The Anthelmintic Quassinoids Ailanthone and Bruceine a Induce Infertility in the Model Organism Caenorhabditis elegans by an Apoptosis-like Mechanism Induced in Gonadal and Spermathecal Tissues

In continuation of the search for new anthelmintic natural products, the study at hand investigated the nematicidal effects of the two naturally occurring quassinoids ailanthone and bruceine A against the reproductive system of the model nematode Caenorhabditis elegans to pinpoint their anthelmintic mode of action by the application of various microscopic techniques. Differential Interference Contrast (DIC) and the epifluorescence microscopy experiments used in the presented study indicated the genotoxic effects of the tested quassinoids (c ailanthone = 50 µM, c bruceine A = 100 µM) against the nuclei of the investigated gonadal and spermathecal tissues, leaving other morphological key features such as enterocytes or body wall muscle cells unimpaired. In order to gain nanoscopic insight into the morphology of the gonads as well as the considerably smaller spermathecae of C. elegans, an innovative protocol of polyethylene glycol embedding, ultra-sectioning, acridine orange staining, tissue identification by epifluorescence, and subsequent AFM-based ultrastructural data acquisition was applied. This sequence allowed the facile and fast assessment of the impact of quassinoid treatment not only on the gonadal but also on the considerably smaller spermathecal tissues of C. elegans. These first-time ultrastructural investigations on C. elegans gonads and spermathecae by AFM led to the identification of specific quassinoid-induced alterations to the nuclei of the reproductive tissues (e.g., highly condensed chromatin, impaired nuclear membrane morphology, as well as altered nucleolus morphology), altogether implying an apoptosis-like effect of ailanthone and bruceine A on the reproductive tissues of C. elegans.

Shikonin Induced Program Cell Death through Generation of Reactive Oxygen Species in Renal Cancer Cells

Shikonin mitigated tumor cell proliferation by elevating reactive oxygen species (ROS) levels. Herein, we investigated the effects of shikonin on renal cancer cell (RCC) cell proliferation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that shikonin dose-dependently reduced the proliferation of Caki-1 and ACHN cells. Shikonin remarkably triggered necrosis and apoptosis in Caki-1 and ACHN cells in proportion to its concentration. Moreover, necrostatin-1 recovered cell viability in the presence of shikonin. Elevated ROS levels and mitochondrial dysfunction were also found in shikonin treatment groups. Pretreatment with N-acetyl cysteine remarkably mitigated shikonin-induced cell death and ROS generation. Western blot analysis revealed that shikonin reduced pro-PARP, pro-caspase-3, and Bcl-2 expression and increased cleavage PARP expression. Enhanced autophagy was also found in the shikonin-treated group as evidenced by acridine orange staining. Moreover, light chain 3B (LC3B)-II accumulation and enhanced p62 expression indicated that autophagy occurred in the shikonin-treated group. LC3B knockdown considerably recovered cell viability in the presence of shikonin. Shikonin treatment elevated p38 activity in a dose-dependent manner. In conclusion, our results revealed that shikonin triggered programmed cell death via the elevation of ROS level and p38 activity in different types of RCC cells. These findings suggested that shikonin may be a potential anti-RCC agent.

1275. Dynamics of Enterococcus faecalis Cardiolipin Synthase Gene Expression Reveal Compensatory Roles in Daptomycin Resistance

Background Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum, and resistance (DAP-R) has been associated with activation of the E. faecalis (Efs) LiaFSR response and redistribution of APL microdomains (predicted to contain cardiolipin) away from the septum. Efs encodes two putative cardiolipin synthase genes, cls1 and cls2. While changes in Cls1 are associated with DAP-R, the exact roles of each enzyme in resistance are unknown. This work aims to establish the contributions for both enzymes in the development of DAP-R. Methods cls1 and cls2 were deleted individually and in tandem from Efs OG117∆liaX (a DAP-R strain with an activated LiaFSR response). Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange staining. Quantitative PCR (qRT-PCR) was used to study gene expression profiles of cls1 and cls2 in Efs OG117∆liaX relative to Efs OG117. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS). Results cls1 was highly upregulated in stationary phase concurrent with a decrease in cls2 expression. However, independent deletion of cls1 or cls2 in the DAP-R background resulted in no significant phenotypic changes from the parent strain. Interestingly, qRT-PCR showed that cls2 expression was upregulated upon deletion of cls1 (and vice-versa), suggesting a compensatory role for one enzyme upon deletion of the other (Fig 1). When comparing membrane lipid content between Efs OG117∆liaX∆cls1 and Efs OG117∆liaX∆cls2, there were no significant differences in both the overall amount or species of cardiolipin generated, further supporting a potential redundancy between the cardiolipin synthases (Fig 2). Ultimately, double deletion of both cls genes lowered the DAP MIC relative to the parent strain and restored septal localization of APL microdomains. Conclusion Overall, Cls1 has a predominant role in the development of DAP-R in E. faecalis. However, here, we describe a novel compensatory role for Cls2 under conditions in which there is no functional Cls1 to maintain the DAP-R phenotype. Disclosures Truc T. Tran, PharmD, Merck (Grant/Research Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

The potential of sperm bovine protamine as a protein marker of semen production and quality at the National Artificial Insemination Center of Indonesia

Background and Aim: Protamine (PRM) is the major protein in the sperm nucleus and plays an essential role in its normal function. Moreover, PRM has great potential as a protein marker of semen production and quality. This study aimed to assess the potential of sperm bovine PRM as a protein marker of semen production and quality in bulls at the National Artificial Insemination (AI) Center of Indonesia. Materials and Methods: The semen production capacity of each bull was collected from frozen semen production data at the Singosari AI Center for 6 months, and was then divided into two groups (high and low). A total of 440 frozen semen straws from six Limousin (LIM), six Friesian Holstein (FH), six Peranakan Ongole (PO), and four Aceh bulls aged 4-5 years were used in the study. The frozen semen was used to measure the concentration of PRM1, PRM2, and PRM3 using the enzyme immunoassay method. The frozen semen was also used to assess the quality of the semen, including progressive motility (PM) through computer-assisted semen analysis, sperm viability through eosin–nigrosin analysis, and the DNA fragmentation index through Acridine Orange staining. Results: PRM1 was significantly higher in all bull breeds included in the study (p<0.00), followed by PRM2 (p<0.00) and PRM3 (p<0.00). PRM1 significantly affected semen production in LIM, FH, PO, and Aceh bulls (p<0.05). Moreover, PRM2 significantly affected semen production only in FH and Aceh bulls (p<0.05), whereas PRM3 affected this parameter in PO and Aceh bulls exclusively (p<0.05). Consistently and significantly, PRM1 was positively correlated with the PM and viability of sperm and negatively associated with its DNA fragmentation in LIM, FH, PO, and Aceh bulls (p<0.05; p<0.01). The correlation analysis between PRM2 and PRM3 and semen quality parameters varied across all bull breeds; some were positively and negatively correlated (p<0.05; p<0.01), and some were not correlated at all. Conclusion: PRM1 has excellent potential as a protein marker of semen production and quality in bulls at the National AI Center of Indonesia.

Cepharanthine Regulates Autophagy via Activating the p38 Signaling Pathway in Lung Adenocarcinoma Cells

Background: Cepharanthine (CEP) is an alkaloid extracted from Stephania cepharantha Hayata. This compound has been reported as a promising anti-tumor drug, although its potential molecular mechanism is not fully understood. Here, we studied the anti-tumor effect of CEP on human lung cancer cells and evaluated its molecular mechanism. Methods: The A549 cells were treated with CEP, the cell viability was measured by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, and formation of autophagosome was observed by acridine orange staining under a fluorescence microscope. The cell migration and invasion were determined by wound healing and transwell assay. The protein levels of autophagy-associated molecules, light chain 3 (LC3)、p38、and phospho-p38 in A549 cells, were determined by western blot analysis. Result: The results showed that CEP inhibited cell proliferation, migration and invasion in A549 cells. Moreover, we found that CEP resulted in significant increases in levels of the autophagy marker protein LC3 in A549 cells. The number of intracellular acid dye follicular bright red fluorescence in A549 cells was significantly increased after CEP treatment. At the molecular levels, CEP markedly increased the phosphorylation of p38 in A549 cells. The knockdown of p38 expression by siRNA-p38 impaired the autophagy-regulating effect of CEP. Our results indicated that CEP-regulated autophagy was an anti-tumor effect and not a protective response to CEP. Conclusion: Taken together, these results demonstrated that CEP regulated autophagy by activating the p38 signaling pathway, which could be provided a potential application for preventing lung cancer.

Developmental toxicity of cryptotanshinone on the early-life stage of zebrafish development

Cryptotanshinone (Cry) has multiple potential functions in treating different diseases. Most studies on Cry focus on its pharmacological effects and mechanisms, but toxicological reports on Cry are rare. Zebrafish is used as a model organism in drug development as it saves costs and time. This work aimed to investigate the toxicity of Cry on zebrafish. Results showed that growth retardation, pericardial edema, and scoliosis occurred when zebrafish embryos were exposed to Cry, indicating its teratogenic effects. Cell apoptosis was observed in the brainstem area of embryos using acridine orange staining, and qPCR showed that caspase-3 was increased in Cry-exposed embryos. The results of locomotor activity and touched-evoke escape reaction experiments showed that Cry significantly reduced the swimming speed and escape reaction time of larvae.

Effects of homeopathic medicines on macrophage - E. cuniculi interaction in vitro

Encephalitozoon cuniculi (E. cuniculi) is a fungus that behaves as an intracellular parasite infecting different types of cells. In rabbits and immunosuppressed animals of other species, including humans, it parasites neural tissues causing a highly characteristic neurological syndrome, for which repertory analysis indicates Phosphorus. Successful treatment of 7 naturally infected rabbits with Phosphorus confirmed that this medicine probably is the remedy of the ‘epidemic genius’ of microsporidiosis. In the present study, an in vitro model was used to evidence the intracellular relationship between macrophages and E. cuniculi after treatment of co-cultures with different potencies of Phosphorus. RAW 254.7 macrophages were co-cultivated with E. cuniculi and treated with Phosphorus 6cH, 30cH and 200cH. Controls were untreated co-cultures and cultures treated with vehicle (0.06% final alcohol concentration). Phagocytosis and lysosome activity were after after 1 and 24 hours of incubation by means of the calcofluor and acridine orange staining methods, followed by automatic image analysis (Metamorph®). Cytokine production was assessed with the MAGPIX®-Luminex system. The vehicle increased IL-6, MCP-1 and MIP1 production (p

Rhamnolipid Biosurfactants—Possible Natural Anticancer Agents and Autophagy Inhibitors

Background/Aim: A number of biologically active substances were proved as an alternative to conventional anticancer medicines. The aim of the study is in vitro investigation of the anticancer activity of mono- and di-Rhamnolipids (RL-1 and RL-2) against human breast cancer. Additionally, the combination with Cisplatin was analyzed. Materials and Methods: Breast cell lines (MCF-10A, MCF-7 and MDA-MB-231) were treated with RLs and in combination with Cisplatin. The viability was analyzed using MTT assay, and investigation of autophagy was performed via acridine orange staining. Results: In contrast to the healthy cells, both tested cancer lines exhibited sensitivity to RLs treatment. This effect was accompanied by an influence on the autophagy-related acidic formation process. Only for the triple-negative breast cancer cell line (MDA-MB-231) the synergistic effect of the combined treatment (10 µM Cisplatin and 1 µg/mL RL-2) was observed. Conclusion: Based on studies on the reorganization of membrane models in the presence of RL and the data about a higher amount of lipid rafts in cancer cell membranes than in non-tumorigenic, we suggest a possible mechanism of membrane remodelling by formation of endosomes. Shortly, in order to have a synergistic effect, it is necessary to have Cisplatin andRL-2 as RL2 is a molecule inducingpositive membrane curvature.

Mvda is required for zebrafish early development

Background The MVD gene mutations are identified in porokeratosis, which is considered a skin-specific autoinflammatory keratinization disease. However, the biological function of MVD gene remains largely unknown. Therefore, we analyzed the function of mvda gene, orthologous to the human MVD gene, in developing zebrafish. Methods Morpholino antisense oligonucleotide technique was used to generate mvda loss-of-function phenotypes. Knockdown of mvda was confirmed by RT-PCR and Sanger sequencing. Scanning and transmission electron microscopy were performed to analyze the morphology of the epidermis. Angiogenesis study was presented using the Tg(fli1a:EGFP)y1 transgenic strain. In addition, acridine orange staining was used to examine the apoptotic cells in vivo. Results As expected, the mvda morphants showed abnormal morphology of the epidermis. Moreover, we observed ectopic sprouts in trunk angiogenesis and impaired formation of the caudal vein plexus in the mvda-deficient zebrafish. Besides, increased apoptosis was found throughout the tail, heart, and eyes in mvda zebrafish morphants. Conclusions These findings indicated the essential role of mvda in the early development of zebrafish. This was the first in vivo knockdown study of the zebrafish mvda gene, which might offer insight into the biological function of the human MVD gene.

Human Adipose Derived Stem Cell Exosomes Enhance the Neural Differentiation of PC12 Cells

Background: Human adipose stem cells (hADSCs) are proper cell sources for tissue regeneration. They mainly mediate their therapeutic effects through paracrine factors as exosomes. The exosomes contents are protein, lipid and RNA. Exosomes are effective in restoring the function of neurons and astrocytes in neurodegenerative diseases, and improve the therapeutic outcomes. We investigated the effect of hADSCs derived exosomes on survival and neural differentiation of PC12 cells in vitro.Methods and Results: The isolated hADSCs, were characterized by flow cytometry. Exosomes were separated from hADSC-condition medium using Exo-spinTM kit and characterized by DLS and TEM. Then acridine orange staining was performed to confirm entrance of exosomes into PC12 cells. PC12 cells were treated with culture medium containing NGF and exosome. Cell viability was assessed by MTT assay, and neural differentiation by ICC technique and qRT-PCR. TEM and DLS data confirmed the isolation of exosomes according to their size (30-100nm) and acridine orange staining indicated entrance of exosomes to target cells. MTT assay showed that cell viability was significantly increased in exosome treated group. ICC technique revealed that the expression of Map2 was superior in the exosome treated group. Based on qRT-PCR data, Map2 and β-tub III gene expression was increased in the exosome treated group. Significant expression of Gfap was seen in the NGF and NGF/EXO treated groups.Conclusions: Present study indicated that hADSCs derived exosomes might enhance cell viability and promote neuronal differentiation and expression of mature neural marker in PC12 cells.