Generation of “Virtual” Control Groups for Single Arm Prostate Cancer Adjuvant Trials

It is difficult to construct a control group for trials of adjuvant therapy (Rx) of prostate cancer after radical prostatectomy (RP) due to ethical issues and patient acceptance. We utilized 8 curve-fitting models to estimate the time to 60%, 65%, … 95% chance of progression free survival (PFS) based on the data derived from Kattan post-RP nomogram. The 8 models were systematically applied to a training set of 153 post-RP cases without adjuvant Rx to develop 8 subsets of cases (reference case sets) whose observed PFS times were most accurately predicted by each model. To prepare a virtual control group for a single-arm adjuvant Rx trial, we first select the optimal model for the trial cases based on the minimum weighted Euclidean distance between the trial case set and the reference case set in terms of clinical features, and then compare the virtual PFS times calculated by the optimum model with the observed PFSs of the trial cases by the logrank test. The method was validated using an independent dataset of 155 post-RP patients without adjuvant Rx. We then applied the method to patients on a Phase II trial of adjuvant chemo-hormonal Rx post RP, which indicated that the adjuvant Rx is highly effective in prolonging PFS after RP in patients at high risk for prostate cancer recurrence. The method can accurately generate control groups for single-arm, post-RP adjuvant Rx trials for prostate cancer, facilitating development of new therapeutic strategies.     

Reduced Expression of Uroplakin 1A Is Associated with the Poor Prognosis of Gastric Adenocarcinoma Patients

Background

The aim of this study was to investigate the expression and prognostic significance of Uroplakin1A (UPK1A) in gastric adenocarcinoma patients. Functional studies were also analyzed in vitro.

Methodology/Principal Findings

Real-time quantitative PCR (RT-qPCR), western blotting, and immunohistochemical (IHC) staining methods were used to analyze the expression of UPK1A in primary gastric adenocarcinoma tissue samples. Compared with matched adjacent non-tumor, the expression of UPK1A in fresh surgical specimens was reduced, which was confirmed by RT-qPCR (P<0.01) and western blotting analysis (P<0.01). The paraffin specimens from a consecutive series of 445 gastric adenocarcinoma patients who underwent surgery between 2003 and 2006 were analyzed by IHC staining. The relationship between UPK1A expression, clinicopathological factors, and survival were evaluated. IHC staining analysis revealed that the reduced expression of UPK1A was observed in 224 cases (50.3%). Additionally, the correlation analysis of clinicopathological factors demonstrated that reduced expression of UPK1A was significantly associated with histological grade (P = 0.022), node metastasis (P<0.001) and tumor node metastasis (TNM) stage (P = 0.008) (7^th edition of the International Union Against Cancer (UICC)). Furthermore, Kaplan-Meier survival analysis revealed that the reduced expression of UPK1A was significantly associated with poor prognosis (P = 0.043). Cox hazards model analysis indicated that UPK1A expression was an independent risk factor at the 0.1 level (P = 0.094). The function of UPK1A in cell cycle, migration, and invasion was investigated by overexpressing UPK1A in the MKN45 gastric cancer cell line. The elevated expression of UPK1A cells induced G1 phase arrest and significantly inhibited migration and invasion.

Conclusions/Significance

The reduced expression of UPK1A might play a role in the progression of gastric cancer. Thus, UPK1A could be a potential favorable biomarker associated with gastric cancer prognosis.     

The relationship between vascular endothelial growth factor and spermatogenesis disturbance in an experimentally-induced unilateral cryptorchidism murine model

Molecular Biology Reports - This study is to explore the relationship between vascular endothelial growth factor (VEGF) and pathological changes in cryptorchidism by using murine model of...     

Functional divergence analysis of vertebrate neuronal nicotinic acetylcholine receptor subunits

Nicotinic acetylcholine receptors (nAChRs) are pentamers formed by subunits from a large multigene family and are highly variable in kinetic, electrophysiological and pharmacological properties. Due to the essential roles of nAChRs in many physiological procedures and diversity in function, identifying the function-related sites specific to each subunit is not only necessary to understand the properties of the receptors but also useful to design potential therapeutic compounds that target these macromolecules for treating a series of central neuronal disorders. By conducting a detailed function divergence analysis on nine neuronal nAChR subunits from representative vertebrate species, we revealed the existence of significant functional variation between most subunit pairs. Specifically, 44 unique residues were identified for the α7 subunit, while another 22 residues that were likely responsible for the specific features of other subunits were detected. By mapping these sites onto the 3?D structure of the human α7 subunit, a structure-function relationship profile was revealed. Our results suggested that the functional divergence related sites clustered in the ligand binding domain, the β2–β3 linker close to the N-terminal α-helix, the intracellular linkers between transmembrane domains, and the “transition zone” may have experienced altered evolutionary rates. The former two regions may be potential binding sites for the α7* subtype-specific allosteric modulators, while the latter region is likely to be subtype-specific allosteric modulations of the heteropentameric descendants such as the α4β2* nAChRs.

Communicated by Ramaswamy H. Sarma     

Covalently linking the Escherichia coli global anaerobic regulator FNR in tandem allows it to function as an oxygen stable dimer

Glutathione (GSH) serves as an important anti-oxidant in the brain by scavenging harmful reactive oxygen species that are generated during different molecular processes. The GSH level in the brain provides indirect information on oxidative stress of the brain. We report in vivo detection of GSH non-invasively from various brain regions (frontal cortex, parietal cortex, hippocampus and cerebellum) in bilateral hemispheres of healthy male and female subjects and from bi-lateral frontal cortices in patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). All AD patients who participated in this study were on medication with cholinesterase inhibitors. Healthy young male (age 26.4±3.0) and healthy young female (age 23.6±2.1) subjects have higher amount of GSH in the parietal cortical region and a specific GSH distribution pattern (parietal cortex>frontal cortex>hippocampus ~ cerebellum) has been found. Overall mean GSH content is higher in healthy young female compared to healthy young male subjects and GSH is distributed differently in two hemispheres among male and female subjects. In both young female and male subjects, statistically significant (p=0.02 for young female and p=0.001 for young male) difference in mean GSH content is found when compared between left frontal cortex (LFC) and right frontal cortex (RFC). In healthy young female subjects, we report statistically significant positive correlation of GSH content between RFC and LFC (r=0.641, p=0.004) as well as right parietal cortex (RPC) and left parietal cortex (LPC) (r=0.797, p=0.000) regions. In healthy young male subjects, statistically significant positive correlation of GSH content was observed between LFC and LPC (r=0.481, p=0.032) regions. This statistical analysis implicates that in case of a high GSH content in LPC of a young male, his LFC region would also contain high GSH and vice versa. The difference in mean of GSH content between healthy young female control and female AD patients in RFC region (p=0.003) and difference in mean of GSH content between healthy young male control and male AD patients (p=0.05) in LFC region is found to be statistically significant. It is the first scientific report correlating alteration (in selective brain regions) of GSH level with clinical status of male and female subjects using non-invasive imaging technique.     

Up-regulation of Cavβ3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.     

Efficient expression and purification of recombinant alcohol oxidase in Pichia pastoris

In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD₆₀₀ 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H₂O₂ inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas.     

Biotransformation of phenylpyruvic acid to phenyllactic acid by growing and resting cells of a <Emphasis Type="Italic">Lactobacillus</Emphasis> sp.

Phenyllactic acid (PLA) is a novel antimicrobial compound derived from phenylalanine (Phe). Lactobacillus sp. SK007, having high PLA-producing ability, was isolated from Chinese traditional pickles. When 6.1 mM phenylpyruvic acid (PPA) was used to replace Phe as substrate at the same concentration, PLA production increased 14-fold and the fermentation time decreased from 72 h to 24 h with growing cells. With resting cells, however, 6.8 mM PLA could be obtained as optimal yield using the following conditions: 12 mM PPA, 55 mM glucose, pH 7.5, 35°C and 4 h.