scholarly journals 602. Mechanism of LiaY-Mediated Daptomycin Resistance in Enterococcus faecalis

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S282-S282
Author(s):  
April Nguyen ◽  
Truc T Tran ◽  
Diana Panesso ◽  
Ayesha Khan ◽  
Eugenia Mileykovskaya ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Transmembrane Protein ◽  
Logarithmic Phase ◽  
Positive Interaction ◽  
Anionic Phospholipid ◽  
Biochemical Basis ◽  
Double Deletion ◽  
Genes Encoding ◽  
Lipopeptide Antibiotic ◽  
Two Hybrid

Abstract Background Daptomycin (DAP) is a lipopeptide antibiotic that targets the cell membrane (CM) at the division septum. DAP resistance (DAP-R) in E. faecalis (Efs) has been linked to mutations in genes encoding the LiaFSR stress response system and lipid biosynthetic enzymes, including cardiolipin synthase (Cls). The signature phenotype of DAP-R is redistribution of CM anionic phospholipid (APL) microdomains. Using a genetic approach, we have identified a transmembrane protein (LiaY) as a major mediator of cell membrane APL redistribution associated with DAP-R. Here, we explore the mechanism of LiaY-mediated changes in the CM under the hypothesis that CM remodeling occurs through interactions with Cls. Methods Efs encodes two cls genes (cls1 and cls2). Deletion mutants of both cls genes were generated using the Crispr/cas9 system in the daptomycin-sensitive strain Efs OG117 and Efs OG117∆liaX (a DAP-R derivative of OG117). DAP minimum inhibitory concentration (MIC) was determined using E-test on Mueller–Hinton II agar. Visualization of APL microdomains was performed by staining mid-logarithmic phase cells with 1 µM of 10-N-nonyl-acridine orange (NAO) and fluorescence microscopy. Bacterial two-hybrid system was used to study interactions between LiaY with Cls1 or Cls2. Results Single or double deletion of cls1 or cls2 in Efs OG117 did not affect DAP MIC, and no changes in CM architecture were seen by NAO staining. In contrast,deletion of cls1 (alone or in conjunction with a deletion of cls2) in a DAP-R derivative of OG117 OG117∆liaX, resulted in a marked decrease in DAP MIC, and NAO staining of Efs OG117∆liaX∆cls1∆cls2 shows a restoration of septal APL microdomain localization.In the same DAP-R background, deletion of cls2 alone did not have any effect on DAP MIC or APL microdomain distribution. Additionally, bacterial two-hybrid assays showed a positive interaction of LiaY with Cls1 but not with Cls2. Conclusion We have identified the biochemical basis for DAP-R associated CM remodeling. In a proposed model, the LiaR-mediated activation of the LiaY triggers specific interactions with Cls1 displacing the protein away from the septum, resulting in local generation of APL microdomains that prevents DAP-mediated damage to the CM. Disclosures All authors: No reported disclosures.

scholarly journals 599. LiaF is an Activator of the LiaR-Mediated Response Against Daptomycin and Antimicrobial Peptides in Multidrug-Resistant Enterococcus faecalis (Efs)

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S281-S281
Author(s):  
Laura C Ortiz-Velez ◽  
Sandra L Rincon ◽  
Jesse Degani ◽  
Yousif Shamoo ◽  
Truc T Tran ◽  
...  
Keyword(s):  
Stress Response ◽  
Stop Codon ◽  
Response Regulator ◽  
Transmembrane Protein ◽  
Premature Stop Codon ◽  
Regulatory System ◽  
Laboratory Strain ◽  
Positive Interaction ◽  
Anionic Phospholipid ◽  
Membrane Architecture

Abstract Background Daptomycin (DAP) is a key first-line agent for the treatment of vancomycin-resistant enterococcal infections. Resistance to DAP in enterococci is regulated by the liaFSR three-component regulatory system that consists of a histidine kinase sensor (LiaS), a response regulator (LiaR) and a transmembrane protein of unknown function (LiaF). Previous studies indicate that deletion of isoleucine in position 177 of LiaF results in DAP tolerance and is sufficient to change membrane architecture. Here, we dissect the role of LiaF in DAP resistance Methods We generated three liaF mutants in OG1RF, a DAP-susceptible laboratory strain of Efs (DAP MIC = 2 µg/mL): (i) a non-polar, C-terminal truncation of liaF (OG1RFliaF∆152), (ii) a null liaF mutant with a premature stop-codon (OG1RFliaF*11), and (iii) an isoleucine deletion at position 177 (OG1RFliaF177). We determined DAP MIC by Etest and characterized the localization of anionic phospholipids microdomains using 10-nonyl-acridine-orange (NAO). The expression of the liaXYZ (the main target of LiaR) and liaFSR clusters were evaluated by qRT-PCR and relative expression ratios (Log2 fold change) were calculated by normalizing to gyrB expression. We assessed activation of LiaFSR by evaluating surface exposure of LiaX by ELISA. We used the bacterial adenylate cyclase two-hybrid system (BACTH) to evaluate the protein-protein interaction between LiaF and LiaS. Results Full deletion of liaF or the C-terminal truncation of LiaF did not have any effect on DAP MICs, membrane architecture or a significant increase in LiaX surface exposure compared with parental strain OG1RF. In contrast, deletion of the codon encoding isoleucine in position 177 of LiaF caused a major increase (8-fold) in LiaX exposure and redistribution of anionic phospholipid microdomains away from the septum without changes in the actual DAP MIC. Transcriptional analyses indicated upregulation (>2 log2-fold) in the liaXYZ gene cluster indicating activation of the stress response. We also observed a positive interaction between LiaF and LiaS. Conclusion LiaF is likely a key activator of the LiaFSR stress response and the critical regulatory domain appears to be located in a stretch of four isoleucines toward the C-terminal of the protein. Disclosures All authors: No reported disclosures.

scholarly journals Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

2020 ◽  
Vol 31 (1-2) ◽  
pp. 2-16 ◽  
Author(s):  
Nobuyo Maeda-Smithies ◽  
Sylvia Hiller ◽  
Sharlene Dong ◽  
Hyung-Suk Kim ◽  
Brian J. Bennett ◽  
...  
Keyword(s):  
Promoter Region ◽  
Transmembrane Protein ◽  
Scavenger Receptor ◽  
Ectopic Expression ◽  
Complete Suppression ◽  
Liver Sinusoidal Endothelial Cells ◽  
Normal Expression ◽  
Number Of Genes ◽  
Genes Encoding ◽  
Intracisternal A Particle

AbstractStabilin2 (Stab2) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor for various macromolecules including hyaluronans (HA). In DBA/2J mice, plasma HA concentration is ten times higher than in 129S6 or C57BL/6J mice, and this phenotype is genetically linked to the Stab2 locus. Stab2 mRNA in the LSECs was significantly lower in DBA/2J than in 129S6, leading to reduced STAB2 proteins in the DBA/2J LSECs. We found a retrovirus-derived transposable element, intracisternal A particle (IAP), in the promoter region of Stab2DBA which likely interferes with normal expression in the LSECs. In contrast, in other tissues of DBA/2J mice, the IAP drives high ectopic Stab2DBA transcription starting within the 5′ long terminal repeat of IAP in a reverse orientation and continuing through the downstream Stab2DBA. Ectopic transcription requires the Stab2-IAP element but is dominantly suppressed by the presence of loci on 59.7–73.0 Mb of chromosome (Chr) 13 from C57BL/6J, while the same region in 129S6 requires additional loci for complete suppression. Chr13:59.9–73 Mb contains a large number of genes encoding Krüppel-associated box-domain zinc-finger proteins that target transposable elements-derived sequences and repress their expression. Despite the high amount of ectopic Stab2DBA transcript in tissues other than liver, STAB2 protein was undetectable and unlikely to contribute to the plasma HA levels of DBA/2J mice. Nevertheless, the IAP insertion and its effects on the transcription of the downstream Stab2DBA exemplify that stochastic evolutional events could significantly influence susceptibility to complex but common diseases.

A novel nucleolar G-protein conserved in eukaryotes

2001 ◽  
Vol 114 (1) ◽  
pp. 173-185 ◽  
Author(s):  
J.H. Park ◽  
B.C. Jensen ◽  
C.T. Kifer ◽  
M. Parsons
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Biological Significance ◽  
Binding Region ◽  
Founding Member ◽  
Nucleolar Proteins ◽  
Evolutionarily Conserved ◽  
Genes Encoding ◽  
Sequence Characteristics ◽  
Two Hybrid

We describe here a novel, evolutionarily conserved set of predicted G-proteins. The founding member of this family, TbNOG1, was identified in a two-hybrid screen as a protein that interacts with NOPP44/46, a nucleolar phosphoprotein of Trypanosoma brucei. The biological relevance of the interaction was verified by co-localization and co-immunoprecipitation. TbNOG1 localized to the trypanosome nucleolus and interacted with domains of NOPP44/46 that are found in several other nucleolar proteins. Genes encoding proteins highly related to TbNOG1 are present in yeast and metazoa, and related G domains are found in bacteria. We show that NOG1 proteins in humans and Saccharomyces cerevisae are also nucleolar. The S. cerevisae NOG1 gene is essential for cell viability, and mutations in the predicted G motifs abrogate function. Together these data suggest that NOG1 may play an important role in nucleolar functions. The GTP-binding region of TbNOG1 is similar to those of Obg and DRG proteins, which, together with NOG, form a newly recognized family of G-proteins, herein named ODN. The ODN family differs significantly from other G-protein families, and shows several diagnostic sequence characteristics. All organisms appear to possess an ODN gene, pointing to the biological significance of this family of G-proteins.

scholarly journals An intramembrane sensory circuit monitors sortase A–mediated processing of streptococcal adhesins

2019 ◽  
Vol 12 (580) ◽  
pp. eaas9941 ◽  
Author(s):  
Jeffrey W. Hall ◽  
Bruno P. Lima ◽  
Gaetan G. Herbomel ◽  
Tata Gopinath ◽  
LeAnna McDonald ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Sortase A ◽  
Gram Positive Bacteria ◽  
Regulatory Circuit ◽  
Bacterial Adhesins ◽  
Genes Encoding ◽  
Quality Control Mechanism ◽  
Cognate Response Regulator

Bacterial adhesins mediate adhesion to substrates and biofilm formation. Adhesins of the LPXTG family are posttranslationally processed by the cell membrane–localized peptidase sortase A, which cleaves the LPXTG motif. This generates a short C-terminal peptide (C-pep) that remains in the cell membrane, whereas the mature adhesin is incorporated into the cell wall. Genes encoding adhesins of the oral bacteriumStreptococcus gordoniiwere differentially expressed depending on whether the bacteria were isolated from saliva or dental plaque and appeared to be coordinately regulated. Deletion ofsspAandsspB (sspAB), both of which encode LPXTG-containing adhesins, unexpectedly enhanced adhesion and biofilm formation. C-peps produced from a model LPXTG-containing adhesin localized to the cell membrane and bound to and inhibited the intramembrane sensor histidine kinase SGO_1180, thus preventing activation of the cognate response regulator SGO_1181. The absence of SspAB C-peps induced the expression of thescaCBAoperon encoding the lipoprotein adhesin ScaA, which was sufficient to preserve and even enhance biofilm formation. This C-pep–driven regulatory circuit also exists in pathogenic streptococci and is likely conserved among Gram-positive bacteria. This quality control mechanism ensures that the bacteria can form biofilms under diverse environmental conditions and may play a role in optimizing adhesion and biofilm formation.

scholarly journals Identification of Factors Regulating Poly(A) Tail Synthesis and Maturation

2004 ◽  
Vol 24 (10) ◽  
pp. 4196-4206 ◽  
Author(s):  
David A. Mangus ◽  
Mandy M. Smith ◽  
Jennifer M. McSweeney ◽  
Allan Jacobson
Keyword(s):  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Genes Encoding ◽  
Mitogen Activated Protein ◽  
Regulatory Complex ◽  
Cleavage And Polyadenylation ◽  
Mrna Polyadenylation ◽  
Two Hybrid ◽  
Polyadenylation Factor

ABSTRACT Posttranscriptional maturation of the 3′ end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length. While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized. Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation. To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts. Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3′-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene. In vitro polyadenylation reactions utilizing extracts derived from fir1Δ and pbp1Δ cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA. Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis. We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).

scholarly journals Glucan Synthase Complex of Aspergillus fumigatus

2001 ◽  
Vol 183 (7) ◽  
pp. 2273-2279 ◽  
Author(s):  
A. Beauvais ◽  
J. M. Bruneau ◽  
P. C. Mol ◽  
M. J. Buitrago ◽  
R. Legrand ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Germ Tube ◽  
Transmembrane Protein ◽  
Homologous Gene ◽  
Rho Proteins ◽  
Glucan Synthase ◽  
Consensus Sequences ◽  
Genes Encoding ◽  
First Time ◽  
Hydrophilic Domain

ABSTRACT The glucan synthase complex of the human pathogenic moldAspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins ofA. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for β(1–3) glucans, showing that AfFks1p colocalized with the newly synthesized β(1–3) glucans.AfRHO1, the most homologous gene to RHO1 ofSaccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H+-ATPase, and a 160-kDa protein which was labeled by an anti-β(1–3) glucan antibody and was homologous to ABC bacterial β(1–2) glucan transporters.

scholarly journals Two Active Forms of UDP-N-Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria

2000 ◽  
Vol 182 (15) ◽  
pp. 4146-4152 ◽  
Author(s):  
Wensheng Du ◽  
James R. Brown ◽  
Daniel R. Sylvester ◽  
Jianzhong Huang ◽  
Alison F. Chalker ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Gene Duplication ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Double Deletion ◽  
Allelic Replacement ◽  
Genes Encoding ◽  
Bacterial Sources

ABSTRACT Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The twomurA genes of the gram-positive organismStreptococcus pneumoniae were studied further. Each of themurA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.

scholarly journals Bending of the BST-2 coiled-coil during viral budding

2017 ◽  
Author(s):  
Kadir A. Ozcan ◽  
Christopher E. Berndsen
Keyword(s):  
Cell Membrane ◽  
Host Cell ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Membrane Anchor ◽  
Mouse Homolog ◽  
Host Cell Membrane ◽  
Structural Mechanism ◽  
Viral Budding ◽  
Hiv 1

AbstractBST-2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV-1 and other viruses by inhibiting viral spreading. Structurally, BST-2 is a homodimeric coiled-coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C-terminal membrane anchor of BST-2 is inserted into the budding virus while the N-terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST-2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST-2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated BST-2 and the cell-virus membrane bridging form. We observed that BST-2 did not transition as a rigid structure, but instead bent at sites with a reduced interface between the helices of the coiled-coil. The simulations for the human BST-2 were then compared with simulations on the mouse homolog, which has a more stable coiled-coil. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled-coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.

scholarly journals IFITM3-Mediated Regulation of Cell Membrane Dynamics Is Essential for Malignant B-Cell Transformation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 552-552
Author(s):  
Jae-Woong Lee ◽  
Huimin Geng ◽  
Derek S Dinson ◽  
Gang Xiao ◽  
Kadriye Nehir Cosgun ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Membrane ◽  
B Cell ◽  
Lipid Rafts ◽  
Transmembrane Protein ◽  
Cell Receptor ◽  
Mrna Levels ◽  
Viral Immunity ◽  
Oncogenic Signaling ◽  
Ras Pathway

Abstract Background & Hypothesis: B cell receptor (BCR) signaling and oncogenic tyrosine kinases that mimic BCR-signaling in B-lineage leukemia and lymphoma depend on assembly of membrane proximal signaling complexes. Signalosomes in normal BCR- and oncogene (e.g. BCR-ABL1, RAS-pathway lesions) signal transduction are recruited to phospholipid anchors in lipid rafts. The robustness of these complexes depends on cholesterol accumulation in lipid rafts. Here we identified the interferon-induced transmembrane protein IFITM3 as a central regulator of cholesterol in lipid rafts. Results: IFITM3 is mostly localized to endosomal compartments. By antagonizing VAP-A and oxysterol-binding protein 1 (OSBP1), IFITM3 promotes cholesterol accumulation and solidifies the endosomal membrane. This mechanism is particular important in anti-viral immunity, to "trap" intraluminal viral particles for lysosomal degradation. In B-cells, IFITM3 can translocate to the cell membrane and form a complex with the BCR and its co-receptors CD19, CD81 and CD21. While the functional significance of membrane expression of IFITM3 on B-cells was not known, we found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.006; n=207) and adults (ECOG E2993; P=0.014; n=215) with B-ALL. In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of relapse and positive MRD status at the end of induction chemotherapy in B-ALL and other B-cell malignancies. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B- ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion. While its membrane-topology can vary in different cell types, we found that IFITM3 functions as a dual-pass transmembrane protein in tight association with CD19 and the Iga and Igb signaling chains of the BCR in B-ALL and B-cell lymphoma cells. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of IFITM3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In mechanistic studies, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT (see schematic, left). Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe induced IFITM3 gain of function and forced accumulation of IFITM3 on the cell membrane, constitutive CD19-PI3K signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells (see schematic, right). Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of membrane-associated oncogenic signaling. Through Filipin-based cholesterol staining, we found Ifitm3-/- pre-B cells have reduced levels of cholesterol in lipid rafts, which causes disruption of lipid rafts formation, as reflected by decreased levels of ganglioside GM1. Notably, the homeostatic cholesterol fluidity by presence of IFITM3 on plasma membrane was also required for initiation of B- and T cell receptor signaling in mature B- and T cell lymphoma to induce Ca2+ mobilization. Conclusions: These findings identify novel role of the viral immunity IFITM3 surface receptor as a central regulator of cell membrane cholesterol fluidity and critical mediator of sustained oncogenic tyrosine kinase (BCR-ABL1) and RAS (NRASG12D) signaling in B cell malignancies. In promoting cholesterol aggregates in lipid rafts, IFITM3 protects healthy individuals from potentially lethal viral infections, but also enables oncogenic signaling by providing a robust membrane scaffold for tyrosine kinase and RAS-pathway oncogenes. Figure Figure. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

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