Design and fabrication of chemically robust three-dimensional microfluidic valves

A current problem in microfluidics is that poly(dimethylsiloxane) (PDMS), used to fabricate many microfluidic devices, is not compatible with most organic solvents. Fluorinated compounds are more chemically robust than PDMS but, historically, it has been nearly impossible to construct valves out of them by multilayer soft lithography (MSL) due to the difficulty of bonding layers made of non-stick fluoropolymers necessary to create traditional microfluidic valves. With our new three-dimensional (3D) valve design we can fabricate microfluidic devices from fluorinated compounds in a single monolithic layer that is resistant to most organic solvents with minimal swelling. This paper describes the design and development of 3D microfluidic valves by molding of a perfluoropolyether, termed Sifel, onto printed wax molds. The fabrication of Sifel-based microfluidic devices using this technique has great potential in chemical synthesis and analysis

Near infra-red photoimmunotherapy with anti-CEA-IR700 results in extensive tumor lysis and a significant decrease in tumor burden in orthotopic mouse models of pancreatic cancer.

Photoimmunotherapy (PIT) of cancer utilizes tumor-specific monoclonal antibodies conjugated to a photosensitizer phthalocyanine dye IR700 which becomes cytotoxic upon irradiation with near infrared light. In this study, we aimed to evaluate the efficacy of PIT on human pancreatic cancer cells in vitro and in vivo in an orthotopic nude mouse model. The binding capacity of anti-CEA antibody to BxPC-3 human pancreatic cancer cells was determined by FACS analysis. An in vitro cytotoxicity assay was used to determine cell death following treatment with PIT. For in vivo determination of PIT efficacy, nude mice were orthotopically implanted with BxPC-3 pancreatic tumors expressing green fluorescent protein (GFP). After tumor engraftment, the mice were divided into two groups: (1) treatment with anti-CEA-IR700 + 690 nm laser and (2) treatment with 690 nm laser only. Anti-CEA-IR700 (100 μg) was administered to group (1) via tail vein injection 24 hours prior to therapy. Tumors were then surgically exposed and treated with phototherapy at an intensity of 150 mW/cm2 for 30 minutes. Whole body imaging was done subsequently for 5 weeks using an OV-100 small animal imaging system. Anti-CEA-IR700 antibody bound to the BxPC3 cells to a high degree as shown by FACS analysis. Anti-CEA-IR700 caused extensive cancer cell killing after light activation compared to control cells in cytotoxicity assays. In the orthotopic models of pancreatic cancer, the anti-CEA-IR700 group had significantly smaller tumors than the control after 5 weeks (p<0.001). There was no significant difference in the body weights of mice in the anti-CEA-IR700 and control groups indicating that PIT was well tolerated by the mice

Site-specific modification of Shigella flexneri virF mRNA by tRNA-guanine transglycosylase in vitro

Shigella flexneri is an enteropathogen responsible for severe dysentery in humans. VirF is a key transcriptional regulator that activates the expression of the downstream virulence factors required for cellular invasion and cell-to-cell spread of this pathogen. There are several environmental factors that induce the translation of VirF including temperature, pH, osmolarity and post-transcriptional RNA modification. Durand and colleagues (vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase of Escherichia coli K-12. J. Bacteriol., 176, 4627–4634) have demonstrated a correlation between VirF and tRNA-guanine transglycosylase (TGT), which catalyzes the exchange of the hypermodified base queuine for the guanine in the wobble position of certain tRNAs. They characterized tgt- mutant S. flexneri strains in which the translation of VirF is markedly reduced and the bacteria are unable to invade host cells. Although the function of TGT is to modify tRNA, we report that the virF mRNA is recognized by the Escherichia coli TGT (99% identity to the S. flexneri TGT) in vitro. Further, we show that this recognition results in the site-specific modification of a single base in the virF mRNA. In the context of previous reports that small molecule binding motifs (‘riboswitches’) in mRNAs modulate mRNA conformation and translation, our observations suggest that TGT may modulate the translation of VirF by base modification of the VirF encoding mRNA

Bayesian online learning for energy-aware resource orchestration in virtualized RANs

Proceedings of: IEEE International Conference on Computer Communications, 10-13 May 2021, Vancouver, BC, Canada.Radio Access Network Virtualization (vRAN) will spearhead the quest towards supple radio stacks that adapt to heterogeneous infrastructure: from energy-constrained platforms deploying cells-on-wheels (e.g., drones) or battery-powered cells to green edge clouds. We perform an in-depth experimental analysis of the energy consumption of virtualized Base Stations (vBSs) and render two conclusions: (i) characterizing performance and power consumption is intricate as it depends on human behavior such as network load or user mobility; and (ii) there are many control policies and some of them have non-linear and monotonic relations with power and throughput. Driven by our experimental insights, we argue that machine learning holds the key for vBS control. We formulate two problems and two algorithms: (i) BP-vRAN, which uses Bayesian online learning to balance performance and energy consumption, and (ii) SBP-vRAN, which augments our Bayesian optimization approach with safe controls that maximize performance while respecting hard power constraints. We show that our approaches are data-efficient and have provably performance, which is paramount for carrier-grade vRANs. We demonstrate the convergence and flexibility of our approach and assess its performance using an experimental prototype.This work was supported by the European Commission through Grant No. 856709 (5Growth) and Grant No. 101017109 (DAEMON); and by SFI through Grant No. SFI 17/CDA/4760

Structural basis for rifamycin resistance of bacterial RNA polymerase by the three most clinically important RpoB mutations found in Mycobacterium tuberculosis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136512/1/mmi13606.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136512/2/mmi13606_am.pd

tRNA-guanine transglycosylase from Escherichia coli: Recognition of dimeric, unmodified tRNATyr

In order to probe the interaction between tRNA and the tRNA hypermodifying enzyme, tRNA-guanine transglycosylase (TGT) from Escherichia coli, we have undertaken the generation of E coli tRNATyr and analogues. During efforts to adapt currently available in vitro transcription techniques we encountered difficulties attributable to dimerization of the tRNA products. E coli tRNATyr has previously been characterized for its ability to form a dimer in solutions of suitable salt concentrations at appropriate temperatures (Yang SK, Soll DG, Crothers DM (1972) Biochemistry 11, 2311-2320; Rordorff BF, Kearns DR (1976) Biochemistry 15, 3320-3330). We have applied similar techniques to our unmodified analogue of E coli tRNATyr and produced both monomeric and dimeric forms of E coli tRNATyr. In this report we find that the dimer does serve as a substrate for modification by TGT. While both the conformers are equal in terms of Vmax (within experimental error) a 2.5-fold increase in KM occurs when going from monomer to dimer. This suggests that TGT prefcrentially binds the monomer but once either conformer is bound will catalyze the modification reaction equally well. We have also compared the results for the two conformers to our previous data of an RNA minihelix corresponding to the anticodon arm of E coli tRNATyr. Here we find that our earlier conclusion, that the recognition elements for TGT are localized within the anticodon arm of cognate tRNAs, is supported.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31951/1/0000904.pd

Cysteine 265 Is in the Active Site of, But Is Not Essential for Catalysis by tRNA-Guanine Transglycosylase (TGT) from Escherichia coli

Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry 34, 3694–3701; Garcia et al. (1966), Biochemistry 35, 3133–3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents ( e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850–2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45082/1/10930_2004_Article_425322.pd

Activation of the JAK-STAT pathway by olanzapine is necessary for desensitization of serotonin2A receptor-stimulated phospholipase C signaling in rat frontal cortex but not serotonin2A receptor-stimulated hormone release

Chronic treatment with olanzapine causes desensitization of serotonin2A receptor signaling. The purpose of the current study is to further understand the mechanisms underlying this desensitization response of serotonin2A receptor signaling in vivo. We now report that desensitization of serotonin2A receptor stimulated-phospholipase C activity in rat frontal cortex induced by olanzapine is dependent on activation of the JAK-STAT pathway. Olanzapine treatment for 7 days significantly increased the levels of the regulator of G protein signaling (RGS7) protein, RGS7 mRNA levels, and activation of JAK2 in rat frontal cortex. Pretreatment with a JAK2 inhibitor AG490, significantly attenuated the olanzapine-induced reductions in serotonin2A receptor-stimulated phospholipase C activity and prevented the olanzapine-induced increases in RGS7 mRNA and protein levels. In contrast, inhibition of the JAK-STAT pathway with AG490 did not reverse the olanzapine-induced desensitization of the serotonin2A receptor pathway in the hypothalamic paraventricular nucleus mediating increases in plasma hormone levels. AG490 dose-dependently inhibited serotonin2A receptor-stimulated oxytocin and corticosterone release. Taken together, these results suggest that the olanzapine-induced increase in RGS7 expression is mediated by activation of JAK-STAT and is necessary for olanzapine-induced desensitization of serotonin2A receptor-stimulated phospholipase C activity in the frontal cortex but not serotonin2A receptor-stimulated hormone release

Modeling the Low State Spectrum of the X-Ray Nova XTE J1118+480

Based on recent multiwavelength observations of the new X-ray nova XTE J1118+480, we can place strong constraints on the geometry of the accretion flow in which a low/hard state spectrum, characteristic of an accreting black hole binary, is produced. We argue that the absence of any soft blackbody-like component in the X-ray band implies the existence of an extended hot optically-thin region, with the optically-thick cool disk truncated at some radius R_{tr} > 55 R_{Schw}. We show that such a model can indeed reproduce the main features of the observed spectrum: the relatively high optical to X-ray ratio, the sharp downturn in the far UV band and the hard X-ray spectrum. The absence of the disk blackbody component also underscores the requirement that the seed photons for thermal Comptonization be produced locally in the hot flow, e.g. via synchrotron radiation. We attribute the observed spectral break at 2 keV to absorption in a warm, partially ionized gas.Comment: 6 pages, including 1 figure; LaTeX (emulateapj5.sty), to appear in Ap