Statistical Analysis of Variation in the Human Plasma Proteome

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery

Characterization and Reconstruction of Nanolipoprotein Particles (Nlps) by Cryo-EM and Image Reconstruction

Nanolipoprotein particles (NLPs) are small 10-20 nm diameter assemblies of apolipoproteins and lipids. At Lawrence Livermore National Laboratory (LLNL), they have constructed multiple variants of these assemblies. NLPs have been generated from a variety of lipoproteins, including apolipoprotein Al, apolipophorin III, apolipoprotein E4 22K, and MSP1T2 (nanodisc, Inc.). Lipids used included DMPC (bulk of the bilayer material), DMPE (in various amounts), and DPPC. NLPs were made in either the absence or presence of the detergent cholate. They have collected electron microscopy data as a part of the characterization component of this research. Although purified by size exclusion chromatography (SEC), samples are somewhat heterogeneous when analyzed at the nanoscale by negative stained cryo-EM. Images reveal a broad range of shape heterogeneity, suggesting variability in conformational flexibility, in fact, modeling studies point to dynamics of inter-helical loop regions within apolipoproteins as being a possible source for observed variation in NLP size. Initial attempts at three-dimensional reconstructions have proven to be challenging due to this size and shape disparity. They are pursuing a strategy of computational size exclusion to group particles into subpopulations based on average particle diameter. They show here results from their ongoing efforts at statistically and computationally subdividing NLP populations to realize greater homogeneity and then generate 3D reconstructions

High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol

Expression and Localization of Mitochondrial Ferritin mRNA in Alzheimer's Disease Cerebral Cortex

Mitochondrial ferritin (MtF) has been identified as a novel ferritin encoded by an intron-lacking gene with specific mitochondrial localization located on chromosome 5q23.1. MtF has been associated with neurodegenerative disorders such as Friedreich ataxia and restless leg syndrome. However, little information is available about MtF in Alzheimer's disease (AD). In this study, therefore, we investigated the expression and localization of MtF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) as well as in situ hybridization histochemistry. We also examined protein expression using western-blot assay. In addition, we used in vitro methods to further explore the effect of oxidative stress and β-amyloid peptide (Aβ) on MtF expression. To do this we examined MtF mRNA and protein expression changes in the human neuroblastoma cell line, IMR-32, after treatment with Aβ, H2O2, or both. The neuroprotective effect of MtF on oxidative stress induced by H2O2 was measured by MTT assay. The in situ hybridization studies revealed that MtF mRNA was detected mainly in neurons to a lesser degree in glial cells in the cerebral cortex. The staining intensity and the number of positive cells were increased in the cerebral cortex of AD patients. Real-time PCR and western-blot confirmed that MtF expression levels in the cerebral cortex were significantly higher in AD cases than that in control cases at both the mRNA and the protein level. Cell culture experiments demonstrated that the expression of both MtF mRNA and protein were increased by treatment with H2O2 or a combination of Aβ and H2O2, but not with Aβ alone. Finally, MtF expression showed a significant neuroprotective effect against H2O2-induced oxidative stress (p<0.05). The present study suggests that MtF is involved in the pathology of AD and may play a neuroprotective role against oxidative stress

Menstruation and the cycle of poverty: a cluster quasi-randomised control trial of sanitary pad and puberty education provision in Uganda

BACKGROUND: Poor menstrual knowledge and access to sanitary products have been proposed as barriers to menstrual health and school attendance. In response, interventions targeting these needs have seen increasing implementation in public and private sectors. However, there has been limited assessment of their effectiveness. // OBJECTIVES: Assess the impact of providing reusable sanitary pads and puberty education on girls’ school attendance and psychosocial wellbeing outcomes. // METHODS: A cluster quasi-randomised controlled trial was conducted across 8 schools, including 1124 girls, in rural Uganda. Schools were allocated to one of four conditions: the provision of puberty education alone; reusable sanitary pads alone; puberty education and reusable sanitary pads; and a control (no intervention). The primary outcome was school attendance. Secondary outcomes reflected psychosocial wellbeing. // RESULTS: At follow-up, school attendance had worsened for girls across all conditions. Per-protocol analysis revealed that this decline was significantly greater for those in the control condition d = 0.52 (95%CI 0.26–0.77), with those in control schools having a 17.1% (95%CI: 8.7–25.5) greater drop in attendance than those in any intervention school. There were no differences between the intervention conditions. High rates of school drop-out and transfer meant the trial suffered from substantial participant drop-out. Intention-to-treat analyses using two different imputation strategies were consistent with the main results, with mean differences of 5.2% attendance in best-case and 24.5% in worst-case imputations. Results were robust to adjustments for clustering. There was no impact of the interventions on girls’ self-reported shame or insecurity during menstruation. // CONCLUSION: Results of the trial support the hypothesised positive impact of providing sanitary pads or puberty education for girls’ school attendance in a developing country context. Findings must be interpreted with caution in light of poor participant retention, intervention fidelity, and the attendance measures used

Estimated GFR reporting is not sufficient to allow detection of chronic kidney disease in an Italian regional hospital

<p>Abstract</p> <p>Background</p> <p>Chronic kidney disease (CKD) is an emerging worldwide problem. The lack of attention paid to kidney disease is well known and has been described in previous publications. However, little is known about the magnitude of the problem in highly specialized hospitals where serum creatinine values are used to estimate GFR values.</p> <p>Methods</p> <p>We performed a cross-sectional evaluation of hospitalized adult patients who were admitted to the medical or surgical department of Santa Maria della Misericordia Hospital in 2007. Information regarding admissions was derived from a database. Our goal was to assess the prevalence of CKD (defined as an estimated glomerular filtration rate [eGFR] < 60 mL/min/1.73 m²) and detection of CKD using diagnostic codes (Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM]). To reduce the impact of acute renal failure on the study, the last eGFR obtained during hospitalization was the value used for analysis, and intensive care and nephrology unit admissions were excluded. We also excluded patients who had ICD-9-CM codes for renal replacement therapy, acute renal failure, and contrast administration listed as discharge diagnoses.</p> <p>Results</p> <p>Of the 18,412 patients included in the study, 4,748 (25.8%) had reduced eGFRs, falling into the category of Kidney Disease Outcomes Quality Initiative (KDOQI) stage 3 (or higher) CKD. However, the diagnosis of CKD was only reported in 19% of these patients (904/4,748). It is therefore evident that there was a "gray area" corresponding to stage 3 CKD (eGFR 30-59 ml/min), in which most CKD diagnoses are missed. The ICD-9 code sensitivity for detecting CKD was significantly higher in patients with diabetes, hypertension, and cardiovascular disease (26.8%, 22.2%, and 23.7%, respectively) than in subjects without diabetes, hypertension, or cardiovascular disease (p < 0.001), but these values are low when the widely described relationship between such comorbidities and CKD is considered.</p> <p>Conclusion</p> <p>Although CKD was common in this patient population at a large inpatient regional hospital, the low rates of CKD detection emphasize the primary role nephrologists must play in continued medical education, and the need for ongoing efforts to train physicians (particularly primary care providers) regarding eGFR interpretation and systematic screening for CKD in high-risk patients (i.e., the elderly, diabetics, hypertensives, and patients with CV disease).</p

DNA-Free Recombinant SV40 Capsids Protect Mice from Acute Renal Failure by Inducing Stress Response, Survival Pathway and Apoptotic Arrest

Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses

Altering APP Proteolysis: Increasing sAPPalpha Production by Targeting Dimerization of the APP Ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers—which bind to dimers of the ectodomain— and Aβ fragments—which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP695 328–332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease