Terminal Mutants Research Articles

In vitro development of resistance against antipseudomonal agents: comparison of novel β-lactam/β-lactamase inhibitor combinations and other β-lactam agents.

We subjected seven P. aeruginosa isolates to a 10-day serial passaging against five antipseudomonal agents to evaluate resistance levels post-exposure and putative resistance mechanisms in terminal mutants were analyzed by whole-genome sequencing analysis. Meropenem (mean, 38-fold increase), cefepime (14.4-fold), and piperacillin-tazobactam (52.9-fold) terminal mutants displayed high minimum inhibitory concentration (MIC) values compared to those obtained after exposure to ceftolozane-tazobactam (11.4-fold) and ceftazidime-avibactam (5.7-fold). Fewer isolates developed elevated MIC values for other β-lactams and agents belonging to other classes when exposed to meropenem in comparison to other agents. Alterations in nalC and nalD, involved in the upregulation of the efflux pump system MexAB-OprM, were common and observed more frequently in isolates exposed to ceftazidime-avibactam and meropenem. These alterations, along with ones in mexR and amrR, provided resistance to most β-lactams and levofloxacin but not imipenem. The second most common gene altered was mpl, which is involved in the recycling of the cell wall peptidoglycan. These alterations were mainly noted in isolates exposed to ceftolozane-tazobactam and piperacillin-tazobactam but also in one cefepime-exposed isolate. Alterations in other genes known to be involved in β-lactam resistance (ftsI, oprD, phoP, pepA, and cplA) and multiple genes involved in lipopolysaccharide biosynthesis were also present. The data generated here suggest that there is a difference in the mechanisms selected for high-level resistance between newer β-lactam/β-lactamase inhibitor combinations and older agents. Nevertheless, the isolates exposed to all agents displayed elevated MIC values for other β-lactams (except imipenem) and quinolones tested mainly due to alterations in the MexAB-OprM regulators that extrude these agents.

Exploring phenazine electron transfer interaction with elements of the respiratory pathways of Pseudomonas putida and Pseudomonas aeruginosa

Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox balances. This electron discharge is also attractive to be used for bioelectrochemical applications. However, elements of the respiratory pathways that interact with phenazines are not well understood. Five terminal oxidases are involved in the aerobic electron transport chain (ETC) of Pseudomonas putida and P. aeruginosa. The latter bacterium also includes four reductases that allow for denitrification. Here, we explored if phenazine-1-carboxylic acid interacts with those elements to enhance anodic electron discharge and drive bacterial growth in oxygen-limited conditions. Bioelectrochemical evaluations of terminal oxidase-deficient mutants of both Pseudomonas strains and P. aeruginosa with stimulated denitrification pathways indicated no direct beneficial interaction of phenazines with ETC elements for extracellular electron discharge. However, the single usage of the Cbb3-2 oxidase increased phenazine production, electron discharge, and cell growth. Assays with purified periplasmic cytochromes NirM and NirS indicated that pyocyanin acts as their electron donor. We conclude that phenazines play an important role in electron transfer to, between, and from terminal oxidases under oxygen-limiting conditions and their modulation might enhance EET. However, the phenazine-anode interaction cannot replace oxygen respiration to deliver energy for biomass formation.

125. Induction of Resistance Against Antipseudomonal Agents: Comparison of Novel b-Lactam/b-Lactamase Inhibitor (BL/BLI) Combinations and Other b-lactam Agents

Abstract Background The acquisition of mutations is the main driver of β-lactam resistance in Pseudomonas aeruginosa isolates. New BL/BLIs, such as ceftazidime-avibactam (CAZ-AVI), ceftolozane-tazobactam (C/T), and imipenem-relebactam (IMI-REL), are active against most P. aeruginosa isolates from US hospitals, but the ability of these agents to induce resistance have not been explored. We subjected 8 P. aeruginosa isolates, including ATCC 27853 and 7 clinical isolates, to a 10-day serial passage with 6 antipseudomonal agents to evaluate resistance levels and mechanisms in terminal mutant strains. Fold change in MIC results from parent isolate to terminal mutant Methods Serial passaging was performed in broth microdilution (BMD) for CAZ-AVI, IMI-REL, C/T, meropenem (MEM), cefepime (FEP), and piperacillin-tazobactam (P/T). The MIC of the terminal mutants was determined after 2X passaging on drug-free agar. Parent strains and terminal mutants were subjected to short-read whole genome sequencing (WGS) at 100X coverage. Parent isolates were sequenced using long-read WGS and the data was combined with short-reading sequencing for single nucleotide polymorphism (SNP) analysis. Results Overall, IMI-REL (1- to 4-fold) and CAZ-AVI (2- to 8-fold) displayed lower fold increases in MIC values when compared to other agents tested (Figure). Of the CAZ-AVI terminal mutants, 3 displayed a nalD regulator alteration, and 1 of these had a clpA chaperone missense substitution. FEP terminal mutants exhibited alterations in ampD, mexB, and the TetR family transcriptional regulator AmrR. C/T mutants had ampG and ftsI missense alterations. MEM mutants had nalC, ftsI, and phoP missense alterations. Mutations in merR, nalC, and ampD were observed in the P/T terminal mutants. Among 2 IMI-REL terminal mutants displaying a SNP alteration, 1 displayed a nonsense mutation in pilF, a pilus forming protein. Many terminal mutants displayed alterations in genes not commonly associated to β-lactam resistance. Conclusion MEM, FEP, and P/T terminal mutants displayed high MIC values compared to those obtained after exposure to C/T, CAZ-AVI, and IMI-REL. This data might indicate a benefit of using these newer agents to prevent the emergence of high-level resistance. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support Timothy Doyle, MS, AbbVie: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support Jessica Ewald, PhD, AbbVie: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support.

H2O2 Induces Calcium and ERMES Complex-Dependent Mitochondrial Constriction and Division as Well as Mitochondrial Outer Membrane Remodeling in Aspergillus nidulans.

The dynamin-like protein DnmA and its receptor FisA are essential for H2O2-induced mitochondrial division in Aspergillus nidulans. Here, we show that in the absence of DnmA or FisA, mitochondria show few spontaneous transient constrictions, the frequency of which is extensively increased by H2O2 or the carbonyl cyanide m-chlorophenyl hydrazone (CCCP). While H2O2-induced constrictions are transient, CCCP induces a drastic and irreversible alteration of mitochondrial filaments. H2O2 induces a gradual mitochondrial depolarization, while CCCP-induced depolarization is abrupt. The calcium chelator BAPTA-AM prevents the formation of mitochondrial constrictions induced by either H2O2 or CCCP. H2O2 also induces major rearrangements of the mitochondrial outer membrane, which remain after constrictions dissipate, as well as changes in endoplasmic reticulum (ER) and nuclear morphology. Similar mitochondrial constriction, ER and nuclear morphology changes are detected during the early stages of asexual development. ER and ER-Mitochondria encounter structure (ERMES) complex—composed of proteins Mdm10, Mmm1, Mdm43 and Mdm12—are important for mitochondrial division in Saccharomyces cerevisiae. As the Mdm10 ortholog MdmB was found to be essential in A. nidulans, we evaluated its functions in ΔmdmB terminal mutants and ΔmdmB heterokaryons. ΔmdmB conidia produce a short germ tube that fails to grow further, in which inherited mitochondria become gigantic and round shaped, lacking clear contacts with the ER. In slow-growing ΔmdmB heterokaryotic mycelia, multiple hyphae contain very long mitochondria with high ROS levels, as occur in ΔdnmA and ΔfisA mutants. In this hyphae, H2O2 fails to induce mitochondrial constrictions but not outer mitochondrial membrane reshaping, indicating that these are two separate effects of H2O2. Our results indicate that H2O2 induces a generalized mitochondrial constriction response, prior to actual division, involving gradual depolarization; they also indicate that Ca2+ and the ERMES complex are critical for both mitochondrial constriction and division. This supports a view of mitochondrial dynamics as the result of a cascade of signaling events that can be initiated in vivo by H2O2.

In-Vitro Selection of Ceftazidime/Avibactam Resistance in OXA-48-Like-Expressing Klebsiella pneumoniae: In-Vitro and In-Vivo Fitness, Genetic Basis and Activities of β-Lactam Plus Novel β-Lactamase Inhibitor or β-Lactam Enhancer Combinations.

Ceftazidime/avibactam uniquely demonstrates activity against both KPC and OXA-48-like carbapenemase-expressing Enterobacterales. Clinical resistance to ceftazidime/avibactam in KPC-producers was foreseen in in-vitro resistance studies. Herein, we assessed the resistance selection propensity of ceftazidime/avibactam in K. pneumoniae expressing OXA-48-like β-lactamases (n = 10), employing serial transfer approach. Ceftazidime/avibactam MICs (0.25–4 mg/L) increased to 16–256 mg/L after 15 daily-sequential transfers. The whole genome sequence analysis of terminal mutants showed modifications in proteins linked to efflux (AcrB/AcrD/EmrA/Mdt), outer membrane permeability (OmpK36) and/or stress response pathways (CpxA/EnvZ/RpoE). In-vitro growth properties of all the ceftazidime/avibactam-selected mutants were comparable to their respective parents and they retained the ability to cause pulmonary infection in neutropenic mice. Against these mutants, we explored the activities of various combinations of β-lactams (ceftazidime or cefepime) with structurally diverse β-lactamase inhibitors or a β-lactam enhancer, zidebactam. Zidebactam, in combination with either cefepime or ceftazidime, overcame ceftazidime/avibactam resistance (MIC range 0.5–8 mg/L), while cefepime/avibactam was the second best (MIC: 0.5–16 mg/L) in yielding lower MICs. The present work revealed the possibility of ceftazidime/avibactam resistance in OXA-48-like K. pneumoniae through mutations in proteins involved in efflux and/or porins without concomitant fitness cost mandating astute monitoring of ceftazidime/avibactam resistance among OXA-48 genotypes.

Leukemogenic Second Hits in CEBPA-Mutated AML Result in Diverse Effects on Haematopoiesis

Acute myeloid leukaemia (AML) is thought to develop through the stepwise acquisition of genetic aberrations within a haematopoietic stem or progenitor cell (HSPC) resulting in clonal expansion. N terminal mutations of the myeloid transcription factor CEBPA are not only a frequent event in sporadic AML, but they are also inherited in the germ line, conveying familial AML predisposition and thus can be assured to be an initiating event in leukaemogenesis. CEBPA-mutated AML also shows a remarkably conserved landscape of secondary mutations. Around a third of cases harbour mutations in the epigenetic regulator tet methylcytosine dioxygenase 2 (TET2), a gene frequently implicated in the pathogenesis of myeloid malignancy. A further third of cases are found to have mutations in the transcription factor, GATA2, moreover this latter association appears to be almost exclusive to CEBPA-mutated cases in AML.We have used a zebrafish model to interrogate the effects of these signature mutation patterns on haematopoiesis in juvenile and adult fish. A CEBPA N terminal mutant line (CebpaNterm) was generated using TALEN pairs, with a 5bp deletion leading to a frameshift between the first and second reading frame of this single exon gene, emulating that seen in both familial and sporadic AML cases. In homozygosity cebpaNTerm leads to complete absence of mature myeloid cells until 4 weeks of age. However, at 6 weeks of age all surviving cebpaNterm/Nterm fish had developed granulated myeloid cells on assessment of whole kidney marrow (WKM), with a dosage dependent increase in numbers with loss of wild-type (WT) Cebpa. By contrast in the peripheral blood (PB), a dose dependent reduction in both thrombocyte and myeloid cell numbers was observed with loss of WT Cebpa, but no evidence of anaemia. No homozygotes have survived past 2 months, due to susceptibility to infection and development of leukaemia with primitive features.To assess the effects of Tet2 loss in combination with cebpaNterm/+ we utilised a Tet2 null mutant line (tet2zdf20) crossed to cebpaNterm/+. Tet2zdf20/+ showed on haematopoietic lineages at 3 and 5 months of age as previously described {Gjini:2015bs}. However, when combined with heterozygosity for cebpaNterm, loss of Tet2 showed a cumulative deficit in both HSPC and myeloid cell numbers. Further evidence of cooperation between the two mutations was seen by assessment of thrombocyte number in PB, with platelet numbers steadily trending to their nadir at the maximum allelic loss i.e. cebpaWT/Nterm ; tet2zdf20/zdf20 noting that cebpaNterm/Nterm do not survive to this age. Whilst no anaemia was observed at 3 months in any genotype, by 5 months cebpaWT/Nterm ; tet2zdf20/+ fish showed a significant reduction in erythroid progenitors, suggesting an anticipatory effect of cebpa mutation on the tet2zdf20/+ myelodysplastic phenotype not usually observed until 8 months of age.Combination of cebpaNterm/+ with a gata2a (one of 2 zebrafish GATA2 orthologues) mutant carrying a truncating mutation prior to the zinc finger domains (gata2aum27) revealed a different picture. On assessment at 5 months gata2aum27/+ showed a reduction in both HSPC and myeloid cell numbers in the WKM, while cebpaNterm/WT showed an increase. In keeping with each individual mutation's influence on cell numbers, cebpaNterm/WT; gata2aum27/+ compound heterozygotes appeared showed near WT levels in both lineages. No abnormalities in erythroid progenitors or peripheral anaemia was evident in any genotype.In summary, we have described diverse haematopoietic effects of two known secondary mutations in cebpaNterm mutated AML. Mutations in Tet2 cooperate with cebpaNterm resulting in reduced cellularity across lineages. In the case of Gata2a, the addition of heterozygous cebpaNterm mutation appears to rescue a myeloid progenitor defect present in gata2aum27/+. Finally, in contrast to cebpaNterm/Nterm, we did not observe any cases of frank leukaemia in cebpaNterm combined with Gata2a or Tet2 loss. We speculate this is because the most common second hit in cebpaNterm, is a cebpa mutation in the carboxy terminal of the second cebpa allele, and this may be requisite for the development of AML. We have now generated a zebrafish with C terminal Cebpa mutation in order to test this hypothesis. DisclosuresNo relevant conflicts of interest to declare.

Optogenetic Rescue of a Patterning Mutant

Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.

Histone 1 Mutations Drive Lymphomagenesis By Inducing Primitive Stem Cell Functions and Epigenetic Instructions through Profound 3D Re-Organization of the B-Cell Genome

Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.

A Zebrafish Model of Cooperating C and N Terminal CEBPA Mutations Reveals Defects in Early Myelopoeisis and HSPCs Leading to Leukaemogenesis

Acute Myeloid Leukaemia (AML) is thought to occur due to stepwise accumulation of mutations in haematopoietic stem and progenitor cells (HSPCs) or early myeloid progenitor cells (EMPs) to either block differentiation or increase proliferation. In around 10% of sporadic AML cases and several known germline predisposition kindreds the myeloid transcription factor CEBPA is mutated. 50% of cases possess biallelic mutations in both C and N terminae (dm) and 50% monoallelic mutation (sm). Using TALENs we have created both C and N terminal mutants in zebrafish to study their cooperating effects in the development of AML.Our first observation was of striking defects in mature myelocytes and monocytes in all double mutants (cebpamut/mut) as assessed by Sudan black staining at 5 days post fertilisation (dpf) and apoeb whole mount in situ hybridisation (WISH) at 3dpf. In situ hybridisation revealed defects in myelopoeisis in all cebpamut/mut fry as early as 28hpf, with markedly decreased expression of coronin and l-plastin.We then interrogated the model further to assess when and where the differentiation block was occurring. We utilised the transcription factor pu.1 both as an in vivo transgenic marker Tg(pu.1:GFP) and WISH probe at earlier time points. In primitive haematopoiesis (until 22hpf) pu.1 expression showed no defects in dm mutants. However, pu.1 expressing cells were markedly reduced in dm mutants in establishment of myelopoeisis in the caudal haematopoietic tissue (CHT), fetal liver equivalent. Following formation of the CHT at around 30hpf, differing patterns of pu.1 expression were seen in sm mutants, with cebpaWT/Cterm having significantly increased staining, which was reduced in cebpaWT/Nterm (see figure 1).HSPC numbers in cebpamut/mut were seen to be normal by WISH for gata2b and runx1 and flow cytometry in Tg(CD41:GFP) fry during early haematopoeisis (2-5dpf). However, myb expression was markedly increased at 36hpf and 3 days but only in cebpaCterm/Nterm and cebpaNterm/Nterm dm mutants (see figure 2). This suggests an accumulation of early progenitors with myeloid potential but not commitment, as implied by absence of pu.1 expression but normal CD41lo numbers and other stem cell marker expression.Absence of MCherry expression in the transgenic LysC:MCherry, which highlights mature myeloid cells in vivo, was sufficiently reliable to genotype all cebpamut/mut fry and observe them for impaired survival compared to wild-type and heterozygous siblings and for the development of leukaemia. Similarly to the increase in myb staining, leukaemic transformation was only observed in dm mutants with at least one N terminal mutation, as assessed by development of anaemia and florid Tg(CD41:GFP) expression. Flow cytometry in juvenile fish (4-6weeks) also identifies a developing pre-leukaemic phenotype with moderate yet significant expansion of HSPCs and continuing absence LysC expression in cebpaNterm/Cterm and cebpaNTerm/NTerm. However, survival is poor in all dm fish, likely due to the metabolic role of cebpa and vulnerability to infection secondary to the severe myelomonocytic defect.Our results show that absence of WT cebpa has dramatic effects myelopoiesis from early stages of differentiation in definitive haematopoiesis. In addition, accumulation of myb expressing progenitors occurs as they arise from the aorta in dm mutants, identifying this as a sub-population of HSPC vulnerable to further leukaemogenic hits. Ongoing work will define the mechanism of these effects in sm and dm mutants and include comparative expression profiling in myb and pu.1 positive cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Clonal heterogeneity of acute myeloid leukemia treated with the IDH2 inhibitor enasidenib.

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.

Electron cryotomography of Mycoplasma pneumoniae mutants correlates terminal organelle architectural features and function.

The Mycoplasma pneumoniae terminal organelle functions in adherence and gliding motility and is comprised of at least eleven substructures. We used electron cryotomography to correlate impaired gliding and adherence function with changes in architecture in diverse terminal organelle mutants. All eleven substructures were accounted for in the prkC, prpC and P200 mutants, and variably so for the HMW3 mutant. Conversely, no terminal organelle substructures were evident in HMW1 and HMW2 mutants. The P41 mutant exhibits a terminal organelle detachment phenotype and lacked the bowl element normally present at the terminal organelle base. Complementation restored this substructure, establishing P41 as either a component of the bowl element or required for its assembly or stability, and that this bowl element is essential to anchor the terminal organelle but not for leverage in gliding. Mutants II-3, III-4 and topJ exhibited a visibly lower density of protein knobs on the terminal organelle surface. Mutants II-3 and III-4 lack accessory proteins required for a functional adhesin complex, while the topJ mutant lacks a DnaJ-like co-chaperone essential for its assembly. Taken together, these observations expand our understanding of the roles of certain terminal organelle proteins in the architecture and function of this complex structure.

The impacts of nineteen mutations on the enzymatic activity of USP26

ObjectiveThe association between mutations in the USP26 gene and male infertility has been studied intensively. However, the biological function of the mutant proteins remains to be elucidated. To confirm the effects of the reported mutations, we analyse the enzyme activity of USP26 between the wild-type and the variants from a molecular perspective. MethodsUsing pGEX-USP26 as a template, site-directed mutagenesis was conducted to generate nineteen USP26 mutant plasmids. Using Ub-Met-β-gal and GST-Ub52 as model substrates, a USP cleavage assay was conducted to assess the enzymatic activities of the mutants. ResultsThe enzyme activity of the Q156H mutant disappeared, but the other 18 mutants had the same activity as the wild type. E174# and E189# were terminal mutants, but they still had the same activity as the wild type. When we constructed the transcription terminal mutants E174#(1-522 bp), E174#(523-2742 bp), E189#(1-567 bp) and E189#(568-2742 bp) artificially, the enzyme activity of these four mutants disappeared. ConclusionsWe have successfully constructed nineteen mutants of USP26. The enzyme activity of the Q156H mutant disappeared, but the enzyme activities of the other 18 mutants were the same as that of the wild type.

Enhanced antitumor effect of biodegradable cationic heparin-polyethyleneimine nanogels delivering FILIP1LΔC103 gene combined with low-dose cisplatin on ovarian cancer.

FILIP1LΔC103 (COOH terminal truncation mutant 1-790 of Filamin A Interacting Protein 1-Like) has been identified to hold therapeutic potential for suppressing tumor growth. Cisplatin (DDP) is commonly used as a first-line drug in the treatment for ovarian cancer. The usage of polymeric nanoparticles to deliver functional genes intraperitoneally holds much promise as an effective therapy for ovarian cancer. In this study, a recombinant plasmid expressing FILIP1LΔC103 (FILIP1LΔC103-p) was constructed, and HPEI nanogels were prepared to deliver FILIP1LΔC103-p into SKOV3 cells. The expression of FILIP1LΔC103 in vitro and in vivo was determined using RT-PCR and Western Blotting. Moreover, in vivo treatment experiments were conducted on nude mice bearing SKOV3 ovarian cancer. The mice were treated with 5% glucose, HPEI+E-p, HPEI+FILIP1LΔC103-p, DDP or HPEI+FILIP1LΔC103-p plus DDP, respectively. Tumor weights were evaluated throughout the treatment duration. The cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay respectively, and the anti-angiogenic effect was assessed by CD31 immunochemical staining and alginate-encapsulated tumor cell assay. FILIP1LΔC103-p could be efficiently transfected into SKOV3 cells by HPEI nanogels. The combination of HPEI+FILIP1LΔC103-p with DDP exerted enhanced antitumor activity compared with HPEI+FILIP1LΔC103-p or DDP alone. Significant reduction of tumor cells proliferation, augmentation of tumor cells apoptosis and suppression of angiogenesis were observed in the combination group compared with controls. Our results demonstrated synergistic antineoplastic activity of combined FILIP1LΔC103 and low-dose DDP with no apparent toxicity, indicating a potential application of the combined approach in the treatment of ovarian cancer.

Identification of human somatostatin receptor 2 domains involved in internalization and signaling in QGP-1 pancreatic neuroendocrine tumor cell line.

Somatostatin exerts inhibitory effects on hormone secretion and cell proliferation via five receptor subtypes (SST1-SST5), whose internalization is regulated by β-arrestins. The receptor domains involved in these effects have been only partially elucidated. The aim of the study is to characterize the molecular mechanism and determinants responsible for somatostatin receptor 2 internalization and signaling in pancreatic neuroendocrine QGP-1 cell line, focusing on the third intracellular loop and carboxyl terminal domains. We demonstrated that in cells transfected with somatostatin receptor 2 third intracellular loop mutant, no differences in β-arrestins recruitment and receptor internalization were observed after somatostatin receptor 2 activation in comparison with cells bearing wild-type somatostatin receptor 2. Conversely, the truncated somatostatin receptor 2 failed to recruit β-arrestins and to internalize after somatostatin receptor 2 agonist (BIM23120) incubation. Moreover, the inhibitory effect of BIM23120 on cell proliferation, cyclin D1 expression, P-ERK1/2 levels, apoptosis and vascular endothelial growth factor secretion was completely lost in cells transfected with either third intracellular loop or carboxyl terminal mutants. In conclusion, we demonstrated that somatostatin receptor 2 internalization requires intact carboxyl terminal while the effects of SS on cell proliferation, angiogenesis and apoptosis mediated by somatostatin receptor 2 need the integrity of both third intracellular loop and carboxyl terminal.

Importance of the N-terminal proline for the promiscuous activity of 4-oxalocrotonate tautomerase (4-OT)

Michael addition of aldehydes to nitroolefins is an effective method to obtain useful chiral ?-nitroaldehydes. ?-Nitroaldehydes are precursors for chiral ?-aminobytiric acid analogues which have numerous pharmacological activities and are used for treatment of neurological disorders. A whole-cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) has been developed and shown to be an effective biocatalyst for Michael addition of branched aldehydes to ?-nitrostyrenes. The aim of this study was to investigate the influence of the substitution of the N-terminal proline with lysine and arginine both containing the reactive ?-amino group, on the Michael addition catalyzed by 4-OT. Firstly, the effect of these mutations was examined by in silico analysis, followed by generation of three terminal ly-sine mutants. The generated mutants, 4-OT_K, 4-OT_PK and 4-OT_KK were tested for the ability to utilise ?-nitrostyrene (1), (E)-2-(thiophene-2-yl)nitroethen (2) and p-chloro-trans-?-nitrostyrene (3) as Michael acceptors with isobutanal as the donor. For comparison, the lithium salt of lysine was used in the same organocatalytic reactions. In general, the introduction of lysine had a negative effect on Michael additions based on overall product yields. However, additional lysine residues at the N-terminus of the protein resulted in structural changes that enhanced the activity towards 2 and 3. Therefore, the N-terminal proline is important for the 4-OT catalysed Michael-additons, but it is not essential.

Functional conservation of the pre-sensor one beta-finger hairpin (PS1-hp) structures in mini-chromosome maintenance proteins of Saccharomyces cerevisiae and archaea.

Mini-chromosome maintenance (MCM) proteins form complexes that are required for DNA replication and are highly conserved throughout evolution. The replicative helicase of eukaryotic organisms is composed of the six paralogs MCM2-7, which form a heterohexameric ring structure. In contrast, the structure of the archaean replicative MCM helicase is a single Mcm protein that forms a homohexameric complex. Atomic structures of archaeal MCMs have identified multiple beta-finger structures in Mcm proteins whose in vivo function is unknown. In the present study, we have investigated the physiological role of the pre-sensor 1 beta-hairpin (PS1-hp) beta-fingers of Saccharomyces cerevisiae Mcm4p and Mcm5p in DNA replication initiation and elongation in vivo. The PS1-hp beta-finger mutant of Mcm5p (mcm5-HAT K506A::URA3) has a growth defect at both 18° and 37°. Mutation of the Mcm4p PS1-hp beta-finger (mcm4-HA K658A::TRP1) does not have a growth defect, indicating different functional contributions of the PS1-hp beta-finger structures of different MCM helicase subunits. Both Mcm4p and Mcm5p PS1-hp beta-finger mutants can coimmunoprecipitate Mcm2p, indicating the formation of the hexameric MCM helicase complex. Both PS1-hp beta-finger mutants have a plasmid loss phenotype that is suppressible by origin dosage, indicating a defective replication initiation. Surprisingly, a defect in the binding of PS1-hp MCM mutants to origins of DNA replication was not found by chromatin immunoprecipitation, suggesting a novel interpretation in which the defect is in a subsequent step of DNA strand separation by the MCM helicase. The double mutant mcm4-HA K658A::TRP1mcm5-HAT K506A::URA3 is lethal, displaying a terminal MCM mutant phenotype of large budded cells.

RNA Sequencing Of Adult Bone Marrow Erythroid Populations Reveals Significant Transcriptional Deregulation In Foxo3-/- Mutant Cells

Transcriptional control of last stages of erythropoiesis is a complex and well orchestrated process controlled by lineage-specific transcription factors. The precise contribution of the different transcription factors to this multistep process has not been fully elucidated. Foxo3 is a transcription factor that is required for terminal erythroid maturation and Foxo3 mutant mice exhibit ineffective erythropoiesis. In order to gain further insight into the contribution of Foxo3 to the control of adult terminal erythroid maturation we analyzed the transcriptome of three adult bone marrow erythroid precursor populations: pro-, basophilic and polychromatophilic erythroblasts from wild type and Foxo3-/- mice. Populations were FACS sorted according to their TER119 and CD44 cell surface expression and FSC properties. RNA was then isolated and sequenced using the Illumina GaII platform. Genes were grouped into 3 categories according to their expression during erythroid cell maturation using the Short Time Series Expression Miner (STEM) program: no change (4577 genes), down-regulated (2868 genes) or up-regulated (2637) (Figure 1). Enrichment analysis of groups of genes using the ChEA database identified Myb, Meis1, Runx1, Fli1 and PU.1 as the main transcription factors regulating gene repression over erythroid maturation. In contrast, ChEA identified known erythroid transcription factors like Gata1, Eklf and Tal1 to drive the up-regulation of many of the erythroid-specific genes. This analysis also enabled the identification of putative novel transcription factors implicated in erythroid cell maturation. Interestingly, the difference between WT and Foxo3-/- cells increased gradually from pro- to polychromatophilic erythroblasts in correlation with increased Foxo3 expression during these steps of maturation. Strikingly, pathway enrichment analysis detected several immune-related pathways such as Toll-like receptors, TGF-β and IL-1 signaling as expressed in maturing wild type erythroid cells and significantly deregulated in Foxo3-/- cells. The expression of a number of these immune genes in erythroid cells has been validated by qRT-PCR. In addition, among others, a cluster of genes from the autophagy pathway was noted to be significantly down-regulated in Foxo3 mutant erythroid cells. In order to better dissect Foxo3 transcriptional control during erythroid maturation, STEM analysis of Foxo3-/- samples revealed an unexpected number of differences compared to WT. Most remarkably the STEM analysis identified that 90% of the 1198 genes that are continuously up-regulated during erythroid maturation from pro- to polychromatophilic are highly compromised in their level of expression during erythroid maturation in the absence of Foxo3. Interestingly, this group was also enriched for Foxo3 direct target genes as determined by ChIP-seq studies. We also identified a subset of genes whose expression increased from pro- to basophilic erythroblasts but decreased thereafter in the absence of Foxo3 in contrast to wild type cells. Interestingly, ChEA analysis on this group identified a subset of genes that are targets of Gata1, Eklf and Tal1 that may require Foxo3 for their full expression at the last stages of erythroid cell maturation. In conclusion, we present an unbiased genome-wide approach using RNA sequencing of adult bone marrow erythroid cells to study the contribution of Foxo3 to the regulation of gene expression at the last stages of erythroid cell maturation. This analysis enabled us to identify novel genes and pathways whose function in the control of red cell generation requires further investigations. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Differences in β‐arrestin dependent signaling by PAR2 and NK1R are directed by their C terminal tails

PAR2 and NK1R are 7 transmembrane receptors (7TMRs), which signal by Gαq leading to Ca2+ release and protein kinase C (PKC) activation. Both receptors are desensitized by β‐arrestin binding to their C termini. They can also activate ERK1/2 through β‐arrestin scaffolding complexes. They differ in ERK1/2 mediated physiological outcomes: cell migration versus proliferation. Using β‐arrestins, PAR2 activates cofilin, to promote chemotaxis. We hypothesized that the differences in β‐arrestin dependent signaling by 7TMRs can be attributed to how the molecular scaffolds bind the C‐tail of the receptors. Using wild type and C‐tail chimeras, we showed that the rate of desensitization, internalization, subcellular localization post endocytosis and ERK1/2 mediated responses depend on the nature of interaction of β‐arrestins with the C‐termini. BRET assays showed that PAR2 recruits both β‐arrestin 1/2 faster and with greater affinity than NK1R. We further show that initial Gαq signaling is necessary for β‐arrestin 1/2 recruitment to NK1R. PAR2 recruits β‐arrestin 1/2 even when Gαq pathway is blocked. Assays with C terminal phosphorylation mutants of PAR2 indicated that, phosphorylation of certain residues are necessary for β‐arrestin 1/2 recruitment. Phosphorylation at putative PKC sites determine stability of PAR2‐β‐arrestin 1/2 complex. This in turn ensures downstream cofilin activation and cell migration

Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities.

Cyanobacteria perform photosynthesis and respiration in the thylakoid membrane, suggesting that the two processes are interlinked. However, the role of the respiratory electron transfer chain under natural environmental conditions has not been established. Through targeted gene disruption, mutants of Synechocystis sp. PCC 6803 were generated that lacked combinations of the three terminal oxidases: the thylakoid membrane-localized cytochrome c oxidase (COX) and quinol oxidase (Cyd) and the cytoplasmic membrane-localized alternative respiratory terminal oxidase. All strains demonstrated similar growth under continuous moderate or high light or 12-h moderate-light/dark square-wave cycles. However, under 12-h high-light/dark square-wave cycles, the COX/Cyd mutant displayed impaired growth and was completely photobleached after approximately 2 d. In contrast, use of sinusoidal light/dark cycles to simulate natural diurnal conditions resulted in little photobleaching, although growth was slower. Under high-light/dark square-wave cycles, the COX/Cyd mutant suffered a significant loss of photosynthetic efficiency during dark periods, a greater level of oxidative stress, and reduced glycogen degradation compared with the wild type. The mutant was susceptible to photoinhibition under pulsing but not constant light. These findings confirm a role for thylakoid-localized terminal oxidases in efficient dark respiration, reduction of oxidative stress, and accommodation of sudden light changes, demonstrating the strong selective pressure to maintain linked photosynthetic and respiratory electron chains within the thylakoid membrane. To our knowledge, this study is the first to report a phenotypic difference in growth between terminal oxidase mutants and wild-type cells and highlights the need to examine mutant phenotypes under a range of conditions.