Substrate Reaction Research Articles

Tau seeding activity in skin biopsy differentiates tauopathies from synucleinopathies

Most neurodegenerative diseases lack definitive diagnostic tests, and the identification of easily accessible and reliable biomarkers remains a critical unmet need. Since tau protein is highly expressed in skin of tauopathies patients, we aimed to exploit the ultrasensitive seeding activity assay (SAA) to assess tau seeding activity in skin of patients with tauopathies. In this multicentric, case-control study, patients with tauopathies and synucleinopathies were consecutively recruited and sex-matched to healthy controls (HC). Subjects underwent a double 3 mm skin biopsy in cervical area and ankle. Skin tau-SAA, using TauK18 and TauK19 as reaction substrates for 4R and 3R isoforms, seeding score, clinical scales, biochemical and morphological characterization of SAA end-products were evaluated. We analyzed 58 subjects: 24 tauopathies (18 progressive supranuclear palsy, PSP, and 6 corticobasal degeneration, CBD), 20 synucleinopathies (14 Parkinson’s disease, PD, and 6 multiple system atrophy, MSA), and 14 HC. PSP and CBD showed higher tau seeding activity at both anatomical sites. A greater sensitivity of 4R-SAA than 3R-SAA was observed. 4R tau-SAA identified tauopathies with 71% sensitivity and 93% specificity. Accuracy was higher for PSP than CBD: PSP vs HC / PD (AUC 0.825), while CBD vs HC / PD (AUC 0.797), and PSP vs MSA (AU 0.778). SAA end-products showed differences in biochemical and morphological characterization according to the anatomical site. Skin tau-SAA identifies tauopathies with good accuracy and can be used to implement the in-vivo clinical diagnosis of patients with neurodegenerative diseases. Further characterization of peripheral tau seed in skin may elucidate the structure of tau deposits in brain.

Thiazolium salt mimics the non-coenzyme effects of vitamin B1 in rat synaptosomes

Long-term studies have confirmed a causal relationship between the development of neurodegenerative processes and vitamin B1 (thiamine) deficiency. However, the biochemical mechanisms underlying the high neurotropic activity of thiamine are not fully understood. At the same time, there is increasing evidence that vitamin B1, in addition to its coenzyme functions, may have non-coenzyme activities that are particularly important for neurons. To elucidate which effects of vitamin B1 in neurons are due to its coenzyme function and which are due to its non-coenzyme activity, we conducted a comparative study of the effects of thiamine and its derivative, 3-decyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium chloride (DMHT), on selected processes in synaptosomes. The ability of DMHT to effectively compete with thiamine for binding to thiamine-binding sites on the plasma membrane of synaptosomes and to participate as a substrate in the thiamine pyrophosphokinase reaction was demonstrated. In experiments with rat brain synaptosomes, unidirectional effects of DMHT and thiamine on the activity of the pyruvate dehydrogenase complex (PDC) and on the incorporation of radiolabeled [2–14C]pyruvate into acetylcholine were demonstrated. The observed effects of thiamine and DMHT on the modulation of acetylcholine synthesis can be explained by suggesting that both compounds, which interact in cells with enzymes of thiamine metabolism, are phosphorylated and exert an inhibitory/activating effect (concentration-dependent) on PDC activity by affecting the regulatory enzymes of the complex. Such effects were not observed in the presence of structural analogues of thiamine and DMHT without a 2-hydroxyethyl substituent at position 5 of the thiazolium cycle. The effect of DMHT on the plasma membrane Ca-ATPase was similar to that of thiamine. At the same time, DMHT showed high cytostatic activity against neuroblastoma cells.

Correlating Substrate Reactivity at Electrified Interfaces with the Electrolyte Structure in Synthetically Relevant Organic Solvent/Water Mixtures.

Optimizing electrosynthetic reactions requires fine tuning of a vast chemical space, including charge transfer at electrocatalyst/electrode surfaces, engineering of mass transport limitations, and complex interactions of reactants and products with their environment. Hybrid electrolytes, in which supporting salt ions and substrates are dissolved in a binary mixture of organic solvent and water, represent a new piece of this complex puzzle as they offer a unique opportunity to harness water as the oxygen or proton source in electrosynthesis. In this work, we demonstrate that modulating water-organic solvent interactions drastically impacts the solvation properties of hybrid electrolytes. Combining various spectroscopies with synchrotron small-angle X-ray scattering (SAXS) and force field-based molecular dynamics (MD) simulations, we show that the size and composition of aqueous domains forming in hybrid electrolytes can be controlled. We demonstrate that water is more reactive for the hydrogen evolution reaction (HER) in aqueous domains than when strongly interacting with solvent molecules, which originates from a change in reaction kinetics rather than a thermodynamic effect. We exemplify novel opportunities arising from this new knowledge for optimizing electrosynthetic reactions in hybrid electrolytes. For reactions proceeding first via the activation of water, fine tuning of aqueous domains impacts the kinetics and potentially the selectivity of the reaction. Instead, for organic substrates reacting prior to water, aqueous domains have no impact on the reaction kinetics, while selectivity may be affected. We believe that such a fine comprehension of solvation properties of hybrid electrolytes can be transposed to numerous electrosynthetic reactions.

Problem-Based Learning for Calculating Kinetic Parameters from Michaelis-Menten Equation

In “Advanced Enzymology” at the University of Barcelona, we teach various methods for identifying uni-uni irreversible reactions. Kinetic constants such as maximum velocity (Vmax) and Michaelis constant (Km) are calculated using non-linear regression with the Michaelis-Menten equation. However, if a computer is not available, students must calculate these kinetic parameters using linear regressions with Lineweaver-Burk, Eadie-Hofstee, and Hanes-Woolf plots. Michaelis-Menten equation: v = (Vmax [S]) / (Km + [S]), Lineweaver-Burk equation: 1/v = (Km/Vmax) (1/[S]) + 1/Vmax, Eadie-Hofstee equation: v = – Km (v/[S]) + Vmax, Hanes-Woolf equation: [S]/v = (1/Vmax) [S] + (Km/Vmax). The objective of this work was that students explore various methods for calculating kinetic constants and determine the most effective approach. A problem was prepared with data on substrate concentrations ([S]) and reaction rates (v). Data points with lower substrate concentration, and also with lower velocities had higher errors. The Michaelis-Menten plot provided only an approximate estimation of the kinetic constants, as it is not a linear graph. The Lineweaver-Burk plot generated maximum velocities that were too low due to the higher errors for low substrate concentrations, rendering impossible values of the kinetic parameters. The Eadie-Hofstee plot provided better results, as the values of v/[S] compensated their errors. Furthermore, kinetic constants were obtained directly from the slope and intersection of the line equation. The Hanes-Woolf plot also produced correct values for kinetic parameters, since high substrate concentrations were less erroneous than lower ones. After completing this computer class, students were very satisfied and learned the different methods for obtaining kinetic parameters. Non-linear regression is undoubtedly the most accurate method for obtaining kinetic parameters values. However, in the absence of computers, the Eadie-Hofstee plot is the best and most commonly used plot in kinetic papers.

Molecular Wiring of Electrocatalytic Nitrate reduction to Ammonia and Water Oxidation by Iron-Coordinated Macroporous Conductive Networks.

Developing stable electrocatalysts with accessible isolated sites is desirable but highly challenging due to metal agglomeration and low surface stability of host materials. Here we report a general approach for synthesis of single-site Fe electrocatalysts by integrating a solvated Fe complex in conductive macroporous organic networks through redox-active coordination linkages. Electrochemical activation of the electrode exposes high-density coordinately unsaturated Fe sites for efficient adsorption and conversion of reaction substrates such as NO3- and H2O. Using the electrode with isolated active Fe sites, electrocatalytic NO3- reduction and H2O oxidation can be coupled in a single cell to produce NH3 and O2 at Faradaic efficiencies of 97% and 100%, respectively. The electrode exhibits excellent robustness in electrocatalysis for 200 hours with small decrease in catalytic efficiencies. Both the maximized Fe-site efficiency and the microscopic localization effect of the conductive organic matrix contribute to the high catalytic performances, which provides new understandings in tuning the efficiencies of metal catalysts for high-performance electrocatalytic cells.

Molecular Wiring of Electrocatalytic Nitrate reduction to Ammonia and Water Oxidation by Iron‐Coordinated Macroporous Conductive Networks

AbstractDeveloping stable electrocatalysts with accessible isolated sites is desirable but highly challenging due to metal agglomeration and low surface stability of host materials. Here we report a general approach for synthesis of single‐site Fe electrocatalysts by integrating a solvated Fe complex in conductive macroporous organic networks through redox‐active coordination linkages. Electrochemical activation of the electrode exposes high‐density coordinately unsaturated Fe sites for efficient adsorption and conversion of reaction substrates such as NO3− and H2O. Using the electrode with isolated active Fe sites, electrocatalytic NO3− reduction and H2O oxidation can be coupled in a single cell to produce NH3 and O2 at Faradaic efficiencies of 97 % and 100 %, respectively. The electrode exhibits excellent robustness in electrocatalysis for 200 hours with small decrease in catalytic efficiencies. Both the maximized Fe‐site efficiency and the microscopic localization effect of the conductive organic matrix contribute to the high catalytic performances, which provides new understandings in tuning the efficiencies of metal catalysts for high‐performance electrocatalytic cells.

Wettability of molten CaO-Al2O3-MgO slag on some refractory materials at 1773K

The interface characteristic of molten slag has an important effect on the interface phenomenon and reaction between molten slag and molten metal/refractory. The wettability of molten CaO-MgO-Al2O3 slag on some refractory materials (including Mo, graphite, boron nitride (BN), MgO and Al2O3) was studied in argon atmosphere at 1773 K. The contact angle between molten slag and substrate material was measured by the sessile drop method; the interfacial tension and work of adhesion were calculated by Young equation and Young-Dupré equation, respectively. The results showed that, except for graphite, the molten slag can well wet these materials, and the contact angle decreases with increasing temperature and eventually converges to a stable equilibrium value. The interfacial tension of different substrate materials is very different, and the interfacial tension between the slag and reactive substrate MgO, Al2O3 and graphite is small, ranging from 71 to 205 mN/m. The molten slag-Mo of the non-reactive substrate is 1323 mN/m and the molten slag-BN is 7297 mN/m. The adhesion work between molten slag and substrate is in the range of 800–1100 mJm−2 except graphite, which is 400 mJm−2.

Cry Toxins from <i>Bt</i> Bollgard II® Cotton Seeds: An Affordable Source for Insect Bioassays

The relative expression of Bacillus thuringiensis (Bt) Cry1Ac and Cry2Ab toxins from seeds of Bt Bollgard II® cotton was quantified using Enzyme Linked Immunosorbent Assays (ELISA) method. The ELISA technique involves binding a sample or calibrator to a solid-phase antibody, forming a "sandwich complex" with an enzyme-labeled antibody. The resulting enzyme substrate reaction produces a product proportional to the antigen concentration. The study reveals that each gram of Bt Bollgard II® seed powder contains 3.25 μg Cry1Ac and 182.32 μg Cry2Ab, with Cry2Ab expression 56 times higher. Variability in Cry protein expression is influenced by factors like temperature, genetic background, and plant maturity. The research emphasizes the importance of Bt plants, particularly Bt Bollgard II® seed powder as a valuable source for studying insect responses to combined Cry1Ac + Cry2Ab expression in bioassay studies.

Synthesis, Characterization, and Catalytic Performance of a New Heterobimetallic Y/Tb Metal-Organic Framework with High Catalytic Activity.

A three-dimensional heterobimetallic porous structure with the formula {[Y3.5Tb1.5L6(OH)3(H2O)1.5 (DMF)1.5] n ·1.5H2O·DMF} n (L = 3-amino-4-hydroxybenzoate) (Y/Tb-MOF) has been synthesized and characterized by single crystal and powder X-ray diffraction, scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX), inductively coupled plasma mass spectrometry (ICP-MS), electrophoretic mobility, and Fourier transform infrared (FTIR) spectroscopy. The structure presents two metal environments: a bioaugmented isosceles wedge (mm2) MO8 and a tricapped trigonal prism (-6m2) MN3O6. These configurations facilitate the creation of channels with a diameter of 10.7 Å, enabling its utilization as an active catalyst where the heterobimetallic nature of the assembly will be explored. This mixed-metal metal-organic framework has been tested in the cycloaddition of epoxides with carbon dioxide as well as in the cyanosilylation and hydroboration reactions of carbonylic substrates. Additionally, a monometallic Tb-MOF analogue has been synthesized for comparative evaluation of their catalytic performances. Both the mixed metal and monometallic variants exhibit outstanding activity in the cyanosilylation and hydroboration of carbonyls and in the synthesis of carbonates under CO2 pressure. However, only the latter exhibits high recyclability.

The stable sulfur isotope and abundance fluxes of reduced inorganic sulfur and organic sulfur phases recorded in the Permian-Triassic transition of the Meishan type section

Sulfur cycle fluxes implicated in the Permian-Triassic mass extinction have traditionally been studied by the sulfur phase abundances in sedimentary rocks and the stable sulfur isotopic value (δ34S) of seawater sulfate inferred from mineral sulfate analyses. This information might be complemented by studies of the reduced inorganic sulfur and organic sulfur produced following bacterial sulfate reduction. To explore this potential the δ34S and concentration analyses of total reduced inorganic sulfur (TRIS) and organic sulfur – separately in the forms of kerogen (Ker) and individual organosulfur compounds, specifically dibenzothiophenes (DBTs) – has been conducted on sediments across the Late Permian to Early Triassic marine type section of Meishan-1 (South China). The relatively steady δ34S profiles (e.g., < 5 ‰ variation) of all sulfur phases measured through much of the late Permian were indicative of a primary seawater sulfate control, but other biogeochemical modulators caused prominent δ34S fluctuations of TRIS and DBT adjacent to the extinction event. The late Triassic δ34STRIS profile of Meishan-1 displayed a notable 34S enrichment (+15 ‰ increase) in bed 22–24 sediments concomitant with lower δ34SDBT values (−7 ‰ decrease), whereas co-eval δ34SKerS values remained relatively constant. The contrasting δ34SDBT and δ34SKerS data suggests the dynamic behavior of specific diagenetic sulfurisation processes may be resolved by the δ34S of discrete organic sulfur compounds (i.e., dibenzothiophenes, DBTs), but dissipated by the sulfurisation collective represented by the bulk kerogen fraction. The inverse isotopic trend observed between DBT and TRIS resulted in negative Δδ34SDBT-TRIS values identifying an organic sulfurisation pathway(s) with an unusual preference over pyrite (FeS2) for the lighter stable sulfur isotope. A redox control of the δ34SDBTs and δ34STRIS deviations in the bed 22–24 extinction interval was confirmed by coincident variation in TRIS/(TRIS + KerS) and pyrite (Py) and highly reactive (HR) iron ratios (FePy/FeHR). The iron (Fe) speciation data indicated a transition to ferruginous conditions, ruling out Fe2+ limitation as a factor in the bias against 34S evident in DBT formation. The 34S depletion of the DBTs promoted by the ferruginous setting may arise from the rapid and irreversible reaction of organic substrates with labile sulfur anions (e.g. HS-) or be supported by an especially localised sediment–water depositional microenvironment. Our study highlights the potential of incorporating stable sulfur isotope analytics of reduced and organic sulfur phases, particularly of specific organic compounds, into a holistic assessment of the dynamic sulfur biochemical periods of Earth’s past.

Tailoring the Active Sites of Nanosheet NiSe/NiSe2 Catalyst by Pulse Electrodeposition on the 3D Microporous Ni-Cu/NF Substrate for both Hydrogen and Oxygen Evolution Reactions

Establishing proper intrinsic catalysts with nanostructured high active surfaces endows the paramount electrocatalytic activity. A Ni-Se@Cu-Ni/NF catalyst for hydrogen and oxygen evolution reactions (HER and OER) is prepared via an efficient two-step pulse current (PC) electrodeposition method. The initial 3D film of Cu-Ni is synthesized via the dynamic hydrogen bubble template (DHBT) method to attain further active surface area. Then, Ni-Se film is prepared by direct current (DC) and PC electrodeposition. Morphological, chemical, and electrocatalytic characteristics of the Ni-Se electrodeposited films are evaluated. X-ray photoelectron spectroscopy and high-resolution transmission electron microscopy results show a NiSe/NiSe2 nanofilm on the 3D microporous nanostructured Cu-Ni substrate which reveals an efficient bifunctional electrocatalytic behavior with overpotentials of 74 and 272 mV in the current density of 10 mA cm−2, and Tafel slopes of 78 and 50 mV dec−1 for HER and OER, respectively. The two-electrode examination with NiSe/NiSe2@Cu-Ni/NF catalyst in overall water splitting indicates a required potential of 1.57 V in the current density of 10 mA cm−2.

Gold-Catalyzed Cascade Reaction of Yne-Enones with Iminooxindoles, Access to 3,2'-Pyrrolidinyl-Spirooxindole Derivatives.

Herein, a gold-catalyzed cascade reaction of yne-enones with iminooxindoles has been developed through a cascade cycloisomerization/(3 + 2) annulation process. This approach provides a straightforward and efficient route for the synthesis of functionalized 3,2'-pyrrolidinyl-spirooxindoles in high reactivity and broad substrate scope with excellent cis-selectivity. Moreover, the subsequent functionalization of furan units allows for the diverse synthesis of spirooxindole derivatives, which have demonstrated good antitumoral activity.

Extending PROXIMAL to predict degradation pathways of phenolic compounds in the human gut microbiota.

Despite significant advances in reconstructing genome-scale metabolic networks, the understanding of cellular metabolism remains incomplete for many organisms. A promising approach for elucidating cellular metabolism is analysing the full scope of enzyme promiscuity, which exploits the capacity of enzymes to bind to non-annotated substrates and generate novel reactions. To guide time-consuming costly experimentation, different computational methods have been proposed for exploring enzyme promiscuity. One relevant algorithm is PROXIMAL, which strongly relies on KEGG to define generic reaction rules and link specific molecular substructures with associated chemical transformations. Here, we present a completely new pipeline, PROXIMAL2, which overcomes the dependency on KEGG data. In addition, PROXIMAL2 introduces two relevant improvements with respect to the former version: i) correct treatment of multi-step reactions and ii) tracking of electric charges in the transformations. We compare PROXIMAL and PROXIMAL2 in recovering annotated products from substrates in KEGG reactions, finding a highly significant improvement in the level of accuracy. We then applied PROXIMAL2 to predict degradation reactions of phenolic compounds in the human gut microbiota. The results were compared to RetroPath RL, a different and relevant enzyme promiscuity method. We found a significant overlap between these two methods but also complementary results, which open new research directions into this relevant question in nutrition.

Production of fermentable glucose from the hydrolysis process of cellulose algae waste using fungal cellulases

Kappaphycus alvarezii is the most widely cultivated algae species globally because of its main use in carrageenan production. Waste resulting from carrageenan production still contains high levels of cellulose, which is potentially convertible into fermentable sugar. However, the commercial viability of sugar conversion from algae waste is limited by the high cost of the hydrolysis required to break down the algae waste. This study aims to develop a new hydrolysis method, through the adoption of fungal algal cell disruption to produce a fermentable sugar. The fungus Trichoderma reseei was developed as a new pretreatment method. It was chosen for its superior inherent properties, including low chemical input and environmental friendliness. This study develops a method for fungal pretreatment of algae processing waste. We investigated the effects of substrate concentration, inoculum size, and reaction time on sugar production using full factorial design. The results showed that substrate concentration, inoculum size, and reaction time affect sugar production. This study demonstrates that sugar production from algae proccessing waste can reach more than 70% under optimum conditions. These results indicate the potential of microbial cellullase as an economically friendly pretreatment for sugar production from algae processing waste.

Textural Precursor Compositions Harvested for Independent Signal Generators: Scaling Micron-Sized Flower-Like Metal-Organic Frameworks as Amplifying Units for Dual-Mode Glycoprotein Assay.

In this work, a micron-sized flower-like metal-organic frameworks (MOFs)-based boronate-affinity sandwich-type immunoassay was fabricated for the dual-mode glycoprotein assay. For proof of concept, the flower-like MOFs were synthesized from transition Cu nodes and tetrakis (4-carboxyphenyl) porphyrin (TCPP) ligands by spontaneous standing assembly. In addition, the specificity toward glycoprotein involved the antigen recognition as well as covalent bonding via the boronate-glycan affinity, and the immediate signal responses were initiated by textural decomposition of the flower-like MOFs. Intriguingly, Cu nodes, of which the valence state is dominant by CuI species, can endow the Fenton-like catalytic reaction of the fluorogenic substrate for generating fluorescence signals. For benefits, TCPP ligands, in which each TCPP molecule has four guest donors, can provide multiple valences for the assembly of cyclodextrin-capped gold nanoparticles via host-guest interaction for colorimetry output. Albeit important, the scaling micrometer patterns for the flower-like MOFs carrying numerous Cu nodes and TCPP ligands can also function as amplifying units, signifying the output signal. The detection limit of the dual-mode glycoprotein assay can reach 10.5 nM for the fluorescence mode and 18.7 nM for the colorimetry mode, respectively. Furthermore, the merits of harvesting different signal generators toward the multimodal readout patterns can allow the mutual verification and make the analytical results more reliable. Collectively, our proposed assay may offer a new idea in combining the inherent textural merits from MOFs for dual signal generators, which can also emphasize accurate detection capability for glycoprotein assay.

Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase.

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

Skeletal muscle hypertrophy rewires glucose metabolism: An experimental investigation and systematic review.

Proliferating cancer cells shift their metabolism towards glycolysis, even in the presence of oxygen, to especially generate glycolytic intermediates as substrates for anabolic reactions. We hypothesize that a similar metabolic remodelling occurs during skeletal muscle hypertrophy. We used mass spectrometry in hypertrophying C2C12 myotubes in vitro and plantaris mouse muscle in vivo and assessed metabolomic changes and the incorporation of the [U-13C6]glucose tracer. We performed enzyme inhibition of the key serine synthesis pathway enzyme phosphoglycerate dehydrogenase (Phgdh) for further mechanistic analysis and conducted a systematic review to align any changes in metabolomics during muscle growth with published findings. Finally, the UK Biobank was used to link the findings to population level. The metabolomics analysis in myotubes revealed insulin-like growth factor-1 (IGF-1)-induced altered metabolite concentrations in anabolic pathways such as pentose phosphate (ribose-5-phosphate/ribulose-5-phosphate: +40%; P=0.01) and serine synthesis pathway (serine: -36.8%; P=0.009). Like the hypertrophy stimulation with IGF-1 in myotubes in vitro, the concentration of the dipeptide l-carnosine was decreased by 26.6% (P=0.001) during skeletal muscle growth in vivo. However, phosphorylated sugar (glucose-6-phosphate, fructose-6-phosphate or glucose-1-phosphate) decreased by 32.2% (P=0.004) in the overloaded muscle in vivo while increasing in the IGF-1-stimulated myotubes in vitro. The systematic review revealed that 10 metabolites linked to muscle hypertrophy were directly associated with glycolysis and its interconnected anabolic pathways. We demonstrated that labelled carbon from [U-13C6]glucose is increasingly incorporated by ~13% (P=0.001) into the non-essential amino acids in hypertrophying myotubes, which is accompanied by an increased depletion of media serine (P=0.006). The inhibition of Phgdh suppressed muscle protein synthesis in growing myotubes by 58.1% (P<0.001), highlighting the importance of the serine synthesis pathway for maintaining muscle size. Utilizing data from the UK Biobank (n=450243), we then discerned genetic variations linked to the serine synthesis pathway (PHGDH and PSPH) and to its downstream enzyme (SHMT1), revealing their association with appendicular lean mass in humans (P<5.0e-8). Understanding the mechanisms that regulate skeletal muscle mass will help in developing effective treatments for muscle weakness. Our results provide evidence for the metabolic rewiring of glycolytic intermediates into anabolic pathways during muscle growth, such as in serine synthesis.

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Optical properties of low-pressure DC Ar plasma torch with N2–SiH4–B2H6 gas mixture injection for Si–B–N thin film deposition

Si–B–N ceramic hard films were prepared by injecting N2+SiH4+B2H6 reaction gas into an argon DC arc plasma jet at reduced pressure. XPS, FT-IR, SEM, and microhardness tester were used to characterize the samples, and the optical properties of Ar discharge plasma jet with and without reaction gas injection were studied by a multichannel high-resolution spectroscopy and a wide spectral range spectroscopy. It is found that the deposited films are mainly composed of Si, B, N and O. The film surface presents a dense structure with hundred-nanometer-sized clusters, and the hardness of sample is greatly enhanced. When the reaction gas is injected into the plasma jet, the emission of excited atoms in the background plasma is greatly reduced, and the growth rate of Si–B–N film shows a significant downward trend as the reaction gas injection ring or substrate holder is moved away from the torch nozzle. A slight enhancement of background plasma emission is observed near the substrate surface with increasing the distance between gas injection ring and torch nozzle, which indicates that the suppression effect of the reactive gas injection on the background plasma emission and the activation efficiency of the reaction gas are weakened, thereby reducing the deposition rate.

Ultrasensitive and Highly Selective Detection of Staphylococcus aureus at the Single-Cell Level Using Bacteria-Imprinted Polymer and Vancomycin-Conjugated MnO2 Nanozyme.

Pathogenic bacterial infections, even at extremely low concentrations, pose significant threats to human health. However, the challenge persists in achieving high-sensitivity bacterial detection, particularly in complex samples. Herein, we present a novel sandwich-type electrochemical sensor utilizing bacteria-imprinted polymer (BIP) coupled with vancomycin-conjugated MnO2 nanozyme (Van@BSA-MnO2) for the ultrasensitive detection of pathogenic bacteria, exemplified by Staphylococcus aureus (S. aureus). The BIP, in situ prepared on the electrode surface, acts as a highly specific capture probe by replicating the surface features of S. aureus. Vancomycin (Van), known for its affinity to bacterial cell walls, is conjugated with a Bovine serum albumin (BSA)-templated MnO2 nanozyme through EDC/NHS chemistry. The resulting Van@BSA-MnO2 complex, serving as a detection probe, provides an efficient catalytic platform for signal amplification. Upon binding with the captured S. aureus, the Van@BSA-MnO2 complex catalyzes a substrate reaction, generating a current signal proportional to the target bacterial concentration. The sensor displays remarkable sensitivity, capable of detecting a single bacterial cell in a phosphate buffer solution. Even in complex milk matrices, it maintains outstanding performance, identifying S. aureus at concentrations as low as 10 CFU mL-1 without requiring intricate sample pretreatment. Moreover, the sensor demonstrates excellent selectivity, particularly in distinguishing target S. aureus from interfering bacteria of the same genus at concentrations 100-fold higher. This innovative method, employing entirely synthetic materials, provides a versatile and low-cost detection platform for Gram-positive bacteria. In comparison to existing nanozyme-based bacterial sensors with biological recognition materials, our assay offers distinct advantages, including enhanced sensitivity, ease of preparation, and cost-effectiveness, thereby holding significant promise for applications in food safety and environmental monitoring.