Design of a novel multiepitope vaccine with CTLA-4 extracellular domain against Mycoplasma pneumoniae: A vaccine-immunoinformatics approach.

BACKGROUND: Community-acquired pneumonia often stems from the macrolide-resistant strain of Mycoplasma pneumoniae, yet no effective vaccine exists against it. METHODS: This study proposes a vaccine-immunoinformatics strategy for Mycoplasma pneumoniae and other pathogenic microbes. Specifically, dominant B and T cell epitopes of the Mycoplasma pneumoniae P30 adhesion protein were identified through immunoinformatics method. The vaccine sequence was then constructed by coupling with CTLA-4 extracellular region, a novel molecular adjuvant for antigen-presenting cells. Subsequently, the vaccine's physicochemical properties, antigenicity, and allergenicity were verified. Molecular dynamics modeling was employed to confirm interaction with TLR-2, TLR-4, B7-1, and B7-2. Finally, the vaccine underwent in silico cloning for expression. RESULTS: The vaccine exhibited both antigenicity and non-allergenicity. Molecular dynamics simulation, post-docking with TLR-2, TLR-4, B7-1, and B7-2, demonstrated stable interaction between the vaccine and these molecules. In silico cloning confirmed effective expression of the vaccine gene in insect baculovirus vectors. CONCLUSION: This vaccine-immunoinformatics approach holds promise for the development of vaccines against Mycoplasma pneumoniae and other pathogenic non-viral and non-bacterial microbes.

DCES-PA: Deformation-controllable elastic shape model for 3D bone proliferation analysis using hand HR-pQCT images.

Bone proliferation is an important pathological feature of inflammatory rheumatic diseases. Although recent advance in high-resolution peripheral quantitative computed tomography (HR-pQCT) enables physicians to study microarchitectures, physicians' annotation of proliferation suffers from slice inconsistency and subjective variations. Also, there are only few effective automatic or semi-automatic tools for proliferation detection. In this study, by integrating pathological knowledge of proliferation formation with the advancement of statistical shape analysis theory, we present an unsupervised method, named Deformation-Controllable Elastic Shape model, for 3D bone Proliferation Analysis (DCES-PA). Unlike previous shape analysis methods that directly regularize the smoothness of the displacement field, DCES-PA regularizes the first and second-order derivative of the displacement field and decomposes these vector fields according to different deformations. For the first-order elastic metric, DCES-PA orthogonally decomposes the first-order derivative of the displacement field by shearing, scaling and bending deformation, and then penalize deformations triggering proliferation formation. For the second-order elastic metric, DCES-PA encodes both intrinsic and extrinsic surface curvatures into the second-order derivative of the displacement field to control the generation of high-curvature regions. By integrating the elastic shape metric with the varifold distances, DCES-PA achieves correspondence-free shape analysis. Extensive experiments on both simulated and real clinical datasets demonstrate that DCES-PA not only shows an improved accuracy than other state-of-the-art shape-based methods applied to proliferation analysis but also produces highly sensitive proliferation annotations to assist physicians in proliferation analysis.

Susceptibility of bovine to SARS-CoV-2 variants of concern: insights from ACE2, AXL, and NRP1 receptors.

The possibilities of cross-species transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) between humans and important livestock species are not yet known. Herein, we used the structural and genetic alignment and surface potential analysis of the amino acid (aa) in angiotensin-converting enzyme 2 (ACE2), tyrosine kinase receptor UFO (AXL), and neuropilin 1 (NRP1) in different species with substantial public health importance. The residues interfacing with the N-terminal domain (NTD) or receptor-binding domain (RBD) of S were aligned to screen the critical aa sites that determined the susceptibility of the SARS-CoV-2 to the host. We found that AXL and NRP1 proteins might be used as the receptors of SARS-CoV-2 in bovines. However, ACE2 protein may not be considered to be involved in the cross-species transmission of SARS-CoV-2 VOCs in cattle because the key residues of the ACE2-S-binding interface were different from those in known susceptible species. This study indicated that emerging SARS-CoV-2 variants potentially expand species tropism to bovines through AXL and NRP1 proteins.

Design of a novel multiepitope vaccine against Chlamydia pneumoniae using the extracellular protein as a target.

Chlamydia pneumoniae (C. pneumoniae) infection in humans is universal and causes various respiratory infectious diseases, making a safe and effective preventive vaccine essential. In this study, a multi-epitope vaccine with CTLA-4 extracellular structure was constructed by an immunoinformatics approach. Since MOMP protein is the major extracellular protein in C. pneumoniae and has good immunogenicity and high conservation, we selected the MOMP protein of C. pneumoniae as the antigen target, predicted the T and B cell epitopes of the MOMP protein and then connected the CTLA-4 extracellular structure with the predicted dominant epitopes by various linkers to construct a multi-epitope vaccine. The biochemical characterization of the multi-epitope vaccine showed its immunogenicity and anti-allergic properties. The tertiary structure of this vaccine, along with molecular docking, molecular dynamics simulation, and principal component analysis, showed that the multi-epitope vaccine structure interacted with B7 (B7-1, B7-2) and toll-like receptors (TLR-2, TLR-4). Ultimately, the vaccine was cloned and effectively expressed in silico on an insect baculovirus expression vector (pFastBac1). These analyses showed that the designed vaccine could potentially target antigen-presenting cells and was immune to C. pneumoniae, which provided novel strategies for developing the vaccine.

Emerging SARS-CoV-2 variants of concern potentially expand host range to chickens: insights from AXL, NRP1 and ACE2 receptors.

BACKGROUND: The possibilities of cross-species transmission of SARS-CoV-2 variants of concern (VOCs) between humans and poultry species are unknown. The analysis of the structure of receptor was used to investigate the potential of emerging SARS-CoV-2 VOCs to expand species tropism to chickens based on the interaction between Spike (S) protein and tyrosine kinase receptor UFO (AXL), angiotensin-converting enzyme 2 (ACE2), and neuropilin 1 (NRP1) with substantial public health importance. METHODS: The structural and genetic alignment and surface potential analysis of the amino acid (aa) in ACE2, AXL, and NRP1 in human, hamster, mouse, mink, ferret, rhesus monkey and chickens were performed by Swiss-Model and pymol software. The critical aa sites that determined the susceptibility of the SARS-CoV-2 to the host were screened by aligning the residues interfacing with the N-terminal domain (NTD) or receptor-binding domain (RBD) of Spike protein. RESULTS: The binding modes of chickens AXL and ACE2 to S protein are similar to that of the ferret. The spatial structure and electrostatic surface potential of NRP1 showed that SARS-CoV-2 VOCs could not invade chickens through NRP1 easily. CONCLUSION: These results suggested that emerging SARS-CoV-2 VOCs potentially expand the host range to chickens mainly through ACE2 and AXL receptors, while NRP1 receptor may rarely participate in the future epidemic of coronavirus disease 2019 in chickens.

BushenHuoxue decoction suppresses M1 macrophage polarization and prevents LPS induced inflammatory bone loss by activating AMPK pathway.

Abnormal bone metabolism and subsequence osteoporotic fractures are common complications of chronic inflammatory diseases. No effective treatment for these bone-related complications is available at present. The chronic inflammatory state in these diseases has been considered as a key factor of bone loss. Therefore, the combination of inflammation inhibition and bone loss suppression may be an important strategy for reducing bone damage associated with inflammatory diseases. Bushen Huoxue Decoction (BSHXD) is a traditional Chinese herbal compound that has demonstrated the ability to improve bone quality and increase bone density. However, the efficacy of BSHXD on inflammatory bone loss and its underlying mechanisms remain unclear. This study aimed to investigate whether BSHXD inhibits inflammatory bone loss in mice and its potential molecular mechanisms. In the present study, the effect of BSHXD on lipopolysaccharide (LPS)-induced M1 polarization of RAW264.7 macrophage and on local inflammatory bone loss model of mouse skull was determined. The results showed that after treating RAW264.7 cells with LPS for 24 h, the expression levels of IL-1ß (39.42 ± 3.076 ng/L, p < 0.05), IL-6 (49.24 ± 1.766 mg/L, p < 0.05) and TNF-α (286.3 ± 27.12 ng/L, p < 0.05) were significantly increased. The addition of BSHXD decreased the expression levels of IL-1ß, IL-6, and TNF-α to 31.55 ± 1.296 ng/L, 37.94 ± 0.8869 mg/L, and 196.4 ± 25.25 ng/L, respectively (p < 0.05). The results of immunofluorescence staining, Western blotting (WB) and flow cytometry indicated that the proportion of M1 macrophages in RAW264.7 cells treated with BSHXD for 24 h was significantly lower than that in the LPS group (13.36% ± 0.9829% VS 24.80% ± 4.619%, p < 0.05). The evidence from in-vitro experiments showed that the immunomodulatory ability of BSHXD may be associated with the activation of AMP-dependent protein kinase (AMPK) pathway in LPS-treated macrophages. In addition, the results of micro-CT, H&E staining, immunohistochemical staining and immunofluorescence staining of mouse skull further demonstrated that BSHXD treatment significantly alleviated LPS-induced local bone loss and inflammatory damage in mouse skull model. All results indicated that BSHXD significantly inhibited inflammatory factors release and M1 polarization of macrophage through AMPK signaling pathway. Therefore, BSHXD may be a promising drug for the treatment of inflammatory bone loss.

Detecting the skewness of data from the five-number summary and its application in meta-analysis.

For clinical studies with continuous outcomes, when the data are potentially skewed, researchers may choose to report the whole or part of the five-number summary (the sample median, the first and third quartiles, and the minimum and maximum values) rather than the sample mean and standard deviation. In the recent literature, it is often suggested to transform the five-number summary back to the sample mean and standard deviation, which can be subsequently used in a meta-analysis. However, if a study contains skewed data, this transformation and hence the conclusions from the meta-analysis are unreliable. Therefore, we introduce a novel method for detecting the skewness of data using only the five-number summary and the sample size, and meanwhile, propose a new flow chart to handle the skewed studies in a different manner. We further show by simulations that our skewness tests are able to control the type I error rates and provide good statistical power, followed by a simulated meta-analysis and a real data example that illustrate the usefulness of our new method in meta-analysis and evidence-based medicine.

Aryl hydrocarbon receptor is a proviral host factor and a candidate pan-SARS-CoV-2 therapeutic target.

The emergence of a series of SARS-CoV-2 variants has necessitated the search for broad-spectrum antiviral targets. The aryl hydrocarbon receptor (AhR) senses tryptophan metabolites and is an immune regulator. However, the role of AhR in SARS-CoV-2 infection and whether AhR can be used as the target of antiviral therapy against SARS-CoV-2 and its variants are yet unclear. Here, we show that infection with SARS-CoV-2 activates AhR signaling and facilitates viral replication by interfering with IFN-I-driven antiviral immunity and up-regulating ACE2 receptor expression. The pharmacological AhR blockade or AhR knockout reduces SARS-CoV-2 and its variants' replication in vitro. Drug targeting of AhR with AhR antagonists markedly reduced SARS-CoV-2 and its variants' replication in vivo and ameliorated lung inflammation caused by SARS-CoV-2 infection in hamsters. Overall, AhR was a SARS-CoV-2 proviral host factor and a candidate host-directed broad-spectrum target for antiviral therapy against SARS-CoV-2 and its variants, including Delta and Omicron, and potentially other variants in the future.

Global expression and functional analysis of human piRNAs during HSV-1 infection.

Piwi-interacting RNAs (piRNAs) are a class of non-coding RNAs that play a key role in spermatogenesis. However, little is known about their expression characterization and role in somatic cells infected with herpes simplex virus type 1 (HSV-1). In this study, we systematically investigated the cellular piRNA expression profiles of HSV-1-infected human lung fibroblasts. Compared with the control group, 69 differentially expressed piRNAs were identified in the infection group, among which 52 were up-regulated and 17 were down-regulated. The changes in the expression of 8 piRNAs were further verified by RT-qPCR with a similar expression trend. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the target genes of piRNAs were mainly involved in antiviral immunity and various human disease-related signaling pathways. Furthermore, we tested the effects of four up-regulated piRNAs on viral replication by transfecting piRNA mimics. The results showed that the virus titers of the group transfected with piRNA-hsa-28,382 (alias piR-36,233) mimic decreased significantly, and that of the group transfected piRNA-hsa-28,190 (alias piR-36,041) mimic significantly increased. Overall, our results revealed the expression characteristics of piRNAs in HSV-1-infected cells. We also screened two piRNAs that potentially regulate HSV-1 replication. These results may promote a better understanding of the regulatory mechanism of pathophysiological changes induced by HSV-1 infection.

Metabolomics Profiles Reveal New Insights of Herpes Simplex Virus Type 1 Infection.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen that can cause significant morbidity, primarily facial cold sores and herpes simplex encephalitis. Previous studies have shown that a variety of viruses can reprogram the metabolic profiles of host cells to facilitate self-replication. In order to further elucidate the metabolic interactions between the host cell and HSV-1, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze the metabolic profiles in human lung fibroblasts KMB17 infected with HSV-1. The results showed that 654 and 474 differential metabolites were identified in positive and negative ion modes, respectively, and 169 and 114 metabolic pathways that might be altered were screened. These altered metabolites are mainly involved in central carbon metabolism, choline metabolism, amino acid metabolism, purine and pyrimidine metabolism, cholesterol metabolism, bile secretion, and prolactin signaling pathway. Further, we confirmed that the addition of tryptophan metabolite kynurenine promotes HSV-1 replication, and the addition of 25-Hydroxycholesterol inhibits viral replication. Significantly, HSV-1 replication was obviously enhanced in the ChOKα (a choline metabolic rate-limiting enzyme) deficient mouse macrophages. These results indicated that HSV-1 induces the metabolic reprogramming of host cells to promote or resist viral replication. Taken together, these observations highlighted the significance of host cell metabolism in HSV-1 replication, which would help to clarify the pathogenesis of HSV-1 and identify new anti-HSV-1 therapeutic targets.

Avicularin alleviates osteoporosis-induced implant loosening by attenuating macrophage M1 polarization via its inhibitory effect on the activation of NF-κB.

Currently, the failure rate for internal fixation in patients with osteoporosis can be reduced by antiosteoporosis therapy alone. However, the administration of anti-osteoporotic drugs is not a complete solution. Therefore, it is necessary to investigate other causes of surgical failure, such as inflammation. In recent years, the inflammation caused by macrophage M1 polarization has garnered wide attention. The purpose of this research is to explore the inhibitory effect of avicularin (AL) on macrophage M1 polarization, by which it ameliorates inflammation, thus alleviating implant instability. We established an osteoporosis mouse model of implant loosening. The mouse tissues were taken out for morphological analysis, staining analysis and bone metabolic index analysis. In in vitro experiments, bone marrow derived macrophages (BMDM) and RAW264.7 cells were polarized to M1 macrophages using lipopolysaccharide (LPS), and analyzed by immunofluorescence (IF) staining, Western blot (WB) and flow cytometry. WB was also used to analyze the nuclear factor kappa-B (NF-κB) pathway. In addition, the expression levels of inflammatory cytokines were detected in cell supernatant using ELISA kits. Through observation of this experiments, we found that AL can inhibit M1 polarization of macrophages. Moreover, it can significantly inhibit the release of inflammatory factors to improve multiple mouse femur parameters. Furthermore, AL inhibited the phosphorylation of IKBα and P65 in the NF-κB pathway. The above data indicate that AL ameliorates inflammatory responses by inhibiting macrophage M1 polarization via its inhibitory effect on the NF-κB pathway, thus alleviating the instability of implants in mice with osteoporosis.

Alterations of serine racemase expression determine proliferation and differentiation of neuroblastoma cells.

Although the role of serine racemase (SR) in neuropsychiatric disorders has been extensively studied, its role in cell proliferation and differentiation remains unclear. Deletion of Srr, the encoding gene for SR, has been shown to reduce dendritic arborization and dendritic spine density in the brains of adult mice, whereas increased SR levels have been associated with differentiation in cell cultures. Previously, we demonstrated that valproic acid induces differentiation in the N2A neuroblastoma cell line, and that this differentiation is associated with increased SR expression. These observations suggest that SR may have a role in cell proliferation and differentiation. We herein found that both valproic acid and all-trans retinoic acid induced N2A differentiation. In contrast, knockdown of SR reduced levels of differentiation, increased N2A proliferation, promoted cell cycle entry, and modulated expression of cell cycle-related proteins. To further evaluate the effects of SR expression on cell proliferation and differentiation, we used an in vivo model of neuroblastoma in nude mice. N2A cells stably expressing scramble shRNA (Srrwt -N2A) or specific Srr shRNA (Srrkd -N2A) were subcutaneously injected into nude mice. The weights and volumes of Srrwt -N2A-derived tumors were lower than Srrkd -N2A-derived tumors. Furthermore, Srrwt -N2A-derived tumors were significantly mitigated by intraperitoneal injection of valproic acid, whereas Srrkd -N2A-derived tumors were unaffected. Taken together, our findings demonstrate for the first time that alterations in SR expression determine the transition between proliferation and differentiation in neural progenitor cells. Thus, in addition to its well-established roles in neuropsychiatric disorders, our study has highlighted a novel role for SR in cell proliferation and differentiation.

Functional analysis and expression profile of human platelets infected by EBV in vitro.

Platelet activation is commonly detected after infection by multiple viruses such as human immunodeficiency virus (HIV), H1N1 influenza, Hepatitis C virus (HCV), Ebola virus (EBV), and Dengue virus (DENV). Non-coding RNAs (ncRNAs) constitute the majority of the human transcribed genome, but the biology of platelet ncRNAs is largely unexplored. In this study, we performed microarray profiling to characterize the expression profile of human platelets infected with EBV in vitro after 2 h. A total of 187 long non-coding RNAs (lncRNAs) displayed differences, of which 114 were upregulated and 73 were downregulated; 78 microRNAs (miRNAs) showed differences, including 73 upregulated and 5 downregulated; 808 mRNAs displayed differences, among which 367 were upregulated and 441 were downregulated. Gene ontology (GO) analysis mostly related to G protein-coupled receptor signaling pathway, detection of chemical stimulus involved in sensory perception of smell and regulation of transcription by RNA polymerase II. Pathway analysis showed that the differentially expressed genes were mainly enriched in cell metabolism and immune-related response. A ceRNA network was established based on predicting regulatory pairs in differentially expressed genes, in which hsa-miR-6877-3p had the highest regulatory capability (degree = 31), FAM230A was the lncRNA with the highest regulatory capability (degree = 28). According to the EBV related miRNA regulation network, it revealed that ebv-miR-BART19-3p had the most target genes and BRWD1, FAM126B, TFRC and JMY were the genes most regulated by EBV-related miRNAs. After overlapping the three networks, we found that the EIFAK2 gene was strongly correlated with autologous ncRNAs, including hsa-miR-1972, hsa-miR-504-3p and hsa-miR-6825-5p, as well as with EBV ncRNAs, including EBER1, EBER2, miR-BART7-3p and miR-BART16. The present study contributes to a better understanding of the expression profiling of ncRNAs and their functions in platelets activated by EBV in vitro, and paves the way to further study on platelet function.

Urolithin B suppressed osteoclast activation and reduced bone loss of osteoporosis via inhibiting ERK/NF-κB pathway.

OBJECTIVES: The main target of current drugs for alleviating bone loss is osteoclasts. However, the long-term application of such drugs will also cause side effects. Therefore, it is of great need to develop new and safer therapeutics for osteoporosis. In recent years, drug development based on gut microbiota has gradually attracted attention. This manuscript investigates the inhibitory effect of urolithin B (UB) on osteoclastogenesis and differentiation in vitro and in ovariectomized (OVX) mice. MATERIALS AND METHODS: CCK-8 was used to analyse the cytotoxicity of UB; BMMs cells were differentiated into osteoclasts by RANKL, and respectively treated with 1, 5, and 25 µmol/L UB during this process. After one week of intervention, tartrate-resistant acid phosphatase (TRAP) staining was used to analyse the number and average area of osteoclasts. F-actin staining and immunofluorescence staining were conducted to evaluate the morphology and function of osteoclasts. Bone resorption function of osteoclasts was detected by Pit Formation Assay. The expression of osteoclast-related protein genes in RAW264.7 cells were investigated via western blot and RT-PCR assays. Western blot analysis of RANKL-mediated activation of MAPK/NF-κB pathway after 0, 5, 15, 30, 60 min of intervention. For in vivo experiments, OVX mice received intraperitoneal injection of 10, 50 mg/kg every two days, 8 weeks later, the femurs of mice were taken for morphological analysis, and the serum content of CTX-1, a bone metabolism index, was analysed. RESULTS: UB could inhibit the osteoclast differentiation of rankl-induced bone marrow macrophages (BMMs) and RAW264.7 cells in vitro, suppress the uptake activity of hydroxyapatite and expression of osteoclast-related gene MMP9, CTSK, NFATc1 and c-fos. Furthermore, UB repressed the rankl-induced phosphorylation and degradation of IκB and the phosphorylation of P65 in the NF-κB pathway of RAW264.7 cells, and also down-regulated the phosphorylation level of ERK in the MAPK pathway. For in vivo studies, UB-treated OVX mice showed more significant improved various parameters of distal femur compared with the control group, with fewer NFATc1, MMP9 and TRAP-positive osteoclasts in bone tissues, and less serum content of CTX-1. CONCLUSION: Urolithin B attenuated bone loss in OVX mice by inhibiting the formation and activation of osteoclasts via down-regulation of the ERK/NF-κB signalling pathway.

Detection of Structural Variations and Fusion Genes in Breast Cancer Samples Using Third-Generation Sequencing.

Background: Structural variations (SVs) are common genetic alterations in the human genome that could cause different phenotypes and diseases, including cancer. However, the detection of structural variations using the second-generation sequencing was limited by its short read length, which restrained our understanding of structural variations. Methods: In this study, we developed a 28-gene panel for long-read sequencing and employed it to Oxford Nanopore Technologies and Pacific Biosciences platforms. We analyzed structural variations in the 28 breast cancer-related genes through long-read genomic and transcriptomic sequencing of tumor, para-tumor, and blood samples in 19 breast cancer patients. Results: Our results showed that some somatic SVs were recurring among the selected genes, though the majority of them occurred in the non-exonic region. We found evidence supporting the existence of hotspot regions for SVs, which extended our previous understanding that they exist only for single nucleotide variations. Conclusion: In conclusion, we employed long-read genomic and transcriptomic sequencing to identify SVs from breast cancer patients and proved that this approach holds great potential in clinical application.

Whole genome analysis identifies intra-serotype recombinants and positive selection sites of dengue virus in mainland China from 2015 to 2020.

Immune pressure can select for escape mutants that can become epidemiologically relevant. Thus, surveillance of recombinants and positively selected mutants of the dengue virus (DENV) are vital for preventing and controlling the dengue fever outbreak. However, little is known about recombinants and positively selected mutants of circulating DENV strains in mainland China. In this study, those variants with recombination and adaptive evolutionary sites of circulating DENV strains were identified during 2015-2020. Phylogenetic analysis showed that the DENV-2 was the dominant epidemic serotype, and the dengue epidemic in China was closely related to the imported virus from Southeast Asian countries. Recombination analysis based on 291 complete genomes of naturally circulating DENV identified 10 new intra-serotype recombinant variants. Two or three recombination regions in a single dengue isolate were also observed. The breakpoints of recombinants were distributed in different regions of the genome. In particular, two recombinant strains (strain DENV-4/China/YN/15DGR394 (2015) and XLLM10666) with extremely large exchange fragments were detected. This large-scale gene fragment exchange (eight genomic regions) of strain DENV-4/China/YN/15DGR394 (2015) with substitutions at both the 5' and 3' ends of the genome, had never been described before. Moreover, selection pressure analyses revealed seven positive selection sites located in regions encoding the NS1, NS3 and NS5 proteins. Overall, this study is the first to report ten specific intra-serotype recombinants and seven positive selection sites of Chinese epidemic strains of DENV, which highlight their significance for DENV surveillance and effective control.

Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway.

BACKGROUND: The inhibition of osteogenic differentiation is a major factor in glucocorticoid-induced bone loss, but there is currently no effective treatment. Dopamine, a major neurotransmitter, transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. Although the relevance of the neuroendocrine system in bone metabolism has emerged, the precise effects of dopamine receptor signaling on osteoblastogenesis remain unknown. METHODS: In vitro, western blotting and immunofluorescence staining were used to observe the expression of dopamine receptors in MC3T3-E1 and BMSCs cells treated with dexamethasone (Dex). In addition, Alizarin red S (ARS) and alkaline phosphatase (ALP) staining and western blotting were used to evaluate the effect of D1R activation on osteogenic differentiation in Dex-induced MC3T3-E1 cells via the ERK1/2 signaling pathway. In vivo, micro-CT and hematoxylin and eosin (H&E), toluidine blue and immunohistochemical staining were used to determine the effect of D1R activation on Dex-induced bone loss. RESULTS: We demonstrated that the trend in D1R but not D2-5R was consistent with that of osteogenic markers in the presence of Dex. We also demonstrated that the activation of D1R promoted Dex-induced osteogenic differentiation by activating the ERK1/2 pathway in vitro. We further demonstrated that a D1R agonist could reduce Dex-induced bone loss, while pretreatment with a D1R inhibitor blocked the effect of a D1R agonist in vivo. CONCLUSIONS: Activation of D1R promotes osteogenic differentiation and reduces Dex-induced bone loss by activating the ERK1/2 pathway. Hence, D1R could serve as a potential therapeutic target for glucocorticoid-induced osteoporosis.

Meta-analysis with zero-event studies: a comparative study with application to COVID-19 data.

BACKGROUND: Meta-analysis is a statistical method to synthesize evidence from a number of independent studies, including those from clinical studies with binary outcomes. In practice, when there are zero events in one or both groups, it may cause statistical problems in the subsequent analysis. METHODS: In this paper, by considering the relative risk as the effect size, we conduct a comparative study that consists of four continuity correction methods and another state-of-the-art method without the continuity correction, namely the generalized linear mixed models (GLMMs). To further advance the literature, we also introduce a new method of the continuity correction for estimating the relative risk. RESULTS: From the simulation studies, the new method performs well in terms of mean squared error when there are few studies. In contrast, the generalized linear mixed model performs the best when the number of studies is large. In addition, by reanalyzing recent coronavirus disease 2019 (COVID-19) data, it is evident that the double-zero-event studies impact the estimate of the mean effect size. CONCLUSIONS: We recommend the new method to handle the zero-event studies when there are few studies in a meta-analysis, or instead use the GLMM when the number of studies is large. The double-zero-event studies may be informative, and so we suggest not excluding them.

The Effect of Immunosuppressive Adjuvant Kynurenine on Type 1 Diabetes Vaccine.

Inducing antigen-specific tolerance is a promising treatment for preventing or reversing Type 1 diabetes (T1D). In contrast to a vaccine that induces immune responses against pathogens, a tolerogenic vaccine can suppress immunity against antigens causing diseases by administrating a mixture of self-antigens with an adjuvant that decreases the strength of antigen-specific response. Kynurenine (Kyn) is an endogenous substance that can inhibit the natural killer cell and T cell proliferation and promote the differentiation of naïve T cells into regulatory T cells (Tregs). In this study, we evaluated the efficacy of Kyn as a novel suppressive adjuvant. Kyn was co-immunized with GAD65 phage vaccine to induce Treg cells and tolerogenic responses for the prevention of T1D in NOD mouse model. Mice were subcutaneously immunized two times with 1011 Pfu (100µL,1012 Pfu/ml) GAD65 phage vaccine doses mixed with 200 µg of Kyn. Serum antibodies and cytokines were detected by ELISA and electrochemiluminescence, respectively. Flow cytometry assay was used to analyze DC and Treg. MTS was used for the analysis of spleen lymphocyte proliferation. RNA sequencing was used to investigate mRNA and miRNA expression profiles in spleen lymphocytes. Compared to GAD65 phage vaccine alone, co-immunization of Kyn and GAD65 phage vaccine resulted in the prevention of hyperglycemia in 60% of mice for at least one month. Further, Kyn enhances GAD65-specific Th2-mediated immune responses; regulates the Th1/Th2 imbalance and increases the secretion of Th2 cytokines and the number of CD4+CD25+Foxp3+T cells; suppresses DC maturation and GAD65-specific T lymphocyte proliferation. Moreover, we integrated Kyn related miRNA and mRNA expression profiles obtained from the spleen lymphocyte RNA-sequencing which was stimulated by Kyn in vitro. These data provide an important basis for understanding the mechanisms underlying Kyn as an immunosuppressive adjuvant which regulated the immune response. These findings suggest that Kyn can serve as an effective suppressive adjuvant candidate for Type 1 diabetes vaccines.

Bioinformatics Analysis of Neuroblastoma miRNA Based on GEO Data.

OBJECTIVE: To analyze the changes in downstream genes, signaling pathways, and proteins based on the difference of microRNA (miRNA) expression in neuroblastoma (NB). METHODS: GSE128004 second-generation sequencing expression data were downloaded from GEO, and Limma package of R language was used to analyze differential expression, and a volcano map and heat map were drawn; the target genes corresponding to the differential miRNA were found using the miWalk web tool, and GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were performed. The key genes were identified and verified in the TCGA database. RESULTS: A total of 34 differentially expressed miRNAs were screened out. Among them, 22 up-regulated miRNAs predicted 1163 target genes and 12 down-regulated miRNAs predicted 1474 target genes. Target genes were enriched and analyzed by KEGG to find the FOXO signal pathway, mTOR signal pathway, AMPK signal pathway, and other signal pathways. After GO analysis, axon formation, regulation of chemical synaptic transmitters, regulation of nerve synapses, regulation of cross-synaptic signals, and other physiological processes were assessed. A total of 16 key genes were obtained by PPI analysis, and the survival analysis of TP53 and ATM genes verified in the TCGA database showed statistical significance. CONCLUSION: The 34 differential miRNAs may be related to the occurrence and development of NB. TP53 and ATM are related to the prognosis of NB. The role and mechanism of TP53 and ATM in NB need to be further verified.