A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).     

Metaprotein expression modeling for label-free quantitative proteomics

Background

Label-free quantitative proteomics holds a great deal of promise for the future study of both medicine and biology. However, the data generated is extremely intricate in its correlation structure, and its proper analysis is complex. There are issues with missing identifications. There are high levels of correlation between many, but not all, of the peptides derived from the same protein. Additionally, there may be systematic shifts in the sensitivity of the machine between experiments or even through time within the duration of a single experiment.

Results

We describe a hierarchical model for analyzing unbiased, label-free proteomics data which utilizes the covariance of peptide expression across samples as well as MS/MS-based identifications to group peptides??a strategy we call metaprotein expression modeling. Our metaprotein model acknowledges the possibility of misidentifications, post-translational modifications and systematic differences between samples due to changes in instrument sensitivity or differences in total protein concentration. In addition, our approach allows us to validate findings from unbiased, label-free proteomics experiments with further unbiased, label-free proteomics experiments. Finally, we demonstrate the clinical/translational utility of the model for building predictors capable of differentiating biological phenotypes as well as for validating those findings in the context of three novel cohorts of patients with Hepatitis C.

Conclusions

Mass-spectrometry proteomics is quickly becoming a powerful tool for studying biological and translational questions. Making use of all of the information contained in a particular set of data will be critical to the success of those endeavors. Our proposed model represents an advance in the ability of statistical models of proteomic data to identify and utilize correlation between features. This allows validation of predictors without translation to targeted assays in addition to informing the choice of targets when it is appropriate to generate those assays.     

Immediate and one-year outcome of patients presenting with Acute Coronary Syndrome complicated by stroke: Findings from the 2nd Gulf Registry of Acute Coronary Events (Gulf RACE-2)

ABSTRACT: BACKGROUND: Stroke is a potential complication of acute coronary syndrome (ACS). The aim of this study was to identify the prevalence, risk factors predisposing to stroke, in-hospital and 1-year mortality among patients presenting with ACS in the Middle East. METHODS: For a period of 9 months in 2008 to 2009, 7,930 consecutive ACS patients were enrolled from 65 hospitals in 6 Middle East countries. RESULTS: The prevalence of in-hospital stroke following ACS was 0.70%. Most cases were ST segment elevation MI-related (STEMI) and ischemic stroke in nature. Patients with in-hospital stroke were 5 years older than patients without stroke and were more likely to have hypertension (66% vs. 47.6%, P = 0.001). There were no differences between the two groups in regards to gender, other cardiovascular risk factors, or prior cardiovascular disease. Patients with stroke were more likely to present with atypical symptoms, advanced Killip class and less likely to be treated with evidence-based therapies. Independent predictors of stroke were hypertension, advanced killip class, ACS type --STEMI and cardiogenic shock. Stroke was associated with increased risk of in-hospital (39.3% vs. 4.3%) and one-year mortality (52% vs. 12.3%). CONCLUSION: There is low incidence of in-hospital stroke in Middle-Eastern patients presenting with ACS but with very high in-hospital and one-year mortality rates. Stroke patients were less likely to be appropriately treated with evidence-based therapy. Future work should be focused on reducing the risk and improving the outcome of this devastating complication.     

Astrogliosis during acute and chronic cuprizone demyelination and implications for remyelination

In multiple sclerosis, microglia/macrophage activation and astrocyte reactivity are important components of the lesion environment that can impact remyelination. The current study characterizes these glial populations relative to expression of candidate regulatory molecules in cuprizone demyelinated corpus callosum. Importantly, periods of recovery after acute or chronic cuprizone demyelination are examined to compare conditions of efficient versus limited remyelination, respectively. Microglial activation attenuates after early demyelination. In contrast, astrocyte reactivity persists throughout demyelination and a 6-week recovery period following either acute or chronic demyelination. This astrocyte reaction is characterized by (a) early proliferation, (b) increased expression of GFAP (glial fibrillary acidic protein), Vim (vimentin), Fn1 (fibronectin) and CSPGs (chondroitin sulphate proteoglycans) and (c) elaboration of a dense network of processes. Glial processes elongated in the axonal plane persist throughout lesion areas during both the robust remyelination that follows acute demyelination and the partial remyelination that follows chronic demyelination. However, prolonged astrocyte reactivity with chronic cuprizone treatment does not progress to barrier formation, i.e. dense compaction of astrocyte processes to wall off the lesion area. Multiple candidate growth factors and inflammatory signals in the lesion environment show strong correlations with GFAP across the acute cuprizone demyelination and recovery time course, yet there is more divergence across the progression of chronic cuprizone demyelination and recovery. However, differential glial scar formation does not appear to be responsible for differential remyelination during recovery in the cuprizone model. The astrocyte phenotype and lesion characteristics in this demyelination model inform studies to identify triggers of non-remyelinating sclerosis in chronic multiple sclerosis lesions.     

Phospholipids of Clostridium perfringens: a reexamination

We have identified phosphatidylethanolamine as one of the major phospholipids of Clostridium perfringens by two dimensional thin layer chromatography of the intact lipids and of their deacylation products and by liquid chromatography followed by mass spectrometry of the intact neutral phospholipid fraction. The principal fatty acids of phosphatidylethanolamine are myristic acid (14:0), lauric acid (12:0), and palmitic acid (16:0) and the major molecular species are 14:0,14:0 (26.3%); 12:0,14:0 (19.0%); 14:0,16:0 (22.4%) and 16:0,16:0 (17.6%). A similar distribution of molecular species was found in the other major phospholipid, O-alanyl phosphatidylglycerol.     

A Clinical Decision Support System for Integrating Tuberculosis and HIV Care in Kenya: A Human-Centered Design Approach

With the aim of integrating HIV and tuberculosis care in rural Kenya, a team of researchers, clinicians, and technologists used the human-centered design approach to facilitate design, development, and deployment processes of new patient-specific TB clinical decision support system for medical providers. In Kenya, approximately 1.6 million people are living with HIV and have a 20-times higher risk of dying of tuberculosis. Although tuberculosis prevention and treatment medication is widely available, proven to save lives, and prioritized by the World Health Organization, ensuring that it reaches the most vulnerable communities remains challenging. Human-centered design, used in the fields of industrial design and information technology for decades, is an approach to improving the effectiveness and impact of innovations that has been scarcely used in the health field. Using this approach, our team followed a 3-step process, involving mixed methods assessment to (1) understand the situation through the collection and analysis of site observation sessions and key informant interviews; (2) develop a new clinical decision support system through iterative prototyping, end-user engagement, and usability testing; and, (3) implement and evaluate the system across 24 clinics in rural West Kenya. Through the application of this approach, we found that human-centered design facilitated the process of digital innovation in a complex and resource-constrained context.     

PSMA Antibody-Conjugated Pentablock Copolymer Nanomicellar Formulation for Targeted Delivery to Prostate Cancer

The main purpose of this study was to develop a prostate-specific membrane antigen (PSMA) antibody-conjugated drug-loaded nanomicelles using MPEG--PLA-PCL-PLA-PEG-NH2 pentablock copolymer for targeted delivery of hydrophobic anticancer drugs to prostate cancer cells. During this experiment, monomers of L-lactide, ε-caprolactone, poly(ethylene glycol)-methyl ether, and poly(ethylene glycol)-NH2 were used to prepare pentablock copolymer using the ring opening technique. The pentablock nanomicellar (PBNM) formulation was prepared by the evaporation-rehydration method. The resultant pentablock nanomicelles were then conjugated with PSMA antibody resulting in PSMA-Ab-PTX-PBNM. Both the block copolymers and the nanomicelles were analyzed by hydrogen nuclear magnetic resonance (H-NMR), Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The obtained nanomicelles (NM) were then analyzed for size and zeta potential using dynamic light scattering-dynamic laser scattering (DLS) and then further submitted to H-NMR and TEM analyses. The XRD, FTIR, and the H-NMR analyses confirmed the structure of the pentablock copolymers. The average size for conjugated nanomicellar was 45 nm?±?2.5 nm. The average (ζ-potential) was around ??28 mV. H-NMR and FTIR analysis done on PSMA-coupled paclitaxel-loaded PBNM showed peaks characteristic of the drug (paclitaxel) and the polymer, confirming the successful encapsulation. TEM analysis showed well-defined spherical morphology and confirmed the size range obtained by the DLS. In vitro release studies revealed sustained slow of PTX in phosphate buffer solution (PBS). Confocal scanning microscopy (TEM) of coumarin6-loaded in PBNM indicated that pentablock nanomicelles were internalized into the prostate cancer (PC-3) cells. Cell proliferation assay showed that nanomicelles ferried paclitaxel into the PC-3 cells and subsequently reduced the cell proliferation. The results depict PTX-PBNM-Ab as a suitable carrier for targeted delivery of drugs to prostate cancer cells.