Ferroptosis: a new hunter of hepatocellular carcinoma.

Ferroptosis is an iron ion-dependent, regulatory cell death modality driven by intracellular lipid peroxidation that plays a key role in the development of HCC. Studies have shown that various clinical agents (e.g., sorafenib) have ferroptosis inducer-like effects and can exert therapeutic effects by modulating different key factors in the ferroptosis pathway. This implies that targeting tumor cell ferroptosis may be a very promising strategy for tumor therapy. In this paper, we summarize the prerequisites and defense systems for the occurrence of ferroptosis and the regulatory targets of drug-mediated ferroptosis action in HCC, the differences and connections between ferroptosis and other programmed cell deaths. We aim to summarize the theoretical basis, classical inducers of ferroptosis and research progress of ferroptosis in HCC cells, clued to the treatment of HCC by regulating ferroptosis network. Further investigation of the specific mechanisms of ferroptosis and the development of hepatocellular carcinoma and interventions at different stages of hepatocellular carcinoma will help us to deepen our understanding of hepatocellular carcinoma, with a view to providing new and more precise preventive as well as therapeutic measures for patients.

Predicting potential therapeutic targets and small molecule drugs for early-stage lung adenocarcinoma.

Lung cancer is a leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) constituting the majority, and its main subtype being lung adenocarcinoma (LUAD). Despite substantial advances in LUAD diagnosis and treatment, early diagnostic biomarkers inadequately fulfill clinical requirements. Thus, we conducted bioinformatics analysis to identify potential biomarkers and corresponding therapeutic drugs for early-stage LUAD patients. Here we identified a total of 10 differentially expressed genes (DEGs) with survival significance through the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). Subsequently, we identified a promising small molecule drug, Aminopurvalanol A, based on the 10 key genes using the L1000FWD application, which was validated by molecular docking followed by in vivo and in vitro experiments. The results highlighted TOP2A, CDH3, ASPM, CENPF, SLC2A1, and PRC1 as potential detection biomarkers for early LUAD. We confirmed the efficacy and safety of Aminopurvalanol A, providing valuable insights for the clinical management of LUAD.

CP-AFM Molecular Tunnel Junctions with Alkyl Backbones Anchored Using Alkynyl and Thiol Groups: Microscopically Different Despite Phenomenological Similarity.

In this paper, we report results on the electronic structure and transport properties of molecular junctions fabricated via conducting probe atomic force microscopy (CP-AFM) using self-assembled monolayers (SAMs) of n-alkyl chains anchored with acetylene groups (CnA; n = 8, 9, 10, and 12) on Ag, Au, and Pt electrodes. We found that the current-voltage (I-V) characteristics of CnA CP-AFM junctions can be very accurately reproduced by the same off-resonant single-level model (orSLM) successfully utilized previously for many other junctions. We demonstrate that important insight into the energy-level alignment can be gained from experimental data of transport (processed via the orSLM) and ultraviolet photoelectron spectroscopy combined with ab initio quantum chemical information based on the many-body outer valence Green's function method. Measured conductance GAg < GAu < GPt is found to follow the same ordering as the metal work function ΦAu < ΦAu < ΦPt, a fact that points toward a transport mediated by an occupied molecular orbital (MO). Still, careful data analysis surprisingly revealed that transport is not dominated by the ubiquitous HOMO but rather by the HOMO-1. This is an important difference from other molecular tunnel junctions with p-type HOMO-mediated conduction investigated in the past, including the alkyl thiols (CnT) to which we refer in view of some similarities. Furthermore, unlike in CnT and other junctions anchored with thiol groups investigated in the past, the AFM tip causes in CnA an additional MO shift, whose independence of size (n) rules out significant image charge effects. Along with the prevalence of the HOMO-1 over the HOMO, the impact of the "second" (tip) electrode on the energy level alignment is another important finding that makes the CnA and CnT junctions different. What ultimately makes CnA unique at the microscopic level is a salient difference never reported previously, namely, that CnA's alkyne functional group gives rise to two energetically close (HOMO and HOMO-1) orbitals. This distinguishes the present CnA from the CnT, whose HOMO stemming from its thiol group is well separated energetically from the other MOs.

Targeted inhibition of gut bacterial ß-glucuronidases by octyl gallate alleviates mycophenolate mofetil-induced gastrointestinal toxicity.

Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20 mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20 mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.

A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.

Genomic Analysis and Taxonomic Characterization of Seven Bacteriophage Genomes Metagenomic-Assembled from the Dishui Lake.

Viruses in aquatic ecosystems exhibit remarkable abundance and diversity. However, scattered studies have been conducted to mine uncultured viruses and identify them taxonomically in lake water. Here, whole genomes (29-173 kbp) of seven uncultured dsDNA bacteriophages were discovered in Dishui Lake, the largest artificial lake in Shanghai. We analyzed their genomic signatures and found a series of viral auxiliary metabolic genes closely associated with protein synthesis and host metabolism. Dishui Lake phages shared more genes with uncultivated environmental viruses than with reference viruses based on the gene-sharing network classification. Phylogeny of proteomes and comparative genomics delineated three new genera within two known viral families of Kyanoviridae and Autographiviridae, and four new families in Caudoviricetes for these seven novel phages. Their potential hosts appeared to be from the dominant bacterial phyla in Dishui Lake. Altogether, our study provides initial insights into the composition and diversity of bacteriophage communities in Dishui Lake, contributing valuable knowledge to the ongoing research on the roles played by viruses in freshwater ecosystems.

Metavirome-assembled genome sequence of a new aquatic RNA virus expands the genus Locarnavirus.

RNA viruses in marine environments have long been recognized as major players in the virosphere. In this study, the complete genome sequence of an RNA virus from Yangshan Harbor, named marine RNA virus Yangshan-LWW (YS-LWW), was obtained based on metavirome assembly. The genome of YS-LWW is 8839 nt in length and contains two open reading frames (ORFs). Both RdRP- and whole-genome-based phylogenetic analysis showed that YS-LWW, together with 45 viral isolates with sequences in public datasets, represents a new species in the genus Locarnavirus of the family Marnaviridae. PCR and public dataset mining indicate that YS-LWW and YS-LWW-like viruses have been widely detected in coastal and freshwater environments, suggesting that they might play a significant role in aquatic ecosystems.

Pseudomonas composti isolate from oyster digestive tissue specifically binds with norovirus GII.6 via Psl extracellular polysaccharide.

Oysters are recognized as important vectors for human norovirus transmission in the environment. Whether norovirus binds to bacteria in oyster digestive tissues (ODTs) remains unknown. To shed light on this concern, ODT-54 and ODT-32, positive for histo-blood group antigen (HBGA) -like substances, were isolated from ODTs and identified as Pseudomonas composti and Enterobacter cloacae, respectively. The binding of noroviruses (GII.4 and GII.6 P domains) to bacterial cells (ODT-32 and ODT-54; in situ assay) as well as extracted extracellular polysaccharides (EPSs; in vitro assay) was analyzed by flow cytometry, confocal laser scanning microscopy, ELISA, and gene knock-out mutants. ODT-32 bound to neither GII.4 nor GII.6 P domains, while ODT-54 specifically binds with GII.6 P domain through Psl, an exopolysaccharide encoded by the polysaccharide synthesis locus (psl), identified based on gene annotation, gene transcription, Psl specific staining, and ELISAs. These findings attest that ODT bacteria specifically bind with certain norovirus genotypes in a strain-dependent manner, contributing to a better understanding of the transmission and enrichment of noroviruses in the environment.

Diversity, Relationship, and Distribution of Virophages and Large Algal Viruses in Global Ocean Viromes.

Virophages are a group of small double-stranded DNA viruses that replicate and proliferate with the help of the viral factory of large host viruses. They are widely distributed in aquatic environments but are more abundant in freshwater ecosystems. Here, we mined the Global Ocean Viromes 2.0 (GOV 2.0) dataset for the diversity, distribution, and association of virophages and their potential host large viruses in marine environments. We identified 94 virophage sequences (>5 kbp in length), of which eight were complete genomes. The MCP phylogenetic tree showed that the GOV virophages were widely distributed on the global virophage tree but relatively clustered on three major branches. The gene-sharing network divided GOV virophages into 21 outliers, 2 overlaps, and 14 viral clusters, of which 4 consisted of only the GOV virophages. We also identified 45 large virus sequences, 8 of which were >100 kbp in length and possibly involved in cell-virus-virophage (C-V-v) trisome relationships. The potential eukaryotic hosts of these eight large viruses and the eight virophages with their complete genomes identified are likely to be algae, based on comparative genomic analysis. Both homologous gene and codon usage analyses support a possible interaction between a virophage (GOVv18) and a large algal virus (GOVLV1). These results indicate that diverse and novel virophages and large viruses are widespread in global marine environments, suggesting their important roles and the presence of complicated unknown C-V-v relationships in marine ecosystems.

YAP1 as a Novel Negative Biomarker of Immune Checkpoint Inhibitors for EGFR-Mutant Non-Small-Cell Lung Cancer.

Background: Immune checkpoint inhibitors (ICIs) have become a standard care in non-small-cell lung cancer (NSCLC). However, its application to epidermal growth factor receptor (EGFR)-mutant NSCLC patients is confronted with drug resistance. This study aimed to clarify the potential role of Yes1-associated transcriptional regulator (YAP1) in ICIs treatment for EGFR-mutant NSCLC population. Methods: All the clinical data of NSCLC were downloaded from Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) for GSE11969 and GSE72094. Based on YAP1 expression, all the NSCLC patients including the EGFR-mutant and EGFR-wildtype (WT) patients were divided into two groups, YAP1_High and YAP1_Low. Using cBioPortal, genetic alterations were analyzed for identification of immunogenicity in EGFR-mutant NSCLC. MR analysis was used to analyze the hub gene of EGFR. The infiltration of immune cells and the expression of the identified tumor-associated antigens were identified with TIMER. By graph learning-based dimensionality reduction analysis, the immune landscape was visualized. Moreover, survival analysis was performed to verify the predictive value of YAP1 in ICIs treatment for EGFR-mutant NSCLC population using Ren's research data (NCT03513666). Results: YAP1 was a poor prognostic factor of EGFR-mutant NSCLC population rather than lung adenocarcinoma (LUAD) patients. MR analysis revealed that the EGFR gene regulated YAP1 expression. YAP1 was identified as a hub gene closely associated with immunosuppressive microenvironment and poor prognosis in EGFR-mutant NSCLC population in TCGA LUAD. Tumors with YAP1_High showed an immune-"cold" and immunosuppressive phenotype, whereas those with YAP1_Low demonstrated an immune-"hot" and immunoactive phenotype. More importantly, it was verified that YAP1_High subpopulation had a significantly shorter progression-free survival (PFS) and overall survival (OS) after ICIs treatment in EGFR-mutant NSCLC patients in the clinical trial. Conclusions: YAP1 mediates immunosuppressive microenvironment and poor prognosis in EGFR-mutant NSCLC population. YAP1 is a novel negative biomarker of ICIs treatment in EGFR-mutant NSCLC population. Clinical Trials. This trial is registered with NCT03513666.

[Olecranon osteotomy approach for Dubberley type â ¢ coronal fractures of the distal humerus].

OBJECTIVE: To retrospectively analyze the clinical efficacy of olecranon osteotomy approach in the treatment of Dubberley type Ⅲ coronal fractures of the distal humerus and summarize the treatment experience. METHODS: From January 2016 to June 2020, 17 patients (5 males and 12 females) with Dubberley type Ⅲ coronal fractures of the distal humerus were treated by olecranon osteotomy approach. The age ranged from 37 to78 years old with an average of (58.5±12.9) years old. According to Dubberley classification, there were 5 cases of type Ⅲ A and 12 cases of type Ⅲ B. The curative effect was evaluated using the Borberg-Morrey elbow function score. The flexion, extension and rotation range of motion of the elbow joint, complications and postoperative imaging evaluation were recorded. RESULTS: All the 17 patients got bony union. The follow-up time ranged from 12 to 33 months with an average of (15.6±5.6) months. There was 1 case of ischemic necrosis of capitulum humeri, 2 cases of traumatic arthritis and 1 case of heterotopic ossification, 1 case of malunion of fracture. The range of motion was (114.80±19.50) °. The Broberg-Morrey score was 85.3±8.2, excellent in 5 cases, good in 9 cases, fair in 3 cases and poor in 0 case. CONCLUSION: Through olecranon osteotomy approach, the articular surface of distal humerus could be fully exposed, and the operation is convenient. Anatomical reduction and rigid fixation of the articular surface of distal humerus are the key factors for the succesful outcome.

Accidental acquisition of a rescued Japanese encephalitis virus with unspliced introns in the viral genome when using an intron-based stabilization approach.

The intron-based stabilization approach is a very useful strategy for construction of stable flavivirus infectious clones. SA14-14-2 is a highly attenuated Japanese encephalitis (JE) live vaccine strain that has been widely used in China since 1989. To develop safe and effective recombinant vaccines with SA14-14-2 as a backbone vector, we constructed the DNA-based infectious clone pCMW-JEV of SA14-14-2 using the intron-based stabilization approach and acquired the rescued virus rDJEV, which retained the biological properties of the parental virus. Unexpectedly, a rescued virus strain with altered virulence, designated rHV-DJEV, was accidentally acquired in one of the transfection experiments. rHV-DJEV showed up to 105-fold increased neurovirulence compared with the SA14-14-2 parental strain. Genome sequencing showed that the inserted introns were still present in the genome of rHV-DJEV. Therefore, we think that the intron-based stabilization approach should be used with caution in vaccine development and direct iDNA immunization.

Three-dimensional quantitative structural-activity relationship and molecular dynamics study of multivariate substituted 4-oxyquinazoline HDAC6 inhibitors.

3D-QSAR models were established by collecting 46 multivariate-substituted 4-oxyquinazoline HDAC6 inhibitors. The relationship of molecular structure and inhibitory activity was studied by comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA). The results showed the models established by CoMFA (q2 = 0.590, r2 = 0.965) and CoMSIA (q2 = 0.594, r2 = 0.931) had good prediction ability. At the same time, 3D-QSAR models met the internal verification, external verification and AD test. Ten new compounds were designed based on CoMFA and CoMSIA contour maps and their pharmacokinetic/toxic properties (ADME/T) were evaluated. It was found that most compounds have well safety profile and pharmacokinetic property. Then, we explored the interaction between HDAC6 and compounds by molecular docking. The results showed that the binding mode of the new compounds with HDAC6 was the same as the template compound 46, and the hydrogen bond and hydrophobic bond played a vital role in the binding process. Molecular dynamics simulation results showed that residues Ser531, His574 and Tyr745 played key roles in the binding process. All newly designed compounds had lower energy gap and binding energy than compound 46 according to DFT analysis and free energy analysis. This study provided a theoretical reference for designing compounds of higher activity and a new idea for the development of novel HDAC6 inhibitors.

The histone deacetylase SIRT6 promotes glycolysis through the HIF-1α/HK2 signaling axis and induces erlotinib resistance in non-small cell lung cancer.

Erlotinib is a first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). Overcoming erlotinib resistance is crucial to improve the survival of advanced non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. It is also an important clinical problem that urgently needs a solution. In this study, we explored strategies to overcome erlotinib resistance from the perspective of energy metabolism. SIRT6 is a histone deacetylase. Here, we found that high expression of SIRT6 is associated with poor prognosis of lung adenocarcinoma, especially in EGFR-mutated NSCLC patients. The next cell experiment found that SIRT6 expression increased in erlotinib-resistant cells, and SIRT6 expression was negatively correlated with the sensitivity of NSCLC to erlotinib. Inhibition of SIRT6 promoted erlotinib-induced apoptosis in erlotinib-resistant cells, and glycolysis in drug-resistant cells was also inhibited. Functional studies have shown that SIRT6 increases glycolysis through the HIF-1α/HK2 signaling axis in drug-resistant cells and inhibits the sensitivity of NSCLC cells to erlotinib. In addition, the HIF-1α blocker PX478-2HCL attenuated the glycolysis and erlotinib resistance induced by SIRT6. More importantly, we confirmed the antitumor effect of SIRT6 inhibition combined with erlotinib in NSCLC-bearing mice. Our findings indicate that the cancer metabolic pathway regulated by SIRT6 may be a new target for attenuating NSCLC erlotinib resistance and has potential as a biomarker or therapeutic target to improve outcomes in NSCLC patients.

Study on an attenuated rabies virus strain CTN181-3.

We have obtained an attenuated rabies virus CTN181-3. In this paper, we make a comprehensive studies on CTN181-3. CTN181-3 showed no pathogenicity by i. c. or o. i. inoculation in 3-week-old mice, lower pathogenic in 2-week-old mice, and no virulence by o. i. inoculation in 8-week-old golden hamsters. CTN181-3 showed high immunogenicity, which produced high level neutralizing antibodies, 100% sero-conversation and >5.0 IU/ml GMT by one dose i. m. or o. i. vaccination in mice and golden hamsters. Cellular immune response by one dose i. m. or o. i. inoculation was detected. Especially in PEP, reduced dose of vaccination resulted in 50% (one dose) and 100% (2 doses) protections in golden hamsters. Molecular basis of the attenuation indicated that eight substitutions compared to its parental virus strain CTN-1, among them the two substitutions at the G276 (Leu→Val) and L1496 (Met→Trp) were the critical attenuated site. The phenotypic and genotypic characteristics of CTN181-3 were highly stable, no reversion was occurred when the virus was multiple passaged in suckling mice brains, guinea pig submandibular glands or BSR/Vero cell cultures. The gene homology compared to the Chinese rabies isolates showed much higher than rabies vaccine strains used in China, suggesting CTN181-3 is a promising and suitable oral rabies vaccine candidate strain.

Construction of a Dengue NanoLuc Reporter Virus for In Vivo Live Imaging in Mice.

Since the first isolation in 1943, the dengue virus (DENV) has spread throughout the world, but effective antiviral drugs or vaccines are still not available. To provide a more stable reporter DENV for vaccine development and antiviral drug screening, we constructed a reporter DENV containing the NanoLuc reporter gene, which was inserted into the 5' untranslated region and capsid junction region, enabling rapid virus rescue by in vitro ligation. In addition, we established a live imaging mouse model and found that the reporter virus maintained the neurovirulence of prototype DENV before engineering. DENV-4 exhibited dramatically increased neurovirulence following a glycosylation site-defective mutation in the envelope protein. Significant mice mortality with neurological onset symptoms was observed after intracranial infection of wild-type (WT) mice, thus providing a visualization tool for DENV virulence assessment. Using this model, DENV was detected in the intestinal tissues of WT mice after infection, suggesting that intestinal lymphoid tissues play an essential role in DENV pathogenesis.

Analysis of the evolution, infectivity and antigenicity of circulating rabies virus strains.

Rabies virus has existed for thousands of years and is circulating in many species. In the present study, a total of 2896 rabies viruses isolated worldwide were phylogenetically classified into ten clusters based on the G gene sequence, and these clusters showed a close relationship with the hosts and regions that they were isolated from. Eighty-three representative G sequences were selected from ten clusters and were used to construct pseudoviruses using the VSV vector. The phylogenetic relationships, infectivity and antigenicity of the representative 83 pseudotyped rabies viruses were comprehensively analyzed. Eighty three pseudoviruses were divided into four antigentic clusters (GAgV), of which GAgV4 showed poor neutralization to all immunized sera. Further analysis showed that almost all strains in the GAgV4 were isolated from wild animals in the America, especially bats and skunks. No significant relationship in terms of phylogeny, infectivity and antigenicity was proved. Amino acid mutations at residues 231and 436 can affect the infectivity, while mutations at residues 113, 164 and 254 may affect the sensitivity to immunized animal sera, especially residue 254. We recommend close monitoring of infectivity and antigenicity, which should be more precise than simple genetic analysis.

Investigation of immune response induction by Japanese encephalitis live-attenuated and chimeric vaccines in mice.

The Japanese encephalitis (JE) live-attenuated vaccine SA14-14-2 and the chimeric vaccine IMOJEV (JE-CV) are two kinds of vaccines available for use worldwide. JE-CV was previously known as ChimeriVax-JE, that consists of yellow fever vaccine 17D (YFV-17D) from which the structural genes (prM/E) have been replaced with those of SA14-14-2. This study aimed to investigate the neutralizing antibody, protection efficacy, and specific T-cell response elicited by both vaccines in mice. The neutralizing antibodies produced by JE-CV were slightly lower than those produced by SA14-14-2, but the protection conferred by JE-CV was considerably lower in the low vaccine dose immunization group. Furthermore, the JE-CV did not induce a specific T-cell response against JEV NS3, while it did induce a potent antigen-specific T-cell response against the viral backbone vaccine YFV. In conclusion, this study is the first detailed investigation of the cellular immune response to the two vaccines. Enzyme-linked immunospot (ELISPOT) and flow staining suggest a more potent specific T-cell response against the JEV antigen was elicited in mice immunized with SA14-14-2 but not JE-CV. Using heterologous flaviviruses as a live-attenuated vaccine backbone may unlikely generate an optimal T-cell response against the vaccine strain virus and might affect the protective efficacy.

Immune Response on Optimal Timing and Fractionation Dose for Hypofractionated Radiotherapy in Non-Small-Cell Lung Cancer.

Background: The intervention timing of immune checkpoint inhibitors (ICIs) and radiotherapy fractionations are critical factors in clinical efficacy. This study aims to explore dynamic changes of the tumor immune microenvironment (TIME) after hypofractionated radiotherapy (HFRT) at different timepoints and fractionation doses in non-small-cell lung cancer (NSCLC). Methods: In the implanted mouse model, the experimental groups received HFRT 3.7 Gy × 4 F, 4.6 Gy × 3 F, 6.2 Gy × 2 F, and 10 Gy × 1 F, respectively, with the same biological equivalent dose (BED) of 20Gy. Tumor volume and survival time were compared with those of the control group. Flow cytometry was performed to detect immune cells and their PD-1/PD-L1 expressions using tail-tip blood at different timepoints and tumor tissues at 48 h after radiotherapy. In NSCLC patients, immune cells, PD-1/PD-L1, and cytokines were detected in peripheral blood for 4 consecutive days after different fractionation radiotherapy with the same BED of 40Gy. Results: Tumor volumes were significantly reduced in all experimental groups compared with the control group, and the survival time in 6.2 Gy × 2 F (p < 0.05) was significantly prolonged. In tail-tip blood of mice, CD8+ T counts increased from 48 h to 3 weeks in 4.6 Gy × 3 F and 6.2 Gy × 2 F, and CD8+ PD-1 shortly increased from 48 h to 2 weeks in 6.2 Gy × 2 F and 10 Gy × 1 F (p < 0.05). Dentritic cells (DCs) were recruited from 2 to 3 weeks (p < 0.01). As for NSCLC patients, CD8+ T counts and PD-1 expression increased from 24 h in 6.2 Gy × 4 F, and CD8+ T counts increased at 96 h in 10 Gy × 2 F (p < 0.05) in peripheral blood. DC cells were tentatively recruited at 48 h and enhanced PD-L1 expression from 24 h in both 6.2 Gy × 4 F and 10 Gy × 2 F (p < 0.05). Besides, serum IL-10 increased from 24 h in 6.2 Gy × 4 F (p < 0.05). Conversely, serum IL-4 decreased at 24 and 96 h in 10 Gy × 2 F (p < 0.05). Conclusion: HFRT induces the increase in CD8+ T cells and positive immune cytokine response in specific periods and fractionation doses. It was the optimal time window from 48 h to 2 weeks for the immune response, especially in 6.2 Gy fractionation. The best immune response was 96 h later in 10 Gy fractionation, delivering twice instead of a single dose. During this time window, the intervention of immunotherapy may achieve a better effect.

Updating a New Semi-nested PCR Primer Pair for the Specific Detection of GII Norovirus in Oysters.

Oysters are major transmission vectors of noroviruses (NoVs) in the environment. Outbreaks of NoVs are often associated with the consumption of NoV-contaminated oysters. Laboratory confirmation of suspected oyster samples is a critical step in the surveillance and control of NoVs. Because of non-specific amplification, false-positive results are frequently obtained by semi-nested RT-PCR with the presently widely used primer set (G2SKF/G2SKR). Here, a novel universal PCR primer set N (NG2OF/NG2OR) specific for genogroup II (GII) NoVs was designed based on all GII NoV sequences available in public databases. Specific products were obtained with the primer set N when the NoV-positive oysters, spiked with each of five representative genotypes of GII NoVs (GII.17, GII.13, GII.4, GII.3, and GII.12), were subjected to analyzing. No products were detected with the primer set N for the NoV-negative oysters, while the primer set C gave various non-specific bands. Twenty-three out of 156 fresh oyster samples were NoV-positive with both the primer set N and the classic primer set, while eight were NoV-positive solely with the primer set N. Compared with the classic primer set, the newly designed primer set N had a higher detection rate and improved specificity for GII NoVs in oyster samples. These results show that the novel PCR primer pair is specific and applicable for the detection of GII NoVs in oysters.