Direct Observation of Covalently Bound Clusters in Resonantly Stabilized Radical Reactions and Implications for Carbonaceous Particle Growth.

Based on quantum mechanically guided experiments that observed elusive intermediates in the domain of inception that lies between large molecules and soot particles, we provide a new mechanism for the formation of carbonaceous particles from gas-phase molecular precursors. We investigated the clustering behavior of resonantly stabilized radicals (RSRs) and their interactions with unsaturated hydrocarbons through a combination of gas-phase reaction experiments and theoretical calculations. Our research directly observed a sequence of covalently bound clusters (CBCs) as key intermediates in the evolution from small RSRs, such as benzyl (C7H7), indenyl (C9H7), 1-methylnaphthyl (1-C11H9), and 2-methylnaphthyl (2-C11H9), to large polycyclic aromatic hydrocarbons (PAHs) consisting of 28 to 55 carbons. We found that hydrogen abstraction and RSR addition drive the formation and growth of CBCs, leading to progressive H-losses, the generation of large PAHs and PAH radicals, and the formation of white smoke (incipient carbonaceous particles). This mechanism of progressive H-losses from CBCs (PHLCBC) elucidates the crucial relationship among RSRs, CBCs, and PAHs, and this study provides an unprecedentedly seamless path of observed assembly from small RSRs to large nanoparticles. Understanding the PHLCBC mechanism over a wide temperature range may enhance the accuracy of multiscale models of soot formation, guide the synthesis of carbonaceous nanomaterials, and deepen our understanding of the origin and evolution of carbon within our galaxy.

Screening, expression and anti-tumor functional identification of anti-LAG-3 nanobodies.

OBJECTIVE: To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells. METHODS: Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjµgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc. RESULTS: One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 µg/mL. CONCLUSION: The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.

Cas-based bacterial detection: recent advances and perspectives.

Persistent bacterial infections pose a formidable threat to global health, contributing to widespread challenges in areas such as food safety, medical hygiene, and animal husbandry. Addressing this peril demands the urgent implementation of swift and highly sensitive detection methodologies suitable for point-of-care testing and large-scale screening. These methodologies play a pivotal role in the identification of pathogenic bacteria, discerning drug-resistant strains, and managing and treating diseases. Fortunately, new technology, the CRISPR/Cas system, has emerged. The clustered regularly interspaced short joint repeats (CRISPR) system, which is part of bacterial adaptive immunity, has already played a huge role in the field of gene editing. It has been employed as a diagnostic tool for virus detection, featuring high sensitivity, specificity, and single-nucleotide resolution. When applied to bacterial detection, it also surpasses expectations. In this review, we summarise recent advances in the detection of bacteria such as Mycobacterium tuberculosis (MTB), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), Salmonella and Acinetobacter baumannii (A. baumannii) using the CRISPR/Cas system. We emphasize the significance and benefits of this methodology, showcasing the capability of diverse effector proteins to swiftly and precisely recognize bacterial pathogens. Furthermore, the CRISPR/Cas system exhibits promise in the identification of antibiotic-resistant strains. Nevertheless, this technology is not without challenges that need to be resolved. For example, CRISPR/Cas systems must overcome natural off-target effects and require high-quality nucleic acid samples to improve sensitivity and specificity. In addition, limited applicability due to the protospacer adjacent motif (PAM) needs to be addressed to increase its versatility. Despite the challenges, we are optimistic about the future of bacterial detection using CRISPR/Cas. We have already highlighted its potential in medical microbiology. As research progresses, this technology will revolutionize the detection of bacterial infections.

Hepatitis C virus subtype diversity and transmission clusters characteristics among drug users in Zhuhai, South China.

BACKGROUND: Hepatitis C virus (HCV) infection poses a major public health challenge globally, especially among injecting drug users. China has the world's largest burden of HCV infections. However, little is known about the characteristics of transmission networks among drug user populations. This study aims to investigate the molecular epidemiology and transmission characteristics of HCV infections among drug users in Zhuhai, a bustling port city connecting Mainland China and its Special Administrative Regions. METHODS: Participants enrolled in this study were drug users incarcerated at Zhuhai's drug rehabilitation center in 2015. Their sociodemographic and behavioral information, including gender, promiscuity, drug use method, and so forth, was collected using a standardized questionnaire. Plasmas separated from venous blood were analyzed for HCV infection through ELISA and RT-PCR methods to detect anti-HCV antibodies and HCV RNA. The 5'UTR fragment of the HCV genome was amplified and further sequenced for subtype identifications and phylogenetic analysis. The phylogenetic tree was inferred using the Maximum Likelihood method based on the Tamura-Nei model, and the transmission cluster network was constructed using Cytoscape3.8.0 software with a threshold of 0.015. Binary logistic regression models were employed to assess the factors associated with HCV infection. RESULTS: The overall prevalence of HCV infection among drug users was 44.37%, with approximately 19.69% appearing to clear the HCV virus successfully. Binary logistic regression analysis revealed that those aged over 40, engaging in injecting drug use, and being native residents were at heightened risk for HCV infection among drug user cohorts. The predominant HCV subtypes circulating among those drug users were 6a (60.26%), followed by 3b (16.7%), 3a (12.8%), 1b (6.41%) and 1a (3.85%), respectively. Molecular transmission network analysis unveiled the presence of six transmission clusters, with the largest propagation cluster consisting of 41 individuals infected with HCV subtype 6a. Furthermore, distinct transmission clusters involved eight individuals infected with subtype 3b and seven with subtype 3a were also observed. CONCLUSION: The genetic transmission networks revealed a complex transmission pattern among drug users in Zhuhai, emphasizing the imperative for a targeted and effective intervention strategy to mitigate HCV dissemination. These insights are pivotal for shaping future national policies on HCV screening, treatment, and prevention in port cities.

Spectrophotometric Detection of the BRCA1 Gene via Exponential Isothermal Amplification and Hybridization Chain Reaction of Surface-Bound Probes.

In this work, we demonstrated an ultrasensitive approach with a dual-amplification strategy for DNA assay based on isothermal exponential amplification (EXPAR) and the hybridization chain reaction (HCR). In the presence of target DNA, the hairpin probe DNA (HP1) recognized and partially hybridized with the target DNA to form double-stranded structures containing the full recognition sequences for nicking endonuclease and then initiated EXPAR. Under the reaction of EXPAR, a large number of single-stranded DNA (ssDNA) was produced in the circle of nicking, polymerization, and strand displacement. The resulting ssDNA can bind to the surface-bound probe on the well of the microplate and trigger the hybridization chain reaction, resulting in the production of numerous double-stranded DNA concatamers with biotin labeling. In the presence of streptavidin-conjugated horseradish peroxidase (HRP), the amplified signal can be detected by a spectrophotometer via HRP-catalyzed substrate 3,3'5,5'-tetramethylbenzidine (TMB). This proposed dual-amplification method provides a detection limit of 74.48 aM, which also exhibits good linearity ranging from 0.1 fM to 100 pM.

The underlying molecular mechanisms and prognostic factors of RNA binding protein in colorectal cancer: a study based on multiple online databases.

BACKGROUND: RNA binding protein (RBP) is an active factor involved in the occurrence and development of colorectal cancer (CRC). Therefore, the potential mechanism of RBP in CRC needs to be clarified by dry-lab analyses or wet-lab experiments. METHODS: The differential RBP gene obtained from the GEPIA 2 (Gene Expression Profiling Interactive Analysis 2) were performed functional enrichment analysis. Then, the alternative splicing (AS) events related to survival were acquired by univariate regression analysis, and the correlation between RBP and AS was analyzed by R software. The online databases were conducted to analyze the mutation and methylation of RBPs in CRC. Moreover, 5 key RBP signatures were obtained through univariate and multivariate Cox regression analysis and established as RBP prognosis model. Subsequently, the above model was verified through another randomized group of TCGA CRC cohorts. Finally, multiple online databases and qRT-PCR analysis were carried to further confirm the expression of the above 5 RBP signatures in CRC. RESULTS: Through a comprehensive bioinformatics analysis, it was revealed that RBPs had genetic and epigenetic changes in CRC. We obtained 300 differentially expressed RBPs in CRC samples. The functional analysis suggested that they mainly participated in spliceosome. Then, a regulatory network for RBP was established to participate in AS and DDX39B was detected to act as a potentially essential factor in the regulation of AS in CRC. Our analysis discovered that 11 differentially expressed RBPs with a mutation frequency higher than 5%. Furthermore, we found that 10 differentially expressed RBPs had methylation sites related to the prognosis of CRC, and a prognostic model was constructed by the 5 RBP signatures. In another randomized group of TCGA CRC cohorts, the prognostic performance of the 5 RBP signatures was verified. CONCLUSION: The potential mechanisms that regulate the aberrant expression of RBPs in the development of CRC was explored, a network that regulated AS was established, and the RBP-related prognosis model was constructed and verified, which could improve the individualized prognosis prediction of CRC.

Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells.

BACKGROUND: Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells. METHODS: CD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function. RESULTS: The nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%. CONCLUSIONS: Nanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Upregulation of TIGIT and PD-1 in Colorectal Cancer with Mismatch-repair Deficiency.

Background: The role of T cell Ig and ITIM domain (TIGIT) and programmed cell death-1 (PD-1) in colorectal cancer (CRC) with mismatch repair deficiency is unknown.Methods: This was a study of 60 CRC patients with mismatch repair deficiency and 30 healthy controls between June 2015 and October 2015.Results: The expression of Foxp3, PD-1, and TIGIT was higher in cancer tissues compared with adjacent mucosa (all P < .05). Patients with advanced TNM stage had a significantly higher expression of TIGIT (P = .025) and PD-1 (P = .020) than patients with early-stage CRC. The disease-free survival (DFS) of patients with high TIGIT (HR = 3.96, 95%CI: 1.34-11.69, P = .013) or PD-1 (HR = 214.8, 95%CI: 49.88-925.2, P < .001) expression were better. The overall survival (OS) of the patients with CRC and high expression of PD-1 was worse than those with low expression (HR = 4.01, 95%CI:1.26-12.69, P = .019).Conclusion: TIGIT and PD-1 are upregulated in CRC with mismatch repair deficiency and associated with TNM stage and DFS.

MiR-129-3p favors intracellular BCG survival in RAW264.7 cells by inhibiting autophagy via Atg4b.

Autophagy plays an important role in the fight against Mycobacterium tuberculosis infection. Massive researches proved that miRNAs could be the regulators of autophagy, which implied miRNAs could favor MTB invasion or latent infection. In our study, multiple bioinformatics databases and software were used to seek and lock the miRNAs associating with regulation of autophagy. Notably, a novel miR-129-3p was found and its target gene Atg4b showed grand potential in mediation of autophagy. Moreover, BCG infection triggered miR-129-3p overexpression in RAW264.7 cells. Up-regulation of miR-129-3p decreased mRNA or protein level of Atg4b and resulted in the inhibition of autophagy. The antagomir of miR-129-3p had the opposite impact. The LC3 puncta formation in RAW264.7 cells were also affected after transfection of miR-129-3p mimic or antagomir. The mRFP-GFP-LC3 analysis indicated that mimic of miR-129-3p impaired autophagic flux while antagomir improved autophagy. The CFU assay results showed that miR-129-3p promoted the intracellular survival of BCG in macrophages. Consequently, these data suggested that miR-129-3p could favor MTB survival by inhibiting autophagy via Atg4b.

Development and In Vitro Release of Isoniazid and Rifampicin-Loaded Bovine Serum Albumin Nanoparticles.

BACKGROUND Bovine serum albumin nanoparticles loaded with isoniazid and rifampicin (INH-RFP-BSA-NPs) were prepared and their release characteristics were studied in vitro. MATERIAL AND METHODS The INH-RFP-BSA-NPs were prepared by a modified self-emulsion solvent diffusion method, with albumin and polylactic acid used as carriers and to form the nanoparticles structure. Transmission electron microscopy was used to observe the morphology of the INH-RFP-BSA-NPs. The size distribution of the INH-RFP-BSA-NPs were assessed using a submicron particle-size analyzer for drug loadings, and the coating rate of the INH-RFP-BSA-NPs was measured by high-performance liquid chromatography. A dynamic membrane dialysis method was used to study the in vitro release characteristics of the INH-RFP-BSA-NPs. RESULTS The INH-RFP-BSA-NPs were smooth, sphere-like, relatively uniform in size, and well-dispersed, and the average diameter was 60.5±4.6 nm. Drug loading and entrapment efficiencies were high, at 19.8% and 87.8% for isoniazid, respectively, and 20.1% and 98.0% for rifampicin, respectively. Drug release was slow and sustained with 97.02% INH cumulative release at 6 days, and full release of RFP requiring 5 days. CONCLUSIONS INH-RFP-BSA-NPs exhibit uniform NP diameter, good dispersion, high drug loading and encapsulation rates, and have sustained release properties.

Immunologic properties and therapeutic efficacy of a multivalent epitope-based vaccine against four Helicobacter pylori adhesins (urease, Lpp20, HpaA, and CagL) in Mongolian gerbils.

BACKGROUND: Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and adhesins, which are on the surface of H. pylori, play a pivotal role in binding to human gastric mucosa. MATERIALS AND METHODS: In the present study, we constructed a multivalent epitope-based vaccine named CFAdE with seven carefully selected antigenic fragments from four H. pylori adhesins (urease, Lpp20, HpaA and CagL). The specificity, immunogenicity and ability to produce neutralizing antibodies of CFAdE were evaluated in BALB/c mice. After that, its therapeutic efficacy and protective immune mechanisms were explored in H. pylori-infected Mongolian gerbils. RESULTS: The results indicated that CFAdE could induce comparatively high levels of specific antibodies against urease, Lpp20, HpaA and CagL. Additionally, oral therapeutic immunization with CFAdE plus polysaccharide adjuvant (PA) significantly decreased H. pylori colonization compared with oral immunization with urease plus PA, and the protection was correlated with IgG and sIgA antibody and antigen-specific CD4+ T cells. CONCLUSIONS: This study indicated that the multivalent epitope-based vaccine, which targeted multiple adhesins in adherence of H. pylori to the gastric mucosa, is more effective than the univalent vaccine targeting urease only. This multivalent epitope-based vaccine may be a promising therapeutic candidate vaccine against H. pylori infection.

MicroRNA-149 negatively regulates TLR-triggered inflammatory response in macrophages by targeting MyD88.

MicroRNAs (miRNAs) have been shown to be important regulators of TLR signaling pathway at the post-transcriptional level. In this study, the potential role of miR-149 was explored in murine alveolar macrophage RAW264.7 cells. Our results demonstrated a dynamic change of the expressions of miR-149 expression and MyD88 in macrophage RAW264.7 upon Mycobacterium bovis Bacillus Calmette-Guerlin (BCG) infection or lipopolysaccharide (LPS) stimulation. The presence of BCG or LPS dynamically reduced the miR-149 expression, along with a substantially striking increase of MyD88 expression in these cells. More importantly, overexpression of miR-149 in RAW264.7 cells was associated with a significant decrease of MyD88 protein expression, as well as a reduced production of inflammatory mediator NF-κB 1, TNF-α and IL-6 in response to BCG infection or LPS stimulation. Further studies using immunoblotting assay against anti-MyD88 antibody and microRNA targeting luciferase reporter assay revealed that miR-149 was able to directly target the 3'-UTR of MyD88 mRNA and post-transcriptionally regulated MyD88 protein expression. These data suggested that miR-149 might be a key player of immune modulator for TLR/MyD88 signaling pathway in macrophages, which may through a mechanism of negatively regulating MyD88-dependent Toll-like receptors signaling pathway.

MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3.

BACKGROUND: microRNA 21 (miR-21) has been demonstrated to be significantly elevated in many types of cancers, including the hepatocellular carcinoma (HCC). In the present study, we investigated the role of miR-21 in HCC by identifying its novel targets, as well as its underlying molecular mechanism. METHODS: The expression of mitogen-activated protein kinase-kinase 3 (MAP2K3) in human HCC tumor tissues and adjacent non-tumor tissues was determined by immunohistochemistry staining (IHC) analysis. The 3'-untranslated region (3'-UTR) of MAP2K3 combined with miR-21 was experimentally verified by a miRNA luciferase reporter approach. Moreover, the role of miR-21 in regulating HCC cell proliferation was analyzed by an MTT assay infected with miR-21mimics/sponge inhibitor Adenoviral viral vectors. RESULTS: By immunohistochemistry staining analysis, we found that mitogen-activated protein kinase-kinase 3 (MAP2K3) was strikingly repressed in the human HCC tumor tissues, in comparison with the adjacent non-tumor tissues in clinical settings. More importantly, the repression of MAP2K3 was inversely correlated with the expression of miR-21 in HCC. Further study demonstrated that the MAP2K3 was a novel direct target of miR-21, which was experimentally validated by a miRNA luciferase reporter approach. In HepG2 cells, inhibition of miR-21 expression with an adenoviral miR-21 sponge vector profoundly suppressed cell proliferation by up-regulating MAP2K3 expression at both mRNA and protein levels. CONCLUSIONS: These results provide a clinical evidence that MAP2K3 may be a tumor repressor gene, and it is a direct target of miR-21 in HCC, indicating an underlying mechanism by which miR-21 is able to directly target MAP2K3 and inhibit its expression during the carcinogenesis of HCC, at both transcriptional and post-translational levels. This study also suggests that targeting miR-21-MAP2K3 pathway may be a promising strategy in the prevention and treatment of HCC.

Synthesis of Polyvinyl Alcohol/Coal Fly Ash Hybrid Nano-Fiber Membranes for Adsorption of Heavy Metals in Diesel Fuel.

Some studies have shown that the heavy metal emissions (HMEs) emitted from diesel engines can seriously threaten human health. HMEs are mainly related to the content of heavy metal ions in diesel fuel. Therefore, in order to reduce HMEs from diesel engines, a nano-fiber membrane filtration technology for diesel fuel was investigated. Herein, coal fly ash (CFA) from coal-fired power plants combined with polyvinyl alcohol (PVA) was successfully fabricated into nano-fibrous membranes using green electrospinning technology. In order to evaluate the adsorption properties, various hybrid membranes with different mixing ratios (PVA/CFA = 10/0, 10/1, 10/3, 10/5, and 10/7 by weight) were fabricated. The results show that eight metal ions with different concentrations are found in the diesel fuel, including Pb, Cu, Zn, Al, Fe, Cr, Ba, and Ni. All PVA/FA membranes have different adsorption capacities for metal ions, following the order: Cu > Fe > Pb > Al > Zn > Cr > Ba > Ni. In addition, the adsorption capacity of CFA3 (PVA/CFA = 10/3) is the largest. The super lipophilicity of the PVA/FA membranes also provide more adsorption sites for the contact of HMs with the membranes. The above research results provide guidance for development of ultra-fine filters in the future.

Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.

Background: The present study aimed to investigate the regulation of miR-25-3p on macrophage autophagy and its effect on macrophage clearance of intracellular Mycobacterium bovis Bacillus Calmette-Guerin (BCG) retention based on the previous findings on the differential expression of exosomal miRNA in macrophages infected with BCG. Methods: Through enrichment analysis and Hub gene analysis, key differentially expressed miRNA and its target genes were selected. The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the TargetScan database, and this was further verified by dual luciferase reporter gene assay. mmu-miR-25-3p mimics, mmu-miR-25-3p inhibitor, si-DUSP10, miR-NC,si-NC and PD98059 (ERK Inhibitor) were used to intervene macrophages Raw264.7. Rt-qPCR was used to detect the expression levels of mmu-miR-25-3p and DUSP10 mRNA. Western blot was used to detect the expression levels of DUSP10, LC3-II, p-ERK1/2, beclin1, Atg5 and Atg7. The autophagy flux of macrophage Raw264.7 in each group was observed by confocal laser microscopy, and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy. Finally, the intracellular BCG load of macrophage Raw264.7 was evaluated by colony-forming unit (CFU) assay. Results: Bioinformatics analysis filtered and identified the differentially expressed exosomal miRNAs. As a result, mmu-miR-25-3p expression was significantly increased, and dual specificity phosphatase 10 (DUSP10) was predicted as its target gene that was predominantly involved in autophagy regulation. The dual luciferase reporter gene activity assay showed that mmu-miR-25-3p was targeted to the 3'-untranslated region (UTR) of DUSP10. The infection of BCG induced the upregulation of mmu-miR-25-3p and downregulation of DUSP10 in RAW264.7 cells, which further increased the expression of LC3-II and promoted autophagy. Upregulated mmu-miR-25-3p expression decreased the level of DUSP10 and enhanced the phosphorylation of ERK1/2, which in turn upregulated the expression of LC3-II, Atg5, Atg7, and Beclin1. Immuno-electron microscopy, transmission electron microscopy, and autophagic flux analysis further confirmed that the upregulation of mmu-miR-25-3p promotes the autophagy of macrophages after BCG infection. The CFU number indicated that upregulated mmu-miR-25-3p expression decreased the mycobacterial load and accelerated residual mycobacteria clearance. Conclusion: mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thus enhancing the BCG-induced autophagy of macrophages. These phenomena reduce the bacterial load of intracellular Mycobacterium and facilitate the clearance of residual mycobacteria. mmu-miR-25-3p has great potential as a target for anti-tuberculosis immunotherapy and can be the optimal miRNA loaded into exosomal drug delivery system in future studies.

LncRNA DANCR restrained the survival of mycobacterium tuberculosis H37Ra by sponging miR-1301-3p/miR-5194.

Tuberculosis is a worldwide contagion caused by Mycobacterium tuberculosis (MTB). MTB is characterized by intracellular parasitism and is semi-dormant inside host cells. The persistent inflammation caused by MTB can form a granuloma in lesion regions and intensify the latency of bacteria. In recent years, several studies have proven that long non-coding RNAs (lncRNAs) play critical roles in modulating autophagy. In our study, the Gene Expression Omnibus (GEO) databases were searched for lncRNAs that are associated with tuberculosis. We found that lncRNA differentiation antagonizing non-protein coding RNA (DANCR) increased in the peripheral blood samples collected from 54 pulmonary tuberculosis patients compared to 23 healthy donors. By constructing DANCR overexpression cells, we analyzed the possible cellular function of DANCR. After analyzing our experiments, it was found that the data revealed that upregulation of DANCR facilitated the expression of signal transducer and activator of transcription 3, autophagy-related 4D cysteine peptides, autophagy-related 5, Ras homolog enriched in the brain, and microtubule-associated protein 1A/1B light chain 3 (STAT3, ATG4D, ATG5, RHEB, and LC3, respectively) by sponging miR-1301-3p and miR-5194. Immunofluorescence analysis indicated that DANCR played a positive role in both autophagosome formation and fusion of autolysosomes in macrophages. The colony-forming unit (CFU) assay data also showed that the cells overexpressing DANCR were more efficient in eliminating the intracellular H37Ra strain. Consequently, these data suggest that DANCR restrained intracellular survival of M. tuberculosis by promoting autophagy via miR-1301-3p and miR-5194.

Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model.

Epitope vaccine based on the enzyme urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In our previous study, the epitope vaccine CTB-UA, which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA183₋203) from the H. pylori urease A subunit (UreA) was constructed. This particular vaccine was shown to have good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies, which exhibited effectively inhibitory effects on the enzymatic activity of H. pylori urease. In this study, the prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA was evaluated in a BALB/c mice model. The experimental results indicated that oral prophylactic or therapeutic immunization with CTB-UA significantly decreased H. pylori colonization compared with oral immunization with PBS. The results also revealed that the protection was correlated with antigen-specific IgG, IgA, and mucosal secretory IgA antibody responses. CTB-UA may be a promising vaccine candidate for the control of H. pylori infection.

Potential and functional prediction of six circular RNAs as diagnostic markers for colorectal cancer.

Background: Circular RNAs (circRNAs) have been discovered in colorectal cancer (CRC), but there are few reports on the expression distribution and functional mining analysis of circRNAs. Methods: Differentially expressed circRNAs in CRC tissues and adjacent normal tissues were screened and identified by microarray and qRT-PCR. ROC curves of the six circRNAs were analyzed. A series of bioinformatics analyses on differentially expressed circRNAs were performed. Results: A total of 207 up-regulated and 357 down-regulated circRNAs in CRC were screened, and three top up-regulated and down-regulated circRNAs were chosen to be verified in 33 pairs of CRCs by qRT-PCR. 6 circRNAs showed high diagnostic values (AUC = 0.6860, AUC = 0.8127, AUC = 0.7502, AUC = 0.9945, AUC = 0.9642, AUC = 0.9486 for hsa_circRNA_100833, hsa_circRNA_103828, hsa_circRNA_103831 and hsa_circRNA_103752, hsa_circRNA_071106, hsa_circRNA_102293). A circRNA-miRNA-mRNA regulatory network (cirReNET) including six candidate circRNAs, 19 miRNAs and 210 mRNA was constructed, and the functions of the cirReNET were predicted and displayed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on these mRNAs and protein-protein interaction (PPI) network of the hub genes acquired by string and CytoHubba. Conclusion: A cirReNET containing potential diagnostic and predictive indicators of CRCs and several critical circRNA-miRNA-mRNA regulatory axes (cirReAXEs) in CRC were mined, and may provide a novel route to study the mechanism and clinical targets of CRC.

Screening and identification of an anti-PD-1 nanobody with antitumor activity.

Blocking of PD-1 or PD-L1 with corresponding antibody to enhance T cell response and mediate antitumor activity has been successfully applied in clinical practice. Several immune checkpoint inhibitors including monoclonal antibodies targeting PD-1 have been approved by the Food and Drug Administration (FDA) in cancer immunotherapy. However, the application of traditional antibodies has limited due to their drawbacks of large molecular weight and low tissue penetration. As the high specificity and strong tissue penetration of nanobodies (Nbs), efforts have been taken to develop Nbs for cancer therapy. Herein, we aim to screen a specific Nb against human PD-1 derived from a naïve camel Nb phage display library and further study its biological characteristic and anti-tumor activity. Finally, an anti-PD-1 Nb with high specificity and affinity was screened and generated, its cytotoxicity and antitumor effect was also confirmed in vitro and vivo. All of these indicate that the anti-PD-1 Nb may provide an alternative and appealing therapeutic agent for cancer immunotherapy.

Spermidine Promotes Nb CAR-T Mediated Cytotoxicity to Lymphoma Cells Through Elevating Proliferation and Memory.

Purpose: Due to the natural advantages of spermidine in immunity, we investigated the effects of spermidine pretreatment on nanobody-based CAR-T cells (Nb CAR-T) mediated cytotoxicity and potential mechanism. Patients and Methods: The optimal concentration of spermidine was determined by detecting its impact on viability and proliferation of T cells. The phenotypic characteristic of CAR-T cells, which were treated with spermidine for 4 days, was examined by flow cytometry. The expansion ability of CAR-T cells was monitored in being cocultured with tumor cells. Additionally, CAR-T cells were stimulated by lymphoma cells to test its cytotoxicity in vitro, and the supernatant in co-culture models were collected to test the cytokine production. Furthermore, xenograft models were constructed to detect the anti-tumor activity of CAR-T cells in vivo. Results: The optimal concentration of spermidine acting on T cells was 5µM. The antigen-dependent proliferation of spermidine pretreatment CD19 CAR-T cells or Nb CAR-T cells was increased compared to control. Central memory T cells(TCM) dominated the CAR-T cell population in the presence of spermidine. When spermidine pretreatment CAR-T cells were stimulated with Daudi cells, the secretion of IL-2 and IFN-γ has been significantly enhanced. The ability of CAR-T cells to lysis Daudi cells was enhanced with the help of spermidine, even at higher tumor loads. Pre-treated Nb CAR-T cells with spermidine were able to control tumor cells in vivo, and therefore prolong mice survival. Conclusion: Our results revealed that spermidine could promote Nb CAR-T mediated cytotoxicity to lymphomas cells through enhancing memory and proliferation, and provided a meaningful approach to strengthen the anti-tumor effect of CAR-T cells.