Identification and Quantification of AKT Isoforms and Phosphoforms in Breast Cancer Using a Novel Nanofluidic Immunoassay

Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoPro^TM 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.Activation of the PI3K-AKT signaling pathway is one of the most common events in cancer (1, 2). Pathway activation can confer a number of advantages to the cancer cells, including enhanced proliferation and survival (1, 2). Multiple mechanisms exist by which the pathway may become activated, including amplification or activation of receptor tyrosine kinases (e.g. ERBB2 in breast and EGFR in lung tumors), mutation of the catalytic or regulatory subunits of PI3K (e.g. PIK3CA in colorectal and breast tumors), loss of the negative regulator PTEN (e.g. mutation in prostate and melanoma), and gain of function of AKT (e.g. amplification or mutation in breast and pancreatic tumors) (reviewed in Refs. 1 and 2).AKT represents a central node in the PI3K signaling cascade (3). AKT is recruited to the cell membrane via its pleckstrin homology domain when PI3K phosphorylates PIP₂ to form PIP₃ (4, 5). Following recruitment, AKT is phosphorylated by PDK1 and the rictor-mTOR complex, resulting in conformational changes and activation of the protein (5–8). Multiple studies have shown that the phosphorylation of AKT leads to the phosphorylation and activation of downstream effectors of the signaling pathway, such as mTOR complex 1 and S6K (reviewed in Ref. 1). The central role of this pathway in cancer is further underscored by the efforts of multiple pharmaceutical companies that have developed inhibitors against AKT as potential anti-oncogenic therapeutics (9).Despite the importance of AKT in growth and survival signaling in cancer, there are surprisingly few data that address the specific roles played in growth and survival by the multiple AKT family members (AKT-1, -2, and -3) and different phosphorylation and putative phosphorylation sites that can potentially activate the protein. Western blot analysis has been the foundation of most AKT studies, but in many cases pan-AKT antibodies have been employed that fail to distinguish between the different AKT isoforms. Recent siRNA silencing studies have indicated distinct functions for different AKT family members within a cell (10, 11). Moreover, there is evidence in breast cancer that the three isoforms exhibit different localizations and therefore must have at least partially distinct functions (12). Similarly, evidence is mounting for multiple phosphorylation sites in AKT beyond the two most studied phosphorylation events (Thr-308 and Ser-473) (5–8). Phosphorylation at serine and threonine residues at Thr-72 and Ser-246 may be required for the activation or regulation of kinase activity (13). The functional significance of constitutive phosphorylation of Ser-124 and Thr-450 is still unknown (14). Finally, there is evidence that phosphorylation of tyrosine residues at Tyr-315 and Tyr-326 is required for full kinase activity (15).Analysis of such phospho- and isoform-specific activation often requires complicated in-depth analyses using large quantities of proteins, purified recombinant protein, immunoprecipitation, incorporation of ³²P isotopes, and/or mass spectroscopy, which makes such studies more difficult to perform and not easily adaptable to clinical specimens. Thus, better methods are required for the accurate assessment of both phosphoform and isoform usage in cells with an activated PI3K-AKT pathway and the effects of pathway inhibitors using relatively small amounts of starting material. We describe here the development of such an assay using nanocapillary-based isoelectric focusing (16). This approach allows the separation of AKT into distinct peaks that correspond to different iso- and phosphoforms using a small amount of starting material and a single pan-specific antibody. This approach should allow for more accurate determinations of isoform usage in different cell types, as well as of changes in phosphorylation states in response to pathway inhibition, including in clinical specimens.     

Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5′-tggccaatatcattggtggt-3′ targeting signal peptide sequence of pediocin structural gene and reverse primer: 5′-ctactaacgcttggctggca-3′ encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E. faecium NCDC124 and Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.     

Is There an Association between Work Stress and Diurnal Cortisol Patterns? Findings from the Whitehall II Study

Objective

The evidence on whether there is work stress related dysregulation of the hypothalamic-pituitary-adrenal axis is equivocal. This study assessed the relation between work stress and diurnal cortisol rhythm in a large-scale occupational cohort, the Whitehall II study.

Methods

Work stress was assessed in two ways, using the job-demand-control (JDC) and the effort-reward-imbalance (ERI) models. Salivary cortisol samples were collected six times over a normal day in 2002–2004. The cortisol awakening response (CAR) and diurnal cortisol decline (slope) were calculated.

Results

In this large occupational cohort (N = 2,126, mean age 57.1), modest differences in cortisol patterns were found for ERI models only, showing lower reward (β = −0.001, P-value = 0.04) and higher ERI (β = 0.002, P-value = 0.05) were related to a flatter slope in cortisol across the day. Meanwhile, moderate gender interactions were observed regarding CAR and JDC model.

Conclusions

We conclude that the associations of work stress with cortisol are modest, with associations apparent for ERI model rather than JDC model.     

Resilience to Freezing in the Vegetative Cells of the Microalga Lobosphaera incisa (Trebouxiophyceae,Chlorophyta)

The chlorophyte microalga Lobosphaera incisa was isolated from the snowy slopes of Mt. Tateyama in Japan. This microalga stores exceptionally high amounts of the omega-6 LC-PUFA arachidonic acid in triacylglycerols, and therefore represents a potent photosynthetic source for this essential LC-PUFA. Assuming that freezing tolerance may play a role in adaptation of L. incisa to specific ecological niches, we examined the capability of L. incisa to tolerate extreme sub-zero temperatures. We report here, that the vegetative cells of L. incisa survived freezing at −20°C and −80°C (over 1 month), without cryoprotective agents or prior treatments. Cells successfully recovered upon thawing and proliferated under optimal growth conditions (25°C). However, cells frozen at −80°C showed better recovery and lower cellular ROS generation upon thawing, compared to those preserved at −20°C. Photosynthetic yield of PSII, estimated by F_v/F_m, temporarily decreased at day 1 post freezing and resumed to the original level at day 3. Interestingly, the thawed algal cultures produced a higher level of chlorophylls, exceeding the control culture. The polar metabolome of the vegetative cells comprised a range of compatible solutes, dominated by glutamate, sucrose, and proline. We posit that the presence of endogenous cryoprotectants, a rigid multilayer cell wall, the high LC-PUFA content in membrane lipids, and putative cold-responsive proteins may contribute to the retention of functionality upon recovery from the frozen state, and therefore for the survival under cryospheric conditions. From the applied perspective, this beneficial property holds promise for the cryopreservation of starter cultures for research and commercial purposes.     

Effect of nitric oxide signaling in bacterial-treated soybean plant under salt stress

To understand protective roles of nitric oxide against salt stress, the effects of exogenous sodium nitroprusside on activities of lipoxygenase, peroxidase, phenylalanine ammonialyase, catalase, superoxide dismutase enzymes, proline accumulation, and distribution of sodium in soybean plants under salt were determined. Application of sodium nitroprusside + bacterium enhanced plant growth-promotion characteristics, activities of different enzymes, and proline accumulation in the presence of sodium nitroprusside under salt stress. Treatment with NaCl at 200 mM and sodium nitroprusside (0.1 mM) reduced Na⁺ levels but increased K⁺ levels in leaves in comparison with the NaCl-treated plants. Correspondingly, the plants treated with exogenous sodium nitroprusside and NaCl maintained a lower ratio of [Na⁺]/[K⁺] in NaCl-stressed plants.     

Regulation of β-catenin stabilization in human platelets

The Wnt/β-catenin pathway controls developmental processes and homeostasis; however, abnormal activation of this pathway has been linked to several human diseases. Recent reports have demonstrated regulation of platelet function by canonical and non-canonical Wnt signalling. Platelet aggregation plays a crucial role in haemostasis and thrombosis. Here we report for the first time that, induction of sustained aggregation of platelets by a strong agonist in the presence of calcium was associated with nearly complete proteolysis of β-catenin, which was abrogated upon depletion of calcium from platelet suspension. β-catenin cleavage was disallowed in absence of aggregation, thus implicating integrin α_IIbβ₃ engagement in β-catenin proteolysis. Degradation of β-catenin was blocked partially by inhibitors of either proteasome or calpain and completely when cells were exposed to both the inhibitors. Protein kinase C inhibition, too, abolished β-catenin degradation. Thus activities of proteasome, calpain and protein kinase C regulate stabilization of β-catenin in aggregated human platelets.     

Impaired male meiosis, morphology and distribution pattern of different cytotypes of Bupleurum lanceolatum Wall. (Apiaceae) from the Western Himalayas

Detailed male meiosis, critical morphological observations and distribution pattern of diploid as well as tetraploid cytotypes of the Western Himalayan species, Bupleurum lanceolatum have been evaluated at present. A diploid (n = 8) cytotype is reported from Kashmir, whereas, both diploid (n = 8) and tetraploid (n = 16) cytotypes are available from two districts Kangra and Sirmaur of Himachal Pradesh. Out of these, the tetraploid cytotype makes new addition for the species on a worldwide basis. As per behavior, a tetraploid cytotype is characterized by abnormal meiosis leading to high pollen sterility and size variation of the pollen grains. Morphologically, tetraploids are noted to be luxuriant in comparison to the diploids.     

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. ¹HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CI_xrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.     

Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.