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.     

Influence of sequential oligopeptide carriers on the bioactive structure of conjugated epitopes: comparative study of the conformation of a Herpes simplex virus glycoprotein gD-1 epitope in the free and conjugated form, and protein "built-in" crystal structure

Synthetic carriers play an important role in immunogen presentation, due to their ability of inducing improved and specific responses to conjugated epitopes. Their influence on the bioactive conformation of the epitope, though admittedly crucial for relevant in vitro and in vivo applications, is difficult to evaluate, given the usual lack of information on the complex conformational features determined by the nature of the carrier and the mode of ligation. Using the Herpes simplex virus glycoprotein D-1 epitope (Leu(9)-Lys-Nle-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu(22)) as a model, we have performed a detailed conformational analysis on the free epitope peptide in solution and on three constructs in which the epitope was conjugated to sequential oligopeptide carriers {Ac-[Lys-Aib-Gly](4)-OH (SOC(4))} (through either a thioether or an amide bond; Ac: acetyl) and polytuftsin oligomers {H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2) (T20)}, (through a thioether bond). The analysis of the epitope conformation in the parent protein, in carrier-conjugated and free form, suggests that the beta-turn structure of the -Asp(13)-Pro-Asn-Arg(16)- segment is highly conserved and independent of the epitope form. However, small conformational variations were observed at the C-terminal part of the epitope, depending on the nature of the carrier.     

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.      

Blood oxidative stress generation after intraperitoneal administration of functionalized single-walled carbon nanotubes in rats

Single-walled carbon nanotubes (SWCNTs) have been proposed for various medical applications. However, their safety for human administration has not been yet fully demonstrated. In vitro studies have pointed oxidative stress as a mechanism involved in their cytotoxic effects. In the present study we have evaluated the capacity of DNA functionalized SWCNTs to induce oxidative stress in blood after intraperitoneal (ip) administration in rats. The presence of SWCNTs in blood was confirmed by Raman spectroscopy 30 minutes after their ip administration. Oxidative stress parameters (malondialdehyde - MDA, protein carbonyls - PC, antioxidant capacity measured as hydrogen donating capacity - HD, sulfhydryl groups - SH, glutathione - GSH and nitrites - NO) were assessed in blood at 3, 6, 24, respectively, and 48 hours after ip injection. MDA, PC and NO exhibited a significant increase at 3-6 hours interval from exposure, followed by a recovery trend. The levels of HD reached a bottom level at 6 hours after administration, while SH strongly decreased at 3 hours interval and increased slightly up to 48 hours without attending the initial values. GSH level recorded an increasing tendency at the 3rd hour, an incomplete recovery process at 24 hours followed by a secondary significant increase following a 48-hour interval. Significant inverse correlations were obtained between the PC and SH levels and between the NO and HD values. In conclusion, the ip administration of DNA functionalized SWCNT in rats results in oxidative stress generation in plasma, with a transient pattern of evolution.     

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.     

Genotoxicity of TiO(2) anatase nanoparticles in B6C3F1 male mice evaluated using Pig-a and flow cytometric micronucleus assays

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were conducted to evaluate the genotoxicity of 10 nm titanium dioxide anatase nanoparticles (TiO(2)-NPs) in mice. Groups of five 6-7-week-old male B6C3F1 mice were treated intravenously for three consecutive days with 0.5, 5.0, and 50 mg/kg TiO(2)-NPs for the two assays; mouse blood was sampled one day before the treatment and on Day 4, and Weeks 1, 2, 4, and 6 after the beginning of the treatment; Pig-a mutant frequencies were determined at Day -1 and Weeks 1, 2, 4 and 6, while percent micronucleated-reticulocyte (%MN-RET) frequencies were measured on Day 4 only. Additional animals were treated intravenously with three daily doses of 50 mg.kg TiO(2)-NPs for the measurement of titanium levels in bone marrow after 4, 24, and 48 h of the last treatment. The measurement indicated that the accumulation of the nanoparticles reached the peak in the tissue 4 h after the administration and the levels were maintained for a few days. No increase in either Pig-a mutant frequency of the frequency of %MN-RETs was detected, although the %RETs was reduced in the treated animals on Day 4 in a dose-dependent manner indicating cytotoxicity of TiO(2)-NPs in the bone marrow. These results suggest that although TiO(2)-NPs can reach the mouse bone marrow and are capable of inducing cytotoxicity, the nanoparticles are not genotoxic when assessed with in vivo micronucleus and Pig-a gene mutation tests.     

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.     

Disorder-order folding transitions underlie catalysis in the helicase motor of SecA

SecA is a helicase-like motor that couples ATP hydrolysis with the translocation of extracytoplasmic protein substrates. As in most helicases, this process is thought to occur through nucleotide-regulated rigid-body movement of the motor domains. NMR, thermodynamic and biochemical data show that SecA uses a novel mechanism wherein conserved regions lining the nucleotide cleft undergo cycles of disorder-order transitions while switching among functional catalytic states. The transitions are regulated by interdomain interactions mediated by crucial 'arginine finger' residues located on helicase motifs. Furthermore, we show that the nucleotide cleft allosterically communicates with the preprotein substrate-binding domain and the regulatory, membrane-inserting C domain, thereby allowing for the coupling of the ATPase cycle to the translocation activity. The intrinsic plasticity and functional disorder-order folding transitions coupled to ligand binding seem to provide a precise control of the catalytic activation process and simple regulation of allosteric mechanisms.