Application of Differential Network Enrichment Analysis for Deciphering Metabolic Alterations

Modern analytical methods allow for the simultaneous detection of hundreds of metabolites, generating increasingly large and complex data sets. The analysis of metabolomics data is a multi-step process that involves data processing and normalization, followed by statistical analysis. One of the biggest challenges in metabolomics is linking alterations in metabolite levels to specific biological processes that are disrupted, contributing to the development of disease or reflecting the disease state. A common approach to accomplishing this goal involves pathway mapping and enrichment analysis, which assesses the relative importance of predefined metabolic pathways or other biological categories. However, traditional knowledge-based enrichment analysis has limitations when it comes to the analysis of metabolomics and lipidomics data. We present a Java-based, user-friendly bioinformatics tool named Filigree that provides a primarily data-driven alternative to the existing knowledge-based enrichment analysis methods. Filigree is based on our previously published differential network enrichment analysis (DNEA) methodology. To demonstrate the utility of the tool, we applied it to previously published studies analyzing the metabolome in the context of metabolic disorders (type 1 and 2 diabetes) and the maternal and infant lipidome during pregnancy

Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens

Maternal lipid levels across pregnancy impact the umbilical cord blood lipidome and infant birth weight

Abstract Major alterations in metabolism occur during pregnancy enabling the mother to provide adequate nutrients to support infant development, affecting birth weight (BW) and potentially long-term risk of obesity and cardiometabolic disease. We classified dynamic changes in the maternal lipidome during pregnancy and identified lipids associated with Fenton BW z-score and the umbilical cord blood (CB) lipidome. Lipidomics was performed on first trimester maternal plasma (M1), delivery maternal plasma (M3), and CB plasma in 106 mother-infant dyads. Shifts in the maternal and CB lipidome were consistent with the selective transport of long-chain polyunsaturated fatty acids (PUFA) as well as lysophosphatidylcholine (LysoPC) and lysophosphatidylethanolamine (LysoPE) species into CB. Partial correlation networks demonstrated fluctuations in correlations between lipid groups at M1, M3, and CB, signifying differences in lipid metabolism. Using linear models, LysoPC and LysoPE groups in CB were positively associated with BW. M1 PUFA containing triglycerides (TG) and phospholipids were correlated with CB LysoPC and LysoPE species and total CB polyunsaturated TGs. These results indicate that early gestational maternal lipid levels influence the CB lipidome and its relationship with BW, suggesting an opportunity to modulate maternal diet and improve long-term offspring cardiometabolic health

Average temperature profiles of 74 runs with nine prototype non-instrumented nucleic acid amplification (NINA) heaters.

<p>Error bars show one standard deviation. Four devices were removed from testing when a failure mode rendered them no longer able to maintain temperature within specification. Final run data are not included in the graph. Mean time between failures (MTBF) for the failed devices is 14 runs.</p

A comparison of the effects on the limit of detection (LOD) of assays heated by the non-instrumented nucleic acid amplification (NINA) heater.

<p>Combination of assays run with and without warm-up ramp and with or without oil are evaluated. Table entries show the number of replicates that returned a positive result as the numerator of a fraction showing the total number of replicates run in the denominator.</p><p>A comparison of the effects on the limit of detection (LOD) of assays heated by the non-instrumented nucleic acid amplification (NINA) heater.</p

Melt curve analysis of the HIV-1 and β-actin biplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay.

<p>Melt analysis showed products specific to HIV-1 at 78°C and β-actin at 89°C as seen in the singleplex assays. Specificity was confirmed in the presence of normal human plasma (NHP) without HIV-1 or β-actin (negative control) and showed no amplification (n = 3).</p

HIV LAMP amplicon detection via Milenia test strips.

<p>(A) Milenia strips used for the detection of fluorescein isothiocyanate (FITC)/biotin-labeled amplicons. Image to the right shows a positive result while image to the left is a negative result. (B) Best cassettes for the dual detection of FITC/biotin (HIV assay) and digoxigenin (DIG)/biotin (β-actin assay)–labeled amplicons. Image shows a positive result for both FITC/biotin amplicons and DIG/biotin amplicons.</p

Details of the primer sets used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) including the primers tagged for nucleic acid lateral flow (NALF) detection.

<p>The HIV and β-actin primer sets are modified for lateral flow detection by the addition of biotin, fluorescein isothiocyanate (FITC), and digoxigenin (DIG).</p><p>Details of the primer sets used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) including the primers tagged for nucleic acid lateral flow (NALF) detection.</p

Results for normal human plasma (NHP) containing HIV-1 RNA diluted to 580, 290, 145, 73, and 0 (negative control) total copies in a 25 µl reaction.

<p>Extracted genomic nucleic acid at 2.2 ng/µL was in all samples except the negative control and HIV-positive control. In this comparison, samples and controls assayed by the non-instrumented nucleic acid amplification (NINA)/nucleic acid lateral flow (NALF) system and the Stratagene system were aliquoted from the same sample extraction and handled identically up to amplification. Since the NINA heater does not have top-heating, oil was added to these samples for amplification to control for any condensation artifacts. The samples in the top-heated Stratagene were amplified as per the manufacturer’s instructions (no mineral oil) to reflect a standardized reference method.</p><p>Results for normal human plasma (NHP) containing HIV-1 RNA diluted to 580, 290, 145, 73, and 0 (negative control) total copies in a 25 µl reaction.</p

Three non-instrumented nucleic acid amplification (NINA) heaters were conditioned in an environmental chamber, and the performance of each heater was determined at multiple temperatures (12°C, 14°C, 16°C, 30°C, 31°C, and 32°C).

<p>Averages are shown. The green lines show averages that were within the specification of 61.5°C +/−1.5°C. The red lines show runs that over-heated, and the blue lines show runs that fell below the test specifications.</p