Influence of a prudent diet on circulating cathepsin S in humans.

BACKGROUND: Increased circulating cathepsin S levels have been linked to increased risk of cardiometabolic diseases and cancer. However, whether cathepsin S is a modifiable risk factor is unclear. We aimed to investigate the effects of a prudent diet on plasma cathepsin S levels in healthy individuals. FINDINGS: Explorative analyses of a randomized study were performed in 88 normal to slightly overweight and hyperlipidemic men and women (aged 25 to 65) that were randomly assigned to ad libitum prudent diet, i.e. healthy Nordic diet (ND) or a control group (habitual Western diet) for 6 weeks. Whereas all foods in the ND were provided, the control group was advised to consume their habitual diet throughout the study. The ND was in line with dietary recommendations, e.g. low in saturated fats, sugars and salt, but high in plant-based foods rich in fibre and unsaturated fats.The ND significantly decreased cathepsin S levels (from 20.1 (+/-4.0 SD) to 19.7 µg/L (+/-4.3 SD)) compared with control group (from 18.2 (+/-2.9 SD) to 19.1 µg/L (+/-3.8 SD)). This difference remained after adjusting for sex and change in insulin sensitivity (P = 0.03), and near significant after adjusting for baseline cathepsin S levels (P = 0.06), but not for change in weight or LDL-C. Changes in cathepsin S levels were directly correlated with change in LDL-C. CONCLUSIONS: Compared with a habitual control diet, a provided ad libitum healthy Nordic diet decreased cathepsin S levels in healthy individuals, possibly mediated by weight loss or lowered LDL-C. These differences between groups in cathepsin S were however not robust and therefore need further investigation.

A tunable spherical cap microfluidic electrically small antenna.

A highly efficient microfluidic 3D electrically small antenna is created using a simple fabrication technique. It is easy to construct simply by pneumatically inflating a planar microfluidic antenna into a spherical cap. It has premium performance around its hemispherical shape, combining a wide working band with high efficiency.

Quantitative and multiplex detection of pathogenic fungi using padlock probes, generic qPCR, and suspension array readout.

The multiplexing qualities of padlock probes and Luminex™ technology combined with the well-established quantitative feature of qPCR were the base for a ten-plex fungal detection protocol that quantitatively reveals ten different fungal species in a single experiment. Padlock probes are oligonucleotides designed to form circular DNA when hybridizing to specific target DNA. The 5' and 3' regions of the probes meet and ligate only when a specific target sequence is present in the examined sample. The region of the padlock probes that separates the target-specific 5' and 3' ends contains general primer sequences for amplification of circularized probes by means of rolling circle amplification (RCA) and qPCR. The interspersed region also contains specific tag sequences for subsequent Luminex™ recognition.

Serum cathepsin S is associated with decreased insulin sensitivity and the development of type 2 diabetes in a community-based cohort of elderly men.

OBJECTIVE: To investigate associations between serum cathepsin S, impaired insulin sensitivity, defective insulin secretion, and diabetes risk in a community-based sample of elderly men without diabetes. RESEARCH DESIGN AND METHODS: Serum cathepsin S, insulin sensitivity (euglycemic-hyperinsulinemic clamp), and insulin secretion (early insulin response during an oral glucose tolerance test) were measured in 905 participants of the Uppsala Longitudinal Study of Adult Men (mean age, 71 years). Thirty participants developed diabetes during 6 years of follow-up. RESULTS: After adjustment for age, anthropometric variables, and inflammatory markers, higher cathepsin S was associated with decreased insulin sensitivity (regression coefficient per SD increase -0.09 [95% CI -0.14 to -0.04], P = 0.001), but no association with early insulin response was found. Moreover, higher cathepsin S was associated with a higher risk for developing diabetes (odds ratio per SD increase 1.48 [1.08-2.01], P = 0.01). CONCLUSIONS: Cathepsin S activity appears to be involved in the early dysregulation of glucose and insulin metabolism.

Liquid alloy printing of microfluidic stretchable electronics.

Recently, microfluidic stretchable electronics has attracted great interest from academia since conductive liquids allow for larger cross-sections when stretched and hence low resistance at longer lengths. However, as a serial process it has suffered from low throughput, and a parallel processing technology is needed for more complex systems and production at low costs. In this work, we demonstrate such a technology to implement microfluidic electronics by stencil printing of a liquid alloy onto a semi-cured polydimethylsiloxane (PDMS) substrate, assembly of rigid active components, encapsulation by pouring uncured PDMS on-top and subsequent curing. The printing showed resolution of 200 µm and linear resistance increase of the liquid conductors when elongated up to 60%. No significant change of resistance was shown for a circuit with one LED after 1000 times of cycling between a 0% and an elongation of 60% every 2 s. A radio frequency identity (RFID) tag was demonstrated using the developed technology, showing that good performance could be maintained well into the radio frequency (RF) range.

Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes.

In contrast to ordinary PCRs, which have a limited multiplex capacity and often return false-negative results due to target variation or inhibition, our new detection strategy, VOCMA (variation-tolerant capture multiplex assay), allows variation-tolerant, target-specific capture and detection of many nucleic acids in one test. Here we demonstrate the use of a single-tube, dual-step amplification strategy that overcomes the usual limitations of PCR multiplexing, allowing at least a 22-plex format with retained sensitivity. Variation tolerance was achieved using long primers and probes designed to withstand variation at known sites and a judicious mix of degeneration and universal bases. We tested VOCMA in situations where enrichment from a large sample volume with high sensitivity and multiplexity is important (sepsis; streptococci, enterococci, and staphylococci, several enterobacteria, candida, and the most important antibiotic resistance genes) and where variation tolerance and high multiplexity is important (gastroenteritis; astrovirus, adenovirus, rotavirus, norovirus genogroups I and II, and sapovirus, as well as enteroviruses, which are not associated with gastroenteritis). Detection sensitivities of 10 to 1,000 copies per reaction were achieved for many targets. VOCMA is a highly multiplex, variation-tolerant, general purpose nucleic acid detection concept. It is a specific and sensitive method for simultaneous detection of nucleic acids from viruses, bacteria, fungi, and protozoa, as well as host nucleic acid, in the same test. It can be run on an ordinary PCR and a Luminex machine and is suitable for both clinical diagnoses and microbial surveillance.

No evidence for xenotropic murine leukemia-related virus infection in Sweden using internally controlled multiepitope suspension array serology.

Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.

Association between serum cathepsin S and mortality in older adults.

CONTEXT: Experimental data suggest that cathepsin S, a cysteine protease, is involved in the complex pathways leading to cardiovascular disease and cancer. However, prospective data concerning a potential association between circulating cathepsin S levels and mortality are lacking. OBJECTIVE: To investigate associations between circulating cathepsin S levels and mortality in 2 independent cohorts of elderly men and women. DESIGN, SETTING, AND PARTICIPANTS: Prospective study using 2 community-based cohorts, the Uppsala Longitudinal Study of Adult Men (ULSAM; n = 1009; mean age: 71 years; baseline period: 1991-1995; median follow-up: 12.6 years; end of follow-up: 2006) and the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS; n = 987; 50% women; mean age: 70 years; baseline period: 2001-2004; median follow-up: 7.9 years; end of follow-up: 2010). Serum samples were used to measure cathepsin S. MAIN OUTCOME MEASURE: Total mortality. RESULTS: During follow-up, 413 participants died in the ULSAM cohort (incidence rate: 3.59/100 person-years at risk) and 100 participants died in the PIVUS cohort (incidence rate: 1.32/100 person-years at risk). In multivariable Cox regression models adjusted for age, systolic blood pressure, diabetes, smoking status, body mass index, total cholesterol, high-density lipoprotein cholesterol, antihypertensive treatment, lipid-lowering treatment, and history of cardiovascular disease, higher serum cathepsin S was associated with an increased risk for mortality (ULSAM cohort: hazard ratio [HR] for 1-unit increase of cathepsin S, 1.04 [95% CI, 1.01-1.06], P = .009; PIVUS cohort: HR for 1-unit increase of cathepsin S, 1.03 [95% CI, 1.00-1.07], P = .04). In the ULSAM cohort, serum cathepsin S also was associated with cardiovascular mortality (131 deaths; HR for quintile 5 vs quintiles 1-4, 1.62 [95% CI, 1.11-2.37]; P = .01) and cancer mortality (148 deaths; HR for 1-unit increase of cathepsin S, 1.05 [95% CI, 1.01-1.10]; P = .01). CONCLUSIONS: Among elderly individuals in 2 independent cohorts, higher serum cathepsin S levels were associated with increased mortality risk. Additional research is needed to delineate the role of cathepsin S and whether its measurement might have clinical utility.

The combined contribution of albuminuria and glomerular filtration rate to the prediction of cardiovascular mortality in elderly men.

BACKGROUND: Cardiovascular risk prediction is particularly important in the primary prevention of cardiovascular disease (CVD). Yet, data on whether the combined addition of albuminuria and estimated glomerular filtration rate (eGFR) improves cardiovascular risk prediction in individuals without CVD in the community is scarce. METHODS: We investigated associations between urinary albumin excretion rate (UAER), cystatin C-based eGFR and cardiovascular mortality in a community-based cohort of elderly men (ULSAM study; n = 1113, mean age 71 years, 208 cardiovascular deaths, median follow-up 12.9 years) with prespecified analyses in participants without CVD (n = 649, 86 cardiovascular deaths). RESULTS: Using multivariable Cox regression, higher UAER and lower eGFR were associated with increased risk for cardiovascular mortality independently of established cardiovascular risk factors in the whole sample and in men without CVD at baseline [subsample without CVD: UAER; hazard ratio (HR) per 1 SD 1.26, 95% confidence interval (CI) 1.05-1.51, P = 0.01; eGFR: HR per 1 SD 0.74, 95% CI 0.59-0.92, P = 0.007]. Analyses of model discrimination, calibration, reclassification and global fit suggested that UAER and eGFR also add relevant prognostic information beyond established cardiovascular risk factors in participants without prevalent CVD. Interestingly, established cutoffs used to diagnose microalbuminuria (UAER > 20 µg/min) and chronic kidney disease Stage 3 (eGFR < 60 mL/min/1.73 m(2)), appeared less suitable for cardiovascular risk prediction [integrated discrimination improvement (IDI) 0.006, P = 0.11], while cutoffs UAER > 6 µg/min and eGFR < 45 mL/min/1.73 m(2) significantly improved IDI (0.047, P < 0.001). CONCLUSIONS: UAER and eGFR improved cardiovascular risk prediction beyond established cardiovascular risk factors, suggesting that these kidney biomarkers may be useful in predicting cardiovascular death in elderly men.

Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm.

One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.

A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identification of pestiviruses.

The genus pestivirus contains four recognized species: classical swine fever virus, border disease virus, bovine viral diarrhoea virus types 1 and 2. All are economically important and globally distributed but classical swine fever is the most serious, concerning losses and control measures. It affects both domestic pigs and wild boars. Outbreaks of this disease in domestic pigs call for the most serious measures of disease control, including a stamping out policy in Europe. Since all the members of the pestivirus genus can infect swine, differential diagnosis using traditional methods poses some problems. Antibody tests may lack specificity due to cross-reactions, antigen capture ELISAs may have low sensitivity, and virus isolation may take several days or even longer time to complete. PCR-based tests overcome these problems for the most part, but in general lack the multiplexing capability to detect and differentiate all the pestiviruses simultaneously. The assay platform described here addresses all of these issues by combining the advantages of real-time PCR with the multiplexing capability of microarray technology. The platform includes a TaqMan real-time PCR designed for the universal detection of pestiviruses and a microarray assay that can use the amplicons produced in the real-time PCR to identify the specific pestivirus.

Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout.

A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.

Insulin sensitivity measured with euglycemic clamp is independently associated with glomerular filtration rate in a community-based cohort.

OBJECTIVE: To investigate the association between insulin sensitivity and glomerular filtration rate (GFR) in the community, with prespecified subgroup analyses in normoglycemic individuals with normal GFR. RESEARCH DESIGN AND METHODS: We investigated the cross-sectional association between insulin sensitivity (M/I, assessed using euglycemic clamp) and cystatin C-based GFR in a community-based cohort of elderly men (Uppsala Longitudinal Study of Adult Men [ULSAM], n = 1,070). We also investigated whether insulin sensitivity predicted the incidence of renal dysfunction at a follow-up examination after 7 years. RESULTS: Insulin sensitivity was directly related to GFR (multivariable-adjusted regression coefficient for 1-unit higher M/I 1.19 [95% CI 0.69-1.68]; P < 0.001) after adjusting for age, glucometabolic variables (fasting plasma glucose, fasting plasma insulin, and 2-h glucose after an oral glucose tolerance test), cardiovascular risk factors (hypertension, dyslipidemia, and smoking), and lifestyle factors (BMI, physical activity, and consumption of tea, coffee, and alcohol). The positive multivariable-adjusted association between insulin sensitivity and GFR also remained statistically significant in participants with normal fasting plasma glucose, normal glucose tolerance, and normal GFR (n = 443; P < 0.02). In longitudinal analyses, higher insulin sensitivity at baseline was associated with lower risk of impaired renal function (GFR <50 ml/min per 1.73 m(2)) during follow-up independently of glucometabolic variables (multivariable-adjusted odds ratio for 1-unit higher of M/I 0.58 [95% CI 0.40-0.84]; P < 0.004). CONCLUSIONS: Our data suggest that impaired insulin sensitivity may be involved in the development of renal dysfunction at an early stage, before the onset of diabetes or prediabetic glucose elevations. Further studies are needed in order to establish causality.

DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays.

Genotyping technologies need to be continually improved in terms of their flexibility, cost-efficiency, and throughput, to push forward genome variation analysis. To this end, we have leveraged the inherent simplicity of dynamic allele-specific hybridization (DASH) and coupled it to recent innovations of centrifugal arrays and iFRET. We have thereby created a new genotyping platform we term DASH-2, which we demonstrate and evaluate in this report. The system is highly flexible in many ways (any plate format, PCR multiplexing, serial and parallel array processing, spectral-multiplexing of hybridization probes), thus supporting a wide range of application scales and objectives. Precision is demonstrated to be in the range 99.8-100%, and assay costs are 0.05 USD or less per genotype assignment. DASH-2 thus provides a powerful new alternative for genotyping practice, which can be used without the need for expensive robotics support.

iFRET: an improved fluorescence system for DNA-melting analysis.

Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.

Creating arrays by centrifugation.

We describe afast, low-cost, and reliable way of creating arrays from sample molecules of interest present within microformatted sample vessels (such as 1536-well microplates). The principle involves simple centrifugal transfer of molecules of interest onto a solid planar or membrane surfaces placed over the initial sample vessel. Tools and procedures are presented that validate the robustness and precision of this facile solution to an otherwise difficult problem in modern molecular genetics. The availability of transferred DNA molecules for hybridization is also demonstrated. In conclusion, this "centrifugal-array" concept should help research studies to be applied on ever-greater scales with very simple machinery.

Effect of oligonucleotide truncation on single-nucleotide distinction by solid-phase hybridization.

Oligonucleotide microarrays are used to analyze target sequences on the basis of differences in hybridization stability between matched and mismatched probe-target duplexes. DNA microarray manufacture via photolithographic synthesis generates a minority of full-length oligonucleotide probes along with a series of 5'-truncated contaminants. In a model experiment, we now investigate the effect of truncated oligonucleotides on the ability to distinguish target sequence variants that differ in a single nucleotide position. A series of oligonucleotides, mixed in proportions simulating stepwise synthetic yields of between 82 and 100%, were bound to a solid support and allowed to hybridize to a target molecule. The extent of hybridization was monitored over a range of temperatures via the fluorescence of a double-strand-specific dye. The discriminatory power of pure oligonucleotide probes was found to be significantly greater than that of a population of truncated probes, but only over a limited temperature interval. We conclude that at optimal temperatures greater oligonucleotide quality can improve the performance of oligonucleotide hybridization microarrays.