The two TORCs and Akt

The regulatory circuits that control the activities of the two distinct target of rapamycin (TOR) complexes, TORC1 and TORC2, and of Akt have been a focus of intense research in recent years. It has become increasingly evident that these regulatory circuits control some of the most fundamental aspects of metabolism, cell growth, proliferation, survival, and differentiation at both the cellular and organismal levels. As such, they also play a pivotal role in the genesis of diseases including cancer, diabetes, aging, and degenerative diseases. This review highlights recent developments aimed at deciphering the interplay between Akt and mTORCs as well as their role in embryonic development and in cancer.     

Restoration of Spermatogenesis and Male Fertility Using an Androgen Receptor Transgene

Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5'' and 3'' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3’ to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR-null background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues.     

Molecular targeting of Akt by thymoquinone promotes G1 arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells

Aim

Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQ's mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells.

Main methods

MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies.

Key findings

Studies revealed G₁ phase arrest till 24 h incubation with TQ while extended exposure showed phase shift to subG₁ indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K.

Significance

Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.     

Gene selection for tumor classification using a novel bio-inspired multi-objective approach

Identifying the informative genes has always been a major step in microarray data analysis. The complexity of various cancer datasets makes this issue still challenging. In this paper, a novel Bio-inspired Multi-objective algorithm is proposed for gene selection in microarray data classification specifically in the binary domain of feature selection. The presented method extends the traditional Bat Algorithm with refined formulations, effective multi-objective operators, and novel local search strategies employing social learning concepts in designing random walks. A hybrid model using the Fisher criterion is then applied to three widely-used microarray cancer datasets to explore significant biomarkers which reveal the effectiveness of the proposed method for genomic analysis. Experimental results unveil new combinations of informative biomarkers have association with other studies.     

Comparative proteomic analysis of Staphylococcus aureus strains with differences in resistance to the cell wall-targeting antibiotic vancomycin

Three isogenic strains derived from a clinical vancomycin-intermediate Staphylococcus aureus isolate were examined by comparative protein abundance analysis. Subcellular fractionation was followed by protein separation in 2-DE gels and spot identification by MALDI-TOFTOF-MS and LC-MS/MS. Sixty-five significant protein abundance changes were determined. Numerous enzymes participating in the purine biosynthesis pathway were dramatically increased in abundance in strain VP32, which featured the highest minimal inhibitory concentration for vancomycin, compared to strains P100 and HIP5827. Peptidoglycan hydrolase LytM (LytM) and the SceD protein, a putative transglycosylase, were increased in abundance in the cell envelope fraction of strain VP32, whereas the enzyme D-Ala-D-Ala ligase was decreased in its cytosol fraction. Furthermore, penicillin-binding protein 2 (PBP2) had substantially higher activity in strain VP32 compared to that in strain HIP5827. LytM, PBP2 and D-Ala-D-Ala ligase catalyze reactions in the biosynthesis or the metabolism of cell wall peptidoglycan. It is plausible that expression and activity changes of these enzymes in strain VP32 are responsible for an altered cell wall turnover rate, which has been observed, and an altered peptidoglycan structure, which has yet to be elucidated for this highly vancomycin-resistant strain.     

Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

Biophysical techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2′-CMP + RNASE and BH3I-1 + hBCL_XL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.