Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Introduction: Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations.

Evaluation of three rapid low-resource molecular tests for Nipah virus.

Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal amplification coupled with lateral flow detection.

Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices.

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology.

Rapid sample preparation and low-resource molecular detection of hepatopancreatic parvoviruses (HPV) by recombinase polymerase amplification lateral flow detection assay in shrimps (Fenneropenaeus merguiensis).

Background: Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming.

Rapid detection of kdr mutation F1534C in Aedes aegypti using recombinase polymerase amplification and lateral flow dipsticks.

Insecticide resistance monitoring is essential in assessing the efficacy of vector control measures. However, gold standard PCR-based molecular analyses for insecticide resistance detection are often hindered by time-consuming sample processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step sample preparation reagent with a rapid isothermal molecular test that detects a knock down resistance (kdr) mutation (F1534C) that enables pyrethroid resistance in Aedes aegypti mosquitoes.

Complexing deoxyribozymes with RNA aptamers for detection of the small molecule theophylline.

Biointegrative information processing systems offer a great advantage to autonomous biodevices, as their capacity for biological computation provides the ability to sense the state of more complex environments and better integrate with downstream biological regulation systems. Deoxyribozymes (DNAzymes) and aptamers are of interest to such computational biosensing systems due to the enzymatic properties of DNAzymes and the ligand-inducible conformational structures of aptamers. Herein, we describe a novel method for providing ligand-responsive allosteric control to a DNAzyme using an RNA aptamer.

Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care.

Background: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories.

Repeated Reuse of Deoxyribozyme-Based Logic Gates.

Deoxyribozymes (DNAzymes) have demonstrated a significant capacity for biocomputing and hold promise for information processing within advanced biological devices if several key capabilities are developed. One required capability is reuse-having DNAzyme logic gates be cyclically, and controllably, activated and deactivated. We designed an oligonucleotide-based system for DNAzyme reuse that could (1) remove previously bound inputs by addition of complementary oligonucleotides via toe-hold mediated binding and (2) diminish output signal through the addition of quencher-labeled oligonucleotides complementary to the fluorophore-bound substrate.

Multiplex Detection of Nucleic Acids Using Recombinase Polymerase Amplification and a Molecular Colorimetric 7-Segment Display.

Nucleic acid analysis has become highly relevant for point-of-care (POC) diagnostics since the advent of isothermal amplification methods that do not require thermal cycling. In particular, recombinase polymerase amplification (RPA) combined with lateral flow detection offers a rapid and simple solution for field-amenable low-resource nucleic acid testing. Expanding POC nucleic acid tests for the detection of multiple analytes is vital to improve diagnostic efficiency because increased multiplexing capacity enables higher information density combined with reduced assay time and costs.

Cardiac myocyte KLF5 regulates body weight via alteration of cardiac FGF21.

Cardiac metabolism affects systemic energetic balance. Previously, we showed that Krüppel-like factor (KLF)-5 regulates cardiomyocyte PPARα and fatty acid oxidation-related gene expression in diabetes. We surprisingly found that cardiomyocyte-specific KLF5 knockout mice (αMHC-KLF5) have accelerated diet-induced obesity, associated with increased white adipose tissue (WAT).

Krüppel-like factors: Crippling and un-crippling metabolic pathways.

Krüppel-like factors (KLFs) are DNA-binding transcriptional factors that regulate various pathways that control metabolism and other cellular mechanisms. Various KLF isoforms have been associated with cellular, organ or systemic metabolism. Altered expression or activation of KLFs has been linked to metabolic abnormalities, such as obesity and diabetes, as well as with heart failure.

Ebolavirus diagnosis made simple, comparable and faster than molecular detection methods: preparing for the future.

Background: The 2014/2015 Ebolavirus outbreak resulted in more than 28,000 cases and 11,323 reported deaths, as of March 2016. Domestic transmission of the Guinea strain associated with the outbreak occurred mainly in six African countries, and international transmission was reported in four countries. Outbreak management was limited by the inability to rapidly diagnose infected cases.

Cardiac Myocyte KLF5 Regulates Ppara Expression and Cardiac Function.

Rationale: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown.

Fasting-induced G0/G1 switch gene 2 and FGF21 expression in the liver are under regulation of adipose tissue derived fatty acids.

Background & Aims: Adipose tissue (AT)-derived fatty acids (FAs) are utilized for hepatic triacylglycerol (TG) generation upon fasting. However, their potential impact as signaling molecules is not established. Herein we examined the role of exogenous AT-derived FAs in the regulation of hepatic gene expression by investigating mice with a defect in AT-derived FA supply to the liver.

The interplay of protein kinase A and perilipin 5 regulates cardiac lipolysis.

Defective lipolysis in mice lacking adipose triglyceride lipase provokes severe cardiac steatosis and heart dysfunction, markedly shortening life span. Similarly, cardiac muscle (CM)-specific Plin5 overexpression (CM-Plin5) leads to severe triglyceride (TG) accumulation in cardiomyocytes via impairing TG breakdown. Interestingly, cardiac steatosis due to overexpression of Plin5 is compatible with normal heart function and life span indicating a more moderate impact of Plin5 overexpression on cardiac lipolysis and energy metabolism.

Fibroblast growth factor 21 is induced upon cardiac stress and alters cardiac lipid homeostasis.

Fibroblast growth factor 21 (FGF21) is a PPARα-regulated gene elucidated in the liver of PPARα-deficient mice or PPARα agonist-treated mice. Mice globally lacking adipose triglyceride lipase (ATGL) exhibit a marked defect in TG catabolism associated with impaired PPARα-activated gene expression in the heart and liver, including a drastic reduction in hepatic FGF21 mRNA expression. Here we show that FGF21 mRNA expression is markedly increased in the heart of ATGL-deficient mice accompanied by elevated expression of endoplasmic reticulum (ER) stress markers, which can be reversed by reconstitution of ATGL expression in cardiac muscle.

Functional cardiac lipolysis in mice critically depends on comparative gene identification-58.

Efficient catabolism of cellular triacylglycerol (TG) stores requires the TG hydrolytic activity of adipose triglyceride lipase (ATGL). The presence of comparative gene identification-58 (CGI-58) strongly increased ATGL-mediated TG catabolism in cell culture experiments. Mutations in the genes coding for ATGL or CGI-58 in humans cause neutral lipid storage disease characterized by TG accumulation in multiple tissues.

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier.

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels.

ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-α and PGC-1.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate genes involved in energy metabolism and inflammation. For biological activity, PPARs require cognate lipid ligands, heterodimerization with retinoic X receptors, and coactivation by PPAR-γ coactivator-1α or PPAR-γ coactivator-1β (PGC-1α or PGC-1β, encoded by Ppargc1a and Ppargc1b, respectively). Here we show that lipolysis of cellular triglycerides by adipose triglyceride lipase (patatin-like phospholipase domain containing protein 2, encoded by Pnpla2; hereafter referred to as Atgl) generates essential mediator(s) involved in the generation of lipid ligands for PPAR activation.