Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV

Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. Human Papillomaviruses (HPVs) which are heterogeneous groups of small double stranded DNA viruses are considered as the primary cause of cervical cancer, involved in 90% of all Cervical Cancers. Two early HPV genes, E6 and E7, are known to play crucial role in tumor formation. E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes. The strategy of the research work was to target the site of interaction of Rb1 -E7 & p53-E6. A total of 88 compounds were selected for molecular screening, based on comprehensive literature survey for natural compounds with anti-cancer activity. Molecular docking analysis was carried out with Molegro Virtual Docker, to screen the 88 chosen compounds and rank them according to their binding affinity towards the site of interaction of the viral oncoproteins and human tumor suppressor proteins. The docking result revealed that Nicandrenone a member of Withanolides family of chemical compounds as the most likely molecule that can be used as a candidate drug against HPV induced cervical cancer. ABBREVIATIONS: HPV - Human Papiloma Virus, HTSP - Human Tumor Suppressor Proteins, VOP - Viral oncoproteins.     

产S-酰胺酶培养基统计学筛选与响应面优化

利用Design Expert软件中的两水平实验设计和响应面法,对发酵生产S-酰胺酶(可用于拆分2,2-二甲基环丙甲酰胺外消旋体)的培养基进行了优化。采用Plackett-Burman(PB)设计对培养基中相关影响因素的效应进行评价并筛选出了有显著效应的葡萄糖、酵母粉及2,2-二甲基环丙甲酰胺浓度,其他因素对酰胺酶产量的影响不显著。然后用旋转中心组和实验设计及响应面分析确定了主要影响因素的最佳条件,在优化的培养基中,酰胺酶产量达到168 U/L,比优化前的80 U/L提高了110.0%。     

Population ecology of the endangered butterflies Maculinea teleius and M. nausithous and the implications for conservation

Butterflies of the genus Maculinea are highly endangered in Europe. The cuckoo species M. rebeli has been thoroughly investigated through both empirical and modelling studies, but less is known about the population ecology of predatory Maculinea. We present the findings of a 2-year research study on sympatric populations of two endangered butterflies: Maculinea teleius and M. nausithous in the Kraków region, southern Poland. The study comprised mark–release–recapture sampling and laboratory rearing of butterflies from larvae collected in the field. For both species the sex ratio was slightly, but consistently, female-biased and there was little year-to-year change in the seasonal population sizes. Daily numbers showed greater variation between the 2 years of the study due to the differences in daily survival rate. The average life span of laboratory-raised butterflies kept in ideal conditions was more than 6 days, compared to only 2–3 days in the field. The recruitment of both males and females consistently followed a bimodal pattern. A small proportion of individuals (maximum 25%) changed sites, in spite of relatively short distances of ca. 100 m separating them. The results indicate that populations of both species are typically stable within their sites, possibly due to larval polymorphism, but there is little inter-site mobility and thus landscape corridors seem necessary to enhance metapopulation viability. A further problem to be considered in the conservation of Maculinea butterflies is the fact that their very short life span in relation to flight-period length reduces the effective population size.     

Seasonal and storm event nutrient removal by a created wetland in an agricultural watershed

This study examines the effectiveness of a 1.2-ha created/restored emergent marsh at reducing nutrients from a 17.0 ha agricultural and forested watershed in the Ohio River Basin in west central Ohio, USA, during base flow and storm flow conditions. The primary source of water to the wetland was surface inflow, estimated in water year 2000 (October 1999–September 2000) to be 646 cm/year. The wetland also received a significant amount of groundwater discharge at multiple locations within the site that was almost the same in quantity as the surface flow. The surface inflow had 2-year averages concentrations of 0.79, 0.033, and 0.16 mg L⁻¹ for nitrate + nitrite (as N), soluble reactive phosphorus (SRP), and total phosphorus (TP), respectively. Concentrations of nitrate–nitrite, SRP, and TP were 40, 56, and 59% lower, respectively, at the outflow than at the inflow to the wetland over the 2 years of the study. Concentrations of SRP and TP exported from the wetland increased significantly (α = 0.05) during precipitation events in 2000 compared to dry weather flows, but concentrations of nitrate–nitrite did not increase significantly. During these precipitation events the wetland retained 41% of the nitrate–nitrite, 74% of the SRP, and 28% of the TP (by mass). The wetland received an average of 50 g N m⁻² per year of nitrate–nitrite and 7.1 g m⁻² per year of TP in 2000. Retention rates for the wetland were 39 g N m⁻² per year of nitrates and 6.2 g P m⁻² per year. These are close to rates suggested in the literature for sustainable non-point source retention by wetlands. The design of this wetland appears to be suitable as it retained a significant portion of the influent nutrient load and did not lose much of its retention capacity during heavy precipitation events. Some suggestions are given for further design improvements.     

Reactor Design for the Novozym 435 ® -catalysed Enantioselective Esterification of 4-methyloctanoic Acid

The bench scale Novozym 435 ® catalysed esterification of 4-methyloctanoic acid with ethanol was studied at 35°C. Esterification in a batch reactor (molar ratio of 1:8 (acid:EtOH)) resulted in the isolation of the enantiomerically enriched product (ee p =81%) and substrate (ee s =93%). In order to integrate reaction and separation, liquid-vapour equilibria calculations were performed showing that an excess of ethanol results in a very low ester fraction in the vapour phase. Since this is undesirable for an integrated process of reaction and product removal, a repeated batch reaction was performed using a molar ratio of 10:1 (acid:EtOH). After six cycles (45% conversion) the ee of 4-methyloctanoic acid ethyl ester turned out to be 80%. For different E values the ee p was calculated for batch and repeated batch reactions. It was shown that in all cases the ee p was higher for the repeated batch reaction. However, the product is not enantiopure since the E value of the reaction is rather low at the low ethanol concentration used. An alternative approach would be the continuous separation of the product during the reaction. A mathematical model was developed to describe esterification in a packed bed reactor integrated with product separation. This model shows that integration of reaction and product removal in advance is not suitable either to obtain an enantiomerically pure product. Since the optimal reaction conditions (high ethanol concentration) and the optimal separation system (low ethanol concentration) do not match in this reaction, the preference is given to the batch reaction at high ethanol concentrations because in that case the highest enantioselectivity of the enzyme is obtained.     

Bacterial versus human sphingosine-1-phosphate lyase (S1PL) in the design of potential S1PL inhibitors

A series of potential active-site sphingosine-1-phosphate lyase (S1PL) inhibitors have been designed from scaffolds 1 and 2, arising from virtual screening using the X-ray structures of the bacterial (StS1PL) and the human (hS1PL) enzymes. Both enzymes are very similar at the active site, as confirmed by the similar experimental kinetic constants shown by the fluorogenic substrate RBM13 in both cases. However, the docking scoring functions used probably overestimated the weight of electrostatic interactions between the ligands and key active-site residues in the protein environment, which may account for the modest activity found for the designed inhibitors. In addition, the possibility that the inhibitors do not reach the enzyme active site should not be overlooked. Finally, since both enzymes show remarkable structural differences at the access channel and in the proximity to the active site cavity, caution should be taken when designing inhibitors acting around that area, as evidenced by the much lower activity found in StS1PL for the potent hS1PL inhibitor D.     

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (R_Select) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of R_Select proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.