Defining synonymous codon compression schemes by genome recoding

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome

Approximate probabilistic verification of hybrid systems

Hybrid systems whose mode dynamics are governed by non-linear ordinary differential equations (ODEs) are often a natural model for biological processes. However such models are difficult to analyze. To address this, we develop a probabilistic analysis method by approximating the mode transitions as stochastic events. We assume that the probability of making a mode transition is proportional to the measure of the set of pairs of time points and value states at which the mode transition is enabled. To ensure a sound mathematical basis, we impose a natural continuity property on the non-linear ODEs. We also assume that the states of the system are observed at discrete time points but that the mode transitions may take place at any time between two successive discrete time points. This leads to a discrete time Markov chain as a probabilistic approximation of the hybrid system. We then show that for BLTL (bounded linear time temporal logic) specifications the hybrid system meets a specification iff its Markov chain approximation meets the same specification with probability $1$. Based on this, we formulate a sequential hypothesis testing procedure for verifying -approximately- that the Markov chain meets a BLTL specification with high probability. Our case studies on cardiac cell dynamics and the circadian rhythm indicate that our scheme can be applied in a number of realistic settings

Defining synonymous codon compression schemes by genome recoding

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome

On the Other Side: Manipulating the Immune Checkpoint Landscape of Dendritic Cells to Enhance Cancer Immunotherapy

Monoclonal antibodies targeting co-inhibitory immune checkpoint molecules have been successful in clinical trials of both solid and hematological malignancies as acknowledged by the 2018 Nobel Prize in Medicine, however improving clinical response rates is now key to expanding their efficacy in areas of unmet medical need. Antibodies to checkpoint inhibitors target molecules on either T cells or tumor cells to stimulate T cells or remove tumor mediated immunosuppression, respectively. However, many of the well-characterized T cell immune checkpoint receptors have their ligands on antigen presenting cells or exert direct effects on those cells. Dendritic cells are the most powerful antigen presenting cells; they possess the ability to elicit antigen-specific responses and have important roles in regulation of immune tolerance. Despite their theoretical benefits in cancer immunotherapy, the translation of DC therapies into the clinic is yet to be fully realized and combining DC-based immunotherapy with immune checkpoint inhibitors is an attractive strategy. This combination takes advantage of the antigen presenting capability of DC to maximize specific immune responses to tumor antigens whilst removing tumor-associated immune inhibitory mechanisms with immune checkpoint inhibition. Here we review the expression and functional effects of immune checkpoint molecules on DC and identify rational combinations for DC vaccination to enhance antigen-specific T cell responses, cytokine production, and promotion of long-lasting immunological memory

A genetically modified adenoviral vector with a phage display-derived peptide incorporated into fiber fibritin chimera prolongs survival in experimental glioma

The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as “GliomaFF.” We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy

Search for the decay B+→K‾∗0K∗+B^+\rightarrow\overline{K}{}^{*0}K^{*+} at Belle

We report a search for the rare charmless decay $B^+\rightarrow\overline{K}{}^{*0}K^{*+}$ using a data sample of $772\times10^6$ $B\bar{B}$ pairs collected at the $\Upsilon(4S)$ resonance with the Belle detector at the KEKB asymmetric-energy $e^+e^-$ collider. No statistically significant signal is found and a 90% confidence-level upper limit is set on the decay branching fraction as $\mathcal{B}(B^+\rightarrow\overline{K}{}^{*0}K^{*+}) <1.31\times 10^{-6}$.Comment: 8 pages, 3 figures, submitted to PRD(RC

Measurement of the CP-violation Parameter sin2ϕ1\phi_1 with a New Tagging Method at the Υ(5S)\Upsilon(5S) Resonance

We report a measurement of the CP-violation parameter sin2$\phi_1$ at the $\Upsilon(5S)$ resonance using a new tagging method, called "$B$-$\pi$ tagging." In $\Upsilon(5S)$ decays containing a neutral $B$ meson, a charged $B$, and a charged pion, the neutral $B$ is reconstructed in the $J/\psi K_S^0$ CP-eigenstate decay channel. The initial flavor of the neutral $B$ meson at the moment of the $\Upsilon(5S)$ decay is opposite to that of the charged $B$ and may thus be inferred from the charge of the pion without reconstructing the charged $B$. From the asymmetry between $B$-$\pi^+$ and $B$-$\pi^-$ tagged $J/\psi K_S^0$ yields, we determine sin2$\phi_1$ = 0.57 $\pm$ 0.58(stat) $\pm$ 0.06(syst). The results are based on 121 fb$^{-1}$ of data recorded by the Belle detector at the KEKB $e^+ e^-$ collider.Comment: 6 pages, 3 figures (submitted to PRL

Evidence for a new resonance and search for the Y(4140) in γγ→ϕJ/ψ\gamma \gamma \to \phi J/\psi

The process \gamma \gamma \to \phi \jpsi is measured for \phi \jpsi masses between threshold and 5 GeV/${\it c}^2$, using a data sample of 825 fb$^{-1}$ collected with the Belle detector. A narrow peak of $8.8^{+4.2}_{-3.2}$ events, with a significance of 3.2 standard deviations including systematic uncertainty, is observed. The mass and natural width of the structure (named X(4350)) are measured to be $(4350.6^{+4.6}_{-5.1}(\rm{stat})\pm 0.7(\rm{syst})) \hbox{MeV}/{\it c}^2$ and $(13^{+18}_{-9}(\rm{stat})\pm 4(\rm{syst})) \hbox{MeV}$, respectively. The product of its two-photon decay width and branching fraction to \phi\jpsi is $(6.7^{+3.2}_{-2.4}(\rm{stat}) \pm 1.1(\rm{syst})) \hbox{eV}$ for $J^P=0^+$, or $(1.5^{+0.7}_{-0.6}(\rm{stat}) \pm 0.3(\rm{syst})) \hbox{eV}$ for $J^P=2^+$. No signal for the Y(4140)\to \phi \jpsi structure reported by the CDF Collaboration in B\to K^+ \phi \jpsi decays is observed, and limits of \Gamma_{\gamma \gamma}(Y(4140)) \BR(Y(4140)\to\phi \jpsi)<41 \hbox{eV} for $J^P=0^+$ or $<6.0 \hbox{eV}$ for $J^P=2^+$ are determined at the 90% C.L. This disfavors the scenario in which the Y(4140) is a $D_{s}^{\ast+} {D}_{s}^{\ast-}$ molecule.Comment: 9 pages, 3 figures, publication in Phys. Rev. Lett. 104, 112004, 201

Search for Lepton Flavor and Lepton Number Violating tau Decays into a Lepton and Two Charged Mesons

We search for lepton flavor and lepton number violating tau decays into a lepton (ell = electron or muon) and two charged mesons (h,h' = pi^\pm or K^\pm), tau- -> ell-h+h'- and tau- -> ell+h-h'-, using 671 fb^{-1} of data collected with the Belle detector at the KEKB asymmetric-energy e+e- collider. We obtain 90% C.L. upper limits on the branching fractions in the range (4.4-8.8)x10^{-8} for tau -> ehh', and (3.3-16)x10^{-8} for tau -> muhh' processes. These results improve upon previously published upper limits by factors between 1.6 to 8.8.Comment: 15 page, 7 figures, submitted to Phys. Lett.

Search for leptonic decays of D0 mesons

We search for the flavor-changing neutral current decays D0\to mu+mu- and D0\to e+e-, and for the lepton-flavor violating decays D0\to e\pm mu\mp using 660 fb^-1 of data collected with the Belle detector at the KEKB asymmetric-energy e+e- collider. We find no evidence for any of these decays. We obtain significantly improved upper limits on the branching fractions: B(D0\to mu+mu-)<1.4x10-7, B(D0\to e+e-)<7.9x10-8, and B(D0\to e+mu-)+B(D0\to mu+e-)<2.6x10-7 at 90% confidence level.Comment: 6 pages, 3 figure