Natl Acad Research Articles

Measuring Droplets Expelled During Endoscopy to Investigate COVID-19 Transmission Risk

Measuring Droplets Expelled During Endoscopy to Investigate COVID-19 Transmission Risk

Murine models of Duchenne muscular dystrophy: is there a best model?

Murine models of Duchenne muscular dystrophy: is there a best model?

Hamiltonicity of a Coprime Graph

The k-coprime graph of order n is the graph with vertex set \(\{k, k+1, \ldots , k+n-1\}\) in which two vertices are adjacent if and only if they are coprime. We characterize Hamiltonian k-coprime graphs. As a particular case, two conjectures by Tout et al. (Natl Acad Sci Lett 11:365–368, 1982) and by Schroeder (Graph Comb 38:119–140, 2019) on prime labeling of 2-regular graphs follow. A prime labeling of a graph with n vertices is a labeling of its vertices with distinct integers from \(\{1, 2,\ldots , n\}\) in such a way that the labels of any two adjacent vertices are relatively prime.

Abstract 1234: G4 binders as potential immunostimulatory compounds for cancer therapy

Abstract G-quadruplex (G4) is non-canonical nucleic acid structure involved in a plethora of fundamental biological processes. In past decades, it has been studied as a promising pharmaceutical target for anticancer therapy. However, no G4-targeting agent has shown significant clinical effects up to date. Pyridostatin (PDS), a well-known G4 binder, can induce double-stranded DNA breaks and genome instability. We have recently shown that PDS and other G4 binders can trigger micronuclei formation in cancer cells at non-cytotoxic concentrations [1]. As micronuclei can play a crucial role in linking genome instability to innate immunity, we have here wondered whether G4 binders can induce immune gene activation in human MCF-7 breast cancer cells. By using RNA-Seq technology, we show that non-cytotoxic doses of PDS can activate Interferon beta- and IRF3-dependent genes leading to an innate immune gene response in human cancer cells. In addition, immune gene activation follows the induction of micronuclei formation supporting cytoplasmic DNA as a trigger of immune gene activation. Immunofluorescence assays showed that cGAS (a cytoplasmic DNA sensor) can bind to and is activated by micronuclei. In addition, cytostatic PDS concentrations activate STING and IRF3 factors leading to activated immune gene expression. STING inhibition with siRNAs, CRISPR knockout or a chemical inhibitor resulted in a suppression of immune gene expression and Interferon-beta (IFN-B) production in human MCF-7 and murine B16 cell lines, showing that STING is required for PDS-induced immune gene activation in cancer cells. We have then extended the study to a structurally-unrelated G4 binder, PhenDC3, and the results show that PhenDC3 can induce micronuclei formation and IFN-B production in a STING-dependent manner. Then, using TCGA-BRCA dataset, we show that specific gene expression patterns mediated by PDS correlate with immunological hot features of human tumors, such as anti-tumor immune cell infiltration. We could also define a specific PDS gene signature that can predict a better survival of breast cancer patients, suggesting an impact of immunomodulation potential of PDS or other G4 binders in human patients. Thus, our results provide strong evidence that G4 binders can activate innate immune genes in human and murine cancer cells with therapeutic potentials, and that G4 binder-induced micronuclei accumulation can trigger the cGAS-STING signaling pathway. The findings may open to the development of novel immunotherapeutic anticancer strategies based on G4-targeting compounds.

A new cytokine target for chronic obstructive pulmonary disease?

A new cytokine target for chronic obstructive pulmonary disease?

Responsive and agile vaccination strategies against COVID-19 in India

Responsive and agile vaccination strategies against COVID-19 in India

The Antibiotic Efflux Protein TolC Is a Highly Evolvable Target under Colicin E1 or TLS Phage Selection.

Bacteriophages and bacterial toxins are promising antibacterial agents to treat infections caused by multidrug-resistant (MDR) bacteria. In fact, bacteriophages have recently been successfully used to treat life-threatening infections caused by MDR bacteria (Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, Barr JJ, Reed SL, Rohwer F, Benler S, et al. 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother. 61(10); Chan BK, Turner PE, Kim S, Mojibian HR, Elefteriades JA, Narayan D. 2018. Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol Med Public Health. 2018(1):60–66; Petrovic Fabijan A, Lin RCY, Ho J, Maddocks S, Ben Zakour NL, Iredell JR, Westmead Bacteriophage Therapy Team. 2020. Safety of bacteriophage therapy in severe Staphylococcus aureus infection. Nat Microbiol. 5(3):465–472). One potential problem with using these antibacterial agents is the evolution of resistance against them in the long term. Here, we studied the fitness landscape of the Escherichia coli TolC protein, an outer membrane efflux protein that is exploited by a pore forming toxin called colicin E1 and by TLS phage (Pagie L, Hogeweg P. 1999. Colicin diversity: a result of eco-evolutionary dynamics. J Theor Biol. 196(2):251–261; Andersen C, Hughes C, Koronakis V. 2000. Chunnel vision. Export and efflux through bacterial channel-tunnels. EMBO Rep. 1(4):313–318; Koronakis V, Andersen C, Hughes C. 2001. Channel-tunnels. Curr Opin Struct Biol. 11(4):403–407; Czaran TL, Hoekstra RF, Pagie L. 2002. Chemical warfare between microbes promotes biodiversity. Proc Natl Acad Sci U S A. 99(2):786–790; Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, Riley M, Slatin S, Cavard D. 2007. Colicin biology. Microbiol Mol Biol Rev. 71(1):158–229). By systematically assessing the distribution of fitness effects of ∼9,000 single amino acid replacements in TolC using either positive (antibiotics and bile salts) or negative (colicin E1 and TLS phage) selection pressures, we quantified evolvability of the TolC. We demonstrated that the TolC is highly optimized for the efflux of antibiotics and bile salts. In contrast, under colicin E1 and TLS phage selection, TolC sequence is very sensitive to mutations. Finally, we have identified a large set of mutations in TolC that increase resistance of E. coli against colicin E1 or TLS phage without changing antibiotic susceptibility of bacterial cells. Our findings suggest that TolC is a highly evolvable target under negative selection which may limit the potential clinical use of bacteriophages and bacterial toxins if evolutionary aspects are not taken into account.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Superiority of Obinutuzumab over Rituximab in a New Molecular Follicular Lymphoma-like Subgroup of DLBCL: Results from an Exploratory Analysis of the Phase 3 GOYA Trial

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a biologically heterogeneous diagnostic category with important prognostic subgroups, including activated B-cell (ABC) and germinal center B-cell (GCB) cell-of-origin (COO) subgroups. The GCB COO subgroup shares more molecular features with follicular lymphoma (FL) than other DLBCL subpopulations, and is itself heterogeneous, with a subgroup dependent on activation of the PI3K/AKT pathway (via loss of PTEN). When combined with chemotherapy, obinutuzumab (GA101; G) was clinically superior to rituximab (R) in first-line (1L) FL in the GALLIUM trial. However, the combination of G and CHOP chemotherapy (G-CHOP) was not more effective than R plus CHOP (R-CHOP) in 1L DLBCL in the GOYA trial (NCT01287741) (Vitolo et al. J Clin Oncol 2017). Here, we performed exploratory biomarker analyses to evaluate if a subgroup of patients (pts) derived benefit from G-CHOP in the GOYA 1L DLBCL population. Treatment effect was assessed using the Linear Predictor Score (LPS) from the Nanostring Lymphoma Subtyping Test (LST), as this continuous variable informs on the likelihood of a DLBCL pt having a germinal center-derived tumor (which has a similar biological origin to FL) based on the gene expression profile [Wright et al. Proc Natl Acad Sci USA 2003].Methods: Biomarker testing was performed on formalin-fixed, paraffin-embedded tumor tissue collected prior to treatment and tested retrospectively in central laboratories. COO was assessed using the NanoString Research Use Only LST (NanoString Technologies Inc., Seattle, WA, USA) in 933 pts (representing 66% of the intent-to-treat population in GOYA). Additional biomarker analyses used for molecular characterization included DNA-targeted sequencing of 467 genes using the FoundationOne® Heme (FOH) panel (n=499) and identification of BCL2 translocations using Vysis LSI Dual Color Break Apart FISH Probes (n=644; FISH cut-off, 50%). Multivariate Cox regression and elastic net penalized regression were used to evaluate biomarker treatment effects. In addition, bootstrap simulations were conducted to identify the optimal LPS to reflect the robustness of the observed treatment effect in GOYA and the generalizability of treatment effect to independent study populations. Multiple testing adjustment was done by estimating false-discovery rates (FDRs) using the Benjamini-Hochberg procedure (significance, <5% FDR). Pathway enrichment analysis was performed using a hypergeometric test. All pts consented to the biomarker analyses.Results: Strong expression of a germinal-center gene expression profile (by LPS) was linked with a benefit in outcome from treatment with G-CHOP vs R-CHOP among pts in GOYA. Bootstrap simulations identified an optimal LPS cut-off for predicting G-CHOP benefit as the 25% (233/933) of GOYA pts with the lowest LPS scores. These pts are referred to as ‘strong-GCB’ pts, and comprised 43% (233/540) of evaluable GCB pts in GOYA. Strong-GCB pts treated with G-CHOP achieved significantly better clinical outcomes in terms of investigator-assessed progression-free (HR=0.33, p=0.0007), event-free (HR=0.47, p=0.003), and overall survival (HR=0.41, p=0.019; but not end-of-treatment PET-complete response) than those treated with R-CHOP (Table 1; Figure 1). In multivariate analyses, the observed benefit was independent of baseline demographics and disease characteristics. Safety was similar with either regimen. In gene-set analyses on the FOH data, compared with other GCB pts, strong-GCB pts were significantly enriched for mutations that are characteristic for FL pts. In particular, BCL2 translocations and mutations in several m7-FLIPI genes were highly enriched in strong-GCB pts vs other DLBCL subgroups (Figure 2). There was no evidence for transformed indolent non-Hodgkin lymphoma in the strong-GCB subset on central pathology review.Conclusions: Analyzing the data from the GOYA trial, we have identified a new distinct molecular subgroup of GCB DLBCL that comprises around 25% of all DLBCL pts, referred to as “strong-GCB”. This distinct subgroup is identifiable by gene expression profiling and characterized by mutations that are also commonly identified in FL pts [Morin et al. Nature 2011]. Strikingly, treatment with G-CHOP appears to confer substantial clinical benefit over R-CHOP in this new subgroup of DLBCL. [Display omitted] DisclosuresOestergaard:F. Hoffmann-La Roche Ltd: Employment. Bolen:Genentech: Employment, Equity Ownership. Mattiello:Roche: Employment. Sehn:Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Trněný:Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Vitolo:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria. Nielsen:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Morariu-Zamfir:Roche: Employment. Fingerle-Rowson:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Lenz:Celgene: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria.

A Novel Phthalimide-Derivative, TC11, Induced G2/M Arrest and Apoptosis of High-Risk Myeloma Cells By Interacting with Nucleophosmin1 and α-Tubulin, but Not By Binding to Celeblon

Background and Purpose:Emergence of novel drugs including immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have markedly improved the survival of multiple myeloma (MM) patients. However, IMiDs have substantial limitation in the treatment of MM; little effect on MM patients with high-risk cytogenetic changes and their teratogenicity. Recently it was reported that IMiDs showed anti-tumor activity as well as teratogenicity by directly binding to cereblon (CRBN).We have previously reported that a novel phthalimide-derivative, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), induced apoptosis of high-risk MM cells in vivo and also showed anti-osteoclastogenic activity. At the same time, we observed abnormal α-tubulin fragmentation in TC11-treated MM cell (Matsushita et. al. PLoS One 2015). We have also demonstrated that TC11 bound to nucleophosmin 1(NPM1) and α-tubulin using in vitro virus (IVV) method (Shiheido et. al. PLoS One 2012). Interestingly, lenalidomide also bound to NPM1. The purpose of this study is to clarify molecular mechanisms of anti-myeloma effects of TC11.Results and Discussion:(1) Effect of TC11 is CRBN-independent:Recently it was reported that lenalidomide bound to CRBN, resulting in degradation of its substrates, IKZF1/3 and CK1α. In this study, we found that lenalidomide caused degradation of IKZF1/3 and CK1α in the high-risk MM cell line, KMS34. However, TC11 didn't down-regulate CRBN or its substrates, IKZF1/3 and CK1α. Taken together with the result of Biacore assay, TC11 induced MM cell death via CRBN-independent pathway.(2) Cell cycle arrest by TC11 vs. lenalidomide:Then we tried to elucidate molecular mechanisms of TC11 by the microarray analysis of KMS21, which is highly sensitive to TC11 and lenalidomide. Microarray Gene Set Enrichment Analysis (GSEA) indicated that TC11 upregulated the gene set of G2/M checkpoint, however lenalidomide did not. These results were also confirmed by analyzing the DNA contents of MM cells using flow cytometry and by western blot analysis of cell cycle regulators, phosphorylated cdc2 and p21. These data suggest that TC11 induced apoptosis of MM cells by distinct molecular mechanism from lenalidomide probably by regulating G2/M cell cycle. TP53 gene regulates G1/S cell cycle and occasionally deleted in high-risk MM cells. However, TC11 is able to induce apoptosis of TP53 -deleted high-risk MM cells by causing G2/M arrest.(3) Inhibition of tubulin assembly by TC11:Since we have previously showed that TC11 bound to α-tubulin by IVV method, we speculated that TC11 affected tubulin polymerization or depolymerization. Treatment of MM cells with TC11 significantly reduced polymerized fraction of α-tubulin, indicating TC11 inhibited tubulin polymerization. In contrast, lenalidomide did not inhibit either tubulin polymerization or depolymerization. In this regard, the mechanism of TC11 is different from that of lenalidomide.(4) Inhibition of oligomerization of NPM1 by TC11:We have also showed that TC11 bound to NPM1 by IVV method (Shiheido et. al. PLoS One 2012). It has been reported that oligomerized NPM1 had pleiotropic biological functions. Thus we examined whether TC11 affected NPM1 oligomerization using blue native polyacrylamide gel electrophoresis. As a result, TC11 treatment significantly reduced the amount of oligomerized NPM1 fraction. At the same time, we found that TC11 significantly increased in phosphorylation of NPM1 at Ser4, Thr95 and Thr199. It has been reported that phosphorylation of NPM1 inhibited its oligomerization (Mitrea et. al. Proc Natl Acad Sci U S A 2014). Thus, we speculated that TC11-induced phosphorylation resulted in inhibition of oligomerization of NPM1.Conclusion:It was shown that TC11 bound to α-tubulin and NPM1, but not to CRBN. TC11 inhibited tubulin assembly as well as NPM1 oligomerization, leading to G2/M arrest and mitotic catastrophe. These novel molecular events by TC11 induced apoptosis of TP53 -deleted high-risk MM cells. [Display omitted] DisclosuresYonemura:IDAC Theranostics Inc.: Employment. Tabata:IDAC Theranostics Inc.: Employment. Yanagawa:IDAC Theranostics Inc.: Employment. Hattori:Cosmic Corporation Co., Ltd.: Research Funding; Merck Sharp & Dohme: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding.

A Novel JAK2 Mutation Confers Cytokine-Independence in the Absence of a Type I Dimeric Cytokine Receptor

JAK2 fusions reported in hematologic malignancies are often associated with eosinophilia. In addition to JAK2 fusions such as PCM1-JAK2, BCR-JAK2 and ETV6-JAK2, the JAK2V617Fpoint mutant has been recurrently observed in patients with hypereosinophilic syndrome (HES), occurring in ~4% of patients with hypereosinophilia of unknown significance (Schwaab, Am J Hematol, 2015).Here, we describe a novel JAK2 mutation in a 69 year-old woman with a history of immune thrombocytopenia referred for steroid-refractory HES. Peripheral blood eosinophilia was present for at least five years, with absolute eosinophil counts ranging up to 17,500/μL. Bone marrow biopsy revealed hypercellular marrow with trilineage hematopoiesis, increased atypical megakaryocytes, and markedly increased eosinophils with abnormal morphology, consistent with myeloproliferative neoplasm. Results from an eosinophilia FISH panel for FIP1L1/CHIC2/PDGFRα, FGFR, PDGFRβ, and CBFβ fusions were negative, as were T-cell clonality studies by next-generation sequencing (NGS). An NGS-based myeloid malignancies panel revealed a novel mutation in JAK2 consisting of a ten base deletion and insertion of a single thymine (JAK2 c.1747_1756delinsT, p.Leu583_Ala586delinsSer;JAK2indel). Treatment with ruxolitinib improved the peripheral blood eosinophilia, but dose intensity was limited by thrombocytopenia (Table 1).For transformation of cells to cytokine-independence, JAK2V617F requires co-expression of a type I dimeric cytokine receptor, such as Epo-R, to enable downstream signaling in the absence of growth factors (Lu et al, Proc Natl Acad Sci, 2005). To characterize the signaling properties of the new JAK2indel mutant, we cloned JAK2WT, JAK2V617F, and JAK2indel into the MSCV-IRES-GFP (MIG) retroviral vector. IL-3-dependent Ba/F3 cells lacking type I cytokine receptors were infected with retrovirus to assess cytokine independence.Parental Ba/F3 cells and those transduced with JAK2WT, JAK2V617F, or JAK2indel proliferated at similar rates when grown in WEHI conditioned medium (CM). However, upon IL-3 (WEHI) withdrawal, only GFP-positive JAK2indel Ba/F3 cells demonstrated growth factor independence (n=3). Furthermore, parental and GFP-sorted JAK2WT- and JAK2V617F-expressingBa/F3 cells display acceleration of growth when cultured in graded dilutions of WEHI CM, while growth of JAK2indel Ba/F3 cells is not further enhanced by WEHI CM. JAK2indel-expressing Ba/F3 cells also demonstrated cytokine-independent growth in colony assays, whereas parental, JAK2WT-, and JAK2V617F-expressing cells displayed little to no colony formation in the absence of cytokines. Immunoblot analyses revealed that after 4 hours of culture ± IL-3, all cells grown in IL-3 demonstrated activation of STAT5 and ERK, but only JAK2indel Ba/F3 cells demonstrated cytokine-independent signaling activation.JAK2indel activates STAT5 and ERK without co-expression of a type I cytokine receptor, implicating a mechanism of JAK2 kinase activation that is distinct from JAK2V617F. Unexpectedly, preliminary structural analysis suggests that the conformational change induced by JAK2indel resembles that of JAK2V617F. Mutation of glutamic acid 596 to arginine (E596R) has been shown to abolish the constitutive activity of JAK2V617F via modulation of the C-terminal residues of the SH2-JH2 linker, thereby restoring pseudokinase domain autoinhibitory function (Leroy et al, Biochem J, 2016). To test whether this regulatory mechanism is preserved in JAK2indel, we introduced E596R into JAK2indel and are currently characterizing JAK2indel/E596R in cellular and biochemical assays. Additional modeling and biochemical work is underway to clarify whether JAK2indel confers a new functionality that supports signaling activation in the absence of a type I cytokine receptor.In summary we report a novel JAK2 mutation in a patient with myeloproliferative-variant HES that results in cytokine-independent growth, is associated with STAT5 and ERK phosphorylation and may be employing an alternative mechanism to achieve constitutive activation of signaling. The fact that the patient's eosinophilia improved with ruxolitinib treatment illustrates the importance of performing NGS in patients with HES to identify potentially targetable genetic lesions. [Display omitted] DisclosuresDeininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; ARIAD: Consultancy.

Heme Synthesis Is Inhibited in Erythroid Cells Derived from Beta-Thalassemic Mice

Thalassemias are a heterogeneous group of red blood cell disorders characterized by ineffective erythropoiesis, peripheral hemolysis and anemia. Transfusion-dependent thalassemia (TDT) includes thalassemia major patients who require lifelong transfusions for survival. Non-transfusion dependent thalassemia (NTDT) patients do not need constant transfusions to survive, although their survival may depend on occasional or even frequent transfusions during defined periods of time. NTDT comprises three different forms of thalassemias: β-thalassemia intermedia, hemoglobin E/β-thalassemia (mild and moderate forms) and α-thalassemia intermedia (hemoglobin H disease). Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia.The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. In β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation. The defective production of α-globin chains would be expected to cause the accumulation of uncommitted heme in thalassemic erythroblasts. However, we have observed that mice β-thalassemic erythroblasts have decreased heme levels when compared to wild type cells. This observation led us to hypothesize that heme synthesis pathway is inhibited in β-thalassemic erythroblasts.To test this hypothesis, we used a β-thalassemic mice model, Th3,1 which has a deletion of both βmajor and βminor genes. Mice homozygous for the deletion (Th3/Th3) die late in gestation and heterozygotes (Th3/+) survive and develop a phenotype similar to β-thalassemia intermedia in humans (anemia, tissue iron overload and ineffective erythropoiesis). We isolated reticulocytes from wild type (Wt) and Th3/+ mice and measured the rate of heme synthesis in them. Reticulocytes are an invaluable model to study heme synthesis and hemoglobin production in mice, since they are pure erythroid cells and responsible for a significant amount of all hemoglobin produced in mice during stress erythropoiesis.2-4We found that when Wt or Th3/+-derived reticulocytes were incubated with increasing amounts of 59Fe-Tf (0.5-4 µM), the total iron uptake increased by the same percentage (51.9±5.4% and 63.3±6.4% [p>0.05; n=3] between 0.5 and 4.0 µM) in both Wt- and Th3/+-derived cells, while the amount of 59Fe incorporated into heme was already maximal at 0.5 µM. Importantly, in Th3/+-derived reticulocytes the percentage of 59Fe incorporated into heme was significantly lower than in Wt cells. Therefore, compared to Wt reticulocytes, Th3/+ reticulocytes are less efficient in utilizing iron for heme synthesis. We then examined whether heme synthesis could be stimulated in Th3/+ reticulocytes by supplying 5-aminolevulinic acid (ALA), which is the product of the first enzyme of heme biosynthesis. The incubation of Wt or Th3/+ reticulocytes with different amounts of ALA led to a significant increase in the 59Fe incorporation into heme only in Th3/+ cells; there was a 48.31±4% increase in the presence of 2 mM ALA. Therefore, the deficiency of Th3/+ reticulocytes in synthesizing heme is not related to a lack of iron.Our data suggest that there is a yet to be identified factor, which may, or not, be related to globin synthesis, which is responsible for decreased heme synthesis in Th3/+ erythroid cells. Additionally, the ALA-mediated increase in heme synthesis in Th3/+ reticulocytes also indicates that heme synthesis is inhibited in these cells prior to or at the step catalyzed by erythroid-specific ALA synthase.1Yang B, Kirby S, Lewis J, Detloff PJ, Maeda N, and Smithies O. A mouse model for beta 0-thalassemia. Proc Natl Acad Sci U S A. 1995;92(25):11608-12.2Sassa A, and Bernstein SE. Levels of delta-aminolevulinate dehydratase, uroporphyrinogen-I synthase, and protoporphyrin IX in erythrocytes from anemic mutant mice. Proc Natl Acad Sci U S A. 1977;74(3): 1181-4.3Sassa S, Kappas A, Bernstein SE and Alvares AP. Heme biosynthesis and drug metabolism in mice with hereditary hemolytic anemia. J Biol Chem . 1979; 254(3):729-35.4Abdulla M and Svensson S. Effect of phenylhydrazine on delta-aminolevulinic acid dehydratase in red blood cells. Enzyme . 1978;23(3):164-9. DisclosuresNo relevant conflicts of interest to declare.

CD19-Specific Chimeric Antigen Receptor-Modified T Cells with Safety Switch Produced Under “Point-of-Care” Using the Sleeping Beauty System for the Very Rapid Manufacture and Treatment of B-Cell Malignancies

Chimeric antigen receptor (CAR)-modified autologous T cells have shown remarkable clinical responses targeting B-cell malignancies. However, a majority of manufacturing approaches rely on expensive viral vectors and ex vivo activation of clinical-grade T cells for gene transfer with subsequent 2-3 week-long numeric expansion to achieve necessary dosing. We have engineered our clinically-validated Sleeping Beauty (SB) system for stable non-viral integration of CAR, cytokine signaling, and safety switch transgenes into T cells to eliminate need for viral vectors. We previously demonstrated that co-expression of membrane bound IL-15 (mbIL15, Proc Natl Acad Sci U S A. 2016 Nov 29;113(48):E7788-E7797) on CD19-specific CAR+ T cells using SB system allows for very rapid (<2 days) manufacturing with point-of-care (P-O-C) technology (Blood 2016 128:2807). This approach to production avoids the need for activation and propagation of CAR+ T cells leading to augmented persistence and anti-tumor activity in an in vivo model of leukemia. We now update this technology by co-expressing a safety switch, HER1t, on CD19-specific CAR+ mbIL15+ T cells. HER1t improves the safety profile by providing a mechanism for selective in vivo depletion of P-O-C-produced T cells through the administration of cetuximab, a clinically-available monoclonal antibody. We confirmed co-expression of CAR, mbIL15, and HER1t in P-O-C-manufactured T cells and demonstrated that these cells exhibited a memory-like phenotype, potent antigen-specific cytotoxicity, and produced IFNγ, TNFα, and IL-2 when co-cultured with CD19+ tumor cells. Cetuximab specifically and efficiently killed mbIL15+ HER1t+ CAR+ T cells via antibody dependent cellular cytotoxicity and complement dependent cytotoxicity in a dose-dependent manner. Adoptive transfer of mbIL15+HER1t+CAR+ T cell generated in less than 2 days under P-O-C into immunocompromised (NSG) mice bearing established CD19+ leukemia (NALM-6) resulted in significant reduction in tumor burden and improvement in overall survival, compared to control mice (Figure). Blood analysis demonstrated improved expansion and persistence of mbIL15+ HER1t+ CAR+ T cells compared to mbIL15negHER1tnegCAR+ T-cell control. In summary, we have generated T cells co-expressing mbIL15 and the HER1t safety switch in less than two days using P-O-C that demonstrated superior efficacy and persistence compared to P-O-C T cells that expressed CAR without mbIL15. These pre-clinical data demonstrate the effectiveness of very rapidly manufactured mbIL15+ HER1t+ CAR+ T cells in targeting CD19+ tumors and provide a rationale for clinical evaluation. [Display omitted] DisclosuresChan:Intrexon Corporation: Employment. Gallagher:Intrexon Corporation: Employment. Cheng:Intrexon Corporation: Employment. Carvajal-Borda:Intrexon Corporation: Employment. Plummer:Intrexon Corporation: Employment. Govekung:Intrexon Corporation: Employment. Barrett:ZIOPHARM Oncology: Employment. Khare:Ziopharm Oncology: Employment. Cooper:Immatics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Miltenyi Biotec: Honoraria; Ferring: Consultancy; Intrexon Corporation: Equity Ownership, Patents & Royalties; Argos Therapeutics: Equity Ownership; Targazyme, Inc.: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Research Funding; Organovo Holdings: Equity Ownership; Procter & Gamble: Equity Ownership; AmpliPhi Biosciences: Equity Ownership; Sangamo Biosciences: Patents & Royalties. Shah:Intrexon Corporation: Employment.

Spectral features of cortical auditory evoked potentials inform hearing threshold and intensity percepts in acoustic and electric hearing

Objective. Stimulus-elicited changes in electroencephalography (EEG) recordings can be represented using Fourier magnitude and phase features (Makeig et al (2004 Trends Cogn. Sci. 8 204–10)). The present study aimed to quantify how much information about hearing responses are contained in the magnitude, quantified by event-related spectral perturbations (ERSPs); and the phase, quantified by inter-trial coherence (ITC). By testing if one feature contained more information and whether this information was mutually exclusive to the features, we aimed to relate specific EEG magnitude and phase features to hearing perception. Approach. EEG responses were recorded from 20 adults who were presented with acoustic stimuli, and 20 adult cochlear implant users with electrical stimuli. Both groups were presented with short, 50 ms stimuli at varying intensity levels relative to their hearing thresholds. Extracted ERSP and ITC features were inputs for a linear discriminant analysis classifier (Wong et al (2016 J. Neural. Eng. 13 036003)). The classifier then predicted whether the EEG signal contained information about the sound stimuli based on the input features. Classifier decoding accuracy was quantified with the mutual information measure (Cottaris and Elfar (2009 J. Neural. Eng. 6 026007), Hawellek et al (2016 Proc. Natl Acad. Sci. 113 13492–7)), and compared across the two feature sets, and to when both feature sets were combined. Main results. We found that classifiers using either ITC or ERSP feature sets were both able to decode hearing perception, but ITC-feature classifiers were able to decode responses to a lower but still audible stimulation intensity, making ITC more useful than ERSP for hearing threshold estimation. We also found that combining the information from both feature sets did not improve decoding significantly, implying that ERSP brain dynamics has a limited contribution to the EEG response, possibly due to the stimuli used in this study. Significance. We successfully related hearing perception to an EEG measure, which does not require behavioral feedback from the listener; an objective measure is important in both neuroscience research and clinical audiology.

Fast and efficient Rmap assembly using the Bi-labelled de Bruijn graph

Genome wide optical maps are high resolution restriction maps that give a unique numeric representation to a genome. They are produced by assembling hundreds of thousands of single molecule optical maps, which are called Rmaps. Unfortunately, there are very few choices for assembling Rmap data. There exists only one publicly-available non-proprietary method for assembly and one proprietary software that is available via an executable. Furthermore, the publicly-available method, by Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006), follows the overlap-layout-consensus (OLC) paradigm, and therefore, is unable to scale for relatively large genomes. The algorithm behind the proprietary method, Bionano Genomics’ Solve, is largely unknown. In this paper, we extend the definition of bi-labels in the paired de Bruijn graph to the context of optical mapping data, and present the first de Bruijn graph based method for Rmap assembly. We implement our approach, which we refer to as rmapper, and compare its performance against the assembler of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) and Solve by Bionano Genomics on data from three genomes: E. coli, human, and climbing perch fish (Anabas Testudineus). Our method was able to successfully run on all three genomes. The method of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) only successfully ran on E. coli. Moreover, on the human genome rmapper was at least 130 times faster than Bionano Solve, used five times less memory and produced the highest genome fraction with zero mis-assemblies. Our software, rmapper is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/Rmapper.

Parental energy-sensing pathways control intergenerational offspring sex determination in the nematode Auanema freiburgensis

BackgroundEnvironmental stimuli experienced by the parental generation influence the phenotype of subsequent generations (Demoinet et al., Proc Natl Acad Sci U S A 114:E2689-E2698, 2017; Burton et al., Nat Cell Biol 19:252–257, 2017; Agrawal et al., Nature 401:60-63, 1999). The effects of these stimuli on the parental generation may be passed through the germline, but the mechanisms at the basis of this non-Mendelian type of inheritance, their level of conservation, how they lead to adaptive vs non-adaptive, and intergenerational vs transgenerational inheritance are poorly understood. Here we show that modulation of nutrient-sensing pathways in the parental generation of the nematode Auanema freiburgensis regulates phenotypic plasticity of its offspring.ResultsIn response to con-specific pheromones indicative of stress, AMP-activated protein kinase (AMPK), mechanistic target of rapamycin complex 1 (mTORC1), and insulin signaling regulate stress resistance and sex determination across one generation, and these effects can be mimicked by pathway modulators. The effectors of these pathways are closely associated with the chromatin, and their regulation affects the chromatin acetylation status in the germline.ConclusionThese results suggest that highly conserved metabolic sensors regulate phenotypic plasticity through regulation of subcellular localization of their effectors, leading to changes in chromatin acetylation and epigenetic status of the germline.

Expression of Hedgehog signalling molecules inmicrocystic adnexal carcinoma.

Microcystic adnexal carcinoma (MAC) is a rare skin neoplasm that has not been characterized on a molecular basis. To assess expression profiles of Hedgehog (HH) signalling molecules in MAC and control tumours. Immunohistochemistry was performed for Sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched 1 (PTCH1) and Smoothened (SMO) on patient MAC tissue (n=26) and control tumour tissue, including syringoma (SyG; n=11), trichoepithelioma (TE; n=11) and basal cell carcinoma (BCC; n=12) tissues. Patched 1 and SMO immunoreactivity was significantly higher in BCC than in SyG, TE or MAC (P<0.001 and P<0.03, respectively). The highest IHH expression was observed in BCC and TE compared with SyG and MAC (P<0.04). Notably, the highest SHH protein expression was observed in SyG compared with MAC, TE and even BCC (P<0.001). In patients with MAC, SMO immunoreactivity significantly (r=0.51; P<0.01) correlated with PTCH1 expression. Further correlation studies did not show significant associations between the HH expression markers assessed (P>0.05). Our results indicate that alterations of the HH signalling are unlikely to play a major role in the pathogenesis of MAC, which is in contrast to the morphologically similar BCC and TE. Our observation provides additional information to the limited molecular pathology knowledge on this rare tumour.

Kinetic Efficiency of R252L and D60N Mutants of C. elegans Glucose‐6‐Phosphate Dehydrogenase Relative to the Wild Type

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme present in all species that have thus far been tested (Vuillamy et al., 1988). G6PD is the rate limiting enzyme in the pentose phosphate pathway (PPP) (Wang and Engel, 2009) and reduces NADP+ to NADPH through the oxidation of glucose-6-phosphate (G6P) to 6-phospho-gluconolactone (Hirono et al. 1989). The effectiveness of the G6PD enzyme is vital to the health and survival of an organism, as the PPP is one of the major NADPH production mechanisms, and NADPH protects cells from succumbing to oxidative stress (Wang and Engel, 2009). In the present study, kinetic characterization of the C. elegans orthologs of two known deficient human G6PD variants was performed. Production of the R252L and D60N C. elegans G6PD variants was done via E. coli plasmid expression. Purification of the mutant enzymes was performed on a nickel column, and enzyme kinetics parameters of the mutant G6PD enzymes were determined and compared to those for the wild type enzyme. From the Michaelis-Menten kinetics assay results, it was determined that the Vmax value for the R252L mutant enzyme was 5.9 * 10-3 ± 7.5 * 10-4 µmol/min, the KM value was 1050 ± 200 µM, and the specific activity was 1.2 µmol/min/mg. For the D60N enzyme variant it was determined that the Vmax value was 3.2 * 10-3 ± 2.1 * 10 -4 µmol/min, the KM value was 80 ± 22 µM, and the specific activity was 6.5 µmol/min/mg. Lastly, the Vmax value for the wild type enzyme was 2.2 * 10-3 ± 1.4 * 10 -4 µmol/min, the KM value was 100 ± 27 µM, and the specific activity was 11.1 µmol/min/mg. These results diverge slightly from those previously published on the human orthologs of the two mutants. A 2013 study by Bendaoud et al. in which the human ortholog of the R252L GSPD mutant was first cited, as G6PD Tunisia, found that the mutant retained an estimated 25% of normal enzymatic activity while this study found that the R252L mutant had about 11% activity compared to the wild type. A 1988 study by Vuillamy et al. found that the Metaponto variant (the human ortholog of the D60N mutant) had a KM of 47 µM relative to G6P concentration, which is a little over half that observed in this study. The same study found that the enzymatic activity of the mutant within red blood cells was between 14 and 39% of the wild type while the present study found the activity of the D60N mutant to be about 59% of the wild type. This novel data presents evidence of the deficiency of the C. elegans orthologs of the Tunisia and Metaponto variants of human G6PD, thus illustrating their promise as model systems for their human counterparts. Bendaoud B, Hosni I, Mosbahi I, Hafsia R, Prehu C, Abbes S. 2013. Pathol Biol (Paris). 61(2):64–69. Hirono A, Kuhl W, Gelbart T, Forman L, Fairbanks VF, Beutler E. 1989. Proc Natl Acad Sci USA. 86(24):10015–10017. Vulliamy TJ, D'Urso M, Battistuzzi G, Estrada M, Foulkes NS, Martini G, Calabro V, Poggi V, Giordano R, Town M. 1988. Proc Natl Acad Sci U S A. 85(14):5171–5175. Wang X-T, Engel PC. 2009. Biochim Biophys Acta. 1792(8):804–809.

Metabolic regulation via ketone body receptors under ketogenic conditions

Ketone bodies, including β-hydroxybutyrate and acetoacetate, are important alternative energy sources during energy shortage. β-Hydroxybutyrate also acts as a signaling molecule via specific G protein-coupled receptors (GPCRs) GPR41 and GPR109A (1, 2); however, the specific associated GPCRs and physiological functions of acetoacetate remain unknown. Here we identified acetoacetate as an endogenous agonist for short-chain fatty acid (SCFA) receptor GPR43 by ligand screening in a heterologous expression system (3). Under ketogenic conditions, such as starvation and low-carbohydrate diets, plasma ketone body levels increased markedly, whereas plasma and cecal SCFA levels decreased dramatically, along with an altered gut microbiota composition. In addition, ketone body receptors-deficient mice showed reduced weight loss and suppressed plasma lipoprotein lipase activity during fasting and eucaloric ketogenic diet feeding. Moreover, these mice exhibited minimal weight decrease after intermittent fasting. These observations provide insight into the role of ketone bodies in energy metabolism under shifts in nutrition and may contribute to the development of preventive medicine via diet and foods. (1); Kimura et al., Proc Natl Acad Sci U S A. 2011 (2); Kimura et al., Physiol Rev. 2020 (3); Miyamoto et al., Proc Natl Acad Sci U S A. 2019

Continual learning in medical devices: FDA's action plan and beyond

Continual learning in medical devices: FDA's action plan and beyond