HEp-2 Cells Research Articles

Photostability, cytotoxicity, and photothermal impact of AgNPs, CoAgNC, and IOAgNC on HEp-2 laryngeal carcinoma cells

Magnetic nanoparticles have shown promise in various medical applications, including cancer treatment and diagnostics. The objective of this study is to attain mastery over the synthesis of silver nanoparticles (AgNPs), Cobalt silver nanocomposite (CoAgNC) and magnetite silver nanocomposite (IOAgNC), while also examining their photostability, cytotoxicity, and photothermal impact on Human Laryngeal Carcinoma Cell line (HEp-2). The preparation and characterization of AgNPs, CoAgNC, and IOAgNC were described, including the assessment of their photostability and cytotoxicity on HEp-2 cell line. The photostability of these nanomaterials was determined by subjecting them to different light sources, while their morphology and magnetic properties were characterized using a spectrophotometer, Transmission Electron Microscopy (TEM), and Vibrating Sample Magnetometer (VSM). The results of the characterization of AgNPs, CoAgNC, and IOAgNC showed that the prepared nanomaterials have distinct optical and morphological properties. The synthesized nanomaterials were stable in aqueous solutions and exhibited magnetic properties. The cytotoxicity tests on HEp-2 cells showed that the nanomaterials were not toxic in the dark, but their toxicity increased under light exposure, with IOAgNC showing the most significant toxicity. CoAgNC was found to be photostable under UV light and Light Emitting Diode (LED) irradiation due to the little lattice mismatch between cobalt and silver. Nanocomposite systems of high magnetic moment, such as IOAgNC and CoAgNC, have potential for tumor treatment through magnetic fluid hyperthermia. The photostability, chemical stability, and biocompatibility of AgNPs, CoAgNC, and IOAgNC were investigated and found to be non-toxic even at high concentrations. These nanomaterials have multiple optical and magnetic functionalities and are promising candidates for biomedical applications.

Characterization and comparative analysis of the Escherichia marmotae M-12 isolate from bank vole (Myodes glareolus)

The Escherichia marmotae is a bacterium of the Enterobacterales order, which was first isolated from the Himalayan marmot (Marmota himalayana). Recently E. marmotae has been shown to cause severe infections in humans. Wild animals were suggested to be a natural reservoir of this bacterium. The present study describes the first case of E. marmotae isolation from an apparently healthy wild bank vole (Myodes glareolus). Phenotype, as well as genotype-based techniques, were applied to characterize E. marmotae M-12 isolate. E. marmotae M-12 had the capsule-positive phenotype, high adhesion to human erythrocytes and HEp-2 cells as well as a low invasion into HEp-2 cells. E. marmotae M-12 was avirulent in mice. The phylogenomic analyses of E. marmotae showed dispersed phylogenetic structure among isolates of different origins. Virulome analysis of M-12 isolate revealed the presence of the following factors: siderophores, heme uptake systems, capsule synthesis, curli and type I fimbriae, flagella proteins, OmpA porin, etc. Comparative virulome analysis among available E. marmotae genomes revealed the presence of capsule K1 genes mostly in pathogenic isolates and OmpA porin presence among all strains. We assume that the K1 capsule and OmpA porin play a key role in the virulence of E. marmotae. Pathogenesis of the latter might be similar to extraintestinal pathogenic E. coli.

Microwave assists synthesis of spirothiazolidine derivatives to induce cervical cancer cell death-mediated apoptosis in vitro

Herein, a new series of spirothiazolidine derivatives 1–14 are effectively produced using microwave irradiation in a quick and efficient manner. IR, NMR, and mass spectra were used to characterize all new formed substances. Newly created substances based on spirothiazolidinone ring system were tested for their anticancer effects on the common human cervical cancer cell lines HEp-2 and HeLa to produce effective initiative anti-cervical cancer analogs in order to complement or replace the existing anti-cervical cancer chemicals. Among the suggested compounds, compounds 7, 9d, 12, and 13 were found to be the most effective. In HeLa and HEp-2 cells, compounds 7 and 13 significantly increased the expression of CAS3 and Bax while significantly decreasing the expression of KRAS, NRAS, and Bcl-2. We are able to conclude that Compounds 7 and 13 inhibited the RAS gene in order to cause apoptosis.

Characterisation of typical enteropathogenic Escherichia coli (tEPEC) lineages and novel bfpA variants detected in Australian fruit bats (Pteropus poliocephalus)

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC strains are defined by the presence of specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp). Bfp is encoded by the bfp operon and includes the bfpA gene for the major pilus subunit. By definition, Bfp are only present in typical EPEC (tEPEC), for which, humans are considered to be the only known natural host. This study detected tEPEC in faecal samples from a wild Australian fruit bat species, the grey-headed flying-fox (Pteropus poliocephalus). Whole genome sequencing of 61 E. coli isolates from flying-foxes revealed that 21.3 % (95%CI: 13 %–33 %) were tEPEC. Phylogenetic analyses showed flying-fox tEPEC shared evolutionary lineages with human EPEC, but were predominantly novel sequence types (9 of 13) and typically harboured novel bfpA variants (11 of 13). HEp-2 cell adhesion assays showed adherence to human-derived epithelial cells by all 13 flying-fox tEPEC, indicating that they all carried functional Bfp. Using an EPEC-specific duplex PCR, it was determined that tEPEC comprised 17.4 % (95%CI: 13 %–22 %) of 270 flying-fox E. coli isolates. Furthermore, a tEPEC-specific multiplex PCR detected the eae and bfpA virulence genes in 18.0 % (95%CI: 8.0 %–33.7 %) of 506 flying-fox faecal DNA samples, with occurrences ranging from 1.3 % to 87.0 % across five geographic areas sampled over a four-year period. The identification of six novel tEPEC sequence types and five novel bfpA variants suggests flying-foxes carry bat-specific tEPEC lineages. However, their close relationship with human EPEC and functional Bfp, indicates that flying-fox tEPEC have zoonotic potential and that dissemination of flying-fox tEPEC into urban environments may pose a public health risk. The consistent detection of tEPEC in flying-foxes over extensive geographical and temporal scales indicates that both wild grey-headed flying-foxes and humans should be regarded as natural tEPEC hosts.

Significane of determining antinuclear antibodies in systemic connective tissue disorders in children

Systemic connective tissue diseases (SCTD) are characterized by systemic autoimmune inflammation and are accompanied by development of antinuclear antibodies (ANA). Our aim was a comparative analysis of ANA in blood serum in children with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). The study included 50 patients with SLE, 50 patients with RA who were treated at the National Medical Research Center of Childrens Health. Median age was 12,2 y. o. (9-15.5). The titers of ANA and the cell fluorescence type were determined with the indirect immunofluorescence reaction (IFR) using the HEp-2 cell line (Immco Diagnostics, Inc, USA), as well as the concentration of antibodies to double-stranded DNA (adsDNA) in blood serum samples of the children detected by immunochemiluminescence (ICM) woth Elia dsDNA (Thermo Fisher Scientific, USA). A positive ANA titer and adsDNA were found, respectively, in 98% and in 48% in children with SLE. A positive ANA titer and adsDNA was detected in 100% and in 4% of children with RA, respectively. Highly positive ANA titers ( 1/1280) have been detected in 68% of children with SLE, and in 30% of children with RA. None of the RA patients with highly positive ANA titers had adsDNA. But, in patients with SLE, highly positive ANA titers and a positive dsDNA level were simultaneously detected in 16% of cases. There are both single types of cell fluorescence and their combinations in children with SLE and RA. Nuclear dot-like fluorescence was more common in children with SLE, cytoplasmic type, in children with RA, nucleolar type of glow was found only in children with RA. The revealed combinations of ANA and adsDNA titers in children with SLE and RA confirm the need for simultaneous use of RNIF and ICM.

Expression of hes, iha, and tpsA codified in locus of adhesion and autoaggregation and their involvement in the capability of shiga toxin-producing Escherichia coli strains to adhere to epithelial cells

ObjectivesShiga toxin-producing Escherichia coli strains LAA-positive are important cause of human infection. The capability to adhere to epithelial cells is a key virulence trait, and genes codified in LAA pathogenicity island could be involved in the adhesion during the pathogenesis of LAA-positive STEC strains. Thus, our objectives were to compare hes-negative and hes-positive STEC strains in their adherence capability to epithelial cells (HEp-2) and to evaluate the expression levels of the hes, iha, and tpsA in the bacteria adhered and non-adhered to HEp-2 cells. These genes are encoded in LAA, and are virulence factors that participate in adhesion and autoaggregation.ResultsWe could not observe differences between the adhesion of strains but also in the expression level of of hes, iha, and tpsA. Genes encoded in LAA contribute to the adhesion phenotype though the expression of STEC adhesins is a coordinated event that depends not only the strain but also on the environment as well as its genetic background. Therefore, the results of this study suggest that LAA ,the most prevalent PAI among LEE-negative STEC strains, plays a role in pathogenesis.

Structure-Activity Relationship Prediction-Based Synthesis and Cytotoxicity Evaluation against the HEp-2 Laryngeal Carcinoma Cell of Isoflavone-Cytisine Mannich Bases.

QSAR analysis of previously synthesized and nature-inspired virtual isoflavone-cytisine hybrids against the HEp-2 laryngeal carcinoma cell lines was performed using the OCHEM web platform. The validation of the models using an external test set proved that the models can be used to predict the activity of newly designed compounds such as 8-cytisinylmethyl derivatives of 5,7- and 6,7-dihydroxyisoflavones. The synthetic procedure for selective aminomethylation of 5,7-dihydroxyisoflavones with cytisine was developed. In vitro testing identified compound 7 f with cisplatin-level cytotoxicity against HEp-2 cell lines and compound 10 which was twice active than cisplatin after 72 h of incubation.

Ultrastructural and Functional Characterization of Mitochondrial Dynamics Induced by Human Respiratory Syncytial Virus Infection in HEp-2 Cells.

Human respiratory syncytial virus (hRSV) is the leading cause of acute lower respiratory tract infections in children under five years of age and older adults worldwide. During hRSV infection, host cells undergo changes in endomembrane organelles, including mitochondria. This organelle is responsible for energy production in the cell and plays an important role in the antiviral response. The present study focuses on characterizing the ultrastructural and functional changes during hRSV infection using thin-section transmission electron microscopy and RT-qPCR. Here we report that hRSV infection alters mitochondrial morphodynamics by regulating the expression of key genes in the antiviral response process, such as Mfn1, VDAC2, and PINK1. Our results suggest that hRSV alters mitochondrial morphology during infection, producing a mitochondrial phenotype with shortened cristae, swollen matrix, and damaged membrane. We also observed that hRSV infection modulates the expression of the aforementioned genes, possibly as an evasion mechanism in the face of cellular antiviral response. Taken together, these results advance our knowledge of the ultrastructural alterations associated with hRSV infection and might guide future therapeutic efforts to develop effective antiviral drugs for hRSV treatment.

Enhanced therapeutic efficacy of biogenic nanosilver-conjugated thymol: In vitro and in vivo evaluation against emerging multi-drug resistant microbes

Phytochemicals and nanoparticles (NPs) despite having strong antibacterial activity, their limited stability and safety prevent their widespread biomedical and environmental applications. Therefore, the bio-efficacy and environmental safety of both can be increased by fabricating and conjugating them using eco-friendly processes. Herein, the impact of a novel green nanomedicine, nanosilver-conjugated thymol (AgT), was evaluated against multi-drug resistant enteroaggregative Escherichia coli (MDR-EAEC) strains using in vitro and in vivo (Galleria mellonella) assays. Characterization of AgT by UV–Vis and FTIR spectroscopy demonstrated the conjugation of thymol with biogenic nanosilver with a loading capacity of 16.60 ± 0.24%. Electron microscopy revealed mono-dispersed spherical morphology with a size of 15.077 ± 3.34 nm. The minimum inhibitory concentration (4–16 μg/mL) and minimum bactericidal concentration (8–16 μg/mL) were five to six-fold times lower than thymol. The antibacterial efficacy of AgT was at par with meropenem in the in vitro time-kill assay. Moreover, AgT was tested safe against gut lactobacilli, sheep erythrocytes, RAW 264.7 and HEp-2 cell lines; stable at various high-end temperatures, pH, proteases, cationic salts, and host sera. G. mellonella larvae treated with AgT exhibited higher survival rates and safety with reduced MDR-EAEC counts. Overall, compared to thymol, AgT showed promising therapeutic potential with improved safety.

A deep learning self-attention cross residual network with Info-WGANGP for mitotic cell identification in HEp-2 medical microscopic images

BackgroundThe identification of human epithelial type-2 mitotic cell patterns in the indirect immunofluorescence images (IIF HEp-2) is a critical step for autoimmune diseases computer-aided diagnosis (CAD) systems. Recognition of HEp-2 cells in the mitotic phase is clinically vital for validating the HEp-2 samples and assisting in diagnosing specimens with mixed patterns. Typically, mitotic cells are rarely observed in the HEp-2 specimen images, resulting in a significant skewness of the available medical datasets towards the majority of interphase (non-mitotic) patterns. MethodsIn this paper, a deep learning framework is proposed based on a self-attention deep cross-residual network with an efficient generative adversarial network (GAN). The proposed framework consists of two consecutive steps. First, the problem of imbalanced data of minority mitotic against the majority interphase cells is remedied using Info-WGANGP approach in combination with the conventional data augmentation methods. Second, a downstream end-to-end deep learning Att-DCRNet is developed to classify the mitotic and interphase cell patterns of the IIF HEp-2 medical images. A comprehensive experimental study is performed to validate the effectiveness of the proposed framework against other state-of-the-art methods over the public medical dataset of UQ-SNP_HEp-2 Task-3. ResultsThe proposed framework demonstrates competitive classification results attaining a maximum performance of 84.10% F1-score, 84.70% Matthew's correlation coefficient (MCC), and 99.0% balanced class accuracy (BcA), which proves its applicability for automatically supporting an accurate diagnosis decision regarding the HEp-2 mitotic and interphase cell patterns. The source code is available at this link: https://github.com/Anaam-dl/AttDCRNet_with_InfoWGANGP.

Retraction.

The article "The circRNA-MYLK plays oncogenic roles in the Hep-2 cell line by sponging microRNA-145-5p" by Yao Chen, Yanmei Wang, Congcong Li, Xuechang Li, Tiejun Yuan, Shuqin Yang and Xiaoyan Sun, published in Gen. Physiol. Biophys. 39(3), 2020, pp. 229-237 (doi: 10.4149/gpb_2019060) has been retracted by agreement between the author(s) and journal's Editor in Chief, Prof. Dr. Lubica Lacinova, and AEPresss, s.r.o.. The corresponding author Xiaoyan Sun asked to retract this manuscript as there were some substantial problems in it, which needed more time and research to solve and can more fully re-examine and revise his research results.The authors were not available for a final confirmation of the retraction.

In English

Today, the antiviral activity of oxide nanomaterials can be used in the fight against the viral disease COVID-19. It is thought that Ag nanoparticles may bind to the surface glycoprotein of the virus and interfere with the virus’s interaction with epithelial cells, and inhibit virus reproduction by releasing silver ions in the cell. The viruses’ inhibition with RNA (ribonucleic acid) and DNA (deoxyribonucleic acid) genomes by oxide nanocomposites action was presented. In this research, the surface structure of doped CeO2 (La2O3) was studied by nitrogen adsorption-desorption based on BET method. The silver atom’s existence in CeO2 - Ag0 can facilitate the transport of more holes to the surface and can enhance the optical, antivirus activity. The primary particle size of pure cerium dioxide is 7 nm, for CeO2 - Ag composite at 2 and 4 wt. % of silver is 6.5 and 6.9 nm; for La2O3 - Ag 27 and 35 nm, respectively. Cell viability was assessed using an MTT (3-(4,5-Dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide) assay after NPs (nanoparticles) exposure, since only viable cells have functional mitochondrial dehydrogenase enzymes that can reduce MTT to formazan. Nanoparticles were non-toxic for BHK-21(Syrian hamster kidney), Hep-2 (Human larynx carcinoma), and MDCK (Canine kidney) cells in concentrations of 10 and 100 μg/ml, while cell viability was within 76÷100 %. La2O3 and CeO2, which contained 4 wt. % of Ag at a concentration of 1000 μg/ml had a lower toxic effect: for BHK-21 cells 68 and 76 % of viable cells, respectively; for Hep-2 - 40 and 36 %, for MDCK - 42 and 48 %; La2O3 and CeO2 with 2 and 5 wt. % of Ag at a concentration of 1000 µg/ml were highly toxic. The level of ВНК-21, Нер-2, and MDCK cells viability was in a range of 7 to 37 %. It has been stated that oxides of cerium and lanthanum have a pronounced virucidal action against the Herpes simplex virus and Influenza A virus by completely inhibiting the development of its cytopathic action. The lanthanum and cerium oxides with 2 and 5 wt. % of silver inhibited the development of the virus’s CPE by more than 5.0 log10 compared to the virus control. The results show that lanthanum and cerium oxides with 2 and 5 wt. % silver have a high virucidal effect against herpes simplex virus type 1. A 1.0÷4.0 log10 reduction in the infectious titer of the Herpes virus synthesized “de novo” in the presence of lanthanum and cerium oxide nanocomposites has been shown.

Anti-dense fine speckled 70 (DFS70) autoantibodies: correlates and increasing prevalence in the United States.

Recent studies report high-titer anti-dense fine speckled 70 (DFS70) autoantibodies in persons with inflammatory conditions, but the clinical significance remains unclear. Our goals were to estimate anti-DFS70 autoantibody prevalence, identify correlates, and assess time trends. Serum antinuclear antibodies (ANA) were measured by indirect immunofluorescence assay on HEp-2 cells in 13,519 participants ≥12 years old from three time periods (1988-1991, 1999-2004, 2011-2012) of the National Health and Nutrition Examination Survey. ANA-positive participants with dense fine speckled staining were evaluated for anti-DFS70 antibodies by enzyme-linked immunosorbent assay. We used logistic models adjusted for survey-design variables to estimate period-specific anti-DFS70 antibody prevalence in the US, and we further adjusted for sex, age, and race/ethnicity to identify correlates and assess time trends. Women were more likely than men (odds ratio (OR)=2.97), black persons were less likely than white persons (OR=0.60), and active smokers were less likely than nonsmokers (OR=0.28) to have anti-DFS70 antibodies. The prevalence of anti-DFS70 antibodies increased from 1.6% in 1988-1991 to 2.5% in 1999-2004 to 4.0% in 2011-2012, which corresponds to 3.2 million, 5.8 million, and 10.4 million seropositive individuals, respectively. This increasing time trend in the US population (P<0.0001) was modified in some subgroups and was not explained by concurrent changes in tobacco smoke exposure. Some, but not all, anti-DFS70 antibody correlates and time trends resembled those reported for total ANA. More research is needed to elucidate anti-DFS70 antibody triggers, their pathologic or potentially protective influences on disease, and their possible clinical implications.

Photodynamic Treatment of Titanium Dioxide Nanoparticles is a Convenient Method of Adenoviral Inactivation

Today, the search for safe ways to inactivate pathogens is becoming especially relevant in connection with the coronavirus pandemic. Standard methods using chlorides and ultraviolet irradiation have disadvantages related to toxicity and low efficiency. Photodynamic inactivation involving nanoparticles is already used to disinfect water and air from microorganisms and enveloped viruses such as human herpes simplex virus, vesicular stomatitis virus, human immunodeficiency virus, and hepatitis B and C viruses. The aim of this work was to evaluate the possibility of the inactivation of human adenovirus type 5 in an organic medium using titanium dioxide irradiated with ultraviolet light. Methods. The nanosized titanium dioxide material was obtained by the thermal decomposition of a suspension of hydrated titanium dioxide TiO(OH)2 (metatitanic acid). The analysis of the morphology of the TiO2 nanopowder was carried out using electron scanning microscopy (SEM), which showed that TiO2 nanopowder contains soft aggregates of nanoparticles mostly 20‒30 nm in size. Cytotoxicity, virulicidal and antiviral action of titanium dioxide were determined by standard methods using (3-(4,5-dimathylthiazol-2-yl)-2,5-dipheniltetrazolium bromide (MTT). The titanium dioxide suspension was irradiated at a distance of 20 cm from 1 to 30 min with a bactericidal UV lamp (OBB15P, BactoSfera, Poland (254 nm)). The concentration of nanoparticles for irradiation was 1.0 mg/mL. Adenovirus suspension with titer 6.0 log10 TCID50 /mL was added to the nanoparticles immediately after irradiation. The titer of virus synthesized in the presence of titanium dioxide was determined by the end of the virus dilution, which causes 50% of the cytopathic effect of the virus on cells. All studies were performed in three replicates; the number of parallel determinations was three. Results. A dose-dependent effect of titanium dioxide nanoparticles on the viability of Hep-2 cells was revealed. At the NPs concentration of 1 mg/mL, quite a low cell viability was observed (32—39%), with a decrease in concentration to 0.1 and 0.01 mg/mL, the NPs were less toxic (cell viability was in the range of 62—90%). The TiO2 NPs dissolved in glycerin-water had no virulicidal effect, as the virus titer was similar to the control values. Instead, NPs dissolved in propanediol-ethanol reduced the infectious titer of the virus by 6.0 log10, which indicates their high virulicidal effect. The absence of an antiviral effect was shown when NPs were added to infected cells. A decrease in the virus titer by 4.5‒5.0 log10 was recorded uponitsinteracting with irradiated NPs for 1‒30 min. The effect persisted for 3 h after exposure to NPs. Conclusions. The cytotoxic, virulicidal, and antiviral effects of optically active TiO2 nanoparticles were determined in optimal conditions. Regardless of the solvent, NPs had low toxicity at a concentration of 0.1 mg/mL. The TiO2 NPs dissolved in glycerin-water had no virulicidal effect; but dissolved in propanediol-ethanol reduced the infectious titer of the virus by 6.0 log10, which indicates its high virulicidal effect. NPs in a propanediol-ethanol solution, irradiated with UV for 1‒30 min, completely inhibited adenovirus reproduction. NPs in a glycine-water solution reduced the virus titer by 0.5 log10. The control with NPs without irradiation slightly reduced the virus titer (by 0.45 log10). The ability of NPs to completely inactivate adenovirus was maintained for 3 h. It was shown for the first time that the non-enveloped HAdV5 virus could be efficiently inactivated by UV-induced TiO2 photocatalysis.

The Aqueous Leaf Extract of the Medicinal Herb Costus speciosus Suppresses Influenza A H1N1 Viral Activity under In Vitro and In Vivo Conditions.

This study investigated the antiviral activity of aqueous leaf extract of Costus speciosus (TB100) against influenza A. Pretreatment of TB100 in RAW264.7 cells enhanced antiviral activity in an assay using the green fluorescence-expressing influenza A/Puerto Rico/8/1934 (H1N1) virus. The fifty percent effective concentration (EC50) and fifty percent cytotoxic concentration (CC50) were determined to be 15.19 ± 0.61 and 117.12 ± 18.31 µg/mL, respectively, for RAW264.7 cells. Based on fluorescent microscopy, green fluorescence protein (GFP) expression and viral copy number reduction confirmed that TB100 inhibited viral replication in murine RAW264.7 and human A549 and HEp2 cells. In vitro pretreatment with TB100 induced the phosphorylation of transcriptional activators TBK1, IRF3, STAT1, IKB-α, and p65 associated with interferon pathways, indicating the activation of antiviral defenses. The safety and protective efficacy of TB100 were assessed in BALB/c mice as an oral treatment and the results confirmed that it was safe and effective against influenza A/Puerto Rico/8/1934 (H1N1), A/Philippines/2/2008 (H3N2), and A/Chicken/Korea/116/2004 (H9N2). High-performance liquid chromatography of aqueous extracts led to the identification of cinnamic, caffeic, and chlorogenic acids as potential chemicals for antiviral responses. Further confirmatory studies using these acids revealed that each of them confers significant antiviral effects against influenza when used as pretreatment and enhances the antiviral response in a time-dependent manner. These findings suggest that TB100 has the potential to be developed into an antiviral agent that is effective against seasonal influenza.

Synergistic effect of Ru(II)-based type II photodynamic therapy with cefotaxime on clinical isolates of ESBL-producing Klebsiella pneumoniae

Multidrug-resistant bacteria, such as ESBL producing-Klebsiella pneumoniae, have increased substantially, encouraging the development of complementary therapies such as photodynamic inactivation (PDI). PDI uses photosensitizer (PS) compounds that kill bacteria using light to produce reactive oxygen species. We test Ru-based PS to inhibit K. pneumoniae and advance in the characterization of the mode of action. The PDI activity of PSRu-L2, and PSRu-L3, was determined by serial micro dilutions exposing K. pneumoniae to 0.612 J/cm 2 of light dose. PS interaction with cefotaxime was determined on a collection of 118 clinical isolates of K. pneumoniae. To characterize the mode of action of PDI, the bacterial response to oxidative stress was measured by RT-qPCR. Also, the cytotoxicity on mammalian cells was assessed by trypan blue exclusion. Over clinical isolates, the compounds are bactericidal, at doses of 8 µg/mL PSRu-L2 and 4 µg/mL PSRu-L3, inhibit bacterial growth by 3 log10 (>99.9%) with a lethality of 30 min. A remarkable synergistic effect of the PSRu-L2 and PSRu-L3 compounds with cefotaxime increased the bactericidal effect in a subpopulation of 66 ESBL-clinical isolates to > 6 log10 with an FIC-value of 0.16 and 0.17, respectively. The bacterial transcription response suggests that the mode of action occurs through Type II oxidative stress. The upregulation of the extracytoplasmic virulence factors mrkD, magA, and rmpA accompanied this response. Also, the compounds show little or no toxicity in vitro on HEp-2 and HEK293T cells. Through the type II effect, PSs compounds are bactericidal, synergistic on K. pneumoniae, and have low cytotoxicity in mammals.

A novel magnetic bead-based immunoprecipitation method for anti-dense fine speckled 70 antibodies

The indirect immunofluorescence assay (IIFA) on HEp-2 cells has been widely used for screening anti-nuclear antibodies (ANA) that are associated with systemic autoimmune rheumatic diseases (SARD). Sera containing ANA display multiple distinct fluorescence patterns on HEp-2 cells. Among them, a dense fine speckled (DFS) pattern caused by anti-DFS70 antibodies has been reported to have higher prevalence in healthy individuals than in patients with SARD. This DFS pattern is often difficult to distinguish amongst other SARD-associated ANA patterns, in particular a mixed homogeneous and speckled pattern. Furthermore, a strong DFS pattern can mask other SARD-associated patterns. Hence, we developed a novel immunoprecipitation method using magnetic beads to remove anti-DFS70 antibodies in serum prior to running IIFA. We also aimed to confirm the presence of anti-DFS70 and to uncover any SARD-associated ANA patterns masked by a strong DFS pattern. The sera used in our study were from 70 individuals who had routine ANA screen, of which 35 sera had an isolated DFS pattern with monospecific anti-DFS70 antibodies confirmed by a complementary assay, and 35 were control sera without a DFS pattern. An immunoprecipitation method using magnetic beads coated with recombinant human full length DFS70 protein was developed. The performance of this new method was evaluated in comparison to an immunoadsorption method using the same DFS70 protein. Our newly developed immunoprecipitation method demonstrated excellent sensitivity (91.4%) and specificity (100%) in confirming the DFS pattern associated with anti-DFS70 in sera. Additionally, our method was able to remove anti-DFS70 and uncover SARD-associated ANA patterns masked by a strong DFS pattern. It also showed a clearer background on IIFA than that of the immunoadsorption method. The novel magnetic bead-based immunoprecipitation method demonstrated satisfactory performance in removing anti-DFS70 without interfering with the detection of other antibodies. It can be easily integrated with IIFA to confirm anti-DFS70 associated DFS pattern. Additionally, it can simultaneously unmask other ANA patterns, which cannot be achieved by a conventional protocol that requires a complementary anti-DFS70 specific assay to be performed. Therefore, the novel method provides a more effective and accurate solution for ANA screening.

Automated Classification of Cell Level of HEp-2 Microscopic Images Using Deep Convolutional Neural Networks-Based Diameter Distance Features

Abstract: To identify autoimmune diseases in humans, analysis of HEp-2 staining patterns at cell level is the gold standard for clinical practice research communities. An automated procedure is a complicated task due to variations in cell densities, sizes, shapes and patterns, overfitting of features, large-scale data volume, stained cells and poor quality of images. Several machine learning methods that analyse and classify HEp-2 cell microscope images currently exist. However, accuracy is still not at the level required for medical applications and computer aided diagnosis due to those challenges. The purpose of this work to automate classification procedure of HEp-2 stained cells from microscopic images and improve the accuracy of computer aided diagnosis. This work proposes Deep Convolutional Neural Networks (DCNNs) technique to classify HEp-2 cell patterns at cell level into six classes based on employing the level-set method via edge detection technique to segment HEp-2 cell shape. The DCNNs are designed to identify cell-shape and fundamental distance features related with HEp-2 cell types. This paper is investigated the effectiveness of our proposed method over benchmarked dataset. The result shows that the proposed method is highly superior comparing with other methods in benchmarked dataset and state-of-the-art methods. The result demonstrates that the proposed method has an excellent adaptability across variations in cell densities, sizes, shapes and patterns, overfitting features, large-scale data volume, and stained cells under different lab environments. The accurate classification of HEp-2 staining pattern at cell level helps increasing the accuracy of computer aided diagnosis for diagnosis process in the future.

MiR-125 family improves the radiosensitivity of head and neck squamous cell carcinoma

BackgroundMiRNAs can affect the radiosensitization of head and neck squamous cell carcinoma (HNSCC). We aimed to analyze the function of miR-125 family members in HNSCC using The Cancer Genome Atlas (TCGA) and determine their effect on radiation in laryngeal squamous cell cancer (LSCC).MethodsFirst, we systematically analyzed the role of the miR-125 family in HNSCC using the TCGA database and found that miR-125a-5p is associated with radiotherapy. We then performed comprehensive enrichment analysis of miR-125a-5p and predicted target genes. Then, we performed transfection, cell proliferation assays, reverse transcription polymerase chain reaction, apoptosis assays, micronucleus tests, and western blotting on hep-2 cells selected with puromycin.ResultsMiR-125 family members exhibited significantly different expression in HNSCC. They were significantly associated with tumor–node–metastasis staging, clinical stages, and histological grades. Radiation therapy had a statistically effect on miR-125 family members, except miR-125a-3p. Moreover, miR-125a-5p was related to overall survival in LSCC. Thus, we predicted 110 target genes and seven hub genes of miR-125a-5p. The proliferation rate of cells transfected with lentivirus vector expressing miR-125a-5p was significantly reduced compared to the other groups. The radiation effect was enhanced in cells transfected with miR-125a-5p. The ratio of apoptotic cells transfected and exposed to X-rays (10 Gy) was distinctly higher than that of the Ad-control group. Western blotting analysis revealed that miR-125a-5p upregulated the apoptotic regulators P53 and rH2AX. Thus, miR-125a-5p may increase radiosensitivity in LSCC via upregulation of pro-apoptotic genes.ConclusionsMiR-125 family members could be prognostic biomarkers of HNSCC and improve HNSCC sensitivity to radiotherapy by activating P53. Upregulating miR-125a-5p via lentivirus vectors may be a novel strategy to strengthen the effect of radiotherapy on LSCC.

The Efficiency of p27 Cleavage during In Vitro Respiratory Syncytial Virus (RSV) Infection Is Cell Line and RSV Subtype Dependent.

Respiratory syncytial virus (RSV) fusion protein (F) is highly conserved between subtypes A and B (RSV/A and RSV/B). To become fully active, F precursor undergoes enzymatic cleavage to yield F1 and F2 subunits and releases a 27-amino-acid peptide (p27). Virus-cell fusion occurs when RSV F undergoes a conformational change from pre-F to post-F. Previous data show that p27 is detected on RSV F, but questions remain regarding if and how p27 affects the conformation of mature RSV F. Monoclonal antibodies against p27, site Ø (pre-F specific), and site II were used to monitor RSV F conformation by enzyme-linked immunosorbent assay (ELISA) and imaging flow cytometry. Pre-F to post-F conformational change was induced by a temperature stress test. We found that p27 cleavage efficiency was lower on sucrose-purified RSV/A (spRSV/A) than on spRSV/B. In addition, cleavage of RSV F was cell line dependent: HEp-2 cells had higher retention of p27 than did A549 cells when infected with RSV. Higher levels of p27 were also found on RSV/A-infected cells than on RSV/B-infected cells. We observed that RSV/A F with higher p27 levels could better sustain the pre-F conformation during the temperature stress challenge in both spRSV- and RSV-infected cell lines. Our findings suggest that despite F sequence similarity, p27 of RSV subtypes was cleaved with different efficiencies, which were also dependent on the cell lines used for infection. Importantly, the presence of p27 was associated with greater stability of the pre-F conformation, supporting the possibility that RSV has more than one mechanism for fusion to the host cell. IMPORTANCE RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F undergoes proteolytic cleavages releasing a 27-amino-acid peptide (p27) to become fully functional. The role of p27 in viral entry and the function of the partially cleaved F containing p27 has been overlooked. p27 is thought to destabilize the F trimers, and thus, there is need for a fully cleaved F. In this study, we detected p27 on purified RSV virions and on the surface of virus-infected HEp-2 and A549 cells for circulating RSV strains of both subtypes. Higher levels of partially cleaved F containing p27 better sustained the pre-F conformation during the temperature stress challenge. Our findings highlight that the cleavage efficiency of p27 is different between RSV subtypes and among cell lines and that the presence of p27 contributes to the stability of the pre-F conformation.