Wild-type Rates Research Articles

Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells.

The Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.

Refinement of Risk-Stratification of Cytogenetically Normal Acute Myeloid Leukemia Adult Patients by MN1 Expression.

Acute myeloid leukemia with normal cytogenetics (CN-AML) represents a heterogeneous group having diverse genetic mutations. Understanding the significance of each of these mutations is necessary. In this study, we evaluated the prognostic role of MN1 expression in adult CN-AML patients. One hundred and sixty-three de-novo adult AML patients were evaluated for MN1 expression by real-time PCR. MN1 expression was correlated with the clinical characteristics of the patients and their outcomes. Higher MN1 expression was associated with NPM1 wild-type (p<0.0001), CD34 positivity (p=0.006), and lower clinical remission rate (p=0.027). FLT3-ITD and CEBPA mutations had no association with MN1 expression. On survival analysis, a high MN1 expression was associated with poor event-free survival (Hazard Ratio 2.47, 95% Confidence Interval: 1.42-4.3; p<0.0001) and overall survival (Hazard Ratio 4.18, 95% Confidence Interval: 2.17-8.08; p<0.0001). On multivariate analysis, the MN1 copy number emerged as an independent predictor of EFS (p<0.0001) and OS (p<0.0001). MN1 expression is an independent predictor of outcome in CN-AML.

FOLFOXIRI ± molecular targeting agent (bevacizumab or panitumumab) for conversion from unresectable to resectable in the advanced/recurrent colorectal cancer.

99 Background: As treatment for unresectable advanced/recurrent colorectal cancer (ARCC), the introduction of doublet chemotherapy, represented by FOLFOX/FOLFIRI, and molecular-targeting agents, such as anti-VEGF antibody and anti-EGFR antibody, has given ARCC patients a more than three years’ survival. Triplet chemotherapy, such as FOLFOXIRI ± Bevacizumab or Panitumumab, has already been used in daily practice. These regimens with powerful anti-tumor effects have enabled conversion from unresectable to resectable, giving patients a longer survival than those without conversion. Therefore, we explored the ideal regimen for conversion, focusing on FOLFOXIRI ± X. Methods: We retrospectively reviewed unresectable ARCC patients treated with FOLFOXIRI ± X (X:Bmab, n=24; Pmab, n=1; alone, n=2; ) between February 2015 and December 2022. The results were compared to those of patients who received other regimens (OTHERS; n=212). Results: The median overall survival (OS) was 1072 days. Forty-nine patients acquired conversion. The patients with conversion had a longer progression-free survival and OS than those without conversion (p&lt;0.0001 and &lt;0.0001, respectively). Conversion rates were 37.0% in FOLFOXIRI ± X vs. 18.4% in OTHERS (p=0.0238), while response rates were 59.3% in FOLFOXIRI ± X vs. 35.8% in OTHERS (p=0.0185). The RAS wild-type group conversion rates were 30.0% in FOLFOXIRI ± X vs. 20.6% in OTHERS, while 50.0% in FOLFOXIRI ± X vs. 15.3% in OTHERS in RAS mutant group. Anti-EGFR antibody +L-OHP-based doublet chemotherapy accounted for conversion in 35.3% of the RAS wild-type group. Anti-VEGF antibody + L-OHP based doublet chemotherapy attained 29.1% conversion rate in RAS mutant group. Molecular targeting agent+doublet or triplet regimens were essential for conversion. FOLFOXIRI ± X achieved a 88.9% disease control rate, with palliation of symptoms due to a high tumor burden in 10 of 13 cases. Conclusions: Based on these results, FOLFOXIRI ± X may be an ideal candidate for achieving conversion in first-line treatment of unresectable ARCC.

Antifungal susceptibility of clinically isolated invasive Candida tropicalis in East China from 2017 to 2021

Objective: To explore the epidemiological characteristics of sample distribution and antifungal susceptibilities of clinically invasive C. tropicalis isolates from 2017 to 2021 in East China. Methods: Using a retrospective analysis, the East China Invasive Fungal Infection Group (ECIFIG) collected C. tropicalis clinically isolated from 32 hospitals in East China between January 2017 and December 2021. The identification results of the strains were reviewed using mass spectrometry by the central laboratory of the Shanghai East Hospital. The minimum inhibitory concentrations (MICs) of the strains against fluconazole (FLU), voriconazole (VOR), itraconazole (ITR), Posaconazole (POS), isavuconazole (ISA), anidulafungin (ANI), caspofungin (CAS), micafungin (MICA) and 5-fluorocytosine (FCT) were tested by the ThermoFisher CMC1JHY colorimetric microdilution method. The MIC of amphotericin B (AMB) was tested by the broth microdilution method. The MIC results were analyzed based on the clinical breakpoints and epidemiological cutoff values (ECV) published by the Clinical and Laboratory Standards Institute (CLSI) M27 Ed3 and M57 Ed4 documents. Data analysis was conducted using the Kruskal-Wallis test and paired t-test. Results: In total, 305 C. tropicalis isolates were collected. There were 38.0% (116/305) strains isolated from blood, 11.5% (35/305) ascites, 8.9% (27/305) catheter and 8.9% (27/305) drainage fluid. The resistance rate of C. tropicalis to FLU was 32.5%, to VOR was 28.5%, and the cross-resistance rate to FLU and VOR was 28.5%. The wild-type proportions for ITR and POS were 79.3% and 29.2% respectively. There was no significant difference in resistance rates, MIC50, and MIC90 of FLU and VOR, or in the wild-type rates of ITR and POS over five years. More than 95.0% of the isolates were susceptible to echinocandins. However, one strain was identified as being multi-drug resistant. In azole antifungals, voriconazole, itraconazole, posaconazole, and isavuconazole have similar GM MIC values. The GM MIC of fluconazole is significantly higher than that of itraconazole (t=9.95, P<0.05), posaconazole (t=9.99, P<0.05), and voriconazole (t=10.01, P<0.05), Meanwhile, among echinocandins, the GM MIC of ANI was comparable to that of CAS (t=1.17, P>0.05), both of which were significantly higher than MICA (t=11.56, P<0.05; t=4.15, P<0.05). Conclusion: The clinical isolates of C. tropicalis in East China from 2017 to 2021 were relatively susceptible to echinocandins. However, there was consistently high resistance to fluconazole and voriconazole. More intensive efforts should be made on the monitoring of drug resistance in C. tropicalis.

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii.

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

Abstract 1480: ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for the acquisition of malignant cells, which causes epithelial cells with specialized cell-cell contacts to gain mesenchymal migratory capacity. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated with poor clinical outcomes of cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a calculated molecular weight of 125kDa; however, numerous groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutant-expressing NSCLC cell lines. We hypothesize that ZEB1 dimerization is regulated by K811 acetylation to promote protein stability and lung adenocarcinoma metastasis. We determined that the K811R ZEB1 (125 kDa) has a shorter half-life wild-type and high ubiquitination rate than (WT) ZEB1 and K811Q ZEB1 (~225 kDa), suggesting that disruption of ZEB1 acetylation hinders its dimerization and protein stability. Further, ZEB1 dimer recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that unlike K811R ZEB1, WT ZEB1 and K811Q ZEB1 downregulate the expression of gene sets involved in tight junction, apical junction, and cell-cell junction formation. We also identified six novel ZEB1 acetylation-dependent gene candidates that function in signaling pathways and epithelial cell maintenance; CBLN3, STAP2, PLEKHG6, LAD1, RNF43, and LLGL2. In vitro 2D migration Boyden chamber, 3D invasion assays, and in vivo studies demonstrated that ZEB1 acetylation promotes the invasive and metastatic potential of the NSCLC cell line. Our findings suggest that K811 acetylation regulates ZEB1 dimerization and stability to promote NuRD interaction and lung adenocarcinoma via EMT. Future studies are focused on assessing changes in gene expression programs and chromatin accessibility that are ZEB1 dimerization dependent and that could contribute to NSCLC metastasis. Citation Format: Mabel Giselle Perez-Oquendo, Roxsan Manshouri, Yanhua Tian, Jared Fradette, Don Gibbons. ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1480.

Poly(A) polymerase 1 contributes to competence acquisition of pollen tubes growing through the style in Arabidopsis thaliana.

Polyadenylation of mRNAs is critical for their export from the nucleus, stability, and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, previous studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the other two isoforms. Such functional specialization raises the possibility of an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression at the transcriptional, but not detectably at the protein level compared with in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes grow almost at the wild-type rate, they are compromised in locating the micropyles of ovules. Previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A) tail lengths of transcripts suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 plays a key role in the acquisition of competence and underline the importance of functional specialization between PAPS isoforms throughout different developmental stages.

Contractile actomyosin cables and pulling forces from adjacent tissue cooperate to drive Drosophila germ-band extension.

Germ-band extension in Drosophila is a major embryogenesis event, when convergent-extension dramatically reshapes the germ-band. Fluid-like cell motions narrow the germ-band in one direction (dorsal-ventral axis, DV), elongating in the other (anterior-posterior axis, AP). What forces drive germ-band extension? This is controversial. (1) One force is from planar polarized myosin at cell-cell junctions parallel to DV. Blocking myosin activation with optogenetic RhoGAP (optoGAP) activation reduced germ-band elongation ∼1/3 and suppressed cell intercalation (Herrera-Perez et al., 2021). (2) Pulling forces from the invaginating posterior midgut, adjacent to the germ-band posterior end, also contribute. Blocking invagination with the torso mutation reduced germ-band elongation ∼1/3 (Collinet et al., 2015). Further, DV cell elongation is reduced with the torso-like mutation blocking pulling forces (Lye et al., 2015), but little-affected by optoGAP-mediated myosin blocking. Thus, experiment implicates both myosin cables and pulling forces from adjacent tissue, but their contributions and the mechanisms are poorly understood. Here, we developed a biophysical model of Drosophila germ-band extension, with junctional actomyosin contractility, elastic restoring forces, internal viscous forces, extracellular drag, forces from adjacent tissues and cell intercalation (T1 transitions or rosette formation). Our results quantitatively reveal that planar polarized myosin and posterior midgut pulling are driving forces for convergent extension in the DV and AP directions, respectively. Actomyosin cables shrink junctions and intercalate cells, but cannot elongate tissue at wild-type rates, consistent with the phenotype of torso mutants (Collinet et al., 2015). Normal tissue elongation required both myosin cables and posterior midgut pulling, and in simulations with both, the profile of cell lengths reproduced the experimental monotonic increase in the AP direction (Lye et al., 2015). These results suggest convergent extension is cooperatively driven by myosin contractility and active pulling from adjacent tissue.

Two cyclic electron flows around photosystem I differentially participate in C4 photosynthesis.

C4 plants assimilate CO2 more efficiently than C3 plants because of their C4 cycle that concentrates CO2. However, the C4 cycle requires additional ATP molecules, which may be supplied by cyclic electron flow (CEF) around photosystem I. One CEF route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C4 plants despite the low activity in C3 plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1) and is considered a major CEF route to generate the proton gradient across the thylakoid membrane in C3 plants. However, its contribution to C4 photosynthesis is still unclear. In this study, we investigated the contribution of the two CEF routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that suppressing the NDH-dependent route drastically delayed growth and decreased the CO2 assimilation rate to approximately 30% of the wild-type rate. On the other hand, suppressing the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO2 assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent CEF substantially contributes to the NADP-malic enzyme subtype of C4 photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during C4 evolution, photosynthetic electron flow may have been optimized to provide the energy required for C4 photosynthesis.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

Temperature instability of a mutation at a multidomain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome

ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited. It had half the turnover rate of wildtype, reduced affinity for Na+, and increased affinity for K+. There was modest endoplasmic reticulum retention during biosynthesis at 37 °C but little benefit from the folding drug phenylbutyrate (4-PBA), suggesting a tolerated level of misfolding. When cells were incubated at just 39 °C, however, α3 protein level dropped without loss of β subunit, paralleled by an increase of endogenous α1. Elevated temperature resulted in internalization of α3 from the surface along with some β subunit, accompanied by cytoplasmic redistribution of a marker of lysosomes and endosomes, lysosomal-associated membrane protein 1. After return to 37 °C, α3 protein levels recovered with cycloheximide-sensitive new protein synthesis. Heating invitro showed activity loss at a rate 20- to 30-fold faster than wildtype, indicating a temperature-dependent destabilization of protein structure. Arg756 appears to confer thermal resistance as an anchor, forming hydrogen bonds among four linearly distant parts of the Na,K-ATPase structure. Taken together, our observations are consistent with fever-induced symptoms in patients.

Characterising diflunisal as a transthyretin kinetic stabilizer at relevant concentrations in human plasma using subunit exchange

Transthyretin (TTR) dissociation is the rate limiting step for both aggregation and subunit exchange. Kinetic stabilisers, small molecules that bind to the native tetrameric structure of TTR, slow TTR dissociation and inhibit aggregation. One such stabiliser is the non-steroidal anti-inflammatory drug (NSAID), diflunisal, which has been repurposed to treat TTR polyneuropathy. Previously, we compared the efficacy of diflunisal, tafamidis, tolcapone, and AG10 as kinetic stabilisers for transthyretin. However, we could not meaningfully compare diflunisal because we were unsure of its plasma concentration after long-term oral dosing. Herein, we report the diflunisal plasma concentrations measured by extraction, reversed phase HPLC separation, and fluorescence detection after long-term 250 mg BID oral dosing in two groups: a placebo-controlled diflunisal clinical trial group and an open-label Japanese polyneuropathy treatment cohort. The measured mean diflunisal plasma concentration from both groups was 282.2 M 143.7 M (mean standard deviation). Thus, quantification of TTR kinetic stabilisation using subunit exchange was carried out at 100, 200, 300, and 400 concentrations, all observed in patients after 250 mg BID oral dosing. A 250 M diflunisal plasma concentration reduced the wild-type TTR dissociation rate in plasma by 95%, which is sufficient to stop transthyretin aggregation, consistent with the clinical efficacy of diflunisal for ameliorating transthyretin polyneuropathy.

Lack of Specificity in Geobacter Periplasmic Electron Transfer.

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple ΔppcABCDE ΔpgcA mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. IMPORTANCE Many inner and outer membrane cytochromes used by Geobacter for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that Geobacter periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of Geobacter electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.

SETDB1 regulates microtubule dynamics.

SETDB1 is a methyltransferase responsible for the methylation of histone H3-lysine-9, which is mainly related to heterochromatin formation. SETDB1 is overexpressed in various cancer types and is associated with an aggressive phenotype. In agreement with its activity, it mainly exhibits a nuclear localization; however, in several cell types a cytoplasmic localization was reported. Here we looked for cytoplasmic functions of SETDB1. SETDB1 association with microtubules was detected by immunofluorescence and co-sedimentation. Microtubule dynamics were analysed during recovery from nocodazole treatment and by tracking microtubule plus-ends in live cells. Live cell imaging was used to study mitotic kinetics and protein-protein interaction was identified by co-immunoprecipitation. SETDB1 co-sedimented with microtubules and partially colocalized with microtubules. SETDB1 partial silencing led to faster polymerization and reduced rate of catastrophe events of microtubules in parallel to reduced proliferation rate and slower mitotic kinetics. Interestingly, over-expression of either wild-type or catalytic dead SETDB1 altered microtubule polymerization rate to the same extent, suggesting that SETDB1 may affect microtubule dynamics by a methylation-independent mechanism. Moreover, SETDB1 co-immunoprecipitated with HDAC6 and tubulin acetylation levels were increased upon silencing of SETDB1. Taken together, our study suggests a model in which SETDB1 affects microtubule dynamics by interacting with both microtubules and HDAC6 to enhance tubulin deacetylation. Overall, our results suggest a novel cytoplasmic role for SETDB1 in the regulation of microtubule dynamics.

Antiprion systems in yeast cooperate to cure or prevent the generation of nearly all [PSI+] and [URE3] prions

[PSI+] and [URE3] are prions of Saccharomyces cerevisiae based on amyloids of Sup35p and Ure2p, respectively. In normal cells, antiprion systems block prion formation, cure many prions that arise, prevent infection by prions, and prevent toxicity of those prions that escape the other systems. The upf1Δ, ssz1Δ, and hsp104T160M single mutants each develop [PSI+] at 10- to 15-fold, but the triple mutant spontaneously generates [PSI+] at up to ∼5,000-fold the wild-type rate. Most such [PSI+] variants are cured by restoration of any one of the three defective antiprion systems, defining a previously unknown type of [PSI+] variant and proving that these three antiprion systems act independently. Generation of [PSI+] variants stable in wild-type cells is also increased in upf1Δ ssz1Δ hsp104T160M strains 25- to 500-fold. Btn2 and Cur1 each cure 90% of [URE3] prions generated in their absence, but we find that btn2Δ or cur1Δ diminishes the frequency of [PSI+] generation in an otherwise wild-type strain. Most [PSI+] isolates in a wild-type strain are destabilized on transfer to a btn2Δ or cur1Δ host. Single upf1Δ or hsp104T160M mutants show the effects of btn2Δ or cur1Δ but not upf1Δ ssz1Δ hsp104T160M or ssz1Δ hsp104T160M strains. The disparate action of Btn2 on [URE3] and [PSI+] may be a result of [PSI+]'s generally higher seed number and lower amyloid structural stability compared with [URE3]. Thus, prion generation is not a rare event, but the escape of a nascent prion from the surveillance by the antiprion systems is indeed rare.

Phenotypic analyses, protein localization, and bacteriostatic activity of Drosophila melanogaster transferrin-1

Transferrin-1 (Tsf1) is an extracellular insect protein with a high affinity for iron. The functions of Tsf1 are still poorly understood; however, Drosophila melanogaster Tsf1 has been shown to influence iron distribution in the fly body and to protect flies against some infections. The goal of this study was to better understand the physiological functions of Tsf1 in D. melanogaster by 1) investigating Tsf1 null phenotypes, 2) determining tissue-specific localization of Tsf1, 3) measuring the concentration of Tsf1 in hemolymph, 4) testing Tsf1 for bacteriostatic activity, and 5) evaluating the effect of metal and paraquat treatments on Tsf1 abundance. Flies lacking Tsf1 had more iron than wild-type flies in specialized midgut cells that take up iron from the diet; however, the absence of Tsf1 had no effect on the iron content of whole midguts, fat body, hemolymph, or heads. Thus, as previous studies have suggested, Tsf1 appears to have a minor role in iron transport. Tsf1 was abundant in hemolymph from larvae (0.4 μM), pupae (1.4 μM), adult females (4.4 μM) and adult males (22 μM). Apo-Tsf1 at 1 μM had bacteriostatic activity whereas holo-Tsf1 did not, suggesting that Tsf1 can inhibit microbial growth by sequestering iron in hemolymph and other extracellular environments. This hypothesis was supported by detection of secreted Tsf1 in tracheae, testes and seminal vesicles. Colocalization of Tsf1 with an endosome marker in oocytes suggested that Tsf1 may provide iron to developing eggs; however, eggs from mothers lacking Tsf1 had the same amount of iron as control eggs, and they hatched at a wild-type rate. Thus, the primary function of Tsf1 uptake by oocytes may be to defend against infection rather than to provide eggs with iron. In beetles, Tsf1 plays a role in protection against oxidative stress. In contrast, we found that flies lacking Tsf1 had a typical life span and greater resistance to paraquat-induced oxidative stress. In addition, Tsf1 abundance remained unchanged in response to ingestion of iron, cadmium or paraquat or to injection of iron. These results suggest that Tsf1 has a limited role in protection against oxidative stress in D. melanogaster.

Stability across the Whole Nuclear Genome in the Presence and Absence of DNA Mismatch Repair.

We describe the contribution of DNA mismatch repair (MMR) to the stability of the eukaryotic nuclear genome as determined by whole-genome sequencing. To date, wild-type nuclear genome mutation rates are known for over 40 eukaryotic species, while measurements in mismatch repair-defective organisms are fewer in number and are concentrated on Saccharomyces cerevisiae and human tumors. Well-studied organisms include Drosophila melanogaster and Mus musculus, while less genetically tractable species include great apes and long-lived trees. A variety of techniques have been developed to gather mutation rates, either per generation or per cell division. Generational rates are described through whole-organism mutation accumulation experiments and through offspring–parent sequencing, or they have been identified by descent. Rates per somatic cell division have been estimated from cell line mutation accumulation experiments, from systemic variant allele frequencies, and from widely spaced samples with known cell divisions per unit of tissue growth. The latter methods are also used to estimate generational mutation rates for large organisms that lack dedicated germlines, such as trees and hyphal fungi. Mechanistic studies involving genetic manipulation of MMR genes prior to mutation rate determination are thus far confined to yeast, Arabidopsis thaliana, Caenorhabditis elegans, and one chicken cell line. A great deal of work in wild-type organisms has begun to establish a sound baseline, but far more work is needed to uncover the variety of MMR across eukaryotes. Nonetheless, the few MMR studies reported to date indicate that MMR contributes 100-fold or more to genome stability, and they have uncovered insights that would have been impossible to obtain using reporter gene assays.

Identification of a Toxin-Antitoxin System That Contributes to Persister Formation by Reducing NAD in Pseudomonas aeruginosa.

Bacterial persisters are slow-growing or dormant cells that are highly tolerant to bactericidal antibiotics and contribute to recalcitrant and chronic infections. Toxin/antitoxin (TA) systems play important roles in controlling persister formation. Here, we examined the roles of seven predicted type II TA systems in the persister formation of a Pseudomonas aeruginosa wild-type strain PA14. Overexpression of a toxin gene PA14_51010 or deletion of the cognate antitoxin gene PA14_51020 increased the bacterial tolerance to antibiotics. Co-overexpression of PA14_51010 and PA14_51020 or simultaneous deletion of the two genes resulted in a wild-type level survival rate following antibiotic treatment. The two genes were located in the same operon that was repressed by PA14_51020. We further demonstrated the interaction between PA14_51010 and PA14_51020. Sequence analysis revealed that PA14_51010 contained a conserved RES domain. Overexpression of PA14_51010 reduced the intracellular level of nicotinamide adenine dinucleotide (NAD+). Mutation of the RES domain abolished the abilities of PA14_51010 in reducing NAD+ level and promoting persister formation. In addition, overproduction of NAD+ by mutation in an nrtR gene counteracted the effect of PA14_51010 overexpression in promoting persister formation. In combination, our results reveal a novel TA system that contributes to persister formation through reducing the intracellular NAD+ level in P. aeruginosa.

Abstract A23: A second site KrasG12D mutation that impairs PI3K binding rescues embryonic lethality, abrogates myeloproliferative disease, and delays lung tumorigenesis

Abstract Phosphatidylinositol 3-kinase (PI3K) signaling is essential for RAS-driven transformation. To directly investigate the role of oncogenic K-Ras binding to PI3K in development and tumorigenesis, we generated KrasG12D/+,Y64G/+ mice that express, from the endogenous locus, a K-Ras oncoprotein that also contains a “second site” amino acid substitution at tyrosine 64 (Y64G) that disrupts the interaction between oncogenic K-Ras and PI3K. Surprisingly, KrasG12D,Y64G mice are viable, fertile, and phenotypically unremarkable, although they are born at a lower than expected Mendelian frequency (26% versus the expected 50% on a C57BL/6 strain background). As opposed to the enhanced proliferative rate observed in mouse embryonic fibroblasts (MEFs) from K-RasG12D mice, KrasG12D,Y64G MEFs exhibit wild-type rates of proliferation. Detailed analysis of the hematopoietic compartment in KrasG12D,Y64G mice reveals a reduced proportion of long-term hematopoietic stem cells, but no evidence of the aggressive myeloproliferative neoplasm observed when a conditional mutant KrasG12D is activated in hematopoietic cells. Consistent with these observations, bone marrow cells isolated from K-RasG12D,Y64G mice exhibit a normal pattern of myeloid progenitor colony growth in response to cytokine stimulation. In contrast to the lack of observed hematologic malignancy, 100% of KrasG12D,Y64G mice we examined at 1 year of age showed lung lesions, ultimately succumbing to lung tumors with a median survival of 496 days. Despite the ubiquitous KrasG12D,Y64G expression, these mice survive longer than models with mosaic, adenoviral-Cre recombinase-controlled KrasG12D (median survival of 185 days). The majority of the lung lesions that arise in KrasG12D, Y64G mice are low grade, classified pathologically as atypical lymphoid proliferation or papillary adenomas; a few adenocarcinomas are also observed. These studies reinforce the importance of oncogenic KRas-mediated activation of PI3K for transformation and demonstrate that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D normalizes cell proliferation, rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis. Beyond the bone marrow and lung, this mutant strain is a potent genetic tool for dissecting the role of aberrant PI3K signaling in pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and also have implications for treating human cancers with KRAS mutations. Citation Format: Jasmine C. Wong, Pedro A. Perez-Mancera, Jangkyung Kim, Kuang-Yu Jen, Scott C. Kogan, Ari J. Firestone, Eric A. Collisson, David A. Tuveson, Kevin Shannon. A second site KrasG12D mutation that impairs PI3K binding rescues embryonic lethality, abrogates myeloproliferative disease, and delays lung tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A23.

Elucidating molecular interactions of L-nucleotides with HIV-1 reverse transcriptase and mechanism of M184V-caused drug resistance

Emtricitabine (FTC) and lamivudine (3TC), containing an oxathiolane ring with unnatural (−)-stereochemistry, are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in anti-HIV therapy. Treatment with FTC or 3TC primarily selects for the HIV-1 RT M184V/I resistance mutations. Here we provide a comprehensive kinetic and structural basis for inhibiting HIV-1 RT by (−)-FTC-TP and (−)-3TC-TP and drug resistance by M184V. (−)-FTC-TP and (−)-3TC-TP have higher binding affinities (1/Kd) for wild-type RT but slower incorporation rates than dCTP. HIV-1 RT ternary crystal structures with (−)-FTC-TP and (−)-3TC-TP corroborate kinetic results demonstrating that their oxathiolane sulfur orients toward the DNA primer 3′-terminus and their triphosphate exists in two different binding conformations. M184V RT displays greater (>200-fold) Kd for the L-nucleotides and moderately higher (>9-fold) Kd for the D-isomers compared to dCTP. The M184V RT structure illustrates how the mutation repositions the oxathiolane of (−)-FTC-TP and shifts its triphosphate into a non-productive conformation.