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Laminin subunit alpha 4 (LAMA4),a member of the laminin family,is present in the intercellular matrix of adult tissues as a major component of basement membrane.LAMA4 is involved in the adhesion of cells and can bind to corresponding integrins to activate relevant signaling pathways,playing an essential role in the growth,proliferation,and migration of cells.It has been demonstrated that LAMA4 is associated with the occurrence and development of a variety of diseases including tumors,and the expression of LAMA4 can be used as a biomarker of tumor diagnosis and prognosis.This paper summarizes the current research progress in LAMA4 with the focus on the relationship between LAMA4 and diseases,especially tumor,with a view to provide new directions for the future research.
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Adult , Humans , Laminin , Extracellular MatrixABSTRACT
ObjectiveTo investigate the pharmacodynamic characteristics and explore the molecular mechanism of Honghua oral liquid （HOL） in relieving neuropathic pain （NP）. MethodHealthy male SD rats were randomly assigned into sham group， model group， low-， medium-， high-dose （0.5， 1.0， 2.0 mL·kg-1·d-1， respectively） HOL groups， and a positive drug （pregabalin， 25 mg·kg-1·d-1） group， with 6 rats in each group. Spinal nerve ligation （SNL） of L5 was conducted in other groups except the sham group. Drug administration was performed 3 days after the SNL surgery for 2 consecutive weeks， and samples were collected after the end of the administration. During the treatment period， the mechanical pain threshold and cold pain threshold were determined to measure the pain-relieving effect of HOL. Transcriptome sequencing was performed on hippocampal tissue samples from the sham， model， and high-dose HOL groups， and differentially expressed genes between the sham group and the model group as well as the model group and HOL high-dose group were obtained. After pathway enrichment analysis， we selected the targets which were closely related to neuroinflammation for validation， and predicted the specific binding sites of the major active components in HOL with the targets through molecular docking. In addition， the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-10 （IL-10） were determined by enzyme-linked immunosorbent assay （ELISA） to evaluate the effect of HOL on neuroinflammation in NP rats. ResultCompared with the sham group， SNL decreased the mechanical pain threshold and cold pain threshold （P<0.05）. Compared with the model group， HOL recovered the mechanical pain threshold and cold pain threshold （P<0.05）. The transcriptome data showed that 376 differentially expressed genes （DEGs） were identified between the model group and the sham group， including 124 upregulated genes and 252 downregulated genes， and 194 DEGs between the model group and the high-dose HOL group， including 33 upregulated genes and 161 downregulated genes. Among them， insulin-like growth factor 1（IGF1）， matrix metallopeptidase-2 （MMP-2）， matrix metallopeptidase-14 （MMP-14）， erb-B2 receptor tyrosine kinase 2 （ERBB2）， and integrin subunit alpha 5 （ITGA5） associated with NP were selected for further validation. The Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） results showed that compared with the sham group， the modeling up-gurelated the mRNA levels of the above five molecules in the hippocampus （P<0.01）. Compared with model group， HOL down-regulated the mRNA levels of these molecules （P<0.01）. The molecular docking results showed that the main active components of safflower， hydroxysafflor yellow A， kaempferol， and quercetin， formed stable hydrogen bonds with the amino acid residues of IGF1， MMP-2， MMP-14， ERBB2， and ITGA5. The enzyme-linked immunosorbent assay（ELISA） results showed that compared with those in the sham group， the serum levels of TNF-α and IL-10 were out of balance in the model rats （P<0.01）. Compared with the model group， HOL lowered the level of the pro-inflammatory cytokine TNF-α （P<0.01） and elevated that of the anti-inflammatory cytokine IL-10 （P<0.05）. ConclusionHOL exerts analgesic effect on SNL rats by inhibiting neuroinflammation.
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Mammalian target of rapamycin (mTOR) controls cellular anabolism, and mTOR signaling is hyperactive in most cancer cells. As a result, inhibition of mTOR signaling benefits cancer patients. Rapamycin is a US Food and Drug Administration (FDA)-approved drug, a specific mTOR complex 1 (mTORC1) inhibitor, for the treatment of several different types of cancer. However, rapamycin is reported to inhibit cancer growth rather than induce apoptosis. Pyruvate dehydrogenase complex (PDHc) is the gatekeeper for mitochondrial pyruvate oxidation. PDHc inactivation has been observed in a number of cancer cells, and this alteration protects cancer cells from senescence and nicotinamide adenine dinucleotide (NAD+) exhaustion. In this paper, we describe our finding that rapamycin treatment promotes pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) phosphorylation and leads to PDHc inactivation dependent on mTOR signaling inhibition in cells. This inactivation reduces the sensitivity of cancer cells' response to rapamycin. As a result, rebooting PDHc activity with dichloroacetic acid (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, promotes cancer cells' susceptibility to rapamycin treatment in vitro and in vivo.
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Humans , Sirolimus/pharmacology , Dichloroacetic Acid/pharmacology , Pyruvate Dehydrogenase Complex , TOR Serine-Threonine Kinases , Mechanistic Target of Rapamycin Complex 1 , Neoplasms/drug therapyABSTRACT
@#Objective To develop and verify a multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity.Methods Specific reverse transcription primers,two pairs of quantitative primers and probes were designed for the CDS sequence of telomerase catalytic subunit telomerase reverse transcriptase(TERT).After optimization of the reverse transcription primers(specific reverse transcription primers and random primers) and quantitative primers(two pairs of quantitative primer probes used alone or in combination) in the reaction system,with the primer probe of internal reference gene GAPDH,multiplex fluorescence quantitative PCR was performed in a single tube.In addition,telomerase positive standard and negative standard were prepared with 293T and MRC-5 cells respectively,and the stability and precision of the method were verified.The telomerase activity in 19 normal mesenchymal cell samples and 32 breast cancer cell samples were detected by the developed method.Results The optimum reaction system was as follows:using cDNA synthesized with specific reverse transcription primers as the template,2 pairs of quantitative primer probes of TERT gene were mixed with internal reference gene GAPDH primer probes for multiplex fluorescence quantitative PCR reaction in a single tube.After optimization,the sensitivity and TERT fluorescence signal quantity of the system were greatly improved,and the ΔRn was enhanced by 3 times.The amplification curve of positive standard TERT gene was normal,and the ΔCt between TERT gene and GAPDH gene remained stable.The amplification curve of GAPDH gene in negative standard was normal,while there was no amplification curve of TERT gene.There was a little difference in ΔCt between TERT and GAPDH genes in the positive standard frozen and thawed for 3 and 5 times repeatedly and the positive standard without freezing and thawing,and the CVs of precision in intra-and inter-groups were all less than 1%.Telomerase activity was negative in 19 normal mesenchymal cell samples and positive in 32 breast cancer cell samples,and significant difference in Ct value of TERT gene between them was observed(t=4.236,P <0.001).Conclusion The developed multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity has good stability and precision,which is expected to be used in early diagnosis and gene therapy of tumors.
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@#Subunit vaccines are developed from the immunologically active fragments of pathogens and do not have genetic materials,which have been the center of research attention,guiding the direction of the development for new generations of vaccines.Given their safety advantages and high immunological activities,subunit vaccines play important roles in prevention of the spread of pathogenic viruses.This paper mainly introduces the research progress and applications of the subunit vaccines in common viral diseases,aiming to establish a theoretical basis for the development of the subunit vaccines on new viral diseases.
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Objective: To investigate the analgesic mechanism of Tuina (Chinese therapeutic massage) by observing the effect of the N-methyl-D-aspartate receptor subunit 2B (NR2B)/postsynaptic density-95 (PSD-95) pathway on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation. Methods: Fifty Sprague-Dawley rats were randomly divided into a blank group, a model group, a Tuina group, a blocker agent group, and a blocker agent + Tuina group. The sciatic nerve chronic constriction injury (CCI) model was prepared by the sciatic nerve ligation method. From the 4th day after modeling, rats in the Tuina group and the blocker agent + Tuina group were subject to daily Tuina intervention, and those in the blocker agent group and the blocker agent + Tuina group were daily intrathecally injected with NR2B blocker agent (MK-801). The spontaneous pain score was used to observe the pain behavior of all rats. The expression levels of NR2B and downstream PSD-95 were measured by immunohistochemistry, and the dendritic structure changes were observed by Golgi staining for rat spinal cord dorsal horn after 14 d of continuous intervention. Results: Compared with the blank group, the degree of rat spontaneous pain after CCI was elevated in both the model and the Tuina groups (P<0.01) and was reduced in the Tuina group after the Tuina intervention compared with the model group (P<0.05). Compared with the model group, the rat spontaneous pain level after blocking NR2B was reduced in both the blocker agent group and the blocker agent + Tuina group (P<0.05). The NR2B and PSD-95 protein levels were significantly higher in the model group compared with the blank group (P<0.01); the total number of dendritic branches was increased (P<0.01), and the total dendritic length became longer (P<0.01) in the spinal cord dorsal horn. The rat NR2B and PSD-95 protein levels were significantly decreased in the Tuina group compared with the model group (P<0.01); the total dendritic branch number was reduced (P<0.01) and the total length was shortened (P<0.01) in the spinal cord dorsal horn. After blocking NR2B, the expression levels of NR2B and downstream PSD-95 protein were significantly lower in both the blocker agent group and the blocker agent + Tuina group compared to the model group (P<0.01). The total branch number was significantly reduced (P<0.01), and the total length was significantly shortened (P<0.01) of the dendrites in the spinal cord dorsal horn. Conclusion: Tuina may exert an analgesic effect by remodeling the dendritic structure in the spinal cord dorsal horn in rats with lumbar disc herniation, and its mechanism may be related to the inhibition of NR2B/PSD-95 signaling pathway.
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Objective:To investigate the role of protein phosphatase 4 catalytic subunit (PP4C) in regulating hepatitis B virus X protein (HBx) levels and its effects on the biological functions of HBx, thus to provide a potential therapeutic targets for hepatitis B virus (HBV)-related hepatocellular carcinoma.Methods:In vivo and in vitro interactions between HBx and PP4C were analyzed by co-immunoprecipitation (Co-IP) and GST pull-down assay. Recombinant plasmids of PP4C and HBx were co-transfected with Lipofectamine 3000 reagents into hepatoma cells to detect the protein levels of HBx by Western blot. The half-life of HBx in the transfected cells treated with cycloheximide (CHX) were detected. The phosphorylation assay was used to evaluate the effects of PP4C on HBx phosphorylation. CCK8 assay, wound healing assay and Matrigel invasion chamber assay were used to analyze the effects of PP4C on the biological functions of HBx. Results:PP4C interacted with HBx in vivo and in vitro. PP4C overexpression significantly increased the protein level and stability of HBx and the phosphorylation assay confirmed that PP4C overexpression decreased the serine phosphorylation of HBx in hepatoma cells. PP4C overexpression enhanced the migration and invasion of hepatoma cells, but had no significant effects on the proliferation. Conclusions:The interactions between HBx and PP4C promoted the stability of HBx and ultimately enhanced the migration and invasion of hepatoma cells, and the mechanisms might be related to the decrease of HBx serine phosphorylation by PP4C. This study provided a theoretical basis for further investigation of the pathogenic mechanisms of HBx, and targeting PP4C and HBx interaction might provide insights for developing novel treatment for HBV-related hepatocellular carcinoma.
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Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.
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Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.
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Objective:To observe the expression of angiopoietin-like protein 4 (ANGPTL4) signaling pathway in adenine-induced chronic kidney disease (CKD) rat model, and to explore the role of this pathway in renal fibrosis.Methods:Thirty-six male Sprague-Dawley rats were randomly divided into control group (saline, intragastric administration, n=15) and CKD group (250 mg·kg -1·d -1 2.5% adenine, intragastric administration, n=21). At the end of the 1st, 2nd and 4th week, 5 rats were randomly selected from each group. Renal function and 24-hour urinary protein quantity were measured. HE and Masson staining were used to observe the pathological changes of kidneys. Immunohistochemistry and real-time PCR were used to detect renal protein and mRNA expressions of hypoxia-inducible factor-1α (HIF-1α), ANGPTL4, bone morphogenetic protein 7 (BMP7), Smad1, α-smooth muscle actin (α-SMA) and type Ⅰ collagen (Col-Ⅰ). Pearson correlation analysis was used to analyze the correlation between the different indicators. Results:(1) Compared with the control group, the expression levels of serum creatinine and blood urea nitrogen in CKD group were higher at each time point, and the expression levels of 24-hour urinary protein quantity were higher at the end of the 2nd and 4th week (all P < 0.05). (2) HE and Masson staining showed that there were obvious renal structural disorders and collagen fiber deposition at each time point in CKD group compared with the control group, which got worse with time. (3) The results of immunohistochemistry and real-time PCR showed that compared with the control group, the protein and mRNA expression levels of ANGPTL4, α-SMA and Col-Ⅰ were higher, while the protein and mRNA expression levels of BMP7 and Smad1 were lower at the end of the 1st, 2nd and 4th week, and the protein and mRNA expression levels of HIF-1α were higher at the end of 2nd and 4th week in CKD group (all P < 0.05). (4) Correlation analysis results showed that HIF-1α and ANGPTL4 mRNA expression were positively correlated with α-SMA mRNA ( r=0.919, P < 0.001; r=0.757, P < 0.001), and also positively correlated with Col-Ⅰ mRNA ( r=0.925, P < 0.001; r=0.777, P < 0.001). HIF-1α mRNA expression was positively correlated with ANGPTL4 mRNA ( r=0.766, P < 0.001). There were significant negative correlations between HIF-1α, ANGPTL4 mRNA and BMP7 mRNA ( r=-0.652, P < 0.001; r=-0.741, P < 0.001). Conclusions:ANGPTL4 signaling pathway may be activated in adenine-induced CKD rat model, and involved in the renal fibrosis process of CKD.
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ObjectiveTo explore the intervention mechanism of Xiangsha Liujunzi Tang in rats with functional dyspepsia （FD） based on the Ras homolog gene family member A （RhoA）/Rho-associated coiled-coil containing protein kinase 2 （ROCK2）/Myosin phosphatase target Subunit 1 （MYPT1） pathway. MethodSixty male SD suckling rats in SPF grades were randomly divided into blank group （n=10） and model group （n=50）. The comprehensive modeling method （gavage administration of iodoacetamide+exhaustion of swimming+disturbance of hunger and satiety） was used to replicate the rat model of FD. After successful replication of the model， the rats in the model group were randomly divided into model group， mosapride group， and high， middle， and low-dose Xiangsha Liujunzi Tang groups， with 10 rats in each group. Rats in the blank group and model group were given 10 mL kg-1·d-1 normal saline， those in the mosapride group were given 1.35 mg·kg-1·d-1 mosapride， and those in the high， middle， and low-dose Xiangsha Liujunzi Tang groups were given 12， 6， and 3 g·kg-1·d-1 Xiangsha Liujunzi Tang， respectively. The intervention lasted 14 days. The general living conditions of rats were observed before and after modeling and administration， and the 3-hour food intake and body mass of rats were measured. After intervention， the intestinal propulsion rate of rats was measured， and the pathological changes in the gastric tissue were observed by hematoxylin-eosin （HE） staining. The content of choline acetyl transferase （ChAT） and vasoactive intestinal peptide （VIP） in the medulla oblongata and gastric tissue homogenate was determined by enzyme-linked immunosorbent assay （ELISA）， the distribution of adenosine triphosphate （ATP） enzyme in gastric antrum smooth muscle was observed by frozen section staining， and the protein expression levels of RhoA， ROCK2， and phosphorylated-myosin phosphatase target subunit 1 （p-MYPT1） in the gastric tissue were detected by Western blot. ResultCompared with the blank group， the model group had withered hair， lazy movement， slow action， poor general living condition， lower 3-hour food intake， body mass， and lower intestinal propulsion rate （P<0.05）， whereas no obvious abnormality in gastric histopathology. In the model group， the content of ChAT in the medulla oblongata and gastric tissue decreased， the content of VIP in gastric tissue increased， the distribution of ATP enzyme in gastric antrum smooth muscle decreased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue decreased significantly （P<0.05）. As compared with the model group， the general living condition of rats in each intervention group was significantly improved， and the 3-hour food intake， body mass， and intestinal propulsion rate were significantly increased （P<0.05）. There was no significant difference in gastric pathology in the intervention groups. The content of ChAT in the medulla oblongata and gastric tissue increased significantly， the content of VIP in the gastric tissue decreased， the distribution of ATP enzyme in gastric antrum smooth muscle increased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue increased significantly （P<0.05）. The intervention effect of Xiangsha Liujunzi Tang group on the above indexes was dose-dependent. ConclusionXiangsha Liujunzi Tang can effectively improve the general living condition and gastric motility of rats with FD， and its specific mechanism may be related to the activation of the RhoA/ROCK2/MYPT1 pathway in the gastric tissue to regulate smooth muscle relaxation and contraction and promote gastric motility.
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Objective To investigate the role of MLLT1 in hepatocellular carcinoma (HCC)and its impact on the tumor immune microenvironment. Methods Multivariate Cox regression analysis and tumor gene analysis tools such as GEPIA and UALCAN were used to explore the expression of the MLLT1 gene and its prognostic significance in different tumors. Real-time PCR, Western blotting, and immunohistochemistry were used to investigate the differential expression of MLLT1 between HCC tumor tissue and normal tissue. MTT assay and cell cycle analysis were performed to assess the effect of MLLT1 knockdown on cell proliferation and cell cycle. The correlation between MLLT1 and immune cells, as well as immune infiltrates in the tumor microenvironment, and their correlation with immune neoantigens, immune checkpoints, tumor mutation burden, and microsatellite instability were also explored. Results The MLLT1 gene was found to be aberrantly expressed in various solid tumors including HCC, and its high expression was associated with poor prognosis in HCC. Knockdown of MLLT1 inhibited HCC cell proliferation and blocked the cell cycle. High expression of MLLT1 was found to affect the content of multiple immune cells, including CD4
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@#Entamoeba gingivalis is present in the oral cavity of humans and is associated with periodontal disease. Consequently, this study aimed to comprehensively investigate the E. gingivalis infection and the associated risk factors among individuals suffering from periodontal conditions. A cross-sectional descriptive study was carried out within a cohort of periodontal patients. Dental plaque specimens were meticulously collected and subsequently subjected to thorough examination using the polymerase chain reaction (PCR)-based technique targeting the small subunit ribosomal RNA (SrRNA) gene of the organism. The occurrence of risk factors for E. gingivalis infection was analyzed by the chi-square test and binary logistic regression. Out of the 230 participants, 60 were clinically diagnosed with periodontitis, while 170 were afflicted with gingivitis. Out of the 230 patients, 25 (10.9%) tested positive for E. gingivalis infections. An in-depth analysis unveiled that a significant majority of infections were recorded within subgroups characterized by a marital status (15.45%), manifestation of periodontitis (25.00%), and concomitant presence of underlying disease (20.83%). Furthermore, the high risk factor associated with E. gingivalis infection was the female (ORadj = 13.65, 95% CI = 1.08-173.21), followed by periodontitis (ORadj = 3.30, 95% CI = 1.21-9.00), respectively. The study employs a molecular diagnostic approach to screen for E. gingivalis enrichment within a subset of periodontal patients with advancing disease. The findings emphasize the necessity for further research to elucidate the pathogenesis of E. gingivalis and advocate for vigilant surveillance within a substantial population of periodontal patients.
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Designing and manufacturing safe and effective vaccines is a crucial challenge for human health worldwide. Research on adjuvant-based subunit vaccines is increasingly being explored to meet clinical needs. Nevertheless, the adaptive immune responses of subunit vaccines are still unfavorable, which may partially be attributed to the immune cascade obstacles and unsatisfactory vaccine design. An extended understanding of the crosstalk between vaccine delivery strategies and immunological mechanisms could provide scientific insight to optimize antigen delivery and improve vaccination efficacy. In this review, we summarized the advanced subunit vaccine delivery technologies from the perspective of vaccine cascade obstacles after administration. The engineered subunit vaccines with lymph node and specific cell targeting ability, antigen cross-presentation, T cell activation properties, and tailorable antigen release patterns may achieve effective immune protection with high precision, efficiency, and stability. We hope this review can provide rational design principles and inspire the exploitation of future subunit vaccines.
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The rise of nanotechnology has opened new horizons for cancer immunotherapy. However, most nanovaccines fabricated with nanomaterials suffer from carrier-related concerns, including low drug loading capacity, unpredictable metabolism, and potential systemic toxicity, which bring obstacles for their clinical translation. Herein, we developed an antigen self-assembled nanovaccine, which was resulted from a simple acryloyl modification of the antigen to induce self-assembly. Furthermore, a dendritic cell targeting head mannose monomer and a mevalonate pathway inhibitor zoledronic acid (Zol) were integrated or absorbed onto the nanoparticles (denoted as MEAO-Z) to intensify the immune response. The synthesized nanovaccine with a diameter of around 70 nm showed successful lymph node transportation, high dendritic cell internalization, promoted costimulatory molecule expression, and preferable antigen cross-presentation. In virtue of the above superiorities, MEAO-Z induced remarkably higher titers of serum antibody, stronger cytotoxic T lymphocyte immune responses and IFN-γ secretion than free antigen and adjuvants. In vivo, MEAO-Z significantly suppressed EG7-OVA tumor growth and prolonged the survival time of tumor-bearing mice. These results indicated the translation promise of our self-assembled nanovaccine for immune potentiation and cancer immunotherapy.
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Colorectal cancer (CRC) is a type of malignant tumor that seriously threatens human health and life, and its treatment has always been a difficulty and hotspot in research. Herein, this study for the first time reports that antipsychotic aripiprazole (Ari) against the proliferation of CRC cells both in vitro and in vivo, but with less damage in normal colon cells. Mechanistically, the results showed that 5-hydroxytryptamine 2B receptor (HTR2B) and its coupling protein G protein subunit alpha q (Gαq) were highly distributed in CRC cells. Ari had a strong affinity with HTR2B and inhibited HTR2B downstream signaling. Blockade of HTR2B signaling suppressed the growth of CRC cells, but HTR2B was not found to have independent anticancer activity. Interestingly, the binding of Gαq to HTR2B was decreased after Ari treatment. Knockdown of Gαq not only restricted CRC cell growth, but also directly affected the anti-CRC efficacy of Ari. Moreover, an interaction between Ari and Gαq was found in that the mutation at amino acid 190 of Gαq reduced the efficacy of Ari. Thus, these results confirm that Gαq coupled to HTR2B was a potential target of Ari in mediating CRC proliferation. Collectively, this study provides a novel effective strategy for CRC therapy and favorable evidence for promoting Ari as an anticancer agent.
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Objective To investigate the expression and prognostic significance of mediator complex subunit 8 (MED8) in gastric cancer and its impact on the cell cycle.Methods The expression of MED8 in gastric cancer and adjacent tissues and its correlation with patients' prognosis were analyzed using public databases.A validation cohort of 104 patients who underwent radical resection for gastric cancer in the First Affiliated Hospital of Bengbu Medical College from June 2012 to July 2017 was included.The receiver operating characteristic curve was established to evaluate the predictive value of MED8 for postoperative 5-year survival.Bioinformatics tools were used to predict the biological roles of MED8 in gastric cancer.The effect of the MED8 level on the G1/S phase transition of gastric cancer cells (MGC-803) was analyzed via lentivirus transduction and flow cytometry.Western blotting was carried out to assess the impact of MED8 expression on the protein levels of cyclin-dependent kinase 4(Cdk4) and G1/S-specific cyclin-D1(CyclinD1) in MGC-803 cells.Results The high expression of MED8 in the gastric cancer tissue was associated with poor prognosis (P<0.001) and had prognostic significance (area under curve=0.733,P<0.001).Gene enrichment analysis suggested that MED8 may participate in the cell cycle process.Flow cytometry results revealed that the upregulation of MED8 expression promoted the transition of MGC-803 cells from the G1 phase to the S phase (P<0.001),while the downregulation of MED8 had the opposite effect (P<0.001).Western blotting showed increases in the protein levels of Cdk4 and CyclinD1 in MGC-803 cells with upregulated MED8 expression (all P<0.001),and decreases in the cells with downregulated MED8 expression (all P<0.001).Conclusion MED8 is highly expressed in gastric cancer and may affect its progression and prognosis by regulating the G1/S phase transition of gastric cancer cells.
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Humans , Stomach Neoplasms , Prognosis , Cell Proliferation , Cell Cycle , Mediator Complex/metabolism , Cell Line, TumorABSTRACT
Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.
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Cattle , Animals , Mice , Bacterial Proteins/metabolism , Bacillus cereus/metabolism , Hemolysin Proteins/metabolism , Virulence Factors/metabolism , Enterotoxins/metabolismABSTRACT
With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.
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Animals , Male , Goats , Stem Cells , Spermatogonia/metabolism , Cell Proliferation , Flow Cytometry , Testis/metabolismABSTRACT
To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.