The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury

Although repetitive Transcranial Magnetic Stimulation (rTMS) in treatment of stroke in humans has been explored over the past decade the data remain controversial in terms of optimal stimulation parameters and the mechanisms of rTMS long-term effects. This study aimed to explore the potential of different rTMS protocols to induce changes in gene expression in rat cortices after acute ischemic-reperfusion brain injury. The stroke was induced by middle cerebral artery occlusion (MCAO) with subsequent reperfusion. Changes in the expression of 96 genes were examined using low-density expression arrays after MCAO alone and after MCAO combined with 1Hz, 5Hz, continuous (cTBS) and intermittent (iTBS) theta-burst rTMS. rTMS over the lesioned hemisphere was given for two weeks (with a 2-day pause) in a single daily session and a total of 2400 pulses. MCAO alone induced significant upregulation in the expression of 44 genes and downregulation in 10. Two weeks of iTBS induced significant increase in the expression of 52 genes. There were no downregulated genes. 1Hz and 5Hz had no significant effects on gene expression, while cTBS effects were negligible. Upregulated genes included those involved in angiogenesis, inflammation, injury response and cellular repair, structural remodeling, neuroprotection, neurotransmission and neuronal plasticity. The results show that long-term rTMS in acute ischemic-reperfusion brain injury induces complex changes in gene expression that span multiple pathways, which generally promote the recovery. They also demonstrate that induced changes primarily depend on the rTMS frequency (1Hz and 5Hz vs. iTBS) and pattern (cTBS vs. iTBS). The results further underlines the premise that one of the benefits of rTMS application in stroke may be to prime the brain, enhancing its potential to cope with the injury and to rewire. This could further augment its potential to favorably respond to rehabilitation, and to restore some of the loss functions.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of rivastigmine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of rivastigmine in human plasma. Rivastigmine was extracted from human plasma by using solid-phase extraction technique. Zolpidem was used as the internal standard. A Betabasic-8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 251.20-->206.10, 86.20 for rivastigmine and m/z 308.10-->235.10 for zolpidem. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatographic conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.2-20.0 ng/ml with a correlation coefficient > or =0.9988. The intra-run and inter-run precision and accuracy were within 10.0%. The overall recoveries for rivastigmine and zolpidem were 86.28% and 87.57%, respectively. The total run time was 2.0 min. The developed method was applied for the determination of the pharmacokinetic parameters of rivastigmine following a single oral administration of a 3 mg rivastigmine capsule in 20 healthy male volunteers.     

Finding the Wolf in Sheep’s Clothing: The 4Kscore Is a Novel Blood Test That Can Accurately Identify the Risk of Aggressive Prostate Cancer

Better biomarkers that can discriminate between aggressive and indolent phenotypes of prostate cancer are urgently needed. In the first 20 years of the prostate-specific antigen (PSA) era, screening for prostate cancer has successfully reduced prostate cancer mortality, but has led to significant problems with overdiagnosis and overtreatment. As a result, many men are subjected to unnecessary prostate biopsies and overtreatment of indolent cancer in order to save one man from dying of prostate cancer. A novel blood test known as the 4Kscore® Test (OPKO Lab, Nashville, TN) incorporates a panel of four kallikrein protein biomarkers (total PSA, free PSA, intact PSA, and human kallikrein-related peptidase 2) and other clinical information in an algorithm that provides a percent risk for a high-grade (Gleason score ≥ 7) cancer on biopsy. In 10 peer-reviewed publications, the four kallikrein biomarkers and algorithm of the 4Kscore Test have been shown to improve the prediction not only of biopsy histopathology, but also surgical pathology and occurrence of aggressive, metastatic disease. Recently, a blinded prospective trial of the 4Kscore Test was conducted across the United States among 1012 men. The 4Kscore Test replicated previous European results showing accuracy in predicting biopsy outcome of Gleason score ≥ 7. In a recent case-control study nested within a population-based cohort from Västerbotten, Sweden, the four kallikrein biomarkers of the 4Kscore Test also predicted the risk for aggressive prostate cancer that metastasized within 20 years after the test was administered. These results indicate that men with an abnormal PSA or digital rectal examination result, and for whom an initial or repeat prostate biopsy is being considered, would benefit from a reflex 4Kscore Test to add important information to the clinical decision-making process. A high-risk 4Kscore Test result may be used to select men with a high probability of aggressive prostate cancer who would benefit from a biopsy of the prostate to prevent an adverse and potentially lethal outcome from prostate cancer. Men with a low 4Kscore Test result may safely defer biopsy.Key words: Prostate cancer, Biomarker, High-grade prostate cancer, ScreeningProstate cancer is the most common cancer in men in the United States, accounting for an estimated 27% of all newly diagnosed cancers in 2014.¹ Since the advent of screening for prostate cancer with serum prostate-specific antigen (PSA), we have seen a significant decline in prostate cancer mortality.¹ Randomized clinical trials have reported a 20% to 40% reduction in death from prostate cancer in men undergoing routine screening compared with those who are not screened.^2,3 However, these trials, and a trial showing little difference between opportunistic and systematic screening,⁴ have raised the concern for overdiagnosis and overtreatment of indolent prostate cancer. The fundamental concern is that an overwhelming number of men are subjected to interventions such as prostate biopsy in order to prevent one man’s death from prostate cancer.^2,3Prostate biopsy is an invasive procedure with significant complications, such as bleeding, urinary retention, and life-threatening infection. A recent population-based study from Ontario, Canada, revealed a fourfold increase to 4.1% for the rate of hospital admissions after prostate biopsy from 1996 to 2005, with 72% of admissions being due to infection.⁵ These risks, combined with the enormous anxiety involved in undergoing the procedure, present a significant burden to any man considering prostate cancer screening.Today, most men diagnosed with prostate cancer have a tumor that is unlikely to pose a threat to their life expectancies. A recent systematic analysis suggested that up to 60% of prostate cancers diagnosed in contemporary studies can be safely observed without a need for immediate intervention.⁶ However, in the United States, because of the concern for possible undergrading of prostate cancer due to biopsy sampling error, 90% of men diagnosed with prostate cancer undergo treatment and approximately 66% will be confirmed to have indolent Gleason score 6 prostate cancer,⁷ suggesting a significant problem with overtreatment. Although treatment for localized prostate cancer provides excellent cancer control,^8,9 it comes at a significant detriment to health-related quality of life (HRQoL). Previous studies have reported significant changes in HRQoL after primary treatment for prostate cancer, primarily in the domains of sexual and urinary function and bother.^10–12 Given the physical and psychological burden of these secondary adverse events, many government agencies and patients are beginning to question the risks and benefits of prostate cancer screening and treatment.¹³The United States Preventive Services Task Force recently advised against routine screening for prostate cancer, claiming that the risks of screening outweigh the benefits.¹³ However, 20% to 30% of men who are diagnosed with prostate cancer are found to have high-grade disease at presentation¹⁴; without screening, these men would lose their opportunity for cure. It is clear that new biomarkers or tests that promote the detection of both indolent and aggressive prostate cancer are unlikely to be helpful. We need tests that focus on the detection of aggressive tumors, not the indolent ones that are better left alone. Aggressive prostate cancer, for purposes of this review, is defined as cancer with a Gleason score ≥ 7 and tumors that are most likely to progress to metastatic disease and death. Targeted detection of aggressive prostate cancer would allow urologists to diagnose and treat those men most likely to benefit from aggressive intervention to avoid premature death. Conversely, those men harboring non-life-threatening disease would be able to avoid unnecessary interventions. The 4Kscore® Test (OPKO Lab, Nashville, TN) is a new blood test that accurately identifies the risk of aggressive prostate cancer. The 4Kscore Test plays an important clinical role as a reflex test prior to proceeding with initial prostate biopsy in men with an elevated PSA level or abnormal digital rectal examination (DRE) results, or after a prior negative biopsy and persistently abnormal PSA levels.     

Expression, purification and structural analysis of the Pyrococcus abyssi RNA binding protein PAB1135

Background

Epistatic interactions of multiple single nucleotide polymorphisms (SNPs) are now believed to affect individual susceptibility to common diseases. The detection of such interactions, however, is a challenging task in large scale association studies. Ant colony optimization (ACO) algorithms have been shown to be useful in detecting epistatic interactions.

Findings

AntEpiSeeker, a new two-stage ant colony optimization algorithm, has been developed for detecting epistasis in a case-control design. Based on some practical epistatic models, AntEpiSeeker has performed very well.

Conclusions

AntEpiSeeker is a powerful and efficient tool for large-scale association studies and can be downloaded from http://nce.ads.uga.edu/~romdhane/AntEpiSeeker/index.html.     

Differential partitioning of Gαi1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells

Background

Taxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance.

Results

Utilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Gαi1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the α-subunit of the G protein have been identified and the Gαi (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Gαi1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Gαi1 in these cells. Moreover, Gαi1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Gαi1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.

Conclusion

Based on the opposing effects of taxol and the Gαi1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Gαi1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.     

Evidence that glycoprotein 96 (B2), a stress protein,functions as a Th2-specific costimulatory molecule

After the engagement of Ag receptor, most of the Th cells for their optimal activation require a second (costimulatory) signal provided by the APCs. We demonstrate the isolation and characterization of a 99- to 105-kDa protein (B2), from LPS-activated B cell surface, and its function as a Th2-specific costimulatory molecule. Appearance of B2 as a single entity on two-dimensional gel electrophoresis and as a distinct peak in reverse-phase HPLC ascertains the fact that B2 is homogeneous in preparation. Electron microscopy as well as competitive binding studies reveal that (125)I-labeled B2 specifically binds anti-CD3-activated T cell surface and also competes with its unlabeled form. Internal amino acid sequences of B2 are found to be identical with stress protein gp96. The identity of B2 as gp96 is also revealed by immunological characterization and by confocal microscopic colocalization studies of B2 and gp96 on LPS-activated B cells. Confocal imaging studies also demonstrate that gp96 can be induced on B cell surface without association of MHC molecules. Furthermore, the novel role of gp96 in Th cell proliferation skewing its differentiation toward Th2 phenotype has also been established. Ab-mediated blocking of gp96-induced signaling not only abrogates in vitro proliferation of CD4(+) T cells, but also diminishes the secretion of Th2-specific cytokines. Notably, the expression of CD91 (receptor of gp96/B2) is up-regulated on anti-CD3-activated Th cells and also found to be present on Th1 and Th2 subsets.