Intracellular Assay Research Articles

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 μM; LRL-TP-94: 2.9 μM; and LRL-TP-101: 35.3 μM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 μM in extracellular promastigote assay and 34.0, 53.7, and 11.4 μM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Azoxystrobin increases the infection of spring viraemia of carp virus in fish

Azoxystrobin (AZ) has entered aquatic ecosystems and produced serious damages to fish associated with potentially increasing the susceptibility to pathogens. This study characterized the defense abilities of fish by exposed to AZ on challenging with the infection of spring viraemia of carp virus (SVCV). The results showed that SVCV replication increased significantly in EPC cells and zebrafish that were exposed to up to 50 μg/L of AZ at 3, 5, 7, and 14 d. Intracellular biochemical assays indicated that AZ at 5 and 50 μg/L inhibited the activation of Nrf2-ARE pathway including a decrease in Nrf2 expression, Nrf2 phosphorylation, HO-1 content, and three antioxidant activities. While no significant difference in ERK1/2 and JNK MAPKs in zebrafish was observed, P38 phosphorylation was significantly decreased at 7 and 14 d, and the changes in MAPKs were more evident in EPC cells previously exposed to AZ at 7 d. These results revealed that AZ initially induced low phosphorylation of MAPKs, triggering the attenuation of Nrf2 phosphorylation to weaken Nrf2 translocation into the nucleus in a longer exposure period (more than 5 d). The data in the cells and fish also showed that antioxidant activities were decreased to some extent at 5–7 d for the cells and 7–14 d for the fish. Furthermore, interferon-related factors were decreased in AZ-exposed zebrafish, explaining the reason that fish can't resist the virus infection. Overall, the present study provided a new adverse threat of AZ by amplifying the viral outbreak to endanger ecological safety in aquatic environment.

Design, Synthesis and Activity Evaluation of New Phthalazinone PARP Inhibitors.

Poly(ADP-ribose)polymerase (PARP) is a significant therapeutic target for the treatment of numerous human diseases. Olaparib has been approved as a PARP inhibitor. In this paper, a series of new compounds were designed and synthesized with Olaparib as the lead compound. In order to evaluate the inhibitory activities against PARP1 of the synthesized compounds, in vitro PARP1 inhibition assay and intracellular PARylation assay were conducted. The results showed that the inhibitory activities of the derivatives were related to the type of substituent and the length of alkyl chain connecting the aromatic ring. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)-based assay also proved that these compounds demonstrating strong inhibition to PARP1 also have high anti-proliferative activities against BRCA2-deficient cell line (Capan-1). Analysis of the entire results suggest that compound 23 with desirable inhibitory efficiency may hold promise for further in vivo exploration of PARP inhibition.

Specific recognition, intracellular assay and detoxification of fluorescent curcumin derivative for copper ions

Recognition and excretion of metal ions play an important role in the diagnosis and treatment of various diseases and poisoning. Although copper (Cu) is a cofactor of many key enzymes in the human body, its accumulation caused by genetic ATP7B mutation or environmental pollution can lead to hepatotoxicity, renal failure, Wilson’s disease, inflammation, and even Parkinson’s disease (PD) and Alzheimer’s disease (AD). Therefore, in this work, a difluoroboron curcumin derivative (DF-Cur) was used for the specific recognition of copper ions (Cu2+). DF-Cur could be further used to as a rapid diagnostic agent for the copper detection in cells and zebrafish at the nanomolar level. DF-Cur could significantly reduce the toxic damage caused by high Cu2+ dose. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis indicated that DF-Cur could promote the excretion of copper ions in the urine and bile and reduce the accumulation of copper ions in vivo. In addition, DF-Cur could selectively detect cholesterol in the blood and adipose tissue in vivo by fluorescent staining. These results demonstrated that this molecule might represent a new and promising diagnostic and therapeutic agent to combat diseases related to copper ions accumulation.

The Combination of Ruxolitinib (RUX) with Decitabine (DAC) in Patients (Pts) with Post-Myeloproliferative Neoplasm Acute Myeloid Leukemia (post-MPN AML): Interim Report of a Phase I/II Trial

The Combination of Ruxolitinib (RUX) with Decitabine (DAC) in Patients (Pts) with Post-Myeloproliferative Neoplasm Acute Myeloid Leukemia (post-MPN AML): Interim Report of a Phase I/II Trial

Invariant NKT Are a More Effective and Versatile Platform Than T Cells for CAR Immunotherapy of CD1d-Expressing B Lineage Malignancies: Cellular and Molecular Mechanisms

Anti-CD19 chimeric antigen receptor T cell (CAR19T) immunotherapy has shown promising clinical potential in relapsed/refractory mature B cell malignancies. However, only about half of patients benefit, highlighting the need for more effective CAR-based strategies.iNKT cells are rare but powerful immunoregulatory and cytotoxic T lymphocytes, playing a pivotal anti-tumor role. iNKT cells are restricted by CD1d, a non-polymorphic, glycolipid-presenting HLA I-like molecule. We previously showed that CD1d, as well as on normal B cells, is also expressed on malignant CD19+ B cells in mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL) cells, while in up to 50% of patients with chronic lymphocytic leukemia (CLL) CD1d expression is very low or negative.We tested the hypotheses that a) bi-specific CAR iNKT 19 cells, targeting simultaneously CD19 and CD1d via the CD19-specific CAR and their natural invariant TCR respectively, would be more effective than CART19 cells against CD19+CD1d+ B cell malignancies and b) transcriptional enhancement of CD1d expression would increase the CAR19iNKT cytotoxic effect.We optimized a novel protocol for manufacturing CAR19iNKT cells. Their in vitro reactivity was assessed in cytotoxicity (flow cytometry), cytokine and cytotoxic granule intracellular staining and cytokine release assays (flow cytometry and Luminex technology) as well as real-time Incucyte proliferation assays. In vivo reactivity was assessed in systemic NSG xenograft assays with growth of the C1RCD1d B cells monitored by bioluminescence and magnetic resonance imaging. Transcription factor binding and histone mark enrichment at the promoter of CD1d was assessed by ChiP- and ChIP-re-ChIP-qPCR assays.We manufactured clinical scale 2nd and 3rd generation CAR19iNKT cells using a highly optimized protocol (>75% transduction efficiency, n=8). In vitro validation, using singly- or dual-positive CD1d and CD19 targets, demonstrated that CAR19iNKT cells are CD19-specific, retain their natural CD1d-restricted reactivity and exert additive dual-specific cytotoxicity against CD1d+CD19+ targets. Compared to same donor CAR19T, CAR19iNKT cells display a significantly higher (P<0.0001) expandability and proliferative potential and are equally or more effective in killing CD19+CD1d+ lymphoid cell lines (C1RCD1d and Farage) and consistently more effective against primary MCL, MZL and CLL cells.Since CD1d expression is low/negative in most cases of CLL, we sought to restore CD1d expression on leukemic cells for the purpose of CAR19iNKT cell immunotherapy. We found that surface CD1d expression can be restored by clinically relevant concentrations of ATRA in a time-dependent manner. Mechanistic dissection of CD1d transcriptional regulation in the CD1d-negative B lineage cell line U266 demonstrated the co-existence of bivalent chromatin marks at the promoter of CD1d commensurate with enriched binding and association of EZH2 with RARα. Notably, an EZH2 inhibitor exerted a synergistic effect along with ATRA in restoring CD1d transcription and surface expression and, in line with our hypothesis, 2nd and 3rd generation CAR19iNKT but not CAR19T cells displayed higher cytotoxic activity against ATRA-treated CLL cells.Finally, in an NSG xenograft model of lymphoma, while survival of T and iNKT cell-treated animals was the same as that of untreated animals (n=7, P>0.05), both CAR19T and CAR19iNKT cell-treated animals (n=19) had significantly and comparably improved overall survival (OS; P<0.0001). However, compared to CAR19T, CAR19iNKT cell immunotherapy led to a significantly improved OS (P=0.01) and also better disease control including of CNS lymphoma, with earlier, more profound and sustained complete responses resulting in a significantly improved tumor-free survival (P=0.03).We conclude thatCARiNKT19 are more effective than CART19 cells against CD19+CD1d+ B cell malignancies in vitro and in vivo and ATRA-mediated restoration of CD1d expression enhances the anti-lymphoma effect of CAR19iNKT immunotherapy in vitro . These properties together with the previously demonstrated ability of donor iNKT cells to protect from aGVHD raise the prospect of developing effective ‘off-the-shelf’ CAR19iNKT immunotherapy of lymphomas, which can mitigate against CD19-negative immune escape upon CAR treatment and can be further enhanced by transcriptional and epigenetic treatment. DisclosuresPule:UCL: Patents & Royalties: UCL Business; Autolus Ltd: Employment, Equity Ownership, Research Funding. Maher:Leucid Bio: Other: Chief scientific officer. Karadimitris:GlaxoSmithKline: Research Funding.

Intracellular fluorescence competition assay for inhibitor engagement of histone deacetylase

An intracellular fluorescence competition assay was developed to assess the capability of inhibitor candidates to engage histone deacetylase (HDAC) inside living cells and thus diminish cell uptake and staining by the HDAC-targeted fluorescent probe APS. Fluorescence cell microscopy and flow cytometry showed that pre-incubation of living cells with candidate inhibitors led to diminished cell uptake of the fluorescent probe. The assay was effective because the fluorescent probe (APS) possessed the required performance properties, including bright fluorescence, ready membrane diffusion, selective intracellular HDAC affinity, and negligible acute cytotoxicity. The concept of an intracellular fluorescence competition assay is generalizable and has broad applicability since it obviates the requirement to use the isolated biomacromolecule target for screening of molecular candidates with target affinity.

An intracellular assay for activity screening and characterization of glutathione-dependent oxidoreductases

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Investigation of enzymatic activity traditionally involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. To accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.

Application of Alanine Scanning to Determination of Amino Acids Essential for Peptide Adsorption at the Solid/Solution Interface and Binding to the Receptor: Surface-Enhanced Raman/Infrared Spectroscopy versus Bioactivity Assays.

The article describes the application of the alanine-scanning technique used in combination with Raman, surface-enhanced Raman, attenuated total reflection Fourier transform infrared, and surface-enhanced infrared absorption (SEIRA) spectroscopies, which allowed defining the role of individual amino acid residues in the C-terminal 6–14 fragment of the bombesin chain (BN6–14) on the path of its adsorption on the surface of Ag (AgNPs) and Au nanoparticles (AuNPs). A reliable analysis of the SEIRA spectra of these peptides was possible, thanks to a curve fitting of these spectra. By combining alanine-scanning with biological activity studies using cell lines overexpressing bombesin receptors and the intracellular inositol monophosphate assay, it was possible to determine which peptide side chains play a significant role in binding a peptide to membrane-bound G protein-coupled receptors (GPCRs). Based on the analysis of spectral profiles and bioactivity results, conclusions for the specific peptide–metal and peptide–GPCR interactions were drawn and compared.

Ginkgo Biloba Extract EGB761 Alleviates Warfarin-induced Aortic Valve Calcification Through the BMP2/Smad1/5/Runx2 Signaling Pathway.

Calcific aortic valve disease is a common heart disease that contributes to increased cardiovascular morbidity and mortality. There is a lack of effective pharmaceutical therapy because its mechanisms are not yet fully known. Ginkgo biloba extract (EGB761) is reported to alleviate vascular calcification. However, whether EGB761 protects against aortic valve calcification, a disease whose pathogenesis shares many similarities with vascular calcification, and potential molecular mechanisms remain unknown. In this study, porcine aortic valve interstitial cell (pAVIC) calcification was induced by warfarin with or without the presence of EGB761. Immunostaining was performed to establish and characterize the pAVIC phenotype. Calcium deposition and calcium content were examined by Alizarin Red S staining and an intracellular calcium content assay. Alkaline phosphatase activity was detected by the p-nitrophenyl phosphate method. The expression levels of bone morphogenetic protein-2 (BMP2), Runt-related transcription factor 2 (Runx2), homeobox protein MSX-2, and phosphorylated (p)-Smad1/5 were detected by reverse transcription-quantitative polymerase chain reaction (PCR) and Western blot analysis. Consistent with these in vitro data, we also confirmed the suppression of in vivo calcification by EGB761 in the warfarin-induced C57/Bl6 mice. The results indicated that both pAVICs and aortic valves tissue of mice stimulated with warfarin showed increased calcium deposition and expression of osteogenic markers (alkaline phosphatase, BMP2, homeobox protein MSX-2, and Runx2) and promoted p-Smad1/5 translocation from the cytoplasm to the nucleus. The addition of EGB761 significantly inhibited p-Smad1/5 translocation from the cytoplasm to the nucleus, thus suppressing calcification. In conclusion, EGB761 could ameliorate warfarin-induced aortic valve calcification through the inhibition of the BMP2-medicated Smad1/5/Runx2 signaling pathway.

튀니지 식물 11종 추출물의 HepG2 세포에서 산화적 스트레스 및 지질 축적 억제 효과

This study was undertaken to evaluate the antioxidative and lipid accumulation inhibitory effects in HepG2 cell of 11 Tunisian plants extracts. Total phenolics contents (TPC), and total flavonoid contents (TFC) of 11 plants extracts were measured, and antioxidative activities was analyzed using DPPH, ABTS, FRAP, ORAC and TBA assay. Inhibitory effect of oxidative stress was evaluated by cell viability and lipid peroxidation level in H2O2-treated HepG2 cells. Lipid accumulation inhibitory effect was determined by Oil-Red-O staining and intracellular triglyceride assay in HepG2 cell. M. communis L. (156.73 mgGAE/g) and N. glauca Graham (108.81 mgNAE/g) were the highest TPC and TFC, respectively, among 11 plants. M. communis L. were the highest antioxidant activity in DPPH and ABTS. FRAP and ORAC results revealed that antioxidant activity in 10 species were higher than the positive control. Among the 11 species, 5 species with the lowest malondialdehyde level were selected and HPLC analysis revealed that plants contain caffeic acid, quercetin, and rutin. 5 plants treatment inhibited lipid peroxidation level and protected HepG2 cells from oxidative stress. Moreover 5 plants significantly inhibited the lipid accumulation and triglyceride content. These results imply scientific evidence for the development of functional foods using 11 plants from Tunisia which has oxidative stress and lipid accumulation reduction effects.

Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer's Disease (Mouse Model) through Autophagy Induced by Activating Axl.

Alzheimer’s disease (AD) is thought to be caused by amyloid-β (Aβ) accumulation in the central nervous system due to deficient clearance. The aim of the present study was to investigate the effect of ganoderic acid A (GAA) on Aβ clearance in microglia and its anti-AD activity. Aβ degradation in BV2 microglial cells was determined using an intracellular Aβ clearance assay. GAA stimulated autophagosome formation via the Axl receptor tyrosine kinase (Axl)/RAC/CDC42-activated kinase 1 (Pak1) pathway was determined by Western blot analyses, and fluorescence-labeled Aβ42 was localized in lysosomes in confocal laser microscopy images. The in vivo anti-AD activity of GAA was evaluated by object recognition and Morris water maze (MWM) tests in an AD mouse model following intracerebroventricular injection of aggregated Aβ42. The autophagy level in the hippocampus was assayed by immunohistochemical assessment against microtubule-associated proteins 1A/1B light-chain 3B (LC3B). Intracellular Aβ42 levels were significantly reduced by GAA treatment in microglial cells. Additionally, GAA activated autophagy according to increased LC3B-II levels, with this increased autophagy stimulated by upregulating Axl and Pak1 phosphorylation. The effect of eliminating Aβ by GAA through autophagy was reversed by R428, an Axl inhibitor, or IPA-3, a Pak1 inhibitor. Consistent with the cell-based assay, GAA ameliorated cognitive deficiency and reduced Aβ42 levels in an AD mouse model. Furthermore, LC3B expression in the hippocampus was up-regulated by GAA treatment, with these GAA-specific effects abolished by R428. GAA promoted Aβ clearance by enhancing autophagy via the Axl/Pak1 signaling pathway in microglial cells and ameliorated cognitive deficiency in an AD mouse model.

A toolbox for studying respiratory viral infections using air-liquid interface cultures of human airway epithelial cells

Submerged cultures of primary human airway epithelial cells or human airway epithelial cell lines have been a mainstay of airway epithelial biology research for decades due to their robust in vitro proliferative capacity, relatively low maintenance culture conditions, and clinically translatable results to nasal or bronchial brushings. With the development and improvement of air-liquid interface (ALI) cultures of human airway epithelial cells, such cultures have been considered superior to immortalized cell lines and primary cell monolayers, as such cultures effectively recapitulate in vivo epithelial architecture and cell types. Although ALI culture growth protocols are well-established and widely available, many researchers have avoided their use, as ALI cultures not only take longer to grow but also present technical challenges and limitations that make in vitro intracellular and structural assays taxing. Challenges arise relating to their complex structure, requirements for air exposure, the constraints of transwell growth apparatus, and interference in assays caused by mucus secretion. Although few publications briefly describe technical adaptations for some assays, there is still considerable trial and error required for researchers to establish consistent and reliable assay adaptations, often becoming a deterrent for pursuing mechanistic investigation. We have created a user-friendly toolbox detailing comprehensive protocols for numerous techniques and assay adaptations, particularly focusing on respiratory virus infections. By expanding the repertoire of ALI culture-adapted in vitro assays, we hope to facilitate the widespread adoption of this valuable culture system for mechanistic investigations of respiratory viral infections or other epithelial-pathogen models.

Immunogenicity of COVID-19 mRNA Vaccines in Pregnant and Lactating Women

Pregnant women are at increased risk of morbidity and mortality from COVID-19 but have been excluded from the phase 3 COVID-19 vaccine trials. Data on vaccine safety and immunogenicity in these populations are therefore limited. To evaluate the immunogenicity of COVID-19 messenger RNA (mRNA) vaccines in pregnant and lactating women, including against emerging SARS-CoV-2 variants of concern. An exploratory, descriptive, prospective cohort study enrolled 103 women who received a COVID-19 vaccine from December 2020 through March 2021 and 28 women who had confirmed SARS-CoV-2 infection from April 2020 through March 2021 (the last follow-up date was March 26, 2021). This study enrolled 30 pregnant, 16 lactating, and 57 neither pregnant nor lactating women who received either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) COVID-19 vaccines and 22 pregnant and 6 nonpregnant unvaccinated women with SARS-CoV-2 infection. SARS-CoV-2 receptor binding domain binding, neutralizing, and functional nonneutralizing antibody responses from pregnant, lactating, and nonpregnant women were assessed following vaccination. Spike-specific T-cell responses were evaluated using IFN-γ enzyme-linked immunospot and multiparameter intracellular cytokine-staining assays. Humoral and cellular immune responses were determined against the original SARS-CoV-2 USA-WA1/2020 strain as well as against the B.1.1.7 and B.1.351 variants. This study enrolled 103 women aged 18 to 45 years (66% non-Hispanic White) who received a COVID-19 mRNA vaccine. After the second vaccine dose, fever was reported in 4 pregnant women (14%; SD, 6%), 7 lactating women (44%; SD, 12%), and 27 nonpregnant women (52%; SD, 7%). Binding, neutralizing, and functional nonneutralizing antibody responses as well as CD4 and CD8 T-cell responses were present in pregnant, lactating, and nonpregnant women following vaccination. Binding and neutralizing antibodies were also observed in infant cord blood and breast milk. Binding and neutralizing antibody titers against the SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern were reduced, but T-cell responses were preserved against viral variants. In this exploratory analysis of a convenience sample, receipt of a COVID-19 mRNA vaccine was immunogenic in pregnant women, and vaccine-elicited antibodies were transported to infant cord blood and breast milk. Pregnant and nonpregnant women who were vaccinated developed cross-reactive antibody responses and T-cell responses against SARS-CoV-2 variants of concern.

Investigation of the free fatty acid receptor 2 and its role in the treatment of type 2 diabetes mellitus

The free fatty acid receptor 2 (FFAR2) is a deorphanized G-protein coupled receptor (GPCR) that is widely expressed in various tissues in the body. Previous studies have shown that the short-chain fatty acids (SCFAs) acetate, butyrate and propionate activate FFAR2, leading to changes in cellular function. SCFAs are gut microbial metabolites important in shaping and maintaining gut homeostasis, as well as impacting other cell functions. FFAR2 is found in pancreatic islets and has led to investigational studies for the treatment of metabolic diseases. Our study focuses on FFAR2 as a potential treatment for type 2 diabetes mellitus (T2DM). FFAR2 contributes to glucose-stimulated insulin secretion (GSIS) and its potential in therapy has not been fully explored. This study focused on the effects of GPCR agonists targeting the deorphanized free fatty acid receptor 2 via intracellular calcium assays. We investigated the effects of numerous GPCR agonists on FFAR2, with a specific focus on identifying agonists or allosteric modulators. Further analysis and testing are underway to pinpoint specific agonists as potential treatment options for T2DM.

Impact of Toxoplasma gondii infection on TM3 Leydig cells: Alterations in testosterone and cytokines levels

Leydig cells play pivotal roles in eliciting male characteristics by producing testosterone and any damage to these cells can compromise male fertility Toxoplasma gondii (T. gondii) is an intracellular parasite capable to invade any nucleated cell, including cells from male reproductive system. Herein, we evaluated the capacity of RH strain of T. gondii to infect TM3 Leydig cells and the impact of this infection on testosterone and inflammatory mediators production. We first, by performing adherence, infection, and intracellular proliferation assays, we found a significant increase in the number of infected Leydig cells, peaking 48 h after the infection with T. gondii. Supernatants of TM3 infected cells exhibited, in a time-dependent manner, increased levels of testosterone as well as monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ), which is correlated with the robust T. gondii infection. In conclusion, our study provides new insights regarding the harmful effects of T. gondii infection on male reproductive system.

Asymmetric CorA Gating Mechanism as Observed by Molecular Dynamics Simulations.

The CorA family of proteins plays a housekeeping role in the homeostasis of divalent metal ions in many bacteria and archaea as well as in mitochondria of eukaryotes, rendering it an important target to study the mechanisms of divalent transport and regulation across different life domains. Despite numerous studies, the mechanistic details of the channel gating and the transport of the metal ions are still not entirely understood. Here, we use all-atom and coarse-grained molecular dynamics simulations combined with in vitro experiments to investigate the influence of divalent cations on the function of CorA. Simulations reveal pronounced asymmetric movements of monomers that enable the rotation of the α7 helix and the cytoplasmic subdomain with the subsequent formation of new interactions and the opening of the channel. These computational results are functionally validated using site-directed mutagenesis of the intracellular cytoplasmic domain residues and biochemical assays. The obtained results infer a complex network of interactions altering the structure of CorA to allow gating. Furthermore, we attempt to reconcile the existing gating hypotheses for CorA to conclude the mechanism of transport of divalent cations via these proteins.

Synthesis, characterization and multiple targeting with selectivity: Anticancer property of ternary metal phenanthroline-maltol complexes

The cobalt(II), copper(II) and zinc(II) complexes of 1,10-phenanthroline (phen) and maltol (mal) (complexes 1, 2, 3 respectively) were prepared from their respective metal(II) chlorides and were characterized by FT-IR, elemental analysis, UV spectroscopy, molar conductivity, p-nitrosodimethylaniline assay and mass spectrometry. The X-ray structure of a single crystal of the zinc(II) analogue reveals a square pyramidal structure with distinctly shorter apical chloride bond. All complexes were evaluated for their anticancer property on breast cancer cell lines MCF-7 and MDA-MB-231, and normal cell line MCF-10A, using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and morphological studies. Complex 2 was most potent for 24, 48 and 72 h treatment of cancer cells but it was not selective towards cancer over normal cells. The mechanistic studies of the cobalt(II) complex 1 involved apoptosis assay, cell cycle analysis, dichloro-dihydro-fluorescein diacetate assay, intracellular reactive oxygen species assay and proteasome inhibition assay. Complex 1 induced low apoptosis, generated low level of ROS and did not inhibit proteasome in normal cells. The study of the DNA binding and nucleolytic properties of complexes 1–3 in the absence or presence of H2O2 or sodium ascorbate revealed that only complex 1 was not nucleolytic.

Phenotypic characterization of auxotrophic mutant of nontyphoidal Salmonella and determination of its cytotoxicity, tumor inhibiting cytokine gene expression in cell line models.

An auxotrophic mutant of nontyphoidal Salmonella (NTS) strain (Salmonella Oslo) was phenotypically characterized in this study. The characterization was based on phenotype, morphology, motility, biofilm forming ability, growth kinetics, etc. The phenotypic results from the above experiments determined that the mutant showed variation in phenotypic characters from that of wild-type strain. Subsequently, mutant and wild-type NTS were subjected to epithelial cell invasion and intracellular replication assays. The real-time PCR analysis was also performed to analyse expression of tumor inhibiting cytokine genes and virulence genes post-bacterial infection in cell lines. The mutant showed highest invasion potential than wild-type NTS whereas the replication of mutant was slower in both the cell lines. Similar to the wild-type strain, the mutant also retained the cytotoxic potential when analysed in vitro. Furthermore, the expression of proinflammatory cytokine genes such as TNF-α and IL-1β was upsurged with the downregulation of anti-inflammatory cytokine genes like TGF-β, IL-6 and IL-10 post-infection of the mutant strain in cell lines. In addition, virulence genes of Salmonella pathogenicity island one and two of mutant were downregulated in vitro except invA in HeLa cell line. Therefore, the auxotrophic mutant showed positive attributes of a potential antitumor agent in terms of expressing tumor inhibiting cytokine genes when assessed in vitro. Though the study did not check the tumor inhibitory effect of NTS strain directly, findings of the study emphasizes on the development of a novel strain of NTS with less virulence and more immunogenic traits to inhibit tumor cells.

Expression of protease activated receptor-2 is reduced in renal cell carcinoma biopsies and cell lines.

Expression of the protease sensing receptor, protease activated receptor-2 (PAR2), is elevated in a variety of cancers and has been promoted as a potential therapeutic target. With the development of potent antagonists for this receptor, we hypothesised that they could be used to treat renal cell carcinoma (RCC). The expression of PAR2 was, therefore, examined in human RCC tissues and selected RCC cell lines. Histologically confirmed cases of RCC, together with paired non-involved kidney tissue, were used to produce a tissue microarray (TMA) and to extract total tissue RNA. Immunohistochemistry and qPCR were then used to assess PAR2 expression. In culture, RCC cell lines versus primary human kidney tubular epithelial cells (HTEC) were used to assess PAR2 expression by qPCR, immunocytochemistry and an intracellular calcium mobilization assay. The TMA revealed an 85% decrease in PAR2 expression in tumour tissue compared with normal kidney tissue. Likewise, qPCR showed a striking reduction in PAR2 mRNA in RCC compared with normal kidney. All RCC cell lines showed lower levels of PAR2 expression than HTEC. In conclusion, we found that PAR2 was reduced in RCC compared with normal kidney and is unlikely to be a target of interest in the treatment of this type of cancer.