Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8ß, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rß, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Human Cytomegalovirus Particles Treated with Specific Antibodies Induce Intrinsic and Adaptive but Not Innate Immune Responses.

Human cytomegalovirus (HCMV) persistently infects 40% to 100% of the human population worldwide. Experimental and clinical evidence indicates that humoral immunity to HCMV plays an important role in restricting virus dissemination and protecting the infected host from disease. Specific immunoglobulin preparations from pooled plasma of adults selected for high titers of HCMV antibodies have been used for the prevention of CMV disease in transplant recipients and pregnant women. Even though incubation of HCMV particles with these preparations leads to the neutralization of viral infectivity, it is still unclear whether the antibody-treated HCMV particles (referred to here as HCMV-Ab) enter the cells and modulate antiviral immune responses. Here we demonstrate that HCMV-Ab did enter macrophages. HCMV-Ab did not initiate the expression of immediate early antigens (IEAs) in macrophages, but they induced an antiviral state and rendered the cells less susceptible to HCMV infection upon challenge. Resistance to HCMV infection seemed to be due to the activation of intrinsic restriction factors and was independent of interferons. In contrast to actively infected cells, autologous NK cells did not degranulate against HCMV-Ab-treated macrophages, suggesting that these cells may not be eliminated by innate effector cells. Interestingly, HCMV-Ab-treated macrophages stimulated the proliferation of autologous adaptive CD4+ and CD8+ T cells. Our findings not only expand the current knowledge on virus-antibody immunity but may also be relevant for future vaccination strategies.IMPORTANCE Human cytomegalovirus (HCMV), a common herpesvirus, establishes benign but persistent infections in immunocompetent hosts. However, in subjects with an immature or dysfunctional immune system, HCMV is a major cause of morbidity and mortality. Passive immunization has been used in different clinical settings with variable clinical results. Intravenous hyperimmune globulin preparations (IVIg) are obtained from pooled adult human plasma selected for high anti-CMV antibody titers. While HCMV neutralization can be shown in vitro using different systems, data are lacking regarding the cross-influence of IVIg administration on the cellular immune responses. The aim of this study was to evaluate the effects of IVIg on distinct components of the immune response against HCMV, including antigen presentation by macrophages, degranulation of innate natural killer cells, and proliferation of adaptive CD4+ and CD8+ T cells.

Interfacial Structure and Interaction of Kaolinite Intercalated with N-methylformamide Insight from Molecular Dynamics Modeling.

The evolution of basal spacing and interfacial structure of kaolinite-N-methylformamide (NMF) complexes during the intercalation process were difficult to obtain using experimental methods. In present study, a series of kaolinite-NMF complex models with various numbers of NMF molecules in the interlayer space were constructed to mimic the progressive stage of the intercalation process of kaolinite intercalated by NMF. The MD simulations were performed on these models to explore the evolution of basal spacing and interfacial structure of kaolinite-NMF complexes during the intercalation process. It was found that the basal spacing of complex was stabilized at 11 Å during the intercalation process, where the molecular plane of NMF oriented at small angles with respect to the interlayer surface with the C=O groups and N-H bonds pointing toward the octahedral and tetrahedral surfaces, respectively, due to the hydrogen bonding interactions. The basal spacing can be enlarged to larger values with the prerequisite of overcoming the energy barrier. With the increase of basal spacing during the intercalation process, the NMF were rearranged as a pillar with the molecular planes orienting at higher angles with respect to the interlayer surface, and then developed to disordered bilayer structure. For the interfacial interaction of kaolinite-NMF complex, both the octahedral surface and tetrahedral surface showed binding affinity to the NMF, which is the driving force for the intercalation of NMF in kaolinite. The octahedral surface displays stronger binding affinity to the NMF in terms of the H-bonds and energetics compared to the tetrahedral surface partially due to the highly active surface hydroxyl groups. The present study provides insight into the basal spacing evolution, and interfacial structure and interaction of kaolinite-NMF complexes, which can enhance the understanding of kaolinite intercalated by small molecules.

Naïve T cells are activated by autologous HCMV-infected endothelial cells through NKG2D and can control HCMV transmission in vitro.

Apart from classical antigen-presenting cells (APCs) like dendritic cells and macrophages, there are semiprofessional APCs such as endothelial cells (ECs) and Langerhans' cells. Human cytomegalovirus (HCMV) infects a wide range of cell types including the ECs which are involved in the trafficking and homing of T cells. By investigating the interaction of naïve T cells obtained from HCMV-seronegative umbilical cord blood with autologous HCMV-infected human umbilical vein ECs (HUVECs), we could show that the activation of naïve T cells occurred after 1 day of peripheral blood mononuclear cell (PBMC) exposure to HCMV-infected HUVECs. The percentage of activated T cells increased over time and the activation of naïve T cells was not induced by either autologous uninfected HUVECs or by autologous HCMV-infected fibroblasts. The activation of T cells occurred also when purified T cells were co-cultured with HCMV-infected HUVECs. In addition, in most of the donors only CD8+ T cells were activated, when the purified T cells were exposed to HCMV-infected HUVECs. The activation of naïve T cells was inhibited when the NKG2D receptor was blocked on the surface of T cells and among the different NKG2D ligands, we identified two ligands (ULBP4 and MICA) on HCMV-infected HUVECs which might be the interaction partners of the NKG2D receptor. Using a functional cell culture assay, we could show that these activated naïve T cells specifically inhibited HCMV transmission. Altogether, we identified a novel specific activation mechanism of naïve T cells from the umbilical cord by HCMV-infected autologous HUVECs through interaction with NKG2D.

Phenotypic characterization of human cytomegalovirus strains in cell cultures based on their transmission kinetics.

We established a new 'transmission kinetic assay (TKA)' to quantify the human cytomegalovirus (HCMV) transmission between cells in vitro and to phenotypically characterize HCMV strains based on their mode of transmission by flow cytometric analysis. On one hand we used the genetically modified HCMV strain TB40/E-delUL16-GFP, and on the other hand, clinical isolates. When twofold diluted infecting cells were seeded to a constant number of uninfected cells, the transmission of virus on each day (day 0-5) followed a strictly linear pattern, which was characterized by a linear equation. The slope of this linear equation represents 'the number of newly infected cells per infecting cell'. To standardize the TKA, the slopes of the different days were plotted against the corresponding days. This resulted in a new linear equation with a new slope value, which characterizes the transmission kinetics. To differentiate cell-associated and cell-free modes of transmission, we introduced HCMV neutralizing antibodies into the system. The slope was 0.9 (±0.5) when the virus exhibited only cell-associated transmission and was 4.1 (±0.7) when the virus exhibited both modes of transmission. TKA was then applied to different clinical isolates and they were phenotypically characterized based on their modes of transmission. Apart from the quantitative analysis of HCMV transmission and the phenotypical characterization of clinical isolates, the TKA was applied to quantify the inhibition of clinical isolates transmission by immune cells and to study the effect of cytokine (IL-2) on immune cells inhibiting HCMV transmission.

Interleukin-2 from Adaptive T Cells Enhances Natural Killer Cell Activity against Human Cytomegalovirus-Infected Macrophages.

UNLABELLED: Control of human cytomegalovirus (HCMV) requires a continuous immune surveillance, thus HCMV is the most important viral pathogen in severely immunocompromised individuals. Both innate and adaptive immunity contribute to the control of HCMV. Here, we report that peripheral blood natural killer cells (PBNKs) from HCMV-seropositive donors showed an enhanced activity toward HCMV-infected autologous macrophages. However, this enhanced response was abolished when purified NK cells were applied as effectors. We demonstrate that this enhanced PBNK activity was dependent on the interleukin-2 (IL-2) secretion of CD4(+) T cells when reexposed to the virus. Purified T cells enhanced the activity of purified NK cells in response to HCMV-infected macrophages. This effect could be suppressed by IL-2 blocking. Our findings not only extend the knowledge on the immune surveillance in HCMV-namely, that NK cell-mediated innate immunity can be enhanced by a preexisting T cell antiviral immunity-but also indicate a potential clinical implication for patients at risk for severe HCMV manifestations due to immunosuppressive drugs, which mainly suppress IL-2 production and T cell responsiveness. IMPORTANCE: Human cytomegalovirus (HCMV) is never cleared by the host after primary infection but instead establishes a lifelong latent infection with possible reactivations when the host's immunity becomes suppressed. Both innate immunity and adaptive immunity are important for the control of viral infections. Natural killer (NK) cells are main innate effectors providing a rapid response to virus-infected cells. Virus-specific T cells are the main adaptive effectors that are critical for the control of the latent infection and limitation of reinfection. In this study, we found that IL-2 secreted by adaptive CD4(+) T cells after reexposure to HCMV enhances the activity of NK cells in response to HCMV-infected target cells. This is the first direct evidence that the adaptive T cells can help NK cells to act against HCMV infection.

Natural killer cells can inhibit the transmission of human cytomegalovirus in cell culture by using mechanisms from innate and adaptive immune responses.

UNLABELLED: Human cytomegalovirus (HCMV) transmission within the host is important for the pathogenesis of HCMV diseases. Natural killer (NK) cells are well known to provide a first line of host defense against virus infections. However, the role of NK cells in the control of HCMV transmission is still unknown. Here, we provide the first experimental evidence that NK cells can efficiently control HCMV transmission in different cell types. NK cells engage different mechanisms to control the HCMV transmission both via soluble factors and by cell contact. NK cell-produced interferon gamma (IFN-γ) suppresses HCMV production and induces resistance of bystander cells to HCMV infection. The UL16 viral gene contributes to an immune evasion from the NK cell-mediated control of HCMV transmission. Furthermore, the efficacy of the antibody-dependent NK cell-mediated control of HCMV transmission is dependent on a CD16-158V/F polymorphism. Our findings indicate that NK cells may have a clinical relevance in HCMV infection and highlight the need to consider potential therapeutic strategies based on the manipulation of NK cells. IMPORTANCE: Human cytomegalovirus (HCMV) infects 40% to 100% of the human population worldwide. After primary infection, mainly in childhood, the virus establishes a lifelong persistence with possible reactivations. Most infections remain asymptomatic; however, HCMV represents a major health problem since it is the most frequent cause of infection-induced birth defects and is responsible for high morbidity and mortality in immunocompromised patients. The immune system normally controls the infection by antibodies and immune effector cells. One type of effector cells are the natural killer (NK) cells, which provide a rapid response to virus-infected cells. NK cells participate in viral clearance by inducing the death of infected cells. NK cells also secrete antiviral cytokines as a consequence of the interaction with an infected cell. In this study, we investigated the mechanisms by which NK cells control HCMV transmission, from the perspectives of immune surveillance and immune evasion.

Resistant mutations and quasispecies complexity of hepatitis B virus during telbivudine treatment.

Ultra-deep pyrosequencing (UDPS) was used to analyse the dynamics of quasispecies and resistant mutations during telbivudine (LDT) treatment of hepatitis B patients. Twenty-six HBeAg-positive chronic hepatitis B patients were treated with LDT for a period of 104 weeks and were characterized as 16 responders, six partial responders and four viral breakthrough patients based on hepatitis B virus (HBV) DNA levels. The plasma samples were subjected to UDPS of the reverse transcriptase (RT) region of HBV. Mutations rtM204I, rtL80I and rtL80V were detected in at least three of the four viral breakthrough patients, indicating the significant roles of the mutations in resistance to LDT. The degree of complexity of viral quasispecies remained in a steady state in the absence of selection pressure, but increased after the LDT treatment. The complexity in the responder group at week 12 was significantly higher than that in the group comprising partial responders and viral breakthrough patients. In vitro replication efficiency analyses showed that the RT mutations had different impacts on HBV replication, with a tendency of rtM204I>rtL80V>rtL80I. Furthermore, double mutations rtL80I/M204I and rtL80V/M204V had replication efficiency similar to that of rtL80I and rtL80V, respectively. Consistent with previous studies, mutation rtM204I was found to be highly resistant to LDT. However, in contrast with their sensitivity to lamivudine, rtL80I and rtL80V were moderately resistant to LDT. Our results indicated that rtL80I and rtL80V may not only serve as replication complementary mutations to rtM204I, but also directly contribute to the LDT resistance.

Human cytomegalovirus-induced NKG2C(hi) CD57(hi) natural killer cells are effectors dependent on humoral antiviral immunity.

Recent studies indicate that expansion of NKG2C-positive natural killer (NK) cells is associated with human cytomegalovirus (HCMV); however, their activity in response to HCMV-infected cells remains unclear. We show that NKG2C(hi) CD57(hi) NK cells gated on CD3(neg) CD56(dim) cells can be phenotypically identified as HCMV-induced NK cells that can be activated by HCMV-infected cells. Using HCMV-infected autologous macrophages as targets, we were able to show that these NKG2C(hi) CD57(hi) NK cells are highly responsive to HCMV-infected macrophages only in the presence of HCMV-specific antibodies, whereas they are functionally poor effectors of natural cytotoxicity. We further demonstrate that NKG2C(hi) CD57(hi) NK cells are intrinsically responsive to signaling through CD16 cross-linking. Our findings show that the activity of pathogen-induced innate immune cells can be enhanced by adaptive humoral immunity. Understanding the activity of NKG2C(hi) CD57(hi) NK cells against HCMV-infected cells will be of relevance for the further development of adoptive immunotherapy.

Unsynchronized butyrophilin molecules dictate cancer cell evasion of Vγ9Vδ2 T-cell killing.

Vγ9Vδ2 T cells are specialized effector cells that have gained prominence as immunotherapy agents due to their ability to target and kill cells with altered pyrophosphate metabolites. In our effort to understand how cancer cells evade the cell-killing activity of Vγ9Vδ2 T cells, we performed a comprehensive genome-scale CRISPR screening of cancer cells. We found that four molecules belonging to the butyrophilin (BTN) family, specifically BTN2A1, BTN3A1, BTN3A2, and BTN3A3, are critically important and play unique, nonoverlapping roles in facilitating the destruction of cancer cells by primary Vγ9Vδ2 T cells. The coordinated function of these BTN molecules was driven by synchronized gene expression, which was regulated by IFN-γ signaling and the RFX complex. Additionally, an enzyme called QPCTL was shown to play a key role in modifying the N-terminal glutamine of these BTN proteins and was found to be a crucial factor in Vγ9Vδ2 T cell killing of cancer cells. Through our research, we offer a detailed overview of the functional genomic mechanisms that underlie how cancer cells escape Vγ9Vδ2 T cells. Moreover, our findings shed light on the importance of the harmonized expression and function of gene family members in modulating T-cell activity.

KCTD9 contributes to liver injury through NK cell activation during hepatitis B virus-induced acute-on-chronic liver failure.

We explored the expression of a newly identified potassium channel tetramerisation domain containing 9 (KCTD9) protein in 113 blood and 81 liver samples, from patients with mild chronic hepatitis B (CHB) or HBV-induced acute-on-chronic liver failure (HBV-ACLF). KCTD9 was highly expressed in peripheral and hepatic NK cells from HBV-ACLF patients compared with mild CHB patients, and this correlated positively with the severity of liver injury. The role of KCTD9 was further investigated in NK92 cells in vitro. KCTD9 overexpressed NK92 cells exhibited a marked increase in CD69 expression, cytotoxicity, IFN-γ secretion and a significant decrease in NKG2A receptor expression. Inhibition of KCTD9 by shRNA resulted in reduced cytotoxic function. These results suggest the involvement of KCTD9 in NK cell activation and provide additional insight into a potential therapeutic target for molecular manipulation for HBV-ACLF patients.

The 7-transmembrane protein homologue UL78 of the human cytomegalovirus forms oligomers and traffics between the plasma membrane and different intracellular compartments.

The human cytomegalovirus (HCMV) UL78 ORF is considered to encode an orphan 7-transmembrane receptor. However, until now, the UL78 protein (pUL78) has not been characterized. Here, we have investigated the expression of pUL78 and found it mainly associated with the endoplasmic reticulum. However, we provide evidence that pUL78 is also localized on the cell surface from where it is quickly endocytosed. Colocalization with adaptin and EEA-1 implies that at least a small amount of pUL78 is transported to the trans Golgi network and early endosomes. Using a bimolecular fluorescence complementation assay and co-immunoprecipitation experiments, we were able to find homomeric and heteromeric structure formations of pUL78 and the US28 protein, respectively. However, the absence of pUL78 had no effect on the accumulation of inositol phosphate triggered by the US28 protein. In summary, our results suggest that the UL78 protein of HCMV traffics between the cell surface and cytoplasm, from where it might be recycled via early endosomes.

Increased killing of liver NK cells by Fas/Fas ligand and NKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure.

The role of liver NK cells in virus-induced severe viral hepatitis and, subsequently, hepatic failure is not well defined. In this study, we investigated the role of liver NK cells in the development of hepatocyte necrosis in fulminant hepatic failure (FHF) and acute-on-chronic liver failure (ACLF) because of viral infection. A mouse model of FHF induced by murine hepatitis virus strain 3 (MHV-3) was used to study the role of liver NK cells. Samples from patients with hepatitis B virus-related ACLF (HBV-ACLF) were examined. After MHV-3 infection, the number of NK cells in livers of BALB/cJ mice increased markedly, peaked at 48 h postinfection, and remained at a high level until sacrifice. In peripheral blood, spleen, and bone marrow, this number decreased significantly. Expression of CD69, cytotoxic activity, and intracellular IFN-gamma and TNF-alpha production by liver NK cells at 48 h postinfection were all significantly upregulated. Depletion of NK cells 24 h post-MHV-3 infection increased the mice survival from 0 of 18 (0%) to 4 of 18 (22.2%). Highly activated liver NK cells were cytotoxic to MHV-3-infected hepatocytes and this effect was markedly inhibited by anti-Fas ligand (FasL) plus anti-NKG2D mAbs. Furthermore, the accumulation of hepatic NK cells and increased expression of FasL and natural cytotoxicity receptors (NKp30 and NKp46) on the peripheral NK cells from patients with HBV-ACLF were correlated with disease progression. These results indicate NK cells play a pivotal role in the pathogenesis of FHF and HBV-ACLF, in which process Fas/FasL and NKG2D/NKG2D ligand pathway contribute to the liver NK cell-mediated hepatocyte injury.

Noninvasive measurement of liver fibrosis by transient elastography and influencing factors in patients with chronic hepatitis B-A single center retrospective study of 466 patients.

The noninvasive measurement of liver stiffness (LS) was evaluated by transient elastography (FibroScan) and the possible influencing factors from the patients' clinical situations including age, gender, liver inflammation represented by alanine transaminase (ALT) and total billirubin (TBIL) level, HBV replication (HBV DNA loads), portal vein pressure (portal vessel diameter, PVD), splenic thickness (SPT) and body mass index (BMI) were analyzed in patients with chronic hepatitis B (CHB). A total of 466 patients including 31 patients with acute-on-chronic liver failure (ACLF), and 435 patients with chronic hepatitis B (CHB) among which 82 patients were diagnosed with liver cirrhosis (LC) by clinical manifestations and liver B-type ultrasonic inspection were enrolled at Tongji Hospital from April to December 2009. LS was measured by a FibroScan device (EchoSens, France). Simultaneously, ALT and TBIL levels, HBV DNA loads, PVD, SPT and BMI in all patients were also tested. Forty-one healthy volunteers served as controls. The values of LS were correlated positively with ages of CHB patients and significantly higher in males than in females. In patients with BMI>28 kg/m(2) (obesity) and abnormal levels of ALT and TBIL, LS values were significantly increased as compared with those having normal levels of ALT and TBIL. The patients with ACLF had the highest LS value. Furthermore, LS values in the patients with LC were significantly higher than those in patients without LC. It is concluded that noninvasive measurement of liver fibrosis by FibroScan provides an alternative method to evaluate liver fibrosis of patients with CHB. In order to properly illustrate the stiffness value taken by transient elastography, patients' gender should be taken into consideration and it is also suggested to avoid possible influencing factors including liver inflammation (high levels of ALT and TBIL) and obesity (high BMI).

Gene editing and its applications in biomedicine.

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

Dynamic variability in SHP-1 abundance determines natural killer cell responsiveness.

Interactions between human leukocyte antigen (HLA) molecules on target cells and the inhibitory killer cell immunoglobulin-like receptors (KIRs) and heterodimeric inhibitory receptor CD94-NKG2A on human natural killer (NK) cells shape and program various response capacities. A functionally orthologous system exists in mice, consisting of major histocompatibility complex (MHC) molecules on target cells and the inhibitory Ly49 and CD94-NKG2A receptors on NK cells. Here, we found that the abundance of Src homology 2 domain­containing phosphatase-1 (SHP-1) in NK cells was established by interactions between MHCs and NK cell inhibitory receptors, although phenotypically identical NK cell populations still showed substantial variability in endogenous SHP-1 abundance and NK cell response potential. Human and mouse NK cell populations with high responsiveness had low SHP-1 abundance, and a reduction in SHP-1 abundance in NK cells enhanced their responsiveness. Computational modeling of NK cell activation by membrane-proximal signaling events identified SHP-1 as a negative amplitude regulator, which was validated by single-cell analysis of human NK cell responsiveness. The amount of mRNA and protein varied among responsive NK cells despite their similar chromatin accessibility to that of unresponsive cells, suggesting dynamic regulation of SHP-1 abundance. Low intracellular SHP-1 abundance was a biomarker of responsive NK cells. Together, these data suggest that enhancing NK cell function through the acute loss of SHP-1 abundance or activity may enhance the tumoricidal capacity of NK cells.

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.

Canonical CRISPR-knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR-KO screens in various settings.

[Exploring the Mechanism of Paclitaxel Inhibiting T-cell Lymphoma based on High-throughput Sequencing and Public Databases].

OBJECTIVE: To analyze gene expression profile of T cell lymphoma Jurkat cell line treated with paclitaxel by computational biology based on next generation sequencing and to explore the possible molecular mechanism of paclitaxel resistance to T cell lymphoma at gene level. METHODS: IC50 of paclitaxel on Jurkat cell line was determined by CCK-8 assay. Gene expression profile of Jurkat cells treated with paclitaxel was acquired by next generation sequencing technology. Gene microarray data related to human T cell lymphoma were screened from Gene Expression Omnibus (GEO) database (including 720 cases of T cell lymphoma and 153 cases of normal tissues). Combined with the sequencing data, differential expression genes (DEGs) were intersected and screened. DAVID database was used for enrichment analysis of GO function and KEGG pathway to determine and visualize functional entries of DEGs, and protein-protein interactions network of DEGs was drawn. The levels of gene expression were detected and verified by RT-qPCR. RESULTS: CCK-8 results showed that the proliferation of Jurkat cells was inhibited by paclitaxel depended on the concentration apparently. Treated by paclitaxel for 48 h, P<0.05 and |log2(FC)|≥1 were used as filter criteria on the results of RNA Sequencing (RNA-Seq) and GeoChip, 351 DEGs were found from Jurkat cells, including 323 up-regulated genes and 28 down-regulated genes. The GO functional annotation and KEGG pathway enrichment analysis showed that the role of paclitaxel was mainly concentrated in protein heterodimerization activity, nucleosome assembly and transcriptional dysregulation in cancer, etc. The results of RT-qPCR were consistent with those of the sequencing analysis, which verified the reliability of this sequencing. CONCLUSION: Paclitaxel can affect the proliferation and apoptosis of T-cell lymphoma by up-regulating JUN gene, orphan nuclear receptor NR4A family genes and histone family genes.

Acute liver failure: mechanisms of immune-mediated liver injury.

Acute liver failure (ALF) is a syndrome of diverse aetiology, including hepatic encephalopathy, renal, cardiac and pulmonary failures, which result in a rapid loss of hepatic function. The mechanisms of liver injury contributing to ALF can be summarized into two categories: direct damage and immune-mediated liver injury. This review summarizes current concepts of immune-mediated liver injury from both clinical studies and animal models. We highlight immune responses of ALF from the liver injury perspective, which combines a variety of molecular and cellular mechanisms, particularly, the contribution of cytokines and the innate immune system. Hepatic and circulating inflammatory cytokines play a significant role in the pathophysiology of ALF including hepatocyte necrosis, extrahepatic complications and hepatocyte regeneration. Overproduction of cytokines, if unchecked, is hazardous to the host and may cause severe outcomes. Measuring pro-inflammatory cytokines in ALF may be of value for predictors of outcome. Innate and adaptive immune systems both involved in ALF contribute to immune-mediated liver injury. The innate immune response is activated much more rapidly compared with adaptive immunity, particularly in acute liver injury where the host has little time to trigger an effective adaptive immune response. From this point of view, the innate immune system may make a more profound contribution than the adaptive immune system. Furthermore, immune responses crosstalk with other physiological or pathophysiological factors, for example, coagulation factors which in turn determine the outcome of ALF and these are discussed.