Utilizing proteomics to understand and define hypertension: where are we and where do we go?

Introduction: Hypertension is a complex and multifactorial cardiovascular disorder. With different mechanisms contributing to a different extent to an individual’s blood pressure, the discovery of novel pathogenetic principles of hypertension is challenging. However, there is an urgent and unmet clinical need to improve prevention, detection, and therapy of hypertension in order to reduce the global burden associated with hypertension-related cardiovascular diseases.

Areas covered: Proteomic techniques have been applied in reductionist experimental models including angiotensin II infusion models in rodents and the spontaneously hypertensive rat in order to unravel mechanisms involved in blood pressure control and end organ damage. In humans proteomic studies mainly focus on prediction and detection of organ damage, particularly of heart failure and renal disease. While there are only few proteomic studies specifically addressing human primary hypertension, there are more data available in hypertensive disorders in pregnancy, such as preeclampsia. We will review these studies and discuss implications of proteomics on precision medicine approaches.

Expert commentary: Despite the potential of proteomic studies in hypertension there has been moderate progress in this area of research. Standardized large-scale studies are required in order to make best use of the potential that proteomics offers in hypertension and other cardiovascular diseases.     

Residual tissue repositories as a resource for population-based cancer proteomic studies

Background

Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed as compared to banked samples. Annotated formalin fixed, paraffin embedded (FFPE) tumor tissue specimens are available for research use from a variety of tissue banks, such as from the surveillance, epidemiology and end results (SEER) registries’ residual tissue repositories. Given the wealth of outcomes information associated with such samples, the reuse of archived FFPE blocks for deep proteomic characterization with mass spectrometry technologies would provide a valuable resource for population-based cancer studies. Further, due to the widespread availability of FFPE specimens, validation of specimen integrity opens the possibility for thousands of studies that can be conducted worldwide.

Methods

To examine the suitability of the SEER repository tissues for proteomic and phosphoproteomic analysis, we analyzed 60 SEER patient samples, with time in storage ranging from 7 to 32 years; 60 samples with expression proteomics and 18 with phosphoproteomics, using isobaric labeling. Linear modeling and gene set enrichment analysis was used to evaluate the impacts of collection site and storage time.

Results

All samples, regardless of age, yielded suitable protein mass after extraction for expression analysis and 18 samples yielded sufficient mass for phosphopeptide analysis. Although peptide, protein, and phosphopeptide identifications were reduced by 50, 20 and 76% respectively, from comparable OCT specimens, we found no statistically significant differences in protein quantitation correlating with collection site or specimen age. GSEA analysis of GO-term level measurements of protein abundance differences between FFPE and OCT embedded specimens suggest that the formalin fixation process may alter representation of protein categories in the resulting dataset.

Conclusions

These studies demonstrate that residual FFPE tissue specimens, of varying age and collection site, are a promising source of protein for proteomic investigations if paired with rigorously verified mass spectrometry workflows.

     

Seminal characteristics and spermatozoal morphology of captive Queensland koalas (Phascolarctos cinereus adustus)

Viable koala spermatozoa were successfully collected from 8 of 11 captive Queensland koalas on 52 of 76 attempts using electroejaculation under general anesthesia. Semen characteristics, including sperm concentration, percent progressive sperm motility and nuclear heterogeneity appeared to be similar to those of free-ranging Victoria koalas (P.c . victor). Two new head morphotypes (Type XI and teratoid) were identified, and the incidence of midpiece and principal piece spermatozoal abnormalities were recorded.     

Properties of equine luteinizing hormone alpha subunit alone and in combination with various beta subunits

Previous studies have shown that equine luteinizing hormone (eLH) inhibits production of cyclic adenosine monophosphate (cAMP) induced by follicle-stimulating hormone (FSH) in preparations of seminiferous tubules from immature rats. It was also shown that the inhibitory effect was a function of the equine LH (eLH) alpha subunit. To explore this phenomenon further, the intrinsic FSH-like activities of eLH alpha alone and in combination with ovine (o) LH beta, ovine FSH beta, and equine FSH beta were evaluated in several assay systems. In a radioreceptor assay employing 125I-o-FSH and testis membranes from day-old calves, eLH was twice as active as oFSH, eLH alpha was 6% as active as oFSH, and other subunits showed a lack of activity (less than 1.5%). Whereas oLH was only 0.1% as active as oFSH, the hybrid eLH alpha-oLH beta was 3.0% as active. The binding activity of eLH alpha-FSH beta hybrids tended to be higher than the oFSH alpha-FSH beta hybrids. In the cAMP production assay, eLH alpha-FSH beta hybrids exhibited dampened dose-response curves when compared to the oFSH alpha-FSH beta hybrids. In a plasminogen activator assay (PAA) employing granulosa cells from intact 21-24-day-old female rats primed with diethylstilbestrol, eLH had activity comparable to that of oFSH, while eLH alpha was inactive. When eLH alpha was recombined with oFSH beta, eFSH beta, or oLH beta, the PAA stimulatory activity was not altered compared to that of the hybrids oLH alpha-oFSH beta, oFSH alpha-eFSH beta, and the recombinant oLH alpha-oLH beta, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystalloid inclusions in the Sertoli cell of the koala,Phascolarctos cinereus (Marsupialia)

Summary Crystalloid inclusions are a common feature in the basal region of Sertoli cells in the koala, Phascolarctos cinereus. Generally located near the nucleus, they are non membrane-bounded, slender rectangular structures composed of tubules which are orientated at right angles to the long axis of the crystalloid and regularly arranged in rows parallel to this long axis. The tubules in adjacent rows are offset from one another at definite angles and extensively interconnected by filaments. Neither the composition nor function of the crystalloids has been determined, but their association with tonofilaments and the presence of ribosomes in the vicinity suggests that they are most likely proteinaceous.The authors would like to thank Mr. D. Harbrow, Mr. S. Brown and Ms. B. Canty for technical assistance; the Queensland National Parks and Wildlife Service and the Victorian Ministry of Conservation (Fisheries and Wildlife Division) for providing permits to work on this protected species; the staff of the Lone Pine Koala Sanctuary, Brisbane, for their assistance in obtaining animals. This work was supported by a grant from the Australian Research Grants Committee, Number DI-77/15525     

Spatio-temporal variation of periphyton biomass and accumulation in a temperate spring-fed stream

Spatio-temporal variation of plant populations often can demonstrate synchronous patterns, particularly within highly connected landscapes. Periphyton biomass (chlorophyll a) and net accumulation were measured at five sites in a spring-fed fourth-order stream located in central Pennsylvania with a mixed land-uses watershed (Spring Creek, USA) to characterize longitudinal variation within the stream. Samples were collected at three-week intervals over one year to describe seasonal patterns of periphyton biomass and net production (n = 17 per site). Spring Creek periphyton biomass and net accumulation increased dramatically from the headwaters to downstream (range 10–1,000 mg/m²). The downstream reaches had exceptionally large algal biomass (chlorophyll a > 300 mg/m²) and potential for rapid turnover. Varying degrees of seasonality were observed among the sites, with upstream sites showing more temporal variation but no distinct seasonal pattern. Despite this, large-scale disturbances within the watershed seem to promote synchrony among sites throughout the stream as reflected by close correlations in chlorophyll values (Pearson correlation coefficient r > 0.50).     

Periphyton nutrient status in a temperate stream with mixed land-uses: implications for watershed nitrogen storage

We sampled periphyton communities in a highly productive stream to characterize how longitudinal changes in watershed geology and land use affect periphyton nutrient status and elemental composition. Nutrient status was evaluated from measures of periphyton nutrient composition (carbon, nitrogen, and phosphorus), stable isotope signatures (δ¹⁵N and δ¹³C), and the response of periphyton to experimental enrichment with nitrogen. Biomass and nutrient content increased dramatically from the headwaters to downstream, while tissue nutrient ratios (C:P and C:N) were more consistent and did not indicate strong N- or P-limitation. Nitrogen enrichment experiments did not exhibit a consistent response upstream or downstream, and periphyton C:N:P stoichiometry showed no significant response to N-enrichment. Absolute densities of periphyton N were 5- to 90-fold greater than the overlying N concentrations in stream water (159- to 353-fold greater for P), and the δ¹⁵N signal indicates downstream enrichment from likely watershed sources (urban and agriculture land-use). These results suggest that periphyton in Spring Creek are not N-limited and store large quantities of both N and P, which in turn can be transported downstream during high flow events. Handling editor: David Hamilton     

Potential ‘Ecological Traps’ of Restored Landscapes: Koalas Phascolarctos cinereus Re-Occupy a Rehabilitated Mine Site

With progressively increasing anthropogenic habitat disturbances, restoration of impacted landscapes is becoming a critical element of biodiversity conservation. Evaluation of success in restoration ecology rarely includes faunal components, usually only encompassing abiotic and floral components of the ecosystems. Even when fauna is explicitly included, it is usually only species presence/absence criteria that are considered. If restoration is to have a positive outcome, however, populations in restored habitats should exhibit comparable survival and reproductive rates to populations found in undisturbed surroundings. If a species recolonises restored areas but later experiences decreased fitness, restored areas could become ecological sinks or traps. We investigated this possibility in a case study of koalas Phascolarctos cinereus occupying rehabilitated mining areas on North Stradbroke Island, Australia. Our holistic approach compared rehabilitated and undisturbed areas on the basis of their vegetation characteristics, of koalas'' body condition, roosting trees, diet, as well as predator index. Koalas using rehabilitated areas appeared to be able to access an adequate supply of roosting and fodder trees, were in good condition and had high reproductive output. We did not find any significant differences in predator density between rehabilitated areas and undisturbed surroundings. The results presented in this study showed there was no evidence that the post-mining rehabilitated areas constitute ecological sinks or traps. However, to reach a definitive conclusion as to whether areas rehabilitated post-mining provide at least equivalent habitat to undisturbed locations, additional research could be undertaken to assess foliar nutrient/water/toxin differences and predation risk in rehabilitated areas compared with undisturbed areas. More generally, the evaluation of whether restoration successfully produces a functional ecological community should include criteria on the fitness of faunal populations reoccupying such sites, so as to ensure functioning ecosystems, rather than ecological sinks or traps, are the outcome.     

Electrocardiographic Abnormalities in Trypanosoma cruzi Seropositive and Seronegative Former Blood Donors

Background

Blood donor screening leads to large numbers of new diagnoses of Trypanosoma cruzi infection, with most donors in the asymptomatic chronic indeterminate form. Information on electrocardiogram (ECG) findings in infected blood donors is lacking and may help in counseling and recognizing those with more severe disease.

Objectives

To assess the frequency of ECG abnormalities in T.cruzi seropositive relative to seronegative blood donors, and to recognize ECG abnormalities associated with left ventricular dysfunction.

Methods

The study retrospectively enrolled 499 seropositive blood donors in São Paulo and Montes Claros, Brazil, and 483 seronegative control donors matched by site, gender, age, and year of blood donation. All subjects underwent a health clinical evaluation, ECG, and echocardiogram (Echo). ECG and Echo were reviewed blindly by centralized reading centers. Left ventricular (LV) dysfunction was defined as LV ejection fraction (EF)<0.50%.

Results

Right bundle branch block and left anterior fascicular block, isolated or in association, were more frequently found in seropositive cases (p<0.0001). Both QRS and QTc duration were associated with LVEF values (correlation coefficients −0.159,p<0.0003, and −0.142,p = 0.002) and showed a moderate accuracy in the detection of reduced LVEF (area under the ROC curve: 0.778 and 0.790, both p<0.0001). Several ECG abnormalities were more commonly found in seropositive donors with depressed LVEF, including rhythm disorders (frequent supraventricular ectopic beats, atrial fibrillation or flutter and pacemaker), intraventricular blocks (right bundle branch block and left anterior fascicular block) and ischemic abnormalities (possible old myocardial infarction and major and minor ST abnormalities). ECG was sensitive (92%) for recognition of seropositive donors with depressed LVEF and had a high negative predictive value (99%) for ruling out LV dysfunction.

Conclusions

ECG abnormalities are more frequent in seropositive than in seronegative blood donors. Several ECG abnormalities may help the recognition of seropositive cases with reduced LVEF who warrant careful follow-up and treatment.     

A new approach for the detection and identification of protein impurities using combinatorial solid phase ligand libraries

We propose a novel method for detection of protein impurities present in plasma-derived and recombinant purified injectable biopharmaceuticals by enhancing the concentration of protein impurities, in essence "amplifying" their presence to detectable levels. The method is based on the capture of proteins using a combinatorial solid-phase hexapeptides ligand library previously described for the reduction of protein concentration difference in biological fluids. Three proteins have been investigated: Staphylococcus aureus Protein A, expressed in Escherichia coli and supplied as 99% pure, recombinant human albumin, expressed in Pichia pastoris and certified as 95% pure, and therapeutic albumin supplied as 96-98% pure injectable solution. In all cases, after treatment with the ligand libraries, a number of additional polypeptide chains, not visible in the control, could be detected and obtained in sufficient amounts for MS analysis. In the cases of the two recombinant proteins, it could be demonstrated that a number of these polypeptide chains were host cell proteins still present in the purified product. In addition, a substantial number of these spots were found to be cleavage products of the original recombinant DNA species. Such cleavage products were particularly abundant in the recombinant human albumin preparation. From pure injectable serum albumin, a number of human plasma protein impurities were also identified by LC-MS/MS analysis. Treatment with ligand libraries of purified proteins is thus seen as a very powerful method of capture and concentration of host proteins and cleaved products for further analysis to control better the quality of industrial biotechnology products.