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Severe acute respiratory syndrome coronavirus 2 variants have continued to emerge in diverse geographic locations with a temporal distribution. The Lambda variant containing multiple mutations in the spike protein, has thus far appeared mainly in South America. The variant harbours two mutations in the receptor binding domain, L452Q and F490S, which may change its infectivity and antigenicity to neutralizing antibodies. In this study, we constructed 10 pseudoviruses to study the Lambda variant and each individual amino acid mutation's effect on viral function, and used eight cell lines to study variant infectivity. In total, 12 monoclonal antibodies, 14 convalescent sera, and 23 immunized sera induced by mRNA vaccines, inactivated vaccine, and adenovirus type 5 vector vaccine were used to study the antigenicity of the Lambda variant. We found that compared with the D614G reference strain, Lambda demonstrated enhanced infectivity of Calu-3 and LLC-MK2 cells by 3.3-fold and 1.6-fold, respectively. Notably, the sensitivity of the Lambda variant to 5 of 12 neutralizing monoclonal antibodies, 9G11, AM180, R126, X593, and AbG3, was substantially diminished. Furthermore, convalescent- and vaccine-immunized sera showed on average 1.3–2.5-fold lower neutralizing titres against the Lambda variant. Single mutation analysis revealed that this reduction in neutralization was caused by L452Q and F490S mutations. Collectively, the reduced neutralization ability of the Lambda variant suggests that the efficacy of monoclonal antibodies and vaccines may be compromised during the current pandemic.

Emerging mutations in SARS-CoV-2 cause several waves of COVID-19 pandemic. Here we investigate the infectivity and antigenicity of ten emerging SARS-CoV-2 variants—B.1.1.298, B.1.1.7(Alpha), B.1.351(Beta), P.1(Gamma), P.2(Zeta), B.1.429(Epsilon), B.1.525(Eta), B.1.526-1(Iota), B.1.526-2(Iota), B.1.1.318—and seven corresponding single amino acid mutations in the receptor-binding domain using SARS-CoV-2 pseudovirus. The results indicate that the pseudovirus of most of the SARS-CoV-2 variants (except B.1.1.298) display slightly increased infectivity in human and monkey cell lines, especially B.1.351, B.1.525 and B.1.526 in Calu-3 cells. The K417N/T, N501Y, or E484K-carrying variants exhibit significantly increased abilities to infect mouse ACE2-overexpressing cells. The activities of furin, TMPRSS2, and cathepsin L are increased against most of the variants. RBD amino acid mutations comprising K417T/N, L452R, Y453F, S477N, E484K, and N501Y cause significant immune escape from 11 of 13 monoclonal antibodies. However, the resistance to neutralization by convalescent serum or vaccines elicited serum is mainly caused by the E484K mutation. The convalescent serum from B.1.1.7- and B.1.351-infected patients neutralized the variants themselves better than other SARS-CoV-2 variants. Our study provides insights regarding therapeutic antibodies and vaccines, and highlights the importance of E484K mutation., Li Zhang, Zhimin Cui, and Qianqian Li et al. compare the infectivity, host tropism, and antigenicity of 10 SARS-CoV-2 variants using a VSV-based pseudovirus system. Their results suggest that variants carrying E484K display the most significant reduction in sensitivity to neutralization, and may provide further insight into the development of relevant therapeutics for SARS-CoV-2 infection.

Amyloid aggregation of α-synuclein (α-syn) is closely associated with Parkinson’s disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of α-syn have been identified causative to the early onset of familial PD. Here, we report the cryo-EM structure of an α-syn fibril formed by N-terminally acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a distinct fold of α-syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) α-syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with an enhanced seeding capability than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of α-syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs., The E46K α-synuclein mutation causes familial Parkinson’s disease. Here, the authors present the cryo-EM structure of N-terminally acetylated E46K α-synuclein fibrils and find that it is distinct from other known α-synuclein fibril structures.

Nowadays, immune diseases are a large burden in healthcare. Mesenchymal stem cells (MSCs) have prominent ability in immunomodulation and have been applicated on treating many immune-related diseases. However, the clinical outcomes can be disparate and sometimes completely counterproductive beyond explanation of cell heterogeneity. The theory of immunomodulation plasticity in MSCs has then emerged to explain that MSCs can be induced into proinflammatory MSC1 or anti-inflammatory MSC2 responding to different immune environment. It would be safer and more efficient if we could induce MSCs into a certain immune phenotype, in most cases MSC2, prior to medical treatment. In this study, we screened and identified a classical FDA-approved drug, chlorzoxazone (CZ). Unlike traditional method induced by IFN-γ, CZ can induce MSC into MSC2 phenotype and enhance the immunosuppressive capacity without elevation of immunogenicity of MSCs. CZ-treated MSCs can better inhibit T cells activation and proliferation, promote expression of IDO and other immune mediators in vitro, and alleviate inflammatory infiltration and tissue damage in acute kidney injury rat model more effectively. Moreover, we discovered that CZ modulates phosphorylation of transcriptional factor forkhead box O3 (FOXO3) independent of classical AKT or ERK signaling pathways, to promote expression of downstream immune-related genes, therefore contributing to augmentation of MSCs immunosuppressive capacity. Our study established a novel and effective approach to induce MSC2, which is ready for clinical application.

Backgroud: Adult-derived mesenchymal stem cells (MSCs) can be used in therapies for the treatment of various diseases. However, MSCs derived from aging tissues or long-term MSC cultures could have diminished therapeutic effects compared with MSCs derived from younger tissues, but the underlying mechanism has not been completely established. Dysfunction of energy metabolism is one of the main mechanisms underlying cell senescence. Although cyclic adenosine monophosphate (cAMP) is known to inhibit cell division and proliferation in vitro, its impact on MSC senescence has not been described. Methods: In this study, we used forskolin, an adenylate cyclase agonist and cAMP inducer, to disrupt metabolism in human adipose-derived MSCs and investigate the effects of metabolic dysfunction on MSC senescence. cAMP and ATP levels were identified by its ELISA kits. The identification of MSCs senescence was assessed by MTS assay, cell cycle tests, β-galactosidase staining, and genes evaluation through qRT-PCR and Western blotting. Functions of the forskolin treated-MSCs were evaluated by oil Red O staining and alkaline phosphatase staining, as well as mRNA tests and wound healing assay. Mitochondrial morphology and function tests were performed by transmission electron microscopy and oxygen consumption analysis, mitochondrial superoxide assay, quantification of nitric oxide.Results: Treatment of human MSCs with forskolin resulted in senescence phenotypes, including reduced proliferation, cell-cycle arrest, and enhanced expression of the cell aging markers P16 and P21. Furthermore, the senescent MSCs exhibited increased adipogenesis capacity and decreased osteogenesis capacity as well as a senescence-associated secretory phenotype characterized by increased expression of several inflammatory factors. Forskolin-associated MSC senescence was mainly caused by oxidative stress–induced disruption of mitochondrial metabolism, and the senescent MSCs had high levels of reactive oxygen species and reduced sirtuin gene expression. Lastly, we found that cAMP inhibitor SQ22536 protects MSCs from forskolin-induced senescence and senescence-related-inflammatory-phenotype (SASP). Conclusions: Our results indicate that forskolin can cause senescence of human MSCs through oxidative stress–induced mitochondrial metabolic dysfunction.

This study aimed to investigate whether tumor volume (TV) is better than diameter for predicting the prognosis of patients with early-stage non-small cell lung cancer (NSCLC) after complete resection. This study retrospectively reviewed the clinicopathologic characteristics of 274 patients with early-stage NSCLC who had received pretreatment computed tomography (CT) scans and complete resection. TV was semi-automatically measured from CT scans using an imaging software program. The optimal cutoff of TV was determined by X-tile software. Disease-free survival (DFS) and overall survival (OS) were assessed by the Kaplan–Meier method. The prognostic significance of TV and other variables was assessed by Cox proportional hazards regression analysis. Using 3.046 cm3 and 8.078 cm3 as optimal cutoff values of TV, the patients were separated into three groups. A larger TV was significantly associated with poor DFS and OS in the multivariable analysis. Kaplan–Meier curves of DFS and OS showed significant differences on the basis of TV among patients with stage 1a disease, greatest tumor diameter (GTD) of 2 cm or smaller, and GTD of 2–3 cm, respectively. Using two TV cutoff points, three categories of TV were created. In 54 cases (19.7%), patients migrated from the GTD categories of 2 cm or smaller, 2–3 cm, and larger than 3 cm into the TV categories of 3.046 cm3 or smaller, 3.046–8.078 cm3, and larger than 8.078 cm3. TV is an independent prognostic factor of DFS and OS for early-stage NSCLC. The findings show that TV is better than GTD for predicting the prognosis of patients with early-stage NSCLC.

Background The efficacy of adjuvant targeted therapy for operable lung cancer is still under debate. Comprehensive genetic profiling is needed for detecting co-mutations in resected epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma (ADC), which may interfere the efficacy of adjuvant tyrosine kinase inhibitor (TKI) treatment. Materials and Methods Mutation profiling of 416 cancer-relevant genes was conducted for 139 resected stage I–IIIa lung ADCs with EGFR mutations using targeted next-generation sequencing. Co-mutation profiles were systematically analyzed. Results Rare EGFR alterations other than exon 19 deletion and L858R, such as L861Q (∼3%) and G719A (∼2%), were identified at low frequencies. Approximately 10% of patients had mutations in EGFR exon 20 that could confer resistance to first-generation TKIs. Ninety-one percent of patients harbored at least one co-mutation in addition to the major EGFR mutation. TP53 was the top mutated gene and was found more frequently mutated at later stage. Markedly, NF1 mutations were found only in stage II–III ADCs. Conversely, RB1 mutations were more frequent in stage I ADCs, whereas APC mutations were observed exclusively in this group. Thirty-four percent of patients with EGFR TKI-sensitizing mutations had genetic alterations involving EGFR downstream effectors or bypass pathways that could affect the response to EGFR TKIs, such as PIK3CA, BRCA1, and NOTCH1. Conclusion Operable lung ADCs with EGFR TKI-sensitizing mutations are associated with a high proportion of co-mutations. Mutation profiling of these resected tumors could facilitate in determining the applicability and efficacy of adjuvant EGFR TKI therapeutic strategy. Implications for Practice The efficacy of adjuvant epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy for lung cancer harboring EGFR mutation after surgical resection is still under debate. Next-generation sequencing of 416 cancer-relevant genes in 139 resected lung cancers revealed the co-mutational landscape with background EGFR mutation. Notably, the study identified potential EGFR TKI-resistant mutations in 34.71% of patients with a drug-sensitizing EGFR mutation and who were naive in terms of targeted therapy. A comprehensive mutation profiling of these resected tumors could facilitate in determining the applicability and efficacy of adjuvant EGFR TKI therapeutic strategy for these patients.

The transcriptomic classification of glioblastoma (GBM) has failed to predict survival and therapeutic vulnerabilities. A computational approach for unbiased identification of core biological traits of single cells and bulk tumors uncovered four tumor cell states and GBM subtypes distributed along neurodevelopmental and metabolic axes, classified as proliferative/progenitor, neuronal, mitochondrial and glycolytic/plurimetabolic. Each subtype was enriched with biologically coherent multiomic features. Mitochondrial GBM was associated with the most favorable clinical outcome. It relied exclusively on oxidative phosphorylation for energy production, whereas the glycolytic/plurimetabolic subtype was sustained by aerobic glycolysis and amino acid and lipid metabolism. Deletion of the glucose-proton symporter SLC45A1 was the truncal alteration most significantly associated with mitochondrial GBM, and the reintroduction of SLC45A1 in mitochondrial glioma cells induced acidification and loss of fitness. Mitochondrial, but not glycolytic/plurimetabolic, GBM exhibited marked vulnerability to inhibitors of oxidative phosphorylation. The pathway-based classification of GBM informs survival and enables precision targeting of cancer metabolism.

The treatment of chronic myeloid leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, has yielded clinical success. However, the direct targeting of BCR-ABL does not eradicate CML cells expressing mutant BCR-ABL, especially the T315I mutation in BCR-ABL. Moreover, increasing mutations were identified in BCR-ABL domain, resulting in TKIs resistance recently. It is necessary to find BCR-ABL-independent target for treating CML patients with various mutations, including T315I mutation in BCR-ABL. The dichotomous behavior of CREB binding protein (CBP) and E1A protein (p300), recruited by β-catenin associated with self-renewal and differentiation, have been identified in hematopoietic stem cells, respectively. In this study, CBP was aberrantly expressed in CML cells on the basis of Oncomine dataset. The β-catenin bound with much more CBP than p300 in CML cells. Down-regulation of CBP inhibited cell proliferation capacity and increased the binding of β-catenin to p300, thus promoting cell differentiation and p53-dependent cell senescence in CML cells with either wild type or T315I mutant BCR-ABL in vitro and in vivo models. These demonstrate CBP blockage can be developed for the treatment of CML independent of BCR-ABL mutation status including T315I.

Background: This study aimed to evaluate T staging system for non-small cell lung cancer (NSCLC) using tumor volume (TV) and other prognostic factors. Methods: This study included 1309 cases. The TV and greatest tumor diameter (GTD) were semi-automatically measured. The receiver operating characteristic (ROC) curves of TV and GTD were used to predict survival. The regression analysis was used to describe the correlation between GTD and TV. Overall survival (OS) was analyzed using the Kaplan-Meier method. Cox’s proportional hazards regression model was applied for multivariate analysis. Results: Using the OS in pN0M0 patients (997 cases), we obtained 4 optimal cutoff values and divided all cases into 5 TV groups (V1: TV ≤ 2.80 cm3; V2: TV > 2.80–6.40 cm3; V3: TV > 6.40–12.9 cm3; V4: TV > 12.9–55.01 cm3; V5: TV > 55.01 cm3) with significant OS ( P < 0.001). Multivariate analysis showed that age, visceral pleural invasion (VPI), and all TV cutoff points were independent factors of OS ( P < 0.05). For V3 and V4 groups, the OS in patients without VPI was better than that in patients with VPI. Using the values of TV, VPI, and N stages, we classified all cases into 5 stages from I to V depending on the OS. The OS in I, II, III, IV, and V stages were 71.3%, 65.5%, 59.8%, 47.7%, and 35.1% respectively ( P < 0.001). Conclusions: We proposed a new T staging system using TV as the main prognostic descriptor in NSCLC patients, which may provide a better comprehensive clinical value than GTD in clinical applications.

Background In this study, we sought to evaluate the correlation between TV, GTD, and lymph node metastases in NSCLC patients with tumors of GTD ≤ 3.0 cm. Methods We retrospectively analyzed the characteristics of clinicopathologic variables for lymph node involvement in 285 NSCLC patients with tumors of GTD ≤ 3.0 cm who accepted curative surgical resection. The TVs were semi-automatically measured by a software, and optimal cutoff points were obtained using the X-tile software. The relationship between GTD and TV were described using non-linear regression. The correlation between GTD, TV, and N stages was analyzed using the Pearson correlation coefficient. The one-way ANOVA was used to compare the GTD and TV of different lymph node stage groups. Results The relationship between GTD and TV accorded with the exponential growth model: y = 0.113e1.455x (y = TV, x = GTD). TV for patients with node metastases (4.78 cm3) was significantly greater than those without metastases (3.57 cm3) (P < 0.001). However, there were no obvious GTD differences in cases with or without lymph node metastases (P = 0.054). We divided all cases into three TV groups using the two cutoff values (0.9 cm3 and 3.9 cm3), and there was an obvious difference in the lymphatic involvement rate between the groups (P < 0.001). The tendency to metastasize was greater with higher TV especially when the TV was > 0.9–14.2 cm3 (P = 0.010). Conclusions For NSCLC tumors with GTD ≤ 3.0 cm, TV is a more sensitive marker than GTD in predicting the positive lymph node metastases. The likelihood for metastasis increases with an increasing TV especially when GTD is > 2.0–3.0 cm.

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 A cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.

The bilateral recurrent laryngeal nerve (RLN) lymph nodes are the most common metastatic site for esophageal squamous cell carcinoma (ESCC); however, the RLNs are susceptible to injury during dissection. Clinically, there is an urgent need to determine an effective diagnostic method for RLN nodes to help achieve selective nodal dissection and avoid potential serious complications by performing more conservative surgery for those with nonmetastatic nodes. Here, we innovatively applied endobronchial ultrasonography (EBUS) and investigated its diagnostic performance for preoperative evaluation of RLN nodes in ESCC patients. All 81 enrolled ESCC patients underwent preoperative EBUS and CT examinations. The ability of EBUS and CT to detect RLN node metastasis was evaluated based on the resulting sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV). The diagnostic performance of EBUS was superior to that of CT; in particular, EBUS of the left RLN (L-RLN) nodes presented the best sensitivity, specificity, PPV, NPV, and accuracy compared with EBUS evaluations of the right RLN (R-RLN) nodes, CT of the L-RLN and R-RLN nodes. Moreover, EBUS combined with CT increased the NPV relative to that of EBUS or CT alone, promoting the ability to identify true-negative RLN nodes. In particular, the NPVs of the combined modality were 100% for both the L- and R-RLN nodes in early-T-stage (T1–T2) ESCC. EBUS is an efficient tool for RLN node evaluation, and the combination with CT may provide better guidance for selective RLN node dissection in ESCC patients.

Background Single-cell RNA sequencing is the reference technique for characterizing the heterogeneity of the tumor microenvironment. The composition of the various cell types making up the microenvironment can significantly affect the way in which the immune system activates cancer rejection mechanisms. Understanding the cross-talk signals between immune cells and cancer cells is of fundamental importance for the identification of immuno-oncology therapeutic targets. Results We present a novel method, single-cell Tumor–Host Interaction tool (scTHI), to identify significantly activated ligand–receptor interactions across clusters of cells from single-cell RNA sequencing data. We apply our approach to uncover the ligand–receptor interactions in glioma using 6 publicly available human glioma datasets encompassing 57,060 gene expression profiles from 71 patients. By leveraging this large-scale collection we show that unexpected cross-talk partners are highly conserved across different datasets in the majority of the tumor samples. This suggests that shared cross-talk mechanisms exist in glioma. Conclusions Our results provide a complete map of the active tumor–host interaction pairs in glioma that can be therapeutically exploited to reduce the immunosuppressive action of the microenvironment in brain tumor.

Background: A novel coronavirus (the SARS-CoV-2) has been identified in January 2020 as the causal pathogen for COVID-19, a pandemic started near the end of 2019. The Angiotensin converting enzyme 2 protein (ACE2) utilized by the SARS-CoV as a receptor was found to facilitate the infection of SARS-CoV-2, initiated by the binding of the spike protein to human ACE2. Methods: Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure and dynamics of the ACE2 in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Results: The predicted model is highly consistent with the experimentally determined structures, validating the homology modeling results. Besides the binding interface reported in the crystal structures, novel binding poses are revealed from all-atom MD simulations. The simulation data are used to identify critical residues at the complex interface and provide more details about the interactions between the SARS-CoV-2 RBD and human ACE2. Conclusion: Simulations reveal that RBD binds to both open and closed state of ACE2. Two human ACE2 mutants and rat ACE2 are modeled to study the mutation effects on RBD binding to ACE2. The simulations show that the N-terminal helix and the K353 are very important for the tight binding of the complex, the mutants are found to alter the binding modes of the CoV2-RBD to ACE2.

Background Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. Methods The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. Results Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. Conclusions Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

Brain tumors are among the most challenging human tumors for which the mechanisms driving progression and heterogeneity remain poorly understood. We combined single-cell RNA-seq with multi-sector biopsies to sample and analyze single-cell expression profiles of gliomas from 13 Chinese patients. After classifying individual cells, we generated a spatial and temporal landscape of glioma that revealed the patterns of invasion between the different sub-regions of gliomas. We also used single-cell inferred copy number variations and pseudotime trajectories to inform on the crucial branches that dominate tumor progression. The dynamic cell components of the multi-region biopsy analysis allowed us to spatially deconvolute with unprecedented accuracy the transcriptomic features of the core and those of the periphery of glioma at single-cell level. Through this rich and geographically detailed dataset, we were also able to characterize and construct the chemokine and chemokine receptor interactions that exist among different tumor and non-tumor cells. This study provides the first spatial-level analysis of the cellular states that characterize human gliomas. It also presents an initial molecular map of the cross-talks between glioma cells and the surrounding microenvironment with single-cell resolution.

Cell transplantation holds considerable promise for end-stage liver diseases but identifying a suitable, transplantable cell type has been problematic. Here, we describe a novel type of mesenchymal stem cells (MSCs) from human adipose tissue. These cells are different from previously reported MSCs, they are in the euchromatin state with epigenetic multipotency, and express pluripotent markers MYC, KLF4, and GMNN. Most of the genes associated with germ layer specification are modified by H3K4me3 or co-modified by H3K4me3 and H3K27me3. We named this new type of MSCs as adult multipotent adipose-derived stem cells (M-ADSCs). Using a four-step nonviral system, M-ADSCs can be efficiently Induced into hepatocyte like cells with expression of hepatocyte markers, drug metabolizing enzymes and transporters, and the other basic functional properties including albumin (ALB) secretion, glycogen storage, detoxification, low-density lipoprotein intake, and lipids accumulation. In vivo both M-ADSCs-derived hepatoblasts and hepatocytes could form vascularized liver-like tissue, secrete ALB and express metabolic enzymes. Single-cell RNA-seq was used to investigate the important stages in this conversion. M-ADSCs could be converted to a functionally multipotent state during the preinduction stage without undergoing reprogramming process. Our findings provide important insights into mechanisms underlying cell development and conversion.

α-Synuclein (α-syn) amyloid fibril, as the major component of Lewy bodies and pathological entity spreading in human brain, is closely associated with Parkinson’s disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of α-syn have been identified causative to the early onset of familial PD. Here, we determined the cryo-EM structure of a full-length α-syn fibril formed by N-terminal acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a new fold of α-syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) α-syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with a higher capability of seeding fibril formation than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of α-syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs.

A high rate of disease relapse makes epithelial ovarian cancer (EOC) the leading cause of death among all gynecologic malignancies. These relapses are often due to tumor dormancy. Here we identify the RNA polymerase II transcriptional mediator subunit 12 (MED12) as an important molecular regulator of tumor dormancy. MED12 knockout (KO) induced dormancy of EOC cells in vitro and in vivo, and microarray analysis showed that MED12 KO decreased expression of EGFR. Restoration of EGFR expression in MED12 KO cells restored proliferation. Additionally, MED12 bound to the promoter of EGFR, and correlation studies showed that MED12 expression positively correlated with EGFR expression in EOC patient samples. Clinical data demonstrated that chemotherapy-resistant patients expressed lower levels of MED12 compared with responsive patients. Overall, our data show that MED12 plays an important role in regulating dormancy of EOC through regulation of EGFR. Significance: MED12 is identified as a novel, important regulator of tumor dormancy in human ovarian cancer. Cancer Res; 78(13); 3532–43. ©2018 AACR.

Background/Aims: Multidrug resistance (MDR) induced by the ABC transporter subfamily B member 1 (ABCB1) and subfamilyG member 2 (ABCG2) limits successful cancer chemotherapy and no commercially available MDR modulator is used in the clinic. In the current study, we aimed to investigate the effects of PCI29732 on the enhancement of chemotherapeutic agents. Methods: Cell cytotoxicity and reversal effect were measured with MTT assay. Additionally, flow cytometry was employed to detect the accumulation and efflux of the drugs. We investigated the interaction of PCI29732 and the substrate binding sites of ABCG2 was investigated via the photo-labeling of ABCG2 with the [125I] iodoarylazidoprazosin. The vanadate-sensitive ATPase activity of ABCG2 was measured to identify whether the drug affected the ATPase activity. RT-PCR and Western blot were utilized to analyze mRNA and protein expression respectively. Results: Here, we found that PCI29732 significantly enhanced the efficacy of substrate chemotherapeutic agents in ABCG2-overexpressing cells and also in xenografts harboring the H460/MX20 cell that overexpress ABCG2, but not in their parental sensitive cells and ABCB1-overexpressing cells. Mechanistically, the intracellular accumulations of doxorubicin and Rhodamine 123 were increased in ABCG2-overexpressing S1-MI-80 cells with the presence of PCI29732. PCI29732 stimulated the ATPase activity of ABCG2 at low concentrations. However at the high concentrations, PCI29732 inhibited the ATPase activity, and competed with [125I]-iodoarylazidoprazosin for photo-affinity labeling of ABCG2. PCI29732 did neither alter the mRNA or protein expression levels of ABCG2 nor the phosphorylation levels of AKT and ERK1/2. Conclusion: Our study demonstrates that PCI29732 inhibits the function of ABCG2 by competitively binding to the ATP-binding site of ABCG2 and enhances the anti-tumor efficacy of substrate chemotherapeutic agents, This findings encourages the development of combinational chemotherapy for the treatment of ABCG2- overexpressing cancer patients.

Background Immunotherapy is effective in treating unresectable esophageal squamous cell carcinoma (ESCC), but little is known about its role in the preoperative setting. The aim of this study was to evaluate the safety, feasibility and efficacy of neoadjuvant treatment with camrelizumab plus chemotherapy in locally advanced ESCC. Methods Patients diagnosed with locally advanced ESCC were retrospectively included if they had received neoadjuvant camrelizumab plus nab-paclitaxel and S1 capsule followed by radical esophagectomy between November, 2019 and June, 2020 at Sun Yat-sen University Cancer Center. Primary endpoints were safety and feasibility. In addition, pathological response and the relationship between tumor immune microenvironment (TIME)/tumor mutational burden (TMB) and treatment response were also investigated. Results Twelve patients were included and they all received three courses of preoperative treatment with camrelizumab plus nab-paclitaxel/S1. No grade 3 or higher toxicities occurred. No surgical delay or perioperative death was reported. Nine patients (75%) responded to the treatment, four with a complete pathological response (pCR) and five with a major pathological response (MPR). Neither programmed death-ligand 1 (PD-L1) expression nor TMB was correlated with treatment response. TIME analysis revealed that a higher abundance of CD56dim natural killer cells was associated with better pathological response in the primary tumor, while lower density of M2-tumor-associated macrophages was associated with better pathological response in the lymph nodes (LNs). Conclusions Neoadjuvant camrelizumab plus nab-paclitaxel and S1 is safe, feasible and effective in locally advanced ESCC and is worth further investigation.

The prognostic significance of serum lactate dehydrogenase (LDH) level in thymic carcinoma (TC) remains unclear. Therefore, we evaluated the role of pretreatment serum LDH level in the prognosis for TC in this study.Sixty consecutive surgical patients were analyzed in this study with pathologic confirmed TC in Sun Yat-sen University Cancer Center from June 1996 to June 2014.The cut-off value of LDH was 210.50 IU/L. In both univariate analysis and multivariable analysis, only pretreatment serum LDH level (P=0.027) and pathological Masaoka stage (P=0.041) were associated with overall survival (OS). In univariate analysis, pretreatment serum LDH level, tumor size, postoperative radiotherapy (PORT) and pathological Masaoka stage were associated with disease-free survival (DFS) (all P0.050). Multivariable analysis showed that LDH level (P=0.001), PORT (P=0.001) and pathological Masaoka stage (P=0.038) were independently prognostic factors of DFS. This study also revealed that male patients and larger tumor size had a significantly higher rate of elevated pretreatment serum LDH level than in the other groups.In conclusion, pretreatment serum LDH level was an independent prognosis factor of OS and DFS for patients with TC.

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA–DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.

Subtle changes in protein sequences are able to alter ligand–protein interactions. Unraveling the mechanism of such phenomena is important for understanding ligand–protein interactions, including the DMXAA–STING interaction. DMXAA specifically binds to mouse STING instead of human STING. However, the S162A mutation and a newly discovered E260I mutation endow human STINGAQ with DMXAA sensitivity. Through molecular dynamics simulations, we revealed how these single mutations alter the DMXAA–STING interaction. Compared to mutated systems, structural correlations in the interaction of STINGAQ with DMXAA are stronger, and the correlations are cross-protomers in the dimeric protein. Analyses on correlation coefficients lead to the identification of two key interactions that mediate the strong cross-protomer correlation in the DMXAA–STINGAQ interaction network: DMXAA–267T–162S* and 238R–260E*. These two interactions are partially and totally interrupted by the S162A and E260I mutations, respectively. Moreover, a...

Objectives The purpose of this study was to investigate the prognostic impact of tumor volume (TV) on patients with stage I non-small cell lung cancer (NSCLC) after complete resection. Materials and Methods We retrospectively reviewed the clinicopathological characteristics of 274 patients with stage I NSCLC who had received preoperative chest computed tomography (CT) scans and complete resection. TV was semi-automatically measured from chest CT scans by using an imaging software program. The optimal cutoff values of TV were determined by X-tile software. Disease-free survival (DFS) and overall survival (OS) were compared using Kaplan-Meier analysis. Univariate and multivariate analyses were performed to identify risk factors for DFS and OS. Results By using 3.046cm 3 and 8.078cm 3 as two optimal cutoff values of TV, the patients were separated into three groups. The 5-year DFS and OS for patients with TV≤3.046cm 3 , 3.046–8.078cm 3 , and>8.078cm 3 were 88.0%, 73.6%, and 62.1%, respectively ( P Conclusion TV is an independent risk factor for DFS and OS for stage I NSCLC after complete resection. TV might provide additional prognostic information over tumor diameter in patients with stage I NSCLC.

Background Body mass index (BMI) has a U-shaped association with lung cancer risk. However, the effect of BMI on prognosis is controversial. This retrospective study aimed to investigate the effect of BMI on the survival of patients with stage I non-small cell lung cancer (NSCLC) after surgical resection. Methods In total, 624 consecutive stage I NSCLC patients who underwent radical resection were classified into four groups according to their BMI: underweight (BMI 28.0 kg/m2). The effect of BMI on progression-free survival (PFS) and overall survival (OS) was estimated using the Kaplan–Meier method and Cox proportional hazards model. Postoperative complications in each group were analyzed using the Chi square test or Fisher’s exact test. Results A univariate analysis showed that PFS and OS were longer in the overweight group than in other groups (both P

Single-cell RNA sequencing is the reference technique to characterize the heterogeneity of tumor microenvironment and can be efficiently used to discover cross-talk mechanisms between immune cells and cancer cells. We present a novel method, single cell Tumor-Host Interaction tool (scTHI), to identify significantly activated ligand-receptor interactions across clusters of cells from single-cell RNA sequencing data. We apply our approach to uncover the ligand-receptor interactions in glioma using six publicly available human glioma datasets encompassing 71 patients. We provide a comprehensive map of the signalling mechanisms between malignant cells and non-malignant cells in glioma uncovering potential novel therapeutic targets.

e16076 Background: Immunotherapy is effective in treating advanced esophageal squamous cell carcinoma (ESCC), but little is known about its role in the setting of neoadjuvant therapy. Methods: Data from a retrospective cohort and a prospective cohort of locally advanced ESCC patients were analyzed. All patients included had received neoadjuvant camrelizumab plus nab-paclitaxel and S1 capsule followed by radical esophagectomy. The main purpose of this study was to evaluate the safety and feasibility of the neoadjuvant treatment. In addition, pathological response and the relationship between tumor immune microenvironment features (TIME)/tumor mutational burden (TMB) and treatment response were also explored. Results: A total of 25 locally advanced ESCC patients from both cohorts were included, of which 12 from the retrospective cohort and 13 from the prospective cohort. Only two patients experienced grade 3 or 4 adverse events, including onehad anemia and the other (4%) had limb numbness. No surgical delay or perioperative death was reported. Sixteen patients (64%) responded to the treatment, eight (32%) with a complete pathological response and eight (32%) with a major pathological response. Neither programmed death-ligand 1 expression nor TMB was correlated with treatment response. In terms of TIME analysis, patients with better pathological response in the primary tumor had significantly higher abundance of CD56dim natural killer cells (109 vs 32, P= 0.01), while lower density of M2-tumor-associated macrophages was associated with numerically better pathological response in the lymph nodes (47 vs 163.1, P= 0.55). Conclusions: Neoadjuvant camrelizumab plus nab-paclitaxel and S1 is safe, feasible and effective, and the efficacy could be predicted by TIME, which is worthy of further investigation. Clinical trial information: ChiCTR2000029807.

The HeLa cells are the earliest and mostly used laboratory human cells for biomedical particularly cancer research. They were derived from a patient's cervical cancerous tissue, and are known for their heterogeneous cellular origin and variable genomic landscapes. Single-cell sequencing techniques with faithful linear and uniformly amplified genomes (DNA) and transcriptomes (RNA) may facilitate the study of cellular differences at the individual cell level. In this work, we have performed single-cell DNA and RNA sequencing with HeLa-CCL2 cells to study their heterogeneity. We have studied the complexity of copy number variations (CNVs) of HeLa-CCL2 genome at the single cell level, and revealed the transcriptomic heterogeneity of HeLa-CCL2. We also analyzed the relationship between genome and transcriptome at the single-cell level, and found overall correlation between CNV and transcriptome expression patterns. Finally, we concluded that although single-cell sequencing techniques are applicable to study heterogeneous cells such as HeLa-CCL2, the data analyses need to be more careful and well controlled.

Cyclic dinucleotides act as intracellular second messengers, modulating a variety of cellular activities including innate immune activation. Although phosphodiesterases (PDEs) hydrolyzing c-di-GMP and c-di-AMP have been identified, no PDEs for cGAMPs have been reported. Here we identified the first three cGAMP-specific PDEs in V. cholerae (herein designated as V-cGAP1/2/3). V-cGAPs are HD-GYP domain-containing proteins and specifically break 3′3′-cGAMP, but not other forms of cGAMP. 3′3′-cGAMP is first linearized by all three V-cGAPs to produce 5′-pApG, which is further hydrolyzed into 5′-ApG by V-cGAP1. In this two-step reaction, V-cGAP1 functions as both a PDE and a 5′-nucleotidase. In vivo experiments demonstrated that V-cGAPs play non-redundant roles in cGAMP degradation. The high specificity of V-cGAPs on 3′3′-cGAMP suggests the existence of specific PDEs for other cGAMPs, including 2′3′-cGAMP in mammalian cells. The absolute requirement of the GYP motif for 3′3′-cGAMP degradation suggests that HD domain-containing PDEs in eukaryotes are probably unable to hydrolyze cGAMPs. The fact that all V-cGAPs attack 3′3′-cGAMP on one specific phosphodiester bond suggests that PDEs for other cGAMPs would utilize a similar strategy. These results will provide valuable information for identification and characterization of mammalian 2′3′-cGAMP-specific PDEs in future studies.

Studies of gene rearrangements and the consequent oncogenic fusion proteins have laid the foundation for targeted cancer therapy. To identify oncogenic fusions associated with glioma progression, we catalogued fusion transcripts by RNA-seq of 272 gliomas. Fusion transcripts were more frequently found in high-grade gliomas, in the classical subtype of gliomas, and in gliomas treated with radiation/temozolomide. Sixty-seven in-frame fusion transcripts were identified, including three recurrent fusion transcripts: FGFR3-TACC3, RNF213-SLC26A11, and PTPRZ1-MET (ZM). Interestingly, the ZM fusion was found only in grade III astrocytomas (1/13; 7.7%) or secondary GBMs (sGBMs, 3/20; 15.0%). In an independent cohort of sGBMs, the ZM fusion was found in three of 20 (15%) specimens. Genomic analysis revealed that the fusion arose from translocation events involving introns 3 or 8 of PTPRZ and intron 1 of MET. ZM fusion transcripts were found in GBMs irrespective of isocitrate dehydrogenase 1 (IDH1) mutation status. sGBMs harboring ZM fusion showed higher expression of genes required for PIK3CA signaling and lowered expression of genes that suppressed RB1 or TP53 function. Expression of the ZM fusion was mutually exclusive with EGFR overexpression in sGBMs. Exogenous expression of the ZM fusion in the U87MG glioblastoma line enhanced cell migration and invasion. Clinically, patients afflicted with ZM fusion harboring glioblastomas survived poorly relative to those afflicted with non-ZM-harboring sGBMs (P < 0.001). Our study profiles the shifting RNA landscape of gliomas during progression and reveled ZM as a novel, recurrent fusion transcript in sGBMs.

High expression of fibrinogen and platelets are often observed in non–small cell lung cancer (NSCLC) patients with local regional or distant metastasis. However, the role of these factors remains unclear. The aims of this study were to evaluate the prognostic significance of plasma fibrinogen concentration and platelet count, as well as to determine the overall survival of NSCLC patients with brain metastases. A total of 275 NSCLC patients with brain metastasis were enrolled into this study. Univariate analysis showed that high plasma fibrinogen concentration was associated with age≥65 years (P = 0.011), smoking status (P = 0.009), intracranial symptoms (P = 0.022), clinical T category (P = 0.010), clinical N category (P = 0.003), increased partial thromboplastin time (P < 0.001), and platelet count (P < 0.001). Patients with low plasma fibrinogen concentration demonstrated longer overall survival compared with those with high plasma fibrinogen concentration (median, 17.3 months versus 11.1 months; P≤0.001). A similar result was observed for platelet counts (median, 16.3 months versus 11.4 months; P = 0.004). Multivariate analysis showed that both plasma fibrinogen concentration and platelet count were independent prognostic factors for NSCLC with brain metastases (R2 = 1.698, P < 0.001 and R2 = 1.699, P < 0.001, respectively). Our results suggest that high plasma fibrinogen concentration and platelet count indicate poor prognosis for NSCLC patients with brain metastases. Thus, these two biomarkers might be independent prognostic predictors for this subgroup of NSCLC patients.

Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.

Caspase 6 (CASP6) is a neuron degeneration-related protease and is widely considered to be a potential drug-design target against neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. The N-terminal pro-peptide of CASP6, also referred to as the pro-domain, contains 23 residues and its functional role remains elusive. In this study, the crystal structure of a full-length CASP6 zymogen mutant, proCASP6H121A, was solved. Although the pro-domain was flexible in the crystal, without visible electron density, structural analyses combined with biochemical assays revealed that the pro-domain inhibited CASP6 auto-activation by inhibiting intramolecular cleavage at the intersubunit cleavage site TEVD(193) and also by preventing this site from intermolecular cleavage at low protein concentration through a so-called `suicide-protection' mechanism. Further experiments showed that the length of the pro-domain and the side chain of Asn18 played critical roles in suicide protection. These results disclosed a new inhibitory mechanism of CASP6 and shed light on the pathogenesis and therapeutically relevant study of CASP6-related neurodegenerative diseases.

Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.

Summary Viral infection triggers host innate immune responses that result in the production of various cytokines including type I interferons (IFN), activation of inflammasomes, and programmed cell death of the infected cells. Tight control of inflammatory cytokine production is crucial for the triggering of an effective immune response that can resolve the infection without causing host pathology. In examining the inflammatory response of Asc −/− and Casp1 −/− macrophages, we found that deficiency in these molecules resulted in increased IFN production upon DNA virus infection, but not RNA virus challenge. Investigation of the underlying mechanism revealed that upon canonical and non-canonical inflammasome activation, caspase-1 interacted with cyclic GMP-AMP (cGAMP) synthase (cGAS), cleaving it and dampening cGAS-STING-mediated IFN production. Deficiency in inflammasome signaling enhanced host resistance to DNA virus in vitro and in vivo, and this regulatory role extended to other inflammatory caspases. Thus, inflammasome activation dampens cGAS-dependent signaling, suggesting cross-regulation between intracellular DNA-sensing pathways.

Background: Epidemiological studies have suggested a potential role for sex hormones in esophageal squamous cell carcinoma (ESCC) etiology. However, the significance of gender as an independent prognostic factor remains uncertain. Our retrospective study was designed to investigate the prognostic role of gender for survival after esophagectomy. Methods: Data from 674 patients with ESCC who underwent surgical resection were retrospectively analysed. Age 55 years was selected as a surrogate for menopause. Patients were assigned to age-gender groups: A (female younger than age 55 years), B (female age 55 years and older), C (male younger than age 55 years) and D (male age 55 years and older). Univariate and multivariate analyses were performed to identify prognostic factors for survival. Results: There were 520 males and 154 females with median age was 58 years. The 1-, 3-, 5-year survival rates of group A, B, C and D were 93.6%, 70.2%, 61.7% vs. 86.9%, 47.7%, 40.2% vs. 77.8%, 43.9%, 37.0% and 80.3%, 47.9%, 36.6%, respectively (P=0.003). Multivariate analysis suggested that age-gender groups and pTNM staging were independent prognostic factors. Conclusions: Among patients with ESCC after esophagectomy, women younger than age 55 years attained a favorable prognosis.

The type VI secretion systems (T6SS) have emerging roles in interspecies competition. In order to have an advantage in defense against other organisms, this system in Pseudomonas aeruginosa delivers a peptidoglycan amidase (Tse1) to the periplasmic space of a competitor. An immune protein (Tsi1) is also produced by the bacterium to protect itself from damage caused by Tse1. Tsi1 directly interacts with Tse1. We report that the crystal structure of Tse1 displays a common CHAP protein fold. Strikingly, our structures showed that the third residue in the catalytic triad may be novel as this residue type has not been observed previously.

Saracatinib, a highly selective, dual Src/Abl kinase inhibitor, is currently in a Phase II clinical trial for the treatment of ovarian cancer. In our study, we investigated the effect of saracatinib on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters in vitro and in vivo. Our results showed that saracatinib significantly enhanced the cytotoxicity of ABCB1 substrate drugs in ABCB1 overexpressing HeLa/v200, MCF-7/adr and HEK293/ABCB1 cells, an effect that was stronger than that of gefitinib, whereas it had no effect on the cytotoxicity of the substrates in ABCC1 overexpressing HL-60/adr cells and its parental sensitive cells. Additionally, saracatinib significantly increased the doxorubicin (Dox) and Rho 123 accumulation in HeLa/v200 and MCF-7/adr cells, whereas it had no effect on HeLa and MCF-7 cells. Furthermore, saracatinib stimulated the ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. In addition, the homology modeling predicted the binding conformation of saracatinib within the large hydrophobic drug-binding cavity of human ABCB1. However, neither the expression level of ABCB1 nor the phosphorylation level of Akt was altered at the reversal concentrations of saracatinib. Importantly, saracatinib significantly enhanced the effect of paclitaxel against ABCB1-overexpressing HeLa/v200 cancer cell xenografts in nude mice. In conclusion, saracatinib reverses ABCB1-mediated MDR in vitro and in vivo by directly inhibiting ABCB1 transport function, without altering ABCB1 expression or AKT phosphorylation. These findings may be helpful to attenuate the effect of MDR by combining saracatinib with other chemotherapeutic drugs in the clinic.

Multidrug resistance (MDR), a well defined phenomenon in which cancer cells become resistant to a variety of anticancer drugs with different structures and mechanisms of action, is a major obstacle towards curative cancer chemotherapy. Among the diverse mechanisms responsible for MDR phenotype, a common cause is the overexpression of ATP-binding cassette (ABC) transporters such as ABC subfamily B member 1 (ABCBI; also called P-glycoprotein, P-gp), ABC subfamily C members (ABCCs; also called MDR-associated proteins, MRPs), and ABC subfamily G member 2 (ABCG2; also called breast cancer resistance protein, BCRP)[1]–[3]. ABCG2 is a 655 amino acid glycoprotein that contains 1 ATP-binding domain and 6 transmembrane domains[4]. It is physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier, and various stem cells[5]–[7]. Unlike P-gp and MRP1, which are arranged in 2 repeated halves, ABCG2 is a half-transporter consisting of only 1 nucleotide-binding domain followed by 1 membrane-spanning domain. The half-transporter can transport a diverse array of substrates, including anticancer agents such as mitoxantrone, camptothecin and its analogs (e.g., diflomotecan, a synthetic derivative of camptothecin), and flavopiridol, a promising drug in clinical development[8]–[10]. Numerous studies have shown that elevated ABCG2 expression in clinical specimens of both solid and hematologic cancers, including esophageal squamous cell carcinoma[11] and acute myeloid leukemia[12]–[14], is associated with poor prognosis. In a retrospective study of non-small cell lung cancer, chemotherapy response rate in patients was correlated with ABCG2 expression but not with the expression of ABCBI, ABCC1, or ABCC3, suggesting that specific inhibition of ABCG2 function may help to overcome drug resistance in these patients[15]. These findings have important potential implications for the strategy of targeting ABCG2 as a part of cancer therapy. As an energy-dependent drug efflux pump, ABCG2 extrudes a variety of xenobiotics and drugs from cells, thereby mediating resistance to these agents and affecting their pharmacologic behavior[16]. It is worth noting that mutations of amino acid 482 in ABCG2 facilitate substrate selection. ABCG2 variants containing either R482G or R482T conferred increased mitoxantrone resistance to transfected cells. Moreover, these variants introduced anthracyclin (doxorubicin) resistance and rhodamine-123 extrusion capacity, which was not observed for the wild-type protein. In contrast, the R482G and R482T mutants were not able to extrude methotrexate, which is a transport substrate of wild-type ABCG2[17]. Many in vitro experimental models have been developed to better understand the mechanisms of drug resistance. The functionally relevant in vivo model has been an important method to study drug resistance and evaluate new effective therapies. S1-M1-80 cells derived from human colon carcinoma S1 cells are mitoxantrone-selected ABCG2-overexpressing cells carrying the R482G variant and have been widely used for MDR research in vitro. To determine whether S1-M1-80 cells in xenografts maintained the MDR phenotype and the biological properties observed in vitro, we established the S1-M1-80 cell xenograft model in nude mice and investigated the biological properties of the drug-resistant xenograft cells.

In Escherichia coli, the BAM complex is employed to mediate correct folding of the outer membrane (OM) proteins into β-barrels and their insertion into the OM. BamA, which is an essential component of the complex, consists of a C-­terminal transmembrane region and five N-terminal polypeptide transport-associated (POTRA) domains. Although deletion studies have shown that each of the POTRA domains plays an important role in the process of BAM complex formation, only POTRA5 is essential for cell viability. Here, the crystal structure of POTRA4–5 has been determined to 1.50 A resolution with an R factor of 14.7% and an R free of 18.9%.

Host-derived macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of malaria infection, especially in malarial anemia. Although two Plasmodium parasite-derived MIF orthologs, Plasmodium falciparum MIF and P. berghei MIF were identified recently, the crystal structure and the precise roles of Plasmodium-derived MIFs, particularly in combination with the host MIF, remain unknown. In this study, we identified another MIF ortholog from a rodent-specific P. yoelii (PyMIF). This molecule shares a conserved three-dimensional structure with murine MIF (MmMIF), but with a different substrate binding pattern and much lower tautomerase activity. It could activate host cells via several signaling pathways in vitro, and inhibiting macrophage apoptosis, also similarly to MmMIF. However, we found that PyMIF and MmMIF acted synergistically to activate the MAPK-ERK1/2 signaling pathway at very low concentration but acted antagonistically at higher concentration. Furthermore, we detected PyMIF in the sera of infected mice and found that injection of recombinant PyMIF (rPyMIF) during infection could up-regulate several pro-inflammatory cytokines in vivo and slightly delay the death of infected mice. These data suggest that PyMIF modulates host immune responses together with host MIF and has potential to prolong parasitemia or the chronicity of malaria infection.