Intravital FRET: Probing Cellular and Tissue Function in Vivo

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.     

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4‐Oxoproline Synthase

X‐type actinomycins (Acms) contain 4‐hydroxyproline (Acm X₀) or 4‐oxoproline (Acm X₂) in their β‐pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4‐hydroxyproline‐ or 4‐oxoproline‐containing Acm halves. In turn, we show—using an artificial Acm half analogue (PPL 1) with proline in its peptide chain—their conversion into the 4‐hydroxyproline‐ and 4‐oxoproline‐containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host‐encoded ferredoxin reductase in a NADH (NADPH)‐dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4′′‐oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.     

Wärmeinduzierte Vorbehandlung lignocellulosehaltiger Biomassen – Prozesse,Verfahren und deren Einordnung

In the following, different techniques for a heat‐induced pretreatment of lignocellulosic biomass for the production of solid fuels are described. Thereby, also changes at the molecular level of the biomass are discussed. The discussed techniques (torrefaction, hydrothermal carbonization, vapothermal carbonization, and autoclaving) proceed in a temperature range up to 300 °C. Finally, a classification of the different techniques takes place to evaluate the specific advantages and disadvantages.     

Multifunctional Hybrid Nanoparticles for Traceable Drug Delivery and Intracellular Microenvironment‐Controlled Multistage Drug‐Release in Neurons

Innovative nanoparticles hold promising potential for disease therapy as drug delivery systems. For brain‐disease therapy, a drug delivery system that can sustainably control drug‐release and monitor fluorescence of the drug cargos is highly desirable. In this study, a light‐traceable and intracellular microenvironment‐responsive drug delivery system was developed based on the combination of glutathione‐responsive autoflurescent nanogel, dendrimer‐like mesoporous silica nanoparticles, and gold nanoparticles. The resulting hybrid nanoparticles represent a new class of delivery system that can efficiently load, transport, and control multistage‐release of sulfydryl‐containing drugs into neurons, with light‐traceable monitoring for future brain‐disease therapy.     

A Robust Intrinsically Green Fluorescent Poly(Amidoamine) Dendrimer for Imaging and Traceable Central Nervous System Delivery in Zebrafish

Intrinsically fluorescent poly(amidoamine) dendrimers (IF‐PAMAM) are an emerging class of versatile nanoplatforms for in vitro tracking and bio‐imaging. However, limited tissue penetration of their fluorescence and interference due to auto‐fluorescence arising from biological tissues limit its application in vivo. Herein, a green IF‐PAMAM (FGP) dendrimer is reported and its biocompatibility, circulation, biodistribution and potential role for traceable central nervous system (CNS)‐targeted delivery in zebrafish is evaluated, exploring various routes of administration. Key features of FGP include visible light excitation (488 nm), high fluorescence signal intensity, superior photostability and low interference from tissue auto‐fluorescence. After intravenous injection, FGP shows excellent imaging and tracking performance in zebrafish. Further conjugating FGP with transferrin (FGP‐Tf) significantly increases its penetration through the blood–brain barrier (BBB) and prolongs its circulation in the blood stream. When administering through local intratissue microinjection, including intracranial and intrathecal injection in zebrafish, both FGP and FGP‐Tf exhibit excellent tissue diffusion and effective cellular uptake in the brain and spinal cord, respectively. This makes FGP/FGP‐Tf attractive for in vivo tracing when transporting to the CNS is desired. The work addresses some of the major shortcomings in IF‐PAMAM and provides a promising application of these probes in the development of drug delivery in the CNS.     

Geometrically nonlinear analysis of laminate composite plates and shells using the eight-node hexahedral element with one-point integration

An eight-node hexahedral isoparametric element with one-point quadrature for the geometrically nonlinear static and dynamic analysis of plates and shells of laminate composite materials is formulated and implemented in this work. The element is free of volumetric and shear locking and spurious modes are not detected. Shear locking is avoided using a corotational system for strain and stress components, whereas volumetric locking is controlled using one-point quadrature for the dilatational (or spherical) part of the gradient matrix. The efficiency and potential of the three-dimensional element in the analysis of plates and shells of laminate composite materials undergoing large displacements and rotations are shown solving several numerical examples and comparing with results obtained by other authors using different plate and shells elements.     

Oscillatory Potentials in Achromatopsia as a Tool for Understanding Cone Retinal Functions

Achromatopsia (ACHM) is an inherited autosomal recessive disease lacking cone photoreceptors functions. In this study, we characterize the time-frequency representation of the full-field electroretinogram (ffERG) component oscillatory potentials (OPs), to investigate the connections between photoreceptors and the inner retinal network using ACHM as a model. Time-frequency characterization of OPs was extracted from 52 controls and 41 achromat individuals. The stimulation via ffERG was delivered under dark-adaptation (DA, 3.0 and 10.0 cd·s·m⁻²) to assess mixed rod-cone responses. The ffERG signal was subsequently analyzed using a continuous complex Morlet transform. Time-frequency maps of both DA conditions show the characterization of OPs, disclosing in both groups two distinct time-frequency windows (~70–100 Hz and >100 Hz) within 50 ms. Our main result indicates a significant cluster (p < 0.05) in both conditions of reduced relative power (dB) in ACHM people compared to controls, mainly at the time-frequency window >100 Hz. These results suggest that the strongly reduced but not absent activity of OPs above 100 Hz is mostly driven by cones and only in small part by rods. Thus, the lack of cone modulation of OPs gives important insights into interactions between photoreceptors and the inner retinal network and can be used as a biomarker for monitoring cone connection to the inner retina.