Nonclinical Evaluation of Single-Mutant E. coli Asparaginases Obtained by Double-Mutant Deconvolution: Improving Toxicological, Immune and Inflammatory Responses.

Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes' pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects.

Friedel-Crafts-Type Arylation Strategy for the Conversion of Alkyl 2-((Diphenoxyphosphoryl)oxy)-2-arylacetates to α,α-Diaryl Esters.

An arylation strategy allowing the conversion of alkyl 2-((diphenoxyphosphoryl)oxy)-2-arylacetates to α,α-diaryl esters is reported. This transformation can be promoted by TfOH when the starting organic phosphates do not carry para-alkoxy groups on their aryl rings, but it does not require any additives when such groups are present. These alkyl 2-((diphenoxyphosphoryl)oxy)-2-arylacetates can be readily accessed from the insertion of diphenyl phosphate into aryldiazoacetates.

An aza-Robinson Annulation Strategy for the Synthesis of Fused Bicyclic Amides: Synthesis of (±)-Coniceine and Quinolizidine.

An aza-Robinson annulation strategy is described using a NaOEt-catalyzed conjugate addition of cyclic imides onto vinyl ketones, followed by a TfOH-mediated intramolecular aldol condensation to afford densely functionalized fused bicyclic amides. The potential use of these amides in the synthesis of alkaloids is demonstrated by the sequential conversion of appropriate precursors to (±)-coniceine and quinolizidine in two additional steps, thus allowing their preparation in overall 40 and 44% yields, respectively.

Visible light-mediated photolysis of organic molecules: the case study of diazo compounds.

Considering the recent rapid advancement of synthetic technologies promoted by visible light in the last 15 years, the use of photocatalysts has been rightfully justified based on the fact that organic molecules generally do not absorb visible light. However, an increasing number of different classes of organic molecules is being identified as actually directly absorbing in this region of the electromagnetic spectrum. Among them, diazo compounds are possibly one of these classes whose chemistry has been more explored so far. Indeed, irradiation of these compounds with visible light has been introduced as a mild photolytic strategy generally leading to free carbene intermediates. This strategy not only allows for a more cost-economical approach revealing similar outcomes to some previously reported thermal, metal-catalyzed transformations; but it can also eventually lead to different reactivities. Herein, we will present the contributions of our laboratory and of other groups to this research area, along with some important elements of design behind the development of selected reaction profiles, aiming to provide the reader with an overall view of the current state of the art.

Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters.

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg-1. The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.

A scoping review to compare and contrast quality assurance aspects of l-asparaginase biosimilars.

l-asparaginase is a first-line medicine used for the treatment of acute lymphoblastic leukemia. Differing quality of marketed l-asparaginase biosimilars has been reported to adversely influence treatment outcomes. Herein, the quality of l-asparaginase biosimilars intended for clinical use was reviewed in sight of quality assurance parameters using English and Chinese language database searching, which provided information for possible improvements to the manufacture of this medicine. Ten articles met inclusion criteria, and quality attributes that measured potency, specific activity, purity and host cell proteins (HCPs) were identified. Biosimilars manufactured in high-income countries represented good quality in all aspects. Biosimilars manufactured in high-middle/middle-income countries, however, suggested poorer quality control particularly over removal of HCPs. Future work should now focus on establishing pharmacopeia monographs to establish equivalent quality assurance for l-asparaginase biosimilars manufactured between countries. Standardization of the quality profile, analytical methods and the limits of critical quality parameters, are essential to ensure appropriated efficacy and safety of clinical grade l-asparaginase.

Are the Post-transplant Outcomes of Kasai's Early Failure and Late Failure Comparable to the Primary Liver Transplantation?

It is uncertain whether prior Kasai procedures negatively impact the outcomes of liver transplantation (LT). The prior meta-analysis did not distinguish between Kasai early failure (K-EF) and late failure (K-LF). Numerous studies have been recently published; therefore, we perform a systematic review and meta-analysis. We searched PubMed and Embase databases to identify studies comparing the outcomes of biliary atresia (BA) patients undergoing primary LT versus patients with prior Kasai procedures. Subgroup analysis was done at the time of Kasai failure (early vs. late). Twenty-five studies comprising 6,408 patients receiving LT were included in the analysis. We found a statistically significant increase in one-year graft survival in K-LF versus primary liver transplant (pLT) (P = 0.0003). One-year patient survival was also increased in K-LF, although not statistically significant (P = 0.09). No difference in the one- and five-year graft and patient survival, reoperation rate, infection, and biliary complication was seen in pLT vs overall prior Kasai (K-EF and K-LF). These results suggest that prior kasai procedure does not negatively impact the outcome of LT. In addition, BA patients with prior Kasai undergoing LT later in life tend to perform better than primary liver transplants.

Synthesis of 4-(organoselenyl) oxazolones via cyclization of N-alkynyl ethylcarbamates promoted by organoselenium.

Organoselenyl iodide promoted the intramolecular nucleophilic cyclization of N-alkynyl ethylcarbamates in the synthesis of 4-(organoselenyl) oxazolones. The reaction was regioselective, giving the five-membered oxazolone products as the unique regioisomer via an initial activation of the carbon-carbon triple bond through a seleniranium intermediate, followed by an intramolecular 5-endo-dig cyclization mode. The generality of the methodology has been proven by applying the optimized reaction conditions to different organoselenyl iodides and N-alkynyl ethylcarbamates having different substituents directly bonded to the nitrogen atom and in the terminal position of the alkyne.

Palladium-Catalyzed Cascade 5-endo-dig Cyclization of Ynamides to Form 4-Alkynyloxazolones.

The selective synthesis of 4-alkynyloxazolones and their further applications as substrates to electrophile-promoted nucleophilic cyclization have been developed. The reaction of ynamides with terminal alkynes proceeded smoothly to give 4-alkynyloxazolones in the presence of a catalytic amount of palladium(II) acetate. The products were obtained with the sequential formation of new C-C and C-O bonds via a cascade procedure. The first step involved a carbon-oxygen bond formation, via a 5-endo-dig closure, which was confirmed by X-ray analyses of the crystalline sample. Subsequently, the reaction of 4-alkynyloxazolones with an electrophilic selenium source gave 3-phenylselanyl benzofuran derivatives via an electrophile-promoted nucleophilic cyclization.

Diorganyl Diselenides and Iron(III) Chloride Drive the Regio- and Stereoselectivity in the Selenation of Ynamides.

We report here our results on the application of ynamides as substrates in the reactions with diorganyl dichalcogenides and iron(III) chloride to give selectively three different types of compounds: E-α-chloro-ß-(organoselenyl)enamides, 4-(organochalcogenyl)oxazolones, and vinyl tosylates. The results reveal that the selectivity in the formation of products was obtained by controlling the functional groups directly bonded to the nitrogen atom of the ynamides. Thus, α-chloro-ß-(organoselenyl) enamide derivatives were exclusively obtained when the TsN- and MsN-ynamides were treated with a mixture of diorganyl diselenides (1.0 equiv) and FeCl3 (3.0 equiv) in dichloroethane (DCE, 3 mL), at room temperature. The 4-(organochalcogenyl)oxazolones were selectively obtained with ynamides having an ester group, directly bonded to the nitrogen atom, upon treatment with a solution of FeCl3 (1.5 equiv) and diorganyl dichalcogenides (1.0 equiv) in dichloromethane (3 mL) at room temperature. Finally, vinyl tosylates were obtained from ynamides having an ester group, directly bonded to the nitrogen atom, by reaction with p-toluenesulfonic acid. We also studied the application of the prepared compounds as substrates for Suzuki and Sonogashira cross-coupling reactions.

Microbial lipase: a new approach for a heterogeneous biocatalyst.

Lipases are enzymes employed in several industrial process and their applicability can be increased if these biocatalysts are in the immobilize form. The objective of this work was to study the immobilization of lipase produced by submerged cultivation of Aspergillus sp. by hydrophobic interaction, evaluating its stability and reuse capacity. The immobilization process on octyl-sepharose (C8) and octadecyl-sepabeads (C18) carriers was possible after the removal of oil excess presented in the fermented broth. The results showed that the enzyme was isolated and concentrated in octyl-sepharose with 22% of the initial activity. To increase the amount of enzyme adsorbed on the carrier, 4 immobilization cycles were performed in a row, on the same carrier, with a final immobilization yield of 151.32% and an increase in the specific activity of 136%. The activity test with immobilized lipase showed that the immobilized enzyme maintained 75% of the initial activity after 20 cycles.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

Similar hepatoprotective effectiveness of Diphenyl diselenide and Ebselen against cisplatin-induced disruption of metabolic homeostasis and redox balance in juvenile rats.

Cisplatin is an antineoplastic drug well recognized for its success in the battle against several types of cancer in adult, juvenile, and child populations. Meanwhile, this drug is also famous due to its serious side effects, such as hepatotoxicity. This study evaluated the hepatoprotective effectiveness of Diphenyl Diselenide (PhSe)2 and Ebselen in a model of cisplatin-induced toxicity in juvenile rats. Juvenile Wistar rats received a single intraperitoneal (i.p) injection of cisplatin (6 mg/kg) or saline solution, at postnatal day (PND) 21. Ebselen (11 mg/kg) or (PhSe)2 (12 mg/kg) was intragastrically (i.g) administered in rats from PND 21 to PND 25. At PND 26, the blood and liver were collected for the biochemistry assays. A single administration of cisplatin was enough to alter the makers of hepatic function (an increase of AST activity) and the blood lipid profile (an increase of cholesterol and triglycerides, TG). The cisplatin-induced metabolic disruption was demonstrated by the increase of hepatic glycogen and TG contents and hexokinase, glucose-6-phosphatase, and tyrosine aminotransferase activities; a decrease of citrate synthase activity and the levels of GLUT-2. Cisplatin-induced hepatic oxidative stress was characterized by an increase in reactive oxygen species, TBARS, protein carbonyl, and Nox levels as well as the decrease in NPSH levels. Ebselen and (PhSe)2 were effective against all alterations caused by this chemotherapy medication. The present findings highlight the (PhSe)2 and Ebselen similar hepatoprotective effectiveness against cisplatin-induced disruption of metabolic homeostasis and redox balance in juvenile rats.

Chemogenomic study of gemcitabine using Saccharomyces cerevisiae as model cell-molecular insights about chemoresistance.

Gemcitabine (GEM) is the drug used as first line to treat pancreatic cancer, one of the most devastating human tumors. This peculiar type of tumor develops resistance to several drugs, including GEM, due to its desmoplastic reaction and other features. The GEM chemoresistance has been investigated at molecular level aiming to find a pathway whose inhibition or activation should overcome it. Through next-generation sequencing was performed a chemogenomic assay of GEM using Saccharomyces cerevisiae as model cell and the results showed that more than 40% of genes related to GEM response in yeast possess unknown or dubious function. We choose two yeast mutants to individually validate the fitness defect results observed by chemogenomic assay, Δhmt1 and Δcsi1, and it was found that in addition to some already described pathways involved in GEM resistance, cells deficient in deneddylation enzyme Cop9 Signalosome Interactor 1 (Csi1p) presented a high sensitivity to GEM. This was confirmed by individual growth analyses of Δcsi1 cells exposed to GEM, and this phenotype was reverted with CSI1 complementation gene. Csi1p is a well-characterized homolog equivalent to human Csn6 subunit of COP9 signalosome (CSN) involved in deneddylation process. We highlighted too that epigenetic alterations, such as methylation mediated by protein arginine methyltransferase 1, play an important role in regulating gemcitabine treatment resistance. Our results point out new unexplored molecular pathways that can be used to overcome GEM resistance: the inhibition of CSN and the arginine methyltransferase activities.

Cyclization of Thiopropargyl Benzimidazoles by Combining Iron(III) Chloride and Diorganyl Diselenides.

A practical synthetic approach to the synthesis of 3-(organoselenyl)-imidazothiazines was developed. The methodology involved the regioselective 6-endo-dig cyclization of thiopropargyl benzimidazoles promoted by diorganyl diselenides and iron(III) chloride. The investigation to determine the best reaction conditions indicated the use of thiopropargyl benzimidazoles (0.25 mmol) with diorganyl diselenides (1.0 equiv) and iron(III) chloride (2.0 equiv) in dichloromethane at 40 °C for 30 min to be optimal. Under these conditions, the scope of the substrates was evaluated varying the structures of thiopropargyl benzimidazoles and diorganyl diselenides giving 28 3-(organoselenyl)-imidazothiazines in moderate to good yields. The reaction conditions were also applicable to diorganyl ditellurides; however, they did not work for diorganyl disulfides. The mechanism studies were carried out indicating that the cyclization proceeds via a cooperative action of diorganyl diselenides and iron(III) chloride, but a direct electrophilic cyclization, promoted by the in situ formed electrophilic organoselenium species, cannot be ruled out.

Penicillium and Talaromyces endophytes from Tillandsia catimbauensis, a bromeliad endemic in the Brazilian tropical dry forest, and their potential for L-asparaginase production.

This study was conducted to report the richness of endophytic Penicillium and Talaromyces species isolated from Tillandsia catimbauensis, a bromeliad endemic in the Brazilian tropical dry forest (Caatinga), to verify their ability to produce the enzyme L-asparaginase and to partially optimise the production of biomass and L-asparaginase of the best enzyme producer. A total of 184 endophytes were isolated, of which 52 (29%) were identified through morphological and phylogenetic analysis using ß-tubulin sequences into nine putative species, four in Penicillium and five in Talaromyces. Talaromyces diversus and T. cf. cecidicola were the most frequent taxa. Among the 20 endophytic isolates selected for L-asparaginase production, 10 had the potential to produce the enzyme (0.50-2.30 U/g), especially T. cf. cecidicola URM 7826 (2.30 U/g) and Penicillium sp. 4 URM 7827 (1.28 U/g). As T. cf. cecidicola URM 7826 exhibited significant ability to produce the enzyme, it was selected for the partial optimisation of biomass and L-asparaginase production. Results of the 23 factorial experimental design showed that the highest dry biomass (0.66 g) was obtained under pH 6.0, inoculum concentration of 1 × 108 and 1% L-proline. However, the inoculum concentration was found to be statistically significant, the pH was marginally significant and the concentration of L-proline was not statistically significant. L-Asparaginase production varied between 0.58 and 1.02 U/g and did not reach the optimal point for enzyme production. This study demonstrates that T. catimbauensis is colonised by different Penicillium and Talaromyces species, which are indicated for enzyme production studies.

Microbial cell disruption methods for efficient release of enzyme L-asparaginase.

The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed in this study, via measurements of the release of total protein and L-asparaginase activity. Three different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified the best procedure for each kind of microorganism. Sonication and glass bead stirring led to obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins and specific activity; however, sonication was the best since it obtained 4.61 ± 0.12 IU mg-1 after 3 min of operation time. Mechanical methods were also the most effective for yeast cell disruption, but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication proved to be the best procedure due to getting the highest specific activity (9.01 IU mg-1) and total enzyme activity (61.7 IU). The data presented lead to conclude that the mechanical methods appeared to be the most effective for the disintegration of the all microbial cells studies. This is the first report related to the experimental comparison of L-ASNase extraction procedures from different microorganisms, which can also be used for extracting periplasm located enzymes from other organisms.

Ammoniacal Nitrogen Stripping From Landfill Leachate at Open Horizontal Flow Reactors.

The ammonia nitrogen stripping process at open horizontal flow reactors was evaluated and is described in this paper. The ammonia came from landfill leachate. Superficial loads of 650 kgN-.ha-1.day-1 (phase 1), 750 kgN-.ha-1.day-1 (phase 2), and 850 kgN-.ha-1.day-1 (phase 3) were applied with a hydraulic retention time of 51.2, 64.9, and 55.6 days, respectively. The experimental system setup was designed, built, and monitored at EXTRABES (Estação Experimental de Tratamentos Biológicos de Esgotos Sanitários) and consisted of a series of four open horizontal flow reactors fed with in natura leachate. The study was based on a stripping process with no alkaline chemical species addition, nor air flow for ammonium gas drag, and aimed to reduce the landfill leachate ammoniacal nitrogen concentration. The studied process showed a removal efficiency of 99.0% in the first phase, 99.3% in the second, and 99.5% in the third. Regarding the organic matter expressed in terms of CODtotal, the removal efficiency was 69.20% in phase 1, 40.12% in phase 2, and 29.23% in phase 3. It was noted that ammonia removal efficiency was directly proportional to the applied superficial load and the carbonaceous material removal was proportional to the organic matter applied in influent, since in phase 1 the CODtotal concentration removal efficiency was 1.7 times greater than that of phase 2, and 2.3 times that of phase 3. Briefly, the ammonia nitrogen stripping process by open horizontal flow reactor is a promising technological alternative for ammonia nitrogen reduction, mainly at regions with available area and temperature greater than 25°C. It is promising because it has no costs in terms of chemical reagents to raise the pH, nor does it require electric costs.

Brain-derived neurotrophic factor signaling plays a role in resilience to stress promoted by isoquinoline in defeated mice.

Certain stressful life events have been associated with the onset of depression. This study aims to investigate if 7-fluoro-1,3-diphenylisoquinoline-1-amine (FDPI) is effective against social avoidance induced by social defeat stress model in mice. Furthermore, it was investigated the effects of FDPI in the mouse prefrontal cortical plasticity-related proteins and some parameters of toxicity. Adult Swiss mice were subjected to social defeat stress for 10 days. Two protocols with FDPI were carried out: 1- FDPI (25 mg/kg, intragastric) was administered to mice 24 h after the last social defeat stress episode; 2- FDPI (1-25 mg/kg, intragastric) was administered to mice once a day for 10 days concomitant with the social defeat stress. The mice performed social avoidance and locomotor tests. The prefrontal cortical protein contents of kinase B (Akt), extracellular signal-regulated kinase (ERK), cAMP-response element binding protein (CREB), pro-brain-derived neurotrophic factor (proBDNF), p75NTR, neuronal nuclear protein (NeuN) and nuclear factor-κB (NF-κB) were determined in mice. A single administration of FDPI (25 mg/kg) partially protected against social avoidance induced by stress in mice. Repeated administration of FDPI (25 mg/kg) protected against social avoidance induced by stress in mice. Social defeat stress decreased the protein contents of p75NTR, NeuN and the pERK/ERK ratio but increased those of proBDNF and the pCREB/CREB ratio, without changing that of NF-κB. Repeated administration of FDPI modulated signaling pathways altered by social defeat stress in mice. The present findings demonstrate that FDPI promoted resilience to stress in mice.

Executive Function Is Selectively Impaired in Old Age Bipolar Depression.

Background: Little is known about the cognitive signature of bipolar disorder (BD) in elderly brains. The neuropsychological features of depressive elderly with early-onset BD are largely unknown. This issue is relevant because cognitive impairment can produce an additional impact on the already compromised functionality of elderly with BD. The aim of this study is to assess executive functions (EFs) in the depressive phase of elderly outpatients with early-onset BD. Methods: Forty-nine elderly outpatients with early-onset BD were assessed with several neuropsychological tests for EF in the depressive phase of the disorder. Results: Executive dysfunction is very common in old age bipolar depression. Thirteen patients (26.5%) had a pseudodementia presentation. The worst performances were observed in the following tests: Trail Making B, Stroop Test 3, Backward Digit Span and Wisconsin Card Sorting Test. Conclusion: Executive dysfunction profile in elderly BD is complex and heterogeneous, but most cases display difficulties in working memory, inhibitory control, mental flexibility, and information processing speed. The performance of elderly with bipolar depression in executive assessment can be divided into two main categories: (1) Single EF domain impairment; and (2) Multiple EF domain impairment with or without a pseudodementia syndrome. Executive dysfunction in old age bipolar depression may be explained by lack of sufficient mental energy to run those cognitive processes that require larger amounts of effort to be performed.