Malaysian and Chinese King Cobra Venom Cytotoxicity in Melanoma and Neonatal Foreskin Fibroblasts Is Mediated by Age and Geography.

Snake venoms constitute a complex, rapidly evolving trait, whose composition varies between and within populations depending on geographical location, age and preys (diets). These factors have determined the adaptive evolution for predatory success and link venom heterogeneity with prey specificity. Moreover, understanding the evolutionary drivers of animal venoms has streamlined the biodiscovery of venom-derived compounds as drug candidates in biomedicine and biotechnology. The king cobra (Ophiophagus hannah; Cantor, 1836) is distributed in diverse habitats, forming independent populations, which confer differing scale markings, including between hatchlings and adults. Furthermore, king cobra venoms possess unique cytotoxic properties that are used as a defensive trait, but their toxins may also have utility as promising anticancer-agent candidates. However, the impact of geographical distribution and age on these potential venom applications has been typically neglected. In this study, we hypothesised that ontogenetic venom variation accompanies the morphological distinction between hatchlings and adults. We used non-transformed neonatal foreskin (NFF) fibroblasts to examine and compare the variability of venom cytotoxicity between adult captive breeding pairs from Malaysian and Chinese lineages, along with that of their progeny upon hatching. In parallel, we assessed the anticancer potential of these venoms in human-melanoma-patient-derived cells (MM96L). We found that in a geographical distribution and gender-independent manner, venoms from hatchlings were significantly less cytotoxic than those from adults (NFF; ~Log EC50: 0.5-0.6 vs. 0.2-0.35 mg/mL). This is consistent with neonates occupying a semifossorial habitat, while adults inhabit more above-ground habitats and are therefore more conspicuous to potential predators. We also observed that Malaysian venoms exhibited a slightly higher cytotoxicity than those from the Chinese cobra cohorts (NFF; Log EC50: 0.1-0.3 vs. 0.3-0.4 mg/mL), which is consistent with Malaysian king cobras being more strongly aposematically marked. These variations are therefore suggestive of differential anti-predator strategies associated with the occupation of distinct niches. However, all cobra venoms were similarly cytotoxic in both melanoma cells and fibroblasts, limiting their potential medical applications in their native forms.

Temporal transcriptomic changes in long non-coding RNAs and messenger RNAs involved in the host immune and metabolic response during Toxoplasma gondii lytic cycle.

BACKGROUND: Long non-coding RNAs (lncRNAs) are important regulators of various biological and pathological processes, in particular the inflammatory response by modulating the transcriptional control of inflammatory genes. However, the role of lncRNAs in regulating the immune and inflammatory responses during infection with the protozoan parasite Toxoplasma gondii remains largely unknown. METHODS: We performed a longitudinal RNA sequencing analysis of human foreskin fibroblast (HFF) cells infected by T. gondii to identify differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs), and dysregulated pathways over the course of T. gondii lytic cycle. The transcriptome data were validated by qRT-PCR. RESULTS: RNA sequencing revealed significant transcriptional changes in the infected HFFs. A total of 697, 1234, 1499, 873, 1466, 561, 676 and 716 differentially expressed lncRNAs (DElncRNAs), and 636, 1266, 1843, 2303, 3022, 1757, 3088 and 2531 differentially expressed mRNAs (DEmRNAs) were identified at 1.5, 3, 6, 9, 12, 24, 36 and 48 h post-infection, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DElncRNAs and DEmRNAs revealed that T. gondii infection altered the expression of genes involved in the regulation of host immune response (e.g., cytokine-cytokine receptor interaction), receptor signaling (e.g., NOD-like receptor signaling pathway), disease (e.g., Alzheimer's disease), and metabolism (e.g., fatty acid degradation). CONCLUSIONS: These results provide novel information for further research on the role of lncRNAs in immune regulation of T. gondii infection.

Arene-Ruthenium(II) Complexes with Carbothiamidopyrazoles as a Potential Alternative for Antibiotic Resistance in Human.

To meet the demand for alternatives to commonly used antibiotics, this paper evaluates the antimicrobial potential of arene-ruthenium(II) complexes and their salts, which may be of value in antibacterial treatment. Their antimicrobial activity (MIC, MBC/MFC) was examined in vitro against Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa, Proteus vulgaris and Candida albicans and compared with classic antibiotics used as therapeutics. Selected arene-ruthenium(II) complexes were found to have synergistic effects with oxacillin and vancomycin against staphylococci. Their bactericidal effect was found to be associated with cell lysis and the ability to cut microbial DNA. To confirm the safety of the tested arene-ruthenium(II) complexes in vivo, their cytotoxicity was also investigated against normal human foreskin fibroblasts (HFF-1). In addition, the antioxidant and thus pro-health potential of the compounds, i.e., their nonenzymatic antioxidant capacity (NEAC), was determined by two different methods: ferric-TPTZ complex and DPPH assay.

The Peptide A-3302-B Isolated from a Marine Bacterium Micromonospora sp. Inhibits HSV-2 Infection by Preventing the Viral Egress from Host Cells.

Herpesviruses are highly prevalent in the human population, and frequent reactivations occur throughout life. Despite antiviral drugs against herpetic infections, the increasing appearance of drug-resistant viral strains and their adverse effects prompt the research of novel antiherpetic drugs for treating lesions. Peptides obtained from natural sources have recently become of particular interest for antiviral therapy applications. In this work, we investigated the antiviral activity of the peptide A-3302-B, isolated from a marine bacterium, Micromonospora sp., strain MAG 9-7, against herpes simplex virus type 1, type 2, and human cytomegalovirus. Results showed that the peptide exerted a specific inhibitory activity against HSV-2 with an EC50 value of 14 µM. Specific antiviral assays were performed to investigate the mechanism of action of A-3302-B. We demonstrated that the peptide did not affect the expression of viral proteins, but it inhibited the late events of the HSV-2 replicative cycle. In detail, it reduced the cell-to-cell virus spread and the transmission of the extracellular free virus by preventing the egress of HSV-2 progeny from the infected cells. The dual antiviral and previously reported anti-inflammatory activities of A-3302-B, and its effect against an acyclovir-resistant HSV-2 strain are attractive features for developing a therapeutic to reduce the transmission of HSV-2 infections.

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis.

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology.

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.

In vitro and in vivo efficacy of thiacloprid against Echinococcus multilocularis.

BACKGROUND: Alveolar echinococcosis (AE) is a chronic zoonosis caused by the larval form of Echinococcus multilocularis (E. multilocularis). Current chemotherapy against AE has relied on albendazole and mebendazole, which only exhibit parasitostatic and not parasiticidal efficacy. Therefore, novel compounds for the treatment of this disease are needed. METHODS: Phosphoglucose isomerase (PGI) assays were used for compound screening of seven neonicotinoids. The anti-parasitic effects of thiacloprid were then evaluated on E. multilocularis metacestode vesicles, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFF) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. Glucose consumption in E. multilocularis protoscoleces and germinal cells was assessed by measuring uptake of 2-deoxyglucose (2-DG). Molecular docking was used to evaluate the potential binding sites of thiacloprid to acetylcholine receptors. In vivo efficacy of thiacloprid was evaluated in mice by secondary infection with E. multilocularis. In addition, ELISA and flow cytometry were used to evaluate the effects of cytokines and T lymphocyte subsets after thiacloprid treatment. Furthermore, collagen deposition and degradation in the host lesion microenvironment were evaluated. RESULTS: We found that thiacloprid is the most promising compound, with an IC50 of 4.54 ± 1.10 µM and 2.89 ± 0.34 µM, respectively, against in vitro-cultured E. multilocularis metacestodes and germinal cells. Thiacloprid was less toxic for HFF and RH mammalian cell lines than for metacestodes. In addition, thiacloprid inhibited the acetylcholinesterase activity in protoscoleces, metacestodes and germinal cells. Thiacloprid inhibited glucose consumption by protoscoleces and germinal cells. Subsequently, transmission electron microscopy revealed that treatment with thiacloprid damaged the germinal layer. In vivo, metacestode weight was significantly reduced following oral administration of thiacloprid at 15 and 30 mg/kg. The level of CD4+ T lymphocytes in metacestodes and spleen increased after thiacloprid treatment. Anti-echinococcosis-related cytokines (IL-2, IL-4, IL-10) were significantly increased. Furthermore, thiacloprid inhibited the expression of matrix metalloproteinases (MMPs 1, 3, 9, 13) and promoted collagen deposition in the host lesion microenvironment. CONCLUSIONS: The results demonstrated that thiacloprid had parasiticidal activity against E. multilocularis in vitro and in vivo, and could be used as a novel lead compound for the treatment of AE.

Cancer Associated Fibroblasts Derived from Pancreatic Adenocarcinoma and Their Role in Cell Migration.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) shows poor survival and early systemic dissemination. Cancer associated fibroblasts (CAFs) enhance migration and invasion of cancer cells. We aimed to investigate the role of CAFs in cell migration and their underlying paracrine effects. MATERIALS AND METHODS: Using Transwell® migration assays, PDAC cells (PANC-1) and three distinct types of fibroblasts were analyzed: CAFs, genetically transformed human foreskin-fibroblasts (BJeLR), and non-transformed human foreskin-fibroblasts (VH7). IL6 in the culture supernatant was measured to investigate paracrine communication in monocultures and direct/indirect cocultures. RESULTS: CAFs showed a significantly higher capacity to migrate in vitro when compared to benign fibroblasts (p=0.009). They also facilitated the migration of PDAC cells in coculture (p=0.001). Neither BJeLR, nor VH7 displayed such features. This was accompanied by a significant increase in IL-6 when CAFs were cocultured with PANC-1 (p=0.009). CONCLUSION: CAFs are a key element of intra-tumoral migration and should be further investigated as a potential therapeutic target.

The Molecular Tweezer CLR01 Inhibits Antibody-Resistant Cell-to-Cell Spread of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) uses two major ways for virus dissemination: infection by cell-free virus and direct cell-to-cell spread. Neutralizing antibodies can efficiently inhibit infection by cell-free virus but mostly fail to prevent cell-to-cell transmission. Here, we show that the 'molecular tweezer' CLR01, a broad-spectrum antiviral agent, is not only highly active against infection with cell-free virus but most remarkably inhibits antibody-resistant direct cell-to-cell spread of HCMV. The inhibition of cell-to-cell spread by CLR01 was not limited to HCMV but was also shown for the alphaherpesviruses herpes simplex viruses 1 and 2 (HSV-1, -2). CLR01 is a rapid acting small molecule that inhibits HCMV entry at the attachment and penetration steps. Electron microscopy of extracellular virus particles indicated damage of the viral envelope by CLR01, which likely impairs the infectivity of virus particles. The rapid inactivation of viral particles by CLR01, the viral envelope as the main target, and the inhibition of virus entry at different stages are presumably the key to inhibition of cell-free virus infection and cell-to-cell spread by CLR01. Importance: While cell-free spread enables the human cytomegalovirus (HCMV) and other herpesviruses to transmit between hosts, direct cell-to-cell spread is thought to be more relevant for in vivo dissemination within infected tissues. Cell-to-cell spread is resistant to neutralizing antibodies, thus contributing to the maintenance of virus infection and virus dissemination in the presence of an intact immune system. Therefore, it would be therapeutically interesting to target this mode of spread in order to treat severe HCMV infections and to prevent dissemination of virus within the infected host. The molecular tweezer CLR01 exhibits broad-spectrum antiviral activity against a number of enveloped viruses and efficiently blocks antibody-resistant cell-to-cell spread of HCMV, thus representing a novel class of small molecules with promising antiviral activity.

Requirement of Toxoplasma gondii metacaspases for IMC1 maturation, endodyogeny and virulence in mice.

BACKGROUND: Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated. METHODS: In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. RESULTS: MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection. CONCLUSION: MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo.

Inhibiting cytomegalovirus replication through targeting the host electron transport chain.

Human cytomegalovirus (HCMV) is a near ubiquitous herpesvirus that relies on host cell metabolism for efficient replication. Although it has been shown that HCMV requires functional host cell mitochondria for efficient replication, it is unknown whether mitochondrial targeted pharmacological agents can be repurposed as antivirals. Here we report that treatment with drugs targeting the electron transport chain (ETC) complexes inhibit HCMV replication. Addition of rotenone, oligomycin, antimycin and metformin resulted in decreased HCMV titers in vitro, independent of HCMV strain. This further illustrates the dependence of HCMV replication on functional mitochondria. Metformin, an FDA approved drug, delays HCMV replication kinetics resulting in a reduction of viral titers. Repurposing metformin as an antiviral is advantageous as its safety profile and epidemiological data are well accepted. Our findings provide new insight into the potential for targeting HCMV infection through host cell metabolism and how these pharmacological interventions function.

In vivo and in vitro efficacy of crocin against Echinococcus multilocularis.

BACKGROUND: Alveolar echinococcosis (AE) is a fatal zoonosis caused by the larvae of Echinococcus multilocularis. However, current chemotherapy treatment options are based on benzimidazoles [albendazole (ABZ) and mebendazole], which have limited efficacy. Therefore, novel drugs are necessary for the treatment of this disease. METHODS: The anthelmintic effects of crocin were tested on E. multilocularis metacestodes, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFFs) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. The in vivo efficacy of crocin was investigated in mice following secondary infection with E. multilocularis. Furthermore, collagen deposition and degradation in host tissues around the metacestodes were evaluated. RESULTS: In vitro, crocin had a median effective concentration of 11.36 µM against cultured E. multilocularis metacestodes, while it reduced germinal cell viability at a median inhibitory concentration of 10.05 µM. Crocin was less toxic to HFFs and RH mammalian cell lines than to metacestodes. Transmission electron microscopy revealed that crocin treatment resulted in structural damage in the germinal layer. In addition, 60.33 ± 3.06% of protoscoleces were killed by treatment with 10 µM crocin for 7 days, indicating that crocin has a parasiticidal effect. In vivo, the metacestode weight was significantly reduced after the administration of crocin at 50 mg/kg and 100 mg/kg (55.1 and 68.1%, respectively). Metacestode pathology showed structural disruption of the germinal and laminated layers after crocin treatment. The crocin- and ABZ-treated groups presented significant increases in the levels of interleukin (IL)-2 and IL-4. Furthermore, crocin inhibited the expression of matrix metalloproteinases (MMPs) (MMP2 and MMP9) and promoted collagen deposition in the metacestode. CONCLUSIONS: Crocin was demonstrated to exert parasiticidal activity against E. multilocularis in vitro and in vivo, and can be developed as a novel drug for the treatment of AE.

Phototoxic versus photoprotective effects of tattoo pigments in reconstructed human skin models: In vitro phototoxicity testing of tattoo pigments: 3D versus 2D.

The increasing number of tattooed persons urges the development of reliable test systems to assess tattoo associated risks. The alarming prevalence of 60 % phototoxic reactions in tattoos ask for a more comprehensive investigation of phototoxic reactions in tattooed skin. Here, we aimed to compare the cellular responses of human skin cells to ultraviolet (UV)A and UVB irradiation in doses of short to intermitted sun exposure (3-48 J/cm² and 0.05-5 J/cm², respectively) in the presence of tattoo pigments. Therefore, we used fibroblast monolayer culture (2D), our recently developed three dimensional full-thickness skin model with dermal-located tattoo pigments (TatSFT) and its dermal equivalents (TatSDE) that lack keratinocytes. We tested the most frequently used tattoo pigments carbon black, titanium dioxide (TiO2) anatase and rutile as well as Pigment Orange (P.O.)13 in ranges from 0.067 to 2.7 ng/cell in 2D. For TatSDE and TatSFT, concentrations were 1.3 ng/cell for TiO2, 0.67 ng/cell for P.O.13 and 0.067 ng/cell for carbon black. We assessed cell viability and cytokine release in all systems, and cyclobutane pyrimidine dimer (CPD) formation in TatSFT. Phototoxicity of tattoo pigments was exclusively observed in 2D, where especially TiO2 anatase induced phototoxic effects in all concentrations (0.067-2.7 ng/cell). In contrast, fibroblasts were protected from UV irradiation in TatSDE by TiO2 and carbon black. Neither toxic nor protective effects were recorded in TatSFT. P.O.13 showed altered cytokine secretion in 2D (0.067-1.3 ng/cell) and TatSDE, despite the absence of significant effects on viability in all systems. All pigments reduced the number of CPDs in TatSFT compared to the pigment-free controls. In conclusion, our study shows that within a 3D arrangement, intradermal tattoo pigments may act photoprotective despite intrinsic phototoxic properties in 2D. Thus, dermal 3D equivalents should be considered to evaluate acute tattoo pigment toxicology.

Inhibitory Effects of Bilirubin on Colonization and Migration of A431 and SK-MEL-3 Skin Cancer Cells Compared with Human Dermal Fibroblasts (HDF).

This study evaluated the inhibitory effects of bilirubin on colony formation and cell migration of melanoma and non-melanoma skin cancer cell lines SK-MEL-3 and A431, compared with normal human dermal fibroblasts (HDF). The IC50 obtained from the MTT assay was 125, 100, and 75 µM bilirubin for HDF, A431, and SK-MEL-3 cells, respectively. The colony formation and cell migration of cancer cells, treated with 100 µM bilirubin, were reduced significantly (p < 0.05). Bilirubin decreased cell adhesion and inhibited cell colonization via inducing apoptosis and cell death. Also by interaction with migration main factors, bilirubin caused inhibition the cell migration.

Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97.

The replication of human cytomegalovirus (HCMV) is characterized by a complex network of virus-host interaction. This involves the regulatory viral protein kinase pUL97, which represents a viral cyclin-dependent kinase ortholog (vCDK) combining typical structural and functional features of host CDKs. Notably, pUL97 interacts with the three human cyclin types T1, H and B1, whereby the binding region of cyclin T1 and the region conferring oligomerization of pUL97 were both assigned to amino acids 231-280. Here, we addressed the question of whether recombinant HCMVs harboring deletions in this region were impaired in cyclin interaction, kinase functionality or viral replication. To this end, recombinant HCMVs were generated by traceless BACmid mutagenesis and were phenotypically characterized using a methodological platform based on qPCR, coimmunoprecipitation, in vitro kinase assay (IVKA), Phos-tag Western blot and confocal imaging analysis. Combined data illustrate the following: (i) infection kinetics of all three recombinant HCMVs, i.e., ORF-UL97 ∆231-255, ∆256-280 and ∆231-280, showed impaired replication efficiency compared to the wild type, amongst which the largest deletion exhibited the most pronounced defect; (ii) specifically, this mutant ∆231-280 showed a loss of interaction with cyclin T1, as demonstrated by CoIP and confocal imaging; (iii) IVKA and Phos-tag analyses revealed strongly affected kinase activity for ∆231-280, with strong impairment of both autophosphorylation and substrate phosphorylation, but less pronounced impairments for ∆231-255 and ∆256-280; and (iv) a bioinformatic assessment of the pUL97-cyclin T1 complex led to the refinement of our current binding model. Thus, the results provide initial evidence for the functional importance of the pUL97-cyclin interaction concerning kinase activity and viral replication fitness.

Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage.

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.

Sodium metabisulfite as a cytotoxic food additive induces apoptosis in HFFF2 cells.

Sodium metabisulfite (SMB), an antioxidant agent, is extensively used as a preservative in food industry. The current study was aimed to clarify its potential toxic effects on human fetal foreskin fibroblasts (HFFF2) cells, in vitro. Subsequently, MTT results illustrated that exposure to SMB significantly (p < 0.0001) decreased HFFF2 cell viability in a dose-dependent manner, and the concentration of 25 µM reduced cell survival rates to 50% as the half-maximal inhibitory concentration of SMB. It was further shown that SMB exerted this cytotoxic effect on HFFF2 cells through apoptosis induction. qRT-PCR and western blotting results showed that treatment of HFFF2 cells with this food additive led to significant upregulation of Bax, caspase 8, and caspase 9 pro-apoptotic genes and downregulation of Bcl-2 expression as a pro-survival agent. Furthermore, SMB remarkably increased caspase 3 levels and promoted its activation through cleavage in treated cells. Besides, exposure to SMB increased ROS levels and activated autophagy in treated cells, which are considered as the other indicators for cell damage. Taken together, our findings suggested that SMB could exert remarkable toxic effects on human normal cells through multiple mechanisms, including apoptosis activation, and its widespread usage in food safety should be reconsidered.

Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility.

Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held "linear motor" model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect.

UVB damage response of dermal stem cells as melanocyte precursors compared to keratinocytes, melanocytes, and fibroblasts from human foreskin.

Ultraviolet B (UVB) radiation induces mutagenic DNA photolesions in skin cells especially in form of cyclobutane pyrimidine dimers (CPDs). Protection mechanisms as DNA repair and apoptosis are of great importance in order to prevent skin carcinogenesis. In human skin, neural crest-derived precursors of melanocytes, the dermal stem cells (DSCs), are discussed to be at the origin of melanoma. Although they are constantly exposed to solar UV radiation, it is still not investigated how DSCs cope with UV-induced DNA damage. Here, we report a comparative study of the DNA damage response after irradiation with a physiological relevant UVB dose in DSCs in comparison to fibroblasts, melanocytes and keratinocytes isolated from human foreskin. Within our experimental settings, DSCs were able to repair DNA photolesions as efficient as the other skin cell types with solely keratinocytes repairing significantly faster. Interestingly, only fibroblasts showed significant alterations in cell cycle distribution in terms of a transient S phase arrest following irradiation. Moreover, with the applied UVB dose none of the examined cell types was prone to UVB-induced apoptosis. This may cause persistent genomic alterations and in case of DSCs it may have severe consequences for their daughter cells, the differentiated melanocytes. Altogether, this is the first study demonstrating a similar UV response in dermal stem cells compared to differentiated skin cells.

Flow cytometric quantification of apoptotic and proliferating cells applying an improved method for dissociation of spheroids.

Spheroids are a promising tool for many cell culture applications, but their microscopic analysis is limited. Flow cytometry on a single cell basis, which requires a gentle but also efficient dissociation of spheroids, could be an alternative analysis. Mono-culture and coculture spheroids consisting of human fibroblasts and human endothelial cells were generated by the liquid overlay technique and were dissociated using AccuMax as a dissociation agent combined with gentle mechanical forces. This study aimed to quantify the number of apoptotic and proliferative cells. We were able to dissociate spheroids of differing size, age, and cellular composition in a single-step dissociation protocol within 10 min. The number of single cells was higher than 95% and in most cases, the viability of the cells after dissociation was higher than 85%. Coculture spheroids exhibited a higher sensitivity as shown by lower viability, higher amount of cellular debris, and a higher amount of apoptotic cells. Considerable expression of the proliferation marker Ki67 could only be seen in 1-day-old spheroids but was already downregulated on Day 3. In summary, our dissociation protocol enabled a fast and gentle dissociation of spheroids for the subsequent flow cytometric analysis. The chosen cell type had a strong influence on cell viability and apoptosis. Initially high rates of proliferative cells decreased rapidly and reached values of healthy tissue 3 days after generation of the spheroids. In conclusion, the flow cytometry of dissociated spheroids could be a promising analytical tool, which could be ideally combined with microscopic techniques.