Month: June 2020

Same-visit testing and treatment of infants to greatly improve child health outcomes and substantially reduce loss to follow-up. Previously, we reported on the development of a non-instrumented TH-302 moa nucleic acid amplification platform and we proposed an LRS-appropriate workflow for an electricity-free kit. Since then, we have advanced the design from the proof-of-concept stage to an optimized, robust alpha prototype with significant improvements in performance and usability. The improved NINA design we report on in this study requires fewer activation steps, and accordingly affords significantly less opportunity for user-introduced error and variation. Incorporating magnesium iron alloy into a diagnostic technology has been previously reported. In the experimental device described here, replacing calcium oxide with magnesium iron alloy for the exothermic reaction offers several distinct advantages: MgFe has significantly higher energy density, reducing the mass of the fuel pouch from 20 g to 1 g; MgFe is commercially available at a very low cost with very little batch-to-batch variation; and MgFe can be milled to a specific particlesize range to further control the heat profile of the chemical reaction. Additional improvements in this prototype include packaging the MgFe fuel in a hydrophilic, heat-sealable membrane and containing the liquid reactant in an easy-to-use blow-fill-seal container. These design enhancements greatly improve ease of use by minimizing user steps and eliminating the post-amplification heater cleaning that was required with previous designs. The improved design also incorporates a smaller vacuum-insulated housing to reduce heat loss and decrease the overall size of the heater. Finally, we incorporated an improved phase change material with very high latent heat to meet the thermal requirements of an HIV-1 LAMP assay. To demonstrate the robustness of the improved heater design, we evaluated its thermal performance over a wide ambient temperature range that represents LRS conditions. In addition to testing a new design and alternative heater materials, we further demonstrated the utility of the platform by incorporating a biplexed internal control as well as a nucleic acid lateral flow visual detection method. Multiplexed assays are commonly used in PCR for the detection of an internal control to confirm absence of inhibition. However, only a limited number of studies have examined multiplexed LAMP, and to our knowledge, none have used NALF as the detection method. To demonstrate the utility of this evolving platform, we paired the technology with a NALF-detection cassette and evaluated the performance of the combined components using a biplexed LAMP assay for the detection of HIV-1 and a ß-actin internal control. Performance of the assay in the non-instrumented system was compared to parallel assays assessed in real-time on a thermocyler, with the sensitivity, reproducibility, and repeatability of the assay evaluated via melt curve analysis, NALF detection, and agarose gel electrophoresis of reaction products. Species identification and clear understanding of genetic relationship of Echinochloa are very important to control effectively these weeds.

The most commonly used NAAT method, the polymerase chain reaction, is not ideal for LRS using current commercially available equipment. Traditional PCR-based diagnostics require a thermocycler, a clean laboratory, reliable electricity, cold storage for reagents, a temperature-controlled environment, provisions for amplicon containment, and trained personnel. Many PCR machines are controlled via a computer, which increases purchase cost. Recently, there have been significant developments in isothermal amplification methods that do not require thermocycling. Among these, loop-mediated isothermal amplification is one of the most published methods. A search of the ISI-Web of Knowledge database using the search terms ‘LAMP’ and ‘loop mediated amplification’ returns 1,230 publications since the first description of the method in 2000. LAMP can be used for the amplification of DNA, and when a reverse transcription step is included, LAMP can also amplify from RNA. LAMP is sufficiently sensitive for clinical use and is much less susceptible to inhibitors than PCR. Amplification occurs at one constant temperature, typically in a range between 58˚C and 65˚C. Set-up is relatively simple, and direct turbidity or fluorescence detection is possible, although other naked-eye-detection schemes such as visualization on an immunochromatographic strip have also been evaluated. Furthermore, the complex sample preparation steps required for PCR can be simplified or eliminated with LAMP. Several LAMP tests have been Temozolomide cost commercialized, and LAMP assays for tuberculosis, malaria, and HIV have been developed. There are several reasons why LAMP has had little impact on diagnostics designed for LRS. Foremost, LAMP-based NAATs still require electricity to achieve amplification temperatures. Secondly, nonspecific amplification has been a challenge to assay development, and has only recently been addressed through sequencespecific detection strategies. To further advance the utility of LAMP and other isothermal technologies for LRS, this study builds upon previously published laboratory data and supports the continued development of an electricity-free, self-contained platform that addresses these limitations. Millions of lives and disability-adjusted life years are lost through delays in the correct and timely diagnosis of malaria, HIV, tuberculosis, influenza, and other infectious diseases. While our design is platform-based and therefore pathogen-agnostic, we chose to demonstrate this electricity-free molecular amplification and visual detection system using HIV-1 detection as a model analyte. Currently, no point-of-care molecular tests for HIV-1 exist in LRS, where detection of acute infections would have significant impact in high-diseaseburden areas. Early identification and treatment of infected individuals could lower HIV transmission rates since acutely infective individuals are at a much higher risk of transmitting the virus. Furthermore, the HIV-1 test could be used for POC early infant diagnosis, replacing conventional reference center testing, which has shown significant delays in reporting of results and loss to follow-up.

Genomes were scanned using the Infernal program. The identified candidate RNA regulatory sites for each riboswitch family were uploaded into the RegPredict Web server and the respective RNA regulogs were reconstructed using the same approach as for TF regulogs. New regulatory RNAs were found by groups of genomes. Secondary structures of two alternative RNA conformations were predicted using Zuker’s algorithm of free energy minimization implemented in the Mfold Web server. To assess conservation of regulatory interactions in the reconstructed orthologous regulogs we calculated the conservation score as the number of gene occurrences in a regulog divided by the number of regulons in a regulog. The average of these taxonomy-specific conservation scores was calculated for all taxonomic groups where the regulated gene was observed. For each TF, we ASP1517 plotted the average conservation scores for all regulatory targets against the number of taxonomic groups, in which this target was observed as regulated. These plots were used to determine the core, well-conserved, taxon-specific and genome-specific target genes within the analyzed regulons. The details of all reconstructed TF- and riboswitch-controlled regulogs are deposited in the RegPrecise database. Sequence logos for the derived TFBS motifs were built using the Weblogo 3 package. Biological functions of genes in the reconstructed regulogs were predicted by sequence similarity search against the Swiss-Prot/UniProt database, domain architecture analysis in the Pfam database, and by using functional gene annotations from the SEED and KEGG. Autism is a clinically complex, heterogeneous, and behaviorally-defined neurodevelopmental disorder characterized by impaired social skills, communication, and repetitive behaviors. Substantial effort has been devoted in recent years to uncover the underlying mechanisms of genomic, epigenomic, proteomic, metabolic, and physiological alterations associated with the disease development. Although the role of genetic factors in autism has been extensively studied, the genetic variations are extremely heterogeneous and their phenotypic penetrance is highly variable in different individuals. In addition to genetic alterations, mounting evidence indicates a key role of other molecular and physiological abnormalities, including immune dysregulation, neuroinflammation, epigenetics, oxidative stress, and mitochondrial dysfunction ; however, the underlying molecular pathogenesis of autism remains elusive. In recent years, the cerebellum has emerged as one of the key brain regions affected in autism. This is evidenced by several well-established observations indicating the essential role of autism in the development of basic social capabilities, its involvement in extensive neural networks that govern the social, communication, repetitive/restrictive behaviors, and motor and cognitive deficits impaired in autism. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. Investigation of these mechanisms using human subjects is desirable.

We previously demonstrated that the insertion of the acyl carrier WZ4002 protein tag at the extracellular domain of TrkA makes it possible to specifically label the receptor at the cell surface when the construct is transfected in living cells. The ACP tag belongs to a family of protein and peptide tags, which can be covalently conjugated to virtually any small-probe substituted phosphopantetheinyl arm of Coenzyme A substrate by post-translational modification enzymes named PP transferases. The ACP tag was shown not to interfere with TrkA receptor function. When coupled to various fluorescent probes, this tool made it possible to monitor in living cells single TrkA movements and changes of oligomerization state upon binding of different biologically-relevant ligands including NGF and proNGF. Here we demonstrate the insertion of an 8-amino-acids tag into the sequence of NGF and of 12-amino-acids tags into the sequence of the two NGF receptors, TrkA and P75NTR. These tags derive from in vitro evolution studies committed to the shortening of the ACP and peptidyl carrier protein tags. The tags were inserted by an insertional mutagenesis method, based on a modification of the standard site-directed mutagenesis protocol, that allows their insertion in the protein of interest with no need for any additional linker sequence and thus with minimal interference with protein activity. Here we conjugate biotin to the tags by using PPTases in the presence of CoA-biotin substrates. We demonstrate that upon insertion of these tags, a site-specific biotinylation is achieved for the purified recombinant neurotrophin and for TrkA and P75NTR receptors expressed in the membrane of living cells. Moreover, the properties of orthogonal labeling displayed by the 12-amino-acid A1 and S6 tags are exploited to simultaneously label single TrkA and P75NTR receptors with two spectrally-distinct fluorophores, even when the two receptors are expressed in the same cell. We show that the insertion of such tags in the chosen sites has no measurable impact in NGF functionality or in the correct translocation of TrkA and P75NTR receptors at the cell membrane. Application of these three nanoprobes to the investigation of trafficking and interactions of NGF and its receptors in living cells will be discussed. The proNGF-A4 recombinant construct was expressed and produced in E. coli, collected from inclusion bodies and purified by FPLC ion exchange chromatography with the same procedure adopted for wt recombinant proNGF. ProNGF-A4 purified protein was then digested by trypsin and further purified by FPLC ion exchange chromatography. We were thus able to obtain the purified tagged mature neurotrophin using the same protocol adopted for the wt counterpart. Purified NGF-A4 and proNGF-A4 were incubated with CoA-biotin substrate and AcpS or SfpS PPTases. The same in vitro biotinylation reaction was performed in parallel using untagged wt NGF and wt proNGF as controls. Western blot analyses of all biotinylation reactions are reported in Fig. 2. Data show that specific biotin labeling was achieved for NGF-A4 and proNGF-A4 reacted with AcpS. In order to verify if the modified neurotrophin still maintains its biological function.

We previously demonstrated that the insertion of the acyl carrier protein tag at the extracellular domain of TrkA makes it possible to specifically label the receptor at the cell surface when the DAPT abmole construct is transfected in living cells. The ACP tag belongs to a family of protein and peptide tags, which can be covalently conjugated to virtually any small-probe substituted phosphopantetheinyl arm of Coenzyme A substrate by post-translational modification enzymes named PP transferases. The ACP tag was shown not to interfere with TrkA receptor function. When coupled to various fluorescent probes, this tool made it possible to monitor in living cells single TrkA movements and changes of oligomerization state upon binding of different biologically-relevant ligands including NGF and proNGF. Here we demonstrate the insertion of an 8-amino-acids tag into the sequence of NGF and of 12-amino-acids tags into the sequence of the two NGF receptors, TrkA and P75NTR. These tags derive from in vitro evolution studies committed to the shortening of the ACP and peptidyl carrier protein tags. The tags were inserted by an insertional mutagenesis method, based on a modification of the standard site-directed mutagenesis protocol, that allows their insertion in the protein of interest with no need for any additional linker sequence and thus with minimal interference with protein activity. Here we conjugate biotin to the tags by using PPTases in the presence of CoA-biotin substrates. We demonstrate that upon insertion of these tags, a site-specific biotinylation is achieved for the purified recombinant neurotrophin and for TrkA and P75NTR receptors expressed in the membrane of living cells. Moreover, the properties of orthogonal labeling displayed by the 12-amino-acid A1 and S6 tags are exploited to simultaneously label single TrkA and P75NTR receptors with two spectrally-distinct fluorophores, even when the two receptors are expressed in the same cell. We show that the insertion of such tags in the chosen sites has no measurable impact in NGF functionality or in the correct translocation of TrkA and P75NTR receptors at the cell membrane. Application of these three nanoprobes to the investigation of trafficking and interactions of NGF and its receptors in living cells will be discussed. The proNGF-A4 recombinant construct was expressed and produced in E. coli, collected from inclusion bodies and purified by FPLC ion exchange chromatography with the same procedure adopted for wt recombinant proNGF. ProNGF-A4 purified protein was then digested by trypsin and further purified by FPLC ion exchange chromatography. We were thus able to obtain the purified tagged mature neurotrophin using the same protocol adopted for the wt counterpart. Purified NGF-A4 and proNGF-A4 were incubated with CoA-biotin substrate and AcpS or SfpS PPTases. The same in vitro biotinylation reaction was performed in parallel using untagged wt NGF and wt proNGF as controls. Western blot analyses of all biotinylation reactions are reported in Fig. 2. Data show that specific biotin labeling was achieved for NGF-A4 and proNGF-A4 reacted with AcpS. In order to verify if the modified neurotrophin still maintains its biological function.