Model System For Characterization Research Articles

Comparative genomics of two protozoans Dictyostelium discoideum and Plasmodium falciparum reveals conserved as well as distinct regulatory pathways crucial for exploring novel therapeutic targets for Malaria

Plasmodium falciparum, which causes life-threatening cerebral malaria has rapidly gained resistance against most frontline anti-malarial drugs, thereby generating an urgent need to develop novel therapeutic approaches. Conducting in-depth investigations on Plasmodium in its native form is challenging, thereby necessitating the requirement of an efficient model system. In line, mounting evidence suggests that Dictyostelium discoideum retains both conformational and functional properties of Plasmodium proteins, however, the true potential of Dictyostelium as a host system is not fully explored. In the present study, we have exploited comparative genomics as a tool to extract, compare, and curate the extensive data available on the organism-specific databases to evaluate if D. discoideum can be established as a prime model system for functional characterization of P. falciparum genes. Through comprehensive in silico analysis, we report that despite the presence of adaptation-specific genes, the two display noteworthy conservation in the housekeeping genes, signaling pathway components, transcription regulators, and post-translational modulators. Furthermore, through orthologue analysis, the known, potential, and novel drug target genes of P. falciparum were found to be significantly conserved in D. discoideum. Our findings advocate that D. discoideum can be employed to express and functionally characterize difficult-to-express P. falciparum genes.

Zebrafish as model system for the biological characterization of CK1 inhibitors.

Introduction: The CK1 family is involved in a variety of physiological processes by regulating different signaling pathways, including the Wnt/β-catenin, the Hedgehog and the p53 signaling pathways. Mutations or dysregulation of kinases in general and of CK1 in particular are known to promote the development of cancer, neurodegenerative diseases and inflammation. There is increasing evidence that CK1 isoform specific small molecule inhibitors, including CK1δ- and CK1ε-specific inhibitors of Wnt production (IWP)-based small molecules with structural similarity to benzimidazole compounds, have promising therapeutic potential. Methods: In this study, we investigated the suitability of the zebrafish model system for the evaluation of such CK1 inhibitors. To this end, the kinetic parameters of human CK1 isoforms were compared with those of zebrafish orthologues. Furthermore, the effects of selective CK1δ inhibition during zebrafish embryonic development were analyzed in vivo. Results: The results revealed that zebrafish CK1δA and CK1δB were inhibited as effectively as human CK1δ by compounds G2-2 with IC50 values of 345 and 270 nM for CK1δA and CK1δB versus 503 nM for human CK1δ and G2-3 exhibiting IC50 values of 514 and 561 nM for zebrafish CK1δA and B, and 562 nM for human CK1δ. Furthermore, the effects of selective CK1δ inhibition on zebrafish embryonic development in vivo revealed phenotypic abnormalities indicative of downregulation of CK1δ. Treatment of zebrafish embryos with selected inhibitors resulted in marked phenotypic changes including blood stasis, heart failure, and tail malformations. Conclusion: The results suggest that the zebrafish is a suitable in vivo assay model system for initial studies of the biological relevance of CK1δ inhibition.

Structural and dynamic effects of pseudouridine modifications on noncanonical interactions in RNA.

Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs.

Keeping up with the condensates: The retention, gain, and loss of nuclear membrane-less organelles

In recent decades, a growing number of biomolecular condensates have been identified in eukaryotic cells. These structures form through phase separation and have been linked to a diverse array of cellular processes. While a checklist of established membrane-bound organelles is present across the eukaryotic domain, less is known about the conservation of membrane-less subcellular structures. Many of these structures can be seen throughout eukaryotes, while others are only thought to be present in metazoans or a limited subset of species. In particular, the nucleus is a hub of biomolecular condensates. Some of these subnuclear domains have been found in a broad range of organisms, which is a characteristic often attributed to essential functionality. However, this does not always appear to be the case. For example, the nucleolus is critical for ribosomal biogenesis and is present throughout the eukaryotic domain, while the Cajal bodies are believed to be similarly conserved, yet these structures are dispensable for organismal survival. Likewise, depletion of the Drosophila melanogaster omega speckles reduces viability, despite the apparent absence of this domain in higher eukaryotes. By reviewing primary research that has analyzed the presence of specific condensates (nucleoli, Cajal bodies, amyloid bodies, nucleolar aggresomes, nuclear speckles, nuclear paraspeckles, nuclear stress bodies, PML bodies, omega speckles, NUN bodies, mei2 dots) in a cross-section of organisms (e.g., human, mouse, D. melanogaster, Caenorhabditis elegans, yeast), we adopt a human-centric view to explore the emergence, retention, and absence of a subset of nuclear biomolecular condensates. This overview is particularly important as numerous biomolecular condensates have been linked to human disease, and their presence in additional species could unlock new and well characterized model systems for health research.

Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold

INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the Xenopus laevis oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of X. laevis NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of X. laevis Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text].

Multi-Omic Analysis to Characterize Metabolic Adaptation of the E. coli Lipidome in Response to Environmental Stress.

As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a ‘multi-omics’ strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using ‘shotgun’ direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in ‘odd-numbered’ acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during β-oxidation or lipid degradation.

Model System for Antiviral Peptide Transport Characterization

Model System for Antiviral Peptide Transport Characterization

Developing Chenopodium ficifolium as a potential B genome diploid model system for genetic characterization and improvement of allotetraploid quinoa (Chenopodium quinoa)

BackgroundQuinoa (Chenopodium quinoa) is a high-value grain known for its excellent nutritional balance. It is an allotetraploid species (AABB, 2n = 4x = 36) formed by the hybridization between AA and BB genome diploid (2n = 2x = 18) species. This study reports genetic studies in Chenopodium ficifolium as a potential B genome diploid model system to simplify the genetic studies of quinoa including gene identification and marker-assisted breeding.ResultsPortsmouth, New Hampshire and Quebec City, Quebec accessions of C. ficifolium were used to develop an F2 population segregating for agronomically relevant traits including flowering time, plant height, the number of branches, branch angle, and internode length. Marker-trait associations were identified for the FLOWERING LOCUS T-LIKE 1 (FTL1) marker gene, where the alternate alleles (A1/A2) were segregating among the F2 generation plants in association with flowering time, plant height, and the number of branches. There was a strong correlation of the flowering time trait with both plant height and the number of branches. Thus, a possible multifaceted functional role for FTL1 may be considered. The parental Portsmouth and Quebec City accessions were homozygous for the alternate FTL1 alleles, which were found to be substantially diverged. SNPs were identified in the FTL1 coding sequence that could have some functional significance in relation to the observed trait variation.ConclusionThese results draw further attention to the possible functional roles of the FTL1 locus in Chenopodium and justify continued exploration of C. ficifolium as a potential diploid model system for the genetic study of quinoa. We expect our findings to aid in quinoa breeding as well as to any studies related to the Chenopodium genus.

The Integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila.

Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. In this work, we describe a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. Our experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality.

Anchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites

Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational “anchor extension” methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.

Changes in membrane elasticity caused by the hydrophobic surfactant proteins correlate poorly with adsorption of lipid vesicles.

To establish how the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of lipids to an air/water interface, we used X-ray diffuse scattering (XDS) to determine an order parameter of the lipid chains (Sxray) and the bending modulus of the lipid bilayers (KC). Samples contained different amounts of the proteins with two sets of lipids. Dioleoylphosphatidylcholine (DOPC) provided a simple, well characterized model system. The nonpolar and phospholipids (N&PL) from extracted calf surfactant provided the biological mix of lipids. For both systems, the proteins produced changes in Sxray that correlated well with KC. The dose-response to the proteins, however, differed. Small amounts of protein generated large decreases in Sxray and KC for DOPC that progressed monotonically. The changes for the surfactant lipids were erratic. Our studies then tested whether the proteins produced correlated effects on adsorption. Experiments measured the initial fall in surface tension during adsorption to a constant surface area, and then expansion of the interface during adsorption at a constant surface tension of 40 mN m-1. The proteins produced a sigmoidal increase in the rate of adsorption at 40 mN m-1 for both lipids. The results correlated poorly with the changes in Sxray and KC in both cases. Disordering of the lipid chains produced by the proteins, and the softening of the bilayers, fail to explain how the proteins promote adsorption of lipid vesicles.

Elevated Expression of Mir-130a in t(8,21) AML Reinforces the Aberrant Molecular Program of AML1-ETO

Background: Deregulation of self-renewal and differentiation programs are central to the pathogenesis of hematologic malignancies. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs and are frequently deregulated in acute myeloid leukemia (AML). From our previous in vivo miRNA enforced expression screen in human hematopoietic stem and progenitor cells (HSPC), we identified miR-130a as a regulator of self-renewal and lineage specification. Enforced expression of miR-130a in human cord blood (CB) derived HSPC caused an expansion of HSC, block in erythroid differentiation and abnormal myelopoiesis in xenografts. Thus, we examined miR-130a expression in AML and found miR-130a to be specifically upregulated in t(8,21) AML. The translocation t(8,21) is one of the most common karyotypic abnormalities in AML, accounting up to 10% of all AML cases. The consequence of this translocation is a fusion of AML1 and ETO genes, resulting in a formation of the AML1-ETO (AE) oncofusion protein, which acts as a dominant repressor of the wild type AML1/RUNX1. The ETO moiety mediates the recruitment of the nuclear corepressor (NCoR) and histone deacetylases (HDAC1-3) to block RUNX1 target gene expression. This prevents myeloid maturation, apoptosis and promotes leukemogenesis. Here we investigated the molecular mechanism of miR-130a in t(8,21) AML and how it contributes to the leukemogenesis of this AML subtype. Results: Using the TCGA dataset and our PMCC patient cohort, we identified miR-130a to be upregulated in t(8,21) AML and high miR-130a expression was associated with worse patient overall survival. To interrogate the functional significance of elevated miR-130a in t(8,21) AML, we performed knock-down (KD) experiments in the Kasumi-1 cell line, which represents a well characterized model system for t(8,21) AML. Notably, KD of miR-130a induced a significant reduction in the CD34+ cell population and an increase in differentiated CD11b+CD15+ and pro-apoptotic annexin V+ cells. We next examined the impact of miR-130a KD in CD34+ blasts from primary t(8,21) AML patient samples. In line with our findings in the Kasumi-1 cells, miR-130a KD decreased the proportion of CD34+ cells and increased the proportion of differentiated CD11b+CD15+ blasts. To investigate the effect of miR-130a KD on leukemic engraftment in vivo, we transduced CD34+ blasts from 2 patient samples and transplanted them into NSG-GF mice. miR-130a KD decreased leukemic engraftment and the proportion of transduced cells, corroborating the functional significance of high miR-130a expression in t(8,21) AML. To investigate the mechanistic action of miR-130a, we performed label-free semi-quantitative proteomics in human CB derived HSPC to uncover miR-130a targets. Surprisingly, we found the beta subunit of RUNX1, CBFb, and Transducin Beta Like 1 X-Linked Receptor 1, TBL1XR1, to be among the most repressed targets. TBL1XR1 is a component of the nuclear receptor corepressor (NCoR) complex and is involved in NCoR degradation. Thus, we performed western and immunoprecipitations (IP) assays in Flag-AE Kasumi-1 cells following miR-130a KD to examine changes in the expression of proteins associated with the AE complex. We observed increased expression of CBFβ, TBL1XR1 and CEBPA with miR-130a KD. Notably, miR-130a KD resulted in a dramatic decrease of NCoR protein levels. IP of Flag-AE showed decreased association of CBFβ and NCoR with AE, despite unaltered protein levels of AE. To investigate changes in binding occupancy of Flag-AE after miR-130a KD, we performed Cleavage Under the Targets and Release Using Nuclease (CUT&RUN) assay. Surprisingly, we observed 2-fold gain of AE sites in miR-130a KD sample compared to control. De novo motif enrichment analysis showed loss of motives for ETS and HOX transcription factors known to associate with AE following miR-130a KD. Genomic distribution of the peaks revealed a dramatic shift of AE peaks away from the promoter region to introns in miR-130a KD. Pathway enrichment analysis of the unique peaks gained in miR-130a KD showed stress responses and organelle disassembly, in line with the differentiation phenotype observed with miR-130a KD. Collectively, we uncovered a novel mechanism by which miR-130a reinforces the aberrant AE molecular program by controlling the composition and binding of the AE complex. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Optimization of regeneration and Agrobacterium tumefaciens-mediated transient transformation systems for Australian native extremophile, Tripogon loliiformis

Tripogon loliiformis, an Australian native resurrection grass having an abundant gene pool for combating desiccation, can be the putative model system for functional characterization of stress tolerance genes due to its diploid genome and being a monocotyledonous plant and member of the grass family (Poaceae), like many important cereal crops. For developing callus mediated regeneration from mature grains of Tripogon, Murashige and Skoog medium containing growth regulator, 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0mgL−1 was the optimum concentration for induction and proliferation of healthy cream calli. Successful regeneration of shoots from callus clumps in MS medium supplemented with 2.0mgL−1 6-benzylaminopurine (BAP) and 0.5mgL−1 α-naphthalene acetic acid (NAA) was obtained from 2 consecutive rounds subculturing of the calli at 3 weeks interval. In addition, rooting needed another 2 rounds within the same media with 2.0mgL−1 BAP but with 0.25mgL−1 NAA. The transient expression of UidA gene at 3 days after Tripogon callus transformation, performed with Agrobacterium tumefaciens strains AGL1 and LBA4404 following rice and Brachypodium distachyon transformation protocols, indicates successful Agrobacterium infection and gene delivery in calli. A stable transformation system for Tripogon loliiformis can be developed near future following the protocols in this study.

Association of Branched Dextrin from Nägeli Amylodextrin in Water for Screening of Additives Affecting Starch Gel Properties

AbstractA novel model system for characterization of double helix in starch is developed using branched dextrin (BD) purified from Nägeli amylodextrin by repeated precipitation in 10% ethanol. The solution of one‐time precipitated BD (BD1, 3% in water) starts to develop turbidity in 8 h of incubation at 4 °C. Volume particle size distribution during the association of four‐time precipitated BD sample (BD4) is analyzed using dynamic light scattering. The mean diameter of the particles jumps from 3.8 to 170 nm between 2 and 2.5 h of incubation, suggesting a prerequisite lag time for maturation of intermolecular association of BD4. Flakes of BD4 in water exhibit a B‐type crystal by X‐ray diffraction analysis. A screening system of additives affecting turbidity development of BD1 in water is applied for 82 compounds, and sodium tetraborate (borax) significantly retards the development. Borax affects the rate of turbidity development and maximum turbidity at the plateau stage in a concentration‐dependent manner. Borax is also added to rice starch gel, which reduces the increase of hardness of the gel during retrogradation. This novel assay system with BD can be used for screening and evaluation of additives affecting starch gelation and retrogradation.

Similarities between Initiation Mechanism between Cotton Fiber and Arabidopsis Trichome: Prospects in Improving Cotton Fiber Yield

Cotton fiber is the fundamental material for a textile industry, and currently there is an immense interest in understanding the process of fiber initiation and development. Cotton fiber, also known as seed trichome, is differentiated from the seed coat epidermal cells similar to Arabidopsis leaf trichome, which is differentiated from the leaf epidermal cells. Despite functional characterization of individual cotton fiber initiation genes, currently there is not a comprehensive understanding of the mechanism behind cotton fiber initiation. Since the resemblance in initiation to cotton fiber, the Arabidopsis trichome has been successfully employed as a model system for functional characterization of cotton fiber initiation genes. Knowledge gained from the initiation mechanism of Arabidopsis trichomes will facilitate, as a comparative model system in understanding of the cotton fiber initiation mechanisms.

CHARACTERIZATION OF THERAPY-INSENSITIVE LSC SUBPOPULATIONS IN CHRONIC MYELOID LEUKEMIA

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a sub population is believed to be functional, TKI-insensitive LSCs. Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent LSC-subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017). Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). By comparing LSCs from patients with different therapeutic outcome we describe how LSC heterogeneity at diagnosis correlate with long-term therapy response. We furthermore evaluate the potential for humanized ossicle-niche xenotransplantation (Reinisch et al., 2016) as a model system for functional characterization of LSC subpopulations in vivo.

A Yeast Suppressor Screen Used To Identify Mammalian SIRT1 as a Proviral Factor for Middle East Respiratory Syndrome Coronavirus Replication.

Viral proteins must intimately interact with the host cell machinery during virus replication. Here, we used the yeast Saccharomyces cerevisiae as a system to identify novel functional interactions between viral proteins and eukaryotic cells. Our work demonstrates that when the Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a accessory gene is expressed in yeast it causes a slow-growth phenotype. ORF4a has been characterized as an interferon antagonist in mammalian cells, and yet yeast lack an interferon system, suggesting further interactions between ORF4a and eukaryotic cells. Using the slow-growth phenotype as a reporter of ORF4a function, we utilized the yeast knockout library collection to perform a suppressor screen where we identified the YDL042C/SIR2 yeast gene as a suppressor of ORF4a function. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We found that when SIRT1 was inhibited by either chemical or genetic manipulation, there was reduced MERS-CoV replication, suggesting that SIRT1 is a proviral factor for MERS-CoV. Moreover, ORF4a inhibited SIRT1-mediated modulation of NF-κB signaling, demonstrating a functional link between ORF4a and SIRT1 in mammalian cells. Overall, the data presented here demonstrate the utility of yeast studies for identifying genetic interactions between viral proteins and eukaryotic cells. We also demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in cells.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) initially emerged in 2012 and has since been responsible for over 2,300 infections, with a case fatality ratio of approximately 35%. We have used the highly characterized model system of Saccharomyces cerevisiae to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. We identify a functional link between the MERS-CoV ORF4a proteins and the YDL042C/SIR2 yeast gene. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in mammalian cells.

Structural Characterization of Rosetta Designed Amino Acyl‐tRNA Synthetase Active Sites for Genetic Code Expansion

The site‐specific incorporation of non‐standard and novel amino acid structures enabled by Genetic Code Expansion (GCE) provides access to new avenues of protein research allowed by engineering diverse functionality into proteins. At its core, GCE requires the engineering of an orthogonal amino acyl‐tRNA synthetase/tRNA cognate pair that can function with high catalytic efficiency for a non‐canonical amino acid (ncAA). While the GCE community has generated many new amino acyl‐tRNA synthetase/tRNA pairs, these pairs have kinetics that are orders of magnitude worse than the natural translational components. A major challenge for the GCE field is identifying which amino acyl‐tRNA synthetase residues to target for engineering improved amino acyl‐tRNA synthetase/tRNA pairs.Here we use Rosetta modeling to identify mutations in the amino acyl‐tRNA synthetase active site that will improve the efficiency of the nitroTyr amino acyl‐tRNA synthetase/tRNA pairs. Rosetta guided mutations were successful in generating improved GCE function in vivo.Crystal structures were obtained for the Rosetta improved amino acyl‐tRNA synthetases to understand the structural basis for the enhanced activity as part of a comprehensive kinetic and structural analysis of these improved synthetases. Here, we used the 3‐nitro‐tyrosine amino acyl‐tRNA synthetase/tRNA pair as a model system for synthetase improvement and structural characterization.We observe that the Rosetta‐improved synthetases allow a more planar 3‐nitro‐tyrosine (nitroTyr) amino acid upon binding, which is predicted to contribute to their increased activity. This Rosetta guided approach provides a third generation of more efficient nitroTyr amino acyl‐tRNA synthetase/tRNA pairs that will accelerate the study of this oxidative stress post‐translational modification and its role in human disease.We demonstrate that Rosetta successfully identified key residues for amino acyl‐tRNA synthetase/tRNA pair optimization. This strategy can be utilized to systematically improve the efficiency of all ncAA amino acyl‐tRNA synthetase/tRNA pairs, resulting in more robust GCE technologies and facilitating new applications.Support or Funding InformationNIH Grant #R01‐GM114653‐01, Oregon State University Summer Undergraduate Research Experience (SURE) in Science ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3971: Combination of neratinib and enzalutamide is an effective treatment for HER2-positive breast cancer metastasis to the brain

Abstract An estimated 10% to 30% of all breast cancer patients eventually develop brain metastases. Up to half of the patients with epidermal growth factor receptor 2 (HER2)-positive breast cancer will develop brain metastasis. Treatment options for patients with breast cancer brain metastases are limited to surgical resection and radiotherapy with dismal median survivals of 2 to 24 months. Efforts to identify clinically relevant targeted therapeutics have been limited by the lack of molecularly characterized model systems. We have developed a patient derived xenograft (PDX) model of a HER2-positive breast cancer brain metastasis and have genomically characterized the PDX and its -matched tumor. Whole exome and RNA sequencing data revealed high fidelity of the PDX tumor with its corresponding patient-matched tumor and confirmed retention of driver gene alterations such as the amplification and overexpression of HER2. Additionally, immunohistochemistry of the patient tumor revealed overexpression of the Androgen receptor (AR) (IHC Score 2+), which was also confirmed in the PDX. Given the importance of both HER2 and AR in aggressive tumor behavior, we developed a preclinical study to inhibit both targets. Neratinib and enzalutamide were selected because of their known blood brain barrier penetration properties. As proof of concept we first tested the combination strategy in the flank of NOG mice (n = 10 animals/group). Tumors were allowed to grow to ~250 mm3 in size and animals were subsequently treated with either vehicle control, neratinib (40 mg/Kg) alone, enzalutamide (30 mg/Kg) alone, or combination for 2 weeks. Neratinib alone and thecombination with enzalutamide demonstrated exceptional efficacy in suppressing tumor growth as compared to either the vehicle control or Enzalutamide alone (5 fold reduction in tumor size, p < 0.001). Interestingly, when the treatments were stopped, tumors treated with Neratinib alone grew significantly faster compared to tumor growth with the combination treatment (p < 0.05), suggesting more effective and sustained tumor suppression by the addition of Enzalutamide. Studies in an orthotopic setting and molecular profiling of treated and non-treated tumors are ongoing. In summary, our results demonstrated that Neratinib in combination with Enzalutamide could potentially be developed for patients with HER2 and AR-positive breast cancer metastasis to the brain and warrants further preclinical testing. Citation Format: Harshil D. Dhruv, Lauren Hartman, Chris Gooden, George Reid, Christophe Legendre, George Snipes, Cynthia Osborne, Joyce O'Shaughnessy, Bodour Salhia. Combination of neratinib and enzalutamide is an effective treatment for HER2-positive breast cancer metastasis to the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3971.

Threshold Fluence Measurement for Laser Liftoff of InP Thin Films by Selective Absorption

We explore conditions for achieving laser liftoff in epitaxially grown heterojunctions, in which single crystal thin films can be separated from their growth substrates using a selectively absorbing buried intermediate layer. Because this highly non‐linear process is subject to a variety of process instabilities, it is essential to accurately characterize the parameters resulting in liftoff. Here, we present an InP/InGaAs/InP heterojunction as a model system for such characterization. We show separation of InP thin films from single crystal InP growth substrates, wherein a ≈10 ns, Nd:YAG laser pulse selectively heats a coherently strained, buried InGaAs layer. We develop a technique to measure liftoff threshold fluences within an inhomogeneous laser spatial profile, and apply this technique to determine threshold fluences of the order 0.5 J cm−2 for our specimens. We find that the fluence at the InGaAs layer is limited by non‐linear absorption and InP surface damage at high powers, and measure the energy transmission in an InP substrate from 0 to 8 J cm−2. Characterization of the ejected thin films shows crack‐free, single crystal InP. Finally, we present evidence that the hot InGaAs initiates a liquid phase front that travels into the InP substrate during liftoff.