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Cancer therapy targets specific metabolic pathways or a single gene. This may result in low therapeutic effects due to drug selectivity and drug resistance. Recent studies revealed that the mitochondrial membrane potential and transmembrane permeability of cancerous mitochondria are differed from normal mitochondria. Thus, chemotherapy targeting cancerous mitochondria could be an innovative and competent strategy for cancer therapy. Previously, our work with a novel group of mitochondria targeting small molecules presented promising inhibitory capability toward various cancer cell lines and suppressed adenosine triphosphate (ATP) generation. Therefore, it is critical to understand the anticancer effect and targeting mechanism of these small molecules. This study investigated the inhibitory activity of mitochondria targeting small molecules with human cervical cancer cells – HeLa to further explore their therapeutic potential. HeLa cells were exposed to 10 µM of synthesized compounds and presented elevation in intracellular reactive oxygen species (ROS) level, impaired mitochondrial membrane potential and upregulation of apoptosis as well as necrosis. In vivo, HeLa cell tumor-bearing BALB/c nude mice were treated with mitochondria targeting small molecules for 12 days consecutively. Throughout this chemotherapy study, no deleterious side effects nor the appearance of toxicity was observed. Furthermore, mitochondria targeting small molecules treated groups exhibited significant down-regulation with both tumor volume and tumor weight compared to the Doxorubicin (DOX) treated group. Thus, inhibition of mitochondrial ATP synthesis, activation of intracellular ROS production, down-regulation of mitochondrial membrane potential and upregulation of apoptosis and necrosis rates are the indications of cancer therapy. In this work, we examined the anticancer capability of four mitochondria targeting small molecules in vitro and in vivo, and demonstrated a novel therapeutic approach in cancer therapy with tremendous potential.

Significance Phase separation of proteins is increasingly thought to play a fundamental role in biological processes. Recent studies show that tau protein phase separates, but the biological significance is unknown since artificial crowding agents are typically used and the resulting tau is not toxic. We now demonstrate that TIA1 potentiates RNA-mediated phase separation of tau, thereby enabling a process that occurs at physiological concentrations and also directs the formation of biologically active, highly neurotoxic oligomeric tau. Coordinated phase separation of functionally related proteins provides a general mechanism through which membraneless organelles can direct biological activities., Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

Vibrio parahaemolyticus is a significant seafood-borne pathogen, leading to serious acute gastrointestinal diseases worldwide. In this study, a reliable 4-plex droplet digital PCR (ddPCR) was successfully established and evaluated for the simultaneous detection of V. parahaemolyticus based on tlh, tdh, ureR, and orf8 in food samples using single intact cells. The targets tlh and ureR were labeled with 6-Carboxyfluorescein (FAM), and the targets tdh and orf8 were labeled with 5’-Hexachlorofluorescein (HEX). Due to reasonable proration of primers and probes corresponding into the two fluorescence channels of the ddPCR detecting platforms, the clearly separated 16 (24) clusters based on fluorescence amplitude were obtained. For better results, the sample hot lysis time and the cycle number were optimized. The results showed that the minimum number of “rain” and maximum fluorescence amplification were presented for precise detection in the condition of 25 min of the sample hot lysis time and 55 cycles. The sensitivity of this 4-plex ddPCR assay was 39 CFU/mL, which was in accordance with that of the conventional plate counting and was 10-fold sensitive than that of qPCR. In conclusion, the 4-plex ddPCR assay presented in this paper was a rapid, specific, sensitive, and accurate tool for the detection of V. parahaemolyticus including pandemic group strains and could be applied in the differentiation of V. parahaemolyticus in a wide variety of samples.

Vibrio parahaemolyticus, a worldwide pathogen, has been proved to be capable of entering into viable but nonculturable (VBNC) state and survive under stressful conditions. In this study, a method of real-time quantitative PCR coupled with propidium monoazide (PMA-qPCR) was developed and evaluated for the reliable quantification of the VBNC cells of V. parahaemolyticus. The samples of different ratios of viable and dead cells were detected by PMA-qPCR and qPCR to assess this method. Then the method was used to monitor the VBNC induction process of V. parahaemolyticus and quantify the viable cells along with confirmation of cell integrity and respiratory activity by fluorescence microscopy and confocal laser scanning microscope (CLSM) observation, flow cytometry (FCM) analysis. In addition, the applicability of the method was verified to detect artificially contaminated seafood samples with different states of V. parahaemolyticus. The results showed that the optimal PMA treatment condition was 5 min-PMA incubation in dark followed by 15 min-light exposure. This method can significantly distinguish viable from dead cells and specifically enumerate viable cells. By PMA-qPCR method, the number change of cells during VBNC induction was monitored successfully. The cells incubated at 4 °C in sterile 3% NaCl entered into VBNC state, and 106 cell/mL of VBNC cells (10% of the original viable cells) were detected on the fifth day of induction, then the density of VBNC cells was 5.8 × 104 cell/mL on the 40th day when no culturable cells were observed. FCM analysis showed the viability of the VBNC cells which were also observed red revealing respiratory activity by CTC/DAPI staining under CLSM. For the detection of the spiked seafood samples (shrimp, pomfret and scallop) with different states of V. parahaemolyticus, it could detect as low as 1.2 × 102 cell/g of the viable or VBNC state of V. parahaemolyticus without any interference from food matrices and dead cells. In summary, the PMA-qPCR method developed in this study is rapid and reliable to quantify VBNC cells of V. parahaemolyticus.

Mitochondria are pivotal energy production sources for cells to maintain necessary metabolism activities. Targeting dysfunctional mitochondrial features has been a hotspot for mitochondrial-related disease researches. Investigation with cancerous mitochondrial metabolism is a continuing concern within tumor therapy. Herein, we set out to assess the anti-cancer activities of a novel family of TPP-thiazole derivatives based on our earlier research on mitochondrial targeting agents. Specifically, we designed and synthesized a series of TPP-thiazole derivatives and revealed by the MTT assay that most synthesized compounds effectively inhibited three cancer cell lines (HeLa, PC3 and MCF-7). After structure modifications, we explored the SAR relationships and identified the most promising compound R13 (IC50 of 5.52 μM) for further investigation. In the meantime, we performed ATP production assay to assess the selected compounds inhibitory effect on HeLa cells energy production. The results displayed the test compounds significantly restrained ATP production of cancer cells. Overall, we have designed and synthesized a series of compounds which exhibited significant cytotoxicity against cancer cells and effectively inhibited mitochondrial energy production.

Vibrio parahaemolyticus, a marine food-borne pathogen, has been proved to be a significant cause of human gastrointestinal disorders worldwide. In this study, a method of multiplex droplet digital PCR (ddPCR) based on primer and probe sequences of the tlh, tdh and ureR genes were developed and evaluated for the reliable quantification of V. parahaemolyticus cells in seafoods. The specificities of all primers and probes used in this study were validated on three standard strains of V. parahaemolyticus, 10 strains of Vibrio spp., and 22 strains of other bacteria by ddPCR and quantitative PCR (qPCR). Then the ddPCR system, primers, probe concentration and amplification procedures were optimized, and the templates with cell and genomic DNA (gDNA) were compared. The results showed that using cell as template could benefit for multiplex ddPCR, which performed higher linkage among three genes. This method improved sensitivity, specificity, accuracy, convenience, and reproducibility for the detection of V. parahaemolyticus, and the limit of detection (LOD) was 15 CFU/mL. In addition, the applicability of this method was compared with plate count and qPCR, and then verified to detect artificially contaminated seafood samples containing different concentrations of V. parahaemolyticus. The results indicated that the established method is stable, accurate, sensitive, range-wide, and has the potential to detect the three different genes of V. parahaemolyticus in food samples.