Development and Applications of Mass Spectrometric Methods for Phosphorylation Analysis

Protein phosphorylation modification regulates numerous cellular functions by a reversible and selective control of kinases and phosphatases. To understand the entire dynamic network of phosphorylation requires sensitive and reliable quantification of phosphorylation, measurements that can be achieved by mass spectrometry. In this research, we established efficient MALDI-mass spectrometric methods as strategies for single- or multi-site phosphorylation quantification without the use of isotopes, chromatography and calibration curves. The methods were assessed by analyzing peptide standards with different single-multiple phosphorylation sites, showing a wide dynamic range, good accuracy and reproducibility. This is the first label-free MALDI method without using a calibration methodology proposed for quantification of in vitro phosphorylation in a kinase assay. Moreover, advanced mass spectrometry empowers identification of a highly conserved Cdk2 phosphorylation site of HIV-1 reverse transcriptase (RT) at Thr 261 across thousands of HIV-1 strains. We demonstrated phosphorylation on HIV-1 RT peptides and protein in in vitro assays, and confirmed phosphorylation in vivo with antibodies and mutation studies. Blocking this phosphorylation by p21, a naturally occurring Cdk inhibitor, defines a potential Cdk2-mediated cell-intrinsic mechanism for restricting HIV-replication in a clinically significant way

A Proteomics Approach to Investigate miR-153-3p and miR-205-5p Targets in Neuroblastoma Cells

MicroRNAs are key regulators associated with numerous diseases. In HEK293 cells, miR-153-3p and miR-205-5p down-regulate alpha-synuclein (SNCA) and Leucine-rich repeat kinase 2 (LRRK2), two key proteins involved in Parkinson’s disease (PD). We have used two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) to identify a spectrum of miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We overexpressed and inhibited both microRNAs in SH-SY5Y cells and through comparative proteomics profiling we quantified ~240 protein spots from each analysis. Combined, thirty-three protein spots were identified showing significant (p-value \u3c 0.05) changes in abundance. Modulation of miR-153-3p resulted in seven up-regulated proteins and eight down-regulated proteins. miR-205 modulation resulted in twelve up-regulated proteins and six down-regulated proteins. Several of the proteins are associated with neuronal processes, including peroxiredoxin-2 and -4, cofilin-1, prefoldin 2, alpha-enolase, human nucleoside diphosphate kinase B (Nm23) and 14-3-3 protein epsilon. Many of the differentially expressed proteins are involved in diverse pathways including metabolism, neurotrophin signaling, actin cytoskeletal regulation, HIF-1 signaling and the proteasome indicating that miR-153-3p and miR-205-5p are involved in the regulation of a wide variety of biological processes in neuroblastoma cells

Fluoroquinolone Efficacy against Tuberculosis Is Driven by Penetration into Lesions and Activity against Resident Bacterial Populations.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of "universal" drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens

Prognostic factors associated with the survival of oral and pharyngeal carcinoma in Taiwan

<p>Abstract</p> <p>Background</p> <p>In Taiwan, a distinct ethnic group variation in incidence and mortality rates has been suggested for most carcinomas. Our aim is to identify the role of prognostic factors associated with the survival of oral and pharyngeal carcinoma in Taiwan.</p> <p>Methods</p> <p>Taiwan Cancer Registry records of 9039 subjects diagnosed with oral and pharyngeal carcinoma were analyzed. The population was divided into three ethnic groups by residence, which were Taiwanese aborigines, Hakka and Hokkien communities. Five-year survival rates were estimated by Kaplan-Meier methods. Ethnic curves differed significantly by log-rank test; therefore separate models for Taiwanese aborigines, Hakka and Hokkien were carried out. The Cox multivariate proportional hazards model was used to examine the role of prognostic factors on ethnic survival.</p> <p>Results</p> <p>The five-year survival rates of oral and pharyngeal carcinoma were significantly poorer for Hokkien community (53.9%) and Taiwanese aborigines community (58.1%) compared with Hakka community (60.5%). The adjusted hazard ratio of Taiwanese aborigines versus Hakka was 1.07 (95%CI, 0.86–1.33) for oral and pharyngeal carcinoma mortality, and 1.16 (95%CI, 1.01–1.33) for Hokkien versus Hakka. Males had significantly poor prognosis than females. Subjects with tongue and/or mouth carcinoma presented the worst prognosis, whereas lip carcinoma had the best prognosis. Subjects with verrucous carcinoma had better survival than squamous cell carcinoma. Prognosis was the worst in elderly subjects, and subjects who underwent surgery had the highest survival rate.</p> <p>Conclusion</p> <p>Our study presented that predictive variables in oral and pharyngeal carcinoma survival have been: ethnic groups, period of diagnosis, gender, diagnostic age, anatomic site, morphologic type, and therapy.</p

Genetic diversity and C2-like subgenogroup strains of enterovirus 71, Taiwan, 2008

<p>Abstract</p> <p>Background</p> <p>Human enterovirus 71 (EV-71) is known of having caused numerous outbreaks of hand-foot-mouth disease, and other clinical manifestations globally. In 2008, 989 EV-71 strains were isolated in Taiwan.</p> <p>Results</p> <p>In this study, the genetic and antigenic properties of these strains were analyzed and the genetic diversity of EV-71 subgenogroups surfacing in Taiwan was depicted, which includes 3 previously reported subgenogroups of C5, B5, and C4, and one C2-like subgenogroup. Based on the phylogenetic analyses using their complete genome nucleotide sequences and neutralization tests, the C2-like subgenogroup forms a genetically distinct cluster from other subgenogroups, and the antisera show a maximum of 128-fold decrease of neutralization titer against this subgenogroup. In addition, the subgenogroup C4 isolates of 2008 were found quite similar genetically to the Chinese strains that caused outbreaks in recent years and thus they should be carefully watched.</p> <p>Conclusions</p> <p>Other than to be the first report describing the existence of C2-like subgenogroup of EV-71 in Taiwan, this article also foresees a potential of subgenogroup C4 outbreaks in Taiwan in the near future.</p

A Secured Authentication Protocol for Wireless Sensor Networks Using Elliptic Curves Cryptography

User authentication is a crucial service in wireless sensor networks (WSNs) that is becoming increasingly common in WSNs because wireless sensor nodes are typically deployed in an unattended environment, leaving them open to possible hostile network attack. Because wireless sensor nodes are limited in computing power, data storage and communication capabilities, any user authentication protocol must be designed to operate efficiently in a resource constrained environment. In this paper, we review several proposed WSN user authentication protocols, with a detailed review of the M.L Das protocol and a cryptanalysis of Das’ protocol that shows several security weaknesses. Furthermore, this paper proposes an ECC-based user authentication protocol that resolves these weaknesses. According to our analysis of security of the ECC-based protocol, it is suitable for applications with higher security requirements. Finally, we present a comparison of security, computation, and communication costs and performances for the proposed protocols. The ECC-based protocol is shown to be suitable for higher security WSNs

Enhancing diagnosis of T-cell lymphoma using non-recombined T-cell receptor sequences

Clonality assessment, which can detect neoplastic T cells by identifying the uniquely recombined T-cell receptor (TCR) genes, provides important support in the diagnosis of T-cell lymphoma (TCL). BIOMED-2 is the gold standard clonality assay and has proven to be effective in European TCL patients. However, we failed to prove its sensitivity in Taiwanese TCL patients, especially based on the TCRβ gene. To explore potential impact of genetic background in the BIOMED-2 test, we analyzed TCRβ sequences of 21 healthy individuals and two TCL patients. This analysis suggests that genetic variations in the BIOMED-2 primer sites could not explain the difference in sensitivity. The BIOMED-2 test results of the two TCL patients were positive and negative, respectively. Interestingly, a higher percentage (&gt;81%) of non-recombined TCRβ sequences was observed in the test-negative patient than those of the test-positive patient and all healthy individuals (13~66%). The result suggests a new TCR target for enhancing TCL diagnosis. To further explore the hypothesis, we proposed a cost-effective digital PCR assay that quantifies the relative abundance of non-recombined TCRβ sequences containing a J2-2P~J2-3 segment. With the digital PCR assay, bone marrow specimens from TCL patients (n=9) showed a positive outcome (i.e., the relative abundance of the J2-2P~J2-3 sequences ≧5%), whereas non-TCL patients (n=6) gave a negative result. As five of nine TCL patients had a negative BIOMED-2 test result, the J2-2P~J2-3 sequences may improve TCL detection. This is the first report showing the capability of characterizing non-recombined TCR sequences as a supplementary strategy for the BIOMED-2 clonality test

Progress with the Prime Focus Spectrograph for the Subaru Telescope: a massively multiplexed optical and near-infrared fiber spectrograph

The Prime Focus Spectrograph (PFS) is an optical/near-infrared multi-fiber spectrograph with 2394 science fibers, which are distributed in 1.3 degree diameter field of view at Subaru 8.2-meter telescope. The simultaneous wide wavelength coverage from 0.38 um to 1.26 um, with the resolving power of 3000, strengthens its ability to target three main survey programs: cosmology, Galactic archaeology, and galaxy/AGN evolution. A medium resolution mode with resolving power of 5000 for 0.71 um to 0.89 um also will be available by simply exchanging dispersers. PFS takes the role for the spectroscopic part of the Subaru Measurement of Images and Redshifts project, while Hyper Suprime-Cam works on the imaging part. To transform the telescope plus WFC focal ratio, a 3-mm thick broad-band coated glass-molded microlens is glued to each fiber tip. A higher transmission fiber is selected for the longest part of cable system, while one with a better FRD performance is selected for the fiber-positioner and fiber-slit components, given the more frequent fiber movements and tightly curved structure. Each Fiber positioner consists of two stages of piezo-electric rotary motors. Its engineering model has been produced and tested. Fiber positioning will be performed iteratively by taking an image of artificially back-illuminated fibers with the Metrology camera located in the Cassegrain container. The camera is carefully designed so that fiber position measurements are unaffected by small amounts of high special-frequency inaccuracies in WFC lens surface shapes. Target light carried through the fiber system reaches one of four identical fast-Schmidt spectrograph modules, each with three arms. Prototype VPH gratings have been optically tested. CCD production is complete, with standard fully-depleted CCDs for red arms and more-challenging thinner fully-depleted CCDs with blue-optimized coating for blue arms.Comment: 14 pages, 12 figures, submitted to "Ground-based and Airborne Instrumentation for Astronomy V, Suzanne K. Ramsay, Ian S. McLean, Hideki Takami, Editors, Proc. SPIE 9147 (2014)