Molecular docking and pharmacophore model studies of Rho kinase inhibitors

Molecular docking and pharmacophore model approaches were used to characterise the binding features of four different series of Rho kinase (ROCK) inhibitors. Docking simulation of 20 inhibitors with ROCK was performed. The binding conformations and binding affinities of these inhibitors were obtained using AutoDock 4.0 software. The predicted binding affinities correlate well with the activities of these inhibitors (R ² = 0.904). 3D pharmacophore models were generated for ROCK based on highly active inhibitors implemented in Catalyst 4.11 program. The best pharmacophore model consists of one hydrogen bond acceptor feature and two hydrophobic features, and they all seemed to be essential for inhibitors in terms of their binding activities. It is anticipated that the findings reported in this paper may provide very useful information for designing new ROCK inhibitors.     

Caspase-2 Short Isoform Interacts with Membrane-Associated Cytoskeleton Proteins to Inhibit Apoptosis

Caspase-2 (casp-2) is the most conserved caspase across species, and is one of the initiator caspases activated by various stimuli. The casp-2 gene produces several alternative splicing isoforms. It is believed that the long isoform, casp-2L, promotes apoptosis, whereas the short isoform, casp-2S, inhibits apoptosis. The actual effect of casp-2S on apoptosis is still controversial, however, and the underlying mechanism for casp-2S-mediated apoptosis inhibition is unclear. Here, we analyzed the effects of casp-2S on DNA damage induced apoptosis through “gain-of-function” and “loss-of-function” strategies in ovarian cancer cell lines. We clearly demonstrated that the over-expression of casp-2S inhibited, and the knockdown of casp-2S promoted, the cisplatin-induced apoptosis of ovarian cancer cells. To explore the mechanism by which casp-2S mediates apoptosis inhibition, we analyzed the proteins which interact with casp-2S in cells by using immunoprecipitation (IP) and mass spectrometry. We have identified two cytoskeleton proteins, Fodrin and α-Actinin 4, which interact with FLAG-tagged casp-2S in HeLa cells and confirmed this interaction through reciprocal IP. We further demonstrated that casp-2S (i) is responsible for inhibiting DNA damage-induced cytoplasmic Fodrin cleavage independent of cellular p53 status, and (ii) prevents cisplatin-induced membrane blebbing. Taken together, our data suggests that casp-2S affects cellular apoptosis through its interaction with membrane-associated cytoskeletal Fodrin protein.     

Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115

Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.     

A novel domain-duplicated SlitFAR3 gene involved in sex pheromone biosynthesis in Spodoptera litura

Fatty acyl reductases (FARs) are key enzymes that participate in sex pheromone biosynthesis by reducing fatty acids to fatty alcohols. Lepidoptera typically harbor numerous FAR gene family members. Although FAR genes are involved in the biosynthesis of sex pheromones in moths, the key FAR gene of Spodoptera litura remains unclear. In this work, we predicted 30 FAR genes from the S. litura genome and identified a domain duplication within gene SlitFAR3, which exhibited high and preferential expression in the sexually mature female pheromone glands (PGs) and a rhythmic expression pattern during the scotophase of sex pheromone production. The molecular docking of SlitFAR3, as predicted using a 3D model, revealed a co-factor NADPH binding cavity and 2 substrate binding cavities. Functional expression in yeast cells combined with comprehensive gas chromatography indicated that the SlitFAR3 gene could produce fatty alcohol products. This study is the first to focus on the special phenomenon of FAR domain duplication, which will advance our understanding of biosynthesis-related genes from the perspective of evolutionary biology.     

Surgical Complications of Primary Rhegmatogenous Retinal Detachment: A Meta-Analysis

Background

To investigate the surgical complications of scleral buckling (SB) and pars plana vitrectomy (PPV) performed on primary rhegmatogenous retinal detachment (RRD) and to discover which surgical procedures bring fewer complications.

Methods

An electronic literature search using the PubMed database, ISI Web of Knowledge and the Cochrane Central Register of Controlled Trials to identify randomized controlled trials and observational studies comparing SB with PPV on primary RRD. Outcome measures included intra-operative complications and early and late post-operative complications.

Results

During the operation, significantly less subretinal hemorrhage occurred in the PPV group than in the SB group (OR = 4.71; 95%CI, 1.33–16.64; p = 0.02) and the hypotony incidence was significantly higher in the SB group (OR = 18.24; 95%CI, 2.37–140.44; p = 0.005); however, the occurrence of iatrogenic breaks was significantly lower in the SB group (OR = 0.05; 95%CI, 0.01–0.21; p<0.0001). In the early stage of post-operation, significantly higher incidence of choroidal detachment was identified in the SB group than in the PPV group (OR = 10.19; 95%CI, 2.36–44.09; p = 0.002); patients undergoing SB had significantly higher odds of residual subretinal fluid (OR = 14.71; 95%CI, 1.84–117.32; p = 0.01); the occurrence of high intraocular pressure was significantly lower in the SB group (OR = 0.46; 95%CI, 0.23–0.89; p = 0.02); and no significant difference was shown in the incidence of epithelia defect (p = 0.37) between the two groups. In the late stage of post-operation, the incidence of diplopia/extraocular muscle dysfunction was significantly higher in the SB group (OR = 4.04; 95%CI, 1.30–12.52; p = 0.02); and significantly less cataract was observed in the SB group (OR = 0.20; 95%CI, 0.14–0.30; p<0.00001); no significant difference was found in the incidences of cystoid macular edema (p = 0.65), macular pucker (p = 0.52), post-operative proliferative vitreoretinopathy (p = 0.73) and epiretinal membrane (p = 0.47) in other late post-operative complications.

Conclusions

This meta-analysis suggests that PPV could be considered as potential surgical management on primary RRD.     

Adverse Events of Anti-Tumor Necrosis Factor α Therapy in Ankylosing Spondylitis

Objective

This study aims to investigate the prevalence of short-term and long-term adverse events associated with tumor necrosis factor-α (TNF-α) blocker treatment in Chinese Han patients suffering from ankylosing spondylitis (AS).

Methods

The study included 402 Chinese Han AS patients treated with TNF-α blockers. Baseline data was collected. All patients were monitored for adverse events 2 hours following administration. Long-term treatment was evaluated at 8, 12, 52 and 104 weeks follow-up for 172 patients treated with TNF-α blockers.

Results

Short-term adverse events occurred in 20.15% (81/402), including rash (3.5%; 14/402), pruritus (1.2%; 5/402), nausea (2.2%; 9/402), headache (0.7%; 3/402), skin allergies (4.0%; 16/402), fever (0.5%; 2/402), palpitations (3.0%; 12/402), dyspnea (0.5%; 2/402), chest pain (0.2%; 2/402), abdominal pain (1.0%; 4/402), hypertension (2.2%; 9/402), papilledema (0.5%; 2/402), laryngeal edema (0.2%; 1/402) and premature ventricular contraction (0.2%; 1/402). Long-term adverse events occurred in 59 (34.3%; 59/172) patients, including pneumonia (7.6%; 13/172), urinary tract infections (9.9%; 17/172), otitis media (4.7%; 8/172), tuberculosis (3.5%; 17/172), abscess (1.2%; 2/172), oral candidiasis (0.6%; 1/172), elevation of transaminase (1.7%; 3/172), anemia (1.2%; 2/172), hematuresis (0.6%; 1/172), constipation (2.3%; 4/172), weight loss (0.6%; 1/172), exfoliative dermatitis (0.6%; 1/172). CRP, ESR and disease duration were found to be associated with an increased risk of immediate and long-term adverse events (P<0.05). Long-term treatment with Infliximab was associated with more adverse events than rhTNFR-Fc (P<0.01).

Conclusion

This study reports on the prevalence of adverse events in short-term and long-term treatment with TNF-α blocker monotherapy in Chinese Han AS patients. Duration of disease, erythrocyte sedimentation rate, and c-reactive protein serum levels were found to be associated with increased adverse events with anti-TNF-α therapy. Long-term treatment with Infliximab was associated with more adverse events than rhTNFR-Fc.     

Isolation and characterization of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> LY214, a camptothecin-producing endophytic bacterium from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Camptothecin (CPT) is mainly produced and extracted from Camptotheca acuminata and Nothapodytes foetida for pharmaceutical use, i.e., the starting material for chemical conversion to the clinical CPT-type drugs. As the third largest plant anticancer drug, the heavy demand on CPT from global market leads to many research efforts to identify new sources for CPT production. Herein we report the isolation and characterization of a CPT-producing endophytic bacterium Paenibacillus polymyxa LY214 from Camptotheca acuminata. A 10.7 μg l^?1 of CPT was presented in the fermentation broth of P. polymyxa LY214. Its CPT production decreased sharply when the strain of the 2nd generation of P. polymyxa LY214 was cultured and fermented. However, the CPT production remained relatively constant from 2.8 μg l^?1 of the 2nd generation to 0.8 μg l^?1 of the 8th generation of P. polymyxa LY214 under optimized fermentation conditions. A 15- to 30-fold increase of CPT yield was observed when the optimized fermentation conditions, together with the addition of putative biosynthetic precursors of CPT and adsorbent resin XAD16, were applied to ferment the strains of the 7th and 8th generation of P. polymyxa LY214. Bioinformatics analysis of the relative species of P. polymyxa LY214 indicates its potential to produce CPT, which will be helpful to decipher the mysteries of CPT biosynthesis.