R-Loops Promote Antisense Transcription across the Mammalian Genome.

Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.

Heterochiral modifications enhance robustness and function of DNA in living human cells.

Oligonucleotide therapeutics are becoming increasingly important as more are approved by the FDA, both for treatment and vaccination. Similarly, dynamic DNA nanotechnology is a promising technique that can be used to sense exogenous input molecules or endogenous biomarkers and integrate the results of multiple sensing reactions in situ via a programmed cascade of reactions. The combination of these two technologies could be highly impactful in biomedicine by enabling smart oligonucleotide therapeutics that can autonomously sense and respond to a disease state. A particular challenge, however, is the limited lifetime of standard nucleic acid components in living cells and organisms due to degradation by endogenous nucleases. In this work, we address this challenge by incorporating mirror-image, ʟ-DNA nucleotides to produce heterochiral "gapmers". We use dynamic DNA nanotechnology to show that these modifications keep the oligonucleotide intact in living human cells for longer than an unmodified strand. To this end, we used a sequential transfection protocol for delivering multiple nucleic acids into living human cells while providing enhanced confidence that subsequent interactions are actually occurring within the cells. Taken together, this work advances the state of the art of ʟ-nucleic acid protection of oligonucleotides and DNA circuitry for applications in vivo.

Increased endocytosis rate and enhanced lysosomal pathway of silica-coated superparamagnetic nanoparticles into M-HeLa cells compared with cultured primary motor neurons.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30 s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1 h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

Photosensitizing effects and physicochemical properties of chlorophyll a derivatives with hydrophilic oligoethylene glycol fragments at the macrocycle periphery.

In this work, screening studies of the cytotoxic effect of chlorins with fragments of di-, tri-, and pentaethylene glycol at the macrocycle periphery in relation to HeLa, A549, and HT29 cells were performed. It is shown that, despite different hydrophobicity, all the compounds studied have a comparable photodynamic effect. The conjugate of chlorin e6 with pentaethylene glycol, which has the lowest tendency to association among the studied compounds with tropism for low density lipoproteins and the best characteristics of the formation of molecular complexes with Tween 80, has a significant difference in dark and photoinduced toxicity (ratio IC50(dark)/IC50(photo) approximately 2 orders of magnitude for all cell lines), which allows to hope for a sufficiently large "therapeutic window". A study of the interaction of this compound with HeLa cells shows that the substance penetrates the cell and, after red light irradiation induces ROS appearance inside the cell, associated, apparently, with the photogeneration of singlet oxygen. These data indicate that photoinduced toxic effects are caused by damage to intracellular structures as a result of oxidative stress. Programmed type of cell death characterized with caspase-3 induction is prevailing. So, the conjugate of chlorin e6 with pentaethylene glycol is a promising antitumor PS that can be successfully solubilized with Tween 80, which makes it suitable for further in vivo studies.

Diminazene aceturate-induced cytotoxicity is associated with the deregulation of cell cycle signaling and downregulation of oncogenes Furin, c-MYC, and FOXM1 in human cervical carcinoma Hela cells.

Diminazene aceturate (DIZE) is an FDA-listed small molecule known for the treatment of African sleeping sickness. In vivo studies showed that DIZE may be beneficial for a range of human ailments. However, there is very limited information on the effects of DIZE on human cancer cells. The current study aimed to investigate the cytotoxic responses of DIZE, using the human carcinoma Hela cell line. WST-1 cell proliferation assay showed that DIZE inhibited the viability of Hela cells in a dose-dependent manner and the observed response was associated with the downregulation of Ki67 and PCNA cell proliferation markers. DIZE-treated cells stained with acridine orange-ethidium and JC-10 dye revealed cell death and loss of mitochondrial membrane potential (Ψm), compared with DMSO (vehicle) control, respectively. Cellular immunofluorescence staining of DIZE-treated cells showed upregulation of caspase 3 activities. DIZE-treated cells showed downregulation of mRNA for G1/S genes CCNA2 and CDC25A, S-phase genes MCM3 and PLK4, and G2/S phase transition/mitosis genes Aurka and PLK1. These effects were associated with decreased mRNA expression of Furin, c-Myc, and FOXM1 oncogenes. These results suggested that DIZE may be considered for its effects on other cancer types. To the best of our knowledge, this is the first study to evaluate the effect of DIZE on human cervical cancer cells.

Vaginal prevention of Candida albicans: synergistic effect of lactobacilli and mannan oligosaccharides (MOS).

Vulvovaginal candidiasis (VVC) affects approximately 30-50% of women at least once during their lifetime, causing uncomfortable symptoms and limitations in their daily quality of life. Antifungal therapy is not very effective, does not prevent recurrencies and usually causes side effects. Therefore, alternative therapies are urgently needed. The goal of this work was to investigate the potential benefits of using mannan oligosaccharides (MOS) extracts together with a Lactobacillus sp. pool, composed by the most significant species present in the vaginal environment, to prevent infections by Candida albicans. Microbial growth of isolated strains of the main vaginal lactobacilli and Candida strains was assessed in the presence of MOS, to screen their impact upon growth. A pool of the lactobacilli was then tested against C. albicans in competition and prophylaxis studies; bacterial and yeast cell numbers were quantified in specific time points, and the above-mentioned studies were assessed in simulated vaginal fluid (SVF). Finally, adhesion to vaginal epithelial cells (HeLa) was also evaluated, once again resorting to simultaneous exposure (competition) or prophylaxis assays, aiming to measure the effect of MOS presence in pathogen adherence. Results demonstrated that MOS extracts have potential to prevent vaginal candidiasis in synergy with vaginal lactobacilli, with improved results than those obtained when using lactobacilli alone. KEY POINTS: Potential benefits of MOS extracts with vaginal lactobacilli to prevent C. albicans infections. MOS impacts on growth of vaginal lactobacilli pool and C. albicans in SVF. MOS extracts in synergy with L. crispatus inhibit C. albicans adhesion in HeLa cells.

Curdepsidone A Induces Intrinsic Apoptosis and Inhibits Protective Autophagy via the ROS/PI3K/AKT Signaling Pathway in HeLa Cells.

Among female oncology patients, cervical cancer stands as the fourth most prevalent malignancy, exerting significant impacts on their health. Over 600,000 women received the diagnosis of cervical cancer in 2020, and the illness claimed over 300,000 lives globally. Curdepsidone A, a derivative of depsidone, was isolated from the secondary metabolites of Curvularia sp. IFB-Z10. In this study, we revised the molecular structure of curdepsidone A and investigated the fundamental mechanism of the anti-tumor activity of curdepsidone A in HeLa cells for the first time. The results demonstrated that curdepsidone A caused G0/G1 phase arrest, triggered apoptosis via a mitochondrial apoptotic pathway, blocked the autophagic flux, suppressed the PI3K/AKT pathway, and increased the accumulation of reactive oxygen species (ROS) in HeLa cells. Furthermore, the PI3K inhibitor (LY294002) promoted apoptosis induced by curdepsidone A, while the PI3K agonist (IGF-1) eliminated such an effect. ROS scavenger (NAC) reduced curdepsidone A-induced cell apoptosis and the suppression of autophagy and the PI3K/AKT pathway. In conclusion, our results revealed that curdepsidone A hindered cell growth by causing cell cycle arrest, and promoted cell apoptosis by inhibiting autophagy and the ROS-mediated PI3K/AKT pathway. This study provides a molecular basis for the development of curdepsidone A as a new chemotherapy drug for cervical cancer.

3-Ethoxysalicylaldimine-based symmetrical azine-linked luminogen exhibiting ESIPT and bright orange colour AIE behaviour with live cell bioimaging application.

Herein, we describe the design and development of a new cell-permeable aggregation-induced emission (AIE) active 3-ethoxysalicylaldimine-based symmetrical azine molecule HDBE. The synthesized compound underwent comprehensive investigation of different spectroscopic methods, like NMR, mass and single crystal X-ray diffraction analysis. The fluorophore HDBE exhibited the bright orange colour AIE behaviour in THF-H2O mixture. The drastic enhancement of emission was achieved upon adding the water to the THF solution of HDBE, with a concentration of 90%. Along with the dynamic light scattering (DLS) and quantum yield measurements, the formation of aggregates was also verified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis. Further, HDBE demonstrated excited state intramolecular proton transfer (ESIPT) characteristics in different polarity of solvents, which was corroborated by absorption, emission and lifetime spectroscopical investigations. The detailed scrutiny of X-ray structure of HDBE displayed the two strong intramolecular hydrogen bonding interactions, while solid-state fluorescent spectra showed dual emission that corresponds to enol and keto form confirming the ESIPT feature. Further, the synthesized AIE molecule was non-toxic and cell-permeable, making it easy to label as a biomarker in live HeLa cells via fluorescent bioimaging. These studies offer a quick and easy way to develop both AIE and ESIPT-coupled molecules for live cell bioimaging applications.

Vaginal Lactobacilli Supernatants Protect from Herpes Simplex Virus Type 1 Infection in Cell Culture Models.

A healthy vaginal microbiota hosts Lactobacillus as the most predominant genus. Lactobacilli play a role in human health through the production of diverse antimicrobial substances that can act against human pathogens or modulate the immune system. Previous reports highlighted the ability of vaginal lactobacilli to counteract viruses causing STIs, e.g., HIV-1 and HSV-2. In this report, we analyze the activity of supernatants of vaginal lactobacilli against HSV-1 infection, which is becoming increasingly relevant as a STI. We show that the supernatants of two vaginal Lactobacillus species (i.e., L. crispatus and L. gasseri) were active at neutralizing HSV-1 infection in two different cell lines of human and simian origin. Specifically, we demonstrate that L. crispatus strains are the most effective in antiviral activity, as evidenced by the comparison with a vaginal pathogen taken as reference. The effect was specific and not attributable to the generic toxicity of the supernatants to the cells. Our results pave the way for the development of probiotics to limit the impact of HSV-1 infection on women's health.

Mechanistic insights into the impact of WIN 55, 212-2, a synthetic cannabinoid, on adhesion molecules PECAM-1 and VE-cadherin in HeLa cells: implications on cancer processes.

The endocannabinoid (eCB) system comprises endogenous ligands, cannabinoid receptors (CBRs), and their regulatory proteins; its alteration leads to many diseases including cancer. Thus, becomes a therapeutic target for synthetic cannabinoids aimed to control cancer cell proliferation, migration, adhesion, and invasion. However, little is known about adhesion molecules regulation through CBRs activation. The aim of this study was to evaluate the effects of a CB1/CB2 agonist, WIN-55, 212-2 (WIN), on the regulation of adhesion molecules platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial cadherin (VE-cadherin) in HeLa cells. CBRs expression was evaluated by immunofluorescence staining in HeLa cells and cell viability (thiazolyl blue tetrazolium bromide), cell adhesion (crystal violet), adhesion molecules expression and location (Western blot and immunofluorescence staining assays) were all assessed on cells treated with different WIN concentrations. Receptors CB1, CB2, and G-protein-coupled receptor 55 were expressed in HeLa cells. Additionally, biphasic effects were observed in their metabolic activity and adhesive properties: low WIN concentrations resulted in significant increases whereas, high ones decreased them compared to controls (p < 0.0001), demonstrating that WIN elicits opposite effects depending on the concentration and exposure time. PECAM-1 was detected in HeLa cell's cytoplasm, membrane, and perinuclear region, whereas VE-cadherin had a nuclear distribution. There were no significant differences in PECAM-1 and VE-cadherin expression and location, suggesting that WIN does not modulate these proteins. These findings support the potential use of WIN due to its anticancer properties without dysregulating adhesion molecules. WIN possible contribution to inhibit cancer progression should be further investigated.

Antioxidant and antiproliferative effects of Teucrium polium extract: computational and in vivo study in rats.

The current study aimed to assess the antioxidant and antiproliferative effects of teucrium polium extract: computational and in vivo study in rats. Three groups of animals: Group (i) constitute the control group; Group (ii) HeLa group received an intrafemoral inoculation of HeLa cells and Group (iii) constitue the combination between HeLa + T. polium. The plant was administered by gavage. Our results revealed that HeLa cell injection showed an elevation in aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TB), creatinine, urea, calcium and phosphorus. The pretreatment with the plant extract reduced the level of these parameters. Injection of HeLa cells showed a significant decrease in phosphorus and calcium respectively. However, the pretreatment by T. polium modulated the level of these two minerals. Rats treated with HeLa cells line showed an increase in the level of lipid peroxidation as evaluated by the TBARS substances, at the same time, a significant decreases in SOD, CAT and GPx activities were noted in the HeLa group compared to the control. On the other hand, pretreatment with the plant improved the level of these enzymes. Our results revealed that T.polium has a therapeutic effect on some health problems. HeLa cell line induced a small infiltration in liver and kidney. T. polium reduced the damage in both liver and kidney, but did not reveal any proliferation of tumor cells from trabecular bone tissue. The computational study revealed that T. polium compound bound with high free binding energies and established promising network of molecular interactions with COX-2 and TNF-α macromolecules.

[Effect of total saponins from Panacis Majoris Rhizoma on proliferation, apoptosis and autophagy of human cervical carcinoma HeLa cells].

This paper investigated the effect of total saponins from Rhizoma Panacis Majoris on the proliferation, apoptosis, and autophagy of human cervical carcinoma HeLa cells. The saponin content was detected by ultraviolet-visible spectrophotometry. Cell coun-ting kit-8(CCK-8) assay, 4,6-diamidino-2-phenylindole(DAPI) staining, and flow cytometry were used to detect the effects of total saponins of Panacis Majoris Rhizoma on cell viability, morphology, cell cycle and apoptosis of HeLa cells. Western blot was used to detect the expression of apoptosis-related proteins B cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved caspase-9, and cleaved caspase-3, autophagy-related proteins Beclin-1 and SQSTM1(p62), and the proteins related to the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR) and mitogen-activated protein kinase(MAPK) signaling pathways. It was found that the yield and saponin content of total saponins from Rhizoma Panacis Majoris were 6.3% and 78.3%, respectively. Total saponins from Rhizoma Panacis Majoris could significantly inhibit the proliferation(P<0.001), effect the nuclear morphology, block the G_0/G_1 cycle, and induce cell apoptosis in HeLa cells with a concentration-dependent manner. In addition, total saponins from Rhizoma Panacis Majoris up-regulated the expression of pro-apoptotic proteins Bax, cleaved caspase-9, and cleaved caspase-3, and autophagy-related protein p62(P<0.05), while down-regulated the expression of anti-apoptotic protein Bcl-2 and autophagy-related protein Beclin-1(P<0.01). Total saponins from Rhizoma Panacis Majoris could promote the expression of p-p38/p38, p-Jun N-terminal kinase(JNK)/JNK, p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR proteins in PI3K/Akt/mTOR and MAPK signaling pathways(P<0.05). In contrast, the effect on p-ERK/ERK expression was not obvious. Therefore, total saponins from Rhizoma Panacis Majoris may inhibit autophagy and promote apoptosis of HeLa cells through the activation of the PI3K/Akt/mTOR, c-JNK, and p38 MAPK signaling pathways, which indicates that total saponins from Rhizoma Panacis Majoris may have a potential role in cervical cancer treatment.

Characterization of Vaginal Lactobacilli with Potential Probiotic Properties Isolated from Healthy Women in Northern Iran.

Vaginal lactobacilli protect against bacterial vaginosis and vaginal candidiasis. They may have probiotic properties and help maintain the balance and health of the vaginal ecosystem while the loss of these bacteria predisposes females to urinary and genital infections. The aim of this study was to investigate the probiotic potential of vaginal Lactobacillus among healthy females in northern Iran. The Lactobacillus strains were isolated from vaginal samples and were identified by sequencing of the 16S rRNA fragment. Functional properties such as tolerance to low pH, H2O2 production, adherence ability to Hela cells and antagonistic activity against Candida albicans was examined. A total of 38 vaginal lactobacilli strains from five species, including Lactobacillus crispatus (n = 13), Lactobacillus gasseri (n = 10), Lactobacillus acidophilus (n = 6), Lactobacillus jensenii (n = 5) and Lactobacillus johnsonii (n = 4), were identified. All of the species showed significant tolerance to low pH over 24 h (p < 0.001). The best adherence ability to Hela cells was seen in Lactobacillus gasseri strains. Nearly 17 of the strains had higher anti-candida activity compared to the other strains. According to the findings, four lactobacilli strains isolated in the vaginal samples of healthy Iranian women had the best probiotic potential.

Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity.

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

Cationic Solution and Solid-State Emitters - Robust Imaging Agents for Cells, Bacteria, and Protists.

A library of eight different cationic emitters with emission properties in solution and in solid-state (solution and solid-state emitters - SSSE) is presented. These compounds, bearing either ammonium or pyridinium groups, have been investigated regarding their photophysical properties as well as their potential application in biological imaging. Besides high quantum yields as well as a high degree of stability during the imaging process, it was additionally revealed that a broad range of biological targets can be addressed, such as different bacterial strains, human cells as well as protists. The reported SSSE approach employing the mentioned robust emitters for biological imaging, will contribute to a rapid and facile way to design and apply affordable emitters with outstanding properties. Additionally, these emitters will overcome the drawbacks of classical luminophores and agents featuring well-known aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ) properties.

P2X7 receptor involved in antitumor activity of atractylenolide I in human cervical cancer cells.

Atractylenolide I (Atr-I) was found to sensitize a variety of human cancer cells in previous studies. Purinergic P2X7R plays important role in different cancers. However, whether Atr-I could generate antitumor activity in human cervical cancer cells and P2X7R get involved in this effect remain unclear. In this study, Hela (HPV 18 +) and SiHa (HPV 16 +) cells were treated with different doses of Atr-I. The results indicated that agonist and antagonist of P2X7 receptors, BzATP and JNJ-47965567 (JNJ), could suppress the proliferation of Hela and SiHa cells. Atr-I demonstrated a considerable antitumor effect in both human cervical cancer cells in vitro. Atr-I combined with P2X7R agonist, BzATP, restored Atr-I-induced growth inhibition in Hela cells but not in SiHa cells. However, the combinatorial treatment of P2X7R antagonist JNJ and Atr-I has an additive effect on cell growth inhibition in SiHa cells rather than in Hela cells. It implied that P2X7R would get involved in the anti-human cervical cancer cells effect of Atr-I.

A Simple Selective Probe for Lysine Detection in Tablets, Food Samples and Cells.

A novel probe ITQ (9-(((E)-1 H-inden-1-ylidene)methyl)-8-(3-(((E)-1 H-inden-1-ylidene)methyl)phenoxy)-2,3,6,7-tetrahydro-1 H,5 H-pyrido[3,2,1ij]quinolone) was successfully designed and synthesized to detect amino acid lysine (Lys). The selective sensing behavior of the probe ITQ was observed using absorption and emission spectral results. Further, the probe ITQ exhibits a strong binding affinity for Lys [1.4 × 104 M- 1] and detects and quantifies Lys even in its nanomolar concentration. Moreover, the probe ITQ detects Lys at 1:2 binding stoichiometry with suitable biological pH [4-11]. Furthermore, the probe ITQ was also successfully utilized to detect Lys in tablets, real samples (avocado, soyabean and pork) and in live HeLa cells.

Detection of triclosan using 2-domain hemoglobin promoter of Daphnia magna.

Triclosan has been widely used as an antimicrobial agent. However, triclosan was found to cause toxicity, including muscle contraction disturbances, carcinogenesis, and endocrine disorders. In addition, it was found to affect central nervous system function adversely and even have ototoxic effects. Conventional methods for detecting such triclosan can be performed easily. However, the conventional detection methods are inadequate in precisely reflecting the impact of toxic substances on stressed organisms. Therefore, a test model for the toxic environment at the molecular level through the organism is needed. From that point of view, Daphnia magna is being used as a ubiquitous model. D. magna has the advantages of easy cultivation, a short lifespan and high reproductive capacity, and high sensitivity to chemicals. Therefore, the protein expression pattern of D. magna that appear in response to chemicals can be utilized as biomarkers for detecting specific chemicals. In this study, we characterized the proteomic response of D. magna following triclosan exposure via two-dimensional (2D) gel electrophoresis. As a result, we confirmed that triclosan exposure completely suppressed D. magna 2-domain hemoglobin protein and evaluated this protein as a biomarker for triclosan detection. We constructed the HeLa cells in which the GFP gene was controlled by D. magna 2-domain hemoglobin promoter, which under normal conditions, expressed GFP, but upon triclosan exposure, suppressed GFP expression. Consequently, we consider that the HeLa cells containing the pBABE-HBF3-GFP plasmid developed in this study can be used as novel biomarkers for triclosan detection.

Genetically engineered E. coli invade epithelial cells and transfer their genetic cargo into the cells: an approach to a gene delivery system.

PURPOSE: Despite advances in gene therapy, the lack of safe and efficient gene delivery systems limited the clinical effectiveness of gene therapy. Due to the inherent potential of bacteria, they can be considered as a good option for the gene transfer system. This study aimed to create a genetically engineered bacterium capable of entering epithelial cells and transferring its genetic cargo to the cell's cytoplasm, eventually expressing the gene of interest in the cell. METHODS: The invasin (inv) gene from Yersinia pseudotuberculosis and the listeriolysin (hlyA) gene from Listeria monocytogenes were isolated by PCR assay and inserted into a pACYCDuet-1 vector. The recombinant plasmid was then transformed into E. coli strain BL21. Subsequently, pEGFP-C1 plasmids containing a CMV promoter were transformed into the engineered bacteria. Finally, the engineered bacteria containing the reporter genes were incubated with the HeLa and LNCaP cell lines. Fluorescence microscopy, flow cytometry, and TEM were used to monitor bacterial entry into the cells and gene expression. We used native E. coli strain BL21 as a control. RESULTS: A fluorescence microscope showed that, in contrast to the control group, the manipulated E. coli were able to penetrate the cells and transport the plasmid pEGFP-C1 to the target cells. Flow cytometry also showed fluorescence intensity of 54.7% in HeLa cells and 71% in LNCaP cells, respectively. In addition, electron micrographs revealed the presence of bacteria in the cell endosomes and in the cytoplasm of the cells. CONCLUSION: This study shows that genetically engineered E. coli can enter cells, transport cargo into cells, and induce gene expression in the target cell. In addition, flow cytometry shows that the gene transfer efficiency was sufficient for protein expression.

Detection of Salmonella dublin using the vitellogenin 2 promoter of Daphnia magna.

Salmonella is a well-known bacterium that causes waterborne diseases in humans and primates. The need for test models to detect such pathogens and study the responses of such organisms to induced toxic environments is vital. Daphnia magna has been ubiquitously used in aquatic life monitoring for decades because of outstanding properties, such as facile cultivation, short lifespan, and high reproductive capacity. In this study, the proteomic response of D. magna exposed to four Salmonella strains (Salmonella dublin, Salmonella enteritidis, Salmonella enterica, and Salmonella typhimurium) was characterized. As indicated by two-dimensional gel electrophoresis, vitellogenin fused with superoxide dismutase was completely suppressed under exposure to S. dublin. Thus, we evaluated the feasibility of using the vitellogenin 2 gene as a biomarker for S. dublin detection, particularly in providing rapid, visual detection through fluorescent signals. Accordingly, the applicability of the HeLa cells transfected with pBABE-Vtg2B-H2B-GFP as a biomarker for the detection of S. dublin was evaluated, and it was confirmed that the fluorescence signal decreased only when S. dublin was treated. Therefore, such HeLa cells can be utilized as a novel biomarker for detecting S. dublin.