E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1.

TBK1 is essential for interferon-ß (IFN-ß) production and innate antiviral immunity. Here we identified the T cell anergy-related E3 ubiquitin ligase RNF128 as a positive regulator of TBK1 activation. RNF128 directly interacted with TBK1 through its protease-associated (PA) domain and catalyzed the K63-linked polyubiquitination of TBK1, which led to TBK1 activation, IRF3 activation and IFN-ß production. Deficiency of RNF128 expression attenuated IRF3 activation, IFN-ß production and innate antiviral immune responses to RNA and DNA viruses, in vitro and in vivo. Our study identified RNF128 as an E3 ligase for K63-linked ubiquitination and activation of TBK1 and delineated a previously unrecognized function for RNF128.

Diverse Roles of ScSERF in Modifying the Fibril Growth of Amyloidogenic Proteins.

The aggregation of amyloidogenic proteins is often related to the occurrence of neurodegenerative diseases, including fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Recently, the SERF protein family has been reported to have a significant regulatory effect on amyloid formation, but it is still unclear about the detailed mechanisms of SERF acting on different amyloidogenic proteins. Herein, nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy were used to explore interactions of ScSERF with three amyloidogenic proteins FUS-LC, FUS-Core, and α-Synuclein. NMR chemical shift perturbations reveal them sharing similar interaction sites on the N-terminal region of ScSERF. However, the amyloid formation of α-Synuclein protein is accelerated by ScSERF, while ScSERF inhibits fibrosis of FUS-Core and FUS-LC proteins. Both the primary nucleation and the total amount of fibrils produced are detained. Our results suggest a diverse role of ScSERF in regulating the fibril growth of amyloidogenic proteins.

An analysis of silybin meglumine tablets in the treatment of drug-induced liver injury as assessed for causality with the updated Roussel Uclaf Causality Assessment Method using a nationwide database.

AIM: Drug-induced liver injury (DILI) poses significant challenges to clinical practice. Currently, there is no recommended therapy to treat DILI; therefore, it is vital to explore new therapeutic agents. This study aimed to investigate the efficacy and safety of silybin meglumine tablets in treating DILI. METHODS: This study analysed 34 296 DILI cases assessed by the updated RUCAM from a nationwide database. A total of 301 patients with RUCAM scores ≥6 were directly enrolled in this study, while an additional 340 patients with RUCAM scores <6 who were adjudged as probable DILI by a panel of three hepatologists were also included in the analysis. The enrolled patients were divided into the silybin meglumine group and the control group. The propensity score matching (PSM) method was used to obtain comparable characteristics between the two groups. RESULTS: There were 129 cases in the silybin meglumine group and 512 cases in the control group. After applying PSM, 129 matched pairs were obtained. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) resumption rates in the silybin meglumine group were significantly higher than the control group (58.91% vs. 20.93%, P ≤ .0001 and 63.49% vs. 37.50%, P ≤ .0001). The univariate and multivariate logistic regression analysis revealed that grouping factor (odds raio [OR], 5.42; 95% confidenxe interval [CI], 3.12-9.39; P < .0001 and OR, 6.10; 95% CI, 2.98-12.48; P < .0001) and ALT levels (OR, 0.95; 95% CI, 0.93-0.98; P = .0015 and OR, 0.95; 95% CI, 0.92-0.99; P = .0157) were essential influencing factors for ALT normalization. CONCLUSIONS: Silybin meglumine tablets are safe and effective in DILI treatment. Large-scale and randomized controlled trials are required to further confirm their efficacy.

Tob2 Inhibits TLR-Induced Inflammatory Responses by Association with TRAF6 and MyD88.

Optimal activation of TLR pathways is crucial for the initiation of inflammatory responses and eliminating invading micro-organisms. However, excessive of TLR activation may lead to autoimmune and inflammatory diseases. Thus, TLR pathways should be tightly controlled. In this study, we identify Tob2, a Tob/BTG family member, as a suppressor of TLR pathways. Tob2 deficiency enhances TLR-induced NF-κB and MAPK activation and promotes the expression of proinflammatory cytokines in primary peritoneal macrophages of C57BL/6 mice. Furthermore, Tob2-defective C57BL/6 mice may be more susceptible to endotoxemic shock in vivo. Mechanistically, Tob2 interacts with TRAF6 and MyD88 and thus inhibits signaling from the MyD88-TRAF6 complex in primary peritoneal macrophages and HEK293T cells. Therefore, our results uncover a regulatory mechanism of TLR pathways and provide a potential target for the intervention of diseases with excessive TLR activation.

IL-27 Promotes Human Placenta-Derived Mesenchymal Stromal Cell Ability To Induce the Generation of CD4+IL-10+IFN-γ+ T Cells via the JAK/STAT Pathway in the Treatment of Experimental Graft-versus-Host Disease.

Human mesenchymal stromal cells (MSCs) harbor immunomodulatory properties to induce the generation of suppressive T cells. MSCs have been successfully used in treating graft-versus-host disease (GVHD) accompanied by abundant inflammatory cytokines such as IL-27. This study investigated the effects of IL-27 on the human placenta-derived MSCs (hPMSCs) to induce generation of CD4+IL-10+IFN-γ+ T cells in vitro and in the humanized xenogenic GVHD NOD/SCID model. The results showed that the percentages of CD4+IL-10+IFN-γ+ T cells were significantly increased in activated human PBMC from both healthy donors and GVHD patients with hPMSCs and in the liver and spleen of hPMSC-treated GVHD mice, and the level of CD4+IL-10+IFN-γ+ T cells in the liver was greater than that in the spleen in hPMSC-treated GVHD mice. The serum level of IL-27 decreased and the symptoms abated in hPMSC-treated GVHD. Further, in vitro results showed that IL-27 promoted the regulatory effects of hPMSCs by enhancing the generation of CD4+IL-10+IFN-γ+ T cells from activated PBMC. Activation occurred through increases in the expression of programmed death ligand 2 (PDL2) in hPMSCs via the JAK/STAT signaling pathway. These findings indicated that hPMSCs could alleviate GVHD mice symptoms by upregulating the production of CD4+IL-10+IFN-γ+ T cells in the spleen and liver and downregulating serum levels of IL-27. In turn, the ability of hPMSCs to induce the generation of CD4+IL-10+IFN-γ+ T cells could be promoted by IL-27 through increases in PDL2 expression in hPMSCs. The results of this study will be of benefit for the application of hPMSCs in clinical trials.

The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 upregulates ICAM-1 through a mechanism involving the NF-қB pathway.

Although the Epstein-Barr virus (EBV) infection is usually asymptomatic, a primary encounter with the virus can cause mononucleosis. EBV infection is also strongly associated with lymphoma and epithelial cancers. The structure and infection mechanism of EBV have been well studied, but the EBV-encoded G protein-coupled receptor, BILF1, is not fully understood. Here, it was found that the EBV BILF1 was expressed early in the viral lytic cycle and its ectopic expression strikingly upregulated the ICAM-1 expression in Raji cells. The positive effect of BILF1 on the ICAM-1 promoter was observed and the BILF1 deficiency attenuated ICAM-1 promoter activity. Moreover, NF-κB binding sites were important for the regulation of ICAM-1 promoter by BILF1. Furthermore, BILF1 reduced the constitutive level of the IқB-a protein and increased the amount of nuclear NF-қB in Raji cells. In conclusion, this study determined that BILF1 upregulated ICAM-1 in a mechanism involving NF-қB.

Lnc-NA inhibits proliferation and metastasis in endometrioid endometrial carcinoma through regulation of NR4A1.

Endometrioid endometrial carcinoma (EEC) is the most common gynaecologic malignancy worldwide. Long non-coding RNAs have previously been demonstrated to play important roles in regulating human diseases, particularly cancer. However, the biological functions and molecular mechanisms of long non-coding RNAs in EEC have not been extensively studied. Here, we describe the discovery of Lnc-NA from the promoter of the transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) gene. The role and function of Lnc-NA in EEC remain unknown. In this study, we used quantitative real-time polymerase chain reactions to confirm that Lnc-NA expression was down-regulated in 30 EEC cases (90%) and in EEC cell lines compared with that in the paired adjacent tissues and normal endometrial cells. In vitro experiments further demonstrated that overexpressing Lnc-NA decreased EEC cell proliferation, migration and invasion and promoted apoptosis via inactivation of the apoptosis signalling pathway. Moreover, the results show that Lnc-NA expression was positively correlated with NR4A1. Furthermore, Lnc-NA regulated NR4A1 expression and activated the apoptosis signalling pathway to inhibit tumour progression. In summary, our results demonstrate that the Lnc-NA-NR4A1 axis could be a useful tumour suppressor and a promising therapeutic target for EEC.

Interferon-γ suppresses the proliferation and migration of human placenta-derived mesenchmal stromal cells and enhances their ability to induce the generation of CD4+CXCR5+Foxp3+Treg subset.

We investigate the effects of interferon (IFN)-γ on human placenta-derived mesenchymal stromal cells (hPMSCs), in particular, their adhesion, proliferation and migration and modulatory effects on the CD4+CXCR5+Foxp3+Treg subset. And we compared hPMSCs ability to induce the generation of different Treg subsets in response to treatment with IFN-γ. We found that IFN-γ suppressed the proliferation and migration for hPMSCs. The ability of hPMSCs to induce the generation of CD4+CXCR5+Foxp3+Treg subset was enhanced by IFN-γ. And maximal effectiveness of IFN-γ treated hPMSCs upon inducing the generation of Treg subsets was for CD4+CXCR5+Foxp3+Treg subset as compared with that of CD4+CD25+Foxp3+, CD8+CD25+Foxp3+, CD4+IL-10+ and CD8+IL-10+Treg subsets. These results have important implications for the development and application of hPMSCs in clinical use.

Phosphatase PTPN4 preferentially inhibits TRIF-dependent TLR4 pathway by dephosphorylating TRAM.

TLR4 recruits TRIF-related adaptor molecule (TRAM, also known as TICAM2) as a sorting adaptor to facilitate the interaction between TLR4 and TRIF and then initiate TRIF-dependent IRF3 activation. However, the mechanisms by which TRAM links downstream molecules are not fully elucidated. In this study, we show that TRAM undergoes tyrosine phosphorylation upon TLR4 activation and that is required for TLR4-induced IRF3 activation. Protein tyrosine phosphatase nonreceptor type 4 (PTPN4), a protein tyrosine phosphatase, inhibits tyrosine phosphorylation and subsequent cytoplasm translocation of TRAM, resulting in the disturbance of TRAM-TRIF interaction. Consequently, PTPN4 specifically inhibits TRIF-dependent IRF3 activation and IFN-ß production in TLR4 pathway. Therefore, our results provide new insight into the TLR4 pathway and identify PTPN4 as a specific inhibitor of TRIF-dependent TLR4 pathway. Targeting PTPN4 would be beneficial for the development of new strategy to control TLR4-associated diseases without unwanted side effects.

Improvement of Survival Rate for Patients with Hepatocellular Carcinoma Using Transarterial Chemoembolization in Combination with Three-Dimensional Conformal Radiation Therapy: A Meta-Analysis.

BACKGROUND Transarterial chemoembolization (TACE) has been used alone or in combination with three-dimensional conformal radiation therapy (3DCRT) for treating hepatocellular carcinoma (HCC). The overall survival rate of HCC patients undergoing both treatments, however, has not been systematically studied. The aim of this meta-analysis-based study was to evaluate the overall efficacy of the combined therapy or monotherapy, thereby providing information for clinical treatment. MATERIAL AND METHODS We searched Google Scholar, PubMed, and Chinese National Knowledge Infrastructure (CNKI) for eligible studies, and a total of 17 case-control studies (including HCC patients treated by TACE plus 3DCRT or TACE alone) were included to perform the meta-analysis. Based on the available data, we assessed the improvements of 1-year, 2-year, and 3-year survival rate for the combination therapy of TACE and 3DCRT or TACE alone. Furthermore, the analysis was also stratified by the tumor response: complete response (CR), partial response (PR), no response (NR) and progressive disease (PD). Statistical analysis was performed using STATA 12 (Stata Statistical Software: Release 12). RESULTS The results show that HCC patients receiving combination therapy have significantly increased overall survival rate when compared to those receiving TACE alone (1-year survival rate: OR=1.95, 95% CI 1.54-2.47, p=7.3×10^-8; 2-year survival rate: OR=1.87, 95% CI 1.49-2.34, p=1.6×10^-7; 3-year survival rate: OR=2.00, 95% CI 1.52-2.64, p=1.8×10^-6). CONCLUSIONS Assessment of tumor response demonstrates that the combination therapy can efficiently increase the tumor response rate (CR+PR: OR=2.29, 95% CI 1.70-3.08, p=1.1×10^-7), with a lower rate of subsequent tumor development (PD: OR=0.25, 95% CI 0.15-0.40, p=5.5×10^-8).

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation.

Enterovirus A71 (EV-A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV-A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV-A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV-A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV-A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV-A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID50 ) values calculated in viral proliferations (ranged from 10(7.6) to 10(7.8) ); the TCID50 being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV-A71Jinan1002 amplifies more efficiently in MA104, Hep-2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV-A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV-A71.

Silencing of CD59 enhanced the sensitivity of HT29 cells to 5-Fluorouracil and Oxaliplatin.

Complement regulatory proteins (CD55 and CD59) were known to be expressed in many tumors and tumor cell lines including colorectal carcinoma, and were proposed as immunotherapy targets, however whether knocking down of CD55 and CD59 will affect the sensitivity of HT-29 cells to chemotherapy drugs for example, 5-Fluorouracil and Oxaliplatin and their possible mechanisms haven't been studied. To address this question, SiRNAs targeting CD55 and CD59 were chemically synthesized and transfected into HT-29 cells by lipofectamine. HT-29 growth curves of CD55 and CD59 knockdown cells were detected by MTT assay, HT29 inhibition curves to chemotherapy drugs (5-Fu and Oxaliplatin) were also assayed, in addition, chemotherapy sensitivity changes of HT29 affected by CD55 and CD59 knockdown were equally detected. Complement mediated lysis was examined by calcein-AM. We found that silencing CD59 in HT-29 cells could significantly enhance their sensitivity to 5-FU (P < 0.05) and Oxaliplatin (P < 0.05), and significantly reduced their IC50 concentration. On the contrary, knocking down of CD55 could inhibit HT-29 growth (P < 0.05). Mechanisms included increasing apoptosis rate of HT-29 by CD59 knocking down and G1/G0 blocking by silencing CD55. Our results thus shed light on the novel mechanism of chemotherapy resistance and provide an alternative strategy to overcome the resistance problem.

The treatment of solvent recovery raffinate by aerobic granular sludge in a pilot-scale sequencing batch reactor.

Mature aerobic granular sludge (AGS) was inoculated for the start-up of a pilot-scale sequencing batch reactor for the treatment of high concentration solvent recovery raffinate (SRR). The proportion of simulated wastewater (SW) (w/w) in the influent gradually decreased to zero during the operation, while volume of SRR gradually increased from zero to 10.84 L. AGS was successfully domesticated after 48 days, which maintained its structure during the operation. The domesticated AGS was orange, irregular, smooth and compact. Sludge volume index (SVI), SV30/SV5, mixed liquor volatile suspended solids/mixed liquor suspended solids (MLVSS/MLSS), extracellular polymeric substances, proteins/polysaccharides, average particle size, granulation rate, specific oxygen utilization rates (SOUR)H and (SOUR)N of AGS were about 38 mL/g, 0.97, 0.52, 39.73 mg/g MLVSS, 1.17, 1.51 mm, 96.66%, 47.40 mg O2/h g volatile suspended solids (VSS) and 8.96 mg O2/h g VSS, respectively. Good removal effect was achieved by the reactor. Finally, the removal rates of chemical oxygen demand (COD), total inorganic nitrogen (TIN), NH4+-N and total phosphorus (TP) were more than 98%, 96%, 97% and 97%, respectively. The result indicated gradually increasing the proportion of real wastewater in influent was a useful domestication method, and the feasibility of AGS for treatment of high C/N ratio industrial wastewater.

[Effect of modified zuoguiwan on Th17/Treg subpopulation of estrogen deficiency induced bone loss mice].

OBJECTIVE: To observe the effect of Modified Zuoguiwan (MZ) on the balance between helper T cell subsets 17 (Th17) and regulatory T cell subsets (Treg) in estrogen deficiency induced bone loss mice and to explore its mechanism. METHODS: Totally 50 BALB/c mice were divided into the sham-operation group, the ovariectomy model group, the low dose MZ group, the middle dose MZ group, and the high dose MZ group by random digit table, 10 in each group. Mice in the low, middle, and high dose MZ groups were respectively administered with MZ at the daily dose of 7.25, 14.50, and 29.00 g/kg by gastrogavage, 0.5 mL each time for 12 successive weeks. Meanwhile, mice in the sham-operation group and the ovariectomy model group were administered with equal volume by gastrogavage, 0.50 mL each time. The serum estradiol (E2) level was assessed by enzyme linked immunosorbent assay (ELISA). Bone mineral density (BMD) of thigh bone was measured with dual energy X ray absorptiometry. In addition, the population of Th17/Treg subsets in spleen mononuclear cells was analyzed by extracellular and intracellular staining method using flow cytometry. Moreover, the mRNA expression of IL-17A and TGF-ß in the spleen mononuclear cells was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared with the sham-operation group, both E2 and BMD significantly decreased, the percentage of Th17 subset and Th17/Treg ratio both increased, the percentage of Treg subset obviously decreased, the expression of IL-17A mRNA significantly increased, and the expression of TGF-ß mRNA significantly decreased in the ovariectomy model group (all P < 0.05). Compared with the model group, BMD obviously increased, the percentage of Th17 subset and Th17/Treg ratio both decreased, the percentage of Treg subset obviously increased, the expression of IL-17A mRNA significantly decreased, and the expression of TGF-ß mRNA significantly increased in the middle dose MZ group and the high dose MZ group (all P < 0. 05). Correlation analyses showed that BMD was positively related to both the serum E2 level and the percentage of Treg subset (P < 0.05), but negatively related to the percentage of Th17 subset (P < 0.05). In addition, the serum E2 level was positively related to the percentage of Treg subset, but obviously negatively related to that of Th17 subset (P < 0.05). CONCLUSIONS: There was correlation between Th17/Treg imbalance and E2 deficient bone loss. MZ could decrease the proportion of Th17 subset, but elevate the proportion of Treg subset in E2 deficient bone loss mice. It could achieve therapeutic effect through adjusting the balance of Th17/Treg in E2 deficient bone loss mice.

A method for rapid nanobody screening with no bias of the library diversity.

Nanobody, referred to the variable domain of heavy-chain-only antibodies, has several advantages such as small size and feasible Escherichia coli expression, making them promising for scientific research and therapies. Conventional nanobody screening and expression methods often suffer from the need for subcloning into expression vectors and amplification-induced diversity loss. Here, we developed an integrated method for simultaneous screening and expression. Nanobody libraries were cloned and secretly expressed in the culture medium. Target-specific nanobodies were isolated through 1-3 rounds of dilution and regrowth following the Poisson distribution. This ensured no dismissal of positive clones, with populations of positive clones increasing over 10-fold in each dilution round. Ultimately, we isolated 5 nanobodies against death domain receptor 5 and 5 against Pyrococcus furiosus DNA polymerase directly from their immunized libraries. Notably, our approach enables nanobody screening without specialized instruments, demonstrating broad applicability in routine monoclonal nanobody production for diverse biomedical applications.

The macrophage polarization by miRNAs and its potential role in the treatment of tumor and inflammation (Review).

The characteristics of monocyte/macrophage lineage are diversity and plasticity, mainly manifested by M1 and M2 subtypes in the body tissues, and playing different roles in the immunity. In the polarization process of macrophages, the classic molecular mechanism is related to sequential transcription factors. Whether in tumor or inflammatory local microenvironment, the pathological factors of the local microenvironment often affect the polarization of M1 and M2 macrophages, and participate in the occurrence and development of these pathological processes. In recent years, a growing number of research results demonstrated that non­coding RNA (ncRNA) also participates in the polarization process of macrophages, in addition to traditional cytokines and transcriptional regulation signal pathway molecules. Among numerous ncRNAs, microRNAs (miRNAs) have attracted more attention from scholars both domestically and internationally, and significant progress has been made in basic and clinical research. Therefore, for improved understanding of the molecular mechanism of miRNAs in macrophage polarization and analysis of the potential value of this regulatory pathway in tumor and inflammatory intervention therapy, a comprehensive review of the progress of relevant literature research was conducted and some viewpoints and perspectives were proposed.

Role of miR­let­7c­5p/c­myc signaling axis in the committed differentiation of leukemic THP­1 cells into monocytes/macrophages.

In a preliminary experiment, it was found that c-myc expression was decreased following the differentiation of THP-1 cells into monocytes/macrophages induced by phorbol 12-myristate 13 acetate (PMA) + lipopolysaccharide (LPS) + interferon (IFN)-γ. The expression of miR-let-7c-5p was then found to be elevated by cross-sectional analysis using TargetScan and PubMed and differential microarray analysis. The present study aimed to investigate the role of the miR-let-7c-5p/c-myc signaling axis in the committed differentiation of THP-1 leukemic cells into monocytes/macrophages induced by PMA + LPS + IFN-γ. Human THP-1 leukemic cells were induced to differentiate into monocytes/macrophages by PMA + LPS + IFN-γ. Following induction for 48 h, the growth density of the THP-1 cells was observed directly under an inverted microscope, cell proliferation was measured using Cell Counting Kit-8 assay and the cell cycle and the expression of differentiation-related antigens (CD11b and CD14) were measured using flow cytometry. The mRNA expression of miR-let-7c-5p and c-myc was detected using reverse transcription-quantitative PCR and the protein expression of c-myc was detected using western blot analysis. Dual luciferase reporter gene analysis was used to detect the targeted binding of miR-let-7c-5p on the 3'UTR of c-myc. The relative expression of miR-let-7c-5p and c-myc genes in THP-1 cells induced by PMA + LPS + IFN-γ was found to be up- and downregulated respectively, and expression of miR-let-7c-5p was negatively correlated with the expression of c-myc gene. Dual luciferase reporter gene assays confirmed that miR-let-7c-5p targeted the 3'UTR of c-myc and inhibited luciferase activity. Following transfection with miR-let-7c-5p mimics, the expression of c-myc was markedly downregulated and the proliferative ability of the THP-1 cells was decreased, while the expression rate of CD11b and CD14 was significantly increased. The rescue experiment revealed that the effects of miR-let-7c-5p mimics on the proliferation and differentiation of THP-1 cells were attenuated by transfection with c-myc overexpression vector. Together, the findings of the present study demonstrated that miR-let-7c-5p can target the 3'UTR region of c-myc and that the miR-let-7c-5p/c-myc signaling axis is one of the critical pathways involved in the directional differentiation of leukemic cells into monocytes/macrophages.

Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods have drawbacks such as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/mL) of several target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency from 90 to 97% for different cell lines. Moreover, the free penetration of molecules less than 600 Da across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

Protein Conformational Exchanges Modulated by the Environment of Outer Membrane Vesicles.

Protein function, in many cases, is strongly coupled to the dynamics and conformational equilibria of the protein. The environment surrounding proteins is critical for their dynamics and can dramatically affect the conformational equilibria and subsequently the activities of proteins. However, it is unclear how protein conformational equilibria are modulated by their crowded native environments. Here we reveal that outer membrane vesicle (OMV) environments modulate the conformational exchanges of Im7 protein at its local frustrated sites and shift the conformation toward its ground state. Further experiments show both macromolecular crowding and quinary interactions with the periplasmic components stabilize the ground state of Im7. Our study highlights the key role that the OMV environment plays in the protein conformational equilibria and subsequently the conformation-related protein functions. Furthermore, the long-lasting nuclear magnetic resonance measurement time of proteins within OMVs indicates that they could serve as a promising system for investigating protein structures and dynamics in situ via nuclear magnetic spectroscopy.

Effects of marine environments on methane hydrate formation in clay nanopores: A molecular dynamics study.

In nature, CH4 hydrates are mainly buried in marine sediments. The complex marine environments on the seafloor continuously affect hydrate formation. Herein, systematic molecular simulations have been performed to investigate CH4 hydrate formation in clay nanopore, mainly affected by several marine environmental factors, including seawater salinity, pressure and temperature. Simulation results show that these factors exert different effects on hydrate formation in the nanopore and the outside bulk solutions by affecting the mass transfer and phase separation inside and outside of the nanopore. Specifically, high salinity hinders the diffusion of CH4 molecules from nanopores into the outside bulk solutions, promoting hydrate formation in nanopore and inhibiting hydrate formation in bulk solution; salinity has a dual effect on hydrate formation in the whole system by changing the local CH4 concentration via the formation of the hydration of salt ions. High pressure favors the diffusion of CH4 molecules from nanopore into outside bulk solutions, promoting hydrate formation in bulk solution and inhibiting hydrate formation in nanopore; high pressure promotes hydrate formation at the nanopore throats by increasing CH4 concentration and reducing ion concentration therein. In contrast, temperature significantly affects hydrate formation in the system by causing phase separation, i.e. high temperature promotes the aggregation of CH4 molecules to form nanobubbles and inhibits hydrate formation. Under high temperature conditions, the nanobubble in the nanopore gradually decomposes, while the nanobubble in the outside bulk solution grows an extra-large cylindrical nanobubble. These molecular insights into the formation behavior of CH4 hydrates in clay nanopores are helpful for understanding the formation process of natural gas hydrates in marine sediments and the development and utilization of CH4 hydrates.