Single-cell analysis tools for drug discovery and development

The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed

Dual regulation of a chimeric plant serine/threonine kinase by calcium and calcium/calmodulin

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca2+-binding domain was recently cloned from plants (Patil, S., Takezawa, D., and Poovaiah, B. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4797-4801). The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca2+/calmodulin-dependent manner. The calmodulin-binding region of CCaMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of 32P/mol) that is stimulated 3.4-fold by Ca2+ (0.339 mol of 32P/mol), while calmodulin inhibits Ca2+-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca2+-stimulated autophosphorylation activity but retained Ca2+/calmodulin-dependent protein kinase activity at a reduced level. Ca2+-dependent mobility shift assays using E. coli-expressed protein from residues 358 520 revealed that Ca2+ binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca2+-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca2+/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its Ca2+-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca2+ and Ca2+/calmodulin. The presence of a visinin-like Ca2+-binding domain in CCaMK adds an additional Ca2+-sensing mechanism not previously known to exist in the Ca2+/calmodulin-mediated signaling cascade in plants

Induction heating in domestic cooking and industrial melting applications: A systematic review on modelling, converter topologies and control schemes

In the current scenario, power electronic device-based induction heating (IH) technologies are widely employed in domestic cooking, industrial melting and medical applications. These IH applications are designed using different converter topologies, modulation and control techniques. This review article mainly focuses on the modelling of half-bridge series resonant inverter, electrical and thermal model of IH load. This review also analyses the performance of the converter topologies based on the power conversion stages, switching frequency, power rating, power density, control range, modulation techniques, load handling capacity and efficiency. Moreover, this paper provides insight into the future of IH application, with respect to the adaptation of wide band-gap power semiconductor materials, multi-output topologies, variable-frequency control schemes with mini-mum losses and filters designed to improve source-side power factor. With the identified research gap in the literature, an attempt has also been made to develop a new hybrid modulation technique, to achieve a wide range of power control with high efficiency. A 100 W full-bridge inverter prototype is realised both in simulation and hardware, with various modulation schemes using a PIC16F877A microcontroller. The results are compared with existing techniques and the comparisons reveal that the proposed scheme is highly viable and effective for the rendered applications

Recombinant TLR5 Agonist CBLB502 Promotes NK Cell-Mediated Anti-CMV Immunity in Mice

<div><p>Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.</p></div

rFlagellin increased NK cell lytic activity in the presence and absence of mCMV infection.

<p>Splenocytes were harvested on days 0 and 3 after mCMV infection and NK cell lytic activity was measured using standard 4-hour ⁵¹Cr-release assay by Yac-1 target cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096165#s2" target="_blank">Materials and Methods</a>. A and B. The % cell lytic activity of NK cells of rflagellin- and PBS-treated WT B6 mice on days 0 and 3 after infection. C and D. The % cell lytic activity by NK cells harvested from rflagellin- and PBS-treated TLR5 KO B6 mice on day 0 and 3 post infection. E. Kinetics of NK cells lytic activity from the splenocytes harvested from rflagellin and PBS-treated WT B6 mice on day 0, 1, 2, 3 and 8 after mCMV infection. The data shown in A–D are representative of three independent experiments and data shown in E are from one experiment. 5 mice were used per group per time point. The “*” indicates p value<0.05, Student's T-test.</p

The effect of rflagellin treatment on cytokine production in the presence and absence of mCMV infection.

<p>Splenocytes harvested from rflagellin- and PBS-treated control mice on day 0, 1, 2, 3 and 8 after mCMV infection were stimulated with PMA ionomycin for 4 hours at 37°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096165#s2" target="_blank">Materials and Methods</a>. Cells were stained for intracellular expression of IFN-<b>γ</b> and granzyme B along with NK cell surface markers. A and B represent the numbers of IFN-<b>γ</b> producing NK cells per spleen in the absence and presence of PMA-ionomycin stimulation. C and D represent the numbers of granzyme B producing NK cells per spleen in the absence and presence of PMA ionomycin stimulation. E and F represent the numbers of IFN-<b>γ</b> and granzyme B producing NK cells per spleen in the absence and presence of PMA ionomycin stimulation, harvested on day 0 after mCMV infection. G. Serum IFN-α on day 0, 2 and 3 after mCMV infection determined by Luminex assay. H and I represent serum cytokines determined on day 0 and 3 after mCMV infection, determined by Luminex assay. 5 mice were used per group in each time point. The “*” and “**” represent p values<0.05 and <0.005, respectively, Student's T-test.</p

rFlagellin treatment enhanced maturation and increased expression of Ly49H on NK cells after mCMV infection.

<p>Harvested splenocytes on day 3 after mCMV infection from rflagellin- and PBS-treated control mice were stained with mAbs to NK1.1, CD27, CD11b along with Ly49H as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096165#s2" target="_blank">Materials and Methods</a>. A. FACS plots of % CD3-NK1.1+ NK cells of lymphocyte-gated populations. B. % Ly49H expressed by NK cells. C. CD11b−CD27−(DN), CD11b−CD27+, CD11b+CD27+ (DP) and CD11b+CD27− NK cell populations. D. Ly49H+ NK cells of 4 subsets gated populations described in C. E. The total numbers of Ly49H+ NK cells and all 4 subsets of NK cells (as described in D) per spleen expressed Ly49H on day 3 after mCMV infection. The “*” represents p value<0.05, Student's T-test. 5 mice were used per group.</p

Prophylactic rflagellin administration reduced liver mCMV load in WT B6 mice.

<p>WT B6 and TLR5 KO B6 mice were given 25 µg rflagellin or 0.2 ml PBS i.p 48 hours before a sub-lethal dose (1×10⁵ pfu/mouse i.p) of mCMV. Mice were sacrificed on day 3 and 10 after mCMV infection and viral load per liver was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096165#s2" target="_blank">Materials and Methods</a>. A. Virus titer in livers of rflagellin- and PBS-treated WT B6 mice. B. Virus titer in livers of rflagellin- and PBS-treated TLR5 KO B6 mice. The data are the representative of three similar experiments using 5 mice per group at each time point. C. WT B6 mice were given 25 µg rflagellin/mouse (rFlagellin 48 Hrs, closed circle) or 0.2 ml PBS (open circle) i.p 48 hours before or at the same time as (rFlagellin 0 Hr, closed triangle) a sub-lethal mCMV infection (1×10⁵ pfu/mouse i.p). Weights of individual mice were measured on day 0, 1, 2, 3, 4 and 8 days after mCMV infection. Percent weight changes per group of experimental mice are presented. D. Mice receiving rflagellin (rFlagellin 0 Hr) during mCMV infection became dehydrated and hunched and were sacrificed on day 3 after mCMV infection and liver viral load was determined. The data are the representative of two similar experiments using 5 mice per group. The symbols “*” and “**”represent the <i>p</i> values<0.05 and <0.005, respectively, Students <i>t</i>-Test.</p