Cells transformed by PLC-gamma 1 overexpression are highly sensitive to clostridium difficile toxin A-induced apoptosis and mitotic inhibition

Phospholipase C-γl (PLC-γl) expression is associated with cellular transformation. Notably, PLC-gamma is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-γl overexpression. We found that PLC-γl-transformed cells, but not vectortransformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-γl-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-γl-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-γl is highly up-regulated.     

Efficacy test of Polycan, a beta-glucan originated from Aureobasidium pullulans SM-2001, on anterior cruciate ligament transection and partial medial meniscectomy-induced-osteoarthritis rats

The object of this study was to assess the efficacy of Polycan from Aureobasidium pullulans SM-2001, which is composed mostly of beta-1,3-1,6-glucan, on osteoarthritis (OA)-induced by anterior cruciate ligament transection and partial medial meniscectomy (ACLT&PMM). Three different dosages of Polycan (85, 42.5, and 21.25 mg/kg) were orally administered once a day for 84 days to male rats a week after ACLT&PMM surgery. Changes in the circumference and maximum extension angle of each knee, and in cartilage histopathology were assessed using Mankin scores 12 weeks after Polycan administration. In addition, cartilage proliferation was evaluated using bromodeoxyuridine (BrdU). As the result of ACLT&PMM, classic OA was induced with increases in maximum extension angles, edematous knees changes, and capsule thickness, as well as decreases in chondrocyte proliferation, cartilages degenerative changes, and loss of articular cartilage. However, these changes (except for capsule thickness) were markedly inhibited in all Polycan- and diclofenac sodium-treated groups compared with OA control. Although diclofenac sodium did not influence BrdU uptake, BrdU-immunoreactive cells were increased with all dosages of Polycan, which means that Polycan treatment induced proliferation of chondrocytes in the surface articular cartilage of the tibia and femur. The results obtained in this study suggest that 84 days of continuous oral treatment of three different dosages of Polycan led to lesser degrees of articular stiffness and histological cartilage damage compared with OA controls 91 days after OA inducement, suggesting that the optimal Polycan dosage to treat OA is 42.5 mg/kg based on the present study.     

Multiplexing analysis of the polyspecific intrathecal immune response in multiple sclerosis

Intrathecal synthesis of the antibodies specific to neurotrofic viruses: measles (M), rubella (R), Varicella-Zoster (Z), and/or H. simplex (H), known as "MRZH-reaction" plays important diagnostic role in multiple sclerosis (MS). Whereas the analysis of the oligoclonal IgG bands provides high sensitivity, the MRZH-reaction shows high specificity, and hence these methods complement each other. For the first time we applied multiplexing bead-based technology to simultaneously analyze cerebrospinal fluid (CSF) and serum concentrations of antibodies against these viruses, and to calculate the antibody specific indices (ASI's). The method shows reasonable precision: intra-assay, 2.9-6.7%, and inter-assay, 2.0-3.2%. The results are comparable with these obtained with other methods (ELISAs), including two runs of the certified external quality control schemes. Eighty-one percent of the MS cases (n=27) and none of the sex- and age-matched controls (n=14), except one subject with "borderline" anti-measles ASI of 1.5, showed intrathecal synthesis of IgG against at least one of the viruses discussed. The ratios of the MRZH-positive cases in the MS group were: 12/22 for M, 12/19 for R, 13/26 for Z, and 7/26 for H. We conclude that the multiplexing technology can be applied as a tool to study the intrathecal immune response in the diagnosis of MS.     

An Image-Based Drug Susceptibility Assay Targeting the Placental Sequestration of Plasmodium falciparum-Infected Erythrocytes

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.     

Noninvasive Optical Measurement of Cerebral Blood Flow in Mice Using Molecular Dynamics Analysis of Indocyanine Green

In preclinical studies of ischemic brain disorders, it is crucial to measure cerebral blood flow (CBF); however, this requires radiological techniques with heavy instrumentation or invasive procedures. Here, we propose a noninvasive and easy-to-use optical imaging technique for measuring CBF in experimental small animals. Mice were injected with indocyanine green (ICG) via tail-vein catheterization. Time-series near-infrared fluorescence signals excited by 760 nm light-emitting diodes were imaged overhead by a charge-coupled device coupled with an 830 nm bandpass-filter. We calculated four CBF parameters including arrival time, rising time and mean transit time of a bolus and blood flow index based on time and intensity information of ICG fluorescence dynamics. CBF maps were generated using the parameters to estimate the status of CBF, and they dominantly represented intracerebral blood flows in mice even in the presence of an intact skull and scalp. We demonstrated that this noninvasive optical imaging technique successfully detected reduced local CBF during middle cerebral artery occlusion. We further showed that the proposed method is sufficiently sensitive to detect the differences between CBF status in mice anesthetized with either isoflurane or ketamine–xylazine, and monitor the dynamic changes in CBF after reperfusion during transient middle cerebral artery occlusion. The near-infrared optical imaging of ICG fluorescence combined with a time-series analysis of the molecular dynamics can be a useful noninvasive tool for preclinical studies of brain ischemia.     

The effect on human balance of standing with toe-extension

Background

Postural balance is vital for safely carrying out many daily activities, such as locomotion. The purpose of this study was to determine how changes in normal standing (NS) and standing with toe-extension (SWT) impact postural control during quiet standing. Furthermore, the research aimed to examine the extent to which the effect of these factors differed between genders.

Methodology/Principal Findings

Thirty healthy young adults (age = 21.2±1.3 y; height = 1.63±0.07 m; mass = 56.0±9.3 kg) with no prior lower limb injuries participated in the study. A postural stability test using the Biodex Balance System was used for both NS and SWT conditions. The three measurements from the BBS were Overall Stability Index (OSI), Medial-Lateral Stability Index (MLSI) and Anterior-Posterior Stability Index (APSI). No significant difference was found between NS and SWT in the OSI, MLSI or APSI (F _{2, 28} = 3.357, p = 0.077). The main difference between the stability index scores was significant (F _{2, 28} = 275.1, p<0.001). The Bonferroni post-hoc test showed significant differences between the OSI and MLSI (p<0.001); the OSI and APSI (p<0.001); and the MLSI and the APSI (p<0.001). Significant differences were found during NS (p<0.001), for the MLSI when compared with the APSI, but this was not found during the SWT condition. Additionally, no gender effects were proven to exist that altered postural sway during quiet standing.

Conclusions/Significance

This study reveals significant interaction between the stability indices measured; OSI, APSI and MLSI in both NS and SWT. Standing with toe extended does not have a significant impact on an individual’s ability to control their balance during normal quiet standing. However, the findings revealed that the sway tendency in the medial-lateral direction might serve as a factor in an individual’s ability to regain balance.     

Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper

Infection of virus (such as nodavirus and iridovirus) and bacteria (such as Vibrio anguillarum) in farmed grouper has been widely reported and caused large economic losses to Taiwanese fish aquaculture industry since 1979. The multiplex assay was used to detect dual viral infection and showed that only nervous necrosis virus (NNV) can be detected till the end of experiments (100% mortality) once it appeared. In addition, iridovirus can be detected in a certain period of rearing. The results of real-time PCR and in situ PCR indicated that NNV, in fact, was not on the surface of the eggs but present in the embryo, which can continue to replicate during the embryo development. The virus may be vertically transmitted by packing into eggs during egg development (formation) or delivering into eggs by sperm during fertilization. The ozone treatment of eggs may fail to remove the virus, so a new strategy to prevent NNV is needed.