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BackgroundProteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.ResultsBLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.ConclusionsThe identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

Background: The duration of the QT interval on the standard electrocardiogram (ECG) is measured from the beginning of the QRS complex (depolarization of the cardiac myocyte) to the end of the T-wave (completion of the repolarization phase of the cardiac myocyte). Repolarization is a result of currents generated by the outward flow of K + through the K + channels. Obstruction of ion flow in the channel leads to delayed repolarization, evidenced by a prolonged QT interval. Clinically, this is known as the long QT syndrome (LQTS), which, when expressed, can lead to severe cardiac arrhythmias and sudden death. Obstruction of K + ion flow can result from gene mutations (eg, the human ether-ago go related gene [hERG]) resulting in phenotypic abnormalities in K + channels and/or common structurally diverse drugs. These gene abnormalities or drug-induced changes result in decreased cardiac delayed-rectifier K + current (I Kr , or KV11.1) in congenital or acquired LQTS, respectively. Increased risk of LQTS is a major drug development hurdle, and many drugs have been withdrawn during preclinical development, assigned black box warnings following approval, or withdrawn from the market. Autosomal recessive or dominant LQTS based upon 500 possible mutations in ten different genes coding for K + channels has an incidence of 1:3000 or about 100,000 persons in the USA. Prolonged QT intervals or risk of LQTS occurs in 2.5% of the asymptomatic US population. The probability of cardiac death in patients with asymptomatic congenital LQTS who are concomitantly medicated with LQTS-inducing drugs appears to have increased. Methods: E-4031 (methanesulfanalide), terfenadine (Seldane ®), curcumin, liposomal curcumin, empty liposomes, empty liposomes vortexed with E-4031, or terfenadine and empty liposomes vortexed with curcumin were assayed for their effects on the K +-selective I Kr tail current inhibition using human embryonic kidney (HEK 293) cells stably transfected with the hERG gene via the whole-cell manual patch clamp technique. Results: E-4031, terfenadine, and curcumin inhibit I Kr channel following nM-to-µM exposures. Empty liposomes had no effect on I Kr. Both the liposomal curcumin formulation and vortexed mixtures of empty liposomes and curcumin prevented the I Kr inhibitory effect of curcumin in a dose-dependent manner. Empty liposomes vortexed with E-4031 prevented the effect of E-4031 to a lesser extent, while empty liposomes vortexed with terfenadine did not alter its I Kr inhibitory activity. Conclusion: Curcumin causes an inhibition of the hERG tail current density. The liposomal curcumin formulation, as well as a mixture of empty liposomes with curcumin or with E-4031, blocked drug-induced I Kr inhibition. However, empty liposomes mixed with terfenadine did not alter terfenadine's I Kr inhibitory effects. The liposomes protected against the inhibitory effect of some compounds on the K +-selective I Kr current, independent of their potency.

Background/Aim: Curcumin is being widely investigated for its anticancer properties and studies in the literature suggest that curcumin distributes to a higher degree in tumor versus non-tumor cells. In the current study, we report on investigation of the distribution of curcumin and metabolism to THC in PBMC from healthy individuals and chronic lymphocytic leukemia (CLL) patients following exposure to Lipocurc™ (liposomal curcumin). Materials and Methods: The time and temperature-dependent distribution of liposomal curcumin and metabolism to tetrahydrocurcumin (THC) were measured in vitro in human peripheral blood mononuclear cells (PBMC) obtained from healthy individuals, PBMC HI (cryopreserved and freshly isolated PBMC) and CLL patients (cryopreserved PBMC) with lymphocyte counts ranging from 17-58×10 6 cells/ml (PBMC CLL,Grp 1 ) and >150×10 6 cells/ml (PBMC CLL,Grp 2 ). PBMC were incubated in plasma protein supplemented media with Lipocurc™ for 2-16 min at 37˚C and 4˚C and the cell and medium levels of curcumin determined by LC-MS/MS. Results: PBMC from CLL patients displayed a 2.2-2.6-fold higher distribution of curcumin compared to PBMC HI . Curcumin distribution into PBMCCLL, Grp 1/Grp 2 ranged from 384.75 -574.50 ng/g w.w. of cell pellet and was greater compared to PBMC HI that ranged from 122.27-220.59 ng/g w.w. of cell pellet following incubation for up to 15-16 min at 37˚C. The distribution of curcumin into PBMC CLL,Grp 2 was time-dependent in comparison to PBMC HI which did not display a time-dependence and there was no temperature-dependence for curcumin distribution in either cell type. Curcumin was metabolized to THC in PBMC. The metabolism of curcumin to THC was not markedly different between PBMC HI (range=23.94-42.04 ng/g w.w. cell pellet) and PBMC CLL,Grp 1/Grp 2 (range=23. 08-48.22 ng/g. w.w. cell pellet). However, a significantly greater time and temperature-dependence was noted for THC in PBMC CLL,Grp 2 compared to PBMC HI . Conclusion: Curcumin distribution into PBMC from CLL patients was higher compared to PBMC from healthy individuals, while metabolism to THC was similar. The potential for a greater distribution of curcumin into PBMC from CLL patients may be of therapeutic benefit. Curcumin (diferuloylmethane) is being extensively investigated for its anticancer properties with several studies indicating its potential for therapeutic benefit. Clinical studies with curcumin have indicated that it has limited, but significant anticancer activity in part as a consequence of its poor oral availability. Significant anticancer effects upon oral administration of curcumin powder have been observed in pancreatic and colorectal cancer and leukemia as well as a variety of high risk and pre-malignant lesions (1-3) including altering the regulation of oncogenes (4). In studies in mice, curcumin enhanced the cytotoxicity of antigen-specific CD8 T-cells (5) and prevented the loss of T-cells, expanded central member T cell populations, reversed the type 2 immune bias and attenuated the tumor-induced inh...

In the past, efforts have been made to determine the influence of sleep quantity and its deprivation, on functioning efficiency of human beings. However, determination of sleeping patterns that could improve intellectual performance has been largely neglected. This study is designed to discover the effects of different sleeping patterns on academic performance among medical students. A descriptive study was carried out in King Edward Medical University in Lahore, Pakistan during a six-month time span from May 11 th , 2011 to September 30 th , 2011. Of the total population of 1350 students in King Edward Medical University, 591 undergraduates were included in the study. A questionnaire designed on sleeping patterns and academic performance was distributed in May 2011. What was described as outstanding students were greater in number in group 4 (7/19) 36.8% and group 6 (6/19) 31.6%. Above average students with sleeping patterns were in group 4 (13/37) 35.1% and group 6 (10/37) 27%. Average students were shown to have sleeping patterns of group 4 (11/25) 44% and group 6 (7/25) 28%. Below average students were shown to have sleeping patterns of group 4 (3/3) 100%. Most of our students had a reduction in the total amount of sleeping hours throughout the years. Midnight to 6 o'clock in the morning with an afternoon nap was the sleeping pattern that was most commonly seen in all groups. We concluded that different sleeping patterns do not affect the performance of medical students in the academic prospective. Many other factors may be involved in the lack of significant achievement, in order to prove that the sleeping patterns are not related to the academic performance, and more data would need to be collected.