A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity

Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 106 variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions

Development and management of systemic lupus erythematosus in an HIV-infected man with hepatitis C and B co-infection following interferon therapy: a case report

<p>Abstract</p> <p>Introduction</p> <p>The association of human immunodeficiency virus and immune dysfunction leading to development of autoimmune markers is well described, but human immunodeficiency virus infection is relatively protective for the development of systemic lupus erythematosus. In contrast, development of systemic lupus erythematosus with hepatitis C and with interferon therapy is well described in a number of case reports. We here describe the first case of systemic lupus erythematosus developing in a man infected with human immunodeficiency virus, hepatitis C and hepatitis B co-infection where the onset seems to have been temporally related to interferon therapy.</p> <p>Case presentation</p> <p>We report the occurrence of systemic lupus erythematosus complicating interferon-α therapy for hepatitis C in a 47-year-old asplenic male with haemophilia co-infected with human immunodeficiency virus and hepatitis B. He presented with a truncal rash, abdominal pains and headache and later developed grade IV lupus nephritis requiring haemodialysis, mycophenolate mofetil and steroid therapy. We were able to successfully withdraw dialysis and mycophenolate while maintaining stable renal function.</p> <p>Conclusion</p> <p>Interferon-α is critical in antiviral immunity against hepatitis C but also acts as a pathogenic mediator for systemic lupus erythematosus, a condition associated with activation of plasmacytoid dendritic cells that are depleted in human immunodeficiency virus infection. The occurrence of auto-antibodies and lupus-like features in the coinfections with hepatitis C require careful assessment. Immunosuppressant therapy for lupus risks exacerbating underlying infections in patients with concurrent human immunodeficiency virus, hepatitis B and C.</p

Evaluating the performance of malaria genomics for inferring changes in transmission intensity using transmission modelling

AbstractAdvances in genetic sequencing and accompanying methodological approaches have resulted in pathogen genetics being used in the control of infectious diseases. To utilise these methodologies for malaria we first need to extend the methods to capture the complex interactions between parasites, human and vector hosts, and environment. Here we develop an individual-based transmission model to simulate malaria parasite genetics parameterised using estimated relationships between complexity of infection and age from 5 regions in Uganda and Kenya. We predict that cotransmission and superinfection contribute equally to within-host parasite genetic diversity at 11.5% PCR prevalence, above which superinfections dominate. Finally, we characterise the predictive power of six metrics of parasite genetics for detecting changes in transmission intensity, before grouping them in an ensemble statistical model. The best performing model successfully predicted malaria prevalence with mean absolute error of 0.055, suggesting genetic tools could be used for monitoring the impact of malaria interventions.</jats:p

Detection of concealed and buried chemicals by using multifrequency excitations

This is the published version. Copyright © 2010 American Institute of PhysicsIn this paper, we present a new type of concealed and buried chemical detection system by stimulating and enhancing spectroscopic signatures with multifrequency excitations, which includes a low frequency gradient dcelectric field, a high frequency microwave field, and higher frequency infrared (IR) radiations. Each excitation frequency plays a unique role. The microwave, which can penetrate into the underground and/or pass through the dielectric covers with low attenuation, could effectively transform its energy into the concealed and buried chemicals and increases its evaporation rate from the sample source. Subsequently, a gradient dcelectric field, generated by a Van De Graaff generator, not only serves as a vapor accelerator for efficiently expediting the transportation process of the vapor release from the concealed and buried chemicals but also acts as a vapor concentrator for increasing the chemical concentrations in the detection area, which enables the trace level chemical detection. Finally, the stimulated and enhanced vapors on the surface are detected by the IR spectroscopic fingerprints. Our theoretical and experimental results demonstrate that more than sixfold increase in detection signal can be achieved by using this proposed technology. The proposed technology can also be used for standoff detection of concealed and buried chemicals by adding the remote IR and/or thermal spectroscopic and imaging detection systems

Identification of a Novel “Almost Neutral” Mu Opioid Receptor Antagonist in CHO Cells Expressing the Cloned Human Mu Opioid Receptor

The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists, and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 hr with medium (control) or 10 μM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK-treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [35S]-GTP-γ-S assays were conducted using established methods. We screened 21 MOR “antagonists” using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6β-naltrexol, were inverse agonists. However, LTC-2 7 4 ( (-)-3-cyclopropylmethyl-2,3,4,4aα,5,6,7,7aα-octahydro-1H-benzofuro[3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [35S]-GTP-γ-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-Phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC-2-009), and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC-274 is a promising lead compound for developing a true MOR neutral antagonist