Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this ‘protein microarray on a smartphone’-concept for on-site testing, e.g., in food safety, environment and health monitoring.     

Glutathione S-transferase Mu 3 is associated to in vivo fertility,but not sperm quality,in bovine

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.     

Effect of a single growth hormone (rbST) treatment at breeding on conception rates and pregnancy retention in dairy and beef cattle

Initiation of long-term treatment with rbST (Posilac, Monsanto, St. Louis, MO) coincident with first insemination increased pregnancy rates in dairy cattle, but neither the efficacy of using only the initial injection, nor its effects on retention of pregnancy are known. Lactating dairy cows, dairy heifers, and lactating beef cows were assigned at random to treatment (rbST) or control. Dairy cows, dairy heifers, and beef cows received 500 mg rbST (n = 48, 35, 137 inseminations, respectively) at artificial insemination or were left untreated (n = 62, 33, 130 inseminations, respectively). Pregnancy was diagnosed by ultrasonography at 28-36 days. Treatment with rbST at insemination improved conception rates in dairy cows (60.4% versus 40.3%; P < 0.05), but not in dairy heifers or beef cows. Conception rates did not differ in dairy cows at < or =100 days in milk (DIM), but were improved in cows treated with rbST after 100 DIM (64.3% versus 25.8%; P < 0.05). Retention of pregnancy to approximately 60 days and sizes of CL, diameter of follicles > or =5 mm, and crown-rump lengths of embryos were not affected by treatment. The second objective was to examine the effects of rbST at insemination on birth weight and post-natal calf growth in beef cows. However, birth and weaning weights of beef calves were not affected by treatment. In conclusion, a single treatment with rbST at insemination increased conception rates in dairy cows, specifically in those >100 DIM.     

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.     

Metabolic effects of the major component of bovine growth hormone

Bovine growth hormone, subjected to DEAE-cellulose chromatography, yielded one major and several minor components. The various chromatographic fractions of bovine growth hormone were compared with the parent material for their ability to promote hormone effects in vivo and in vitro. The major component of bovine growth hormone was homogeneous by acrylamide-gel electrophoresis, rechromatography and sedimentation equilibrium. Its amino acid composition was similar to that of the parent hormone. The major component possessed all the qualitative activities present in the original heterogeneous material, including promotion of acute hypoglycaemia and hypolipaemia. In studies in vitro in adipose-tissue segments the major component of the hormone increased entry of glucose and its oxidation to CO₂, conversion of glucose into glyceride glycerol, release of glycerol and incorporation of histidine into adiposetissue protein. Other chromatographic fractions of bovine growth hormone were not homogeneous and possessed some but not all of the metabolic activities attributed to the hormone preparations or its major component. Thus, the metabolic effects obtained with bovine growth-hormone preparations in vivo and in vitro can be obtained with the major homogeneous component of the hormone. This observation precludes the possibility that the metabolic effects obtained with bovine growth-hormone preparations are due to the combined actions of a number of components found therein.     

Proteomic Profiling of Cerebrospinal Fluid by 8-Plex iTRAQ Reveals Potential Biomarker Candidates of Alzheimer’s Disease

Introduction  Alzheimer’s disease (AD) poses specific challenges for drug development. It has a slow and variable clinical course, an insidious onset, and symptom expression is only observed when a significant proportion of neurons are already lost. Discussion  Determinants of clinical course, such as molecular biomarkers, are urgently needed for early detection and diagnosis, or for prognosis and monitoring disease-modifying therapies in stratified patient populations. Due to its proximity to the brain and clinical availability, cerebrospinal fluid (CSF) is likely to have the highest yield of biomarker potential for neurodegenerative diseases. In this study, we examined the feasibility of using of an 8-plex isobaric tagging approach, coupled to two-dimensional liquid chromatography and tandem mass spectrometry using the matrix-assisted laser desorption/ionization time-of-flight/time-of-flight platform, for the discovery of potential biomarker candidates in CSF. Comparative analysis identified a number of statistically significant differences in the level of proteins when comparing AD to nondemented controls. Although the study is statistically underpowered to represent the disease population, the regulation of proteins with involvement in processes such as neuronal loss, synaptic dysfunction, neuroinflammation, and tissue degeneration and remodeling reflects the ability of our method in providing biologically meaningful CSF biomarkers as candidates for larger scale biomarker verification and validation studies.     

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.     

Effect of a prolonged release formulation of recombinant bovine somatotropin on plasma concentrations of hormones and metabolites, and milk production in dairy cows.

The effect of a prolonged release formulation of recombinant DNA derived bovine somatotropin (rbST) on the plasma concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, glucose, blood urea nitrogen (BUN) and nonesterified fatty acids (NEFA), and milk production in lactating dairy cows was studied. Eight cows were divided into two equal groups. One group was the noninjected control, and cows in the other group received a single subcutaneous injection of 640 mg rbST. Plasma GH levels in the rbST-treated cows were higher than in the control cows for 10 days after the injection. Plasma IGF-I concentrations were significantly higher in the rbST-treated cows than in the control for 14 days after the treatment. In the rbST-treated cows, the plasma concentrations of insulin and glucose tended to be higher than those in the control until 7 days after the injection. Also, plasma NEFA levels were higher in the rbST-treated cows for 10 days. In contrast, plasma BUN levels were significantly lower in the rbST-treated cows for 17 days after the treatment. For 28 days after the injection, the mean daily milk yield in rbST-treated cows was 4.5 kg (21.2%) more than that in the control cows. In the rbST-treated cows, a highly positive correlation was observed between the mean daily milk yield and the mean plasma concentration of IGF-I throughout the postinjection period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomarkers of dairy fat intake,incident cardiovascular disease,and all-cause mortality: A cohort study,systematic review,and meta-analysis

BackgroundWe aimed to investigate the association of serum pentadecanoic acid (15:0), a biomarker of dairy fat intake, with incident cardiovascular disease (CVD) and all-cause mortality in a Swedish cohort study. We also systematically reviewed studies of the association of dairy fat biomarkers (circulating or adipose tissue levels of 15:0, heptadecanoic acid [17:0], and trans-palmitoleic acid [t16:1n-7]) with CVD outcomes or all-cause mortality.Methods and findingsWe measured 15:0 in serum cholesterol esters at baseline in 4,150 Swedish adults (51% female, median age 60.5 years). During a median follow-up of 16.6 years, 578 incident CVD events and 676 deaths were identified using Swedish registers. In multivariable-adjusted models, higher 15:0 was associated with lower incident CVD risk in a linear dose–response manner (hazard ratio 0.75 per interquintile range; 95% confidence interval 0.61, 0.93, P = 0.009) and nonlinearly with all-cause mortality (P for nonlinearity = 0.03), with a nadir of mortality risk around median 15:0. In meta-analyses including our Swedish cohort and 17 cohort, case–cohort, or nested case–control studies, higher 15:0 and 17:0 but not t16:1n-7 were inversely associated with total CVD, with the relative risk of highest versus lowest tertile being 0.88 (0.78, 0.99), 0.86 (0.79, 0.93), and 1.01 (0.91, 1.12), respectively. Dairy fat biomarkers were not associated with all-cause mortality in meta-analyses, although there were ≤3 studies for each biomarker. Study limitations include the inability of the biomarkers to distinguish different types of dairy foods and that most studies in the meta-analyses (including our novel cohort study) only assessed biomarkers at baseline, which may increase the risk of misclassification of exposure levels.ConclusionsIn a meta-analysis of 18 observational studies including our new cohort study, higher levels of 15:0 and 17:0 were associated with lower CVD risk. Our findings support the need for clinical and experimental studies to elucidate the causality of these relationships and relevant biological mechanisms.

Kathy Trieu and co-workers study biomarkers of dairy fat intake and associated health outcomes.     

Changes of plasma insulin, urea, amino acids and rumen metabolites in somatotropin treated dairy cows

Summary An experiment was performed to evaluate the effects of somatotropin on plasma free amino acid, urea and insulin concentrations and rumen fermentation pattern and to assess their relationships. Four Italian Friesian dairy cows fitted with rumen cannulae were used in a switch-back design. Slow releasing recombinant bovine somatotropin (640 mg/cow) was injected every 28 days for two consecutive periods. Rumen fluid and blood samples were collected before and after feeding at 0, 7 and 21 days after rbST injection. Exogenous rbST increased plasma insulin concentration and the insulin response to feeding, and decreased plasma urea and free essential and branched chain amino acid concentrations. rbST did not affect rumen fermentation pattern. No correlation was found between rumen and plasma parameters measured after feeding. Our results are consistent with the notion that the main effect of somatotropin is post-absorptive.     

Effect of short-term treatment with bovine somatotropin at estrus on conception rate and luteal function of repeat-breeding dairy cows.

We studied the effect of recombinant bovine somatotropin (rbST) at the time of estrus on progesterone concentrations and conception rates of repeat-breeding Holstein cows. We used repeat-breeding cows of varied parity (n = 510). All the animals were clinically healthy and had had at least three unsuccessful services before entering the study. After detection of estrus, the cows were randomly assigned to either a treated (n = 201) or a control (n = 309) group. The animals in the treated group were given rbST (500 mg s.c.) at the time of estrus and again 10 d later. Artificial insemination was performed 12 h after the first detection of estrus. In order to evaluate the effect of rbST on luteal function, blood samples were taken from 10 cows in each group every 3 d for 18 d, starting on the day of insemination (Day 0) to determine progesterone concentrations. Conception rates were significantly higher (P < 0.05) in the cows treated with rbST (29.3%) than in the control cows (16.9%). The effects of rbST were maximal in cows with 8 or more previous unsuccessful services and in cows with 2 to 4 calvings. Progesterone concentrations tended to be higher in nonpregnant cows that were treated with rbST than in those that were not treated. The difference between groups was significant (p < 0.05) on Day 18 after insemination. In pregnant cows there were no significant differences in progesterone concentrations between treated and nontreated animals at any time. Treatment with rbST at estrus improved the conception rate of repeat-breeding Holstein cows. This effect was associated with an increase in circulating progesterone concentrations on Day 18.     

A multi-herd study shows that saliva is more than a reflection of serum biomarkers in pigs

This study evaluates if biomarkers of porcine health status in saliva samples is a mere reflection of serum to detect disease in pigs under field conditions. Four farms from the same commercial company were included to obtain samples from animals with different pathological conditions. A total of 10 healthy animals and 10–15 animals from each farm with clinical symptoms of the disease were sampled for paired saliva and blood during a veterinary clinical visit. The biomarker panel included acute-phase proteins (APPs), C-reactive protein (CRP), haptoglobin (Hp), an inflammatory marker, adenosine deaminase (ADA), the total antioxidant capacity (TAC), the levels of essential trace elements, copper (Cu) and zinc (Zn), and the measurement of the total protein content (TP). After detailed statistical analysis, the results showed that saliva could replace serum for APP measurements since a good agreement has been observed between the concentrations of APPs in both body fluids. For any other biomarker, no agreement between the concentrations quantified in serum and saliva samples was observed visually. However, salivary ADA and TP concentrations were statistically significantly higher in the diseased, whereas the statistical tests with serum concentrations were inconclusive. Furthermore, greater differentiation between healthy and diseased animals could be observed when the distribution of biomarkers was analysed in saliva than in other serum samples. The diagnostic power to discriminate between healthy and diseased pigs is similar in saliva and in serum samples. Preliminary regression models may offer an optimal combination of biomarkers for disease detection in saliva (Hp, CRP, and TAC) and serum (Hp, CRP, and Cu), which demands less labour, sample, and financial cost for saliva determinations. The contradictory results observed for TAC, Cu, and Zn levels between body fluids indicate a need for further studies. To sum up, saliva-based biomarkers instead of serum-based biomarkers could contribute to more efficient detection of diseased animals.     

Tissue and serum proteomic profiling for diagnostic and prognostic bladder cancer biomarkers

A panel of biomarkers for the early detection of bladder cancer has not yet been identified. Many different molecules, including DNA, RNA or proteins have been reported but none have provided adequate sensitivity for a single-tier screening test or a test to replace cystoscopy. Therefore, multimarker panels are discussed at present to give a more-precise answer to the biomarker quest. Mass spectrometry or 2D gel-electrophoresis have evolved greatly within recent years and are capable of analyzing multiple proteins or peptides in parallel with high sensitivity and specificity. However, transmission of screening results from one laboratory to another is still the main pitfall of those methods; a fact that emphasizes the need for consistent and standardized procedures as suggested by the Human Proteome Organization (HUPO). In this article, recent results in screening approaches and other proteomic techniques used for biomarker evaluation in bladder cancer are discussed with a focus on serum and tissue biomarkers.     

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.     

Multiple Inflammatory Biomarker Detection in a Prospective Cohort Study: A Cross-Validation between Well-Established Single-Biomarker Techniques and an Electrochemiluminescense-Based Multi-Array Platform

Background

In terms of time, effort and quality, multiplex technology is an attractive alternative for well-established single-biomarker measurements in clinical studies. However, limited data comparing these methods are available.

Methods

We measured, in a large ongoing cohort study (n = 574), by means of both a 4-plex multi-array biomarker assay developed by MesoScaleDiscovery (MSD) and single-biomarker techniques (ELISA or immunoturbidimetric assay), the following biomarkers of low-grade inflammation: C-reactive protein (CRP), serum amyloid A (SAA), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1). These measures were realigned by weighted Deming regression and compared across a wide spectrum of subjects’ cardiovascular risk factors by ANOVA.

Results

Despite that both methods ranked individuals’ levels of biomarkers very similarly (Pearson’s r all≥0.755) absolute concentrations of all biomarkers differed significantly between methods. Equations retrieved by the Deming regression enabled proper realignment of the data to overcome these differences, such that intra-class correlation coefficients were then 0.996 (CRP), 0.711 (SAA), 0.895 (sICAM-1) and 0.858 (sVCAM-1). Additionally, individual biomarkers differed across categories of glucose metabolism, weight, metabolic syndrome and smoking status to a similar extent by either method.

Conclusions

Multiple low-grade inflammatory biomarker data obtained by the 4-plex multi-array platform of MSD or by well-established single-biomarker methods are comparable after proper realignment of differences in absolute concentrations, and are equally associated with cardiovascular risk factors, regardless of such differences. Given its greater efficiency, the MSD platform is a potential tool for the quantification of multiple biomarkers of low-grade inflammation in large ongoing and future clinical studies.     

Tissue and serum proteomic profiling for diagnostic and prognostic bladder cancer biomarkers

A panel of biomarkers for the early detection of bladder cancer has not yet been identified. Many different molecules, including DNA, RNA or proteins have been reported but none have provided adequate sensitivity for a single-tier screening test or a test to replace cystoscopy. Therefore, multimarker panels are discussed at present to give a more-precise answer to the biomarker quest. Mass spectrometry or 2D gel-electrophoresis have evolved greatly within recent years and are capable of analyzing multiple proteins or peptides in parallel with high sensitivity and specificity. However, transmission of screening results from one laboratory to another is still the main pitfall of those methods; a fact that emphasizes the need for consistent and standardized procedures as suggested by the Human Proteome Organization (HUPO). In this article, recent results in screening approaches and other proteomic techniques used for biomarker evaluation in bladder cancer are discussed with a focus on serum and tissue biomarkers.     

Between- and within-herd variation in blood and milk biomarkers in Holstein cows in early lactation

Both blood- and milk-based biomarkers have been analysed for decades in research settings, although often only in one herd, and without focus on the variation in the biomarkers that are specifically related to herd or diet. Biomarkers can be used to detect physiological imbalance and disease risk and may have a role in precision livestock farming (PLF). For use in PLF, it is important to quantify normal variation in specific biomarkers and the source of this variation. The objective of this study was to estimate the between- and within-herd variation in a number of blood metabolites (β-hydroxybutyrate (BHB), non-esterified fatty acids, glucose and serum IGF-1), milk metabolites (free glucose, glucose-6-phosphate, urea, isocitrate, BHB and uric acid), milk enzymes (lactate dehydrogenase and N-acetyl-β-D-glucosaminidase (NAGase)) and composite indicators for metabolic imbalances (Physiological Imbalance-index and energy balance), to help facilitate their adoption within PLF. Blood and milk were sampled from 234 Holstein dairy cows from 6 experimental herds, each in a different European country, and offered a total of 10 different diets. Blood was sampled on 2 occasions at approximately 14 days-in-milk (DIM) and 35 DIM. Milk samples were collected twice weekly (in total 2750 samples) from DIM 1 to 50. Multilevel random regression models were used to estimate the variance components and to calculate the intraclass correlations (ICCs). The ICCs for the milk metabolites, when adjusted for parity and DIM at sampling, demonstrated that between 12% (glucose-6-phosphate) and 46% (urea) of the variation in the metabolites’ levels could be associated with the herd-diet combination. Intraclass Correlations related to the herd-diet combination were generally higher for blood metabolites, from 17% (cholesterol) to approximately 46% (BHB and urea). The high ICCs for urea suggest that this biomarker can be used for monitoring on herd level. The low variance within cow for NAGase indicates that few samples would be needed to describe the status and potentially a general reference value could be used. The low ICC for most of the biomarkers and larger within cow variation emphasises that multiple samples would be needed - most likely on the individual cows - for making the biomarkers useful for monitoring. The majority of biomarkers were influenced by parity and DIM which indicate that these should be accounted for if the biomarker should be used for monitoring.