All-Trans-Retinoic Acid-Adjuvanted mRNA Vaccine Induces Mucosal Anti-Tumor Immune Responses for Treating Colorectal Cancer.

Messenger RNA (mRNA) cancer vaccines are a new class of immunotherapies that can activate the immune system to recognize and destroy cancer cells. However, their effectiveness in treating colorectal cancer located on the mucosal surface of the gut is limited due to the insufficient activation of mucosal immune response and inadequate infiltration of cytotoxic T cells into tumors. To address this issue, a new mRNA cancer vaccine is developed that can stimulate mucosal immune responses in the gut by co-delivering all-trans-retinoic acid (ATRA) and mRNA using lipid nanoparticle (LNP). The incorporation of ATRA has not only improved the mRNA transfection efficiency of LNP but also induced high expression of gut-homing receptors on vaccine-activated T cells. Additionally, the use of LNP improves the aqueous solubility of ATRA, eliminating the need for toxic solvents to administer ATRA. Upon intramuscular injections, ATRA-adjuvanted mRNA-LNP significantly increase the infiltration of antigen-specific, cytotoxic T cells in the lamina propria of the intestine, mesenteric lymph nodes, and orthotopic colorectal tumors, resulting in significantly improved tumor inhibition and prolonged animal survival compared to conventional mRNA-LNP without ATRA. Overall, this study provides a promising approach for improving the therapeutic efficacy of mRNA cancer vaccines against colorectal cancer.

Layer-by-layer nanoparticle encapsulating all-trans retinoic acid and CpG as a mucosal adjuvant targeting colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, demanding innovative therapeutic strategies. Immunotherapy, a promising avenue, employs cancer vaccines to activate the immune system against tumors. However, conventional approaches fall short of eliciting robust responses within the gastrointestinal (GI) tract, where CRC originates. Harnessing the potential of all-trans retinoic acid (ATRA) and cytosine-phosphorothioate-guanine (CpG), we developed layered nanoparticles using a layer-by-layer assembly method to co-deliver these agents. ATRA, crucial for gut immunity, was efficiently encapsulated alongside CpG within these nanoparticles. Administering these ATRA@CpG-NPs, combined with ovalbumin peptide (OVA), effectively inhibited orthotopic CRC growth in mice. Our approach leveraged the inherent benefits of ATRA and CpG, demonstrating superior efficacy in activating dendritic cells, imprinting T cells with gut-homing receptors, and inhibiting tumor growth. This mucosal adjuvant presents a promising strategy for CRC immunotherapy, showcasing the potential for targeting gut-associated immune responses in combating colorectal malignancies.

A Synergistic Lipid Nanoparticle Encapsulating mRNA Shingles Vaccine Induces Potent Immune Responses and Protects Guinea Pigs from Viral Challenges.

Shingles is caused by the reactivation of varicella zoster virus (VZV) and manifests as painful skin rashes. While the recombinant protein-based vaccine proves highly effective, it encounters supply chain challenges due to a shortage of the necessary adjuvant. Messenger RNA (mRNA)-based vaccines can be rapidly produced on a large scale, but their effectiveness relies on efficient delivery and sequence design. Here, an mRNA-based VZV vaccine using a synergistic lipid nanoparticle (Syn-LNP) containing two different ionizable lipids is developed. Syn-LNP shows superior mRNA expression compared to LNPs formulated with either type of ionizable lipid and to a commercialized LNP. After encapsulating VZV glycoprotein E (gE)-encoding mRNA, mgE@Syn-LNP induces robust humoral and cellular immune responses in two strains of mice. The magnitude of these responses is similar to that induced by adjuvanted recombinant gE proteins and significantly higher than that observed with live-attenuated VZV. mgE@Syn-LNP exhibits durable humoral responses for over 7 months without obvious adverse effects. In addition, mgE@Syn-LNP protects vaccinated guinea pigs against live VZV challenges. Preliminary studies on the mRNA antigen design reveal that the removal of glycosylation sites of gE greatly reduces its immune responses. Collectively, Syn-LNP encapsulating gE-encoded mRNA holds great promise as a shingles vaccine.

Computational Insights into the Esterification of Palmitic Acid with Methanol in Water.

This work provides quantified explanations for the thermodynamic and kinetic characteristics of esterification in aqueous phase, and how phase transfer catalysts improve water phase esterification of fatty acids in a computational-experimental way. Self-catalyzed reaction mode with or without solvation effects, water participated reaction mode, and catalytic reaction mode (catalyzed by p-dodecyl benzene sulfonic acid, DBSA) are discussed. Our results show that the initial self-catalytic reaction mode undergoes the energy barrier of 100.1 kJ/mol, and rises to 148.9 kJ/mol when water molecule is involved, which hinders the esterification reaction. With the DBSA catalyst, this energy barrier will drop to 97.5 kJ/mol and the water phase esterification is successfully promoted with the yield of 81%. The key kinetic factor of binding energy is discussed as that water molecule has a strong reactant binding competitiveness (with the binding energy of -57.9 kJ/mol, and the value for the non-aqueous phase mode is 3.0 kJ/mol) and DBSA has the binding energy with the value of -45.3 kJ/mol, so it can compete with water to form reactant complexes. This work is a successful practice of a computation-experiment combined scheme, and provides a quantitative basis for the improvement of phase transfer catalysts on water phase esterification reactions. The calculation mode and method of aqueous esterification make it possible to convert bio-based fatty acids into fatty acid esters in fermentation broth.

Splicing forgery localization via noise fingerprint incorporated with CFA configuration.

Noise is the inherent intrinsic fingerprint in digital images and is often used for forgery localization. Most noise-based methods assume that the noise is similar over the whole image and can be considered as white Gaussian noise. However, the noise is different in various regions, which degrade the performance of these noise-based methods. To reduce the impact of impractical assumptions, in this paper, we propose an effective noise fingerprint incorporated with CFA configuration for splicing forgery localization. The noise of interpolated pixels is expected to be suppressed after interpolation, and the relationship between the noise levels of adjacent acquired and interpolated pixels is only related to the interpolation algorithm, which is constant in the original image. We utilize a dual tree wavelet based denoising algorithm to extract the noise from the green channel and compute the standard deviation of the noise for acquired and interpolated pixels, respectively. The noise level of acquired and interpolated pixels are then obtained by the geometric mean of the noise standard deviations. Finally, the ratio of noise levels between acquired and interpolated pixels can be a fingerprint to locate tampered regions. Experiments conducted on publicly available databases demonstrate that the proposed approach outperforms previous methods for detecting splice tampering. Moreover, the proposed method is robust to Gaussian filtering and JPEG compression attacks.

Induction of ligustrazine apoptosis of A549 cells through activation of death receptor pathway.

Background: The aim of this study was to evaluate the effect of ligustrazine on the apoptosis of A549 cells and clarify the mechanism of ligustrazine-induced apoptosis. Methods: Ligustrazine was prepared with medium according to the gradient concentration. Based on a cytotoxicity test, 3 different concentrations of ligustrazine were selected to form low, medium, and high groups, with a 0 mg/mL dose used as the control. The apoptosis degree and Fas (Fas cell surface death receptor) and Fas-L (Fas Ligand) expression were detected by flow cytometry and quantitative polymerase chain reaction (qPCR), respectively; meanwhile, the activity of caspase 8 and caspase 3 was analyzed by enzyme-linked immunosorbent assay (ELISA) and qPCR, respectively. Results: After 24 hours of ligustrazine administration, the survival rate of A549 cells decreased with the increase of drug concentration, while the rate of apoptosis increased with the increase of drug concentration. Meanwhile, Fas and Fas-L expression was found to be significantly increased at both the gene and protein level, which was positively correlated with drug concentration. Furthermore, the expression of caspase 8 and caspase 3 was positively correlated with the concentration of ligustrazine, and there was significant difference compared with the control group. Conclusions: Ligustrazine can induce the apoptosis of A549 cells via the upregulation of Fas- and caspase-activating death receptor pathway expression.

Application of Bayesian network and regression method in treatment cost prediction.

Charging according to disease is an important way to effectively promote the reform of medical insurance mechanism, reasonably allocate medical resources and reduce the burden of patients, and it is also an important direction of medical development at home and abroad. The cost forecast of single disease can not only find the potential influence and driving factors, but also estimate the active cost, and tell the management and reasonable allocation of medical resources. In this paper, a method of Bayesian network combined with regression analysis is proposed to predict the cost of treatment based on the patient's electronic medical record when the amount of data is small. Firstly, a set of text-based medical record data conversion method is established, and in the clustering method, the missing value interpolation is carried out by weighted method according to the distance, which completes the data preparation and processing for the realization of data prediction. Then, aiming at the problem of low prediction accuracy of traditional regression model, this paper establishes a prediction model combined with local weight regression method after Bayesian network interpretation and classification of patients' treatment process. Finally, the model is verified with the medical record data provided by the hospital, and the results show that the model has higher prediction accuracy.

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Virus-Host Interactome and Proteomic Survey Reveal Potential Virulence Factors Influencing SARS-CoV-2 Pathogenesis.

BACKGROUND: The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a global public health concern due to relatively easy person-to-person transmission and the current lack of effective antiviral therapy. However, the exact molecular mechanisms of SARS-CoV-2 pathogenesis remain largely unknown. METHODS: Genome-wide screening was used to establish intraviral and viral-host interactomes. Quantitative proteomics was used to investigate the peripheral blood mononuclear cell (PBMC) proteome signature in COVID-19. FINDINGS: We elucidated 286 host proteins targeted by SARS-CoV-2 and >350 host proteins that are significantly perturbed in COVID-19-derived PBMCs. This signature in severe COVID-19 PBMCs reveals a significant upregulation of cellular proteins related to neutrophil activation and blood coagulation, as well as a downregulation of proteins mediating T cell receptor signaling. From the interactome, we further identified that non-structural protein 10 interacts with NF-κB-repressing factor (NKRF) to facilitate interleukin-8 (IL-8) induction, which potentially contributes to IL-8-mediated chemotaxis of neutrophils and the overexuberant host inflammatory response observed in COVID-19 patients. CONCLUSIONS: Our study not only presents a systematic examination of SARS-CoV-2-induced perturbation of host targets and cellular networks but it also reveals insights into the mechanisms by which SARS-CoV-2 triggers cytokine storms, representing a powerful resource in the pursuit of therapeutic interventions. FUNDING: National Key Research and Development Project of China, National Natural Science Foundation of China, National Science and Technology Major Project, Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, Shanghai Science and Technology Commission, Shanghai Municipal Health Commission, Shanghai Municipal Key Clinical Specialty, Innovative Research Team of High-level Local Universities in Shanghai, Interdisciplinary Program of Shanghai Jiao Tong University, SII Challenge Fund for COVID-19 Research, Chinese Academy of Sciences (CAS) Large Research Infrastructure of Maintenance and Remolding Project, and Chinese Academy of Sciences Key Technology Talent Program.

[Flubendazole Inhibits the Proliferation of A549 and H460 Cells and Promotes Autophagy].

BACKGROUND: Flubendazole is an anthelmintic and categorized in benzimidazole. Previous evidence indicates its suppression on proliferation of colon cancer and breast cancer cells. Our study aims to explore the effects of flubendazole on non-small cell lung cancer A549 and H460 cell lines and the underlying mechanism. METHODS: CCK-8 assay was used to detect the effect of flubendazole at different concentrations on viability of both cell lines A549 and H460. We used western blot to detect the expression levels of autophagy-related proteins p62 and LC3 after flubendazole treatment. Cells were transfected with tandem fluorescent adenovirus (mRFP-GFP-LC3), and the impact of flubendazole treatment on autophagic flux were analyzed. RESULTS: Cell viability analysis showed a dose-dependent inhibitory effect on proliferation of both A549 and H460, comparing to cells without flubendazole treating (P<0.001). Level of p62 decreased and LC3 II/I ratio increased in cells treated with 2 µmol/L flubendazole for 24 h and 48 h, compared to control groups (P<0.005). Red fluorescence signals increased in mRFP-GFP-LC3 transfected cells after flubendazole treating, suggesting an elevation in autophagic flux. CONCLUSIONS: Flubendazole may inhibit the proliferation of A549 and H460 cells and promote autophagy.

STAT3-induced upregulation of lncRNA ABHD11-AS1 promotes tumour progression in papillary thyroid carcinoma by regulating miR-1301-3p/STAT3 axis and PI3K/AKT signalling pathway.

OBJECTIVES: Emerging evidences indicated the importance of long non-coding RNAs (lncRNAs) in the tumorigenesis and deterioration of malignant tumours. To our knowledge, the study about lncRNAs in papillary thyroid carcinoma (PTC) is still inadequate. ABHD11-AS1 was highly expressed in the PTC samples of The Cancer Genome Atlas database. This study focused on the biological function and mechanism of lncRNA ABHD11-AS1 in PTC. MATERIALS AND METHODS: qRT-PCR analysis was used to examine the expression of ABHD11-AS1 in PTC tissues and cell lines. The prognostic significance of ABHD11-AS1 for the patients with PTC was analysed with Kaplan-Meier analysis. The effects of ABHD11-AS1 knockdown on the cell proliferation and metastasis were evaluated by in vitro functional assays and in vivo experiments. The molecular mechanism which contributed to the oncogenic role of ABHD11-AS1 in PTC was explored by conducting mechanism experiments. Rescue assays were carried out for final demonstration. RESULTS: High expression of ABHD11-AS1 predicted poor prognosis for patients with PTC and promoted cell proliferation and metastasis in vitro and in vivo. ABHD11-AS1 was activated by the transcription factor STAT3. ABHD11-AS1 positively regulated PI3K/AKT signalling pathway. ABHD11-AS1 acted as a competitive endogenous (ce) RNA to upregulate STAT3 by sponging miR-1301-3p. CONCLUSIONS: STAT3-induced lncRNA ABHD11-AS1 promoted PTC progression by regulating PI3K/AKT signalling pathway and miR-1301-3p/STAT3 axis.

Metagenomic identification, genetic characterization and genotyping of porcine sapoviruses.

Sapoviruses (SaVs), belonging to the genus Sapovirus of the family Caliciviridae, were known as the enteric pathogen causing acute gastroenteritis. SaVs have been detected in humans and several animals including pigs and some porcine SaVs showed close sequence relationship with human strains suggesting the possibility of interspecies transmission. Here, we sequenced the genomes of two porcine SaVs (with strain names of p38 and SH1703) using the metagenomic analyses and traditional RT-PCR methods. Phylogenetic trees were constructed based on the complete genome, the full-length VP 1 nucleotide and amino acid sequences to group those two strains. The two porcine SaV strains, p38 and SH1703, detected in this study, were classified as genogroup III and genogroup VII, respectively. These two strains showed similar genomic organization with that of other SaVs. We firstly divided SaVs into 51 genotypes within 19 genogroups. Our data are helpful for genetic characterization and classification of newly detected SaVs worldwide.

Plasma virome of cattle from forest region revealed diverse small circular ssDNA viral genomes.

BACKGROUND: Free-range cattle are common in the Northeast China area, which have close contact with farmers and may carry virus threatening to cattle and farmers. METHODS: Using viral metagenomics we analyzed the virome in plasma samples collected from 80 cattle from the forested region of Northeast China. RESULTS: The virome of cattle plasma is composed of the viruses belonging to the families including Parvoviridae, Papillomaviridae, Picobirnaviridae, and divergent viral genomes showing sequence similarity to circular Rep-encoding single stranded (CRESS) DNA viruses. Five such CRESS-DNA genomes were full characterized, with Rep sequences related to circovirus and gemycircularvirus. Three bovine parvoviruses belonging to two different genera were also characterized. CONCLUSION: The virome in plasma samples of cattle from the forested region of Northeast China was revealed, which further characterized the diversity of viruses in cattle plasma.

Viral metagenomics analysis of feces from coronary heart disease patients reveals the genetic diversity of the Microviridae.

Recent studies have declared that members of the ssDNA virus family Microviridae play an important role in multiple environments, as they have been found taking a dominant position in the human gut. The aim of this study was to analyze the overall composition of the gut virome in coronary heart disease (CHD) patients, and try to discover the potential link between the human gut virome and CHD. Viral metagenomics methods were performed to detect the viral sequences in fecal samples collected from CHD inpatients and healthy persons as controls. We present the analysis of the virome composition in these CHD patients and controls. Our data shows that the virome composition may be linked to daily living habits and the medical therapy of CHD. Virgaviridae and Microviridae were the two dominant types of viruses found in the enteric virome of CHD patients. Fourteen divergent viruses belonging to the family Microviridae were found, twelve of which were grouped into the subfamily Gokushovirinae, while the remaining two strains might represent two new subfamilies within Microviridae, according to the phylogenetic analysis. In addition, the genomic organization of these viruses has been characterized.

Genomic organization and recombination analysis of a porcine sapovirus identified from a piglet with diarrhea in China.

BACKGROUND: Sapovirus (SaV), a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. To date, both intra- and inter-genogroup recombinant strains have been reported in many countries except for China. Here, we report an intra-genogroup recombination of porcine SaV identified from a piglet with diarrhea of China. METHODS: A fecal sample from a 15-day-old piglet with diarrhea was collected from Shanghai, China. Common agents of gastroenteritis including porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine SaV, porcine norovirus, and porcine epidemic diarrhea virus were detected by RT-PCR or PCR method. The complete genome of porcine SaV was then determined by RT-PCR method. Phylogenetic analyses based on the structural region and nonstructural (NS) region were carried out to group this SaV strain, and it was divided into different genotypes based on these two regions. Recombination analysis based on the genomic sequence was further performed to confirm this recombinant event and locate the breakpoint. RESULTS: All of the agents showed negative results except for SaV. Analysis of the complete genome sequence showed that this strain was 7387 nt long with two ORFs and belonged to SaV GIII. Phylogenetic analyses of the structural region (complete VP1 nucleotide sequences) grouped this strain into GIII-3, whereas of the nonstructural region (RdRp nucleotide sequences) grouped this strain into GIII-2. Recombination analysis based on the genomic sequence confirmed this recombinant event and identified two parental strains that were JJ259 (KT922089, GIII-2) and CH430 (KF204570, GIII-3). The breakpoint located at position 5139 nt of the genome (RdRp-capsid junction region). Etiologic analysis showed the fecal sample was negative with the common agents of gastroenteritis, except for porcine SaV, which suggested that this recombinant strain might lead to this piglet diarrhea. CONCLUSIONS: P2 strain was an intra-genogroup recombinant porcine SaV. To the best of our knowledge, this study would be the first report that intra-genogroup recombination of porcine SaV infection was identified in pig herd in China.

Identification and genomic characterization of a novel species of feline anellovirus.

Here, a novel feline anellovirus strain (named FelineAV621 and GenBank no. KX262893) was detected in two cats with diarrhea. The complete genome of FelineAV621 is 2409 nt long with a G+C content of 56.67 %, including three open reading frames (ORFs). Phylogenetic analysis based on the amino acid sequence of the putative capsid protein (ORF1) indicated that FelineAV621 belonged to a novel anellovirus species inside a clade containing the seal anellovirus, canine TTVs, and porcine TTVs, but was distant from all the previous feline anelloviruses.

Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China.

Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand, and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST) analysis, and 48 sequence types (STs) were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17, and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these "environmental" pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve.