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  • PCR
    • Real-Time Quantitative PCR
      • SYBR Green Detection
        • DNA
          Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems
          Daniel Rodrigues - 2022
          Abstract
          Background T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. Methods GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. Results Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. Conclusions Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.
          Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit
          Larissa Bessler - 2022
          Abstract
          Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.
      • Probe-based Detection
        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.
        Experimental challenge of flatfishes (Pleuronectidae) with salmonid alphavirus (SAV): Observations on tissue tropism and pathology in common dab Limanda limanda L.
        Linda Andersen - 2022
        Abstract
        Salmonid alphavirus (SAV) is the aetiological agent of pancreas disease (PD), a serious viral disease in salmonids. For several decades, SAV was known to infect salmonid species only, until SAV was detected using real-time PCR in several species of wild-caught flatfishes in Scotland in 2010. The presence of SAV in wild flatfishes has been confirmed by further surveys from Ireland and Scotland. The role of flatfishes in SAV-spread and epizootiology has not been elucidated, and no experimental challenges have been conducted to examine virus tissue tropism, virulence and pathology in flatfishes. Wild-caught flatfishes (common dab; Limanda limanda, European plaice; Pleuronectes platessa, European flounder; Platichthys flesus and lemon sole; Microstomus kitt) were either intramuscularly (i.m.) or intraperitoneally (i.p.) challenged with SAV3 or exposed to SAV3 through cohabitation with i.p. injected salmon. SAV-infections were seen in i.m. and i.p. injected dab and i.p. injected salmon but did not result in a transmissible infection in dab although several routes of entry were assessed (oral route not tested). SAV was detected in several tissues of eight common dab (not from cohabitants), with high SAV-levels in pancreas. No viraemia was detected in the SAV-positive common dab and no virus shedding were detected in the tanks. However, pathology in exocrine pancreas and hearts consistent with SAV-replication were seen. This is the first study reporting SAV-induced pathology in a non-salmonid species. The results from the present challenge study supports evidence for common dab being susceptible hosts for SAV. The study also demonstrates that flatfishes are less susceptible to SAV3-infection than salmon.
        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.
        Efficiency-corrected PCR quantification for identification of prevalence and load of respiratory disease-causing agents in feedlot cattle
        RJ Barnewall - 2022
        Abstract
        Bovine respiratory disease (BRD) is the most prevalent disease in feedlot cattle worldwide with Bovine alphaherpesvirus 1 (BoAHV1), Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida and Trueperella pyogenes accepted to be common etiological agents associated with BRD. Although these agents are common in the upper and lower airways in clinical BRD cases, some also exist as normal flora suggesting their presence in the upper airways alone is not necessarily informative with respect to disease status or risk. To determine the relationship between presence, load and disease status, we investigated the relationship between load in the upper airways at induction and active BRD cases in feedlot cattle using efficiency-corrected PCR quantification. By this approach, we were able to accurately determine the prevalence and load of the key BRD agents in the upper respiratory tract showing that cattle in the hospital pen had a higher prevalence, and load, of these agents both singly and in combination compared to cattle sampled at feedlot induction. A combination of agents was the most accurate indicator of BRD risk with cattle with four or more agents detected in the upper airway more likely to be undergoing treatment for BRD than non-BRD ailments. In addition, M. bovis was rarely detected at feedlot induction but was identified at high prevalence in cattle in the hospital pen. These findings present a potential new technological approach for the investigation, analysis and identification of BRD-associated viral and bacterial agents for Australian feedlot systems as well as for BRD disease management and treatment
        Systematic stepwise screening of new microbial antagonists for biological control of European canker
        G. Elena - 2022
        Abstract
        Neonectria ditissima is the causal agent of European canker. This pathogen causes cankers on apple branches and the main stem, which may lead to the loss of the whole tree. Chemical control is the essential component in disease management and no suitable biocontrol products are commercially available. This study aimed at selecting potential microbial antagonists against N. ditissima through a systematic stepwise screening program for the development of a new biocontrol product. A total of 520 potential candidates were isolated from apple branches showing canker symptoms. Important characteristics for application of Microbial Biological Control Agents were tested per each candidate: spore production, spore survival during storage, cold tolerance, drought tolerance and UV-B resistance. Isolates able to germinate and grow at human body temperature were excluded. A total of 252 candidates fulfilled the stablished criteria. All 520 candidates belonged to 44 different taxonomic groups, being the most abundant Alternaria spp. (22.2%), Aureobasidium pullulans (16.1%), Cladosporium spp. (9.5%) and Fusarium spp. (9.0%). Information on possible pathogenicity and toxicity for humans, animals, plants and the environment and on patents in biocontrol use was collected per each identified species. A total of 158 candidates belonging to species that did not show potential risks or patent conflicts were assessed by their antagonistic behaviour against N. ditissima in bioassays in planta. Each candidate was inoculated in ‘Elstar’ apple branches inoculated with N. ditissima 24 h before. Inoculated branches were incubated at 17 °C, 16 h light per day and RH>90%. After four weeks, canker symptom expression was visually assessed. The capacity of the candidates to reduce colonisation of N. ditissima in the branches was evaluated by quantifying N. ditissima DNA concentration using qPCR. Four candidates of Clonostachys rosea showed antagonistic properties; after four weeks of treatment, no canker symptoms or small bark cracks were observed in the inoculated branches and N. ditissima DNA was reduced by 90-99%. Following them, the branches inoculated with one candidate of Akanthomyces muscarius, A. pullulans and Cladosporium europaeum showed small bark cracks and small swollen bark and N. ditissima DNA was reduced by more than 90%. The systematic stepwise screening approach was a powerful strategy to find new MBCAs against N. ditissima with antagonistic properties that also fulfilled major criteria with regards to commercial production and safety, as well as ecological needs.
        Engineering heterologous enzyme secretion in Yarrowia lipolytica
        Weigao Wang - 2022
        Abstract
        Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and α-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.
        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
        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.
        Experimental challenge of flatfishes (Pleuronectidae) with salmonid alphavirus (SAV): Observations on tissue tropism and pathology in common dab Limanda limanda L.
        Linda Andersen - 2022
        Abstract
        Salmonid alphavirus (SAV) is the aetiological agent of pancreas disease (PD), a serious viral disease in salmonids. For several decades, SAV was known to infect salmonid species only, until SAV was detected using real-time PCR in several species of wild-caught flatfishes in Scotland in 2010. The presence of SAV in wild flatfishes has been confirmed by further surveys from Ireland and Scotland. The role of flatfishes in SAV-spread and epizootiology has not been elucidated, and no experimental challenges have been conducted to examine virus tissue tropism, virulence and pathology in flatfishes. Wild-caught flatfishes (common dab; Limanda limanda, European plaice; Pleuronectes platessa, European flounder; Platichthys flesus and lemon sole; Microstomus kitt) were either intramuscularly (i.m.) or intraperitoneally (i.p.) challenged with SAV3 or exposed to SAV3 through cohabitation with i.p. injected salmon. SAV-infections were seen in i.m. and i.p. injected dab and i.p. injected salmon but did not result in a transmissible infection in dab although several routes of entry were assessed (oral route not tested). SAV was detected in several tissues of eight common dab (not from cohabitants), with high SAV-levels in pancreas. No viraemia was detected in the SAV-positive common dab and no virus shedding were detected in the tanks. However, pathology in exocrine pancreas and hearts consistent with SAV-replication were seen. This is the first study reporting SAV-induced pathology in a non-salmonid species. The results from the present challenge study supports evidence for common dab being susceptible hosts for SAV. The study also demonstrates that flatfishes are less susceptible to SAV3-infection than salmon.
        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.
        Engineering heterologous enzyme secretion in Yarrowia lipolytica
        Weigao Wang - 2022
        Abstract
        Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and α-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.
        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
        Fine-scale spatial variation shape fecal microbiome diversity and composition in black- tailed prairie dogs (Cynomys ludovicianus)
        Sufia A. Neha - 2022
        Abstract
        Background Host associated gut microbiota are important in understanding the coevolution of host-microbe, it’s causes and consequences that may help wildlife population to adapt to its rapid climatic changes. Mammalian gut microbiota composition and diversity may be affected by a variety of factors including geographic variation, seasonal variation in diet, habitat disturbance, environmental conditions, age, and sex. However, there have been few studies that have examined how ecological and environmental factors influence gut microbiota composition in animals' natural environments. In this study, we explore how host habitat, geographical location and environmental factors affect the fecal microbiota of Cynomys ludovicianus at a small spatial scale. We collected fecal samples from five geographically distinct locations in Texas Panhandle occupying habitat classified as urban and rural areas using high throughput 16S rRNA gene amplicon sequencing. Results The results showed that microbiota of fecal samples was largely dominated by phylum Bacteroidetes. Fecal microbiome diversity and composition differed significantly across sampling sites and habitats. Prairie dogs inhabiting urban areas showed reduced fecal diversity due to more homogenous environment and anthropogenic disturbance. Urban prairie dog colonies displayed greater phylogenetic variation than those in rural habitats. Differentially abundant analysis revealed that bacterial species pathogenic to humans and animals were highly abundant in urban areas which indicates that host health and fitness might be negatively affected. Random forest model identified Alistipes shahii as the important species driving the changes in fecal microbiome composition. Despite the effects of habitat and geographic location of host, we found a strong correlation with environmental factors- average maximum temperature was the best predictor of prairie dog fecal microbial diversity. Conclusions Our findings suggest that reduction in alpha diversity in conjunction with greater dispersion in beta diversity could be indicative of declining host health in urban areas which could help determine in future conservation efforts. Moreover, several bacterial species pathogenic to humans and other animals were highly abundant in prairie dog colonies near urban areas, which may in turn adversely affect host phenotype and fitness.
        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.
        DNA methylation in Friedreich ataxia silences expression of frataxin isoform E
        Layne N. Rodden - 2022
        Abstract
        Epigenetic silencing in Friedreich ataxia (FRDA), induced by an expanded GAA triplet-repeat in intron 1 of the FXN gene, results in deficiency of the mitochondrial protein, frataxin. A lesser known extramitochondrial isoform of frataxin detected in erythrocytes, frataxin-E, is encoded via an alternate transcript (FXN-E) originating in intron 1 that lacks a mitochondrial targeting sequence. We show that FXN-E is deficient in FRDA, including in patient-derived cell lines, iPS-derived proprioceptive neurons, and tissues from a humanized mouse model. In a series of FRDA patients, deficiency of frataxin-E protein correlated with the length of the expanded GAA triplet-repeat, and with repeat-induced DNA hypermethylation that occurs in close proximity to the intronic origin of FXN-E. CRISPR-induced epimodification to mimic DNA hypermethylation seen in FRDA reproduced FXN-E transcriptional deficiency. Deficiency of frataxin E is a consequence of FRDA-specific epigenetic silencing, and therapeutic strategies may need to address this deficiency.
        Performance of Conventional Urine Culture Compared to 16S rRNA Gene Amplicon Sequencing in Children with Suspected Urinary Tract Infection
        Christopher W. Marshall - 2021
        Abstract
        Because some organisms causing urinary tract infection (UTI) may be difficult to culture, examination of bacterial gene sequences in the urine may provide a more accurate view of bacteria present during a UTI. Our objective was to estimate how often access to 16S rRNA gene amplicon sequencing alters diagnosis and/or clinical management. The study was designed as a cross-sectional study of a convenience sample of children with suspected UTI. The setting was the emergency department or outpatient clinic at six pediatric centers. Participants included children 2 months to 10 years of age suspected of UTI. We categorized the results of urine culture as follows: “likely UTI” ($100,000 CFU/ml of a single uropathogen), “possible UTI” (10,000 to 99,000 CFU/ml of a uropathogen or $100,000 CFU/ ml of a single uropathogen plus other growth), and “unlikely UTI” (no growth or growth of nonuropathogens). Similarly, we categorized the results of 16S rRNA gene sequencing into the same three categories using the following criteria: likely UTI ($90% relative abundance of a uropathogen), possible UTI (50 to 89% relative abundance of a uropathogen), and unlikely UTI (remainder of samples). The main study outcome was concordance between conventional culture results and 16S rRNA gene sequencing. Concordance between the two methods was high in children with likely and unlikely UTI by conventional culture (95% and 87%, respectively). In children with possible UTI according to conventional culture, 71% had a single uropathogen at a relative abundance of $90% according to 16S rRNA gene sequencing data. Concordance between conventional culture and 16S rRNA gene amplicon sequencing appears to be high. In children with equivocal culture results, 16S rRNA gene results may provide information that may help clarify the diagnosis. IMPORTANCE Concordance between conventional culture and 16S rRNA gene amplicon sequencing appears to be high. In children with equivocal culture results, 16S rRNA gene results may provide information that may help clarify the diagnosis.
        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.
        Exploring the diversity of the deep sea—four new species of the amphipod genus Oedicerina described using morphological and molecular methods
        Anna M. Jazszewska - 2021
        Abstract
        Collections of the amphipod genus Oedicerina were obtained during six expeditions devoted to the study of deep-sea environments of the Pacific Ocean. The material revealed four species new to science. Two species (Oedicerina henrici sp. nov. and sp. nov.) were found at abyssal depths of the central eastern Pacific in the Clarion-Clipperton Zone; one species (sp. nov.) (Oedicerina claudei sp. nov.) was recovered in the Sea of Okhotsk (north-west Pacific), and one (Oedicerina lesci sp. nov.) in the abyss adjacent to the Kuril-Kamchatka Trench (KKT). The four new species differ from each other and known species by the shapes of the rostrum, coxae 1 and 4, basis of pereopod 7, armatures of pereonite 7, pleonites and urosomites. An identification key for all known species is provided. The study of the cytochrome c oxidase subunit I gene of the four new species and Oedicerina ingolfi collected in the North Atlantic confirmed their genetic distinction. However, small intraspecific variation within each of the studied species was observed. In the case of the new species occurring across the KKT, the same haplotype was found on both sides of the trench, providing evidence that the trench does not constitute an insurmountable barrier for population connectivity. None of the species have so far been found on both sides of the Pacific.
  • Next Generation Sequencing (NGS)
    • DNA
      Biodegradation of microplastic in freshwaters: A long-lasting process affected by the lake microbiome
      Sami J. Taipale - 2022
      Abstract
      Plastics have been produced for over a century, but definitive evidence of complete plastic biodegradation in different habitats, particularly freshwater ecosystems, is still missing. Using 13C-labelled polyethylene microplastics (PE-MP) and stable isotope analysis of produced gas and microbial membrane lipids, we determined the biodegradation rate and fate of carbon in PE-MP in different freshwater types. The biodegradation rate in the humic-lake waters was much higher (0.45% ± 0.21% per year) than in the clear-lake waters (0.07% ± 0.06% per year) or the artificial freshwater medium (0.02% ± 0.02% per year). Complete biodegradation of PE-MP was calculated to last 100–200 years in humic-lake waters, 300–4000 years in clear-lake waters, and 2000–20,000 years in the artificial freshwater medium. The concentration of 18:1ω7, characteristic phospholipid fatty acid in Alpha- and Gammaproteobacteria, was a predictor of faster biodegradation of PE. Uncultured Acetobacteraceae and Comamonadaceae among Alpha- and Gammaproteobacteria, respectively, were major bacteria related to the biodegradation of PE-MP. Overall, it appears that microorganisms in humic lakes with naturally occurring refractory polymers are more adept at decomposing PE than those in other waters.
      Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection
      Merrit Romeike - 2022
      Abstract
      Stem cells intrinsically express a subset of genes which are normally associated with interferon stimulation and the innate immune response. However, the expression of these interferon-stimulated genes (ISG) in stem cells is independent from external stimuli such as viral infection. Here, we show that the interferon regulatory factor 1, Irf1, is directly controlled by the murine formative pluripotency gene regulatory network and transiently upregulated during the transition from naive to formative pluripotency. IRF1 binds to regulatory regions of a conserved set of ISGs and is required for their faithful expression upon exit from naive pluripotency. We show that in the absence of IRF1, cells exiting the naive pluripotent stem cell state are more susceptible to viral infection. Irf1 therefore acts as a link between the formative pluripotency network, regulation of innate immunity genes, and defense against viral infections during formative pluripotency.
      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.
      Altered costimulatory signals and hypoxia support chromatin landscapes limiting the functional potential of exhausted T cells in cancer
      View ORCID ProfileB. Rhodes Ford - 2021
      Abstract
      Immunotherapy has changed cancer treatment with major clinical successes, but response rates remain low due in part to elevated prevalence of dysfunctional, terminally exhausted T cells. However, the mechanisms promoting progression to terminal exhaustion remain undefined. We profiled the histone modification landscape of tumor-infiltrating CD8 T cells throughout differentiation, finding terminally exhausted T cells possessed chromatin features limiting their transcriptional potential. Active enhancers enriched for bZIP/AP-1 transcription factor motifs lacked correlated gene expression, which were restored by immunotherapeutic costimulatory signaling. Epigenetic repression was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Our study is the first to profile the precise epigenetic changes during intratumoral differentiation to exhaustion, highlighting their altered function is driven by both improper costimulatory signals and environmental factors. These data suggest even terminally exhausted T cells remain poised for transcription in settings of increased costimulatory signaling and reduced hypoxia.
  • Reverse Transcription
    • First-Strand cDNA Synthesis
      TEMPERATURE REGULATION OF PIPECOLIC ACID-MEDIATED PLANT SYSTEMIC IMMUNITY IN ARABIDOPSIS THALIANA
      Alyssa Shields - 2022
      Abstract
      Significant crop losses are caused by pathogenic infections annually, which are exacerbated by increasing global temperatures due to climate change. One way by which plants respond to pathogenic attacks is through the activation of pattern-triggered immunity (PTI), effector-triggered immunity (ETI), and systemic acquired resistance (SAR), which lead to production of the central defence phytohormone salicylic acid (SA). Accompanying SA release is the putative mobilization of pipecolic acid (Pip), which acts as an immune regulatory plant metabolite that works with and independently from SA. As demonstrated in the model plant Arabidopsis thaliana following infection with the model bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, Pip and its hydroxylated derivative N-hydroxypipecolic acid (NHP) accumulate in local and distal tissues to amplify the plant immune response and prime the plant for future infections. Previous studies have only shown that increased temperature negatively impact PTI, ETI and SA production in the local/primary sites of infection. However, how temperature affects plant systemic immunity has not been fully explored. In this thesis, I showed that systemic immunity in Arabidopsis to Pst DC3000 was significantly reduced at elevated temperatures. Elevated temperature decreased expression of the SAR-associated Pip/NHP biosynthetic genes AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) and FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1) in systemically primed leaf tissues. Remarkably, exogenous Pip application via local leaf infiltration or root-drench restored immunity to Pst DC3000 at elevated temperature; however, local leaf infiltration did not restore immunity in systemic leaves. I have also shown how Pip-induced gene expression locally and systemically were affected by temperature. Finally, because of the interlinked regulation between SA and Pip/NHP by the master transcription factor CAM-BINDING PROTEIN 60-LIKE G (CBP60g), I have shown that Arabidopsis plants constitutively expressing CBP60g (35S:CBP60g) exhibited SAR at both normal and elevated temperatures. My results suggest that CBP60g controls the temperature-sensitivity of plant systemic immunity by modulating NHP biosynthesis. Overall, this thesis contributes to understanding the signaling pathways regulating local and systemic plant immune responses in our warming climate.
      Critical Sites on Ostreolysin Are Responsible for Interaction with Cytoskeletal Proteins
      Nastacia Adler Berke - 2022
      Abstract
      We explored the structural features of recombinant ostreolysin A (rOlyA), a protein produced by Pleurotus ostreatus and responsible for binding to α/β-tubulin. We found that rOlyA cell internalization is essential for the induction of adipocyte-associated activity, which is mediated by the interaction of rOlyA and microtubule proteins. We created different point mutations at conserved tryptophan (W) sites in rOlyA and analyzed their biological activity in HIB-1B preadipocytes. We demonstrated that the protein’s cell-internalization ability and the differentiated phenotype induced, such as small lipid-droplet formation and gene expression of mitogenesis activity, were impaired in point-mutated proteins W96A and W28A, where W was converted to alanine (A). We also showed that an rOlyA homologue, OlyA6 complexed with mCherry, cannot bind to β-tubulin and does not induce mitochondrial biosynthesis-associated markers, suggesting that the OlyA6 region masked by mCherry is involved in β-tubulin binding. Protein–protein docking simulations were carried out to investigate the binding mode of rOlyA with β-tubulin. Taken together, we identified functional sites in rOlyA that are essential for its binding to β-tubulin and its adipocyte-associated biological activity.
      Endocrine and Metabolic Impact of Oral Ingestion of a Carob-Pod-Derived Natural-Syrup-Containing D-Pinitol: Potential Use as a Novel Sweetener in Diabetes
      Juan A. Navarro - 2022
      Abstract
      The widespread use of added sugars or non-nutritive sweeteners in processed foods is a challenge for addressing the therapeutics of obesity and diabetes. Both types of sweeteners generate health problems, and both are being blamed for multiple complications associated with these prevalent diseases. As an example, fructose is proven to contribute to obesity and liver steatosis, while non-nutritive sweeteners generate gut dysbiosis that complicates the metabolic control exerted by the liver. The present work explores an alternative approach for sweetening through the use of a simple carob-pod-derived syrup. This sweetener consists of a balanced mixture of fructose (47%) and glucose (45%), as sweetening sugars, and a functional natural ingredient (D-Pinitol) at a concentration (3%) capable of producing active metabolic effects. The administration of this syrup to healthy volunteers (50 g of total carbohydrates) resulted in less persistent glucose excursions, a lower insulin response to the hyperglycemia produced by its ingestion, and an enhanced glucagon/insulin ratio, compared to that observed after the ingestion of 50 g of glucose. Daily administration of the syrup to Wistar rats for 10 days lowered fat depots in the liver, reduced liver glycogen, promoted fat oxidation, and was devoid of toxic effects. In addition, this repeated administration of the syrup improved glucose handling after a glucose (2 g/kg) load. Overall, this alternative functional sweetener retains the natural palatability of a glucose/fructose syrup while displaying beneficial metabolic effects that might serve to protect against the progression towards complicated obesity, especially the development of liver steatosis.
      Cerebrospinal fluid of progressive multiple sclerosis patients reduces differentiation and immune functions of oligodendrocyte progenitor cells
      Omri Zveik - 2022
      Abstract
      Oligodendrocyte progenitor cells (OPCs) are responsible for remyelination in the central nervous system (CNS) in health and disease. For patients with multiple sclerosis (MS), remyelination is not always successful, and the mechanisms differentiating successful from failed remyelination are not well-known. Growing evidence suggests an immune role for OPCs, in addition to their regenerative role; however, it is not clear if this helps or hinders the regenerative process. We studied the effect of cerebrospinal fluid (CSF) from relapsing MS (rMS) and progressive MS (pMS) patients on primary OPC differentiation and immune gene expression and function. We observed that CSF from either rMS or pMS patients has a differential effect on the ability of mice OPCs to differentiate into mature oligodendrocytes and to express immune functions. CSF of pMS patients impaired differentiation into mature oligodendrocytes. In addition, it led to decreased major histocompatibility complex class (MHC)-II expression, tumor necrosis factor (TNF)-α secretion, nuclear factor kappa-B (NFκB) activation, and less activation and proliferation of T cells. Our findings suggest that OPCs are not only responsible for remyelination, but they may also play an active role as innate immune cells in the CNS.
  • 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
    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.
    Experimental challenge of flatfishes (Pleuronectidae) with salmonid alphavirus (SAV): Observations on tissue tropism and pathology in common dab Limanda limanda L.
    Linda Andersen - 2022
    Abstract
    Salmonid alphavirus (SAV) is the aetiological agent of pancreas disease (PD), a serious viral disease in salmonids. For several decades, SAV was known to infect salmonid species only, until SAV was detected using real-time PCR in several species of wild-caught flatfishes in Scotland in 2010. The presence of SAV in wild flatfishes has been confirmed by further surveys from Ireland and Scotland. The role of flatfishes in SAV-spread and epizootiology has not been elucidated, and no experimental challenges have been conducted to examine virus tissue tropism, virulence and pathology in flatfishes. Wild-caught flatfishes (common dab; Limanda limanda, European plaice; Pleuronectes platessa, European flounder; Platichthys flesus and lemon sole; Microstomus kitt) were either intramuscularly (i.m.) or intraperitoneally (i.p.) challenged with SAV3 or exposed to SAV3 through cohabitation with i.p. injected salmon. SAV-infections were seen in i.m. and i.p. injected dab and i.p. injected salmon but did not result in a transmissible infection in dab although several routes of entry were assessed (oral route not tested). SAV was detected in several tissues of eight common dab (not from cohabitants), with high SAV-levels in pancreas. No viraemia was detected in the SAV-positive common dab and no virus shedding were detected in the tanks. However, pathology in exocrine pancreas and hearts consistent with SAV-replication were seen. This is the first study reporting SAV-induced pathology in a non-salmonid species. The results from the present challenge study supports evidence for common dab being susceptible hosts for SAV. The study also demonstrates that flatfishes are less susceptible to SAV3-infection than salmon.
    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.
    Engineering heterologous enzyme secretion in Yarrowia lipolytica
    Weigao Wang - 2022
    Abstract
    Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and α-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.
    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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