Active Reaction Research Articles

To Reverse Sensitization By Co-Administration of Bortezomib and Rituximab with Conventional Chemotherapy for Patients Who Developed Neutralizing Hypersensitivity Reactions Against Asparaginase

Background: Acute lymphoblastic leukemia (ALL) patients undergoing Pegylated asparaginase (Peg-asp) or Erwinia-asparaginase (Erw-asp) preparation therapy may experience silent inactivation and allergy to asparaginase (ASP), leading to decreased therapeutic efficacy. How to treat ASP sensitized patients, especially those who are allergic to both Peg-asp and Erw-asp, without compromising the treatment outcome remains a problem for hematologists.This study aims to investigate whether the sensitization to Peg-asp can be reversed through the co-treatment of bortezomib and rituximab with conventional chemotherapy for patients hypersensitive to both Peg-asp and Erw-asp. Method: ALL Patients who developed silent inactivation and allergic reaction to both Peg-asp and Erw-asp enrolled in the study. The study featured 2 courses of combined conventional chemotherapy with rituximab and bortezomib co-treatment. In the second course of desensitization, Peg-asp was re-exposed according to the severity of previous allergic reaction against Peg-asp. For those with silent inactivation or previous allergic reactions of grade ≤ III, a split intramuscular injection was administered (the first dose was given at 1/10th of the required dosage, and then the remaining dose was given at an interval of 2 hours if there was no allergic reaction). Patients with grade IV previous allergic reactions underwent a standard 3-bag desensitization procedure. ASP activity was measured on days 1, 3, 7, 10, 14, and 21 after the first Peg-asp re-challenge. If re-exposure to the first dose did not result in an allergic reaction, a subsequent intramuscular single full dose was administered based on the results of ASP activity monitoring. Primary endpoint: to observe the percentage of allergic reaction on re-exposure and the duration of sustained ASP activity. Result: From August 2020 to May 2023, 10 patients of ALL who developed a neutralizing allergy or silent inactivation to both Peg-asp and Erw-asp preparation were recruited to the study. Among them, six cases were B-ALL, and four were T-ALL. The median age of the participants was 8 years, with seven boys and three girls. Seven children experienced Peg-asp neutralizing allergy or silent inactivation during remission induction, while three children experienced it during the interphase treatment. Eight of them had clinical manifestations of allergy and two had silent inactivation. During the second desensitization course, none of the children showed any allergic reaction after the first re-exposure to Peg-asp. Three children maintained >3 weeks of therapeutic activity after the first Peg-asp re-exposure, while one child maintained 14 days, and three children maintained 10 days. Three children detected Asp activity below 100U/L on day 3 monitoring. After two courses of desensitization, the patients returned to the original chemotherapy regimen. Nine patients (90%) maintained activity above 0.1IU/ML for more than three weeks after subsequent Peg-asp administration. One child developed pancreatitis after the last dose of Peg-asp but recovered soon. Another child experienced silent inactivation and was discontinued from all ASP administration. During desensitization therapy, eight children developed febrile neutropenia. Three were recovered with outpatient antibiotic treatment, while five required hospitalization for 5-7 days. As of July 30, 2023, all ten children survived without any adverse events, with a median follow-up time of 28 months from the start of desensitization therapy. Conclusion： Rituximab and bortezomib co-treatment effectively reverse ASP sensitization and restore the sustained potent activity of Peg-asp. This co-treatment is a safe and promising desensitization therapy after ASP sensitization in ALL patients.

Magnetically recoverable Fe3O4/MoS2/BiOI microspheres for visible light water disinfection: Molecular mechanism and transcriptomic insights

Photocatalysis driven by green energy is ideal for water purification. To gain a deeper understanding of the underlying mechanism behind the photocatalytic inactivation of bacteria, a novel visible-light-driven Fe3O4/MoS2/BiOI (FMB) photocatalyst was synthesized, and E. coli disinfection was demonstrated. Complete inactivation of E. coli was achieved in 100 min by FMB exposed to 30 mW/cm2 of > 400 nm light. FMB can also effectively reduce the total number of bacteria and heterotrophic bacteria in actual source water. The photochemical experiments revealed that h+, e-, H2O2, 1O2 and •O2– were responsible for inactivation reactions. The semipermeable membrane experiments provided further evidence that contact between the photocatalyst and the bacteria was necessary to achieve inactivation. During the disinfection process, the zeta potential of the cells first decreased and then increased, while the particle size first increased and then decreased, indicating the rupture of the cells. Scanning electron microscopy, potassium ion leakage, and changes in cell surface hydrophobicity and hydrophilicity all confirmed the destruction of the E. coli cell membrane at the molecular level. The β-GAL activity of E. coli decreased, and the activities of superoxide dismutase (SOD) and catalase (CAT) increased initially, but subsequently decreased, further demonstrating disruption of the cell membrane and the leakage of cell contents. Transcriptomics was employed to understand gene expression and confirm bacterial membrane damage followed by oxidative stress response. This work provides a demonstration of the FMB inactivation by visible light and a mechanistic understanding of inactivation of E. coli.

Design and characterization of dexamethasone loaded microsponges for the management of ulcerative colitis

Ulcerative colitis is an inflammatory condition with ulcerations throughout the colon. The existing remedies have some limitations such as drug inactivation, poor absorption, and adverse reactions. The present study aimed to design novel microsponge formulations to enhance remission of the dexamethasone (as a model pharmaceutical ingredient) in the colon. Microsponges were prepared by using the quasi-emulsion technique. The optimal formulation was selected by applying the design of experiments approach which used methylcellulose (MC) (0.75–2%, w/w), polyvinylalcohol (PVA)(0.5–1%, w/w), and tween 80 (TW80) (1.5–2.5%, w/w). The critical quality attributes were selected as particle size and entrapment efficiency. The particle size and encapsulation efficiency were found as 140.38 ± 9.2 µm and 77.96 ± 3.4 %. After the optimization; morphological, thermal, and physicochemical characterization studies were performed. Ultimately, the optimal formulation was investigated by using the acetic acid-induced ulcerative colitis model in rats. The physicochemical characterization studies confirmed that the formulation components were compatible with each other. The in vitro release mechanisms were fitted to First order kinetics at pH 1.2 (R2:0.9563), and Korsmeyer-Peppas kinetics at pH 4.5 (R2: 0.9877), and pH 6.8 (R2: 0.9706). The medicated microsponges exhibited remarkable recovery compared to the control group of the in vivo ulcerative colitis model (p < 0.05). It could be concluded that microsponges were evaluated as a promising alternative drug delivery system for the management of ulcerative colitis.

State of spatial memory and antioxidant system activity of rats in the dynamics of development of blast-induced traumatic brain injury

The main purpose of this study was to investigate the changes in spatial memory and catalase activity in dynamics in the blast-induced traumatic brain injury (bTBI). The experiment was carried out on 67 albino male Wistar rats, which were randomly divided into three groups: I group – experimental (n=34), animals were subjected to inhalation anesthesia with halothane, fixed and was simulated blast-induced traumatic brain injury was simulated by generating a shock wave with an overpressure of 26.4±3.6 kPa, II group – sham (n=34), animals which were subjected only to inhalation anesthesia and fixation and III group – intact (n=34). After the study of behavior, euthanasia was performed, blood and brain were taken shelter. Histopathological examination of the brain showed disturbances both of hippocampal neurons and microcirculature. In the dynamics of the post-traumatic period of mild bTBI there was observed a significant (p&lt;0.01) impairment of rats spatial memory in the experimental group, which was established as a prolongation of latent time of the search in the Barnes maze: by 39% – on day 1, by 76% – on day 3, by 65% – on day 7 and by 61% – on day 14 of the study. The analysis of catalase activity revealed a significant decrease of this enzyme activity in blood plasma of the rats in the experimental group in comparison with rats of sham and intact groups: on day 1 – by 35% (p&lt;0.01), on day 3 – by 27% (p&lt;0.01) and by 12% on day 7 of the post-traumatic period (p&lt;0.05), which indicates the involvement of catalase in the reaction of hydrogen peroxide inactivation. Correlation analysis between spatial memory and catalase activity in experimental rats showed a negative relationship of medium degree on day 1 (r=-0.3, p&lt;0.01) and a negative relationship (r=-0.2, p&lt;0.01) of weak degree on day 3 of post-traumatic period, indicating increased formation of free radicals during this period. One-way analysis of variance showed a significant (H1, p&lt;0.01) effect of changes in the catalase activity of rat blood plasma on the state of spatial memory in the Barnes maze during 14 days of the post-traumatic period. It was established that oxidative stress is an important link in pathogenesis of the spatial memory disorders in animals with mild bTBI in the first week of the post-traumatic period.

Cell polarisation in a bulk-surface model can be driven by both classic and non-classic Turing instability

The GTPase Cdc42 is the master regulator of eukaryotic cell polarisation. During this process, the active form of Cdc42 is accumulated at a particular site on the cell membrane called the pole. It is believed that the accumulation of the active Cdc42 resulting in a pole is driven by a combination of activation–inactivation reactions and diffusion. It has been proposed using mathematical modelling that this is the result of diffusion-driven instability, originally proposed by Alan Turing. In this study, we developed, analysed and validated a 3D bulk-surface model of the dynamics of Cdc42. We show that the model can undergo both classic and non-classic Turing instability by deriving necessary conditions for which this occurs and conclude that the non-classic case can be viewed as a limit case of the classic case of diffusion-driven instability. Using three-dimensional Spatio-temporal simulation we predicted pole size and time to polarisation, suggesting that cell polarisation is mainly driven by the reaction strength parameter and that the size of the pole is determined by the relative diffusion.

Impact of a Newly Synthesized Molecule (2-chloro-N-(1-(3,4-dimethoxyphenyl) propan-2-yl)-2-phenylacetamide) on the Bioelectrogenesis and the Contractile Activity of Isolated Smooth Muscles.

Examination of the potential possibilities of 2-chloro-N-(1-(3,4-dimethoxyphenyl)propan-2-yl)-2-phenylacetamide (IQP) to affect bioelectrogenesis and the contractile activity of isolated smooth muscles (SM) from stomach. Having in mind the structural similarities between the molecules of papaverine and IQP, the aim of the present study was to examine such features of the newly synthesized molecule that may potentially affect the muscle tonus, spontaneous bioelectrical and contractile activities of smooth muscles isolated from the stomach, basing on specific mechanisms of papaverine. The synthesis of IQP is based on the initially formed aziridine ring by principles of Gilbert's reaction. Impact of IQP on the bioelectrogenesis and the contractile activity of isolated smooth muscles from male Wistar rats was measured by the single sucrose-gap method and isometrically recorded. IQP (1×10-5 - 2.5×10-4 mol/l) causes muscle relaxation, producing changes in two processes that have influence on the mechanical activity of smooth muscles:1. Blocked Ca2+ influx through the potential-dependent membrane Ca2+ channels, followed in turn by lowering the Ca2+ intracellular levels. This effect is proved by the changes in the frequency and amplitude of spike-potentials in sucrose-bridge experiments when IQP is applied.2. Activation of a cAMP-dependent signal cascade. The relaxing effect of IQP was significantly reduced in the presence of KT5720(5×10-6 mol/l), an inhibitor of protein kinase A. We assume that there might be interconnections between these two IQP-dependent processes, because PKA-dependent phosphorylation of the L-type Ca2+ channels in smooth muscles provokes a reaction of inactivation.

Individualized Asparaginase Dosing in Childhood Acute Lymphoblastic Leukemia.

In the DCOG ALL-11 protocol, polyethylene glycol-conjugated Escherichia coli asparaginase (PEGasparaginase) and Erwinia asparaginase treatment of pediatric acute lymphoblastic leukemia are individualized with therapeutic drug monitoring (TDM). The efficacy of TDM and its effect on asparaginase-associated toxicity are reported. After induction with 3 fixed intravenous doses of 1,500 IU/m2 PEGasparaginase, medium-risk patients (n = 243) received 14 individualized doses that targeted trough levels of 100-250 IU/L, standard-risk patients (n = 108) received 1 individualized dose, and high-risk patients (n = 18) received 2-5 fixed administrations (1,500 IU/m2). After a neutralizing hypersensitivity reaction, patients were started with 20,000 IU/m2 Erwinia asparaginase 3 times per week, and l-asparagine was measured to monitor asparaginase efficacy. Several asparaginase-associated toxicities were studied. The final median PEGasparaginase dose was lowered to 450 IU/m2. Overall, 97% of all trough levels of nonallergic patients were > 100 IU/L. Asparagine was < 0.5 μM in 96% and 67% of the PEGasparaginase and Erwinia asparaginase levels > 100 IU/L, respectively. Ten percent developed a neutralizing hypersensitivity reaction to PEGasparaginase, of which 40% were silent inactivations. The cumulative incidence of grade 3-4 pancreatitis, central neurotoxicity, and thromboses was 12%, 4%, and 6%, respectively, and not associated with asparaginase activity levels. During medium-risk intensification, 50% had increased ALT and 3% hyperbilirubinemia (both grade 3/4 and correlated with asparaginase activity levels), and 37% had grade 3/4 hypertriglyceridemia. Hypertriglyceridemia occurred less in intensification compared with ALL-10 (37% v 47%), which is similar to ALL-11 but with higher asparaginase levels during intensification. TDM of asparaginase results in a significant reduction of the PEGasparaginase dose with adequate asparaginase activity levels and sufficient asparagine depletion. In addition, with TDM, silent inactivation and allergic-like reactions were identified. However, the effect of reduced asparaginase activity levels on toxicity is limited.

Regulation of ion transport from within ion transit pathways.

All cells must control the activities of their ion channels and transporters to maintain physiologically appropriate gradients of solutes and ions. The complexity of underlying regulatory mechanisms is staggering, as exemplified by insulin regulation of transporter trafficking. Simpler strategies occur in single-cell organisms, where subsets of transporters act as solute sensors to regulate expression of their active homologues. This Viewpoint highlights still simpler mechanisms by which Na transporters use their own transport sites as sensors for regulation. The underlying principle is inherent to Na/K pumps in which aspartate phosphorylation and dephosphorylation are controlled by occupation of transport sites for Na and K, respectively. By this same principle, Na binding to transport sites can control intrinsic inactivation reactions that are in turn modified by extrinsic signaling factors. Cardiac Na/Ca exchangers (NCX1s) and Na/K pumps are the best examples. Inactivation of NCX1 occurs when cytoplasmic Na sites are fully occupied and is regulated by lipid signaling. Inactivation of cardiac Na/K pumps occurs when cytoplasmic Na-binding sites are not fully occupied, and inactivation is in turn regulated by Ca signaling. Potentially, Na/H exchangers (NHEs) and epithelial Na channels (ENaCs) are regulated similarly. Extracellular protons and cytoplasmic Na ions oppose secondary activation of NHEs by cytoplasmic protons. ENaCs undergo inactivation as cytoplasmic Na rises, and small diffusible molecules of an unidentified nature are likely involved. Multiple other ion channels have recently been shown to be regulated by transiting ions, thereby underscoring that ion permeation and channel gating need not be independent processes.

Body side-specific changes in sensorimotor processing of movement feedback in a walking insect.

Feedback from load and movement sensors can modify timing and magnitude of the motor output in the stepping stick insect. One source of feedback is stretch reception by the femoral chordotonal organ (fCO), which encodes such parameters as the femorotibial (FTi) joint angle, the angular velocity, and its acceleration. Stimulation of the fCO causes a postural resistance reflex, during quiescence, and can elicit the opposite, so-called active reaction (AR), which assists ongoing flexion during active movements. In the present study, we investigated the role of fCO feedback for the difference in likelihood of generating ARs on the inside vs. the outside during curve stepping. We analyzed the effects of fCO stimulation on the motor output to the FTi and the neighboring coxa-trochanter and thorax-coxa joints of the middle leg. In inside and outside turns, the probability for ARs increases with increasing starting angle and decreasing stimulus velocity; furthermore, it is independent of the total angular excursion. However, the transition between stance and swing motor activity always occurs after a specific angular excursion, independent of the turning direction. Feedback from the fCO also has an excitatory influence on levator trochanteris motoneurons (MNs) during inside and outside turns, whereas the same feedback affects protractor coxae MNs only during outside steps. Our results suggest joint- and body side-dependent processing of fCO feedback. A shift in gain may be responsible for different AR probabilities between inside and outside turning, whereas the general control mechanism for ARs is unchanged.NEW & NOTEWORTHY We show that parameters of movement feedback from the tibia in an insect during curve walking are processed in a body side-specific manner, and how. From our results it is highly conceivable that the difference in motor response to the feedback supports the body side-specific leg kinematics during turning. Future studies will need to determine the source for the inputs that determine the local changes in sensory-motor processing.

Kinetic study of Z-scheme C3N4/CuWO4 photocatalyst towards solar light inactivation of mixed populated bacteria

In the present work, Z-scheme composite C3N4/CuWO4 was synthesised by sol-gel method for the photocatalytic degradation of a mixed population of Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli). The effect of the photoinactivation was observed for two different types of bacteria in the same medium together and individually in the absence of the nutrients. The lattice structures and phase purities were determined by X-ray diffraction. For morphological and topographical features, SEM and TEM imaging were performed. The oxidation states of the elements and band edges of the semiconductor were determined by XPS and UPS, respectively. The lifetime of the charge carriers and band gap of the semiconductors were determined by time resolved florescence spectroscopy and diffused reflectance spectroscopy, respectively. The photocatalytic experiments were performed for different weight ratios of C3N4 and CuWO4. Scavenger experiments were performed to investigate the exact mechanism and major responsible radicals for photocatalysis. The rate constants and order of the inactivation reactions were obtained by power law kinetics. The rate followed by the inactivation for E. coli and S. aureus were analysed. For E. coli, the order of inactivation reaction is 1.15 while S. aureus inactivation reaction order is 0.9.

Antimicrobial activity inactivation and toxic immune reactions induce Epilepsy in humans

Antimicrobial activity inactivation and toxic immune reactions induce Epilepsy in humans

Free radical scavenging and COX-2 inhibition by simple colon metabolites of polyphenols: A theoretical approach

Free radical scavenging and inhibitory potency against cyclooxygenase-2 (COX-2) by two abundant colon metabolites of polyphenols, i.e., 3-hydroxyphenylacetic acid (3-HPAA) and 4-hydroxyphenylpropionic acid (4-HPPA) were theoretically studied. Different free radical scavenging mechanisms are investigated in water and pentyl ethanoate as a solvent. By considering electronic properties of scavenged free radicals, hydrogen atom transfer (HAT) and sequential proton loss electron transfer (SPLET) mechanisms are found to be thermodynamically probable and competitive processes in both media. The Gibbs free energy change for reaction of inactivation of free radicals indicates 3-HPAA and 4-HPPA as potent scavengers. Their reactivity toward free radicals was predicted to decrease as follows: hydroxyl>>alkoxyls>phenoxyl≈peroxyls>>superoxide. Shown free radical scavenging potency of 3-HPAA and 4-HPPA along with their high μM concentration produced by microbial colon degradation of polyphenols could enable at least in situ inactivation of free radicals. Docking analysis with structural forms of 3-HPAA and 4-HPPA indicates dianionic ligands as potent inhibitors of COX-2, an inducible enzyme involved in colon carcinogenesis. Obtained results suggest that suppressing levels of free radicals and COX-2 could be achieved by 3-HPAA and 4-HPPA indicating that these compounds may contribute to reduced risk of colon cancer development.

Stereoelectronic explanations for the mechanistic details of transimination and HF elimination reactions

The β-fluoroamines are commonly used as substrate analogs to determine the mechanistic details of enzymatic reactions. Presence of fluorine atom gives rise to the alterations in the electronic profile and the pKa of molecules which results in mechanistic deviations. The fluorine-substituted mechanism-based substrate analogs are widely used in the inactivation of pyridoxal 5′-phosphate (PLP)-dependent enzymes. The presence of fluorine atom also alters the sequence of reactions taking place in PLP-dependent enzymes where the HF elimination reaction appears in between the transimination and inactivation reactions. Despite the amount of the works on β-fluoroamines, the effect of stereoelectronic differences on the transimination and HF elimination reactions taking place in PLP-dependent enzymes has not been investigated yet. A density functional theory study is conducted to elucidate mechanistic details of the reactions occurring in PLP-dependent enzymes. In order to understand the mechanistic insights of different isomers and the effect of the fluorine atom, 4-amino-3-fluorobutanoic acid (3-F-GABA) enantiomers are chosen to be investigated besides 4-aminobutanoic acid (GABA), which is the natural substrate for γ-aminobutyric acid aminotransferase (GABA-AT). The investigated β-fluoroamines are the experimentally proposed potential inhibitors of PLP-dependent enzyme GABA-AT.

Длинные некодирующие РНК – «камертон»

Most of RNA transcripts, instead of serving templates for protein synthesis, perform different functions such as control of embryogenesis, differentiation, imprinting, X-chromosome inactivation, immune response, and stress reactions. A large portion of these RNAs are denoted as long non-coding RNAs (lncRNAs) which function as signaling molecules, navigating systems and platforms for ribonucleic complexes assembly. They also participate in the organization of specific cellular domains. Moreover, they are able to bind regulatory proteins and micro-RNAs, and serve as precursors for small RNAs. It was shown that lncRNAs participate in transcription regulation; they are also involved in alternative splicing, RNA editing, traffic, translation and degradation of RNA. The three-dimensional structure of lncRNAs plays a crucial role in processes of chromatin remodeling and transcription regulation. In this review we discuss various aspects of lncRNAs functioning.

MOLECULAR CLONING AND TRANSCRIPTION EXPRESSION OF 3‐DEHYDROECDYSONE 3α‐REDUCTASE (3DE 3α‐REDUCTASE) IN THE DIFFERENT TISSUES AND THE DEVELOPING STAGE FROM THE SILKWORM, Bombyx mori L

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3-epiecdyson under ecdysone oxidase and 3-dehydroecdysone 3α-reductase (3DE 3α-reductase). In this paper, we cloned and characterized 3-dehydroecdysone 3α-reductase (3DE 3α-reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α-reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α-reductase belongs to SDR family, which has the NAD(P)-binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α-reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α-reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α-reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α-reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis.

INFLUENCE OF PROCESS CONDITIONS ON MEASLES VIRUS STABILITY

Recombinant measles viruses are currently tested in clinical trials as oncolytic agent to be applied i n cancer therapy. Contrary to their use as vaccine where 10 3 infectious virus particles per dose are needed, fo r cancer therapy 10 9 virus particles should be provided per dose. This leads to other challenges for the production process when compared to vaccine production. This study presents measles virus stability with regard t o conditions during production and storage of the vir us. Relevant process parameters such as temperature (437°C), pH (pH 4-11), conductivity (1.5 to 137.5 mS cm -1 ) and oxygen partial pressure were analyzed. The infectivity of measles virus particles decreased hi ghly at 37 and 32°C, while at 22 and 4°C it remaine d stable for several hours or even days, respectively. The t hermal inactivation reactions followed first order kinetics and the thermodynamic parameters enthalpy and entropy were estimated. Towards changes in pH measles virus particles were very sensitive, while no inact ivation could be observed with varying conductivity . Measles virus incubation at an oxygen partial press ure of 100% did not lead to any loss of infectivity . The results show which parameters should be considered and controlled strongly in the production process t o further raise measles virus yields for the high amo unt needed in cancer therapy approaches.

Comparative Study between Neural Network Model and Mathematical Models for Prediction of Glucose Concentration during Enzymatic Hydrolysis

atic hydrolysis of cellulose is a complex process because of a number of inhibition and enzyme inactivation reactions which happen during hydrolysis. Artificial Neural Networks (ANNs) are very effective in developing predictive models for processes involving complex reaction kinetics that would otherwise be difficult to be modeled by more traditional deterministic approaches. The present investigation was carried out to study the application of Artificial Neural Network as a tool for predicting glucose production by enzymatic hydrolysis of pure cellulose and comparison with mathematical models and experimental results. A feed forward neural network with one hidden layer was trained and used to predict the glucose production. Comparing the R 2 (coefficient of determination), MSE (mean square error) and ARD (average relative deviation) values of the neural network model with the mathematical model, it was concluded that the neural network is more accurate than the mathematical models. The obtained results show that the ANN can be a useful method for the design of the enzymatic hydrolysis.

Genotoxicity and inactivation of catechol metabolites of the mycotoxin zearalenone

Zearalenone (ZEN) is a highly estrogenic mycotoxin produced by Fusarium species. The adverse effects of ZEN and its reductive metabolite α-zearalenol (α-ZEL) are often compared to those of 17β-estradiol (E2) and estrone (E1). These endogenous steroidal estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (1) hormonal activity and (2) genotoxic effects after cytochrome P450-catalyzed metabolic activation to catechols. Like E1 and E2, ZEN and α-ZEL exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites. The subsequent methylation of catechols by catechol-O-methyltransferase (COMT) is generally considered as a detoxifying pathway. Imbalances between the activation and inactivation reactions can lead to the formation of reactive semiquinones and quinones, which can alkylate DNA or produce reactive oxygen species by redox cycling. In the present study, the genotoxicity of the catechol metabolites of ZEN, α-ZEL, E1 and E2 was determined in a cell-free system by measuring 8-oxo-2'-deoxyguanosine using a LC-DAD-MS(2) method. Each of the individual catechols of ZEN, α-ZEL, E1 and E2 induced oxidative DNA damage in calf thymus DNA. The ranking order of the DNA damaging activity was 15-hydroxy-ZEN/α-ZEL ≈ 2/4-hydroxy-E1/E2 > 13-hydroxy-ZEN/α-ZEL. When hepatic microsomes from different species were incubated with ZEN, the rat had the highest activity for catechol formation, followed by human, mouse, pig and steer. The amount of catechol metabolites correlated directly with the amount of oxidative damage in calf thymus DNA. The ranking order for the rate of methylation by human hepatic COMT was 2-hydroxy-E1/E2 >> 4-hydroxy-E1/E2 >> 13/15-hydroxy-ZEN/α-ZEL. Thus, the catechol metabolites of the mycoestrogen ZEN and its reductive metabolite α-ZEL exhibit a DNA-damaging potential comparable to that of the catechol metabolites of E1 and E2, but are much poorer substrates for inactivation by human COMT.

The Arrhenius Equation Revisited

The Arrhenius equation has been widely used as a model of the temperature effect on the rate of chemical reactions and biological processes in foods. Since the model requires that the rate increase monotonically with temperature, its applicability to enzymatic reactions and microbial growth, which have optimal temperature, is obviously limited. This is also true for microbial inactivation and chemical reactions that only start at an elevated temperature, and for complex processes and reactions that do not follow fixed order kinetics, that is, where the isothermal rate constant, however defined, is a function of both temperature and time. The linearity of the Arrhenius plot, that is, Ln[k(T)] vs. 1/T where T is in °K has been traditionally considered evidence of the model's validity. Consequently, the slope of the plot has been used to calculate the reaction or processes’ “energy of activation,” usually without independent verification. Many experimental and simulated rate constant vs. temperature relationships that yield linear Arrhenius plots can also be described by the simpler exponential model Ln[k(T)/k(Treference)] = c(T-Treference). The use of the exponential model or similar empirical alternative would eliminate the confusing temperature axis inversion, the unnecessary compression of the temperature scale, and the need for kinetic assumptions that are hard to affirm in food systems. It would also eliminate the reference to the Universal gas constant in systems where a “mole” cannot be clearly identified. Unless proven otherwise by independent experiments, one cannot dismiss the notion that the apparent linearity of the Arrhenius plot in many food systems is due to a mathematical property of the model's equation rather than to the existence of a temperature independent “energy of activation.” If T+273.16°C in the Arrhenius model's equation is replaced by T+b, where the numerical value of the arbitrary constant b is substantially larger than T and Treference, the plot of Ln k(T) vs. 1/(T+b) will always appear almost perfectly linear. Both the modified Arrhenius model version having the arbitrary constant b, Ln[k(T)/k(Treference) = a[1/ (Treference+b)-1/ (T+b)], and the exponential model can faithfully describe temperature dependencies traditionally described by the Arrhenius equation without the assumption of a temperature independent “energy of activation.” This is demonstrated mathematically and with computer simulations, and with reprocessed classical kinetic data and published food results.

Biapenem Inactivation by B2 Metallo β-Lactamases: Energy Landscape of the Post-Hydrolysis Reactions

BackgroundThe first line of defense by bacteria against β-lactam antibiotics is the expression of β-lactamases, which cleave the amide bond of the β-lactam ring. In the reaction of biapenem inactivation by B2 metallo β-lactamases (MβLs), after the β-lactam ring is opened, the carboxyl group generated by the hydrolytic process and the hydroxyethyl group (common to all carbapenems) rotate around the C5–C6 bond, assuming a new position that allows a proton transfer from the hydroxyethyl group to C2, and a nucleophilic attack on C3 by the oxygen atom of the same side-chain. This process leads to the formation of a bicyclic compound, as originally observed in the X-ray structure of the metallo β-lactamase CphA in complex with product.Methodology/Principal FindingsQM/MM and metadynamics simulations of the post-hydrolysis steps in solution and in the enzyme reveal that while the rotation of the hydroxyethyl group can occur in solution or in the enzyme active site, formation of the bicyclic compound occurs primarily in solution, after which the final product binds back to the enzyme. The calculations also suggest that the rotation and cyclization steps can occur at a rate comparable to that observed experimentally for the enzymatic inactivation of biapenem only if the hydrolysis reaction leaves the N4 nitrogen of the β-lactam ring unprotonated.Conclusions/SignificanceThe calculations support the existence of a common mechanism (in which ionized N4 is the leaving group) for carbapenems hydrolysis in all MβLs, and suggest a possible revision of mechanisms for B2 MβLs in which the cleavage of the β-lactam ring is associated with or immediately followed by protonation of N4. The study also indicates that the bicyclic derivative of biapenem has significant affinity for B2 MβLs, and that it may be possible to obtain clinically effective inhibitors of these enzymes by modification of this lead compound.