VITAMIN B1 BIOLOGICAL FUNCTION IMPLEMENTATION IN THE BLOOD OF PATIENTS WITH STOMACH CANCER UNDER SURGICAL INTERVENTION

Background. The probability of undergoing surgery always predetermines the state of stress in a person; therefore, it is advisable to search for ways to optimize and (or) reduce this unfavourable effect. Objective. To find out the mechanism of vitamin B1 antistress activity during surgery. Material and methods. Metabolism intensity was investigated on donors’ blood lysates (n = 19) and those of patients with stage III stomach cancer (n = 64), referred to an elective surgery, aged 51-70. The blood was taken from the cubital vein three days before the operation, after premedication, during the most traumatic moment of the operation, after extubation, as well as on the first and third days of the postoperative period. The surgery was performed under combined multicomponent anesthesia using nitric oxide, sodium hydroxybutyrate, and epidural block. Thiamine and thiamine diphosphate kinase activities were assessed by the concentration of the formed thiamine di- and triphosphates of the vitamin. The activities of thiamine mono-, di- and triphosphatases were determined by the release of inorganic phosphate. The concentration of inorganic phosphate was recorded colorimetrically. The content of B1 and its derivatives in the blood was determined by the method of ion-pair reversed-phase HPLC. Results. There has been observed an increased content of thiamine monophosphate and that of free thiamine in the blood of donors and patients with stomach cancer. The registered rate of the hydrolytic thiamine monophosphatase reaction is not high. At the stages of premedication and maximum trauma of surgical exposure, the concentration of monophosphoric ester rapidly decreases alongside with monophosphatase activation. Therefore, the thiamine monophosphate hydrolysis is the rate-limiting link of vitamin B1 metabolism. The level of free thiamine remains persistently increased at all stages of surgical treatment. Thiamine monophosphatase activity is manifested at two pH optima – of 6.0 and 9.0. Thiamine monophosphate hydrolysis at pH of 9.0 is catalyzed by alkaline phosphatase. At pH of 6.0, in addition to thiamine monophosphoric ester, the enzyme hydrolyzes only p-nitrophenyl phosphate, flavin mononucleotide and phosphotyrosine, that allows it to be classified as hepatic acid phosphatase. The noted changes in B1 metabolism under stress concern mainly non-coenzyme forms - thiamine mono-, triphosphate, and free thiamine, which are used at the stages of thiol reduction as important components of insulin synthesis. Conclusions. The use of vitamin B1 allows to optimize the development of the stress response at all stages of surgical treatment. Its protective effect is achieved through the activation of the insulin-synthetic function of the pancreas, which increases the level of immunoreactive insulin in the blood. The formation of the most favorable physiological conditions for insulin synthesis provides an increased background of free thiamine, which is created due to the hydrolysis of noncoenzyme forms of the vitamin. The relationship between thiamine metabolism and B2 exchange and regulation of intracellular signaling pathways has been traced.

Revealing the unknown aspects of initial steps of prenylated flavin mononucleotide biosynthesis – the role of Lys129 in PaUbiX

Background Prenylated flavin mononucleotide (prFMN) is a recently discovered, heavily modified flavin compound. It is the only known cofactor that enables enzymatic 1,3-dipolar cycloaddition reactions. It is produced by enzymes from UbiX family, from flavin mononucleotide and either dimethylallyl mono- or diphosphate. prFMN biosynthesis is currently reported to be initiated by a protonation of the substrate by Glu140. Methods Computational chemistry methods are applied herein - mostly different flavors of molecular dynamics MD, such as Constant pH MD, hybrid Quantum-Mechanical / Molecular Mechanical MD, and classical MD. Results Glu140 competes for a single proton with Lys129 but it is the latter that adopted a protonated state throughout most of the simulation time. Lys129 plays a key role in the positioning of the DMAP’s phosphate group within the PaUbiX active site. DMAP’s breakdown into a phosphate and a prenyl group can be decoupled from the protona-tion of the DMAP’s phosphate group. Conclusions The role of Lys129 in functioning of PaUbiX is reported for the first time. The severity of interactions between Glu140, Lys129, and DMAP’s phosphate group enables an unusual decoupling of phosphate’s protonation from DMAP’s breakdown. Those findings are most likely conserved throughout the UbiX family to the structural re-semblence of active sites of those proteins. Significance Mechanistic insights into a crucial biochemical process, biosynthesis of prFMN, are provided. This study, alt-hough purely computational, extends and perfectly complements the knowledge obtained in classical laboratory experiments.

Development of nutrient medium for riboflavin biosynthesis by Eremothecium ashbyi ascomycetes

The object of the study is the riboflavin producer Eremothecium ashbyi Guilliermond 1935 VKPM F-340, the subject of the study is the regularities of riboflavin biosynthesis by the E. ashbyi F-340 strain under different cultivation conditions. Riboflavin is an important micronutrient that is a precursor of the coenzymes flavin mononucleotide and flavinadine dinucleotide, it is necessary for biochemical reactions in all living cells. Population growth and an increase in human needs for vitamin-fortified food and agricultural products is the reason for an increase in demand for riboflavin preparations. Considering this, it is important and economically beneficial to improve the technology for the production of vitamin B2. An important factor that affects the yield of the product is the nutrient medium. At present, the influence of agricultural waste on the biosynthesis of riboflavin is being actively studied in the world. However, not all of the studied types of raw materials are typical for the agriculture of this or that country. Therefore, in order to determine whether this direction of research is promising, it is important to check the effect on the biosynthetic activity of the riboflavin producer of the most common wastes of the domestic industry. In this work, this is done on the example of Ukraine. In the course of the study, microbiological (surface and deep cultivation of E. ashbyi F-340), physicochemical (determination of the amount of biomass by the gravimetric method, determination of the concentration of riboflavin by the spectrophotometric method) and mathematical methods were used. The proposed media with the addition of agricultural waste, providing a higher yield of riboflavin compared to conventional media. The influence of different types of agricultural waste on the biosynthesis of riboflavin by the producer E. ashbyi F-340 was evaluated. The efficiency of using sunflower cake as a component of the nutrient medium is shown. The optimal sources of carbon for the nutrient medium with oil cake have been determined, which increases the yield of riboflavin. Due to the large amount of sunflower cake obtained in Ukraine, its use for modifying the nutrient medium in order to increase the yield of riboflavin in the future will lead to a decrease in the cost of the target product due to the use of cheap and ecological raw materials.

Facile Cell-Friendly Hollow-Core Fiber Diffusion-Limited Photofabrication

Bioprinting emerges as a powerful flexible approach for tissue engineering with prospective capability to produce tissue on demand, including biomimetic hollow-core fiber structures. In spite of significance for tissue engineering, hollow-core structures proved difficult to fabricate, with the existing methods limited to multistage, time-consuming, and cumbersome procedures. Here, we report a versatile cell-friendly photopolymerization approach that enables single-step prototyping of hollow-core as well as solid-core hydrogel fibers initially loaded with living cells. This approach was implemented by extruding cell-laden hyaluronic acid glycidyl methacrylate hydrogel directly into aqueous solution containing free radicals generated by continuous blue light photoexcitation of the flavin mononucleotide/triethanolamine photoinitiator. Diffusion of free radicals from the solution to the extruded structure initiated cross-linking of the hydrogel, progressing from the structure surface inwards. Thus, the cross-linked wall is formed and its thickness is limited by penetration of free radicals in the hydrogel volume. After developing in water, the hollow-core fiber is formed with centimeter range of lengths. Amazingly, HaCaT cells embedded in the hydrogel successfully go through the fabrication procedure. The broad size ranges have been demonstrated: from solid core to 6% wall thickness of the outer diameter, which was variable from sub-millimeter to 6 mm, and Young’s modulus ∼1.6 ± 0.4 MPa. This new proof-of-concept fibers photofabrication approach opens lucrative opportunities for facile three-dimensional fabrication of hollow-core biostructures with controllable geometry.

Isopentenyl diphosphate/dimethylallyl diphosphate-specific Nudix hydrolase from the methanogenic archaeon Methanosarcina mazei

Nudix hydrolases typically catalyze the hydrolysis of nucleoside diphosphate linked to moiety X and yield nucleoside monophosphate and X-phosphate, while some of them hydrolyze a terminal diphosphate group of non-nucleosidic compounds and convert it into a phosphate group. Although the number of Nudix hydrolases is usually limited in archaea comparing with those in bacteria and eukaryotes, the physiological functions of most archaeal Nudix hydrolases remain unknown. In this study, a Nudix hydrolase family protein, MM_2582, from the methanogenic archaeon Methanosarcina mazei was recombinantly expressed in Escherichia coli, purified, and characterized. This recombinant protein shows higher hydrolase activity toward isopentenyl diphosphate and short-chain prenyl diphosphates than that toward nucleosidic compounds. Kinetic studies demonstrated that the archaeal enzyme prefers isopentenyl diphosphate and dimethylallyl diphosphate, which suggests its role in the biosynthesis of prenylated flavin mononucleotide, a recently discovered coenzyme that is required, for example, in the archaea-specific modified mevalonate pathway.

MSMEG_3955 from Mycobacterium smegmatis is a FMN bounded homotrimeric NAD(P)H:Flavin mononucleotide (FMN) oxidoreductase

Background Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb. Results In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% β-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways. Conclusion MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Current Strategies to Minimize Ischemia-Reperfusion Injury in Liver Transplantation: A Systematic Review.

Background: Hepatic ischemia reperfusion injury (IRI) is a serious threat that characterizes the liver but also other transplantable organs. The worst effect of long-term IRI on an impaired graft could lead to irreversible damage and organ failure. Several events characterize the cascade that ultimately leads to organ failure. Among all, multiple strategies have been attempted to identify early phenomena of IRI with divergent results, and biomarkers might represent a novel approach to early detect ischemic damage. Methods: A literature review of the current state-of-the-art on IRI was conducted in the present manuscript. Information was collected from worldwide clinical trials conducted in highly specialized institutions. Experiments conducted on IRI animal models and clinical studies were screened. The final outcomes were analyzed and reported in the present review. Results: Matrix Metalloproteinases (MMPs) represent an interesting example of the early detector of neutrophil invasion after acute and chronic hepatic IRI. Neutrophil gelatinase-associated lipocalin (NGAL) is another biomarker that seems more predictable of the IRI gravity phase. Mitochondrial flavin mononucleotide (FMN) was recently discovered and might become a reliable biomarker of hepatic IRI during hypothermic oxygenation machine perfusion (HOPE). Conclusion: The available strategies to avoid IRI, despite constantly improving, are still lacking of a gold standard method. Further studies are still needed to explore new options in the IRI diagnosis and treatment, and to this purpose regenerative medicine and tissue engineering surely can play a pivotal role in the transplantation field.