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High blood glucose is one of the risk factors for metabolic disease and INS (insulin) is the key regulatory hormone for glucose homeostasis. Hypoinsulinemia accompanied with hyperglycemia was diagnosed in mice with pancreatic β-cells exhibiting autophagy deficiency; however, the underlying mechanism remains elusive. The role of secretory autophagy in the regulation of metabolic syndrome is gaining more attention. Our data demonstrated that increased macroautophagic/autophagic activity leads to induction of insulin secretion in β-cells both in vivo and in vitro under high-glucose conditions. Moreover, proteomic analysis of purified autophagosomes from β-cells identified a group of vesicular transport proteins participating in insulin secretion, implying that secretory autophagy regulates insulin exocytosis. RAB37, a small GTPase, regulates vesicle biogenesis, trafficking, and cargo release. We demonstrated that the active form of RAB37 increased MAP1LC3/LC3 lipidation (LC3-II) and is essential for the promotion of insulin secretion by autophagy, but these phenomena were not observed in rab37 knockout (rab37-/-) cells and mice. Unbalanced insulin and glucose concentration in the blood was improved by manipulating autophagic activity using a novel autophagy inducer niclosamide (an antihelminthic drug) in a high-fat diet (HFD)-obesity mouse model. In summary, we reveal that secretory autophagy promotes RAB37-mediated insulin secretion to maintain the homeostasis of insulin and glucose both in vitro and in vivo.

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV.

Ferroptosis is an iron-dependent oxidative cell death with accumulation of lipid peroxidation and abnormal morphology of mitochondria. Although previous studies have shown that ferroptosis inducers have therapeutic potentials in several treatment-naive and drug-resistant cancer types, lacking systematic analysis of the ferroptosis sensitivity in different cancer types makes it difficult to know which cancer type is suitable for clinical usage and critical regulator determining the ferroptosis sensitivity during cancer progression is still not clear. In this study, colon cancer was identified as one of the ferroptosis-insensitive cancer types by dry and wet bench analyses. Importantly, our large-scale analysis also showed that NUDT16L1 was a novel ferroptosis repressor and overexpressed in colon cancer clinical specimens. Similar findings are also observed in the chemical-induced and genetic mouse models of colon cancer. Furthermore, NUDT16L1 loss-of-function and gain-of-function cell models and its conditional knock-in and knock-out mouse models are used to prove its crucial role in the development of colon cancer. In addition, loss of NUDT16L1 induced several key ferroptosis characteristics and impairment of mitochondrial functions while its restoration attenuated those phenomena in colon cancer cells. Mechanistically, our RNA-Seq and RIP-Seq analyses shown that NUDT16L1 directly interacted and positively regulated several crucial negative regulators of ferroptosis. More interestingly, loss of NUDT16L1 not only restores the sensitivity of ferroptosis inducer but also impairs the function of mitochondria. Finally, a specific NUDT16L1 inhibitor was shown to not only repress colon cancer growth in vitro and in vivo but also induce the ferroptosis in colon cancer. In conclusion, this is the first study to demonstrate that overexpression of NUDT16L1 promotes colon cancer progression by inhibition of ferroptosis and maintenance proper function of mitochondria. Furthermore, specific NUDT16L1 inhibitor might be a promising therapeutic strategy for colon cancer patient in the future. Citation Format: Yi-Syuan Lin, Ya-Chuan Tsai, Chia-Jung Li, Tzu-Tang Wei, Ya-Na Wu, Shang-Rung Wu, Shin-Chin Lin, Shaw-Jenq Tsai, Shih-Chieh Lin. NUDT16L1 promotes colon cancer progression via inhibition of ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1372.

The connecting pieces of the sperm neck link the flagellum and the sperm head, and they are important for initiating flagellar beating. The connecting pieces are important building blocks for the sperm neck; however, the mechanism of connecting piece assembly is poorly understood. In the present study, we explored the role of septins in sperm motility and found that Sept12D197N knock-in (KI) mice produce acephalic and immotile spermatozoa. Electron microscopy analysis showed defective connecting pieces in sperm from KI mice, indicating that SEPT12 is required for the establishment of connecting pieces. We also found that SEPT12 formed a complex with SEPT1, SEPT2, SEPT10 and SEPT11 at the sperm neck and that the D197N mutation disrupted the complex, suggesting that the SEPT12 complex is involved in the assembly of connecting pieces. Additionally, we found that SEPT12 interacted and colocalized with γ-tubulin in elongating spermatids, implying that SEPT12 and pericentriolar materials jointly contribute to the formation of connecting pieces. Collectively, our findings suggest that SEPT12 is required for the formation of striated columns, and the capitulum and for maintaining the stability of the sperm head–tail junction.

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m

Group A streptococcus (GAS) is an important human pathogen which can cause fatal diseases after invasion into the bloodstream. Although antibiotics and immune surveillance are the main defenses against GAS infection, GAS utilizes internalization into cells as a major immune evasion strategy. Our previous findings revealed that light chain 3 (LC3)-associated single membrane GAS-containing vacuoles in endothelial cells are compromised for bacterial clearance due to insufficient acidification after fusion with lysosomes. However, the characteristics and the activation mechanisms of these LC3-positive compartments are still largely unknown. In the present study, we demonstrated that the LC3-positive GAS is surrounded by single membrane and colocalizes with NADPH oxidase 2 (NOX2) complex but without ULK1, which are characteristics of LC3-associated phagocytosis (LAP). Inhibition of NOX2 or reactive oxygen species (ROS) significantly reduces GAS multiplication and enhances autolysosome acidification in endothelial cells through converting LAP to conventional xenophagy, which is revealed by enhancement of ULK1 recruitment, attenuation of p70s6k phosphorylation, and formation of the isolation membrane. We also clarify that the inactivation of mTORC1, which is the initiation signal of autophagy, is inhibited by NOX2- and ROS-activated phosphatidylinositol 3-kinase (PI3K)/AKT and MEK/extracellular signal-regulated kinase (ERK) pathways. In addition, streptolysin O (SLO) of GAS is identified as a crucial inducer of ROS for β1 integrin-mediated LAP induction. After downregulation of β1 integrin, GAS multiplication is reduced, accompanied with LAP inhibition and xenophagy induction. These results demonstrate that GAS infection preferentially induces ineffective LAP to evade xenophagic killing in endothelial cells through the SLO/β1 integrin/NOX2/ROS pathway.

Addressing nanomedicine resistance is critical for its ultimate clinical success; despite this, advancing the therapeutic designs for cancer therapy are rarely discussed in the literature. In this study, we discovered that ferroptosis is the central mechanism governing the therapeutic efficacy and resistance of treatment with zero-valent iron nanoparticles (ZVI NPs). In ZVI-sensitive oral cancer cells, ZVI NPs-induced ferroptosis was characterized by mitochondrial lipid peroxidation and reduced levels of glutathione peroxidases (GPx) in subcellular organelles. However, resistant cells could attenuate ZVI-induced oxidative stress and GPx reduction. They also showed stronger mitochondrial respiration ability, thus resisting ZVI NPs-induced mitochondrial membrane potential loss. Transcriptome comparison and quantitative polymerase chain reaction (qPCR) analysis revealed that ZVI-resistant cancer cells expressed a gene set related to enhanced NADPH supply, higher detoxification capacity of reactive oxygen species, and decreased sensitivity to ferroptosis inducers (FINs). Finally, we discovered that certain FINs were able to sensitize ZVI-resistant cancer cells to become treatable without compromising healthy non-malignant cells. These findings suggest that ferroptosis can serve as a druggable target for anti-cancer nanomedicine and therapeutic resistance modulation using ZVI NPs.

Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

Endometriosis is a highly prevalent gynecological disease with severe negative impacts on life quality and financial burden. Unfortunately, there is no cure for this disease, which highlights the need for further investigation about the pathophysiology of this disease to provide clues for developing novel therapeutic regimens. Herein, we identified that vascular endothelial growth factor (VEGF)-C, a potent lymphangiogenic factor, is up-regulated in endometriotic cells and contributes to increased lymphangiogenesis. Bioinformatic analysis and molecular biological characterization revealed that VEGF-C is negatively regulated by an orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Further studies demonstrated that proinflammatory cytokines, via suppression of COUP-TFII level, induce VEGF-C overexpression. More importantly, we show that functional VEGF-C is transported by extracellular vesicles (EVs) to enhance the lymphangiogenic ability of lymphatic endothelial cells. Autotransplanted mouse model of endometriosis showed lenvatinib treatment abrogated the increased lymphatic vessels development in the endometriotic lesion, enlarged retroperitoneal lymph nodes, and immune cells infiltration, indicating that blocking VEGF-C signaling can reduce local chronic inflammation and concomitantly endometriosis development. Evaluation of EV-transmitted VEGF-C from patients' sera demonstrates it is a reliable noninvasive way for clinical diagnosis. Taken together, we identify the vicious cycle of inflammation, COUP-TFII, VEGF-C, and lymphangiogenesis in the endometriotic microenvironment, which opens up new horizons in understanding the pathophysiology of endometriosis. VEGF-C not only can serve as a diagnostic biomarker but also a molecular target for developing therapeutic regimens.

Early dissemination is a unique characteristic and a detrimental process of pancreatic ductal adenocarcinoma (PDAC); however, the underlying mechanism remains largely unknown. Here, we investigate the role of dual-specificity phosphatase-2 (DUSP2)-vascular endothelial growth factor-C (VEGF-C) axis in mediating PDAC lymphangiogenesis and lymphovascular invasion. Expression of DUSP2 is greatly suppressed in PDAC, which results in increased aberrant expression of extracellular vesicle (EV)-associated VEGF-C secretion. EV-VEGF-C exerts paracrine effects on lymphatic endothelial cells and autocrine effects on cancer cells, resulting in the lymphovascular invasion of cancer cells. Tissue-specific knockout of Dusp2 in mouse pancreas recapitulates PDAC phenotype and lymphovascular invasion. Mechanistically, loss-of-DUSP2 enhances proprotein convertase activity and vesicle trafficking to promote the release of the mature form of EV-VEGF-C. Collectively, these findings represent a conceptual advance in understanding pancreatic cancer lymphovascular invasion and suggest that loss-of-DUSP2-mediated VEGF-C processing may play important roles in early dissemination of pancreatic cancer. Abbreviations: DUSP2: dual-specificity phosphatase-2; VEGF-C: vascular endothelial growth factor-C; EV: extracellular vesicles; PDAC: pancreatic ductal adenocarcinoma; KD: knockdown

Ultrasound-targeted microbubble destruction (UTMD) in conjunction with neurotrophic factors (NFs) gene delivery has the potential to facilitate the penetration of therapeutic genes into the brain for neuroprotective therapy against neurodegenerative diseases. We previously presented a gene delivery system that conjugates gene-carrying liposomes with microbubbles (MBs) to open the blood-brain barrier (BBB) for the delivery of genes into the brain. Since both glia cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) can protect dopaminergic neurons from neurotoxicity demonstrated in Parkinson's disease (PD) animal models, the present study seeks (1) to develop a novel gene-nanocarrier MB complex carrying BDNF or GDNF gene and (2) to protect dopaminergic neurons in a mouse model of PD via the proposed UTMD system. In the experimental design, PD animals received treatment that delivered GDNF, BDNF, or combined GDNF/BDNF in conjunction with UTMD treatment, and pathological changes in dopamine neurons were histologically examined. Rotarod assay was employed to evaluate the motor behavior. Our results demonstrate that either BDNF or GDNF gene delivery via the UTMD system provides a neuroprotective effect with evidence of improvements of behavioral deficits, decreased calcium influx, GFAP and caspase 3 expression, and rescued dopaminergic neuronal loss. Simultaneously performing GDNF/BDNF gene delivery did not show additional benefits beyond individually delivering BDNF or GDNF genes, possibly due to a hampering effect of simultaneous GDNF/BDNF competing expressions, thus dampening the overall therapeutic effect. In conclusion, these results suggest that UTMD in conjunction with delivery of GDNF or BDNF gene can synergistically serve as an effective gene therapy strategy for neurodegenerative diseases.

Our previous reports showed that the LC3-associated GAS-containing single membrane vacuoles are inefficient for bacterial clearance in endothelial cells, which may result in bacteremia. However, the characteristics and the induction mechanisms of these LC3-positive vacuoles are still largely unknown. Here we provide the first evidence that these LC3-positive GAS-containing single membrane compartments appear to be LAPosomes, which are induced by NOX2 and ROS. Through NOX2- and ROS-mediated signaling, GAS preferentially induces LAP and inhibits bacteriostatic xenophagy in endothelial cells. We also provide the first demonstration that β1 integrin acts as the receptor for LAP induction through GAS-produced SLO stimulation in endothelial cells. Our findings reveal the underlying mechanisms of LAP induction and autophagy evasion for GAS multiplication in endothelial cells., Group A streptococcus (GAS) is an important human pathogen which can cause fatal diseases after invasion into the bloodstream. Although antibiotics and immune surveillance are the main defenses against GAS infection, GAS utilizes internalization into cells as a major immune evasion strategy. Our previous findings revealed that light chain 3 (LC3)-associated single membrane GAS-containing vacuoles in endothelial cells are compromised for bacterial clearance due to insufficient acidification after fusion with lysosomes. However, the characteristics and the activation mechanisms of these LC3-positive compartments are still largely unknown. In the present study, we demonstrated that the LC3-positive GAS is surrounded by single membrane and colocalizes with NADPH oxidase 2 (NOX2) complex but without ULK1, which are characteristics of LC3-associated phagocytosis (LAP). Inhibition of NOX2 or reactive oxygen species (ROS) significantly reduces GAS multiplication and enhances autolysosome acidification in endothelial cells through converting LAP to conventional xenophagy, which is revealed by enhancement of ULK1 recruitment, attenuation of p70s6k phosphorylation, and formation of the isolation membrane. We also clarify that the inactivation of mTORC1, which is the initiation signal of autophagy, is inhibited by NOX2- and ROS-activated phosphatidylinositol 3-kinase (PI3K)/AKT and MEK/extracellular signal-regulated kinase (ERK) pathways. In addition, streptolysin O (SLO) of GAS is identified as a crucial inducer of ROS for β1 integrin-mediated LAP induction. After downregulation of β1 integrin, GAS multiplication is reduced, accompanied with LAP inhibition and xenophagy induction. These results demonstrate that GAS infection preferentially induces ineffective LAP to evade xenophagic killing in endothelial cells through the SLO/β1 integrin/NOX2/ROS pathway.

BackgroundPathogenic protozoans use extracellular vesicles (EVs) for intercellular communication and host manipulation.Acanthamoeba castellaniiis a free-living protozoan that may cause severe keratitis and fatal granulomatous encephalitis. Although several secreted molecules have been shown to play crucial roles in the pathogenesis ofAcanthamoeba, the functions and components of parasite-derived EVs are far from understood.MethodsPurified EVs fromA. castellaniiwere confirmed by electron microscopy and nanoparticle tracking analysis. The functional roles of parasite-derived EVs in the cytotoxicity to and immune response of host cells were examined. The protein composition in EVs fromA. castellaniiwas identified and quantified by LC-MS/MS analysis.ResultsEVs fromA. castellaniifused with rat glioma C6 cells. The parasite-derived EVs induced an immune response from human THP-1 cells and a cytotoxic effect in C6 cells. Quantitative proteomic analysis identified a total of 130 proteins in EVs. Among the identified proteins, hydrolases (50.2%) and oxidoreductases (31.7%) were the largest protein families in EVs. Furthermore, aminopeptidase activities were confirmed in EVs fromA. castellanii.ConclusionsThe proteomic profiling and functional characterization of EVs fromA. castellaniiprovide an in-depth understanding of the molecules packaged into EVs and their potential mechanisms mediating the pathogenesis of this parasite.

Childhood exanthema caused by different serotypes of coxsackievirus (CV-A) and enterovirus A71 (EV-A71) has become a serious global health problem; it is commonly known as hand, foot, and mouth disease (HFMD). Current EV-A71 vaccine clinical trials have demonstrated that human antibody responses generated by EV-A71 vaccinations do not cross-neutralize coxsackievirus A16 (CV-A16). An effective multivalent HFMD vaccine is urgently needed. From molecular epidemiological studies in Southeast Asia, CV-A6 and CV-A10 are commonly found in HFMD outbreaks. In this study, CV-A6 and CV-A10 were individually cultured in rhabdomyosarcoma (RD) cells grown in medium containing serum, harvested and concentrated. In viral downstream purification, two viral fractions were separated by sucrose gradient zonal ultracentrifugation and detected using a SDS-PAGE analysis and a virus infectivity assay. These two viral fractions were formalin-inactivated, and only the infectious particle fraction was found to be capable of inducing CV-A serotype-specific neutralizing antibody responses in animal immunogenicity studies. These mouse and rabbit antisera also failed to cross-neutralize EV-A71 and CV-A16 infections. Only a combination of formalin-inactivated EV-A71, CV-A6, CV-A10 and CV-A16 multivalent vaccine candidates elicited cross-neutralizing antibody responses in both mouse and rabbit immunogenicity studies. The current results certainly provide important information for multivalent HFMD vaccine development.

Pancreatic cancer is one of the highest causes of mortality throughout the world, thus, it requires an effective treatment strategy. Some chemotherapeutic agents used in the clinics or under clinical trials are hydrophobic and have poor aqueous solubility, consequently, they also have minimal systemic bioavailability. Nanoparticle-based drug delivery tactics have the potential for overcoming these limitations and enhancing their therapeutic efficacy. Herein, a glutathione (GSH)-sensitive micelle (PAH-SS-PLGA) was synthesized for the combined delivery of alpha-tocopheryl succinate (TOS) and curcumin to improve its therapeutic efficacy. The chemical structures of PAH-SS-PLGA were analyzed using Proton Nuclear Magnetic Resonance (1H-NMR) and Fourier Transform Infrared (FTIR) spectroscopy, whereas the particle size, zeta potential, and surface morphology were observed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release results revealed that more TOS and curcumin were released in the presence of GSH (5 mM) than the physiological pH value. Fluorescence microscopy images revealed that nanoformulated curcumin/rhodamine was uptaken by PAN02 pancreatic cancer cells. In vitro cytotoxicity assays showed higher cytotoxicity for nanoformulated TOS and/or curcumin than free TOS and/or curcumin. In addition, higher cytotoxicity was observed for combination drugs than free drugs alone. Most interestingly, at all tested concentrations of nanoformulated drugs (PAH-SS-PLGA, TOS, and curcumin), the calculated combination index (CI) value was less than one, which shows that TOS and curcumin have a synergistic effect on cellular proliferation inhibition. Overall, synthesized co-polymers are the best carriers for combination drugs, TOS, and curcumin, because they enhance the therapeutic efficacy and improve pancreatic cancer treatments.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein we show for the first time that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes through cryo-electron microscopy reconstruction. Mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher cross reactive (CR) neutralization antibodies (NtAbs) and were protected against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. Our results revealed that a “epitope-resurfaced” mature-form dengue VLP has the potential to induce quaternary structure-recognizing broad CR NtAbs.

Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease (HFMD). The current EV71 propagating in Vero (EV-V) or sub-passaged in RD (EV-R) cells was used as a pathogen. Interestingly, EV-R exhibited differential virulence; challenging human scavenger receptor class B2-expressing (hSCARB2-Tg) mice with EV71 revealed that EV-V was more virulent than EV-R: 100% of mice that received lethal amounts of EV-V died, while all the mice that received EV-R survived. Severe pathogenesis correlated with viral burdens and proinflammatory cytokine levels were observed in EV-V-challenged mice, but controversy in EV-R-challenged mice. Consensus sequence analysis revealed EV-R rapidly acquired complete mutations at E145G and S241L and partial mutations at V146I of VP1, and acquired a T to C substitution at nucleotide 494 of the 5'-UTR. EV-R exhibited higher binding affinity for another EV71 receptor, human P-selectin glycoprotein ligand-1 (hPSGL-1), than EV-V. Both EV71s exhibited no significant difference in binding to hSCARB2. The molecular modelling indicate that these mutations might influence EV71 engagement with PSGL-1 and in vivo virulence.

Swine are a critical amplifying host involved in human Japanese encephalitis (JE) outbreaks. Cross-genotypic immunogenicity and sterile protection are important for the current genotype III (GIII) virus-derived vaccines in swine, especially now that emerging genotype I (GI) JE virus (JEV) has replaced GIII virus as the dominant strain. Herein, we aimed to develop a system to generate GI JEV virus-like particles (VLPs) and evaluate the immunogenicity and protection of the GI vaccine candidate in mice and specific pathogen-free swine. A CHO-heparan sulfate-deficient (CHO-HS(-)) cell clone, named 51-10 clone, stably expressing GI-JEV VLP was selected and continually secreted GI VLPs without signs of cell fusion. 51-10 VLPs formed a homogeneously empty-particle morphology and exhibited similar antigenic activity as GI virus. GI VLP-immunized mice showed balanced cross-neutralizing antibody titers against GI to GIV viruses (50% focus-reduction micro-neutralization assay titers 71 to 240) as well as potent protection against GI or GIII virus infection. GI VLP-immunized swine challenged with GI or GIII viruses showed no fever, viremia, or viral RNA in tonsils, lymph nodes, and brains as compared with phosphate buffered saline-immunized swine. We thus conclude GI VLPs can provide sterile protection against GI and GIII viruses in swine.

Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease (HFMD). The current EV71 propagating in Vero (EV-V) or sub-passaged in RD (EV-R) cells was used as a pathogen. Interestingly, EV-R exhibited differential virulence; challenging human scavenger receptor class B2-expressing (hSCARB2-Tg) mice with EV71 revealed that EV-V was more virulent than EV-R: 100% of mice that received lethal amounts of EV-V died, while all the mice that received EV-R survived. Severe pathogenesis correlated with viral burdens and proinflammatory cytokine levels were observed in EV-V-challenged mice, but controversy in EV-R-challenged mice. Consensus sequence analysis revealed EV-R rapidly acquired complete mutations at E145G and S241L and partial mutations at V146I of VP1, and acquired a T to C substitution at nucleotide 494 of the 5′-UTR. EV-R exhibited higher binding affinity for another EV71 receptor, human P-selectin glycoprotein ligand-1 (hPSGL-1), than EV-V. Both EV71s exhibited no significant difference in binding to hSCARB2. The molecular modelling indicate that these mutations might influence EV71 engagement with PSGL-1 and in vivo virulence.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. The vaccine candidates under development require a tetravalent immunogen to induce a balanced immunity against all four serotypes of dengue virus. Herein we show that mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher and broader neutralization antibodies (NtAbs) against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. The cryo-electron microscopy reconstruction showed that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Most importantly, maternally transferred antibodies derived from mD2VLP-vaccinated female mice protected suckling mice from lethal challenge by all four serotypes of DENV. Our results support the fact that a universal dengue vaccine that protects against all four serotypes of dengue viruses can be achieved by using an immunogen such as mD2VLP.

Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens. IMPORTANCE Outbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71 infection mechanism and anti-EV71 drug discovery but also provide a new strategy for virus receptor identification.

Clinical management of Clostridium difficile infection is still far from satisfactory as bacterial spores are resistant to many chemical agents and physical treatments. Certain types of nanoparticles have been demonstrated to exhibit anti-microbial efficacy even in multi-drug resistance bacteria. However, most of these studies failed to show biocompatibility to the mammalian host cells and no study has revealed in vivo efficacy in C. difficile infection animal models. The spores treated with 500 µg/mL Fe3-δO4 nanoparticles for 20 minutes, 64% of the spores were inhibited from transforming into vegetative cells, which was close to the results of the sodium hypochlorite-treated positive control. By cryo-electron micro-tomography, we demonstrated that Fe3-δO4 nanoparticles bind on spore surfaces and reduce the dipicolinic acid (DPA) released by the spores. In a C. difficile infection animal model, the inflammatory level triple decreased in mice with colonic C. difficile spores treated with Fe3-δO4 nanoparticles. Histopathological analysis showed a decreased intense neutrophil accumulation in the colon tissue of the Fe3-δO4 nanoparticle-treated mice. Fe3-δO4 nanoparticles, which had no influence on gut microbiota and apparent side effects in vivo, were efficacious inhibitors of C. difficile spore germination by attacking its surface and might become clinically feasible for prophylaxis and therapy.

Dysfunction of degradation machineries causes cancers, including hepatocellular carcinoma (HCC). Overexpression of cyclin D1 in HCC has been reported. We previously reported that autophagy preferentially recruits and degrades the oncogenic microRNA (miR)-224 to prevent HCC. Therefore, in the present study, we attempted to clarify whether cyclin D1 is another oncogenic factor selectively regulated by autophagy in HCC tumorigenesis. Initially, we found an inverse correlation between low autophagic activity and high cyclin D1 expression in tumors of 147 HCC patients and three murine models, and these results taken together revealed a correlation with poor overall survival of HCC patients, indicating the importance of these two events in HCC development. We found that increased autophagic activity leads to cyclin D1 ubiquitination and selective recruitment to the autophagosome (AP) mediated by a specific receptor, sequestosome 1 (SQSTM1), followed by fusion with lysosome and degradation. Autophagy-selective degradation of ubiquitinated cyclin D1 through SQSTM1 was confirmed using cyclin D1/ubiquitin binding site (K33-238 R) and phosphorylation site (T286A) mutants, lentivirus-mediated silencing autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and Sqstm1 knockout cells. Functional studies revealed that autophagy-selective degradation of cyclin D1 plays suppressive roles in cell proliferation, colony, and liver tumor formation. Notably, an increase of autophagic activity by pharmacological inducers (amiodarone and rapamycin) significantly suppressed tumor growth in both the orthotopic liver tumor and subcutaneous tumor xenograft models. Our findings provide evidence of the underlying mechanism involved in the regulation of cyclin D1 by selective autophagy to prevent tumor formation. Conclusion Taken together, our data demonstrate that autophagic degradation machinery and the cell-cycle regulator, cyclin D1, are linked to HCC tumorigenesis. We believe these findings may be of value in the development of alternative therapeutics for HCC patients. (Hepatology 2018;68:141-154).

The HIV-1 spike is composed of three protomeric units, each containing a peripheral gp120 and a transmembrane gp41 subunit. Binding to the CD4 and the chemokine receptors triggers them to mediate virus entry into cells by membrane fusion. The spikes also represent the major target for neutralizing antibodies (Abs) against the virus. We have studied how two related broadly neutralizing Abs, PG9 and PG16, react with the spike. Unexpectedly, this also suggested how the functions of the individual protomers in the spike depend on each other. The Abs have been shown to bind the V1/V2 loops of gp120, located at the top of the spike. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we show that only single Abs or antigen-binding fragments could bind to the spikes of HIV-1 virus-like particles. Apparently, binding to one gp120 sterically interferes with binding to the other two subunits in the spike top. Despite this constraint, all of the protomers of the spike became resistant to CD4 binding and subsequent formation of the coreceptor binding site. These activities were measured by monitoring the sequential complex formation of the spike first with Abs and then with soluble 2d- or 4d-CD4 or with soluble CD4 and the CD4 inducible coreceptor binding site Ab 17b in BN-PAGE. The inhibition of the spike by single-Ab binding suggested that the activation reactions of the individual protomeric units are linked to each other in a coordinated activation process.

Hand, foot and mouth diseases (HFMD) are mainly caused by Enterovirus A71 (EV-A71) infections. Clinical trials in Asia conducted with formalin-inactivated EV-A71 vaccine candidates produced from serum-free Vero cell culture using either roller bottle or cell factory technology, are found to be safe and highly efficacious. To increase vaccine yields and reduce the production costs, the bioprocess improvement for EV-A71 vaccine manufacturing is currently being investigated. The parameters that could affect and enhance the production yields of EV-A71 virus growth in the microcarrier bioreactor were investigated. The medium replacement culture strategy included a multi-harvested semi-batch process and perfusion technology and was found to increase the production yields more than 7-14 folds. Based on the western blot and cryo-EM analyses of the EV-A71 virus particles produced from either the multi-harvested semi-batch (MHSBC) or perfusion cultures were found to be similar to those virus particles obtained from the single batch culture. Mouse immunogenicity studies indicate that the EV-A71 vaccine candidates produced from the perfusion culture have similar potency to those obtained from single batch bioprocess. The physical structures of the EV-A71 particles revealed by the cryo-EM analysis were found to be spherical capsid particles. These results provide feasible technical bioprocesses for increasing virus yields and the scale up of EV-A71 vaccine manufacturing using the bioreactor cell culture methods.

During the process of autophagy, the autophagy-related proteins are translocated to autophagosome formation sites. Here, we demonstrate that S100A10 is required for ULK1 localization to autophagosome formation sites. Silencing of S100A10 reduces IFN-γ-induced autophagosome formation. We also determined the role of annexin A2 (ANXA2), a binding partner of S100A10, which has been reported to promote phagophore assembly. Silencing of ANXA2 reduced S100A10 expression. However, overexpression of S100A10 in ANXA2-silenced cells was still able to enhance autophagosome formation, suggesting that ANXA2 regulates IFN-γ-induced autophagy through S100A10. We also observed that S100A10 interacted with ULK1 after IFN-γ stimulation, and S100A10 knockdown prevented ULK1 localization to autophagosome formation sites. Finally, the release of high mobility group protein B1, one of the functions mediated by IFN-γ-induced autophagy, was inhibited in S100A10 knockdown cells. These results elucidate the importance of S100A10 in autophagosome formation and reveal the relationship between S100A10 and ULK1 in IFN-γ-induced autophagy.