Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.     

AbrB-like transcription factors assume a swapped hairpin fold that is evolutionarily related to double-psi beta barrels

AbrB is a key transition-state regulator of Bacillus subtilis. Based on the conservation of a betaalphabeta structural unit, we proposed a beta barrel fold for its DNA binding domain, similar to, but topologically distinct from, double-psi beta barrels. However, the NMR structure revealed a novel fold, the "looped-hinge helix." To understand this discrepancy, we undertook a bioinformatics study of AbrB and its homologs; these form a large superfamily, which includes SpoVT, PrlF, MraZ, addiction module antidotes (PemI, MazE), plasmid maintenance proteins (VagC, VapB), and archaeal PhoU homologs. MazE and MraZ form swapped-hairpin beta barrels. We therefore reexamined the fold of AbrB by NMR spectroscopy and found that it also forms a swapped-hairpin barrel. The conservation of the core betaalphabeta element supports a common evolutionary origin for swapped-hairpin and double-psi barrels, which we group into a higher-order class, the cradle-loop barrels, based on the peculiar shape of their ligand binding site.     

Long non‐coding RNAs in melanoma

Melanoma is the most lethal cutaneous cancer with a highly aggressive and metastatic phenotype. While recent genetic and epigenetic studies have shed new insights into the mechanism of melanoma development, the involvement of regulatory non‐coding RNAs remain unclear. Long non‐coding RNAs (lncRNAs) are a group of endogenous non‐protein‐coding RNAs with the capacity to regulate gene expression at multiple levels. Recent evidences have shown that lncRNAs can regulate many cellular processes, such as cell proliferation, differentiation, migration and invasion. In the melanoma, deregulation of a number of lncRNAs, such as HOTAIR, MALAT1, BANCR, ANRIL, SPRY‐IT1 and SAMMSON, have been reported. Our review summarizes the functional role of lncRNAs in melanoma and their potential clinical application for diagnosis, prognostication and treatment.     

Changes in odor quality discrimination following recovery from olfactory nerve transection

Following recovery from olfactory nerve transection, animals regain their ability to discriminate between odors. Odor discrimination is restored after new neurons establish connections with the olfactory bulb. However, it is not known if the new connections alter odor quality perception. To address this question, 20 adult hamsters were first trained to discriminate between cinnamon and strawberry odors. After reaching criterion (> or = 90% correct response), half of the animals received a bilateral nerve transection (BTX) and half a surgical sham procedure. Animals were not tested again until day 40, a point in recovery when connections are re-established with the bulb. When BTX animals were tested without food reinforcement, they could not perform the odor discrimination task. Sham animals, however, could discriminate, demonstrating that the behavioral response had not been extinguished during the 40 day period. When reinforcement was resumed, BTX animals were able to discriminate between cinnamon and strawberry after four test sessions. In addition, their ability to discriminate between these two familiar odors was no different than that of BTX and sham animals tested with two novel odors, baby powder and coffee. These findings suggest that, after recovery from nerve transection, there are alterations in sensory perception and that restoration of odor quality discrimination requires that the animal must again learn to associate individual odor sensations with a behavioral response.      

Binding of TPX2 to Aurora A alters substrate and inhibitor interactions

The Aurora kinases are a family of serine/threonine kinases involved in mitosis. The expression of AurA is ubiquitous and cell cycle regulated. It is overexpressed in many tumor types, including breast, colon, and ovarian. TPX2 is a binding partner and activator of AurA. A fragment of TPX2 (residues 1-43) has been shown to be sufficient for binding, kinase activation, and protection from dephosphorylation. We have shown that the addition of TPX2(1-43) increases the catalytic efficiency of AurA. While TPX2 binding has no effect on the turnover number of AurA and does not change the reaction mechanism (characterized here to be a rapid equilibrium random mechanism), it increases the binding affinity of both ATP and a peptide substrate. We have also demonstrated differences in the inhibitor structure-activity relationship (SAR) in the presence or absence of TPX2(1-43). To better understand the differential SAR, we carried out computer modeling studies to gain insight into the effect of TPX2 on the binding interactions between AurA and inhibitors. Our working hypothesis is that TPX2 binding decreases the size and accessibility of a hydrophobic pocket, adjacent to the ATP site, to inhibitors.     

Modulation of kinase-inhibitor interactions by auxiliary protein binding: crystallography studies on Aurora A interactions with VX-680 and with TPX2

VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 A resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a pi-pi interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggest that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.     

Common evolutionary origin of swapped-hairpin and double-psi beta barrels

The core of swapped-hairpin and double-psi beta barrels is formed by duplication of a conserved betaalphabeta element, suggesting a common evolutionary origin. The path connecting the two folds is unclear as the two barrels are not interconvertible by a simple topological modification, such as circular permutation. We have identified a protein family whose sequence properties are intermediate to the two folds. The structure of one of these proteins, Pyrococcus horikoshii PhS018, is also built by duplication of the conserved betaalphabeta element but shows yet a third topology, which we name the RIFT barrel. This topology is widespread in the structure database and spans three folds of the SCOP classification, including the middle domain of EF-Tu and the N domain of F1-ATPase. We propose that swapped-hairpin beta barrels arose from an ancestral RIFT barrel by strand invasion and double-psi beta barrels by a strand swap. We group the three barrel types into a metafold, the cradle-loop barrels.     

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Background

Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.

Methods

DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).

Results

Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).

Conclusion

This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.     

Young patients with colorectal cancer have poor survival in the first twenty months after operation and predictable survival in the medium and long-term: Analysis of survival and prognostic markers

Introduction

Male breast cancer (MBC) is a rare, yet potentially aggressive disease. Although literature regarding female breast cancer (FBC) is extensive, little is known about the etiopathogenesis of male breast cancer. Studies from our laboratory show that MBCs have a distinct immunophenotypic profile, suggesting that the etiopathogenesis of MBC is different from FBCs. The aim of this study was to evaluate and correlate the immunohistochemical expression of cell cycle proteins in male breast carcinoma to significant clinico-biological endpoints.

Methods

75 cases of MBC were identified using the records of the Saskatchewan Cancer Agency over 26 years (1970-1996). Cases were reviewed and analyzed for the immunohistochemical expression of PCNA, Ki67, p27, p16, p57, p21, cyclin-D1 and c-myc and correlated to clinico-biological endpoints of tumor size, node status, stage of the disease, and disease free survival (DFS).

Results

Decreased DFS was observed in the majority of tumors that overexpressed PCNA (98%, p = 0.004). The overexpression of PCNA was inversely correlated to the expression of Ki67 which was predominantly negative (78.3%). Cyclin D1 was overexpressed in 83.7% of cases. Cyclin D1 positive tumors were smaller than 2 cm (55.6%, p = 0.005), had a low incidence of lymph node metastasis (38.2%, p = 0.04) and were associated with increased DFS of >150 months (p = 0.04). Overexpression of c-myc (90%) was linked with a higher incidence of node negativity (58.3%, p = 0.006) and increased DFS (p = 0.04). p27 over expression was associated with decreased lymph node metastasis (p = 0.04). P21 and p57 positive tumors were related to decreased DFS (p = 0.04). Though p16 was overexpressed in 76.6%, this did not reach statistical significance with DFS (p = 0.06) or nodal status (p = 0.07).

Conclusion

Aberrant cell cycle protein expression supports our view that these are important pathways involved in the etiopathogenesis of MBC. Tumors with overexpression of Cyclin D1 and c-myc had better outcomes, in contrast to tumors with overexpression of p21, p57, and PCNA with significantly worse outcomes. P27 appears to be a predictive marker for lymph nodal status. Such observation strongly suggests that dysregulation of cell cycle proteins may play a unique role in the initiation and progression of disease in male breast cancer. Such findings open up new avenues for the treatment of MBC as a suitable candidate for novel CDK-based anticancer therapies in the future.