Phosphorylation Reaction Research Articles

On the Bromination of Diethyl 3,5-dimethyl-1H-pyrrole-2,4-dicarboxylate: the True Structure of the Halbig’s Product

Pyrrole and its derivatives have attracted the attention of researchers as promising objects of practical importance in various areas of human life. Focusing on Knorr pyrrole as the most readily available pyrrole derivative, our objective was to obtain its bromo derivatives, containing an allyl bromine atom followed by phosphorylation reactions. The reaction involving bromination was carried out with 2 mol of N-bromosuccinimide per 1 mol of Knorr pyrrole 1 in carbon tetrachloride. This reaction was initiated with azobis(isobutyronitrile) and carried out at a boiling point. In acetic acid, the bromination reaction was carried out at a temperature of 38-45°C with a molar ratio of Knorr pyrrole to bromine of 1:4 and another reaction was carried out at 40-50°C with a molar ratio of Knorr pyrrole to bromine of 1:5. The bromination reaction was also performed in chloroform at 27-30°C in the presence of catalytic amounts of aluminum chloride. The same crystalline product was obtained in all cases. X-ray diffraction analysis revealed that the single crystal structure is diethyl 5-(dibromomethyl)-3-methyl-2-oxo-2,3-dihydro-1H-pyrrole-3,4-dicarboxylate 5 with two bromine atoms. This composition is identical to that of the Halbig product. It was found that the bromination of Knorr pyrrole 1 occurs stepwise with excess N-bromosuccinimide and bromine, resulting in the unsaturated lactam 5, commonly known as Halbig’s product. The true structure of product 5 was determined.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. N/A. This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Deletion of the Sodium Glucose Cotransporter 1 (Sglt-1) impairs mouse sperm movement.

The Sodium Glucose Cotransporter Isoform 1 (Sglt-1) is a symporter that moves Na+ and glucose into the cell. While most studies have focused on the role of Sglt-1 in the small intestine and kidney, little is known about this transporter's expression and function in other tissues. We have previously shown that Sglt-1 is expressed in the mouse sperm flagellum and that its inhibition interferes with sperm metabolism and function. Here, we further investigated the importance of Sglt-1 in sperm, using a Sglt-1 knockout mouse (Sglt-1 KO). RNA, immunocytochemistry, and glucose uptake analysis confirmed the ablation of Sglt-1 in sperm. Sglt-1 KO male mice are fertile and exhibit normal sperm counts and morphology. However, Sglt-1 null sperm displayed a significant reduction in total, progressive and other parameters of sperm motility compared to wild type (WT) sperm. The reduction in motility was exacerbated when sperm were challenged to swim in media with higher viscosity. Parameters of capacitation, namely protein tyrosine phosphorylation and acrosomal reaction, were similar in Sglt-1 KO and WT sperm. However, Sglt-1 KO sperm displayed a significant decrease in hyperactivation. The impaired motility of Sglt-1 null sperm was observed in media containing glucose as the only energy substrate. Interestingly, the addition of pyruvate and lactate to the media partially recovered sperm motility of Sglt-1 KO sperm, both in the low and high viscosity media. Altogether, these results support an important role for Sglt-1 in sperm energetics and function, providing sperm with a higher capacity for glucose uptake.

Research new type of cation-exchange resin for waste water treatment

This article presents the studies obtaining and research new polycondensation type phosphoric cation-exchange resin. The conditions for the phosphorylation of this polymer were selected from the experiments accumulated in relation to the phosphorylation reactions of low- and high-molecular compounds. The resulting polymer had an exchange capacity for a 0.1 N NaOH solution – 5,5-5,6 mEq/g and contained 16,5% phosphorus. Based on the studies carried out, the optimal conditions for the synthesis of the styrene-furfural polymer are assumed to be: the reaction temperature is 90°C, the concentration of the ZnCl2 catalyst is 0.07 mol per mole of furfural, and the molar ratio of styrene to furfural is 1:1. There was studied an interaction of the cation exchanger in Na- and H-forms with solutions of salts of copper sulfate, nickel, cobalt, sodium chloride, calcium and uranyl nitrate. In order to elucidate the mechanism of sorption of the cations of these metals, were taken the IR-spectra of the cation exchanger in the H- and Na- form, saturated with copper ions. The less dissociated phosphoric acid groups, the stronger hydrogen bond forms phosphoryl oxygen with OH-groups. Therefore, it can be expected that as the cation exchanger is saturated with sodium, the maximum of the band corresponding to the phosphorus-oxygen bond vibrations will slightly shift to longer wavelengths as a result of the destruction of the weaker hydrogen bond.

A general platform for phosphorylation reactions enabled by photoinduced palladium catalysis

A photoinduced palladium-catalyzed generation of phosphorus radicals using Cl–phosphine oxides and Cl–phosphates is realized to achieve formal P-Heck reaction, C–H phosphinylation of heteroaromatics and difunctionalization of alkenes.

Calcium stress reduces the reproductive capacity and pathogenicity of the pine wood nematode (Bursaphelenchus xylophilus) by inhibiting oxidative phosphorylation reaction

The continuous use of chemical pesticides to control nematodes could result in the developing of pesticide-resistant nematodes. Novel nucleic acid pesticides are becoming the focus of pesticide research due to their strong specificity, high efficiency, and environmental friendliness. However, the limited known biochemical targets restrict the development of target pesticides for nematodes. The calcium stress experiments on pine wood nematodes (PWN) showed that 100 mmol/L Ca2+ resulted in longitudinal depression on the PWN body wall, reduced oviposition, and increased corrected mortality. To enrich the biological targets of nematode pesticides, we further investigated the response mechanism of PWN to calcium stress at the molecular level. Differentially expressed gene analysis showed that genes involved in the oxidative phosphorylation (OXPHOS) pathway were significantly enriched. RNA interference results of 6 key genes belonging to four mitochondrial complex I (BXNDUFA2), III (BXQCR8), IV (BXCOX17), V (BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε) in non-stressed nematodes showed reduction in PWN oviposition, population size, feeding ability, and pathogenicity. The BXNDUFA2 gene interference had the highest inhibitory impact by decreasing the oviposition from 31.00 eggs to 6.75 eggs and PWN population size from 8.27 × 103 nematodes to 1.64 × 103 nematodes, respectively. Interestingly, RNA interference of these 6 key genes in calcium-stressed nematodes also led to increased mortality and decreased oviposition of PWN. In summary, calcium stress inhibited the reproductive capacity of PWN by down-regulating key genes BXNDUFA2, BXQCR8, BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε, and BXCOX17, thereby reducing the pathogenicity. The current results enrich the RNAi targets in PWN and provide a scientific basis for developing novel nucleic nematicides.

Novel In Vitro Multienzyme Cascade for Efficient Synthesis of d-Tagatose from Sucrose

d-Tagatose is a rare sugar with low calories, and is extensively used in food, beverage, and drug additives. In this study, an in vitro multienzyme cascade route for d-tagatose synthesis from sucrose (MCTS) was designed, which contains five enzymes (sucrose phosphorylase, fructokinase, d-fructose 6-phosphate 4-epimerase, d-tagatose 6-phosphate phosphatase, and polyphosphate kinase). The whole MCTS route comprised a sucrose phosphorylation reaction, and a phosphorylation–dephosphorylation reaction coupled with an ATP regeneration system. After optimization, the conversion of d-tagatose from 10 mM sucrose reached 82.3%. At an elevated sucrose concentration of 50 mM, 72.4% of d-tagatose conversion and 0.27 g·L–1·h−1 of space–time yield were obtained. Furthermore, ADP consumption decreased to 1% of the sucrose concentration after introducing the ATP regeneration system. The MCTS strategy is an efficient and cost-effective approach for d-tagatose production.

Graphene oxide enabled efficient multi-enzyme immobilization for thermodynamic-driven biomanufacturing of fructose

Thermodynamic-driven biomanufacturing of fructose catalyzed by phosphorylation and dephosphorylation cascade reactions is considered as the future of industrial fructose production due to its high theoretical yield of 100 %. Co-immobilizing all four cascade enzymes involved on solid materials is crucial for its successful industrial application, as it significantly reduces costs through enzyme reusage. Graphene oxide served as a multi-enzyme carrier in the thermodynamic-driven biosystem of fructose and displayed high loading capacities for the four cascade enzymes. The resulting co-immobilized biosystem based on graphene oxide exhibited superior biocatalytic performance than the other support materials. Additionally, it yielded 54.7 % fructose from maltodextrin. Utilizing two additional enzymes to improve the substrate utilization, an 85.2 % fructose yield was achieved, surpassing the current industrial-scale fructose production process employing monosaccharide isomerization. This study sheds light on the potential industrial applications of thermodynamic-driven biomanufacturing systems for fructose production.

AMP-dependent phosphite dehydrogenase, a phosphorylating enzyme in dissimilatory phosphite oxidation.

Oxidation of phosphite (HPO32-) to phosphate (HPO42-) releases electrons at a very low redox potential (E0'= -690 mV) which renders phosphite an excellent electron donor for microbial energy metabolism. To date, two pure cultures of strictly anaerobic bacteria have been isolated that run their energy metabolism on the basis of phosphite oxidation, the Gram-negative Desulfotignum phosphitoxidans (DSM 13687) and the Gram-positive Phosphitispora fastidiosa (DSM 112739). Here, we describe the key enzyme for dissimilatory phosphite oxidation in these bacteria. The enzyme catalyzed phosphite oxidation in the presence of adenosine monophosphate (AMP) to form adenosine diphosphate (ADP), with concomitant reduction of oxidized nicotinamide adenine dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). The enzyme of P. fastidiosa was heterologously expressed in Escherichia coli. It has a molecular mass of 35.2 kDa and a high affinity for phosphite and NAD+. Its activity was enhanced more than 100-fold by addition of ADP-consuming adenylate kinase (myokinase) to a maximal activity between 30 and 80 mU x mg protein-1. A similar NAD-dependent enzyme oxidizing phosphite to phosphate with concomitant phosphorylation of AMP to ADP is found in D. phosphitoxidans, but this enzyme could not be heterologously expressed. Based on sequence analysis, these phosphite-oxidizing enzymes are related to nucleotide-diphosphate-sugar epimerases and indeed represent AMP-dependent phosphite dehydrogenases (ApdA). A reaction mechanism is proposed for this unusual type of substrate-level phosphorylation reaction.

Prebiotic Syntheses of Organophosphorus Compounds from Reduced Source of Phosphorus in Non-Aqueous Solvents.

Reduced-oxidation-state phosphorus (reduced P, hereafter) compounds were likely available on the early Earth via meteorites or through various geologic processes. Due to their reactivity and high solubility, these compounds could have played a significant role in the origin of various organophosphorus compounds of biochemical significance. In the present work, we study the reactions between reduced P compounds and their oxidation products, with the three nucleosides (uridine, adenosine, and cytidine), with organic alcohols (glycerol and ethanolamine), and with the tertiary ammonium organic compound, choline chloride. These reactions were studied in the non-aqueous solvent formamide and in a semi-aqueous solvent comprised of urea: ammonium formate: water (UAFW, hereafter) at temperatures of 55-68 °C. The inorganic P compounds generated through Fenton chemistry readily dissolve in the non-aqueous and semi-aqueous solvents and react with organics to form organophosphites and organophosphates, including those which are identified as phosphate diesters. This dual approach (1) use of non-aqueous and semi-aqueous solvents and (2) use of a reactive inorganic P source to promote phosphorylation and phosphonylation reactions of organics readily promoted anhydrous chemistry and condensation reactions, without requiring any additive, catalyst, or other promoting agent under mild heating conditions. We also present a comparative study of the release of P from various prebiotically relevant phosphate minerals and phosphite salts (e.g., vivianite, apatite, and phosphites of iron and calcium) into formamide and UAFW. These results have direct implications for the origin of biological P compounds from non-aqueous solvents of prebiotic provenance.

Identification of CryAB as a target of NUAK kinase activity in Drosophila muscle tissue.

Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phospho-mimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis.

Improvement of thermal stability and flame retardancy of beech wood fibers using steam explosion and phosphorylation reaction

An environmentally friendly approach was studied for improving the fire retardancy of beech wood using a steam explosion process coupled with a phosphorylation reaction. Raw beech, steam-exploded, or bleached wood was subjected to phosphorylation employing etidronic acid (HEDP) and urea, and the resulting chemical composition and thermal behavior were analyzed using techniques such as elemental analysis, Fourier transform infrared spectroscopy, ionic chromatography, and pyrolysis combustion flow calorimetry. The thermal performance of beech wood underwent significant enhancements after the steam explosion treatment combined with soda impregnation. It resulted in a striking reduction in heat release rates (HRR), especially its peak (pHRR) and time-to-pHRR (TpHRR) values (from 139.5 W/g and 365 °C to 40 W/g and 247 °C) and an increase in residue from 10.5% to 41.3%. The results demonstrate that the process created a self-extinguishing and non-flammable material that exhibited noteworthy flame retardancy improvement.

HGF/c-MET: A Potential Target for the Treatment of Various Cancers

Background: Cancer is the abnormal growth of cells in the body due to an imbalance in the normal apoptotic pathways. The abnormality in the cancer cells makes them malignant. Various types of treatment, including chemotherapy, radiation therapy, targeted therapy, and immunotherapy (IMT), are used for cancer. Mesenchymal-epithelial transition factor (c-Met) belongs to the tyrosine kinase receptor family and is overexpressed in various types of cancers. c-Met is a proto-oncogene and facilitates a wide range of biological functions, including cell proliferation, growth, migration, invasion, and angiogenesis, through interaction with its sole ligand hepatocyte growth factor (HGF). Currently, various c-mesenchymal-epithelial transition (c-MET) inhibitors and antibodies are in human trials for their anti-cancer activity. Introduction: The c-MET is a kinase receptor for hepatocyte growth factor (HGF). It is wellrecognized for its tumorigenic potential. HGF binding with c-Met leads to c-Met dimerization and c- Met phosphorylation, which in turn activates many intracellular signalling pathways, including ERK1/2, MAPK, STAT3, Rac1, and PI3K/AKT. These pathways regulate the proliferation, invasion, and migration of cancer cells. Upon binding of HGF to c-MET, a series of phosphorylation reactions get started, which leads to transcription and translation of various proteins, followed by abnormal growth of cancerous tissues due to dysregulation of the cell cycle. The HGF/c-MET signalling pathways have shown their potential in the development of many cancers, including gastric cancer (GC). Several clinical trials have evaluated the therapeutic benefits of MET-targeted therapies involving various agents, such as anti-MET antibodies, anti-HGF antibodies, and tyrosine kinase inhibitors (TKIs). Various c-MET inhibitors are in clinical trials. The current review is focussed on the critical role of the HGF/c-MET pathways in the progression of various cancers, including GC. In addition, this review will also focus on the combination potential of c-MET inhibitors with immuno-oncology drugs, such as programmed cell death protein 1 (PD-1) inhibitors, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, tumor necrosis factor (TNF) receptor (OX-40) agonists, etc. Materials and Methods: The method adopted for the study was primarily based on the secondary search through a systematic review of the literature. Results: A study of recent literature and various preclinical and human trials suggests the effectiveness of c-MET inhibitors (alone or in combination) in different cancer types, including GC. In this paper, the results have been elaborated as to how many papers/manuscripts/publications on the topic are present, how many drugs are in Phase I/II/ III/RCT, etc., and that how many papers report on the clinical outcomes of which agent/drug (mentioned in percentage). Conclusion: The use of c-MET inhibitors and antibodies has emerged as a latent therapeutic approach for the treatment of various types of cancer. The c-MET inhibitors can also be used in combination with various immunotherapeutic drugs, like PD-1 inhibitors, OX-40 agonists, etc.

Enol phosphates from 3-diethylphosphonocoumarin: synthesis and transformations under phase-transfer conditions

Enol phosphates were prepared from 3-diethylphosphonocoumarin under phase-transfer conditions as altered conditions for tandem hydrogenation / phosphorylation reactions. The optimized reaction conditions are characterized with high regioselectivity for O-phosphorylation and O-acylation. The newly synthesized enol phosphates were fully characterized by NMR Spectroscopic techniques and the single (Z)-isomers were assigned.

Electro-enzymatic ATP regeneration coupled to biocatalytic phosphorylation reactions

Adenosine-5-triphosphate (ATP) is the main energy vector in biological systems, thus its regeneration is an important issue for the application of many enzymes of interest in biocatalysis and synthetic biology. We have developed an electroenzymatic ATP regeneration system consisting in a gold electrode modified with a floating phospholipid bilayer that allows coupling the catalytic activity of two membrane-bound enzymes: NiFeSe hydrogenase from Desulfovibrio vulgaris and F1Fo-ATP synthase from Escherichia coli. Thus, H2 is used as a fuel for producing ATP. This electro-enzymatic assembly is studied as ATP regeneration system of phosphorylation reactions catalysed by kinases, such as hexokinase and NAD+-kinase for respectively producing glucose-6-phosphate and NADP+.

Reaction acceleration in microdroplet mass spectrometry: Inlet capillary and solvent composition effects.

Microdroplet chemistry has attracted tremendous interest in recent years. We have previously reported that microdroplet mass spectrometry (MS) achieves reaction acceleration. Here we systematically investigated the effect of capillary heating of MS inlet and solvent polarity of microdroplets on the conversion ratios of dehydration and phosphorylation reactions. The micron-sized droplets generated by high-speed gas encapsulated the compounds. The conversion ratios of dehydration and phosphorylation reactions were investigated at different capillary temperatures of MS inlet between 30°C and 300°C. Subsequently, the effects of solvent polarity of different microdroplets (acetonitrile, acetonitrile/water [v/v: 9:1], and water) on microdroplet reactions were investigated. The microdroplets could be used as reaction vessels for rapid dehydration and phosphorylation reactions. Microdroplet MS is characterized by the completion of the reaction in microseconds. The increase in capillary temperature increased the conversion ratio of dehydration reactions but had little effect on phosphorylation reactions. The stability of compounds supports this phenomenon. In addition, the increase in solvent polarity in microdroplets promoted the dehydration reaction but inhibited the nucleophilic substitution reaction (phosphorylation reaction). Microdroplet MS achieved an acceleration of the reaction, which was attributed to capillary temperature, microdroplet solvents, and the stability of reaction products. This finding suggested that the inlet capillary and solvent system should be considered in the study and interpretation of microdroplet MS.

Synthesis of Phosphate Esters by Using Diphenyl Ditelluride as Organocatalyst

AbstractWe report a protocol for the synthesis of new dialkyl(1‐(organolthio)naphthalen‐2‐yl)phosphates and derivatives via a dehydrogenative phosphorylation reaction between functionalized naphthols and H‐phosphonates. Diphenyl ditelluride was employed as an organocatalyst and sonochemistry as an alternative energy source. The reaction conditions were defined through a factorial design approach, and the best condition provided the synthesis of dialkyl(1‐(organolthio)naphthalen‐2‐yl)phosphates in yields that varied from 51–98 % under ultrasound irradiation after 2 h of sonication. The synthetic protocol was also effective for phenol and thiophenol starting materials. All products were characterized by state‐of‐the‐art spectroscopic and spectrometric techniques.

Multiplex Identification of Post‐Translational Modifications at Point‐of‐Care by Deep Learning‐Assisted Hydrogel Sensors

AbstractMultiplex detection of protein post‐translational modifications (PTMs), especially at point‐of‐care, is of great significance in cancer diagnosis. Herein, we report a machine learning‐assisted photonic crystal hydrogel (PCH) sensor for multiplex detection of PTMs. With closely‐related PCH sensors microfabricated on a single chip, our design achieved not only rapid screening of PTMs at specific protein sites by using only naked eyes/cellphone, but also the feasibility of real‐time monitoring of phosphorylation reactions. By taking advantage of multiplex sensor chips and a neural network algorithm, accurate prediction of PTMs by both their types and concentrations was enabled. This approach was ultimately used to detect and differentiate up/down regulation of different phosphorylation sites within the same protein in live mammalian cells. Our developed method thus holds potential for POC identification of various PTMs in early‐stage diagnosis of protein‐related diseases.

Multiplex Identification of Post-Translational Modifications at Point-of-Care by Deep Learning-Assisted Hydrogel Sensors.

Multiplex detection of protein post-translational modifications (PTMs), especially at point-of-care, is of great significance in cancer diagnosis. Herein, we report a machine learning-assisted photonic crystal hydrogel (PCH) sensor for multiplex detection of PTMs. With closely-related PCH sensors microfabricated on a single chip, our design achieved not only rapid screening of PTMs at specific protein sites by using only naked eyes/cellphone, but also the feasibility of real-time monitoring of phosphorylation reactions. By taking advantage of multiplex sensor chips and a neural network algorithm, accurate prediction of PTMs by both their types and concentrations was enabled. This approach was ultimately used to detect and differentiate up/down regulation of different phosphorylation sites within the same protein in live mammalian cells. Our developed method thus holds potential for POC identification of various PTMs in early-stage diagnosis of protein-related diseases.

Impact of Negative Feedbacks on De Novo Pyrimidines Biosynthesis in Escherichia coli.

Earlier studies aimed at investigating the metabolism of endogenous nucleoside triphosphates in synchronous cultures of E. coli cells revealed an auto-oscillatory mode of functioning of the pyrimidine and purine nucleotide biosynthesis system, which the authors associated with the dynamics of cell division. Theoretically, this system has an intrinsic oscillatory potential, since the dynamics of its functioning are controlled through feedback mechanisms. The question of whether the nucleotide biosynthesis system has its own oscillatory circuit is still open. To address this issue, an integral mathematical model of pyrimidine biosynthesis was developed, taking into account all experimentally verified negative feedback in the regulation of enzymatic reactions, the data of which were obtained under in vitro conditions. Analysis of the dynamic modes of the model functioning has shown that in the pyrimidine biosynthesis system, both the steady-state and oscillatory functioning modes can be realized under certain sets of kinetic parameters that fit in the physiological boundaries of the investigated metabolic system. It has been demonstrated that the occurrence of the oscillatory nature of metabolite synthesis depended on the ratio of two parameters: the Hill coefficient, hUMP1-the nonlinearity of the UMP effect on the activity of carbamoyl-phosphate synthetase, and the parameter r characterizing the contribution of the noncompetitive mechanism of UTP inhibition to the regulation of the enzymatic reaction of UMP phosphorylation. Thus, it has been theoretically shown that the E. coli pyrimidine biosynthesis system possesses its own oscillatory circuit whose oscillatory potential depends to a significant degree on the mechanism of regulation of UMP kinase activity.