Magnesium ions (Mg[2+]) play a critical role in RNA structure stabilization by forming various coordinated complexes, preferentially interacting with the backbone phosphate groups. Using extensive atomistic and free energy simulations across simple models and RNA structures of varying complexity, we characterized critical components of the RNA-ion atmosphere. Radial distribution function analysis reveals distinct peak positions for direct (inner) and solvent-separated (outer-sphere) Mg[2+]-phosphate coordination layers, aligning with solution X-ray diffraction data. Addressing force field limitations, the free energy calculations quantify the kinetic barriers for Mg[2+]-phosphate binding and benchmark the results against [25]Mg NMR measurement. Importantly, the free energy differences between inner-sphere and outer-sphere Mg[2+] coordination states, as well as between monocoordinated and bicoordinated (chelated) states, are found to be marginal. These findings suggest a dynamic equilibrium among multiple, barrier-separated Mg[2+] coordination states, consistent with prior simulations, NMR spectroscopy, and other spectroscopic studies. Free energy calculations further explore Mg[2+] chelation in a biphosphate-coordinated system, identifying a dynamic ensemble of prechelate complexes, in addition to a chelated and outer-sphere hexa-hydrated state of Mg[2+]. In the prechelated states, Mg[2+] maintains one inner-sphere interaction while simultaneously coordinating with multiple other phosphates in a solvent-separated manner─a hybrid inner-outer-sphere ion-coordination mode. The prechelated complexes spanning different solvent-separated layers undergo frequent transitions, mediated by a unique oxygen exchange mechanism between phosphate groups and water ligands. Insights into the free energy landscape of SAM-I riboswitch aptamer RNA further emphasize the significance of prechelate complexes for complex RNA structure stabilization, where multiple such solvent-separated dynamic phosphate groups are found to influence Mg[2+]-RNA coordination. The comprehensive thermodynamic analysis of Mg[2+] chelation and quantitative characterizations of various RNA-ion coordination modes provide critical insights for advancing RNA modeling and experimental exploration of complex phosphate networks in the RNA structures.

As part of the work, polymer composites dedicated to rapid prototyping were developed, especially for 3D printing using the material extrusion technique. For this purpose, a polymer matrix was selected, which was an acrylonitrile-butadiene-styrene (ABS) terpolymer and a flame retardant, which was tetrakis (2,6-dimethylphenyl)-m-phenylenebisphosphate, commercially known as PX200. The effect of the presence and amount (5, 10, 15 and 20 wt.%) of the introduced additive on the rheological properties, structural properties, flammability (limiting oxygen index, LOI; UL94) and flame retardant properties (microcone calorimeter, MLC) of ABS-based composites was investigated. In addition, the mechanism of thermal degradation and flame resistance was investigated using thermogravimetric analysis, TGA and Fourier transform infrared spectroscopy, FT-IR of the residue after the MLC test. In the first part of the work, using the author's technological line, filaments were obtained from unfilled ABS and its composites. Samples for testing were obtained by 3D printing in Fused Deposition Modeling (FDM) technology. In order to determine the quantitative and qualitative spread of fire and the effectiveness of the phosphorus flame retardant PX200 in the produced composites, the Maximum Average Rate of Heat Emission (MARHE); Fire Growth Rate Index (FIGRA); Fire Potential Index (FPI) and Flame Retardancy Index (FRI) were determined. Based on the obtained results, it was found that the aryl biphosphate used in this work exhibits activity in the gas phase, which was confirmed by quantitative assessment using data from a microcone calorimeter and non-residues after combustion and thermolysis at 700 °C. As a result, the flammability class did not change (HB40), and the LOI slightly increased to 20% for the composite with 20% flame retardant content. Moreover, this composite was characterized by the following flammability indices: pHRR = 482.9 kW/m2 (-40.3%), MARHE = 234 kW/m2 (-40.7%), FIGRA = 3.1 kW/m2·s (-56.3%), FPI = 0.061 m2·s/kW (+64.9%), FRI = 2.068 (+106.8%).

Energy metabolism homeostasis emerges as a dominant element influencing mesenchymal stem/stromal cells' trajectory of development. The predominant glycolysis activity is a primary driver of cell proliferation and maintenance of the high-energetic state. Here, we examined the functions of two crucial auxiliary pathways: the phosphate-pentose pathway (PPP) and fructose-2,6-biphosphate pathway (FBP) to evaluate their impact on the therapeutic potential of Adipose-Derived Stem/Stromal cells (ASCs) during prolonged culture in various oxygen conditions: 5 % O2 - physiological normoxia or 21 % O2 - atmospheric oxygen. Our findings demonstrate that ASCs cultured in 5 % O2 increased the rate of proliferation, migration, and expression of stemness factors, which is prominent during the initial and middle passages. Additionally, ASCs cultured in a 5 % O2 exhibited heightened protection mechanisms against free radicals, increased LDH gene expression, and elevated extracellular acidification rate (ECAR). By estimating the HIF-1α level, we concluded that 5 % oxygen conditions were insufficient to induce a profound hypoxic state in ASCs. However, at the protein level, both the PPP and FBP pathways appeared to be more active in young (2-passage) cells, regardless of oxygen conditions, and their activity diminished over time. Additionally, the chemical suppression of G6PDH by Polydatin and inhibition of PFKFB3 by PFK-158 in ASCs (passage-2) revealed dose- and time-dependent effect on decreasing migratory capabilities of cells. Nevertheless, our work underscores the adaptable nature of ASC metabolism to prevailing external conditions, with the aging of the culture contributing to the decline in glycolysis-associated auxiliary pathways.

Acetate is a biological anion with many applications in the chemical and food industries. In addition to being a common microbial fermentative end-product, acetate can be produced by photosynthetic cyanobacteria from CO2 using solar energy. Using wild-type cells of the unicellular model cyanobacterium Synechocystis PCC 6803 only low levels of acetate are observed outside the cells. By inserting a heterologous phosphoketolase (PKPa) in the acs locus, encoding acetyl-CoA synthetase responsible for the irreversible conversion of acetate to acetyl-CoA, an increased level of 40 times was observed. Metabolite analyses indicate an enhanced Calvin-Benson-Bassham cycle, based on increased levels of glyceraldehyde 3-phosphate and fructose-1,6-biphosphate, while the decreased levels of 3-phosphoglycerate and pyruvate suggest a quick consumption of the fixed carbon. Acetyl-P and erythrose-4-phosphate showed significantly increased levels, as products of phosphoketolase, while acetyl-CoA remained stable through the experiment. The results of intra- and extra-cellular acetate levels clearly demonstrate an efficient excretion of produced acetate from the cells in the engineered strain. Knock-out of ach and pta showed a reduction in acetate production however, it was not as low as in cells with a single knock-out of ach. Overexpressing acetyl-CoA hydrolase (Ach) and acetate kinase (AckA) did not significantly increase production. In contrast, overexpressing phosphotransacetylase (Pta) in cells containing an inserted PKPa resulted in 80 times more acetate reaching 2.3 g/L after 14 days of cultivation.

Emerging organophosphate flame retardants (E-OPFRs) are a new class of pollutants that have attracted increasing attention, but their bioaccumulation patterns and trophodynamic behaviors in aquatic food webs still need to be validated by comparison with legacy OPFRs (L-OPFRs). In this study, we simultaneously investigated the bioaccumulation, trophic transfer, and dietary exposure of 8 E-OPFRs and 10 L-OPFRs in a tropical estuarine food web from Hainan Island, China. Notably, the Σ10L-OPFRs concentration (16.1-1.18 × 10[5] lipid weight (lw)) was significantly greater than that of Σ8E-OPFRs (nondetectable (nd) - 2.82 × 10[3] ng/g lw) among the investigated organisms, and they both exhibited similar trends: fish<mollusk<crustacean. Significant biomagnification was found only for triphenyl phosphate (TPHP, an L-OPFR) with a trophic magnification factor (TMF) of 1.55, whereas other E- and L-OPFRs showed limited trophic transfer potential. Storage lipid was the dominant adsorption phase for most E-OPFRs and L-OPFRs on the basis of the fugacity approach. The water content controls the main trophic partitioning for the L-OPFRs of triethyl phosphate (TEP) and tris(2-chloroethyl) phosphate (TCEP) in the food web. Storage lipid and structure protein are equally important for 2,2-bis(chloromethyl)-propane-1,3-diyltetrakis(2-chloroethyl) biphosphate (V6, an emerging OPFR). The investigation of metabolites and the biotransformation rate (K M) confirmed the role of biotransformation in offsetting trophic transfer for both legacy and emerging OPFRs. Furthermore, the hazard quotients (HQs) were found to be <1 for South China residents for both the E- and L-OPFRs, but the health risks of these two kinds of OPFRs should be given sustained attention.

Glucose metabolism plays a pivotal role in physiological processes and cancer growth. The final stage of glycolysis, converting phosphoenolpyruvate (PEP) into pyruvate, is catalyzed by the pyruvate kinase (PK) enzyme. Whereas PKM1 is mainly expressed in cells with high energy requirements, PKM2 is preferentially expressed in proliferating cells, including tumor cells. Structural analysis of PKM1 and PKM2 is essential to design new molecules with antitumoral activity. To understand their structural dynamics, we performed extensive high-resolution molecular dynamics (MD) simulations using adaptive sampling techniques coupled to the polarizable AMOEBA force field. Performing more than 6 μs of simulation, we considered all oligomerization states of PKM2 and propose structural insights for PKM1 to further study the PKM2-specific allostery. We focused on key sites including the active site and the natural substrate Fructose Bi-Phosphate (FBP) fixation pocket. Additionally, we present the first MD simulation of biologically active PKM1 and uncover important similarities with its PKM2 counterpart bound to FBP. We also analysed TEPP-46's fixation, a pharmacological activator binding a different pocket, on PKM2 and highlighted the structural differences and similarities compared to PKM2 bound to FBP. Finally, we determined potential new cryptic pockets specific to PKM2 for drug targeting.

Binding phospholipid is a simple, yet flexible, strategy for anchorage of bacterial effectors at cell membrane to manipulate host signaling responses. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are the only two phospholipid species known to direct bacterial effectors to establish inner leaflet localization at the plasma membrane. Here, selectivity of phosphatidic acid (PA) by bacterial effectors for the plasma membrane anchorage and its molecular entity was identified. C-terminal BID domain of Bartonella T4SS effectors (Beps) directed the plasma membrane localization of Beps in host cells through binding with PA. A hydrophobic segment of the 'HOOK' subdomain from BID is inserted into the bilayer to enhance the interaction of positively charged residues with the lipid headgroups. Mutations of a conserved arginine facilitating the electrostatic interaction, a conserved glycine maintaining the stability of the PA binding groove, and hydrophobic residues determining membrane insertion, prevented the anchorage of Beps at the plasma membrane. Disassociation from plasma membrane to cytosol attenuated the BepC capacity to induce stress fiber formation and cell fragmentation in host cells. The substitution of alanine with aspartic acid at the -1 position preceding the conserved arginine residue hindered BepD anchoring at the plasma membrane, a vital prerequisite for its ability to elicit IL-10 secretion in host macrophages. In conclusion, our findings reveal the PA-binding properties of bacterial effectors to establish plasma membrane localization and will shed light on the intricate mechanisms employed by bacterial effectors within host cells.

Seashore paspalum (Paspalum vaginatum) is a warm-season and perennial turfgrass and is known for its cadmium (Cd)-stress tolerance. Here, a Phosphoproteomics analysis was performed to examine the key proteins relating to Cd tolerance in seashore paspalum. Fructose 1,6-biphosphate aldolase, PvFBA1, was identified for its phosphorylated state after exposure to Cd stress. Specifically, the phosphorylation of PvFBA1 was enhanced in several metabolic pathways, including pentose phosphate pathway (PPP), carbon fixation and biosynthesis of amino acids under Cd stress. By transforming PvFBA1 into Arabidopsis, the PvFBA1-OE plants exhibited longer roots, greater FBA activity and higher soluble sugar content than WT under 100 µM CdCl2 treatment. By expressing the PvFBA1 in yeast, a serine 50 phosphorylation site was identified as functional site. By microscale thermophoresis experiment, we indicted that PvFBA1can bind Cd directly enhancing its phosphorylation level to alleviate the damage of Cd. This finding may provide new insights into the molecular mechanisms of plants Cd tolerance.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P2. We observed that the concentration of free PI(4,5)P2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P2.

Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death.

Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging. Herein, a strategy is presented where glycolysis is inhibited in cancer cells using PFK15 (inhibitor of PFKFB3, rate-limiting step in glycolysis), while simultaneously glycolysis and function is rescued in DCs by delivery of fructose-1,6-biphosphate (F16BP, one-step downstream of PFKFB3). To demonstrate the feasibility of this strategy, vaccine formulations are generated using calcium-phosphate chemistry, that incorporate F16BP, poly(IC) as adjuvant, and phosphorylated-TRP2 peptide antigen and tested in challenging and established YUMM1.1 tumours in immunocompetent female mice. Furthermore, to test the versatility of this strategy, adoptive DC therapy is developed with formulations that incorporate F16BP, poly(IC) as adjuvant and mRNA derived from B16F10 cells as antigens in established B16F10 tumours in immunocompetent female mice. F16BP vaccine formulations rescue DCs in vitro and in vivo, significantly improve the survival of mice, and generate cytotoxic T cell (Tc) responses by elevating Tc1 and Tc17 cells within the tumour. Overall, these results demonstrate that rescuing glycolysis of DCs using metabolite-based formulations can be utilized to generate immunotherapy even in the presence of glycolytic inhibitor.

Hot chilli ('Bhut Jolokia') (Capsicum chinense Jacq.) is the hottest chilli widely grown in the North-Eastern region of India for its high pungency. However, little information is available on its physiology, growth and developmental parameters including yield. Therefore, the present research was undertaken to study the physiological responses of Bhut Jolokia under elevated CO2 (eCO2) and temperature. Two germplasms from two different agro-climatic zones (Assam and Manipur) within the North-East region of India were collected based on the pungency. The present study explored the interactive effect of eCO2 [at 380, 550, 750 ppm (parts per million)] and temperature (at ambient, > 2 °C above ambient, and > 4 °C above ambient) on various physiological processes, and expression of some photosynthesis and capsaicin related genes in both the germplasms. Results revealed an increase (> 1-2 fold) in the net photosynthetic rate (Pn), carbohydrate content, and C: N ratio in 'Bhut Jolokia' under eCO2 and elevated temperature regimes compared to ambient conditions within the germplasms. Gene expression studies revealed an up-regulation of photosynthesis-related genes such as Cs RuBPC2 (Ribulose biphosphate carboxylase 2) and Cs SPS (Sucrose phosphate synthase) which, explained the higher Pn under eCO2 and temperature conditions. Both the germplasm showed better performance under CTGT-II (Carbon dioxide Temperature Gradient Tunnel having 550 ppm CO2 and temperature of 2 °C above ambient) in terms of various physiological parameters and up-regulation of key photosynthesis-related genes. An up-regulation of the Cs capsaicin synthase gene was also evident in the study, which could be due to the metabolite readjustment in 'Bhut Jolokia'. In addition, the cultivar from Manipur (cv. 1) had less fruit drop compared to the cultivar from Assam (cv. 2) in CTGT II. The data indicated that 550 ppm of eCO2 and temperature elevation of > 2 °C above the ambient with CTGT-II favored the growth and development of 'Bhut Jolokia'. Thus, results suggest that Bhut Jolokia grown under the elevation of CO2 up to 550 ppm and temperature above 2 °C than ambient may support the growth, development, and yield.

Rubisco activase (RCA) catalyzes the release of inhibitory sugar phosphates from ribulose-1,6-biphosphate carboxylase/oxygenase (Rubisco) and can play an important role in biochemical limitations of photosynthesis under dynamic light and elevated temperatures. There is interest in increasing RCA activity to improve crop productivity, but a lack of understanding about the regulation of photosynthesis complicates engineering strategies. In this review, we discuss work relevant to improving RCA with a focus on advances in understanding the structural cause of RCA instability under heat stress and the regulatory interactions between RCA and components of photosynthesis. This reveals substantial variation in RCA thermostability that can be influenced by single amino acid substitutions, and that engineered variants can perform better in vitro and in vivo under heat stress. In addition, there are indications RCA activity is controlled by transcriptional, post-transcriptional, post-translational, and spatial regulation, which may be important for balancing between carbon fixation and light capture. Finally, we provide an overview of findings from recent field experiments and consider the requirements for commercial validation as part of efforts to increase crop yields in the face of global climate change.