US
array(51) {
  ["SERVER_SOFTWARE"]=>
  string(6) "Apache"
  ["REQUEST_URI"]=>
  string(40) "/resources/?materials%5B%5D=publications"
  ["PATH"]=>
  string(49) "/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin"
  ["PP_CUSTOM_PHP_INI"]=>
  string(48) "/var/www/vhosts/system/quantabio.com/etc/php.ini"
  ["PP_CUSTOM_PHP_CGI_INDEX"]=>
  string(19) "plesk-php74-fastcgi"
  ["SCRIPT_NAME"]=>
  string(10) "/index.php"
  ["QUERY_STRING"]=>
  string(28) "materials%5B%5D=publications"
  ["REQUEST_METHOD"]=>
  string(3) "GET"
  ["SERVER_PROTOCOL"]=>
  string(8) "HTTP/1.1"
  ["GATEWAY_INTERFACE"]=>
  string(7) "CGI/1.1"
  ["REDIRECT_URL"]=>
  string(11) "/resources/"
  ["REDIRECT_QUERY_STRING"]=>
  string(28) "materials%5B%5D=publications"
  ["REMOTE_PORT"]=>
  string(5) "51270"
  ["SCRIPT_FILENAME"]=>
  string(48) "/var/www/vhosts/quantabio.com/httpdocs/index.php"
  ["SERVER_ADMIN"]=>
  string(14) "root@localhost"
  ["CONTEXT_DOCUMENT_ROOT"]=>
  string(38) "/var/www/vhosts/quantabio.com/httpdocs"
  ["CONTEXT_PREFIX"]=>
  string(0) ""
  ["REQUEST_SCHEME"]=>
  string(5) "https"
  ["DOCUMENT_ROOT"]=>
  string(38) "/var/www/vhosts/quantabio.com/httpdocs"
  ["REMOTE_ADDR"]=>
  string(11) "23.20.20.52"
  ["SERVER_PORT"]=>
  string(3) "443"
  ["SERVER_ADDR"]=>
  string(13) "172.31.63.191"
  ["SERVER_NAME"]=>
  string(17) "www.quantabio.com"
  ["SERVER_SIGNATURE"]=>
  string(0) ""
  ["HTTP_ACCEPT_ENCODING"]=>
  string(7) "br,gzip"
  ["HTTP_ACCEPT_LANGUAGE"]=>
  string(14) "en-US,en;q=0.5"
  ["HTTP_ACCEPT"]=>
  string(63) "text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8"
  ["HTTP_USER_AGENT"]=>
  string(40) "CCBot/2.0 (https://commoncrawl.org/faq/)"
  ["HTTP_X_SUCURI_COUNTRY"]=>
  string(2) "US"
  ["HTTP_X_SUCURI_CLIENTIP"]=>
  string(11) "23.20.20.52"
  ["HTTP_X_FORWARDED_PROTO"]=>
  string(5) "https"
  ["HTTP_CONNECTION"]=>
  string(5) "close"
  ["HTTP_X_FORWARDED_FOR"]=>
  string(11) "23.20.20.52"
  ["HTTP_X_REAL_IP"]=>
  string(13) "185.93.229.32"
  ["HTTP_HOST"]=>
  string(17) "www.quantabio.com"
  ["HTTPS"]=>
  string(2) "on"
  ["HTTP_AUTHORIZATION"]=>
  string(0) ""
  ["SCRIPT_URI"]=>
  string(36) "https://www.quantabio.com/resources/"
  ["SCRIPT_URL"]=>
  string(11) "/resources/"
  ["UNIQUE_ID"]=>
  string(27) "YfGwiX25bYJpYFmux25TfQAAAAI"
  ["REDIRECT_STATUS"]=>
  string(3) "200"
  ["REDIRECT_HTTPS"]=>
  string(2) "on"
  ["REDIRECT_HTTP_AUTHORIZATION"]=>
  string(0) ""
  ["REDIRECT_SCRIPT_URI"]=>
  string(36) "https://www.quantabio.com/resources/"
  ["REDIRECT_SCRIPT_URL"]=>
  string(11) "/resources/"
  ["REDIRECT_UNIQUE_ID"]=>
  string(27) "YfGwiX25bYJpYFmux25TfQAAAAI"
  ["FCGI_ROLE"]=>
  string(9) "RESPONDER"
  ["PHP_SELF"]=>
  string(10) "/index.php"
  ["REQUEST_TIME_FLOAT"]=>
  float(1643229321.3764)
  ["REQUEST_TIME"]=>
  int(1643229321)
  ["SUCURIREAL_REMOTE_ADDR"]=>
  string(13) "185.93.229.32"
}
Find the Resources You Need
Clear Search/Filters

Publications

  • PCR
    • Real-Time Quantitative PCR
      • SYBR Green Detection
        • DNA
          Oxidative Stress and X-ray Exposure Levels-Dependent Survival and Metabolic Changes in Murine HSPCs
          Melis Karabulutoglu - 2021
          Abstract
          Haematopoietic bone marrow cells are amongst the most sensitive to ionizing radiation (IR), initially resulting in cell death or genotoxicity that may later lead to leukaemia development, most frequently Acute Myeloid Leukaemia (AML). The target cells for radiation-induced Acute Myeloid Leukaemia (rAML) are believed to lie in the haematopoietic stem and progenitor cell (HSPC) compartment. Using the inbred strain CBA/Ca as a murine model of rAML, progress has been made in understanding the underlying mechanisms, characterisation of target cell population and responses to IR. Complex regulatory systems maintain haematopoietic homeostasis which may act to modulate the risk of rAML. However, little is currently known about the role of metabolic factors and diet in these regulatory systems and modification of the risk of AML development. This study characterises cellular proliferative and clonogenic potential as well as metabolic changes within murine HSPCs under oxidative stress and X-ray exposure. Ambient oxygen (normoxia; 20.8% O2) levels were found to increase irradiated HSPC-stress, stimulating proliferative activity compared to low oxygen (3% O2) levels. IR exposure has a negative influence on the proliferative capability of HSPCs in a dose-dependent manner (0–2 Gy) and this is more pronounced under a normoxic state. One Gy x-irradiated HSPCs cultured under normoxic conditions displayed a significant increase in oxygen consumption compared to those cultured under low O2 conditions and to unirradiated HSPCs. Furthermore, mitochondrial analyses revealed a significant increase in mitochondrial DNA (mtDNA) content, mitochondrial mass and membrane potential in a dose-dependent manner under normoxic conditions. Our results demonstrate that both IR and normoxia act as stressors for HSPCs, leading to significant metabolic deregulation and mitochondrial dysfunctionality which may affect long term risks such as leukaemia.
          The RNA binding protein IMP2 drives a stromal-Th17 cell circuit in autoimmune neuroinflammation
          Rami Bechara - 2021
          Abstract
          Stromal cells are emerging as key drivers of autoimmunity, in part by producing inflammatory chemokines that orchestrate inflammation. Chemokine expression is regulated transcriptionally but also through post-transcriptional mechanisms, the specific drivers of which are still incompletely defined. CCL2 (MCP1) is a multifunctional chemokine that drives myeloid cell recruitment. During experimental autoimmune encephalomyelitis (EAE), an IL-17-driven model of multiple sclerosis, CCL2 produced by lymph node (LN) stromal cells is essential for immunopathology. Here, we show that Ccl2 mRNA upregulation in human stromal fibroblasts in response to IL-17 requires the RNA binding protein (RBP) insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2, IMP2), which is expressed almost exclusively in non-hematopoietic cells. IMP2 binds directly to CCL2 mRNA, markedly extending its transcript half-life and thus required for efficient CCL2 secretion. Consistent with this, Imp2−/− mice showed reduced CCL2 production in LN during EAE, causing impairments in monocyte recruitment and Th17 cell polarization. Imp2-/- mice were fully protected from CNS inflammation. Moreover, deletion of IMP2 after EAE onset was sufficient to mitigate disease severity. These data show that posttranscriptional control of Ccl2 in stromal cells by IMP2 is required to permit IL-17-driven progression of EAE pathogenesis.
      • Probe-based Detection
        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.
        Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice
        Jianxiong Zeng - 2022
        Abstract
        Background The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. Methods We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. Findings We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3−/− mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. Interpretation Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease.
        QUANTITATIVE ASSESSMENT OF METHODS FOR BACTERIAL AND VIRAL PURIFICATION AND CONCENTRATION
        Nina Sara Fraticelli-Guzmán - 2021
        Abstract
        The COVID-19 global pandemic has led to the exploration and implementation of rapid tests for viral load identification and thus, control of the spread. For this, there are varying approaches with the gold standard being quantitative Polymerase Chain Reaction [1]and a potential more rapid alternate approach being the use of an antibody coated biosensor such as a field effect transistor (FET). Analogously, bacterial pathogens such as anthrax need sensing and quantification as well. Therefore, methods for pathogen collection and detection for both viruses and bacteria are needed. Regardless of the analysis technique, it is ideal for samples to contain little to zero non-target particles or contaminants that might interfere with the detection approach. As such, traditional ways of purifying and concentrating samples prior to any type of analysis involve both chemical, physical, physiochemical, or biological approaches such as filtration, centrifugation, affinity chromatography, immunomagnetic separation, etc. Of these, we will focus on the physical approach, filtration, due to its simplicity, low cost, varying options, and ability to process large and small sample volumes. In this work, we investigate how well the method works for virus or bacteria in the presence of high interferent concentrations that could potentially be present in saliva samples or other hydrosol samples taken from an environment to be tested. The research presented here characterizes the applicability of syringe filters and a tangential flow filtration device for the purification and concentration of bacteria and virus samples, respectively. Furthermore, automation of such systems was explored. Varying syringe filter pore sizes yielded different recoveries of bacteria for purifying and concentrating the sample. Furthermore, varying volumes were analyzed for xviii optimal recovery and concentration of the target. We also developed a fully automated method for double filter filtration to enable hands-free purification and concentration in 5.5 minutes for 5 mL of input volume with a 42 ± 13-fold enrichment improvement (n = 3). Furthermore, the purification and concentration of virus using a manually operated tangential flow filtration device was also explored and yielded modest concentration increases of around 2× with an enrichment improvement of up to 1,916 ± 1,839-fold (n = 3) under one configuration. By characterizing and automating these readily available items, we can enhance the detection of samples by decreasing labor time and processing complexity required for the purification and concentration of the target pathogens.
        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.
        Profiling Multiomic Biomarkers using Particle Detection Counters and Spectral-FLIM Microscopy
        Tam Vu - 2021
        Abstract
        Profiling multiomic biomarkers in bulk and in situ provides critical information which enables basic research and clinical applications. Unfortunately, most existing profiling methods are limited due to either low multiplexing, sensitivity, costs, or assay complexity. This thesis aims to develop two core technologies that address 1) bulk profiling issues with sensitivity and low throughput as well as 2) in situ profiling issues with low multiplexing capabilities, costs, and limited throughput. To address the first issue, this work introduces a novel liquid biopsy approach that utilizes a platform technology called Integrated Comprehensive Droplet Digital Detection (IC3D). This integrated approach combines microfluidic droplet partitioning technology, fluorescent multiplexed PCR chemistry, and our own unique and rapid particle counting technology to deliver ultrasensitive and ultrafast detection of colorectal cancer-specific genomic biomarkers from minimally processed blood samples. xv To address the second issue, this work introduces a new spatial multi-omics technology termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) that integrates a) in situ labeling of molecular markers (e.g. mRNA, proteins) in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, and b) spectra and timeresolved fluorescence imaging and analysis to enable rapid, high-throughput, and cost-effective spatial profiling of multi-omics biomarkers. By utilizing both time and intensity domains for labeling and imaging, this technology seeks to discriminate a vast repertoire of lifetime and spectral components simultaneously within the same pixel or image of a sample to enable highly increased multiplexing capabilities with standard optical systems. Overall, these two technologies represent simple, versatile, and scalable tools which enable more rapid, sensitive, and/or multiplexed protein/transcriptomic analysis.
        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.
        Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing
        Shairaz Baksh - 2021
        Abstract
        We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
        Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model
        Anne-Laure Thomas - 2020
        Abstract
        Background Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. Methods Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. Results Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. Conclusions Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of Study Basic science.
        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.
        Iron oxide nanoparticles enhance Toll-like receptor-induced cytokines in a particle size- and actin-dependent manner in human blood
        Susann Wolf-Grosse - 2018
        Abstract
        Aim: To assess the effects of different-sized iron oxide nanoparticles (IONPs) on inflammatory responses in human whole blood. Materials & methods: Human whole blood with and without 10 and 30 nm IONPs was incubated with Toll-like receptor (TLR) ligands. Cytokine levels, complement activation, reactive oxygen species and viability were determined. Results: The 10 nm IONPs enhanced the TLR2/6, TLR4 and partly TLR8-mediated cytokine production, whereas the 30 nm IONPs partly enhanced TLR2/6 and decreased TLR8-mediated cytokine production. Particle-mediated enhancement of TLR4-induced cytokines could not be explained by complement activation, but was dependent on TLR4/MD2 and CD14, as well as actin polymerization. Conclusion: The IONPs differentially affected the TLR ligand-induced cytokines, which has important implications for biomedical applications of IONPs.
      • RNA
        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.
        Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice
        Jianxiong Zeng - 2022
        Abstract
        Background The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. Methods We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. Findings We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3−/− mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. Interpretation Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease.
        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.
        Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing
        Shairaz Baksh - 2021
        Abstract
        We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
        Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model
        Anne-Laure Thomas - 2020
        Abstract
        Background Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. Methods Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. Results Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. Conclusions Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of Study Basic science.
    • Conventional PCR
      • DNA
        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.
        Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase
        Xiaodong Zhang - 2021
        Abstract
        Amylases account for about 30% of the global market of industrial enzymes, and the current amylases cannot fully meet industrial needs. This study aimed to identify a high α-amylase producing bacterium WangLB, to clone its α-amylase coding gene, and to characterize the α-amylase. Results showed that WangLB belonged to Bacillus velezensis whose α-amylase gene was 1980 bp coding 659 amino acids designated as BvAmylase. BvAmylase was a hydrophilic stable protein with a signal peptide and a theoretical pI of 5.49. The relative molecular weight of BvAmylase was 72.35 kDa, and was verified by SDS-PAGE. Its modeled structure displayed that it was a monomer composed of three domains. Its optimum temperature and pH were 70 °C and pH 6.0, respectively. It also showed high activity in a wide range of temperatures (40–75 °C) and a relatively narrow pH (5.0–7.0). It was a Ca2+-independent enzyme, whose α-amylase activity was increased by Co2+, Tween 20, and Triton X-100, and severely decreased by SDS. The Km and the Vmax of BvAmylase were 3.43 ± 0.53 and 434.19 ± 28.57 U/mg. In conclusion, the α-amylase producing bacterium WangLB was identified, and one of its α-amylases was characterized, which will be a candidate enzyme for industrial applications.
        Microbiome Fingerprint as Biomarker for Geographical Origin and Heredity in Crocus sativus: A Feasibility Study
        Nancy Bhagat - 2021
        Abstract
        Host–microbiome interactions are specific and not random, making them defining entities for the host. The hypothesis proposed by various researchers earlier, that both plants and animals harbor specific inheritable core microbiome, is being augmented in the present study. Additionally, a case for using microbial fingerprint as a biomarker, not only for plant identification but also as a geographical indicator, has been investigated, taking Crocus sativus, saffron, as a study material. Crocus sativus, a monogenetic herb, on account of its male sterility and vegetative propagation, is reported to lack genome based molecular markers. Cormosphere microbiome (microbiome associated with corm) has been compared across three geographical locations, in two continents, to identify the core and unique microbiome, during the vegetative phase of its growth. Microbiome analysis done at phylum and genus level, using next generation sequencing technology, revealed that cormosphere at three locations harbored common phyla. At genus level, 24 genera were found common to all three geographical locations, indicating them to be part of the core microbiome of saffron. However, there were some bacterial genera unique to Kashmir, Kishtwar, and Morocco that can be used to develop microbial markers/geographical indicators for saffron grown in these regions. This is a preliminary study, indicating that the location specific bacterial community can be used to develop microbial barcodes but needs further augmentation with high coverage data from other saffron growing geographical regions.
        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.
        Genomic Characterization of the Fruity Aroma Gene, FaFAD1, Reveals a Gene Dosage Effect on γ-Decalactone Production in Strawberry (Fragaria × ananassa)
        Youngjae Oh - 2021
        Abstract
        Strawberries produce numerous volatile compounds that contribute to the unique flavors of fruits. Among the many volatiles, γ-decalactone (γ-D) has the greatest contribution to the characteristic fruity aroma in strawberry fruit. The presence or absence of γ-D is controlled by a single locus, FaFAD1. However, this locus has not yet been systematically characterized in the octoploid strawberry genome. It has also been reported that the volatile content greatly varies among the strawberry varieties possessing FaFAD1, suggesting that another genetic factor could be responsible for the different levels of γ-D in fruit. In this study, we explored the genomic structure of FaFAD1 and determined the allele dosage of FaFAD1 that regulates variations of γ-D production in cultivated octoploid strawberry. The genome-wide association studies confirmed the major locus FaFAD1 that regulates the γ-D production in cultivated strawberry. With the hybrid capture-based next-generation sequencing analysis, a major presence–absence variation of FaFAD1 was discovered among γ-D producers and non-producers. To explore the genomic structure of FaFAD1 in the octoploid strawberry, three bacterial artificial chromosome (BAC) libraries were developed. A deletion of 8,262 bp was consistently found in the FaFAD1 region of γ-D non-producing varieties. With the newly developed InDel-based codominant marker genotyping, along with γ-D metabolite profiling data, we revealed the impact of gene dosage effect for the production of γ-D in the octoploid strawberry varieties. Altogether, this study provides systematic information of the prominent role of FaFAD1 presence and absence polymorphism in producing γ-D and proposes that both alleles of FaFAD1 are required to produce the highest content of fruity aroma in strawberry fruit.
        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
      Microbiome Fingerprint as Biomarker for Geographical Origin and Heredity in Crocus sativus: A Feasibility Study
      Nancy Bhagat - 2021
      Abstract
      Host–microbiome interactions are specific and not random, making them defining entities for the host. The hypothesis proposed by various researchers earlier, that both plants and animals harbor specific inheritable core microbiome, is being augmented in the present study. Additionally, a case for using microbial fingerprint as a biomarker, not only for plant identification but also as a geographical indicator, has been investigated, taking Crocus sativus, saffron, as a study material. Crocus sativus, a monogenetic herb, on account of its male sterility and vegetative propagation, is reported to lack genome based molecular markers. Cormosphere microbiome (microbiome associated with corm) has been compared across three geographical locations, in two continents, to identify the core and unique microbiome, during the vegetative phase of its growth. Microbiome analysis done at phylum and genus level, using next generation sequencing technology, revealed that cormosphere at three locations harbored common phyla. At genus level, 24 genera were found common to all three geographical locations, indicating them to be part of the core microbiome of saffron. However, there were some bacterial genera unique to Kashmir, Kishtwar, and Morocco that can be used to develop microbial markers/geographical indicators for saffron grown in these regions. This is a preliminary study, indicating that the location specific bacterial community can be used to develop microbial barcodes but needs further augmentation with high coverage data from other saffron growing geographical regions.
      Ammonia-oxidizing bacterial communities are affected by nitrogen fertilization and grass species in native C4 grassland soils
      Jialin Hu - 2021
      Abstract
      Background Fertilizer addition can contribute to nitrogen (N) losses from soil by affecting microbial populations responsible for nitrification. However, the effects of N fertilization on ammonia oxidizing bacteria under C4 perennial grasses in nutrient-poor grasslands are not well studied. Methods In this study, a field experiment was used to assess the effects of N fertilization rate (0, 67, and 202 kg N ha−1) and grass species (switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii)) on ammonia-oxidizing bacterial (AOB) communities in C4 grassland soils using quantitative PCR, quantitative reverse transcription-PCR, and high-throughput amplicon sequencing of amoA genes. Results Nitrosospira were dominant AOB in the C4 grassland soil throughout the growing season. N fertilization rate had a stronger influence on AOB community composition than C4 grass species. Elevated N fertilizer application increased the abundance, activity, and alpha-diversity of AOB communities as well as nitrification potential, nitrous oxide (N2O) emission and soil acidity. The abundance and species richness of AOB were higher under switchgrass compared to big bluestem. Soil pH, nitrate, nitrification potential, and N2O emission were significantly related to the variability in AOB community structures (p < 0.05).
      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.
      Six de novo assemblies from pathogenic and non-pathogenic strains of Fusarium oxysporum f. sp. niveum
      James C. Fulton - 2021
      Abstract
      Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (Fon), is a soilborne disease which significantly limits yield in watermelon (Citrullus lanatus) and occasionally causes the loss of an entire year’s harvest. Reference-quality de novo genomic assemblies of pathogenic and non-pathogenic strains were generated using a combination of next-generation and third-generation sequencing technologies. Chromosomal-level genomes were produced with representatives from all Fon races facilitating comparative genomic analysis and the identification of chromosomal structural variation . Syntenic analysis between isolates allowed differentiation of the core and lineage-specific portions of their genomes. This research will support future efforts to refine the scientific understanding of the molecular and genetic factors underpinning the Fon host range, develop diagnostic assays for each of the four races, and decipher the evolutionary history of race 3.
  • Reverse Transcription
    • Quantitative RT-qPCR
      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.
      Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice
      Jianxiong Zeng - 2022
      Abstract
      Background The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. Methods We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. Findings We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3−/− mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. Interpretation Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease.
      Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing
      Shairaz Baksh - 2021
      Abstract
      We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
      Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model
      Anne-Laure Thomas - 2020
      Abstract
      Background Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. Methods Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. Results Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. Conclusions Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of Study Basic science.
    • First-Strand cDNA Synthesis
      Bioreactor and Small Molecule Drug Applications in Hair Cell Differentiation
      Sylvia Yip - 2021
      Abstract
      Hair cells are mechanoreceptors of the inner ear that convert sounds into electrical signals to be perceived by the brain. The gradual loss of hair cells is common after a lifetime of chronic exposure to loud noises. Mammalian hair cells lack the ability to regenerate after embryonic development. Advancements in regenerative medicine strategies have been employed to restore function to damaged organs. The use of stem cells can regenerate and replace damaged hair cells. Understanding how stem cells differentiate into hair cells will provide insight into regeneration. To study hair cell regeneration, we employed an inner ear organoid system. Immortalized multipotent otic progenitor (iMOP) cells that differentiate into spiral ganglion neurons, supporting cells, and hair cells were used to generate organoids. This study tests whether a bioreactor facilitates sensory epithelial differentiation. The bioreactor agitates the medium to promote nutrient diffusion in the cultures. After ten days of differentiation, iMOP-derived organoids were collected, and relative changes in transcripts and protein markers that correspond to neurons, supporting cells, and hair cells were determined. The use of a bioreactor increased the percentage of cells expressing MYO6, a hair cell marker. The percentage of TUBB3 labeled neurons also increased while GFAP labeled supporting cells remained the same. Relative levels of Myo6 mRNA did not increase in any of the culture conditions while relative levels of Tubb3 mRNA significantly increased in all cultures compared to control samples. Although molecular mechanisms are unknown, the study shows that use of the bioreactor improves hair cell differentiation in otic progenitor-derived organoids.
      Muscle-specific functional deficits and lifelongfibrosisin response to paediatric radiotherapy and tumourelimination
      Jacob G. Kallenbach - 2022
      Abstract
      BackgroundAs paediatric cancer survivors are living into adulthood, they suffer from the age-related, accelerateddecline of functional skeletal muscle tissue, termed sarcopenia. With ionizing radiation (radiotherapy) at the core ofpaediatric cancer therapies, its direct and indirect effects can have lifelong negative impacts on paediatric growthand maintenance of skeletal muscle. Utilizing our recently developed preclinical rhabdomyosarcoma mouse model,we investigated the late effects of paediatric radiation treatment on skeletal muscles from late adolescent (8 weeksold) and middle-aged (16 months old) mice.MethodsPaediatric C57BL/6J male mice (3 weeks old) were injected with rhabdomyosarcoma cells into their righthindlimbs, and then fractionated irradiation (3 × 8.2 Gy) was administered to those limbs at 4 weeks old to eliminatethe tumours. Radiation-alone and tumour-irradiated mice were assessed at either 8 weeks (3 weeks post-irradiation) or16 months (14 months post-irradiation) of age for muscle physiology, myofibre characteristics, cell loss, histopathology,fibrosis, inflammatory gene expression, andfibrotic gene expression.ResultsMice that received only paediatric radiation demonstrated reduced muscle mass (17%,P<0.001), musclephysiological function (25%,P<0.01), muscle contractile kinetics (25%,P<0.05), satellite cell number (45%,P<0.05), myofibre cross-sectional area (30%,P<0.0001), and myonuclear number (17%,P<0.001). Paediatricradiation increased inflammatory gene expression, increasedfibrotic gene expression, and induced extracellular matrixprotein deposition (fibrosis) with tumour elimination exacerbating some phenotypes. Paediatric tumour-eliminatedmice demonstrated exacerbated deficits to function (20%,P<0.05) and myofibre size (17%,P<0.001) in somemuscles as well as further increases to inflammatory andfibrotic gene expression. Examining the age-related effectsof paediatric radiotherapy in middle-aged mice, we found persistent myofibre atrophy (20%,P<0.01), myonuclearloss (18%,P<0.001), up-regulated inflammatory andfibrotic signalling, and lifelongfibrosis.ConclusionsThe results from this paediatric radiotherapy model are consistent and recapitulate the clinical and mo-lecular features of accelerated sarcopenia, musculoskeletal frailty, and radiation-inducedfibrosis experienced by paedi-atric cancer survivors. We believe that this preclinical mouse model is well poised for future mechanistic insights andtherapeutic interventions that improve the quality of life for paediatric cancer survivors.
      The Betacoronavirus PHEV Replicates and Disrupts the Respiratory Epithelia and Upregulates Key Pattern Recognition Receptor Genes and Downstream Mediators, Including IL-8 and IFN-l
      Rahul Nelli - 2021
      Abstract
      The upper respiratory tract is the primary site of infection by porcine hemagglutinating encephalomyelitis virus (PHEV). In this study, primary porcine respiratory epithelial cells (PRECs) were cultured in an air-liquid interface (ALI) to differentiate into a pseudostratified columnar epithelium, proliferative basal cells, M cells, ciliated cells, and mucus-secreting goblet cells. ALI-PRECs recreates a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. PHEV replicated actively in this environment, inducing cytopathic changes and progressive disruption of the mucociliary apparatus. The innate immunity against PHEV was comparatively evaluated in ALI-PREC cultures and tracheal tissue sections derived from the same cesarean-derived, colostrum-deprived (CDCD) neonatal donor pigs. Increased expression levels of TLR3 and/or TLR7, RIG1, and MyD88 genes were detected in response to infection, resulting in the transcriptional upregulation of IFN-l1 in both ALI-PREC cultures and tracheal epithelia. IFN-l1 triggered the upregulation of the transcription factor STAT1, which in turn induced the expression of the antiviral IFN-stimulated genes OAS1 and Mx1. No significant modulation of the major proinflammatory cytokines interleukin-1b (IL-1b), IL-6, and tumor necrosis factor alpha (TNF-a) was detected in response to PHEV infection. However, a significant upregulation of different chemokines was observed in ALI-PREC cultures (CCL2, CCL5, CXCL8, and CXCL10) and tracheal epithelium (CXCL8 and CXCL10). This study shed light on the molecular mechanisms driving the innate immune response to PHEV at the airway epithelium, underscoring the important role of respiratory epithelial cells in the maintenance of respiratory homeostasis and on the initiation, resolution, and outcome of the infectious process. IMPORTANCE The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) primarily infects and replicates in the swine upper respiratory tract, causing vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study investigated the modulation of key early innate immune genes at the respiratory epithelia in vivo, on tracheal tissue sections from experimentally infected pigs, and in vitro, on air-liquid interface porcine respiratory cell cultures. The results from the study underscore the important role of respiratory epithelial cells in maintaining respiratory homeostasis and on the initiation, resolution, and outcome of the PHEV infectious process
      Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds
      Ivan Jozic - 2021
      Abstract
      Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MβCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.
  • Sample Preparation
    • DNA
      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.

Product Finder

Select Your Assay

Starting Template

Assay Format

Detection Chemistry

Multiplexing (more than 3 targets)

Is gene-specific priming (GSP) required?

What current Reverse Transcriptase or cDNA kit are you using?

Select the group which contains your real-time PCR cycler

  • Applied Biosystems 7500
  • Applied Biosystems 7500 Fast
  • Stratagene Mx3000P®
  • Stratagene Mx3005P™
  • Stratagene Mx4000™
  • Applied Biosystems ViiA 7
  • Applied Biosystems QuantStudio™
  • Agilent AriaMx
  • Douglas Scientific IntelliQube®
  • Applied Biosystems 5700
  • Applied Biosystems 7000
  • Applied Biosystems 7300
  • Applied Biosystems 7700
  • Applied Biosystems 7900
  • Applied Biosystems 7900HT
  • Applied Biosystems 7900 HT Fast
  • Applied Biosystems StepOne™
  • Applied Biosystems StepOnePlus™
  • Quantabio Q
  • BioRad CFX
  • Roche LightCycler 480
  • QIAGEN Rotor-Gene Q
  • Other
  • BioRad iCycler iQ™
  • BioRad MyiQ™
  • BioRad iQ™5

Choose your application from the categories below

Products