Integration of single-cell RNA sequencing and bulk RNA transcriptome sequencing reveals a heterogeneous immune landscape and pivotal cell subpopulations associated with colorectal cancer prognosis

IntroductionColorectal cancer (CRC) is a highly heterogeneous cancer. The molecular and cellular characteristics differ between the colon and rectal cancer type due to the differences in their anatomical location and pathological properties. With the advent of single-cell sequencing, it has become possible to analyze inter- and intra-tumoral tissue heterogeneities.MethodsA comprehensive CRC immune atlas, comprising 62,398 immune cells, was re-structured into 33 immune cell clusters at the single-cell level. Further, the immune cell lineage heterogeneity of colon, rectal, and paracancerous tissues was explored. Simultaneously, we characterized the TAM phenotypes and analyzed the transcriptomic factor regulatory network of each macrophage subset using SCENIC. In addition, monocle2 was used to elucidate the B cell developmental trajectory. The crosstalk between immune cells was explored using CellChat and the patterns of incoming and outgoing signals within the overall immune cell population were identified. Afterwards, the bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA) were combined and the relative infiltration abundance of the identified subpopulations was analyzed using CIBERSORT. Moreover, cell composition patterns could be classified into five tumor microenvironment (TME) subtypes by employing a consistent non-negative matrix algorithm. Finally, the co-expression and interaction between SPP1+TAMs and Treg cells in the tumor microenvironment were analyzed by multiplex immunohistochemistry.ResultsIn the T cell lineage, we found that CXCL13+T cells were more widely distributed in colorectal cancer tissues, and the proportion of infiltration was increased. In addition, Th17 was found accounted for the highest proportion in CD39+CD101+PD1+T cells. Mover, Ma1-SPP1 showed the characteristics of M2 phenotypes and displayed an increased proportion in tumor tissues, which may promote angiogenesis. Plasma cells (PCs) displayed a significantly heterogeneous distribution in tumor as well as normal tissues. Specifically, the IgA+ PC population could be shown to be decreased in colorectal tumor tissues whereas the IgG+ PC one was enriched. In addition, information flow mediated by SPP1 and CD44, regulate signaling pathways of tumor progression. Among the five TME subtypes, the TME-1 subtype displayed a markedly reduced proportion of T-cell infiltration with the highest proportion of macrophages which was correlated to the worst prognosis. Finally, the co-expression and interaction between SPP1+TAMs and Treg cells were observed in the CD44 enriched region.DiscussionThe heterogeneity distribution and phenotype of immune cells were analyzed in colon cancer and rectal cancer at the single-cell level. Further, the prognostic role of major tumor-infiltrating lymphocytes and TME subtypes in CRC was evaluated by integrating bulk RNA. These findings provide novel insight into the immunotherapy of CRC

Experimental study on effects of gas flow rate on soot characteristics in diffusion flames coupled with plasma

This study examined the evolution of morphology and nanostructure of soot particles from the plasma-flame interaction for various gas flow rates. The current study used both optical diagnostic and sampling methods to explore the soot production and combustion characteristics. Soot particles were characterized at the same positions downstream from the flame zone by thermophoretic sampling and transmission electron microscopy. Moreover, X-ray diffraction analysis, and thermogravimetric analysis were performed to study the nanostructure and oxidation reactivity of soot. A reduction in soot concentration was found with the plasma addition, which illustrated an inhibition effect of plasma on soot emission. The increased gas flow rate promoted soot concentration since a growing number of carbons participated in the combustion process. Depending on the gas flow rate (carbon content) variation and plasma activation, either liquid-like soot material with irregularly shaped protrusions or chain-like structure, or a mixture of both, were generated from the diffusion flames. The soot produced by plasma-flame interaction also demonstrated a high correlation between nanostructure and reactivity. The soot from lower carbon content with plasma activation had a shorter fringe length and larger fringe tortuosity related to higher oxidation reactivity. On the contrary, soot from the highest carbon content without plasma-flame interaction exhibited prevalent fullerene-like nanostructures with evident large or small shells and also had a higher carbonization degree resulting in lower oxidation reactivity

Tolerance of transgenic Arabidopsis thaliana overexpressing apple MdAGO4.1 gene to drought and salt stress

The regulatory role of apple MdAGO4.1 gene in plant drought and salt resistance is unclear. In this study, transgenic A. thaliana in which the apple MdAGO4.1 gene was over-expressed was used to analyze the regulatory effects of the MdAGO4.1 gene on plant drought and salt resistance, to verify the function of the apple MdAGO4.1 gene. The seed germination rate, seedling fresh weight and root length of transgenic Arabidopsis strains in MS medium containing different concentrations of NaCl and mannitol were better than those of the wild type. The transgenic A. thaliana seedlings were more resistant to drought than wild type under drought stress. The transgenic strains were less affected by salt stress than thewild type. Exposure to drought and salt stress reduced the relative elektrolyte leakage, malondialdehyde (MDA), superoxide anion (O2-), and hydrogen peroxide (H2O2) levels of the transgenic strain significantly compared with the levels in the wild type. The levels of proline, protective enzyme activities, and the expression of genes related to drought and salt stress resistance were significantly higher than those of the wild type. These results indicate that MdAGO4.1 overexpression improved drought and salt tolerance in transgenic Arabidopsis. This study can provide a theoretical basis for future research on stress tolerance mechanisms and breeding new varieties of fruit trees resistant to drought and salt

Derivation and Characterization of Hepatic Progenitor Cells from Human Embryonic Stem Cells

The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell–derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation

CYP-omega-hydroxylation-dependent metabolites of arachidonic acid inhibit the basolateral 10pS chloride channel in the rat thick ascending limb

Metabolites of arachidonic acid influence sodium chloride (NaCl) transport in the thick ascending limb. Because a 10pS Cl channel is the major type of chloride channel in the basolateral membrane of this nephron segment, we explored the effect of arachidonic acid on this channel in cell-attached patches. Addition of 5μmol arachidonic acid significantly decreased channel activity (a product of channel number and open probability) while linoleic acid had no effect. To determine if this was mediated by acachidonic acid per se or by its metabolites, we measured channel activity in the presence of the cyclooxygenase inhibitor indomethacin, the selective lipoxygenase inhibitor nordihydroguaiaretic acid, and the cytochrome P-450 (CYP)-ω-hydroxylation inhibitor 17-octadecynoic acid. Neither cyclooxygenase nor lipoxygenase inhibition had an effect on basal chloride channel activity; further they failed to abolish the inhibitory effect of arachidonate on the 10pS channel. However, inhibition of CYP-ω-hydroxylation completely abolished the effect of arachidonic acid. The similarity of the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid suggests that the effect of arachidonic acid was mediated by CYP-ω-hydroxylation-dependent metabolites. We conclude that arachidonic acid inhibits the 10pS chloride channel in the basolateral membrane of the medullary thick ascending limb, an effect mediated by the CYP-ω-hydroxylation-dependent metabolite 20-HETE

Baculovirus Surface Display of SARS Coronavirus (SARS-CoV) Spike Protein and Immunogenicity of the Displayed Protein in Mice Models

ABSTRACT The baculovirus surface display technique has provided an ideal tool to display foreign proteins with natural conformation, functions, and immunogenicity. In this work, we explored the application of this technique on SARS-associated coronavirus (SARS-CoV) spike (S) protein, and further analyzed the immunogenicity of displayed S protein. The entire ectodomain of S protein was fused between the gp64 signal peptide and the VSV-G membrane anchor and successfully displayed on the baculovirus surface. Subcutaneous injection with purified S-displayed baculoviruses without adjuvant elicited highly effective production of specific and neutralizing antibodies against S protein in mice. These results confirmed a successful surface display of S protein on baculoviruse, and suggested a potential role of S-displayed baculoviruses as a novel live virus-based vaccine candidate for SARS-CoV

Efficacy and safety of molnupiravir in patients with Omicron variant vaccine breakthrough COVID-19 infection: a randomized, controlled trial

Introduction: Randomized, controlled trials of molnupiravir in real-world use during the Omicron wave are scarce. The frequency of hospitalization and death is low, so further research is needed to confirm the virological efficacy of molnupiravir.Methods: A single-center, randomized, controlled clinical trial was conducted, and 111 hospitalized coronavirus disease 2019 (COVID-19) patients were randomly assigned at a ratio of 1:1. Fifty-three patients in the molnupiravir group were administered 800 mg of molnupiravir twice daily for 5 days in addition to the standard therapy, and 58 patients in the control group only received the standard therapy in accordance with local guidelines. The antiviral effect and adverse events were evaluated during the follow-up.Results: The median viral clearance time in the molnupiravir group was significantly shorter than that in the control group (p = 0.003). Furthermore, patients who started molnupiravir therapy within 3 days had significantly shorter viral clearance time than the controls (p = 0.003). In the vaccinated subgroup, molnupiravir therapy was also associated with a shorter viral clearance time (p = 0.003). A total of three adverse events, which were minor, were reported in the molnupiravir group. One of the patients had mild liver function abnormalities, and all of them were resolved without intervention. However, the remission time was similar between the two tested groups.Conclusion: Molnupiravir exhibited good viral replication inhibitor efficacy in patients with Omicron variant vaccine breakthrough COVID-19 infection.Clinical Trial Registration: [https://www.chictr.org.cn/], identifier [ChiCTR2200059796]

Safety and efficacy of lentinan nasal drops in patients infected with the variant of COVID-19: a randomized, placebo-controlled trial

Objective: Lentinan has antiviral, anti-tumor, immunomodulatory, stimulating interferon production, and other pharmacological effects. Previous animal experiments have shown that lentinan nasal drops can assist [Corona Virus Disease 2019) COVID-19] vaccine to induce high levels of neutralizing antibodies and can effectively resist the invasion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the safety and efficacy of lentinan nasal drops in patients infected with Omicron (SARS-CoV-2 variant) through a dose-escalation study and a placebo-controlled trial.Methods: A randomized, placebo-controlled trial. The study was divided into two phases: Phase I: a dose escalation trial in which 24 COVID-19 patients were enrolled, that is, 12 in the escalation dose group (50, 75, and 100 µg/day) and 12 in the standard treatment group. The aim was to evaluate the safety and tolerance of lentinan nasal drops. The second stage was a placebo-controlled study. The optimal dose group of the first stage was used as the therapeutic dose, and the sample size was expanded to verify the anti-COVID-19 efficacy of lentinan nasal drops.Results: In the dose-increasing study, lentinan nasal drops showed good safety, and no serious adverse reactions occurred. The virus shedding time of the 100 µg dose group was significantly shorter than that in the control group (7.75 ± 1.71 VS 13.41 ± 3.8 days) (p = 0.01), and the 100 µg/day lentinan nasal drops were tolerated well. The results of the placebo-controlled study showed that compared with that in the placebo group, the time for COVID-19 antigen to turn negative was significantly shorter in the 100 µg lentinan nasal drop group (p = 0.0298), but no significant difference was observed in symptom improvement between the two groups. In the placebo-controlled study, two patients experienced mild nasal discomfort with nasal drops, but the symptoms relieved themselves.Conclusion: Lentinan nasal drops are tolerated well and can shorten the time of virus clearance

Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals

Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5′UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5′UTR and NS3-3′UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5′UTR and 3′UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated “CH6acc”), releasing HCV of 104.3–104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses