Simultaneous Inactivation Research Articles

Performic Acid Disinfection of Municipal Secondary Effluent Wastewater: Inactivation of Murine Norovirus, Fecal Coliforms, and Enterococci.

Performic acid (PFA) is an emerging disinfectant to inactivate bacterial and viral microorganisms in wastewater. In this study, the inactivation kinetics of murine norovirus (MNV) by PFA, in phosphate buffer and municipal secondary effluent wastewater, are reported for the first time. PFA decay followed first-order kinetics and the inactivation of MNV was governed by the exposure of microorganisms to PFA, i.e., the integral of the PFA concentration over time (integral CT or ICT). The extension of the Chick-Watson model, in the ICT domain, described well the reduction of MNV by PFA, with determined ICT-based inactivation rate constants, kd, of 1.024 ± 0.038 L/(mg·min) and 0.482 ± 0.022 L/(mg·min) in phosphate buffer and wastewater, respectively, at pH 7.2. Furthermore, the simultaneous PFA inactivation of MNV and fecal indicators indigenously present in wastewater such as fecal coliforms and enterococci showed that 1-log reduction could be achieved with ICT of 2, 1.5, and 3.5 mg·min/L, respectively. When compared with the most commonly used peracid disinfectant of municipal wastewater, peracetic acid (PAA), the ICT requirements determined using the fitted ICT-based kinetic models were ∼20 times higher for PAA than PFA, indicating a much stronger inactivation power of the PFA molecule.

Preferential use of carbon central metabolism and anaerobic respiratory chains in porcine extraintestinal pathogenic Escherichia coli during bloodstream infection

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is occurring with increasing frequency in China, and leads to significant economic and welfare costs in the swine industry. The underlying mechanisms of porcine ExPEC in blood colonization during systematic infection is poorly understood. Here we measured the gene expression of porcine ExPEC in infected animal bloodstream in vivo and fresh swine blood in vitro. Using comparisons with P values of ≤ 0.01, we identified 354 and 313 genes as being significantly up- or down-regulated at least 2-fold change during bloodstream infection, respectively. Excepting for an array of iron acquisition systems, numerous genes involved in carbon central metabolism and anaerobic respiratory chains were upregulated here. These genes were categorized into several clusters including the TCA-cycle (frdABCD, citCEFXG), d-ribose transporter (rbsDACB), nickel transporter (nikABCDER), NiFe hydrogenase (hybOABCDEF, hycBCDEFG), Hyp-complex (hypABCDE), DMSO reductase (dmsABC and ynfEFGHI), format dehydrogenase (fdnGHI) and NADH dehydrogenase I (nuoA-N). The mutant with simultaneous inactivation of ribose and citrate imports showed significant reduced fitness in host blood, suggesting these two carbohydrates are utilized by central metabolism network as important carbon-source during bloodstream infection. Similar deficiency was also observed in the mutant double deleted NiFe hydrogenase 2 and 3 anaerobic respiratory chains. Further study found that FNR (a global regulator facilitating bacterial adaptation to anaerobic conditions) is an important regulator in response to bloodstream to activate center metabolism and anaerobic respiratory chains, thus contribute to the full-virulence of porcine ExPEC. These findings provide compelling evidence to support the notion that carbon central metabolism network and anaerobic respiratory chains play key roles for porcine ExPEC fitness within host bloodstream.

Regulation of electron transport is essential for photosystem I stability and plant growth.

Photosynthetic electron transport is regulated by cyclic and pseudocyclic electron flow (CEF and PCEF) to maintain the balance between light availability and metabolic demands. CEF transfers electrons from photosystem I to the plastoquinone pool with two mechanisms, dependent either on PGR5/PGRL1 or on the type I NADH dehydrogenase-like (NDH) complex. PCEF uses electrons from photosystem I to reduce oxygen and in many groups of photosynthetic organisms, but remarkably not in angiosperms, it is catalyzed by flavodiiron proteins (FLVs). In this study, Physcomitrella patens plants depleted in PGRL1, NDH and FLVs in different combinations were generated and characterized, showing that all these mechanisms are active in this moss. Surprisingly, in contrast to flowering plants, Physcomitrella patens can cope with the simultaneous inactivation of PGR5- and NDH-dependent CEF but, when FLVs are also depleted, plants show strong growth reduction and photosynthetic activity is drastically reduced. The results demonstrate that mechanisms for modulation of photosynthetic electron transport have large functional overlap but are together indispensable to protect photosystem I from damage and they are an essential component for photosynthesis in any light regime.

1721-P: In Vivo Effect of Perturbations in Hepatic Insulin Signaling on the Synthesis and Export of De Novo Synthesized Fatty Acids and Triglycerides

Hepatic lipid synthesis and Vldl production plays a major role in whole body lipid homeostasis. Insulin imposed regulation of liver metabolism is in large part executed on a transcriptional level. Several published studies focused on the effect of hepatic insulin resistance and the role of mTORC1 in liver lipid metabolism, sometimes with conflicting results. Here we used mouse genetic models with short-term liver specific removal of the insulin receptor (complete signaling pathway inactivation), Tsc1 (mTORC1 activation), Raptor (mTORC1 inactivation), Pten (simultaneous mTORC1 and Akt activation), and Raptor Pten (simultaneous mTORC1 inactivation and Akt activation) to precisely dissect the role of insulin signaling in the regulation of lipid metabolism. In vivo 2H2O administration, 1H and 2H NMR analyses revealed hepatic de novo fatty acid (FA) and triglyceride (TG) synthesis, export, as well as FA profiles both in liver and plasma. By direct examination of in vivo hepatic de novo lipogenesis (DNL), the direction and magnitude of changes observed in lipogenic gene expression did not correlate well with differences observed in DNL flux. For example, PtenLKO mice had a 3-fold increase in DNL flux with little effect on lipogenic gene expression. Our results suggest that in some contexts other factors, possibly including metabolite availability, TCA cycle metabolism, redox state or energy charge impose stronger control over DNL than regulation of gene expression. We also show that triglyceride synthesis and incorporation of de novo synthesized FA into triglycerides is regulated by mTORC1. While mTORC1 positively regulates export of newly synthesized TG it has a negative effect on export of de novo produced FA. Disclosure B. Kucejova: None. S. Deja: None. J. Duarte: None. X. Fu: None. S.C. Burgess: None. Funding National Institutes of Health (R01DK078184)

Abstract A36: Organoid models for deciphering roles of the evolving landscape of epigenetic heterogeneity during aging in cancer development

Abstract Background: Tumor initiation and development occur not necessarily by genetic changes alone, but in the context of the epigenome and cell microenvironment. Growing evidence indicate that cancer risk factors, such as aging and inflammation, may primarily modulate the epigenome, causing predisposition to cancer development by driver mutations. Thus, there is a need to study and model epigenetic changes preceding and associating with cancer development. We have developed ex vivo models for investigating the evolution and roles of DNA methylation changes during normal cell divisions and how these epigenetic changes play critical roles in early stages of tumor development. Results: We showed that spontaneous epigenetic changes, specifically promoter-DNA hypermethylation, have a very important role in tumor initiation by oncogenic driver mutations. We addressed the precursor role of aging-like spontaneous promoter-DNA hypermethylation in initiating tumorigenesis. Using mouse colon-derived organoids, we show promoter hypermethylation spontaneously arises in cells mimicking the human “aging-like” phenotype. The silenced genes activate the Wnt pathway, causing a stemlike state and differentiation defects. These changes render aged organoids profoundly more sensitive than young ones to transformation by BrafV600E producing the typical human proximal BRAFV600E-driven colon adenocarcinomas characterized by extensive, abnormal gene promoter CGI methylation or the methylator phenotype (CIMP). Conversely, CRISPR-mediated simultaneous inactivation of a panel of the silenced genes markedly sensitized to BrafV600E-induced transformation. We showed that “spontaneous” promoter hypermethylation, arising in an “aging-like” scenario, sufficiently affects genes with roles in negative regulation of Wnt pathway. Inactivation of these genes leads to increased stem cell state and defects in differentiation. Conclusion: Our studies tightly link aging-like epigenetic abnormalities to intestinal cell fate changes and predisposition to oncogene-driven colon tumorigenesis. The organoid-based model opens up an opportunity to study mechanisms underlying epigenetic heterogeneity arising during normal cellular processes and their role in cancer predisposition. Citation Format: Yong Tao, Byunghak Kang, Daniel Petkovich, Steve Baylin, Hariharan Easwaran. Organoid models for deciphering roles of the evolving landscape of epigenetic heterogeneity during aging in cancer development [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A36.

Roles of FadRACB system in formaldehyde detoxification, oxidative stress alleviation and antibiotic susceptibility in Stenotrophomonas maltophilia

Formaldehyde toxicity is invariably stressful for microbes. Stenotrophomonas maltophilia, a human opportunistic pathogen, is widely distributed in different environments and has evolved an array of systems to alleviate various stresses. To characterize the role of the formaldehyde detoxification system FadRACB of S. maltophilia in formaldehyde detoxification, oxidative stress alleviation and antibiotic susceptibility. Presence of the fadRACB operon was verified by RT-PCR. Single or combined deletion mutants of the fadRACB operon were constructed for functional assays. Formaldehyde, menadione and quinolone susceptibilities were assessed by observing cell viability in formaldehyde-, menadione- and quinolone-containing media, respectively. Susceptibility to hydrogen peroxide was evaluated by disc diffusion assay. The agar dilution method was used to assess bacterial antibiotic susceptibilities. Expression of fadRACB was assessed by quantitative RT-PCR. The fadR, fadA, fadC and fadB genes were arranged in an operon. Mutants of fadA and/or fadB were more susceptible to formaldehyde and oxidative stress than the WT KJ strain of S. maltophilia. No significant difference was observed in the ability of a fadC single mutant to ameliorate formaldehyde and oxidative stress; however, simultaneous inactivation of fadA, fadB and fadC further enhanced susceptibility to formaldehyde and oxidative stress. In addition, compared with WT KJ, the triple mutant KJΔFadACB was more susceptible to quinolones and more resistant to aminoglycosides. FadR functions as a repressor for the fadRACB operon. The FadRACB operon has moderate expression in aerobically grown WT KJ and is further derepressed by formaldehyde challenge or oxidative stress, but not by antibiotics. The FadACB system contributes to mitigation of formaldehyde toxicity and oxidative stress and cross-protects S. maltophilia from quinolones.

Engineering ethanol production in a marine cyanobacterium Synechococcus sp. PCC7002 through simultaneously removing glycogen synthesis genes and introducing ethanolgenic cassettes

Photosynthetic production of ethanol with cyanobacteria utilizing CO2 and solar energy could serve as a promising route for providing green biofuels. Comparing with Synechocystis sp. PCC6803, the most widely utilized cyanobacteria chassis, a marine cyanobacterium Synechococcus sp. PCC7002 (hereafter termed as PCC7002 for short) displayed better tolerances to high temperature and strong illuminations. To engineer ethanol production in PCC7002, we designed a strategy by simultaneous inactivation of glycogen synthesis pathways and introduction of two ethanolgenic cassettes. Two glgA genes responsible for elongation reaction of glycogen was selected as targets for blocking glycogen synthesis, and the removal of glycogen synthesis in PCC7002 caused growth retardation, which could be rescued by introducing two copies of ethanolgenic pathways. In addition, the synergy effects of blocked glycogen synthesis and gene dosage effects for ethanol synthesis resulted in significantly improved performances for ethanol production in the engineered strain, which synthesized 2.2 g/L ethanol in 10 days. In outdoor conditions, the PCC7002 derived cell factory still maintained considerable ethanolgenic capacities, accumulating 0.8 g/L ethanol in 7 days, with an average productivity of over 100 mg/L/day.

Simultaneous disinfection of E. faecalis and degradation of carbamazepine by sulfate radicals: An experimental and modelling study

The existence of micropollutants (MPs) including pathogens in waters poses great risks to ecological safety and human health. Sulfate radical (SO4•−)‒based advanced oxidation processes (AOPs) have attracted considerable attention in water treatment for both disinfection and removal of emerging MPs. Here, we investigated the SO4•−‒mediated kinetic and mechanistic aspects of simultaneous inactivation of Enterococcus faecalis (E. faecalis) and degradation of carbamazepine (CBZ), a typical MP with high occurrence in wastewater. In the absence of CBZ, (73.8 ± 2.3) % E. faecalis were inactivated after 12 min of treatment, while in the presence of CBZ, (68 ± 1.6) % of E. faecalis were inactivated, exhibiting similar inactivation efficiency with or without MP. The second‒order rate constant (k) of E. faecalis reacting with SO4•− was determined to be (5.42 ± 0.64) × 109 M−1 s−1. In addition, two distinct types of disinfection models, one based on the quenching ratio (Q model) and the other on steady‒state concentration of SO4•− (R model), were developed to predict the inactivation kinetics of E. faecalis. Both models exhibited good performance for describing the disinfection of E. faecalis with RMSE of 0.065 and 0.048, respectively. Our kinetic experimental and modelling results on bacterial disinfection and degradation of CBZ were projected to offer valuable insight into future developments for typical wastewater scenarios where microorganisms and MPs coexist.

XPF-ERCC1 protects liver, kidney and blood homeostasis outside the canonical excision repair pathways.

Loss of the XPF-ERCC1 endonuclease causes a dramatic phenotype that results in progeroid features associated with liver, kidney and bone marrow dysfunction. As this nuclease is involved in multiple DNA repair transactions, it is plausible that this severe phenotype results from the simultaneous inactivation of both branches of nucleotide excision repair (GG- and TC-NER) and Fanconi anaemia (FA) inter-strand crosslink (ICL) repair. Here we use genetics in human cells and mice to investigate the interaction between the canonical NER and ICL repair pathways and, subsequently, how their joint inactivation phenotypically overlaps with XPF-ERCC1 deficiency. We find that cells lacking TC-NER are sensitive to crosslinking agents and that there is a genetic interaction between NER and FA in the repair of certain endogenous crosslinking agents. However, joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice. These analyses indicate that XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage. Failure to resolve such damage leads to loss of tissue homeostasis in mice and humans.

Regulatory circuits controlling Spx levels in Streptococcus mutans.

Spx is a major regulator of stress responses in Firmicutes. In Streptococcus mutans, two Spx homologues, SpxA1 and SpxA2, were identified as mediators of oxidative stress responses but the regulatory circuits controlling their levels and activity are presently unknown. Comparison of SpxA1 and SpxA2 protein sequences revealed differences at the C-terminal end, with SpxA1 containing an unusual number of acidic residues. Here, we showed that a green fluorescence protein (GFP) reporter becomes unstable when fused to the last 10 amino acids of SpxA2 but remained stable when fused to the C-terminal acidic tail of SpxA1. Inactivation of clpP or simultaneous inactivation of clpC and clpE stabilized the GFP::SpxA2tail fusion protein. Addition of acidic amino acids to the GFP::SpxA2tail chimera stabilized GFP, while deletion of the acidic residues destabilized GFP::SpxA1tail . Promoter reporter fusions revealed that spxA1 transcription is co-repressed by the metalloregulators PerR and SloR while spxA2 transcription is largely dependent on the envelope stress regulator LiaFSR. In agreement with spxA2 being part of the LiaR regulon, SpxA2 was found to be critical for the growth of S. mutans under envelope stress conditions. Finally, we showed that redox sensing is essential for SpxA1-dependent activation of oxidative stress responses but dispensable for SpxA2-mediated envelope stress responses.

Unleashing the full potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1

The Hsp90 family proteins Hsp90, Grp94, and TRAP1 are present in the cell cytoplasm, endoplasmic reticulum, and mitochondria, respectively; all play important roles in tumorigenesis by regulating protein homeostasis in response to stress. Thus, simultaneous inhibition of all Hsp90 paralogs is a reasonable strategy for cancer therapy. However, since the existing pan-Hsp90 inhibitor does not accumulate in mitochondria, the potential anticancer activity of pan-Hsp90 inhibition has not yet been fully examined in vivo. Analysis of The Cancer Genome Atlas database revealed that all Hsp90 paralogs were upregulated in prostate cancer. Inactivation of all Hsp90 paralogs induced mitochondrial dysfunction, increased cytosolic calcium, and activated calcineurin. Active calcineurin blocked prosurvival heat shock responses upon Hsp90 inhibition by preventing nuclear translocation of HSF1. The purine scaffold derivative DN401 inhibited all Hsp90 paralogs simultaneously and showed stronger anticancer activity than other Hsp90 inhibitors. Pan-Hsp90 inhibition increased cytotoxicity and suppressed mechanisms that protect cancer cells, suggesting that it is a feasible strategy for the development of potent anticancer drugs. The mitochondria-permeable drug DN401 is a newly identified in vivo pan-Hsp90 inhibitor with potent anticancer activity.

HIPP1 stabilizes the interaction between CP190 and Su(Hw) in the Drosophila insulator complex

Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophila and recruits the CP190 and Mod(mdg4)-67.2 proteins to chromatin, where they form a well-known insulator complex. Recently, HP1 and insulator partner protein 1 (HIPP1), a homolog of the human co-repressor Chromodomain Y-Like (CDYL), was identified as a new partner for Su(Hw). Here, we performed a detailed analysis of the domains involved in the HIPP1 interactions with Su(Hw)-dependent complexes. HIPP1 was found to directly interact with the Su(Hw) C-terminal region (aa 720–892) and with CP190, but not with Mod(mdg4)-67.2. We have generated Hipp1 null mutants (HippΔ1) and found that the loss of Hipp1 does not affect the enhancer-blocking or repression activities of the Su(Hw)-dependent complex. However, the simultaneous inactivation of both HIPP1 and Mod(mdg4)-67.2 proteins resulted in reduced CP190 binding with Su(Hw) sites and significantly altered gypsy insulator activity. Taken together, these results suggested that the HIPP1 protein stabilized the interaction between CP190 and the Su(Hw)-dependent complex.

Functional Complementation between β-Catenin and Hoxa9 Sustains HSC Self-Renewal in a Prmt1-Dependent Manner

Self-renewal is a fundamental property essential for both normal and malignant hematopoietic stem cells (HSCs). While we and others have previously shown that activation of Hoxa9 or β-catenin enhances HSC self-renewal, its inactivation has modest impacts on adult HSCs and their malignant counterparts (Cobas et al., 2004; Lawrence et al., 2005; Siriboonpiputtana et al., 2017; Smith et al., 2011; So et al., 2004; Zhao et al., 2007), suggesting the presence of complementary pathways capable of mediating self-renewal in the absence of either protein. However, simultaneous inactivation of other key Hox genes involved in hematopoietic self-renewal including Hoxb3/b4 did not yield additional hematopoietic phenotypes in the Hoxa9 knockout (KO) background (Magnusson et al., 2007). Similarly, suppression of γ-catenin in β-catenin KO mice did not result in additional hematopoietic defects, and exhibited largely normal hematopoietic functions (Koch et al., 2008). Therefore, the alternative pathways that support hematopoietic self-renewal upon inactivation of Hoxa9 or β-catenin remain largely unknown. Our recent studies examining the regulation of posterior Hoxa loci reveals a key function of long non-coding RNA, HOTTIP in protecting Hoxa9 gene expression and uncovers a co-regulation of canonical Wnt signalling pathways by HOTTIP in HSCs, providing a molecular link between these two previously unrelated pathways. This finding is also consistent with our recent report demonstrating their functional complementation in AML stem cells, where suppression of Hoxa9 sensitizes HSC-derived AML stem cells to β-catenin inhibition and ablates their transformation ability, suggesting a novel crosstalk between β-catenin and Hoxa9in mediating hematopoietic self-renewal. To this end, the current study developed and characterized a novel β-cateninfl/fl Hoxa9-/-Rosa-CreER mouse model. In contrast to single Hoxa9 or β-catenin inactivation where a mild hematopoietic phenotype has been reported in adult HSCs, we found that double inactivation of Hoxa9 and β-catenin resulted in severe hematopoietic defects. In vitroclonogenic assays revealed that bone marrow cells harbouring combined inactivation of Hoxa9/β-catenin generated markedly reduced myeloid colony numbers (>9-fold reduction compared to either single KO alone) of which mature CFU-G (50%) and CFU-E (50%) were the predominant composition. Colonies generated from isolated LSK (Lin-c-kit+Sca1+) populations were equivalent in quantity but devoid of multipotential progenitors (CFU-GEMM) in contrast to the diverse colony composition of control and single KO cells. Transplantation of β-cateninfl/flHoxa9-/-bone marrow to lethally irradiated recipient mice followed by β-catenin inactivation (4 days tamoxifen treatment) resulted in defects in all HSC and progenitor compartments compared to single KO alone at all time points measured (3wk, 6wk and 12 wk post tamoxifen treatment). Immunophenotypic analysis revealed that the defect originated as early as the LT-HSC stage with a drastic 7-fold reduction in LT-HSCs (Lin-c-kit+Sca1+CD150+CD48-) and 3-fold reduction in ST-HSCs (Lin-c-kit+Sca1+CD150-CD48-). Consistent with a stem cell defect, we also observed significant reductions in downstream myeloid progenitor populations including common myeloid progenitor (CMP) (2-fold), granulocyte-monocyte progenitor (GMP) (2-fold) and megakaryocyte and erythrocyte progenitors (MEP) (7-fold). Mechanistically, Hoxa9 and β-catenin co-regulate expression of Prmt1, a key epigenetic regulator with multifaceted functions in mediating RNA splicing and DNA damage response in hematopoietic cells. To further investigate the role of Prmt1 in hematopoietic development, we generated a novel Prmt1 KO model where Prmt1 can be conditionally inactivated in HSCs. Deletion of Prmt1 alone phenocopied simultaneous inactivation of β-catenin and Hoxa9, resulting in severe reductions in LT-HSC (3.5-fold), ST-HSC (4-fold) and downstream hematopoietic progenitor populations (CMP 4.5-fold, GMP 3.5-fold and MEP 4-fold). Together these data suggest that the posterior Hoxa loci and canonical Wnt pathways are developmentally regulated in a complementary manner as a safeguard mechanism to allow efficient hematopoietic self-renewal, which is largely dependent on intact Prmt1 functions. Disclosures No relevant conflicts of interest to declare.

Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast.

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal “cut” phenotype. By implementing an artificial targeting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Interestingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Electrochemical pretreatment for stabilization of waste activated sludge: Simultaneously enhancing dewaterability, inactivating pathogens and mitigating hydrogen sulfide

Stabilization of waste activated sludge (WAS) is an essential step for the disposal or reuse. In this study, WAS stabilization via electrochemical pretreatment (EPT) at 0–15V was evaluated for simultaneous dewaterability enhancement, pathogen removal and H2S mitigation. The mechanism underlying EPT was investigated and discussed based on the changes in the physicochemical (e.g., particle size, zeta potential, hydrophobicity and extracellular polymeric substances) and biological characteristics (i.e. cell morphology, and distribution and percentages of live/dead cells) of WAS with different EPT voltages. The results revealed that EPT disintegrated WAS flocs and disrupted the cell walls leading to a reduction in particle size (by up to 50%), increased release of extracellular and intracellular substances (by up to 4 times) to facilitate WAS stabilization. With EPT at 15V, the capillary suction time of WAS decreased by 42%, and the concentrations of E. coli and indicator pathogens (Salmonella spp. and Streptococcus faecalis) fell by nearly 5 log10 reaching U.S. EPA hygienization levels. Furthermore, EPT at 12V or higher suppressed the amounts of dissolved sulfide and H2S(g) produced from the WAS under anaerobic conditions by over 99%. This study demonstrates the feasibility of EPT for simultaneous WAS dewaterability enhancement, pathogen inactivation and H2S mitigation, providing a one-step alternative for sludge stabilization.

Expression profiles of types 2 and 3 iodothyronine deiodinase genes in relation to vitellogenesis in a tropical damselfish, Chrysiptera cyanea

Thyroid hormone (TH) is involved in regulating the reproduction of vertebrates. Its physiological action in the target tissues is due to the conversion of TH by iodothyronine deiodinases. In this study, we aimed to clone and characterize type 2 (sdDio2) and type 3 (sdDio3) of the sapphire devil Chrysiptera cyanea, a tropical damselfish that undergoes active reproduction under long-day conditions, and to study the involvement of THs in the ovarian development of this species. When the cDNAs of sdDio2 and sdDio3 were partially cloned, they had deduced amino acid sequences of lengths 271 and 267, respectively, both of which were characterized by one selenocysteine residue. Real-time quantitative PCR (qPCR) revealed that both genes are highly expressed in the whole brain, and sdDio2 and sdDio3 are highly transcribed in the liver and ovary, respectively. In situ hybridization analyses showed positive signals of sdDio2 and sdDio3 transcripts in the hypothalamic area of the brain. Little change in mRNA abundance of sdDio2 and sdDio3 in the brain was observed during the vitellogenic phases. It is assumed that simultaneous activation and inactivation of THs occur in this area because oral administration of triiodothyronine (T3), but not of thyroxine (T4), upregulated mRNA abundance of both genes in the brain. The transcript levels of sdDio2 in the liver and sdDio3 in the ovary increased as vitellogenesis progressed, suggesting that, through the metabolism of THs, sdDio2 and sdDio3 play a role in vitellogenin synthesis in the liver and yolk accumulation/E2 synthesis in the ovary. Taken together, these results suggest that iodothyronine deiodinases act as a driver for vitellogenesis in tropical damselfish by conversion of THs in certain peripheral tissues.

Ste20 and Cla4 modulate the expression of the glycerol biosynthesis enzyme Gpd1 by a novel MAPK-independent pathway

p21-activated kinases (PAKs) are important signalling molecules with a wide range of functions. In budding yeast, the main PAKs Ste20 and Cla4 regulate the response to hyperosmotic stress, which is an excellent model for the adaptation to changing environmental conditions. In this pathway, the only known function of Ste20 and Cla4 is the activation of a mitogen-activated protein kinase (MAPK) cascade through Ste11. This eventually leads to increased transcription of glycerol biosynthesis genes, the most important response to hyperosmotic shock. Here, we show that Ste20 and Cla4 not only stimulate transcription, they also bind to the glycerol biosynthesis enzymes Gpd1, Gpp1 and Gpp2. Protein levels of Gpd1, the enzyme that catalyzes the rate limiting step in glycerol synthesis, positively correlate with glucose availability. Using a chemical genetics approach, we find that simultaneous inactivation of STE20 and CLA4 reduces the glucose-induced increase of Gpd1 levels, whereas the deletion of either STE20 or CLA4 alone has no effect. This is also observed for the hyperosmotic stress-induced increase of Gpd1 levels. Importantly, under both conditions the deletion of STE11 has no effect on Gpd1 induction. These observations suggest that Ste20 and Cla4 not only have a role in the transcriptional regulation of GPD1 through Ste11. They also seem to modulate GPD1 expression at another level such as translation or protein degradation.

Inositol pyrophosphates impact phosphate homeostasis via modulation of RNA 3' processing and transcription termination.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3′-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3′-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.

Abstract 2569: Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair

Abstract Genomic instability is one of the enabling hallmarks of cancer and can arise by exposure to several factors: endogenous challenges such as stress to DNA replication forks or exogenous compounds such as exposure to ionizing radiation (IR) and other environmental mutagens. These insults can lead to the induction and propagation of mutations through the generation of multiple DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different repair pathways. One of these pathways is homologous recombination (HR), a relatively faithful DSB repair pathway that relies on the availability of a homologous template for DNA repair synthesis. DNA strand exchange in HR is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. In human cells, RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While RAD54 translocates on double-stranded DNA and promotes DNA strand separation, RAD51AP1 enhances the assembly of the synaptic complex. However, if RAD54 and RAD51AP1 work independently or together in the HR reaction is unclear. Since functional loss of either RAD54 or RAD51AP1 each leads to a moderate defect in HR, we speculate that RAD54 and RAD51AP1 may act in parallel in the promotion of RAD51 activity. We hypothesize that simultaneous inactivation of both RAD54 and RAD51AP1 may lead to a synthetic phenotype in HR impairment. The main focus of this study is to investigate the potential synergism between RAD51AP1 and RAD54 during late stages of the HR reaction to improve targeted cancer therapy. We are investigating if loss of RAD54 in human cells exacerbates the phenotype of RAD51AP1-deficiency and renders cells more sensitive to DNA damaging agents. We have generated RAD51AP1 knockout HeLa cells using CRISPR/Cas9 technology. In these cells, we have also inactivated the RAD54 gene using a very similar approach. Moreover, we have generated RAD54 knockout (KO) cells in a wild type RAD51AP1 background. All cell lines are tested for their response to chemotherapeutic agents in cell survival and DNA replication assays. Our first results show that the double KO cells are more sensitive to mitomycin C exposure than single KO cells, suggesting that RAD51AP1 and RAD54 function largely independently of each other in the HR reaction. The results from our investigation are expected to lead to the potential development of new cancer drugs targeted at ancillary factors of RAD51 in late stages of the HR reaction. Citation Format: Platon Selemenakis, David Maranon, Claudia Wiese. Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2569.