Clinicogenomic characteristics and synthetic lethal implications of germline homologous recombination-deficient hepatocellular carcinoma.

BACKGROUNDS AND AIMS: We performed an in-depth examination of pathogenic germline variants (PGVs) and somatic variants in DNA damage response (DDR) genes in hepatocellular carcinoma (HCC) to explore their clinical and genomic impacts. APPROACH AND RESULTS: We used a merged whole-exome or RNA sequencing data set derived from in-house ( n = 230) and The Cancer Genome Atlas ( n = 362) databases of multiethnic HCC samples. We also evaluated synthetic lethal approaches targeting mutations in homologous recombination (HR) genes using HCC cells selected from five genomic databases of cancer cell lines. A total of 110 PGVs in DDR pathways in 96 patients were selected. Of the PGV carriers, 44 were HR-altered and found to be independently associated with poorer disease-free survival after hepatectomy. The most frequently altered HR gene in both germline and somatic tissues was POLQ , and this variant was detected in 22.7% (10/44) and 23.8% (5/21) of all the corresponding carriers, respectively. PGVs in HR were significantly associated with upregulation of proliferation and replication-related genes and familial risk of HCC. Samples harboring PGVs in HR with loss of heterozygosity were most strongly correlated with the genomic footprints of deficient HR, such as mutation burden and denovoSig2 (analogous to Catalogue of Somatic Mutations in Cancer [COSMIC] 3), and poor outcome. Pharmacologic experiments with HCC cells defective in BRCA2 or POLQ suggested that tumors with this phenotype are synthetic lethal with poly(ADP-ribose) polymerase inhibitors. CONCLUSIONS: Our findings suggest that germline HR defects in HCC tend to confer a poor prognosis and result in distinctive genomic scarring. Tests of the clinical benefits of HR-directed treatments in the affected patients are needed.

Molecular Recognition in Confined Space Elucidated with DNA Nanopores and Single-Molecule Force Microscopy.

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions.

Production, expression, and function of dual-specific monoclonal antibodies in a single plant.

MAIN CONCLUSION: LSC CO17-1AK and anti-HER2 VHH-FcK can be produced in a single plant and exhibit anti-tumor activities comparable to those of their respective parent antibodies. Recombinant monoclonal antibodies (mAbs) which can be applied to treat various cancers, are primarily produced using mammalian, insect, and bacteria cell culture systems. Plant expression systems have also been developed to produce antibodies. Plant expression systems present several advantages, including a lack of human pathogenic agents, efficient production costs, and easy large-scale production. In this study, we generated a transgenic plant expressing anti-colorectal cancer large single chain (LSC) CO17-1AK and anti-human epidermal growth factor receptor 2 (HER2) VHH-FcK mAbs by cross-pollinating plants expressing LSC CO17-1AK and anti-HER2 VHH-FcK, respectively. F1 siblings expressing both LSC CO17-1AK and anti-HER2 VHH-FcK were screened using polymerase chain reaction and Western-blot analyses. The cell enzyme-linked immunosorbent assay (Cell ELISA) confirmed the binding of LSC CO17-1AK and anti-HER2 VHH-FcK to target proteins in the SW620 human colorectal cancer and the SKBR-3 human breast cancer cell lines, respectively. The wound healing assay confirmed the inhibitory activity of both antibodies against SW620 and SKBR-3 cell migration, respectively. In conclusion, both LSC CO17-1AK mAb and anti-HER2 VHH-FcK can be produced in a single plant, achieve binding activities to SW620 and SKBR-3 cancer cells, and inhibitory activity against SW620 and SKBR-3 cell migration similar to their parental antibodies, respectively.

Comprehensive characterization of viral integrations and genomic aberrations in HBV-infected intrahepatic cholangiocarcinomas.

BACKGROUND AND AIMS: Despite the epidemiological association between intrahepatic cholangiocarcinoma (iCCA) and HBV infection, little is known about the relevant oncogenic effects. We sought to identify the landscape and mechanism of HBV integration, along with the genomic architecture of HBV-infected iCCA (HBV-iCCA) tumors. APPROACH AND RESULTS: We profiled a cohort of 108 HBV-iCCAs using whole-genome sequencing, deep sequencing, and RNA sequencing, together with preconstructed data sets of HBV-infected HCC (HBV-HCC; n = 167) and combined hepatocellular cholangiocarcinoma (HBV-cHCC/CCA; n = 59), and conventional (n = 154) and fluke-related iCCAs (n = 16). Platforms based on primary iCCA cell lines to evaluate the functional effects of chimeric transcripts were also used. We found that HBV had inserted at multiple sites in the iCCA genomes in 45 (41.7%) of the tumors. Recurrent viral integration breakpoints were found at nine different sites. The most common insertional hotspot (7 tumors) was in the TERT (telomerase reverse transcriptase) promoter, where insertions and mutations (11 tumors) were mutually exclusive, and were accompanied by promoter hyperactivity. Recurrent HBV integration events (5 tumors) were also detected in FAT2 (FAT atypical cadherin 2), and were associated with enrichment of epithelial-mesenchymal transition-related genes. A distinctive intergenic insertion (chr9p21.3), between DMRTA1 (DMRT like family A1) and LINC01239 (long intergenic non-protein coding RNA 1239), had oncogenic effects through activation of the mammalian target of rapamycin (mTOR)/4EBP/S6K pathway. Regarding the mutational profiles of primary liver cancers, the overall landscape of HBV-iCCA was closer to that of nonviral conventional iCCA, than to HBV-HCC and HBV-cHCC/CCA. CONCLUSIONS: Our findings provide insight into the behavior of iCCAs driven by various pathogenic mechanisms involving HBV integration events and associated genomic aberrations. This knowledge should be of use in managing HBV carriers.

Prognostic Molecular Indices of Resectable Hepatocellular Carcinoma: Implications of S100P for Early Recurrence.

BACKGROUND: Although hepatocellular carcinomas (HCCs) often recur in patients undergoing hepatectomy, there are no reliable biomarkers of this undesirable event. Recent RNA-based efforts have developed valuable genetic indices prognostic of cancer outcomes. We aimed to identify molecular predictors of early recurrence after resection of HCC, and reveal the genomolecular structure of the resected tumors. METHOD: Based on the transcriptomic and genomic datasets of 206 HCC samples surgically resected in the Asan Medical Center (AMC), we performed a differential gene expression analysis to identify quantitative markers associated with early recurrence and used the unsupervised clustering method to classify genomolecular subtypes. RESULTS: Differential gene expression profiling revealed that S100P was the highest-ranked overexpressed gene in HCCs that recurred within 2 years of surgery. This trend was reproduced in immunohistochemical studies of the original cohort and an independent AMC cohort. S100P expression also independently predicted HCC-specific mortality post-resection (adjusted hazard ratio 1.09, 95% confidence interval 1.01-1.19; p = 0.042). Validation in a Chinese cohort and in in vitro experiments confirmed the prognostic value of S100P in HCC. We further identified five discrete molecular subtypes of HCC; a subtype with stem cell features ('AMC-C4') was associated with the worst prognosis, both in our series and another two Asian datasets, and S100P was most strongly upregulated in that subtype. CONCLUSION: We identified a promising prognostic biomolecule, S100P, associated with early recurrence after HCC resection, and established the genomolecular architecture of tumors affecting clinical outcomes, particularly in Asian patients. These new insights into molecular mediators should contribute to effective care for affected patients.

Hepatic STAMP2 mediates recombinant FGF21-induced improvement of hepatic iron overload in nonalcoholic fatty liver disease.

Although previous studies have shown that the administration of fibroblast growth factor 21 (FGF21) reverses hepatic steatosis, the mechanism by which FGF21 exerts a therapeutic effect on nonalcoholic fatty liver disease (NAFLD) is not yet entirely understood. We previously demonstrated that hepatic six transmembrane protein of prostate 2 (STAMP2) may represent a suitable target for NAFLD. We investigated the mechanism underlying the therapeutic effect of recombinant FGF21 on NAFLD, focusing on the involvement of hepatic STAMP2. In this study, we used human nonalcoholic steatosis patient pathology samples, C57BL/6 mice for a high-fat diet (HFD)-induced in vivo NAFLD model, and used human primary hepatocytes and HepG2 cells for oleic acid (OA)-induced in vitro NAFLD model. We observed that recombinant FGF21 treatment ameliorated hepatic steatosis and insulin resistance through the upregulation of STAMP2 expression. We further observed hepatic iron overload (HIO) and reduced iron exporter, ferroportin expression in the liver samples obtained from human NAFLD patients, and HFD-induced NAFLD mice and in OA-treated HepG2 cells. Importantly, recombinant FGF21 improved HIO through the hepatic STAMP2-mediated upregulation of ferroportin expression. Our data suggest that hepatic STAMP2 may represent a suitable therapeutic intervention target for FGF21-induced improvement of NAFLD accompanying HIO.

Ultra-Sensitive and Label-Free Probing of Binding Affinity Using Recognition Imaging.

Reliable quantification of binding affinity is important in biotechnology and pharmacology and increasingly coupled with a demand for ultrasensitivity, nanoscale resolution, and minute sample amounts. Standard techniques are not able to meet these criteria. This study provides a new platform based on atomic force microscopy (AFM)-derived recognition imaging to determine affinity by visualizing single molecular bindings on nanosize dendrons. Using DNA hybridization as a demonstrator, an AFM sensor adorned with a cognate binding strand senses and localizes target DNAs at nanometer resolution. To overcome the limitations of speed and resolution, the AFM cantilever is sinusoidally oscillated close to resonance conditions at small amplitudes. The equilibrium dissociation constant of capturing DNA duplexes was obtained, yielding 2.4 × 10-10 M. Our label-free single-molecular biochemical analysis approach evidences the utility of recognition imaging and analysis in quantifying biomolecular interactions of just a few hundred molecules.

Cilostazol Improves HFD-Induced Hepatic Steatosis by Upregulating Hepatic STAMP2 Expression through AMPK.

Nonalcoholic fatty liver disease (NAFLD) is an increasingly studied condition that can progress to end-stage liver disease. Although NAFLD was first described in 1980, a complete understanding of the mechanism and causes of this disease is still lacking. Six-transmembrane protein of prostate 2 (STAMP2) plays a role in integrating inflammatory and nutritional signals with metabolism. Our previous study suggested that STAMP2 may be a suitable target for treating NAFLD. In the current study, we performed a focused drug-screening and found that cilostazol could be a potential STAMP2 enhancer. Thus, we examined whether cilostazol alleviates NAFLD through STAMP2. The in vivo and in vitro pharmacological efficacies of cilostazol on STAMP2 expression and lipid accumulation were analyzed in NAFLD mice induced by high-fat diet (HFD) and in HepG2 cell lines treated by oleic acid (OA), respectively. Cilostazol increased the expression of STAMP2 through transcriptional regulation in vivo and in vitro. Cilostazol also dampened the STAMP2 downregulation caused by the HFD and by OA in vivo and in vitro, respectively. Cilostazol activated AMP-activated protein kinase (AMPK) in vivo and in vitro, and AMPK functions upstream of STAMP2, and reversed downregulation of STAMP2 expression through AMPK in the NAFLD model. Cilostazol ameliorates hepatic steatosis by enhancing hepatic STAMP2 expression through AMPK. Enhancing STAMP2 expression with cilostazol represents a potential therapeutic avenue for treatment of NAFLD.

Lipoteichoic acid mediates binding of a Lactobacillus S-layer protein.

The Gram-positive lactic acid bacterium Lactobacillus buchneri CD034 is covered by a two-dimensional crystalline, glycoproteinaceous cell surface (S-) layer lattice. While lactobacilli are extensively exploited as cell surface display systems for applied purposes, questions about how they stick their cell wall together are remaining open. This also includes the identification of the S-layer cell wall ligand. In this study, lipoteichoic acid was isolated from the L. buchneri CD034 cell wall as a significant fraction of the bacterium's cell wall glycopolymers, structurally characterized and analyzed for its potential to mediate binding of the S-layer to the cell wall. Combined component analyses and 1D- and 2D-nuclear magnetic resonance spectroscopy (NMR) revealed the lipoteichoic acid to be composed of on average 31 glycerol-phosphate repeating units partially substituted with α-d-glucose, and with an α-d-Galp(1→2)-α-d-Glcp(1→3)-1,2-diacyl-sn-Gro glycolipid anchor. The specificity of binding between the L. buchneri CD034 S-layer protein and purified lipoteichoic acid as well as their interaction force of about 45 pN were obtained by single-molecule force spectroscopy; this value is in the range of typical ligand-receptor interactions. This study sheds light on a functional implication of Lactobacillus cell wall architecture by showing direct binding between lipoteichoic acid and the S-layer of L. buchneri CD034.

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy.

Lipopolysaccharide (LPS) on gram-negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 µg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano-resolution approaches in microbiology.

Polychlorinated biphenyls exposure-induced insulin resistance is mediated by lipid droplet enlargement through Fsp27.

Although epidemiological and experimental studies demonstrated that polychlorinated biphenyls (PCBs) lead to insulin resistance, the mechanism underlying PCBs-induced insulin resistance has remained unsolved. In this study, we examined in vitro and in vivo effects of PCB-118 (dioxin-like PCB) and PCB-138 (non-dioxin-like PCB) on adipocyte differentiation, lipid droplet growth, and insulin action. 3T3-L1 adipocytes were incubated with PCB-118 or PCB-138 during adipocyte differentiation. For in vivo studies, C57BL/6 mice were administered PCB-118 or PCB-138 (37.5 mg/kg) by intraperitoneal injection and we examined adiposity and whole-body insulin action. PCB-118 and PCB-138 significantly promoted adipocyte differentiation and increased the lipid droplet (LD) size in 3T3-L1 adipocytes. In mice, both PCBs increased adipose mass and adipocyte size. Furthermore, both PCBs induced insulin resistance in vitro and in vivo. Expression of fat-specific protein 27 (Fsp27), which is localized to LD contact sites, was increased in PCB-treated 3T3-L1 adipocytes and mice. Depletion of Fsp27 by siRNA resulted in the inhibition of LD enlargement and attenuation of insulin resistance in PCB-treated 3T3-L1 adipocytes. An anti-diabetic drug, metformin, attenuated insulin resistance in PCB-treated 3T3-L1 adipocytes through the reduced expression of Fsp27 protein and LD size. This study suggests that PCB exposure-induced insulin resistance is mediated by LD enlargement through Fsp27.

Calibrated complex impedance of CHO cells and E. coli bacteria at GHz frequencies using scanning microwave microscopy.

The application of scanning microwave microscopy (SMM) to extract calibrated electrical properties of cells and bacteria in air is presented. From the S 11 images, after calibration, complex impedance and admittance images of Chinese hamster ovary cells and E. coli bacteria deposited on a silicon substrate have been obtained. The broadband capabilities of SMM have been used to characterize the bio-samples between 2 GHz and 20 GHz. The resulting calibrated cell and bacteria admittance at 19 GHz were Y cell = 185 µS + j285 µS and Y bacteria = 3 µS + j20 µS, respectively. A combined circuitry-3D finite element method EMPro model has been developed and used to investigate the frequency response of the complex impedance and admittance of the SMM setup. Based on a proposed parallel resistance-capacitance model, the equivalent conductance and parallel capacitance of the cells and bacteria were obtained from the SMM images. The influence of humidity and frequency on the cell conductance was experimentally studied. To compare the cell conductance with bulk water properties, we measured the imaginary part of the bulk water loss with a dielectric probe kit in the same frequency range resulting in a high level of agreement.

Influence of Surface Morphology on the Antimicrobial Effect of Transition Metal Oxides in Polymer Surface.

In this study, the physical properties of transition metal oxide surfaces were examined using scanning probe microscopic (SPM) techniques for elucidating the antimicrobial activity of molybdenum trioxide (MoO3), tungsten trioxide (WO3), and zinc oxide (ZnO) embedded into the polymers thermoplastic polyurethane (TPU) and polypropylene (PP). We utilized atomic force microscopy (AFM) in the contact imaging mode and its derivative single-pass Kelvin probe force microscopy for investigating samples that were presumably identical in their compositions, but showed different antimicrobial activity in bacterial adhesion tests. Our results revealed that surfaces with larger roughness and higher surface potential variation showed stronger antimicrobial activities compared to smoother and homogeneously charge-distributed surfaces. In addition, capacitance gradient (dC/dZ) measurements were performed to elucidate the antimicrobial activity arising from the different dielectric behavior of the transition metal oxides in this heterogeneous polymer surface. We found that the nano-scale exposure of transition metal oxides on polymer surfaces provided strong antimicrobial effects. Applications arising from our studies will be useful for public and healthcare environments.

3D airway geometry analysis of factors in airway navigation failure for lung nodules.

BACKGROUND: This study aimed to quantitatively reveal contributing factors to airway navigation failure during radial probe endobronchial ultrasound (R-EBUS) by using geometric analysis in a three-dimensional (3D) space and to investigate the clinical feasibility of prediction models for airway navigation failure. METHODS: We retrospectively reviewed patients who underwent R-EBUS between January 2017 and December 2018. Geometric quantification was analyzed using in-house software built with open-source python libraries including the Vascular Modeling Toolkit ( http://www.vmtk.org ), simple insight toolkit ( https://sitk.org ), and sci-kit image ( https://scikit-image.org ). We used a machine learning-based approach to explore the utility of these significant factors. RESULTS: Of the 491 patients who were eligible for analysis (mean age, 65 years +/- 11 [standard deviation]; 274 men), the target lesion was reached in 434 and was not reached in 57. Twenty-seven patients in the failure group were matched with 27 patients in the success group based on propensity scores. Bifurcation angle at the target branch, the least diameter of the last section, and the curvature of the last section are the most significant and stable factors for airway navigation failure. The support vector machine can predict airway navigation failure with an average area under the curve of 0.803. CONCLUSIONS: Geometric analysis in 3D space revealed that a large bifurcation angle and a narrow and tortuous structure of the closest bronchus from the lesion are associated with airway navigation failure during R-EBUS. The models developed using quantitative computer tomography scan imaging show the potential to predict airway navigation failure.

Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Fc engineered anti-virus therapeutic human IgG1 expressed in plants with altered binding to the neonatal Fc receptor.

Production of therapeutic monoclonal antibody (mAb) in transgenic plants has several advantages such as large-scale production and the absence of pathogenic animal contaminants. However, mAb with high mannose (HM) type glycans has shown a faster clearance compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of antirabies virus therapeutic mAb SO57 with the endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) in plant cell was conducted to enhance its binding activity to human neonatal Fc receptor (hFcRn), consequently improve its serum half-life. Enzyme-linked immunosorbent assay (ELISA) and Surface plasmon resonance assay showed altered binding affinity of the Fc region of three different mAbpK SO57 variants [M252Y/S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN)] to hFcRn compared to wild type (WT) of mAbpK SO57. Molecular modeling data visualized the structural alterations in these mAbpK SO57. All of the mAbpK SO57 variants had HM type glycan structures similar to the WT mAbpK SO57. In addition, the neutralizing activity of the three variants against the rabies virus CVS-11 was effective as the WT mAbpK SO57. These results indicate that the binding affinity of mAbpK SO57 variants to hFcRn can be modified without alteration of N-glycan structure and neutralization activity. Taken together, this study suggests that Fc-engineering of antirabies virus mAb can be applied to enhance the efficacy of therapeutic mAbs in plant expression systems.

Characterizing the S-layer structure and anti-S-layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X-ray scattering.

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22-nm-thick, 2D crystalline surface (S-) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium-host cross-talk. The S-layer is composed of two intercalating glycoproteins (TfsA-GP, TfsB-GP) that are aligned into a periodic lattice. To characterize this unique S-layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and single-molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild-type and S-layer-deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high-resolution imaging of the outer surface of T. forsythia wild-type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti-TfsA antibody coupled to the AFM tip and the S-layer as present on T. forsythia wild-type and mutant cells, displaying TfsA-GP alone. Unbinding forces between the antibody and wild-type cells were greater than with mutant cells. This indicated that the TfsA-GP is not so strongly attached to the mutant cell surface when the co-assembling TfsB-GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S-layer architecture with two intercalating S-layer glycoproteins and TfsA-GP being mainly outwardly oriented.

The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis.

Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis.

Characterization of curli A production on living bacterial surfaces by scanning probe microscopy.

Curli are adhesive surface fibers produced by many Enterobacteriaceae, such as Escherichia coli and Salmonella enterica. They are implicated in bacterial attachment and invasion to epithelial cells. In this study, atomic force microscopy was used to determine the effects of curli on topology and mechanical properties of live E. coli cells. Young's moduli of both curli-deficient and curli-overproducing mutants were significantly lower than that of their wild-type (WT) strain, while decay lengths of the former strains were higher than that of the latter strain. Surprisingly, topological images showed that, unlike the WT and curli-overproducing mutant, the curli-deficient mutant produced a large number of flagella-like fibers, which may explain why the strain had a lower Young's modulus than the WT. These results suggest that the mechanical properties of bacterial surfaces are greatly affected by the presence of filamentous structures such as curli and flagella.