Infected HeLa Cells Research Articles

Rhesus rotavirus receptor-binding site affects high mobility group box 1 release, altering the pathogenesis ofexperimental biliary atresia.

Biliary atresia (BA) is a neonatal inflammatory cholangiopathy that requires surgical intervention by Kasai portoenterostomy to restore biliary drainage. Even with successful portoenterostomy, most patients diagnosed with BA progress to end‐stage liver disease, necessitating a liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of neonatal mice induces an inflammatory obstructive cholangiopathy that parallels human BA. The model is triggered by RRV viral protein (VP)4 binding to cholangiocyte cell‐surface proteins. High mobility group box 1 (HMGB1) protein is a danger‐associated molecular pattern that when released extracellularly moderates innate and adaptive immune response. In this study, we investigated how mutations in three RRV VP4‐binding sites, RRVVP4‐K187R (sialic acid‐binding site), RRVVP4‐D308A (integrin α2β1‐binding site), and RRVVP4‐R446G (heat shock cognate 70 [Hsc70]‐binding site), affects infection, HMGB1 release, and the murine model of BA. Newborn pups injected with RRVVP4‐K187R and RRVVP4‐D308A developed an obstruction within the extrahepatic bile duct similar to wild‐type RRV, while those infected with RRVVP4‐R446G remained patent. Infection with RRVVP4‐R446G induced a lower level of HMGB1 release from cholangiocytes and in the serum of infected pups. RRV infection of HeLa cells lacking Hsc70 resulted in no HMGB1 release, while transfection with wild‐type Hsc70 into HeLa Hsc70‐deficient cells reestablished HMGB1 release, indicating a mechanistic role for Hsc70 in its release. Conclusion: Binding to Hsc70 contributes to HMGB1 release; therefore, Hsc70 potentially serves as a therapeutic target for BA.

IDO1, FAT10, IFI6, and GILT Are Involved in the Antiretroviral Activity of γ-Interferon and IDO1 Restricts Retrovirus Infection by Autophagy Enhancement.

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.

Human Guanylate-Binding Protein 1 Positively Regulates Japanese Encephalitis Virus Replication in an Interferon Gamma Primed Environment.

RNA virus infection triggers interferon (IFN) receptor signaling, leading to the activation of hundreds of interferon-stimulated genes (ISGs). Guanylate-binding proteins (GBPs) belong to one such IFN inducible subfamily of guanosine triphosphatases (GTPases) that have been reported to exert broad anti-microbial activity and regulate host defenses against several intracellular pathogens. Here, we investigated the role of human GBP1 (hGBP1) in Japanese encephalitis virus (JEV) infection of HeLa cells in both an IFNγ unprimed and primed environment. We observed enhanced expression of GBP1 both at transcript and protein levels upon JEV infection, and GBP1 association with the virus replication membranes. Depletion of hGBP1 through siRNA had no effect on JEV replication or virus induced cell death in the IFNγ unprimed environment. IFNγ stimulation provided robust protection against JEV infection. Knockdown of GBP1 in the primed environment upregulated expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and significantly reduced JEV replication. Depletion of GBP1 in an IFNγ primed environment also inhibited virus replication in human neuroblastoma SH-SH5Y cells. Our data suggests that in the presence of IFNγ, GBP1 displays a proviral role by inhibiting innate immune responses to JEV infection.

Peroxisomal Membrane Protein PMP34 Is Involved in the Human Papillomavirus Infection Pathway

Infection with high-risk human papillomavirus (HPV) types is linked to the onset of several cancers. The mechanism of HPV infection, however, has not yet been fully elucidated. Here, using the newly developed HPV infectious pseudovirion (HPV PsV) and a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening system, we established an experimental system and searched for genes involved in HPV infection. The HPV PsV has the truncated herpes simplex virus thymidine kinase (dTK) to kill PsV-infected cells when combined with ganciclovir. The five rounds of selection of 293FT cells by infection with HPV PsVs identified two candidate genes involved in the HPV infection pathway. The validation experiments showed that SLC25A17, which encodes the peroxisomal membrane protein PMP34, was involved in the HPV infection pathway. The gRNAs against SLC25A17 attenuated the efficiency of HPV PsV infection in 293FT and HeLa cells. Although further experiments are required to determine whether PMP34 acts as the HPV infection pathway, these results indicate that our screening system is useful for identification of the genes involved in the HPV infection pathway.

CD73 promotes cervical cancer growth via EGFR/AKT1 pathway.

BackgroundCervical cancer ranks third in cancer incidence worldwide and is the most frequent gynecological cancer in developing countries. To expore the molecular mechanism of cervical cancer and to find effective treatment have become the focus of medical workers. CD73 has been implicated in the progression of many cancers. However, the study of CD73 in cervical cancer has not been reported. The aim of this study was to identify the effect and mechanism of CD73 overexpression on cervical cancer growth in vitro and in vivo.MethodsCervical cancer cell models with CD73 overexpression were construction by using lentiviruses infection in Hela and SiHa cells. Cell’s proliferation was investigated by using xCELLigence real-time cell analysis (RTCA) system. Murine xenograft models were used to evaluate the effect of CD73 overexpression on tumor growth in vivo. Small interfering RNA (siRNA) transfection were used to suppress expression levels of EGFR and AKT1. Cell cycle and apoptosis were evaluated by flow cytometry (FCM).ResultsCD73 overexpression significantly promoted cervical cancer cells proliferation in vitro and tumor growth in vivo. The expression levels of EGFR and AKT1 were significantly increased in cell models and transplanted tumor tissues with CD73 overexpression. And moreover, knockdown of EGFR and AKT1 could inhibit proliferation of CD73 overexpressed cell models via inducing cell apoptosis and cell cycles increased in G2/M phase and reduction of G1 phase. Furthermore, the expression levels of CDK2, CDK3 and CDKN1A, which are cell cycle regulated molecules, were significantly increased in CD73 overexpressed cells with EGFR/AKT1 knockdown.ConclusionsOur data demonstrated that CD73 overexpression promote cervical cancer growth in vitro and in vivo, via activating EGFR/AKT1 pathway.

A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

Expression and structure of the Chlamydia trachomatis DksA ortholog.

Chlamydia trachomatis is a bacterial obligate intracellular parasite and a significant cause of human disease, including sexually transmitted infections and trachoma. The bacterial RNA polymerase-binding protein DksA is a transcription factor integral to the multicomponent bacterial stress response pathway known as the stringent response. The genome of C. trachomatis encodes a DksA ortholog (DksACt) that is maximally expressed at 15–20 h post infection, a time frame correlating with the onset of transition between the replicative reticulate body (RB) and infectious elementary body (EB) forms of the pathogen. Ectopic overexpression of DksACt in C. trachomatis prior to RB–EB transitions during infection of HeLa cells resulted in a 39.3% reduction in overall replication (yield) and a 49.6% reduction in recovered EBs. While the overall domain organization of DksACt is similar to the DksA ortholog of Escherichia coli (DksAEc), DksACt did not functionally complement DksAEc. Transcription of dksACt is regulated by tandem promoters, one of which also controls expression of nrdR, encoding a negative regulator of deoxyribonucleotide biosynthesis. The phenotype resulting from ectopic expression of DksACt and the correlation between dksACt and nrdR expression is consistent with a role for DksACt in the C. trachomatis developmental cycle.

Integrating lncRNAs and mRNAs Expression Profiles in Penicillin-Induced Persistent Chlamydial Infection in HeLa Cells.

Chlamydia trachomatis (C. trachomatis) is a major etiological agent of sexually transmitted infection. Some stressing conditions can result in persistent chlamydial infection, which is thought to be associated with severe complications including ectopic pregnancy and tubal factor infertility. Long noncoding RNAs (lncRNAs) have been identified as key modulators in many biological processes. Nevertheless, the role of lncRNAs in persistent chlamydial infection is still unclear. In this study, we used lncRNA and mRNA microarray to identify the global lncRNAs and mRNAs expression in penicillin-induced persistent chlamydial infection in HeLa cells as well as the control group (HeLa cells without C. trachomatis infection). Among 1005 differentially expressed lncRNAs, 585 lncRNAs were upregulated and 420 downregulated in persistent chlamydial infection, while 410 mRNAs were identified to express differentially, of which 113 mRNAs were upregulated and 297 downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with differentially expressed genes were performed. We then constructed the lncRNA-miRNA-mRNA competing endogenous RNAs (ceRNAs) network. Four mRNAs were validated to be changed by quantitative real-time PCR which were correlated with the microarray result. Integration of protein-protein interaction network was constructed and hub genes were identified. These findings provide a new perspective on the molecular mechanisms of penicillin-induced persistent chlamydial infection.

Dysregulation of Cytosolic c-di-GMP in Edwardsiella piscicida Promotes Cellular Non-Canonical Ferroptosis.

Programmed cell death plays an important role in modulating host immune defense and pathogen infection. Ferroptosis is a type of inflammatory cell death induced by intracellular iron-dependent accumulation of toxic lipid peroxides. Although ferroptosis has been associated with cancer and other sterile diseases, very little is known about the role of ferroptosis in modulating host-pathogen interactions. We show that accumulation of the secondary messenger bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP) in the pathogenic bacterium Edwardsiella piscicida (E. piscicida) triggers a non-canonical ferroptosis pathway in infected HeLa cells. Moreover, we observed that the dysregulation of c-di-GMP in E. piscicida promotes iron accumulation, mitochondrial dysfunction, and production of reactive oxygen species, all of which that can be blocked by iron chelator. Importantly, unlike classical ferroptosis that is executed via excess lipid peroxidation, no lipid peroxidation was detected in the infected cells. Furthermore, lipoxygenases inhibitors and lipophilic antioxidants are not able to suppress morphological changes and cell death induced by E. piscicida mutant producing excess c-di-GMP, and this c-di-GMP dysregulation attenuates bacterial virulence in vivo. Collectively, our results reveal a novel non-canonical ferroptosis pathway mediated by bacterial c-di-GMP and provide evidence for a role of ferroptosis in the regulation of pathogen infection.

Discovery of potential anti-SARS-CoV-2 drugs based on large-scale screening in vitro and effect evaluation in vivo.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global crisis. Clinical candidates with high efficacy, ready availability, and that do not develop resistance are in urgent need. Despite that screening to repurpose clinically approved drugs has provided a variety of hits shown to be effective against SARS-CoV-2 infection in cell culture, there are few confirmed antiviral candidates in vivo. In this study, 94 compounds showing high antiviral activity against SARS-CoV-2 in Vero E6 cells were identified from 2,580 FDA-approved small-molecule drugs. Among them, 24 compounds with low cytotoxicity were selected, and of these, 17 compounds also effectively suppressed SARS-CoV-2 infection in HeLa cells transduced with human ACE2. Six compounds disturb multiple processes of the SARS-CoV-2 life cycle. Their prophylactic efficacies were determined in vivo using Syrian hamsters challenged with SARS-CoV-2 infection. Seven compounds reduced weight loss and promoted weight regain of hamsters infected not only with the original strain but also the D614G variant. Except for cisatracurium, six compounds reduced hamster pulmonary viral load, and IL-6 and TNF-α mRNA when assayed at 4 d postinfection. In particular, sertraline, salinomycin, and gilteritinib showed similar protective effects as remdesivir in vivo and did not induce antiviral drug resistance after 10 serial passages of SARS-CoV-2 in vitro, suggesting promising application for COVID-19 treatment.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-2031-7. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

The parapoxvirus Orf virus ORF116 gene encodes an antagonist of the interferon response.

Orf virus (ORFV) is the type species of the Parapoxvirus genus of the Poxviridae family. Genetic and functional studies have revealed ORFV has multiple immunomodulatory genes that manipulate innate immune responses, during the early stage of infection. ORF116 is a novel gene of ORFV with hitherto unknown function. Characterization of an ORF116 deletion mutant showed that it replicated in primary lamb testis cells with reduced levels compared to the wild-type and produced a smaller plaque phenotype. ORF116 was shown to be expressed prior to DNA replication. The potential function of ORF116 was investigated by gene-expression microarray analysis in HeLa cells infected with wild-type ORFV or the ORF116 deletion mutant. The analysis of differential cellular gene expression revealed a number of interferon-stimulated genes (ISGs) differentially expressed at either 4 or 6 h post infection. IFI44 showed the greatest differential expression (4.17-fold) between wild-type and knockout virus. Other ISGs that were upregulated in the knockout included RIG-I, IFIT2, MDA5, OAS1, OASL, DDX60, ISG20 and IFIT1 and in addition the inflammatory cytokine IL-8. These findings were validated by infecting HeLa cells with an ORF116 revertant recombinant virus and analysis of transcript expression by quantitative real time-PCR (qRT-PCR). These observations suggested a role for the ORFV gene ORF116 in modulating the IFN response and inflammatory cytokines. This study represents the first functional analysis of ORF116.

Lactobacillus rhamnosus GG attenuates the pathology of Chlamydial muridarium in the upper genital tract in mice.

Chlamydia trachomatis is a pathogen which can cause hydrosalpinx and tubal fibrosis when infecting the urogenital tract. However, the mechanism is still not clear. There is growing evidence that the gut microbiota is associated with the pathogenesis of both intestinal and extra-intestinal disorders, such as cardiovascular disease, hepatocirrhosis, allergy, respiratory tract infection, polycystic ovary syndrome, endometriosis, and bacterial vaginitis. Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria, the benefits of LGG including the treatment in gastrointestinal disorders and immunomodulation are well demonstrated, and it can also alleviate hypersensitivity reaction and diarrhoea, inhibit a variety of respiratory and urogenital diseases. Chlamydia muridarium (Cm) infection is a good model for the study on human Chlamydia pathogenicity in genitourinary tract. The mice infected with Cm were used as animal models to preliminarily explore the mechanism for the effect of LGG on upper reproductive tract infection in the mice, and to provide experimental basis for the pathogenesis of Chlamydia trachomatis genitourinary tract infection and the new idea for the treatment of Chlamydia trachomatis infection. Five to six weeks-old C57BL/6J mice were divided into 2 groups: An experimental group and a control group. The experimental group were administrated with 5×108 colony forming units (CFU) LGG for 19 consecutive days, while the control group were feed PBS. The mice in the 2 group were subcutaneously injected with 2.5 mg progesterone on Day 9 and infected with 1×105 inclusion body forming unit of Cm via the vaginal tract on Day 14. Vaginal and rectal swabs were taken every 7 days to infect HeLa cells for 24 hours, then the indirect immunofluorescence assay was used and the number of inclusion bodies of Chlamydia were calculated. Mice were euthanized on Day 14 and Day 63 after Cm inoculation, the vaginal tracts were dissected, and the tissue homogenates were prepared to culture the pathogens for 24 hours. The Cm bearing capacity in the bilateral uterine horn, tubal ovary, and cervical vaginal tissues in the 2 groups were calculated. The spleen cells were harvested to assay the intracellular IFN-γ, IL-5, and IL-17 by flow cytometry. On Day 63 after the Chlamydia infection, the pathology injury in the bilateral uterine horn and oviduct was observed, and the pathological sections and HE staining in the various part of genital tract were performed. The inflammatory cell infiltration and lumen dilatation was assessed. The specific IgM and IgG in sera were detected by indirect ELISA on Day 14 and 63 after infection. There was no effect of LGG on the clearing of Cm from the urogenital tract, the Chlamydia ascending to fallopian tube or the uterine horn, and the organism dissemination and colonization to the gastrointestinal tract (all P>0.05). On Day 14 after Cm infection via the vagina, the IL-17 expression level in the experimental group was significant decreased than that in the control group (t=2.486, P<0.05), but there was no significant difference between the 2 groups in the CD4+ T rate in spleen and IgM and IgG levels in serum after Cm intravaginal infection (all P>0.05). On Day 63 after Cm infection, there was no difference in the severity of inflammation in the uterine horns and fallopian tubes between the 2 groups (P>0.05), but the dilation of the fallopian tubes and hydrosalpinx was attenuated in the experimental group compared with the control group (P<0.05). Oral administration of LGG has no effect on inhibiting Cm ascending to upper genital tract and preventing the dissemination and colonization of Cm to the gastrointestinal tract, which also cannot affect the secretion of specific IgM and IgG in sera. Oral administration of LGG can suppress the production of IL-17 in the spleen cells and attenuate hydrosalpinx development when following Cm intravaginal infection in mice.

Lactobacillus rhamnosus reduces the cytotoxic effects of group B streptococcus on HeLa cells

Group B Streptococcus (GBS) is an opportunistic pathogen found in the vaginal tract and is a leading cause of preterm birth and neonatal illness. Aside from GBS, the vaginal tract is predominantly colonized by commensal Lactobacillus species that are thought to protect the vaginal tract from pathogens, including GBS. Studies that examined if, and how Lactobacilli modulate GBS pathogenicity remain limited. This study sought to investigate the potential protective role of Lactobacillus rhamnosus against GBS, using an in vitro model system. Immunofluorescence microscopy and Scanning Electron Microscopy (SEM) captured images of infected HeLa cells and were analyzed using the image analysis program ImageJ. Results indicate that GBS causes HeLa cell detachment unless L. rhamnosus is present. SEM images show that GBS reduces length and number of microvilli on HeLa cell surface, as well as size of secreted vesicles. L. rhamnosus partially inhibits GBS-dependent microvilli and vesicle disruption. GBS also disrupts HeLa cell F-actin fibers unless L. rhamnosus is present. These results reveal effects of GBS infection on the host cell cytoskeleton and implies a protective role of L. rhamnosus against GBS colonization.

The impact of TiO2 nanoparticle exposure on transmembrane cholesterol transport and enhanced bacterial infectivity in HeLa cells

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 13 ± 2-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 51 ± 10 times higher, while the total cholesterol content increased only 21 ± 2%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-β-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity. Statement of significanceA great deal of work has been done regarding the toxicology of the particles, especially focusing on detrimental outcomes associated with reactive oxygen species (ROS) production. In this paper we show unambiguously a very surprising result, namely the ability of these particles to enhance bacterial infection even at very small exposure levels, where none of the deleterious effects of ROS products can yet be detected. Using a new imaging technique, we are able to demonstrate, in operando, the effect of the particles on cholesterol generation and distribution in live HeLa cells. This paper also represents the first in a series where we explore other consequences of increased membrane cholesterol, due to particle exposure, which are known to have multiple other consequences on human tissue function and development.

Ocoxin oral solution demonstrates antiviral properties in cellular models.

Ocoxin Oral Solution (OOS) and Viusid (VS) are nutritional supplements that include several natural products which affect different cellular functions, such as proliferation or the redox status. In addition, some of their constituent components have been described to exert an antiviral effect. Considering this, it was hypothesized that treatment with OOS and VS could protect from viral infections. In order to evaluate the impact of OOS and VS on viral infection, lentivirus and retrovirus whose genomes coded for green fluorescent protein were used. In addition, and as a second approach to measure viral infection, a hemagglutinin-tagged form of the mitogen-activated protein kinase ERK5 was also inserted in the retroviral vector. Viral particles produced in 293T cells were used to infect HeLa cells in the presence or absence of OOS or VS. It was observed that VS had a minimal effect on the capacity of either lentivirus or retrovirus to infect HeLa cells. However, OOS significantly reduced the infection of HeLa cells with both of these viruses. The effect was dose-dependent, reaching a maximum at a 1:100 dilution of OOS. These results suggested that, in addition to its well-known antitumoral properties, OOS may also inhibit infection with viruses. This effect is relevant since patients receiving oncological therapies are more susceptible to viral infections, and nutritional supplements such as OOS may help in reducing the severity of these potential pathogenic infections.

Construction and characterization of a novel Tenofovir-loaded PEGylated niosome conjugated with TAT peptide for evaluation of its cytotoxicity and anti-HIV effects

In recent years, nanocarriers have become potent drug delivery system candidates, especially in anti-HIV therapies. Meanwhile, using cell-penetrating peptides such as TAT for improvement of cellular transportation has been widely noticed. In the current study, a novel PEGylated niosomal formulation (PEG-NI) loaded with an anti-HIV drug (Tenofovir) has been prepared by using thin-film hydration method based on cholesterol and Span 60 surfactant. Then, the TAT peptide was incorporated into optimized PEGylated niosome. Further morphological and in vitro studies were performed by TAT conjugated (TAT-NI1) and PEGylated niosomes. The average size, polydispersity index and encapsulation efficiency of Tenofovir-loaded TAT-NI1 were 208 ± 9.004 nm, 0.39 ± 0.008 and 75 ± 2.516%, respectively. The cytotoxicity effects of TAT-NI1 and PEG-NI niosomes were analyzed by MTT assay in comparison with Tenofovir. The IC50 of PEG-NI, empty PEG-NI, TAT-NI1, free Tenofovir and TAT peptide were 65.41, 37.04, 41.02, 43.07, and 37.12, respectively. Furthermore, the inhibitory effects of samples against the HIV infected HeLa cells were evaluated. The results displayed a higher cytotoxicity, lower anti-Scr HIV effect and improved Tenofovir release profile for TAT-NI1 in comparison with PEG-NI. Overall, this study revealed that PEGylation is a superior alternative rather than TAT conjugation in anti-HIV drug delivery systems.

Biogenic silver nanoparticles (AgNp-Bio) reduce Toxoplasma gondii infection and proliferation in HeLa cells, and induce autophagy and death of tachyzoites by apoptosis-like mechanism

Toxoplasma gondii is a protozoan parasite that can cause severe and debilitating diseases, especially in immunocompromised individuals. The available treatment is based on drugs that have low efficacy, high toxicity, several adverse effects, and need long periods of treatment. Thus, the search for therapeutic alternatives is urgently needed. Biogenic silver nanoparticles (AgNp-Bio) have been associated with several biological effects, as antiproliferative, pro-apoptotic, antioxidant, antiviral, antifungal, and antiprotozoal activity. Thus, the objective was evaluating AgNp-Bio effect on HeLa cells infected with T. gondii (RH strain). First, nontoxic AgNp-Bio concentrations for HeLa cells (1.5 – 6 µM) were determined, which were tested on cells infected with T. gondii. A significant reduction in infection, proliferation, and intracellular parasitic load was observed, also an increase in ROS and IL-6. Additionally, the evaluation of the action mechanisms of the parasite showed that AgNp-Bio acts directly on tachyzoites, inducing depolarization of the mitochondrial membrane, ROS increase, and lipid bodies accumulation, as well as triggering an autophagic process, causing damage to the parasite membrane, and phosphatidylserine exposure. Based on this, it was inferred that AgNp-Bio affects T. gondii by inducing immunomodulation and microbicidal molecules produced by infected cells, and acts on parasites, by inducing autophagy and apoptosis.

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice.

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26's role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity.

Differential microRNA Expression in Newcastle Disease Virus-Infected HeLa Cells and Its Role in Regulating Virus Replication.

As an oncolytic virus, Newcastle disease virus (NDV) can specifically kill tumor cells and has been tested as an attractive oncolytic agent for cancer virotherapy. Virus infection can trigger the changes of the cellular microRNA (miRNA) expression profile, which can greatly influence viral replication and pathogenesis. However, the interplay between NDV replication and cellular miRNA expression in tumor cells is still largely unknown. In the present study, we compared the profiles of cellular miRNAs in uninfected and NDV-infected HeLa cells by small RNA deep sequencing. Here we report that NDV infection in HeLa cells significantly changed the levels of 40 miRNAs at 6 h post-infection (hpi) and 62 miRNAs at 12 hpi. Among 23 highly differentially expressed miRNAs, NDV infection greatly promoted the levels of 3 miRNAs and suppressed the levels of 20 miRNAs at both time points. These 23 miRNAs are predicted to target various genes involved in virus replication and antiviral immunity such as ErbB, Jak-STAT, NF-kB and RIG-I-like receptor. Verification of deep sequencing results by quantitative RT-PCR showed that 9 out of 10 randomly selected miRNAs chosen from this 23-miRNA pool were consistent with deep sequencing data, including 6 down-regulated and 3 up-regulated. Further functional research revealed that hsa-miR-4521, a constituent in this 23-miRNA pool, inhibited NDV replication in HeLa cells. Moreover, dual-luciferase and gene expression array uncovered that the member A of family with sequence similarity 129 (FAM129A) was directly targeted by hsa-miR-4521 and positively regulated NDV replication in HeLa cells, indicating that hsa-miR-4521 may regulate NDV replication via interaction with FAM129A. To our knowledge, this is the first report of the dynamic cellular miRNA expression profile in tumor cells after NDV infection and may provide a valuable basis for further investigation on the roles of miRNAs in NDV-mediated oncolysis.

Analyzing Potential Binding Factors of Translation Enhancing Sequence 5d5

The vaccinia virus has been used for years in recombinant vaccine development to target other pathogens. In this development, several attenuated viral strains have been created, however vaccines employing these attenuated viruses are deficient in producing an effective immune response, in part, due to insufficient protein expression. An avenue to increase protein expression in this setting that remains largely unexplored is the use of novel human translation enhancing elements. One such element, termed 5d5, has been shown to increase transcription and translation significantly compared to baseline levels of expression using standard vaccinia promoters. Due to the element's novelty, further investigation of the motifs that govern the sequence's function are still unknown. The goal of this study is to identify potential binding proteins responsible for the function of enhancing sequence 5d5. To find the proteins that govern the sequence, vaccinia infected HeLa cell lysate was incubated with the 5d5 sequence, which was tethered to magnetic beads. After incubation, the proteins were eluted and analyzed via MALDI-TOF mass spectrometry to characterize the proteins that bound to the enhancing element. The characterized proteins were then compared to proteins that bound to control sequences, and the unique proteins found on sequence 5d5 were analyzed. Finally, to confirm the binding sites of the identified proteins and the contribution to 5d5 function, targeted mutational analysis of the 5d5 sequence will be performed. By understanding the motifs that govern 5d5 function, the horizons for application of 5d5 can be broadened and the use of this enhancing motif in vaccine development can potentially improve.