single-cell transcriptome map for hypopharyngeal carcinoma and explored its underlying mechanisms. We systematically studied single-cell transcriptome data of 17,599 cells from hypopharyngeal carcinoma and paracancerous tissues. We identified categories of cells by dimensionality reduction and performed further subgroup analysis. Focusing on the potential mechanism in the cellular communication of hypopharyngeal carcinoma, we predicted ligand-receptor interactions and verified them via immunohistochemical and cellular experiments. In total, seven cell types were identified, including epithelial and myeloid cells. Subsequently, subgroup analysis showed significant tumor heterogeneity. Based on the pathological type of squamous cell carcinoma, we focused on intercellular communication between epithelial cells and various cells. We predicted the crosstalk and inferred the regulatory effect of cellular active ligands on the surface receptor of epithelial cells. From the top potential pairs, we focused on the BMPR2 receptor for further research, as it showed significantly higher expression in epithelial cancer tissue than in adjacent tissue. Further bioinformatics analysis, immunohistochemical staining, and cell experiments also confirmed its cancer-promoting effects. Overall, the single-cell perspective revealed complex crosstalk in hypopharyngeal cancer, in which BMPR2 promotes its proliferation and migration, providing a rationale for further study and treatment of this carcinoma.

Role of the activated microglia in hippocampal synaptic plasticity alteration in in vitro inflammatory models

AbstractBackgroundMicroglia cells are primary players in neuroinflammation, which plays a key role in the pathogenesis of Alzheimer's disease (AD). Under pathological inflammatory conditions, microglia activation leads to synaptic alterations thereby influencing the progression of AD. Increased activation of microglial colony stimulating factor 1 receptor (CSF1R) can lead to pathological microglia activation in experimental models of AD (Olmos‐Alonso et al., 2016). Using electrophysiological and immunohistochemical analysis, we investigated hippocampal synaptic transmission and plasticity in experimental models of neuroinflammation. We then tested whether pharmacological manipulation of microglia activation could restore the observed neuroplasticity alterations.MethodC57BL6/J mices of both sexes aged between 30‐ and 40‐days were used. Employing electrophysiological approaches (field and whole cell patch clamp), we recorded in area CA1 in response to stimulation of Schaffer collaterals. We evaluated Long‐Term Potentiation (LTP) induced by theta‐burst stimulation measuring field Excitatory Post‐Synaptic Potentials (fEPSPs) 1 hour after tetanization. We also investigated paired‐pulse ratio (PPR) and spontaneous EPSCs (sEPSCs). Two in vitro inflammatory models were obtained applying Lipopolysaccharide (LPS) (10 μg/ml) or Aβ 1‐42 oligomers (200 nM). As a control, we tested the effect of scrambled Aβ 1‐42. To inhibit the activation of microglia we used Pexidartinib (PLX‐3397) (10μM) and Minocycline (500 nM). For immunohistochemical experiments free‐floating sections were incubated with primary antibodies overnight at 4°C followed by Alexa‐conjugated secondary antibody and DAPI for 2 hours at room temperature in the dark. VGLUT1, Iba1 and CD68 were used as synaptic marker in a triple staining to study pathological pruning and its reversal by PLX‐3397.ResultBoth inflammatory models induced LTP impairment, that was restored by application of a no‐specific blocker, Minocycline, or a specific inhibitor of activated microglia, PLX‐3397.Using whole cell patch clamp recordings, we observed that Aβ produces a reduction in glutamatergic transmission, evaluated as amplitude or frequency of sEPSCs and PPR, that is prevented by PLX‐3397. The data were confirmed using imaging studies, by which count of CA1 microglia and spine remodeling were evaluated.ConclusionOur results demonstrate a functional role of activated microglia in synaptic plasticity alteration that occurs in neuroinflammation suggesting CSF1R as new pharmacological target for AD treatment.

Identification and validation of transferrin receptor protein 1 for predicting prognosis and immune infiltration in lower grade glioma.

Transferrin receptor protein 1 (TFRC), an ananda molecule associated with ferroptosis, has been identified as affecting a wide spectrum of pathological processes in various cancers, but the prognostic value correlates with the tumor microenvironment of TFRC in lower-grade glioma (LGG) is still unclear. Clinical pathological information and gene expression data of patients with LGG come from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), GTEx, Oncomine, UCSC Xena, and GEO databases. We then used various bioinformatics methods and mathematical models to analyze those data, aiming to investigate the clinical significance of TFRC in LGG and illustrate its association with tumor immunity. In addition, the molecular function and mechanisms of TFRC were revealed by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA). Immunohistochemical experiments and single-cell analysis have been performed. TFRC expression was highly expressed in many tumors and showed a poor prognosis. Including gliomas, it was significantly associated with several poor clinical prognostic variables, tumor immune microenvironment, tumor mutational burden (TMB), m6a modification, and ferroptosis in LGG. TFRC as a key factor was further used to build a prediction nomogram. The C-index, calibration curve, and decision curve analysis showed the nomogram was clinically useful and calibration was accurate. At the same time, we also demonstrated that promoter hypomethylation of DNA upstream of TFRC could lead to high TFRC expression and poor overall survival. There is a significant correlation between TFRC and CD8 + T cell, macrophage cell infiltration, and several immune checkpoints, such as PD-L1(cd274), CTLA4, and PD1, suggesting a novel direction for future clinical application. Functional and molecular mechanism analysis showed an association of TFRC expression with immune-related pathways through GSEA, GO, and KEGG analysis. Finally, immunohistochemical experiments and single-cell analysis confirmed the expression of TFRC in glioma. TFRC may be a potential prognostic biomarker and an immunotherapeutic target for glioma.

Getting sweeter: new evidence for glucose transporters in specific cell types of the airway?

New technologies such as single-cell RNA sequencing (scRNAseq) has enabled identification of the mRNA transcripts expressed by individual cells. This review provides insight from recent scRNAseq studies on the expression of glucose transporters in the epithelial cells of the airway epithelium from trachea to alveolus. The number of studies analyzed was limited, not all reported the full range of glucose transporters and there were differences between cells freshly isolated from the airways and those grown in vitro. Furthermore, glucose transporter mRNA transcripts were expressed at lower levels than other epithelial marker genes. Nevertheless, these studies highlighted that there were differences in cellular expression of glucose transporters. GLUT1 was the most abundant of the broadly expressed transporters that included GLUT8, 10, and 13. GLUT9 transcripts were more common in basal cells and GLUT12 in ionocytes/ciliated cells. In addition to alveolar cells, SGLT1 transcripts were present in secretory cells. GLUT3 mRNA transcripts were expressed in a cell cluster that expressed monocarboxylate (MCT2) transporters. Such distributions likely underlie cell-specific metabolic requirements to support proliferation, ion transport, mucous secretion, environment sensing, and airway glucose homeostasis. These studies have also highlighted the role of glucose transporters in the movement of dehydroascorbic acid/vitamin C/myoinositol/urate, which are factors important to the innate immune properties of the airways. Discrepancies remain between detection of mRNAs, protein, and function of glucose transporters in the lungs. However, collation of the data from further scRNAseq studies may provide a better consensus and understanding, supported by qPCR, immunohistochemistry, and functional experiments.

Expression of IRAK1 in Hepatocellular Carcinoma, Its Clinical Significance, and Docking Characteristics with Selected Natural Compounds.

This study aimed to explore clinical significance of interleukin-1 receptor-associated kinase 1 (IRAK1) in the diagnosis, prognosis, and targeted therapy of hepatocellular carcinoma. A systematic analysis based on the cancer genome atlas (TCGA) indicated that IRAK1 was highly expressed in 18 cancer types (p < 0.01) and may be a pan-cancer biomarker. In hepatocellular carcinoma, the alteration rate of IRAK1 was rather high (62.4%), in which mRNA high relative to normal predominated (58.9%). Higher expression was associated with shorter overall survival (p < 0.01). IRAK1 expression correlated positively with pathology stage and tumor grade (for the latter there was only a slight trend). Interestingly, it correlated positively with TP53 mutation (p < 0.001), suggesting a possible strategy for targeting TP53 via IRAK1. Immunohistochemistry experiments confirmed a higher positive rate of IRAK1 in carcinoma than in para-carcinoma tissues (χ2 = 18.006, p < 0.001). Higher tumor grade correlated with more strongly positive staining. Molecular docking revealed cryptotanshinone, matrine, and harmine as the best hit compounds with inhibition potential for IRAK1. Our findings suggest that IRAK1 may play biologically predictive roles in hepatocellular carcinoma. The suppression of IRAK1/NF-κB signaling via inhibition of IRAK1 by the hit compounds can be a potential strategy for the targeted therapy.