AK-1, a Sirt2 inhibitor, alleviates carbon tetrachloride-induced hepatotoxicity in vivo and in vitro.

Background/Aim: Acute liver injury (ALI) is a life-threatening clinical syndrome that is usually caused by toxic chemicals, drugs, or pathogen infections. Sirtuin2 (Sirt2), an NAD+-dependent deacetylase, appears to play detrimental roles in liver injury. Here, we evaluated the therapeutic application targeting Sirt2 in carbon tetrachloride (CCl4)-induced ALI, by using AK-1 (a Sirt2 inhibitor).Methods: For in vivo experiments, a single injection of CCl4 was used to induce ALI. One hour later, mice were intraperitoneally injected with AK-1 and were sacrificed 24 h after CCl4 administration. For in vitro experiments, primary mouse hepatocytes were used to determine the effects of AK-1 on oxidative stress and hepatocellular death induced by CCl4.Results: AK-1 alleviated CCl4-induced ALI as confirmed by histopathologic analysis, and decreased levels of serum biochemicals and inflammatory cytokines. Although it barely affected the expression of hepatic cytochrome P450 enzymes, AK-1 attenuated CCl4-induced oxidative stress and its related cell death. Mechanistically, Sirt2 inhibition significantly increased the nuclear protein level of nuclear factor erythroid 2-related factor 2 (Nrf2), and meanwhile decreased phosphorylation of c-Jun N-terminal kinases (JNK), in normal and injured livers. Similar results were observed in vitro. AK-1 significantly attenuated CCl4-induced cytotoxicity and oxidative stress by up-regulating the activity of Nrf2, and down-regulating JNK signaling in hepatocytes.Conclusions: Our results suggest that AK-1 treatment attenuated oxidative stress and cell death in the ALI model, at least partially, via activating Nrf2 and inhibiting JNK signaling, and that Sirt2 inhibition might be a potential approach to cure ALI.

Expression Patterns of Host Inflammatory Cytokine Genes during Infestation with Haemaphysalis longicornis, a Zoonotic Vector, in Blood Sucking Periods.

Tick saliva is critically important for continuous attachment to the host, blood feeding for days, and transmission of tick-borne pathogens. To characterize the patterns of inflammatory cytokine gene expression during its attachment and blood sucking time, peripheral blood samples of rabbits infested with Haemaphysalis longicornis ticks were collected at different intervals. Blood histamine concentration was evaluated as well as gene encoding IFN-γ, TNF-α, IL-2, IL-6, IL-4, and IL-10 were compared with non-infested rabbits. Blood histamine concentration of tick-infested rabbits during fast feeding time was significantly higher than that of non-infested rabbits. In both nymph and adult tick infested rabbits, expression of TNF-α and IFN-γ genes were decreased significantly (P<0.05), while expression of IL-4, IL-6, and IL-10 were increased 1.3 to 7 folds in adult infested rabbits with the exception of IL-6 that was significantly (P<0.05) decreased in nymph infested rabbits. IL-2 was not expressed in either nymph or adult infestation. H. longicornis saliva is capable of modulate host responses through a complex correlation with histamine and Th1, Th2 mediated cytokines that suppress the inflammatory responses directed toward inflammatory mediators introduced into the host during tick feeding.

Proteomic screening of antigenic proteins from the hard tick, Haemaphysalis longicornis (Acari: Ixodidae).

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.

Suppression of Eimeria tenella sporulation by disinfectants.

The disinfectant effects (DEs) of 10 types of chemicals, defined by their ability to destroy or inhibit oocysts and consequently prevent sporulation of Eimeria tenella field isolate, were evaluated in vitro. Correct species assignments and sample purities were confirmed by the singular internal transcribed spacer (ITS)-PCR analysis. A total of 18 treatments were performed, and the disinfection suppression levels were 75.9% for 39% benzene + 22% xylene (1:10 dilution), 85.5% for 30% cresol soup (1:1 dilution), and 91.7% for 99.9% acetic acid (1:2 dilution) group. The results indicate that acetic acid, cresol soup, and benzene+xylene are good candidates for suppression of E. tenella oocyst sporulation.

Effects of different sizes of glass beads on the release of sporocysts from Eimeria tenella oocysts.

The oocyst wall is severed by means of mechanical injury or chemical agents. This study reports the percentage of in vitro sporocyst release following mechanical shaking in the presence of varying sizes of glass beads. Glass beads measured 0.5, 1, and 3 mm in diameter and were shaken with the oocysts for different times ranging from 5 sec to 5 min. Approximately 80% of sporocysts were released with 5 min of shaking in the presence of 3 mm glass beads, as well as 30 sec with 0.5 mm beads and 1 mm glass beads. The release of sporocysts of E. tenella was most efficient using 1 mm glass beads and treatment times of 30 sec to 1 min. Therefore, the use of 1 mm glass beads with 30 sec to 1 min of agitation is recommended in order to maximize sporocyst release and recovery and to improve the yield of viable sporozoites for use in biochemical, tissue culture, and immunological applications of coccidia.

Comparative analysis of essential oil efficacy against the Asian longhorned tick Haemaphysalis longicornis (Acari: Ixodidae).

This study evaluated the potential repellent and acaricidal effects of 4 essential oils (clove, eucalyptus, lavender, and mint) against the Asian longhorned tick Haemaphysalis longicornis, a vector of various tick-borne diseases in medical and veterinary contexts. Selected for their potential repellent and acaricidal properties, the 4 essential oils were tested on adult and nymph H. longicornis ticks at different concentrations. The experiment assessed mortality rates and repellency, particularly during tick attachment to host skin. There was a significant increase (p<0.05) in tick mortality and repellency scores across all groups. At a 1% concentration, adult tick mortality ranged from 36% to 86%, while nymph mortality ranged from 6% to 97%. Clove oil exhibited notable efficacy, demonstrating high mortality rates of nymphs and adults. Clove oil also displayed strong repellency properties, with a repellency index of 0.05, surpassing those of mint, eucalyptus, and lavender oils. Clove oil showed the highest effectiveness in deterring nonattached adult ticks (90%) and nymphs (95%) when applied to skin. Clove oil was the most effective against adult and nymph ticks, achieving mortality rates of 86% and 97%, respectively, and led to the highest nonattachment rates when applied to skin. In conclusion, essential oils such as clove, eucalyptus, lavender, and mint oils present promising results for tick population control.

Effect of Silencing subolesin and enolase impairs gene expression, engorgement and reproduction in Haemaphysalis longicornis (Acari: Ixodidae) ticks.

IMPORTANCE: Haemaphysalis longicornis is an obligate blood-sucking ectoparasite that has gained attention due its role of transmitting medically and veterinary significant pathogens and it is the most common tick species in Republic of Korea. The preferred strategy for controlling ticks is a multi-antigenic vaccination. Testing the efficiency of a combination antigen is a promising method for creating a tick vaccine. OBJECTIVE: The aim of the current research was to analyze the role of subolesin and enolase in feeding and reproduction of H. longicornis by gene silencing. METHODS: In this study, we used RNA interference to silence salivary enolase and subolesin in H. longicornis. Unfed female ticks injected with double-stranded RNA targeting subolesin and enolase were attached and fed normally on the rabbit's ear. Real-time polymerase chain reaction was used to confirm the extent of knockdown. RESULTS: Ticks in the subolesin or enolase dsRNA groups showed knockdown rates of 80% and 60% respectively. Ticks in the combination dsRNA (subolesin and enolase) group showed an 80% knockdown. Knockdown of subolesin and enolase resulted in significant depletion in feeding, blood engorgement weight, attachment rate, and egg laying. Silencing of both resulted in a significant (p < 0.05) reduction in tick engorgement, egg laying, egg hatching (15%), and reproduction. CONCLUSIONS AND RELEVANCE: Our results suggest that subolesin and enolase are an exciting target for future tick control strategies.

Molecular cloning, identification, transcriptional analysis, and silencing of enolase on the life cycle of Haemaphysalis longicornis (Acari, Ixodidae) tick.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

Difference in Intraspecies Transmissibility of Severe Fever with Thrombocytopenia Syndrome Virus Depending on Abrogating Type 1 Interferon Signaling in Mice.

Severe fever with thrombocytopenia syndrome (SFTS), a tick-borne zoonotic disease, is caused by infection with SFTS virus (SFTSV). A previous study reported that human-to-human direct transmission of SFTSV can occur. However, potential animal-to-animal transmission of SFTSV without ticks has not been fully clarified. Thus, the objective of this study was to investigate potential mice-to-mice transmission of SFTSV by co-housing three groups of mice [i.e., wild-type mice (WT), mice injected with an anti-type I interferon-α receptor-blocking antibody (IFNAR Ab), and mice with knockout of type I interferon-α receptor (IFNAR KO)] as spreaders or recipients with different immune competence. As a result, co-housed IFNAR Ab and IFNAR KO mice showed body weight loss with SFTS viral antigens detected in their sera, extracorporeal secretions, and various organs. Based on histopathology, white pulp atrophy in the spleen was observed in all co-housed mice except WT mice. These results obviously show that IFNAR Ab and IFNAR KO mice, as spreaders, exhibited higher transmissibility to co-housed mice than WT mice. Moreover, IFNAR KO mice, as recipients, were more susceptible to SFTSV infection than WT mice. These findings suggest that type I interferon signaling is a pivotal factor in mice intraspecies transmissibility of SFTSV in the absence of vectors such as ticks.

Efficacy of recombinant enolase as a candidate vaccine against Haemaphysalis longicornis tick infestation in mice.

Tick infestation causes a significant threat to human and animal health, requiring effective immunological control methods. This study aimed to investigate the potential of recombinant Haemaphysalis longicornis enolase protein for tick vaccine development. The exact mechanism of the recently identified enolase protein from the H. longicornis Jeju strain remains poorly understood. Enolase plays a crucial role in glycolysis, the metabolic process that converts glucose into energy, and is essential for the motility, adhesion, invasion, growth, and differentiation of ticks. In this study, mice were immunized with recombinant enolase, and polyclonal antibodies were generated. Western blot analysis confirmed the specific recognition of enolase by the antiserum. The effects of immunization on tick feeding and attachment were assessed. Adult ticks attached to the recombinant enolase-immunized mice demonstrated longer attachment time, increased blood-sucking abilities, and lower engorgement weight than the controls. The nymphs and larvae had a reduced attachment rate and low engorgement rate compared to the controls. Mice immunized with recombinant enolase expressed in Escherichia coli displayed 90% efficacy in preventing tick infestation. The glycolytic nature of enolase and its involvement in crucial physiological processes makes it an attractive target for disrupting tick survival and disease transmission. Polyclonal antibodies recognize enolase and significantly reduce attachment rates, tick feeding, and engorgement. Our findings indicate that recombinant enolase may be a valuable vaccine candidate for H. longicornis infection in experimental murine model.

Effects of histamine and antihistamine on the hard tick Haemaphysalis longicornis during blood sucking.

At the time of host attachment, ticks are very sensitive to histamine, but during rapid blood sucking they paradoxically require histamine. Using a rabbit model, we studied the effects of histamine and antihistamine during attachment and fast-feeding in different life stages of Haemaphysalis longicorns. We examined how they responded to histamine and antihistamine by analyzing the detachment rate, histology of feeding lesions, and post-feeding behavior. A significant difference (P<0.01) was found in the detachment rate between experimental and control treatments throughout the observation period. Ticks exhibited a higher detachment rate (30.1%) at 12 h after histamine application during attachment time and on antihistamine-treated skin (25.4%) at 96 h during fast-feeding. After feeding on histamine-treated rabbits, the fully engorged body weights of larvae and nymphs were 0.7±0.36 mg and 3.5±0.65 mg, respectively. An average increase in body weight of 0.6±0.05 mg and 3.2±0.30 mg was observed for larvae and nymphs compared to the respective control weights. Nymphs and adults engorged after antihistamine treatment had an average body weight of 1.3±0.54 mg and 54±0.81 mg, respectively. An average decrease in body weight was observed in antihistamine-treated H. longicornis compared with control nymphs (3.3±0.42 mg) and adults (174±1.78 mg). Skin biopsies were collected after treatment, and differential histopathological characteristics were found between the treatment and control groups. Tick-infested skin collected from rabbits in the antihistamine-treated group lacked erythrocytes in the feeding pool, indicating that antihistamine impaired tick fast-feeding stage.

Catheter-related bacteremia caused by multidrug-resistant Leclercia adecarboxylata in a patient with breast cancer.

We report a multidrug-resistant strain of Leclercia adecarboxylata responsible for catheter-related bacteremia in a 47-year-old female with breast cancer. The isolated strain was resistant to several ß-lactams, aminoglycosides, and folate pathway inhibitors and harbored bla(TEM-1) and bla(CTX-M) group 1 and intl1 genes (dfrA12-orfF-aadA2) as genetic determinants for resistance. Based on a review of the L. adecarboxylata literature, there have been only 4 reports of antibiotic-resistant strains. To our knowledge, this is the first report of an L. adecarboxylata strain with simultaneous resistance to ß-lactams, aminoglycosides, and sulfonamides.

The effect of oculo-acupuncture on recovery from ethylene glycol-induced acute renal injury in dogs.

The potential recovery effect by oculo-acupuncture (OA) on ethylene glycol-induced acute renal injury in dogs was investigated. Acute renal damage was induced by ingestion of ethylene glycol in six mongrel dogs. The dogs were assigned to control (three dogs) and experimental (three dogs) groups. The control group did not receive any treatment, while the experimental group was treated with oculo-acupuncture at kidney/urinary bladder region plus zhong jiao region of the eyes after the induction of renal damage. Serum blood urea nitrogen (BUN), creatinine, sodium (Na), chloride (Cl), and potassium (K) were measured in both control and experimental groups. The blood RBC and Hb were also examined. The serum BUN and creatinine activities in the experimental group were lower than those in the control group, the serum Na and Cl had the irregular change in both groups, and the blood Hb in the control and experimental group showed decreasing tendency. Significant differences were observed on the 3rd and 7th day in BUN, 7th day in creatinine, 2nd day in Na and Cl, and 7th day in Hb when compared to the control group. Whereas, serum K concentration and RBC in the experimental group did not change significantly. The recovery findings of the renal injury were also observed in the experimental group histopathologically. In conclusion, OA therapy (kidney/urinary bladder region plus zhong jiao region) was effective for recovery of the renal injury induced by ethylene glycol in dogs.

Drug Induced Sialorrhea and Microfluidic-Chip-Electrophoretic Analysis of Engorged Adult Female Tick Saliva of Haemaphysalis longicornis (Acari: Ixodidae).

BACKGROUND: The aim of the present study was to induce salivation in Haemaphysalis longicornis to increase saliva production and to characterize the collection of proteins present in the collected saliva using on-chip-electrophoresis. METHODS: Saliva of adult female engorged H. longicornis was collected by treatment with 0.2% dopamine hydrochloride. All protein samples were characterized by SDS-PAGE electrophoresis using a microfluidic High Sensitivity Protein Assay 250 kit by 2100 Bioanalyzer (Agilent Technologies, USA) under non-reducing conditions. RESULTS: The average salivary protein concentration was 0.169 µg/µl/tick and saliva secretion decreased with increased time of tick detachment from the host. Saliva secretion volume increased to 3.56 µl in the group of ticks with a body weight between 301-350 mg as compared to higher and lower body weight groups. On-chip-electrophoresis results show 13 distinct bands ranging from 9.9 to 294 kDa. CONCLUSION: Based on molecular weight, the putative salivary proteins are comprised of proline-rich proteins, triabin, apyrase members of the 12-kDa protein family, platelet inhibitors and anti-inflammatory proteins as tick saliva contains anti-inflammatory components.

Convallaria keiskei as a novel therapeutic alternative for salivary gland cancer treatment by targeting myeloid cell leukemia-1.

BACKGROUND: Various chemotherapeutic agents have been used largely for the treatment of salivary gland cancer. However, results are disappointing, and these agents can cause some serious side effects. Therefore, recent studies have focused on the possible roles of natural products to overcome these limitations. METHODS: Salivary gland cancer cells treated with or without Convallaria keiskei (MECK) for 24 hours. Apoptotic changes were evaluated by live/dead assay, immunoblotting, and expression levels of caspase-3 and B-cell lymphoma-2 family member. RESULTS: MECK significantly inhibited salivary gland cancer growth. At the molecular level, MECK dramatically reduced myeloid cell leukemia-1 (Mcl-1) in a translation-dependent manner and thereby induced apoptosis through Bax/Bid. Furthermore, we found that Mcl-1 could be a potential therapeutic target of MECK-induced apoptosis and its stability is regulated by extracellular signal-regulated kinases 1/2 (ERK1/2) signaling CONCLUSION: MECK can be used as a safe and efficient therapeutic alternative for the treatment of salivary gland cancer. © 2015 Wiley Periodicals, Inc. Head Neck 38: E761-E770, 2016.

Immunization of mice with recombinant P27/30 protein confers protection against hard tick Haemaphysalis longicornis (Acari: Ixodidae) infestation.

The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of Haemaphysalis longicornis, and identified the P27/30 is a troponin I-like protein. In this study, the recombinant P27/30 (rP27/30) expressed in Escherichia coli was used to immunize mice and the mice were challenge-infested with ticks at different developmental stages of the same species. The rP27/30 protein stimulated a specific protective anti-tick immune response in mice, evidenced by the statistically significant longer pre-feeding periods in adult ticks. Furthermore, significantly longer feeding periods were noted in both larval and adult ticks. On the other hand, only larval ticks exhibited low attachment rates (31.1%). Immunization of mice with rP27/30 protein confers protection against hard tick Haemaphysalis longicornis infestation. These results demonstrated that the rP27/30 protein might be a useful vaccine candidate antigen for biological control of ticks.

Clostridium tertium bacteremia in a patient with glyphosate ingestion.

BACKGROUND: Clostridium tertium is distributed in the soil and in animal and human gastrointestinal tracts. C. tertium has been isolated from patients with blood diseases, immune disorders, and abdominal surgeries. Glyphosate is toxic, causing cause eye and skin irritation, gastrointestinal pain, and vomiting. Ingestion of herbicides modifies the gastrointestinal environment, which stresses the living organisms. However, there has been little attention to cases of bacteremia in patients recovering from suicide attempt by ingesting herbicide. CASE REPORT: Clostridium tertium was identified in a 44-year-old female who attempted suicide by glyphosate (a herbicide) ingestion. The 16S rRNA sequences from all colonies were 99% identical with that of C. tertium (AB618789) found on a BLAST search of the NCBI database. The bacterium was cultured on TSA under aerobic and anaerobic conditions. Antimicrobial susceptibility tests performed under both aerobic and anaerobic conditions showed that the bacterium was susceptible to penicillin, a combination of ß-lactamase inhibitor and piperacillin or amoxicillin, and first- and second- generation cephalosporins. However, it was resistant to third- and fourth-generation cephalosporins. CONCLUSIONS: Glyphosate herbicide might be a predisposing factor responsible for the pathogenesis of C. tertium. The results highlight the need for careful diagnosis and selection of antibiotics in the treatment of this organism.

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study.

BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.

Detection of four important Eimeria species by multiplex PCR in a single assay.

The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl.

Inhibition of specificity protein 1 by dibenzylideneacetone, a curcumin analogue, induces apoptosis in mucoepidermoid carcinomas and tumor xenografts through Bim and truncated Bid.

OBJECTIVES: Dibenzylideneacetone (DBA), a curcumin analogue that has anti-cancer activity in a variety of tumor cells. In this study, we investigated the apoptotic effects of DBA and its molecular mechanism in human mucoepidermoid carcinoma (MEC) cell lines and tumor xenografts. MATERIAL AND METHODS: The apoptotic effects and related molecular mechanisms of DBA on MEC cell lines were evaluated using cell viability assay, DAPI staining, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and Dual-luciferase Reporter Assay. The anti-tumor activity using in vivo were determined by Nude mouse xenograft assay and histopathological examination. RESULTS: DBA decreased cell viability and induced apoptosis in MEC cells. These events were accompanied by inhibition of specificity protein 1 (Sp1). DBA did not induce major changes in Sp1 mRNA and promoter activity. Furthermore, inhibition of protein synthesis by cycloheximide demonstrated that DBA decreased Sp1 protein stability, but DBA did not attenuate phosphorylation of eIF4E. DBA also increased Bim and truncated Bid (t-Bid) via Sp1. Finally, DBA exhibited significant anti-tumor activity in athymic nude mice xenografts bearing MC-3 cells by regulating Sp1, Bim and t-Bid without any systemic toxicity. CONCLUSION: These results elucidate a crucial apoptotic mechanism of DBA and suggest that DBA may be a potent anticancer drug candidate for MEC.