Lost Activity Research Articles

Complete ammonia oxidation (comammox) at pH 3–4 supports stable production of ammonium nitrate from urine

This study developed a new process that stably produced ammonium nitrate (NH4NO3), an important and commonly used fertilizer, from the source-separated urine by comammox Nitrospira. In the first stage, the complete conversion of ammonium to nitrate was achieved by comammox Nitrospira. In this scenario, the pH was maintained at 6 by adding external alkali, which also provided sufficient alkalinity for full nitrification. In the second stage, the NH4NO3 was produced directly by comammox Nitropsira by converting half of the ammonium in urine into nitrate. In this case, no alkali was added and pH automatically dropped and self-maintained at an extremely acidic level (pH 3–4). In both scenarios, negligible nitrite accumulation was observed, while the final product of the second stage contained ammonium and nitrate at the molar ratio of 1:1. The dominance of comammox Nitrospira over canonical ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) was systematically proved by the combination of 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction, and metagenomics. Notably, metagenomic sequencing suggested that the relative abundance of comammox Nitrospira was over 20 % under the acidic condition at pH 3–4, while canonical AOB and NOB were undetectable. Batch experiments showed that the optimal pH for the enriched comammox Nitrospira was ∼7, which could sustain their activity in a wider pH range from 4 to 8 surprisingly but lost activity at pH 3 and 9. The findings not only present an application potential of comammox Nitrospira in nitrogen recovery from urine wastewater but also report the survivability of comammox bacteria in acidic environments.

Oscillatory network spontaneously recovers both activity and robustness after prolonged removal of neuromodulators.

Robustness of neuronal activity is a property necessary for a neuronal network to withstand perturbations, which may otherwise disrupt or destroy the system. The robustness of complex systems has been shown to depend on a number of features of the system, including morphology and heterogeneity of the activity of the component neurons, size of the networks, synaptic connectivity, and neuromodulation. The activity of small networks, such as the pyloric network of the crustacean stomatogastric nervous system, appears to be robust despite some of the factors not being consistent with the expected properties of complex systems, e.g., small size and homogeneity of the synaptic connections. The activity of the pyloric network has been shown to be stable and robust in a neuromodulatory state-dependent manner. When neuromodulatory inputs are severed, activity is initially disrupted, losing both stability and robustness. Over the long term, however, stable activity homeostatically recovers without the restoration of neuromodulatory input. The question we address in this study is whether robustness can also be restored as the network reorganizes itself to compensate for the loss of neuromodulatory input and recovers the lost activity. Here, we use temperature changes as a perturbation to probe the robustness of the network's activity. We develop a simple metric of robustness, i.e., the variances of the network phase relationships, and show that robustness is indeed restored simultaneously along with its stable network activity, indicating that, whatever the reorganization of the network entails, it is deep enough also to restore this important property.

Evaluation of N-alkyl isatins and indoles as acetylcholinesterase and butyrylcholinesterase inhibitors

Two series of N-alkyl isatins and N-alkyl indoles varying in size of the alkyl group were synthesised and evaluated for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Among the N-alkyl isatins 4a-j, the addition of the N-alkyl group improved inhibition potency towards AChE and BChE compared to isatin. Selectivity towards inhibition of BChE was observed, and the increase in size of the N-alkyl group positively correlated to improved inhibition potency. The most potent inhibitor for BChE was 4i (IC50 = 3.77 µM, 22-fold selectivity for BChE over AChE). N-alkyl indoles 5a-j showed similar inhibition of AChE, the most potent being 5g (IC50 = 35.0 µM), but 5a-j lost activity towards BChE. This suggests an important role of the 3-oxo group on isatin for BChE inhibition, and molecular docking of 4i with human BChE indicates a key hydrogen bond between this group and Ser198 and His438 of the BChE catalytic triad.

Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications.

Pseudomonas aeruginosa PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP+-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in Escherichia coli and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP+ and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD+ while it lost activity towards NADP+, and the Km value (7770.67 μM) of the mutant PaIDH2-L589 I600 for NADP+ was higher than that observed for NAD+ (5824.33 μM), indicating a shift in coenzyme specificity from NADP+ to NAD+ for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium P. aeruginosa.

Epitope Tag Conjugation System Provides Versatility for Multiplex Assay Design

Abstract With the ever-growing use of multiplex assays in research, therapeutics, and diagnostics, the need to conjugate antibodies to dyes, enzymes, and other materials is increasing. While there are many challenges to overcome regarding conjugation, the largest hurdle is maintaining antibody activity after conjugation. To combat these conjugation challenges, Alpha Thera developed a conjugation system that utilizes a small adapter protein to label antibodies quickly and site-specifically, resulting in antibodies that maintain their activity post conjugation. The adapter protein contains a customizable C-terminal region that can be modified to contain diverse labels including fluorescent dyes, click-chemistry, drugs, and epitope tags. Incorporating the epitope tag conjugation system with Fortis Life Sciences anti-epitope tag detection antibodies, we developed an easy and adaptable system for generating conjugated antibodies to use in multiplex assays. We used this system to detect staining of multiple makers including CD3, TIM3, PCNA, and actin via flow cytometry, IHC, ICC, and western blot. One of the tested antibodies retained normal activity post-conjugation even though prior attempts with metal tagging via sulfhydryl groups resulted in lost activity. The vast array of conjugated anti-epitope tag antibodies allows for the selection of specific dyes/materials that are optimized to your desired assays. Additionally, since the secondary antibody is detecting the epitope tag, multiplexing is simplified, and antibody host is rendered irrelevant. This epitope tag system provides a simple, site-specific conjugation method that has incredible adaptability for any multiplex assay.

ODP440 Characterization of the Major Single Nucleotide Polymorphic Variants of Aldo-Keto Reductase 1c3 (Type 5 17β-Hydroxysteroid Dehydrogenase)

Abstract Background Aldo-keto-reductase (AKR) 1C3, also known as type 517β-hydroxysteroid dehydrogenase and prostaglandin F synthase,is a member of the AKR superfamily that reduces aldehydes and ketones to primary and secondary alcohols. It plays an essential role in the peripheral formation of androgens and is associated with several steroid hormone dependent diseases including prostate cancer (PC), breast cancer, and polycystic ovarian syndrome (PCOS). Furthermore, AKR1C3 is implicated in castration resistant prostate cancer (CRPC) drug resistance on the basis that it is upregulated in CRPC tumors and metastases and overexpressed in cell culture & xenograft models of Abiraterone and Enzalutamide resistance, where resistance is surmounted by AKR1C3 knockdown or treatment with the AKR1C3 inhibitor indomethacin. AKR1C3 has 14 nonsynonymous single nucleotide polymorphisms (nsSNPs) with different global frequencies and ethnic distributions. Association studies support their role in PC and other diseases, but detailed functional genomic studies of these variants are lacking. One study examined5 AKR1C3 nsSNPs for their ability to reduce exemestane, an aromatase inhibitor used to treat breast cancer, to 17β-dihydroexemestane, and reported a 17-250-fold reduction in catalytic efficiency of H5Q, E77G, K104D, and R258C variants compared to WT. This observation provided the impetus to examine impact of these variants on AKR1C3 function in the context of PC. We purified AKR1C3 WT, and the top four most frequently occurring nsSNPs, H5Q, E77G, K104D, and R258C, from E. coli to expand upon their characterization and illuminate functional differences that could affect disease outcome and treatment. METHODS: Kinetic measurements were made using either a continuous spectrophotometric assay monitoring the appearance of the reduced cofactor NADH, or a discontinuous RP UV-HPLC assay based on chromatographic separation of substrate and product. Inhibitor sensitivity studies were conducted with a fluorescence-based plate-reader assay monitoring enzyme activity via oxidation of a standard substrate in the presence of increasing inhibitor. Stability was assessed by monitoring the percent of maximum activity remaining via oxidation of a standard substrate over a range of increasing temperatures for each variant. Results We report no major deviations in kinetics in the top four most frequently occurring AKR1C3 variants compared to WT. Additionally, variants did not display differences in inhibitor sensitivity to competitive inhibitor indomethacin. However, the K104D variant lost activity at a lower temperature than WT, suggesting that this mutation results in a decrease in stability. Conclusions Altogether, these findings contrast with previous claims that AKR1C3 variants, mainly H5Q, give rise to significant advantage or disadvantage over WT in PC and other diseases, and suggests that patients with AKR1C3 dependent diseases would likely be overall unaffected by variant status. Presentation: No date and time listed

Adsorption of recalcitrant phosphorus compounds using the phosphate-selective binding-protein PstS.

Currently available wastewater phosphorus (P) treatment technologies target removal of reactive forms of P. Selective adsorption of more recalcitrant soluble non-reactive phosphorus (sNRP) can improve P removal and recovery. A phosphate-selective phosphate-binding protein (PBP), PstS, was immobilized onto NHS-activated beads to assess the ability of this novel bioadsorbent to remove (adsorb) and subsequently recover (desorb) a range of sNRP compounds. Four sNRP compounds representative of wastewater sNRP were selected for use in this study: phytic acid (PA), sodium triphosphate (TrP), beta-glycerol phosphate (BGP), and sodium hexametaphosphate (HMP). Using PBP, adsorption of all sNRP compounds was thermodynamically favorable. The PBP had nearly equivalent binding affinity for PA compared to PBP's typical target, orthophosphate, although it had less affinity for the other sNRP compounds. Adsorption followed pseudo-second order reaction kinetics, with 95% of maximum adsorption occurring within 4min. This was substantially faster sNRP adsorption compared to other adsorbents in the literature. Adsorption was modeled using the Langmuir isotherm, reflecting that one phosphate molecule attached to one PBP binding site. Notably, this selective 1:1 attachment resulted in higher total P removal for sNRP molecules with high P content. The binding site lost activity with increasing pH, and as such, highest desorption was achieved at pH 12, making the system amenable to sNRP removal as well as controlled recovery.

Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture.

Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk-clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk-clotting activity under a solid-state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal-sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost-effective production, milk-clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese-making industry. PRACTICAL APPLICATION: The milk-clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.

Role of ZrO2 and CeO2 support on the In2O3 catalyst activity for CO2 hydrogenation

Methanol synthesis from CO2 hydrogenation has drawn global attention as catalytic CO2 hydrogenation is an attractive choice to mitigate CO2 emissions and lessen dependency on fossil resources. In the present study, we have synthesized ZrO2 and CeO2-supported In2O3 catalysts for methanol synthesis from CO2 hydrogenation and the catalytic performances of Inx/ZrO2, and Inx/CeO2 (x = 1 % and 13 %) were compared. Specifically, the effect of the ZrO2 and CeO2 supports on In2O3 catalyst during CO2 hydrogenation was explored. This study reveals that ZrO2 support increased the catalytic activity while the CeO2 support decreased although both supports have almost the same indium loading and surface area. Various characterizations like XRD, DRIFT, CO2-TPD, H2-TPR and XPS analysis of catalysts provided insight into changes that arise after mixing the two oxides and during the reaction as well as after the reaction. The stabilities of In2O3, In13/ZrO2, and In13/CeO2 were tested for up to 50 h and we found In13/ZrO2 was stable during this time-on-stream, while In13/CeO2 lost activity after 2 h of reaction. XPS results of spent catalysts showed that In(OH)3 was observed significantly over the spent In13/CeO2. OH groups were also verified by DRIFT experiments, however at low levels due to low CO2 conversion at atmospheric pressure. XRD analysis confirmed the sintering of CeO2 support during the reaction. Thus, the hydrophilic nature of CeO2, redox properties of CeO2 and sintering of CeO2 support in the presence of water, were the main reasons for the early deactivation of In13/CeO2. A regeneration study was carried out to regenerate the catalyst and the results showed that partial regeneration of the In13/CeO2 catalyst is possible by Ar flushing. We, therefore, suggest that the build-up of OHgroups deactivate the In13/CeO2 catalyst and some of these OH groups could be removed during flushing with inert gas, causing a partial regeneration. However, the decreased surface area is not reversible, and this results in a continuous decrease in the activity of the catalyst after repeated experiments, even if the catalyst is flushed with Ar between the experiments.

Analysis of the responsible site for favipiravir resistance in RNA-dependent RNA polymerase of influenza virus A/PR/8/34 (H1N1) using site-directed mutagenesis

Favipiravir (T-705, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) selectively and strongly inhibits the replication of influenza virus in vitro and in vivo. Favipiravir is converted to favipiravir-4-ribofuranosyl-5-triphosphate (favipiravir RTP) by intracellular enzymes and functions as a nucleotide analog to selectively inhibit RNA-dependent RNA polymerase (RdRP) of influenza virus. Our previous experiments failed in an attempt to obtain a favipiravir-resistant influenza virus in vitro using influenza virus A/PR/8/34(H1N1). Conversely, Goldhill et al. reported a favipiravir-resistant influenza virus generated by in vitro passage of influenza virus A/England/195/2009 (H1N1), an early isolate from the 2009 H1N1 pandemic (pdm09), in the presence of favipiravir with K229R mutation in PB1. This study focused on K229R mutation near the NTP cross-linked region in PB1 based on the above conflicting findings to confirm whether K229R mutation brings favipiravir resistance to influenza virus A/PR/8/34. Thirty PB1 mutants generated by site-directed mutagenesis of the NTP cross-linked region were evaluated using an influenza virus A/PR/8/34 replicon system. Among the 30 mutants, 10 possessed but 20 lost replicon activity. When susceptibility to favipiravir in 10 mutants was further assessed, the PB1 E491D mutant was five times more sensitive than the wild-type (WT), while only the PB1 K229R mutant was resistant to favipiravir. Results suggested that the evaluated region was essential for polymerase activity, and K229 mutation was responsible for polymerase inhibition of favipiravir in the influenza virus A/PR/8/34. Interestingly, the tested K229X series mutants entirely lost replicon activity, except for K229R. This suggested that the amino acid at position 229 in PB1 of influenza virus may play a pivotal role in polymerase activity. Moreover, this lysine residue is highly conserved among positive- and negative-sense single-stranded RNA viruses, in which favipiravir showed potent activity, suggesting that this mutation may determine the characterization of the in vitro broad-spectrum activity of favipiravir. Additionally, this mutation acquisition greatly influences the viral replication and the susceptibility to favipiravir.

Membrane fluidity, composition, and charge affect the activity and selectivity of the AMP ascaphin-8

Ascaphins are cationic antimicrobial peptides that have been shown to have potential in the treatment of infectious diseases caused by multidrug-resistant pathogens (MDR). However, to date, their principal molecular target and mechanism of action are unknown. Results from peptide prediction software and molecular dynamics simulations confirmed that ascaphin-8 is an alpha-helical peptide. For the first time, the peptide was described as membranotrophic using biophysical approaches including calcein liposome leakage, Laurdan general polarization, and dynamic light scattering. Ascaphin-8’s activity and selectivity were modulated by rearranging the spatial distribution of lysine (Var-K5), aspartic acid (Var-D4) residues, or substitution of phenylalanine with tyrosine (Var-Y). The parental peptide and its variants presented high affinity toward the bacterial membrane model (≤2 μM), but lost activity in sterol-enriched membranes (mammal and fungal models, with cholesterol and ergosterol, respectively). The peptide-induced pore size was estimated to be >20 nm in the bacterial model, with no difference among peptides. The same pattern was observed in membrane fluidity (general polarization) assays, where all peptides reduced membrane fluidity of the bacterial model but not in the models containing sterols. The peptides also showed high activity toward MDR bacteria. Moreover, peptide sensitivity of the artificial membrane models compared with pathogenic bacterial isolates were in good agreement.

A fungal dioxygenase CcTet serves as a eukaryotic 6mA demethylase on duplex DNA.

N6-methyladenosine (6mA) is a DNA modification that has recently been found to play regulatory roles during mammalian early embryo development and mitochondrial transcription. We found that a dioxygenase CcTet from the fungus Coprinopsis cinerea is also a dsDNA 6mA demethylase. It oxidizes 6mA to the intermediate N6-hydroxymethyladenosine (6hmA) with robust activity of 6mA-containing duplex DNA (dsDNA) as well as isolated genomics DNA. Structural characterization revealed that CcTet utilizes three flexible loop regions and two key residues-D337 and G331-in the active pocket to preferentially recognize substrates on dsDNA. A CcTet D337F mutant protein retained the catalytic activity on 6mA but lost activity on 5-methylcytosine. Our findings uncovered a 6mA demethylase that works on dsDNA, suggesting potential 6mA demethylation in fungi and elucidating 6mA recognition and the catalytic mechanism of CcTet. The CcTet D337F mutant protein also provides a chemical biology tool for future functional manipulation of DNA 6mA in vivo.

The Pathways to Create Containers for Bacteriophage Delivery.

Antimicrobial resistance is a global public health threat. One of the possible ways to solve this problem is phage therapy, but the instability of bacteriophages hinders the development of this approach. A bacteriophage delivery system that stabilizes the phage is one of the possible solutions to this problem. This study is dedicated to exploring methods to create encapsulated forms of bacteriophages for delivery. We studied the effect of proteolytic enzymes on the destruction of the polyelectrolyte microcapsule shell and revealed that protease from Streptomyces griseus was able to destroy the membrane of the microcapsule (dextran sulfate/polyarginine)3 ((DS/PArg)3). In addition, the protease decreased the activity of the bacteriophage in the second hour of incubation, and the phage lost activity after 16 h. It was found that a medium with pH 9.02 did not affect the survival of the bacteriophage or E. coli. The bacteriophages were encapsulated into polyelectrolyte microcapsules (DS/PArg)3. It was established that it is impossible to use microcapsules as a means of delivering bacteriophages since the bacteriophages are inactivated. When bacteriophages were included inside a CaCO3 core, it was demonstrated that the phage retained activity before and after the dissolution of the CaCO3 particle. From the results of this study, we recommend using CaCO3 microparticles as a container for bacteriophage delivery through the acidic stomach barrier.

World Economic Prospects Monthly

Overview: Omicron prompts a further growth downgrade ▀ It is unclear how serious the health implications of the new Omicron coronavirus variant will be, let alone how governments, households and firms will respond to it. But against this uncertain backdrop, we have lowered our baseline GDP forecasts. Global GDP growth is now expected to slow from 5.7% this year to 4.3% in 2022, down 0.2pp from a month ago. ▀ It may take a few weeks before the significance of the emergence of the Omicron variant becomes apparent. For now, we assume that there will be a modest reimposition of some activity restrictions — most notably on foreign travel — an increase in voluntary social distancing and some resulting supply‐chain disruption. This will dampen growth around the turn of the year. But the lost activity will be made up in Q2 and Q3 2022. ▀ Beyond the near term, Omicron could pose upside risks to the growth forecast if initial albeit unproven suggestions that it has milder symptoms than prior variants are true. However, if the variant has greater transmissibility or more resistance to existing vaccines, the economic disruption from Omicron would probably be greater and lengthier than assumed in our new baseline. ▀ Omicron may also delay any fall back in inflation if it exacerbates ongoing supplychain problems and slows the speed at which households re‐orientate their spending back towards services from goods. ▀ As a result of this, recent upside inflation surprises in several economies and an upward revision to our oil price forecast, we have raised our CPI forecasts. We have lifted 2022 global CPI inflation by 0.3pp to 4.2%, just a shade below this year's average. But over the next year, inflation is still expected to fall sharply from 5.4% in Q4 2021 to about 3% in Q4 2022.

Therapeutic Targeting of TET-Dioxygenase Deficiency in Myeloid Malignancies

Background: TET-dioxygenases (TET1, TET2, and TET3) are key epigenetic regulators, which require molecular oxygen, αKG, and Fe 2+ to progressively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to the demethylation of mCpGs at promoter and enhancer, a key step for mounting an efficient transcription profile and thereby differentiation. Loss of function TET2 mutation (TET2 MT) is frequently observed in myeloid neoplasms (MN). Despite extensive studies of the biochemical mechanisms underlying distorted myeloid differentiation and neoplastic evolution of TET2 MT HSPCs, targeted therapies are lagging. Here, we report the therapeutic utility of preferential targeting of TET2 mutant and TET-dioxygenase deficient cells either by restoring the lost activity to induce differentiation and death or by inhibiting the residual activity to induce synthetic lethality.Methods: Genetic as well as pharmacologic in vitro and in vivo models of AML were used to validate specific targeting of TET2 mutant cells. Knockout and over-expression in the isogenic background of different cells were used to understand the pro-survival and proliferative mechanism of TET2 loss in myeloid cells. The efficacy of small molecule pharmacophore targeting TET-dioxygenase was evaluated in vitro in cell free and cell culture as well as in vivo in a mouse competitive transplant model to demonstrate the proof of therapeutic concept.Result: Ascorbate, used as an activator of αKG/Fe 2+ dependent dioxygenase including TETs, functions via maintaining the redox state of iron (II) in the dioxygenase catalytic site with a half-maximal effective concentration of 13.8 ± 1.3 µM for enhancing TET2activity as measured by an ELISA for 5hmC. However, the reported intracellular cellular concentrations of ascorbate in human remains in the millimolar range, well above the saturating concentration required for TET-dioxygenases activation. Interestingly, ascorbate failed to activate TET in primary mononuclear cells isolated from myeloid neoplasia patients with TET2 mutations. Consistent with the in-vitro observation, we found that ascorbate treatment in physiologically relevant doses has no significant effect in syngeneic murine models (p=0.1). TET-inactivation is a complex phenomenon that is controlled by context-dependent post-translational modifications including lysine acetylation by acetyltransferases and class I and II deacetylases. Therefore, the anti-leukemic effect of the supraphysiological doses of ascorbate in humans and mice leukemia models may not be dependent on TET activation by ascorbate.On the other hand, a comprehensive analysis of the configurations of TET2 MT in myeloid neoplasia (n= 2617) demonstrated a remarkable exclusivity with 2-hydroxyglutarate (2-HG) producing neomorphic IDH1/2 MT. Ectopic inducible expression of IDH1/2 MT induced synthetic lethality of malignant TET2 MT cells. Therefore, themutual exclusivity of TET2 MT and neomorphic IDH1/2 MT is due to synthetic lethality caused by 2-HG production. In addition, we demonstrate that sequential deletion of TET1 and TET3 leads to growth arrest in cellular models, which has never been observed in any myeloid malignancies. Altogether, these observations suggest that malignant TET2 mutant myeloid cells may be vulnerable to inhibition of residual TET-activity coming from TET1 and TET3.Therefore, we synthesized TET-specific inhibitor on 2HG scaffold using iterative computer-aided design, characterized in cell-free as well as cell culture model systems, and selected TETi76 with no cytotoxic effect in normal bone marrow-derived CD34 + cells. TETi76 selectively inhibits TET-dioxygenase activity with IC50 of 1.5, 9.4, and 8.8 μmol/L for TET1/2/3, and it restricts clonal outgrowth of TET2 MT both in vitro and in vivo in malignant and nonmalignant clonal hematopoiesis of indeterminate potential (CHIP).Conclusion: Taken together, here we demonstrated that anti-cancer activity of ascorbate may not come from its role as a TET activator and that a minimum level of TET-dioxygenase activity is required for cell survival, rendering TET2 -mutant malignant cells selectively vulnerable to inhibitors of TET-function. DisclosuresMaciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

Developing novel anthelmintics: the stability of cysteine proteinase activity in a supernatant extract of papaya latex

Plant derived cysteine proteinases (CPs) have long been known to possess anthelmintic properties but have attracted renewed attention recently because of the acute need to discover novel methods for controlling helminth infections as a result of increasing drug resistance. However, surprisingly little is known about the stability of these proteins under typical storage and in vivo exposure conditions. We found that CPs in a supernatant preparation from papaya latex (PLS) were stable during the initial refinement process and when stored under low temperatures, but lost activity during dialysis and within 7 days of storage when kept at ambient temperature (18–20 °C). The enzyme activity in PLS was not affected by repeated freeze-thaw cycles and was also stable under typical in vitro assay conditions at 37 °C used for quantifying effects on helminths. Active enzyme activity was still detectable in the colon 3–4 h after oral administration in rodent models.

Recombinant production and characterisation of two chitinases from Rasamsonia emersonii, and assessment of their potential industrial applicability.

Rasamsonia emersonii (previously Talaromyces emersonii) is a thermophilic filamentous fungus displaying optimum growth at 45°C. It has a history of use in commercial food enzyme production. Its unfractionated chitinolytic secretome was partially characterised in the early 1990s; however, no individual chitinase from this source has been described in literature previously. This study describes two GH18 chitinases originating from the R. emersonii genome, expressed in the methylotrophic yeast P. pastoris. Chit1 comprises of a GH18 catalytic domain and Chit2 comprises of a GH18 catalytic domain and a chitin-binding motif at the C-terminal. The chitinases were expressed as glycoproteins. The apparent molecular weight of Chit1 was 35.8-42.1kDa with a smearing tail associated with glyco-sidechains visible up to 72.2kDa. This became two bands of 30.8 and 29.0kDa upon de-glycosylation. The apparent molecular weight of Chit2 was 50.4kDa, reducing to 48.2kDa upon de-glycosylation. Both chitinases displayed endo-chitinase and chitobiosidase activity, temperature optima of 50-55°C and low pH optima (pH 4.5 or lower); Chit1 displayed a pH optimum of 3.5, retaining > 60% maximum activity at pH 2.2, a pH range lower than most enzymes of fungal origin. Chit2 displayed the highest chitin-degrading ability at 3456µmol/mg on 4-NP-triacetylchitotriose, but lost activity faster than Chit1, which displayed 403µmol/mg on the same substrate. The predicted D values (time required to reduce the enzyme activity to 10% of its original value at 50°C) were 19.2 and 2.3days for Chit1 and Chit2, respectively. Thus, Chit1 can be considered one of few hyperthermostable chitinase enzymes described in literature to date. Their physicochemical properties render these chitinases likely suitable for shrimp chitin processing including one-step chitin hydrolysis and alternative sustainable protein processing and the attractive emerging application of mushroom food waste valorisation.Key points• Two GH18 chitinases originating from the industrially relevant thermophilic fungus R. emersonii were cloned and expressed in P. pastoris.• The purified recombinant chitinases showed low pH and high temperature optima and appreciable thermostability at industrially relevant temperatures.• The chitinases displayed characteristics that indicate their likely suitability to several industrial applications including sustainable alternative protein processing, food waste valorisation of commercial mushroom production and one-step shrimp chitin processing.

A potently neutralizing SARS-CoV-2 antibody inhibits variants of concern by utilizing unique binding residues in a highly conserved epitope

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

Organoarsenical tolerance in Sphingobacterium wenxiniae, a bacterium isolated from activated sludge.

Organoarsenicals enter the environment from biogenic and anthropogenic sources. Trivalent inorganic arsenite (As(III)) is microbially methylated to more toxic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) that oxidize in air to MAs(V) and DMAs(V). Sources include the herbicide monosodium methylarsenate (MSMA or MAs(V)), which is microbially reduced to MAs(III), and the aromatic arsenical roxarsone (3-nitro-4-hydroxybenzenearsonic acid or Rox), an antimicrobial growth promoter for poultry and swine. Here we show that Sphingobacterium wenxiniae LQY-18T , isolated from activated sludge, is resistant to trivalent MAs(III) and Rox(III). Sphingobacterium wenxiniae detoxifies MAs(III) and Rox(III) by oxidation to MAs(V) and Rox(V). Sphingobacterium wenxiniae has a novel chromosomal gene, termed arsU1. Expressed in Escherichia coli arsU1 confers resistance to MAs(III) and Rox(III) but not As(III) or pentavalent organoarsenicals. Purified ArsU1 catalyses oxidation of trivalent methylarsenite and roxarsone. ArsU1 has six conserved cysteine residues. The DNA sequence for the three C-terminal cysteines was deleted, and the other three were mutated to serines. Only C45S and C122S lost activity, suggesting that Cys45 and Cys122 play a role in ArsU1 function. ArsU1 requires neither FMN nor FAD for activity. These results demonstrate that ArsU1 is a novel MAs(III) oxidase that contributes to S. wenxiniae tolerance to organoarsenicals.

In vitro fermentation and isolation of heparin-degrading bacteria from human gut microbiota

Heparin and its derivative are commonly used as injectable anticoagulants in clinical procedures, but possess poor oral bioavailability. To explore the role of gut microbiota in the poor oral effect of heparin, the degradation profiles of heparin on six human gut microbiota were investigated. The heparin-degradation ability varied significantly among individuals. Furthermore, two strains of heparin-degrading bacteria, Bacteroides ovatus A2 and Bacteroides cellulosilyticus B19, were isolated from the gut microbiota of different individuals and the degradation products of the isolates were profiled. The ΔUA2S-GlcNS6S was the major end product with almost no desulfation. 3-O-sulfo group-containing tetrasaccharides were detected, which indicated that the antithrombin binding site was broken and this explained the lost anticoagulant activity of heparin. Collectively, the present study assessed the degradation profiles of heparin by human gut microbiota and provided references for the development of oral administration of heparin from a gut microbiota perspective.