Combinatorial Strategy for Studying Biochemical Pathways in Double Emulsion Templated Cell-Sized Compartments

Abstract Cells rely upon producing enzymes at precise rates and stoichiometry for maximizing functionalities. The reasons for this optimal control are unknown, primarily because of the interconnectivity of the enzymatic cascade effects within multi-step pathways. Here, an elegant strategy for studying such behavior, by controlling segregation/combination of enzymes/metabolites in synthetic cell-sized compartments, while preserving vital cellular elements is presented. Therefore, compartments shaped into polymer GUVs are developed, producing via high-precision double-emulsion microfluidics that enable: i) tight control over the absolute and relative enzymatic contents inside the GUVs, reaching nearly 100% encapsulation and co-encapsulation efficiencies, and ii) functional reconstitution of biopores and membrane proteins in the GUVs polymeric membrane, thus supporting in situ reactions. GUVs equipped with biopores/membrane proteins and loaded with one or more enzymes are arranged in a variety of combinations that allow the study of a three-step cascade in multiple topologies. Due to the spatiotemporal control provided, optimum conditions for decreasing the accumulation of inhibitors are unveiled, and benefited from reactive intermediates to maximize the overall cascade efficiency in compartments. The non-system-specific feature of the novel strategy makes this system an ideal candidate for the development of new synthetic routes as well as for screening natural and more complex pathways

Association of Mortality and Risk of Epilepsy With Type of Acute Symptomatic Seizure After Ischemic Stroke and an Updated Prognostic Model

IMPORTANCE: Acute symptomatic seizures occurring within 7 days after ischemic stroke may be associated with an increased mortality and risk of epilepsy. It is unknown whether the type of acute symptomatic seizure influences this risk. OBJECTIVE: To compare mortality and risk of epilepsy following different types of acute symptomatic seizures. DESIGN, SETTING, AND PARTICIPANTS: This cohort study analyzed data acquired from 2002 to 2019 from 9 tertiary referral centers. The derivation cohort included adults from 7 cohorts and 2 case-control studies with neuroimaging-confirmed ischemic stroke and without a history of seizures. Replication in 3 separate cohorts included adults with acute symptomatic status epilepticus after neuroimaging-confirmed ischemic stroke. The final data analysis was performed in July 2022. EXPOSURES: Type of acute symptomatic seizure. MAIN OUTCOMES AND MEASURES: All-cause mortality and epilepsy (at least 1 unprovoked seizure presenting >7 days after stroke). RESULTS: A total of 4552 adults were included in the derivation cohort (2547 male participants [56%]; 2005 female [44%]; median age, 73 years [IQR, 62-81]). Acute symptomatic seizures occurred in 226 individuals (5%), of whom 8 (0.2%) presented with status epilepticus. In patients with acute symptomatic status epilepticus, 10-year mortality was 79% compared with 30% in those with short acute symptomatic seizures and 11% in those without seizures. The 10-year risk of epilepsy in stroke survivors with acute symptomatic status epilepticus was 81%, compared with 40% in survivors with short acute symptomatic seizures and 13% in survivors without seizures. In a replication cohort of 39 individuals with acute symptomatic status epilepticus after ischemic stroke (24 female; median age, 78 years), the 10-year risk of mortality and epilepsy was 76% and 88%, respectively. We updated a previously described prognostic model (SeLECT 2.0) with the type of acute symptomatic seizures as a covariate. SeLECT 2.0 successfully captured cases at high risk of poststroke epilepsy. CONCLUSIONS AND RELEVANCE: In this study, individuals with stroke and acute symptomatic seizures presenting as status epilepticus had a higher mortality and risk of epilepsy compared with those with short acute symptomatic seizures or no seizures. The SeLECT 2.0 prognostic model adequately reflected the risk of epilepsy in high-risk cases and may inform decisions on the continuation of antiseizure medication treatment and the methods and frequency of follow-up

Effect of Schistosomiasis and Soil-Transmitted Helminth Infections on Physical Fitness of School Children in Côte d'Ivoire

The burden of parasitic worm infections is considerable, particularly in developing countries. It is acknowledged that parasitic worm infections negatively impact on children's school performance and physical development. A deeper understanding of these linkages is important for updating burden of disease measures. We investigated the relationship between worm infection status and physical fitness of 156 school children from Côte d'Ivoire and controlled for potential confounding of Plasmodium infection (the causative agent of malaria) and environmental parameters (temperature and humidity). Children were diagnosed for parasitic worm and Plasmodium infections, examined by a physician, and participated in a 20 m shuttle run test to assess their maximal oxygen uptake (VO2 max) as a proxy for physical fitness. Most of the children had parasitic worms and a Plasmodium infection. Nevertheless, their physical fitness was excellent (average VO2 max: 52.7 ml kg−1 min−1). The level of VO2 max was only influenced by sex and age, but not by parasitic worms and Plasmodium infections. In future studies, the dynamics of children's physical performance should be assessed before and after control interventions, including the assessment of blood hemoglobin, hematocrit, and nutritional indicators to determine whether physical fitness in worm- and Plasmodium-infected individuals can be further improved

Silicon-based Plasmonic Surfaces and Flow Microreactors

There is a need for streamlining certain laboratory techniques in synthetic chemistry for the discovery of novel molecules. This applies to many fields, especially to drug discovery, which relies on the cost-efficient design and synthesis of drug candidates. As the requirements for new materials and molecules in medicinal chemistry and other fields get more complex, new technologies have received great attention in recent years. New chemical reactor types that benefit from process automation, continuous manufacturing and miniaturisation have been developed for that purpose. Miniaturised flow microreactors can provide exceptionally uniform reaction conditions and possibly increase the space-time yield compared to batch reactors—in particular for large reaction screenings. They also facilitate multistep reactions without the need for working up the intermediates. Significant contributions have been made from microsystems technology in the design and fabrication of Si- and glass-based microfluidic devices as flow microreactors. Due to their high chemical resistance and their compatibility with micro- and nanofabrication processing, they are well suited as materials in lab-on-a-chip systems for synthetic chemistry. The aim of this work was to establish the basis for a self-optimising reaction screening device on a chip. The system consists of multiple connected flow microreactor compartments populated with different heterogeneous organocatalysts. These molecular catalysts are immobilised as monolayers on the channel surfaces and the reactors are therefore called "wall-coated flow microreactors". Several fundamental aspects of this aim were investigated. The viability of wall-coated flow microreactors for this purpose was studied by designing various reactor types and developing a versatile fabrication protocol using microfabrication. This fabrication process involved the metallisation of the microchannel surfaces with a Au thin film. This served to bind thiol-linked organocatalysts as self-assembled monolayers (SAMs) to the reactor surfaces. The detection and characterisation of such SAMs was a major focus of this work. In the context of the wall-coated flow microreactors, methods for the assessment of the presence and stability of the catalyst monolayers are important. Field-enhancing, nanopatterned surfaces can enable the detection of immobilised molecular monolayers, e.g. by surface-enhanced Raman spectroscopy (SERS). Therefore, a fabrication protocol was developed to generate a large-area plasmonic surface patterning in a Au thin film on Si, which consisted of regular nanohole arrays in the metallic layer. This plasmonic surface delivers a uniform field-enhancement over the entire patterned area for fast SERS spectra acquisition of the molecular monolayers. For the deposition of different catalyst monolayers in connected microfluidic compartments, methods for the site-selective functionalisation of the reactor surfaces and the chemical modification of these molecular monolayers on the surface are required. Therefore, this project also focused on detecting the chemical and electrochemical modifications of SAMs. The SERS monitoring of a two-step solid-phase synthesis in a SAM could be demonstrated using the developed plasmonic nanohole arrays. For the site-selective surface functionalisation, a fabrication process was developed to integrate the Au nanohole arrays in thin-film electrodes. This allows first the electrochemically triggered functionalisation of the Au thin-film electrode, followed by characterisation of the deposited molecular layer by SERS. Substantial contributions were thus made with the described research projects towards reaction screening using Si-based wall-coated flow microreactor networks

Monitoring Solid-Phase Reactions in Self-Assembled Monolayers by Surface-Enhanced Raman Spectroscopy

Nanopatterned surfaces enhance incident electromagnetic radiation and thereby enable the detection and characterization of self-assembled monolayers (SAMs), for instance in surface-enhanced Raman spectroscopy (SERS). Herein, Au nanohole arrays, developed and characterized as SERS substrates, are exemplarily used for monitoring a solid-phase deprotection and a subsequent copper(I)-catalyzed azide-alkyne cycloaddition "click" reaction, performed directly on the corresponding SAMs. The SERS substrate was found to be highly reliable in terms of signal reproducibility and chemical stability. Furthermore, the intermediates and the product of the solid-phase synthesis were identified by SERS. The spectra of the immobilized compounds showed minor differences compared to spectra of the microcrystalline solids. With its uniform SERS signals and the high chemical stability, the platform paves the way for monitoring molecular manipulations in surface functionalization applications

Monitoring Solid-Phase Reactions in Self-Assembled Monolayers by Surface-Enhanced Raman Spectroscopy

Nanopatterned surfaces enhance incident electromagnetic radiation and thereby enable the detection and characterization of self-assembled monolayers (SAMs), for instance in surface-enhanced Raman spectroscopy (SERS). Herein, Au nanohole arrays, developed and characterized as SERS substrates, are exemplarily used for monitoring a solid-phase deprotection and a subsequent copper(I)-catalyzed azide-alkyne cycloaddition "click" reaction, performed directly on the corresponding SAMs. The SERS substrate was found to be highly reliable in terms of signal reproducibility and chemical stability. Furthermore, the intermediates and the product of the solid-phase synthesis were identified by SERS. The spectra of the immobilized compounds showed minor differences compared to spectra of the microcrystalline solids. With its uniform SERS signals and the high chemical stability, the platform paves the way for monitoring molecular manipulations in surface functionalization applications

Ruthenium water oxidation catalysts containing the non-planar tetradentate ligand, biisoquinoline dicarboxylic acid (biqaH2_2)

Two ruthenium complexes containing the tetradentate ligand [1,1’-biisoquinoline]-3,3’-dicarboxylic acid, and 4-picoline or 6-bromoisoquinoline as axial ligands have been prepared. The complexes have been fully characterised and initial studies on their potential to function as molecular water oxidation catalysts have been performed. Both complexes catalyse the oxidation of water in acidic media with Ce$^{IV}$ as a stoichiometric chemical oxidant, although turnover numbers and turnover frequencies are modest when compared with the closely related Ru-bda and Ru-pda analogues. Barriers for the water nucleophilic attack and intermolecular coupling pathways were obtained from density functional theory calculations and the crucial influence of the ligand framework in determining the most favourable reaction pathway was elucidated from a combined analysis of the theoretical and experimental results

Sodium Thiosulfate Pharmacokinetics in Hemodialysis Patients and Healthy Volunteers

Vascular calcification is a major cause of morbidity and mortality in dialysis patients. Human and animal studies indicate that sodium thiosulfate (STS) may prevent the progression of vascular calcifications. The pharmacokinetics of STS in hemodialysis patients has not been investigated yet

Ru water oxidation catalysts based on py5 ligands

Ru complexes containing the pentapyridyl ligand 6,6&#39;&#39;-(methoxy(pyridin-2-yl)methylene)di-2,2&#39;-bipyridine (L-OMe) of general formula trans-[RuII(X)(L-OMe-k-N5)]n+ (X = Cl, n = 1, trans-1+; X = H2O, n = 2, trans-22+) have been isolated and characterized in solution (NMR, UV-Vis) and in the solid state by XRD. Both complexes suffer a series of substitution reactions at oxidation state II and III, when dissolved in aqueous triflic acid-trifluroethanol solutions that have been monitored by UV-vis spectroscopy and their corresponding rate constants reported. In particular, aqueous solutions of the RuIII-Cl complex trans-[RuIII(Cl)(L-OMe-k-N5)]2+ (trans-12+) generates a family of Ru-aqua complexes namely trans-[RuIII(H2O)(L-OMe-k-N5)]3+ (trans-23+), [RuIII(H2O)2(L-OMe-k-N4)]3+ (trans-33+) and [RuIII(Cl)(H2O)(L-OMe-k-N4)]3+ (trans-42+). While complex trans-42+ is a powerful water oxidation catalyst, complex trans-23+ has only a moderate activity and trans-33+ is not a catalyst. Further, a parallel work has been carried out with related complexes but containing the methyl substituted ligand 6,6&#39;&#39;-(1-pyridin-2-yl)ethane-1,1-diyl)di-2,2&#39;-bipyridine (L-Me). The behavior of all these catalysts has been rationalized based on substitution kinetics, oxygen evolution kinetics, electrochemical properties and DFT calculations. The best catalysts, trans-42+, reach turnover frequencies of 0.71 s-1 using Ce(IV) as a sacrificial chemical oxidant with oxidative efficiencies above 95%</p