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  • PCR
    • Real-Time Quantitative PCR
      • SYBR Green Detection
        • DNA
          Variable effects of Wolbachia on alphavirus infection in Aedes aegypti
          Brittany L. Dodson - 2023
          Abstract
          Wolbachia pipientis (=Wolbachia) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquitoborne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of somatic infection with two strains of Wolbachia (wAlbB and wMel) on the alphaviruses Sindbis virus (SINV), O’nyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti. We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both wAlbB- and wMel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with wAlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; wMel strongly blocked MAYV infections and suppressed viral titers, while wAlbB did not influence MAYV infection. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit.
          Quantifying the effect of grilling and roasting on the eating quality of lamb leg muscles
          Al Moadhen H - 2022
          Abstract
          Lamb eating quality was measured using consumers to develop a cut by cook method eating quality program for sheepmeat. The current protocol for cooking and serving roast lamb legs does not allow for the differentiation of muscles. Thus, there is no comparison of grilled and roasted lamb leg muscles. Previous work conducted in beef has shown that there is a difference in consumer sensory scores for grilled and roasted m. semimembranosus (Watson et al. 2008). This differentiation has allowed the beef industry to predict the eating quality of individual cuts, for each prescribed cooking method. This study aimed to quantify the effect of cooking method on the eating quality of three different muscles: m.semimembranosus (topside); m.biceps femoris (outside flat); and m.rectus femoris, vastus lateralis and vastus intermedius (knuckle). It was hypothesised that the outside flat, topside and knuckle will have different consumer eating quality when cooked using grill and roast. Eating quality samples were collected from the 2018 drop of information nucleus flock lambs at the University of New England as part of larger study. Sixty carcasses were fabricated into consumer sensory samples for the grill and roast cook methods. The outside, topside and knuckle from one leg were portioned into grill samples, while the opposite leg was portioned into a single roast consisting of topside and outside, with the knuckle removed. Leg (left/right) was rotated between carcasses for grill and roast cook methods. Sensory testing was completed using untrained consumer sensory panels who scored lamb for tenderness (T), juiciness (J), flavour (F) and overall liking (OL) (Watson et al. 2008). Ten consumers tested each sample. Data were analysed using a linear model in R (R Core Team 2020), with muscle and cook method as fixed effects, and the interaction between muscle and cook method, to estimate the variance in eating quality score.
      • Probe-based Detection
        At-home oral bacteria collection using a lollipop-based microfluidic device
        Wan-chen Tu - 2023
        Abstract
        Our previous work introduced the CandyCollect,1 a lollipop-inspired saliva collection device for respiratory disease diagnostics. Here, we performed the first human subjects study using this device to capture bacteria in saliva; we demonstrate that the CandyCollect device can be used to collect salivary bacteria from healthy adults using Streptococcus mutans and Staphylococcus aureus as proof-of-concept commensal bacteria. We enrolled 14 healthy adults in a nationwide (USA) remote study in which participants were sent study packages containing CandyCollect devices and traditional commercially available oral swabs and spit tubes. Participants sampled themselves at home, completed usability and user preference surveys, and mailed the samples back to our laboratory for analysis by qPCR. S. mutans and S. aureus are not universally present in all healthy adults.2-5 Our results showed that for participants in which a given bacterium (S. mutans or S. aureus) was detected in one or both of the commercially available methods, CandyCollect devices had a 100% concordance with those results. Furthermore, the CandyCollect device was ranked the highest preference sampling method among the three sampling methods by 26 participants surveyed. We also showed that the CandyCollect device has a shelf life of up to 1 year at room temperature, a storage period that is convenient for clinics or patients to keep the CandyCollect device and use it any time. Taken together, we have demonstrated that the CandyCollect is a user-friendly saliva collection tool that has the potential to be incorporated into diagnostic assays in clinic visits and telemedicine.
        Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce
        Kannan V. Balan - 2023
        Abstract
        Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.
        Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction
        Laiton-Donato Katherine - 2023
        Abstract
        Introduction Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. Objective To develop and test a control RNA for YFV detection through real-time RT-PCR. Methods A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. Results A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). Conclusion The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.
        Increased Acetylcholinesterase expression in Bumble Bees During Neonicotinoid-Coated Corn sowing
        Olivier samson-Robert - 2015
        Abstract
        While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChe) activity in honey bees at sublethal doses. High AChe levels may therefore act as a biomarker of exposure to neonicotinoids. this two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoidtreated seeds), and maintained for the duration of corn sowing. As determined by Real time qpCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication.
        Sperm DNA methylation alterations from cannabis extract exposure are evident in offspring
        Rose Schrott - 2022
        Abstract
        Background Cannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable. Results Whole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers. Conclusions This is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a “washout” period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly. These results support that paternal preconception exposure to cannabis can influence offspring outcomes.
        Validation of Microchip Based RT-PCR ABC Test (InfA/B & COVID-19) in Clinical Samples
        Gabriel Martinez - 2022
        Abstract
        To contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for influenza A, and 140 copies/μL for influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of influenza A/B, and SARS-CoV-2.
        TIRAP/Mal Positively Regulates TLR8‐Mediated Signaling via IRF5 in Human Cells
        Kaja Elisabeth Nilsen - 2022
        Abstract
        Toll‐like receptor 8 (TLR8) recognizes single‐stranded RNA of viral and bacterial origin as well as mediates the secretion of pro‐inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll‐inter‐ leukin 1 receptor domain‐containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte‐derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine‐gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, re‐ spectively, while nuclear translocation of transcription factors was addressed by immunofluores‐ cence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt‐kinase acti‐ vation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5‐dependent cyto‐ kines IFNβ and IL‐12p70 as well as, to a lesser degree, TNF. These findings reveal a new and un‐ conventional role of TIRAP in innate immune defense
        Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens
        Alexander Y. Trick - 2022
        Abstract
        The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.
        A Sensitive, Portable Microfluidic Device for SARS-CoV-2 Detection from Self-Collected Saliva
        Jianing Yang - 2021
        Abstract
        Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in December 2019, the spread of SARS-CoV2 infection has been escalating rapidly around the world. In order to provide more timely access to medical intervention, including diagnostic tests and medical treatment, the FDA authorized multiple test protocols for diagnostic tests from nasopharyngeal swab, saliva, urine, bronchoalveolar lavage and fecal samples. The traditional diagnostic tests for this novel coronavirus 2019 require standard processes of viral RNA isolation, reverse transcription of RNA to cDNA, then real-time quantitative PCR with the RNA templates extracted from the patient samples. Recently, many reports have demonstrated a direct detection of SARS-Co-V2 genomic material from saliva samples without any RNA isolation step. To make the rapid detection of SARS-Co-V2 infection more accessible, a point-of-care type device was developed for SARS-CoV-2 detection. Herein, we report a portable microfluidic-based integrated detectionanalysis system for SARS-CoV-2 nucleic acids detection directly from saliva samples. The saliva cartridge is self-contained and capable of microfluidic evaluation of saliva, from heating, mixing with the primers to multiplex real-time quantitative polymerase chain reaction, detecting SARSCoV- 2 with different primer sets and internal control. The approach has a detection sensitivity of 1000 copies/mL of SARS-CoV-2 RNA or virus, with consistency and automation, from saliva sample-in to result-out.
        RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury
        Antonia Clarissa Wehn - 2021
        Abstract
        Traumatic brain injury (TBI) causes acute and subacute tissue damage, but is also associated with chronic inflammation and progressive loss of brain tissue months and years after the initial event. The trigger and the subsequent molecular mechanisms causing chronic brain injury after TBI are not well understood. The aim of the current study was therefore to investigate the hypothesis that necroptosis, a form a programmed cell death mediated by the interaction of Receptor Interacting Protein Kinases (RIPK) 1 and 3, is involved in this process. Neuron-specific RIPK1- or RIPK3-deficient mice and their wild-type littermates were subjected to experimental TBI by controlled cortical impact. Posttraumatic brain damage and functional outcome were assessed longitudinally by repetitive magnetic resonance imaging (MRI) and behavioral tests (beam walk, Barnes maze, and tail suspension), respectively, for up to three months after injury. Thereafter, brains were investigated by immunohistochemistry for the necroptotic marker phosphorylated mixed lineage kinase like protein(pMLKL) and activation of astrocytes and microglia. WT mice showed progressive chronic brain damage in cortex and hippocampus and increased levels of pMLKL after TBI. Chronic brain damage occurred almost exclusively in areas with iron deposits and was significantly reduced in RIPK1- or RIPK3-deficient mice by up to 80%. Neuroprotection was accompanied by a reduction of astrocyte and microglia activation and improved memory function. The data of the current study suggest that progressive chronic brain damage and cognitive decline after TBI depend on the expression of RIPK1/3 in neurons. Hence, inhibition of necroptosis signaling may represent a novel therapeutic target for the prevention of chronic post-traumatic brain damage.
        Investigation of an outbreak caused by antibiotic‐susceptible Klebsiella oxytoca in a neonatal intensive care unit in Norway
        Torunn Gresdal Ronning - 2019
        Abstract
        Aim Klebsiella spp. have been stated to be the most frequent cause of neonatal intensive care unit (NICU) outbreaks. We report an outbreak of Klebsiella oxytoca in a NICU at a tertiary care hospital in Norway between April 2016 and April 2017. This study describes the outbreak, infection control measures undertaken and the molecular methods developed. Methods The outbreak prompted detailed epidemiological and microbial investigations, where whole‐genome sequencing (WGS) was particularly useful for both genotyping and development of two new K. oxytoca‐specific real‐time PCR assays. Routine screening of patients, as well as sampling from numerous environmental sites, was performed during the outbreak. A bundle of infection control measures was instigated to control the outbreak, among them strict cohort isolation. Results Five neonates had symptomatic infection, and 17 were found to be asymptomatically colonised. Infections varied in severity from conjunctivitis to a fatal case of pneumonia. A source of the outbreak could not be determined. Conclusion This report describes K. oxytoca as a significant pathogen in a NICU outbreak setting and highlights the importance of developing appropriate microbiological screening methods and implementing strict infection control measures to control the outbreak in a setting where the source could not be identified.
      • RNA
        Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction
        Laiton-Donato Katherine - 2023
        Abstract
        Introduction Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. Objective To develop and test a control RNA for YFV detection through real-time RT-PCR. Methods A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. Results A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). Conclusion The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.
        Increased Acetylcholinesterase expression in Bumble Bees During Neonicotinoid-Coated Corn sowing
        Olivier samson-Robert - 2015
        Abstract
        While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChe) activity in honey bees at sublethal doses. High AChe levels may therefore act as a biomarker of exposure to neonicotinoids. this two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoidtreated seeds), and maintained for the duration of corn sowing. As determined by Real time qpCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication.
        Sperm DNA methylation alterations from cannabis extract exposure are evident in offspring
        Rose Schrott - 2022
        Abstract
        Background Cannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable. Results Whole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers. Conclusions This is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a “washout” period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly. These results support that paternal preconception exposure to cannabis can influence offspring outcomes.
        Validation of Microchip Based RT-PCR ABC Test (InfA/B & COVID-19) in Clinical Samples
        Gabriel Martinez - 2022
        Abstract
        To contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for influenza A, and 140 copies/μL for influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of influenza A/B, and SARS-CoV-2.
        Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens
        Alexander Y. Trick - 2022
        Abstract
        The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.
        A Sensitive, Portable Microfluidic Device for SARS-CoV-2 Detection from Self-Collected Saliva
        Jianing Yang - 2021
        Abstract
        Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in December 2019, the spread of SARS-CoV2 infection has been escalating rapidly around the world. In order to provide more timely access to medical intervention, including diagnostic tests and medical treatment, the FDA authorized multiple test protocols for diagnostic tests from nasopharyngeal swab, saliva, urine, bronchoalveolar lavage and fecal samples. The traditional diagnostic tests for this novel coronavirus 2019 require standard processes of viral RNA isolation, reverse transcription of RNA to cDNA, then real-time quantitative PCR with the RNA templates extracted from the patient samples. Recently, many reports have demonstrated a direct detection of SARS-Co-V2 genomic material from saliva samples without any RNA isolation step. To make the rapid detection of SARS-Co-V2 infection more accessible, a point-of-care type device was developed for SARS-CoV-2 detection. Herein, we report a portable microfluidic-based integrated detectionanalysis system for SARS-CoV-2 nucleic acids detection directly from saliva samples. The saliva cartridge is self-contained and capable of microfluidic evaluation of saliva, from heating, mixing with the primers to multiplex real-time quantitative polymerase chain reaction, detecting SARSCoV- 2 with different primer sets and internal control. The approach has a detection sensitivity of 1000 copies/mL of SARS-CoV-2 RNA or virus, with consistency and automation, from saliva sample-in to result-out.
        RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury
        Antonia Clarissa Wehn - 2021
        Abstract
        Traumatic brain injury (TBI) causes acute and subacute tissue damage, but is also associated with chronic inflammation and progressive loss of brain tissue months and years after the initial event. The trigger and the subsequent molecular mechanisms causing chronic brain injury after TBI are not well understood. The aim of the current study was therefore to investigate the hypothesis that necroptosis, a form a programmed cell death mediated by the interaction of Receptor Interacting Protein Kinases (RIPK) 1 and 3, is involved in this process. Neuron-specific RIPK1- or RIPK3-deficient mice and their wild-type littermates were subjected to experimental TBI by controlled cortical impact. Posttraumatic brain damage and functional outcome were assessed longitudinally by repetitive magnetic resonance imaging (MRI) and behavioral tests (beam walk, Barnes maze, and tail suspension), respectively, for up to three months after injury. Thereafter, brains were investigated by immunohistochemistry for the necroptotic marker phosphorylated mixed lineage kinase like protein(pMLKL) and activation of astrocytes and microglia. WT mice showed progressive chronic brain damage in cortex and hippocampus and increased levels of pMLKL after TBI. Chronic brain damage occurred almost exclusively in areas with iron deposits and was significantly reduced in RIPK1- or RIPK3-deficient mice by up to 80%. Neuroprotection was accompanied by a reduction of astrocyte and microglia activation and improved memory function. The data of the current study suggest that progressive chronic brain damage and cognitive decline after TBI depend on the expression of RIPK1/3 in neurons. Hence, inhibition of necroptosis signaling may represent a novel therapeutic target for the prevention of chronic post-traumatic brain damage.
    • Conventional PCR
      • DNA
        FXN gene methylation determines carrier status in Friedreich ataxia
        Christina Lam - 2023
        Abstract
        Background Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat (GAA-TRE) in intron 1 of the FXN gene. Some patients are compound heterozygous for the GAA-TRE and another FXN pathogenic variant. Detection of the GAA-TRE in the heterozygous state, occasionally technically challenging, is essential for diagnosing compound heterozygotes and asymptomatic carriers. Objective We explored if the FRDA differentially methylated region (FRDA-DMR) in intron 1, which is hypermethylated in cis with the GAA-TRE, effectively detects heterozygous GAA-TRE. Methods FXN DNA methylation was assayed by targeted bisulfite deep sequencing using the Illumina platform. Results FRDA-DMR methylation effectively identified a cohort of known heterozygous carriers of the GAA-TRE. In an individual with clinical features of FRDA, commercial testing showed a paternally inherited pathogenic FXN initiation codon variant but no GAA-TRE. Methylation in the FRDA-DMR effectively identified the proband, his mother and various maternal relatives as heterozygous carriers of the GAA-TRE, thus confirming the diagnosis of FRDA. Conclusion FXN DNA methylation reliably detects the GAA-TRE in the heterozygous state and offers a robust alternative strategy to diagnose FRDA due to compound heterozygosity and to identify asymptomatic heterozygous carriers of the GAA-TRE.
        Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals
        Arianna I. Celis - 2022
        Abstract
        While microbial communities inhabit a wide variety of complex natural environments, in vitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. In vitro assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of in vitro samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates.
        Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest Storage
        Giada Schiavon - 2022
        Abstract
        Botrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils (EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed 100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments on apples of cultivar ‘Opal’ with 1% EOs showed that, after 60 d storage, thyme and savoury EOs significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere inside the cabinets was performed to characterize and quantify the volatile components of EOs released through biofumigation. Though thymol and p-cymene were the main components of thyme EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic microbiome had higher richness and evenness compared to their endophytic counterpart. By the end of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes (from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective in hampering grey mould rot development in vivo.
        NUDT7 regulates total hepatic CoA levels and the composition of the intestinal bile acid pool in male mice fed a Western diet
        Schuyler D. Vickers - 2022
        Abstract
        Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism, but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the β-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.
        Ecological and evolutionary consequences of microbial community responses to environmental change
        Sarai S. Finks - 2022
        Abstract
        Global changes such as increased frequency of fire, drought, and nitrogen deposition, perturb microorganisms and the higher trophic life forms they support. Microorganisms play key roles in carbon and nutrient cycling, which are important to agriculture and ecosystem health. Although microorganisms are pivotal in an ecosystem's response to environmental changes, little is known about how abundant and diverse microbial communities adapt to such changes. The overarching aim of my thesis is to investigate how bacterial communities respond to global change and in particular, their ability to quickly adapt to environmental perturbations. I first investigated how microbial responses to global changes are influenced by interactions with plant communities using the Loma Ridge Global Change Experiment, a decadelong experiment that manipulates rainfall and nitrogen levels across two adjacent ecosystems (Chapter 1). My findings underscore the importance of plant–microbe interactions when considering the transferability of the results of global change experiments across ecosystems. Next, I investigated traits found on plasmids, a type of mobile genetic element (MGE) that can facilitate rapid evolution in bacteria. I asked what are the ecologically-relevant plasmid genes that may serve as reservoirs of environmental-adaptive traits in bacteria (Chapter 2). The findings of this chapter suggest that plasmid traits may contribute to host adaptation in environmental microbiomes. Lastly, I extended this work to a cosmopolitan soil taxon, Curtobacterium, an abundant genus of bacteria in southern California ecosystems. This taxon shows marked shifts in relative abundance in response to simulated drought and is amenable to culturing, providing a tractable system for investigating both genotypic and phenotypic characteristics of this organism. Previous experiments have shown Curtobacterium rapidly evolve via de novo mutations in response to environmental changes. I asked what MGE and associated traits are found in Curtobacterium, and determined whether MGE and traits showed any environment- versus clade- specific genomic signatures (Chapter 3). The findings of this chapter highlight the potential of traits found on plasmids to be mobilized within the bacterial communities where these Curtobacterium were isolated. Overall, my thesis work highlights the importance for considering the intersection of evolution and ecology in understanding how microbial communities adapt to environmental changes.
        Association between gut microbiota and prediabetes in people living with HIV
        Kulapong Jayanama - 2022
        Abstract
        The prevalence of prediabetes is rapidly increasing in general population and in people living with HIV (PLWH). Gut microbiota play an important role in human health, and dysbiosis is associated with metabolic disorders and HIV infection. Here, we aimed to evaluate the association between gut microbiota and prediabetes in PLWH. A cross-sectional study enrolled 40 PLWH who were receiving antiretroviral therapy and had an undetectable plasma viral load. Twenty participants had prediabetes, and 20 were normoglycemic. Fecal samples were collected from all participants. The gut microbiome profiles were analyzed using 16S rRNA sequencing. Alpha-diversity was significantly lower in PLWH with prediabetes than in those with normoglycemia (p<0.05). A significant difference in beta-diversity was observed between PLWH with prediabetes and PLWH with normoglycemia (p<0.05). Relative abundances of two genera in Firmicutes (Streptococcus and Anaerostignum) were significantly higher in the prediabetes group. In contrast, relative abundances of 13 genera (e.g., Akkermansia spp., Christensenellaceae R7 group) were significantly higher in the normoglycemic group. In conclusion, the diversity of gut microbiota composition decreased in PLWH with prediabetes. The abundances of 15 bacterial taxa in the genus level differed between PLWH with prediabetes and those with normoglycemia. Further studies on the effect of these taxa on glucose metabolism are warranted.
        The effect of a mass distribution of insecticide-treated nets on insecticide resistance and entomological inoculation rates of Anopheles gambiae s.l. in Bandundu City, Democratic Repub`lic of Congo
        Emery Metelo-Matubi - 2021
        Abstract
        Introduction insecticide-treated nets (ITNs) remain the mainstay of malaria vector control in the Democratic Republic of Congo. However, insecticide resistance of malaria vectors threatens their effectiveness. Entomological inoculation rates and insecticide susceptibility in Anopheles gambiae s.l. were evaluated before and after mass distribution of ITNs in Bandundu City for possible occurrence of resistance. Methods a cross-sectional study was conducted from 15th July 2015 to 15th June 2016. Adult mosquitoes were collected using pyrethrum spray catches and human landing catches and identified to species level and tested for the presence of sporozoites. Bioassays were carried out before and after distribution of ITNs to assess the susceptibility of adult mosquitoes to insecticides. Synergist bioassays were also conducted and target site mutations assessed using Polymerase chain reaction (PCR). Results a total of 1754 female An. gambiae s.l. were collected before and after deployment of ITNs. Fewer mosquitoes were collected after the distribution of ITNs. However, there was no significant difference in sporozoite rates or the overall entomological inoculation rate before and after the distribution of ITNs. Test-mosquitoes were resistant to deltamethrin, permethrin, and Dichlorodiphenyltrichloroethane but susceptible to bendiocarb. Pre-exposure of mosquitoes to Piperonyl butoxide increased their mortality after exposure to permethrin and deltamethrin. The frequency of the Kinase insert domain receptor (kdr)-West gene increased from 92 to 99% before and after the distribution of nets, respectively. Conclusion seasonal impacts could be a limiting factor in the analysis of these data; however, the lack of decrease in transmission after the distribution of new nets could be explained by the high-level of resistance to pyrethroid.
  • Next Generation Sequencing (NGS)
    • DNA
      Leptospira enrichment culture followed by ONT metagenomic sequencing allows better detection of Leptospira presence and diversity in water and soil samples
      Myranda Gorman - 2022
      Abstract
      Background Leptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. Methodology/Principal findings In this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by PCR and metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Additionally, metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp. from environmental samples. Conclusions/Significance The selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance.
      Genetic structure and historic demography of endangered unarmoured threespine stickleback at southern latitudes signals a potential new management approach
      Rachel Turba - 2022
      Abstract
      Habitat loss, flood control infrastructure, and drought have left most of southern California and northern Baja California's native freshwater fish near extinction, including the endangered unarmoured threespine stickleback (Gasterosteus aculeatus williamsoni). This subspecies, an unusual morph lacking the typical lateral bony plates of the G. aculeatus complex, occurs at arid southern latitudes in the eastern Pacific Ocean and survives in only three inland locations. Managers have lacked molecular data to answer basic questions about the ancestry and genetic distinctiveness of unarmoured populations. These data could be used to prioritize conservation efforts. We sampled G. aculeatus from 36 localities and used microsatellites and whole genome data to place unarmoured populations within the broader evolutionary context of G. aculeatus across southern California/northern Baja California. We identified three genetic groups with none consisting solely of unarmoured populations. Unlike G. aculeatus at northern latitudes, where Pleistocene glaciation has produced similar historical demographic profiles across populations, we found markedly different demographics depending on sampling location, with inland unarmoured populations showing steeper population declines and lower heterozygosity compared to low armoured populations in coastal lagoons. One exception involved the only high elevation population in the region, where the demography and alleles of unarmoured fish were similar to low armoured populations near the coast, exposing one of several cases of artificial translocation. Our results suggest that the current “management-by-phenotype” approach, based on lateral plates, is incidentally protecting the most imperilled populations; however, redirecting efforts toward evolutionary units, regardless of phenotype, may more effectively preserve adaptive potential.
      A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system
      Oleksandr Kondratov - 2021
      Abstract
      Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, deliv- ery routes, and disease indications in a well-powered, compre- hensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild- type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques’ CNS after intrapar- enchymal (putamen) injection, or cerebrospinal fluid (CSF)- mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital- droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of ratio- nally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been devel- oped and made available in the public domain.
      Association between gut microbiota and prediabetes in people living with HIV
      Kulapong Jayanama - 2022
      Abstract
      The prevalence of prediabetes is rapidly increasing in general population and in people living with HIV (PLWH). Gut microbiota play an important role in human health, and dysbiosis is associated with metabolic disorders and HIV infection. Here, we aimed to evaluate the association between gut microbiota and prediabetes in PLWH. A cross-sectional study enrolled 40 PLWH who were receiving antiretroviral therapy and had an undetectable plasma viral load. Twenty participants had prediabetes, and 20 were normoglycemic. Fecal samples were collected from all participants. The gut microbiome profiles were analyzed using 16S rRNA sequencing. Alpha-diversity was significantly lower in PLWH with prediabetes than in those with normoglycemia (p<0.05). A significant difference in beta-diversity was observed between PLWH with prediabetes and PLWH with normoglycemia (p<0.05). Relative abundances of two genera in Firmicutes (Streptococcus and Anaerostignum) were significantly higher in the prediabetes group. In contrast, relative abundances of 13 genera (e.g., Akkermansia spp., Christensenellaceae R7 group) were significantly higher in the normoglycemic group. In conclusion, the diversity of gut microbiota composition decreased in PLWH with prediabetes. The abundances of 15 bacterial taxa in the genus level differed between PLWH with prediabetes and those with normoglycemia. Further studies on the effect of these taxa on glucose metabolism are warranted.
  • Reverse Transcription
    • First-Strand cDNA Synthesis
      Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA Isoforms
      Gjendine Voss - 2023
      Abstract
      icroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-toI editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting cDNA in the following qPCR step. We also give guidelines for primer design and for the evaluation of assays using synthetic oligonucleotides. These tools are expected to be transferable to any A-to-I-edited microRNA and its isoforms
      All together now: Geographically coordinated miticide treatment benefits honey bee health
      Luke Woodford - 2023
      Abstract
      Deformed wing virus (DWV) is a pathogenic virus of honey bees transmitted by the ectoparasitic mite Varroa destructor. Annual overwintering colony losses, accounting for ~25% of all colonies, are associated with high levels of Varroa-DWV infestation. Effective miticide treatments are available to control Varroa. However, the absence of coordinated treatment means environmental transmission of mites continues unchecked. We aimed to determine whether rational, coordinated treatment is beneficial, and characterized the DWV population as an indicator of colony health. This study uses coordinated treatment of Varroa in a geographically isolated environment (Isle of Arran, Scotland) over 3 years. The study area contained 50–84 colonies managed by ~20 amateur beekeepers. Sampling and virus analysis to assess strain diversity and viral loads were conducted before and after treatments, and changes in population diversity were quantified by sequence analysis. Over the 3 years analysis of the virus population revealed that the dominant DWV variant shifted from Type A to Type B in all apiaries, regardless of mite levels or proximity to other colonies. During this period the number of managed colonies increased by 47% (57–84 colonies), but despite this, we estimate total mite numbers decreased by 58%. Synthesis and applications. In this study, the beekeepers in Arran significantly improved the number of colonies they managed, without importing any bees onto the island, indicating that an improved focus on management techniques, through the combination of a coordinated miticide programme and an improved understanding of bee diseases, could yield positive results for bee health and sustainability.
      Gene silencing for invasive paper wasp management: Synthesized dsRNA can modify gene expression but did not affect mortality
      Mariana Bulgarella - 2023
      Abstract
      Invasive paper wasps such as Polistes dominula are a major pest and problem for biodiversity around the globe. Safe and highly targeted methods for the control of these and other social wasp populations are needed. We attempted to identify potentially-lethal gene targets that could be used on adult paper wasps in a gene silencing or RNA interference (RNAi) approach. Double-stranded RNA (dsRNA) was designed to target genes for which silencing has proven lethal in other insects. dsRNA was provided either orally to foragers or directly injected into the wasps. We also provided the dsRNA unprotected or protected from degradation by gut nucleases in two different forms (lipofectamine and carbon quantum dots). The effects of oral delivery of 22 different gene targets to forager wasps was evaluated. The expression of five different genes was successfully reduced following dsRNA ingestion or injection. These gene targets included the FACT complex subunit spt16 (DRE4) and RNA-binding protein fusilli (FUSILLI), both of which have been previously shown to have potential as lethal targets for pest control in other insects. However, we found no evidence of significant increases in adult wasp mortality following ingestion or injection of dsRNA for these genes when compared with control treatments in our experiments. The methods we used to protect the dsRNA from digestive degradation altered gene expression but similarly did not influence wasp mortality. Our results indicate that while many of the same gene targets can be silenced and induce mortality in other insects, dsRNA and RNAi approaches may not be useful for paper wasp control.
      The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in Hydra
      Matthias Christian Vogg - 2022
      Abstract
      he molecular mechanisms that maintain cellular identities and prevent dedifferentiation or transdifferentiation remain mysterious. However, both processes are transiently used during animal regeneration. Therefore, organisms that regenerate their organs, appendages, or even their whole body offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4, whose expression is controlled by Wnt3/β-catenin signaling and the Sp5 transcription factor, plays a key role in tentacle formation and tentacle maintenance. Reducing Zic4 expression suffices to induce transdifferentiation of tentacle epithelial cells into foot epithelial cells. This switch requires the reentry of tentacle battery cells into the cell cycle without cell division and is accompanied by degeneration of nematocytes embedded in these cells. These results indicate that maintenance of cell fate by a Wnt-controlled mechanism is a key process both during homeostasis and during regeneration.
  • Sample Preparation
    • DNA
      Sensitivity of Dried Blood Spot Testing for Detection of Congenital Cytomegalovirus Infection
      Sheila C. Dollard - 2021
      Abstract
      Importance The sensitivity of dried blood spots (DBS) to identify newborns with congenital cytomegalovirus (cCMV) infection has not been evaluated in screening studies using the current, higher-sensitivity methods for DBS processing. Objective To assess the sensitivity of DBS polymerase chain reaction (PCR) for newborn screening for cCMV infection using saliva as the reference standard for screening, followed by collection of a urine sample for confirmation of congenital infection. Design, Setting, and Participants This population-based cohort study took place at 5 newborn nurseries and 3 neonatal intensive care units in the Minneapolis/Saint Paul area in Minnesota from April 2016 to June 2019. Newborns enrolled with parental consent were screened for cCMV using DBS obtained for routine newborn screening and saliva collected 1 to 2 days after birth. Dried blood spots were tested for CMV DNA by PCR at both the University of Minnesota (UMN) and the US Centers for Disease Control and Prevention (CDC). Saliva swabs were tested by CMV DNA PCR at the UMN laboratory only. Newborns who screened positive by saliva or DBS had a diagnostic urine sample obtained by primary care professionals, tested by PCR within 3 weeks of birth. Analysis began July 2019. Exposures Detection of CMV from a saliva swab using a PCR assay. Main Outcomes and Measures Number of children with urine-confirmed cCMV and the proportion of them who were CMV positive through DBS screening. Results Of 12 554 individuals enrolled through June 2019 (of 25 000 projected enrollment), 56 newborns were confirmed to have cCMV (4.5 per 1000 [95% CI, 3.3-5.7]). Combined DBS results from either UMN or CDC had a sensitivity of 85.7% (48 of 56; 95% CI, 74.3%-92.6%), specificity of 100.0% (95% CI, 100.0%-100.0%), positive predictive value (PPV) of 98.0% (95% CI, 89.3%-99.6%), and negative predictive value (NPV) of 99.9% (95% CI, 99.9%-100.0%). Dried blood spot results from UMN had a sensitivity of 73.2% (95% CI, 60.4%-83.0%), specificity of 100.0% (100.0%-100.0%), PPV of 100.0% (95% CI, 91.4%-100.0%), and NPV of 99.9% (95% CI, 99.8%-99.9%). Dried blood spot results from CDC had a sensitivity of 76.8% (95% CI, 64.2%-85.9%), specificity of 100.0% (95% CI, 100.0%-100.0%), PPV of 97.7% (95% CI, 88.2%-99.6%), and NPV of 99.9% (95% CI, 99.8%-99.9%). Saliva swab results had a sensitivity of 92.9% (52 of 56; 95% CI, 83.0%-97.2%), specificity of 99.9% (95% CI, 99.9%-100.0%), PPV of 86.7% (95% CI, 75.8%-93.1%), and NPV of 100.0% (95% CI, 99.9%-100.0%). Conclusions and Relevance This study demonstrates relatively high analytical sensitivity for DBS compared with previous studies that performed population-based screening. As more sensitive DNA extraction and PCR methods continue to emerge, DBS-based testing should remain under investigation as a potential low-cost, high-throughput option for cCMV screening.
      Plaque-associated human microglia accumulate lipid droplets in a chimeric model of Alzheimer’s disease
      Christel Claes - 2021
      Abstract
      Background Disease-associated microglia (DAMs), that surround beta-amyloid plaques, represent a transcriptionally-distinct microglial profile in Alzheimer’s disease (AD). Activation of DAMs is dependent on triggering receptor expressed on myeloid cells 2 (TREM2) in mouse models and the AD TREM2-R47H risk variant reduces microglial activation and plaque association in human carriers. Interestingly, TREM2 has also been identified as a microglial lipid-sensor, and recent data indicates lipid droplet accumulation in aged microglia, that is in turn associated with a dysfunctional proinflammatory phenotype. However, whether lipid droplets (LDs) are present in human microglia in AD and how the R47H mutation affects this remains unknown. Methods To determine the impact of the TREM2 R47H mutation on human microglial function in vivo, we transplanted wild-type and isogenic TREM2-R47H iPSC-derived microglial progenitors into our recently developed chimeric Alzheimer mouse model. At 7 months of age scRNA-seq and histological analyses were performed. Results Here we report that the transcriptome of human wild-type TREM2 and isogenic TREM2-R47H DAM xenografted microglia (xMGs), isolated from chimeric AD mice, closely resembles that of human atherosclerotic foam cells. In addition, much like foam cells, plaque-bound xMGs are highly enriched in lipid droplets. Somewhat surprisingly and in contrast to a recent in vitro study, TREM2-R47H mutant xMGs exhibit an overall reduction in the accumulation of lipid droplets in vivo. Notably, TREM2-R47H xMGs also show overall reduced reactivity to plaques, including diminished plaque-proximity, reduced CD9 expression, and lower secretion of plaque-associated APOE. Conclusions Altogether, these results indicate lipid droplet accumulation occurs in human DAM xMGs in AD, but is reduced in TREM2-R47H DAM xMGs, as it occurs secondary to TREM2-mediated changes in plaque proximity and reactivity.
  • Real-Time qPCR
    Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction
    Laiton-Donato Katherine - 2023
    Abstract
    Introduction Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. Objective To develop and test a control RNA for YFV detection through real-time RT-PCR. Methods A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. Results A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). Conclusion The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.
    Increased Acetylcholinesterase expression in Bumble Bees During Neonicotinoid-Coated Corn sowing
    Olivier samson-Robert - 2015
    Abstract
    While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChe) activity in honey bees at sublethal doses. High AChe levels may therefore act as a biomarker of exposure to neonicotinoids. this two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoidtreated seeds), and maintained for the duration of corn sowing. As determined by Real time qpCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication.
    Sperm DNA methylation alterations from cannabis extract exposure are evident in offspring
    Rose Schrott - 2022
    Abstract
    Background Cannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable. Results Whole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers. Conclusions This is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a “washout” period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly. These results support that paternal preconception exposure to cannabis can influence offspring outcomes.
    Validation of Microchip Based RT-PCR ABC Test (InfA/B & COVID-19) in Clinical Samples
    Gabriel Martinez - 2022
    Abstract
    To contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for influenza A, and 140 copies/μL for influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of influenza A/B, and SARS-CoV-2.
    Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens
    Alexander Y. Trick - 2022
    Abstract
    The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.

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