Targeted modulation of gene expression through receptor-specific delivery of small interfering RNA peptide conjugates.

Small interfering RNA (siRNA) has emerged as a valuable tool to address RNA interference (RNAi) to modulate gene expression also in therapy. However, challenges such as inefficient cell targeting and rapid degradation in biological systems have limited its success. To address these issues, the development of a receptor-specific shuttle system represents a promising solution. [F7,P34]-NPY analogues were modified by solid-phase peptide synthesis, enabling non-covalent conjugation with siRNA. This modification yielded an efficient siRNA vehicle capable of binding and transporting its cargo into target cells without adversely affecting receptor activation or cell viability. Mass spectrometry and gel shift assays confirmed successful and stable siRNA binding under various conditions. Microscopy experiments further demonstrated the co-internalization of labeled peptides and siRNA in Hepa1c1 cells, highlighting the stability of the complex. In vitro quantitative RT-PCR experiments, targeting the TSC22D4 gene to normalize systemic glucose homeostasis and insulin resistance, revealed a functional peptide-based siRNA shuttle system with the ability to decrease mRNA expression to approximately 40%. These findings strengthen the potential of receptor-specific siRNA shuttle systems as efficient tools for gene therapy that offer a possibility for reducing side effects.

Illuminating the Path to Enhanced Bioimaging by Phosphole-based Fluorophores.

As the research of biological systems becomes increasingly complex, there is a growing demand for fluorophores with a diverse range of wavelengths. In this study, we introduce phosphole-based fluorophores that surpass existing options like dansyl chloride. The reactive S-Cl bond in chlorosulfonylimino-5-phenylphosphole derivatives allows rapid and direct coupling to peptides making the fluorophores easily introducible to peptides. This coupling process occurs under mild conditions, demonstrated for [F7 ,P34 ]-NPY and its shorter analogues. Peptides linked with our fluorophores exhibit similar receptor activation to the control peptide, while maintaining high stability and low toxicity, making them ideal biolabeling reagents. In fluorescence microscopy experiments, they can be easily visualized even at low concentrations, without suffering from the typical issue of bleaching. These phosphole-based fluorophores represent a significant leap forward in the field. Their versatility, ease of modification, superior performance, and applicability in biological labeling make them a promising choice for researchers seeking advanced tools to unravel the details of complex biological systems.

Bacillus subtilis spores in probiotic feed quantified via bacterial metabolite using planar chromatography.

Interest in probiotics in animal production has increased due to the European ban on antibiotic growth promoters in 2006. Bacillus subtilis DSM 29784 (B. s. 29784) is one such probiotic feed additive used in poultry. Cell counting has been the most common tool for feed analysis, besides flow cytometry and quantitative polymerase chain reaction. However, quantification of the active probiotic in feed is challenging, since results are influenced by cultivation conditions, viable but non-culturable bacteria and the high contents of feed ingredients. This study presents the first quantitative analysis of a metabolite generated by B. s. 29784 spores in feed to draw conclusions on the amount of active dried spores in the feed. Thus, it is the first quantification of active probiotic bacteria at the trace level in feed based on metabolite production but not cell counting. To generate the calibration standards, solutions with different amounts of dried B. s. 29784 spores were cultured under the same conditions as the feed sample, ensuring independence from growth performance. Upstream cultivation, metabolite extraction and high-performance thin-layer chromatography analysis were proven to be highly reliable and reproducible. The repeatability of the method (RSD 1.9%) and the recovery (111% ± 21% in feed additive matrix, 96% ± 13% in ionized feed matrix) were excellent. The variations during cultivation occurred due to the complex spore germination process and presence of other microbes in the feed. This new procedure, detecting only those cells that produced the metabolite of interest, has several advantages as it takes into account bacterial viability, cultivation conditions, spore germination process, growth behavior and the influence of the nutrient-rich feed matrix. It truly reflects the activity of the probiotic in the feed product, allows side-by-side comparison of characteristic metabolite patterns and nutrient consumptions to understand the metabolism of dried spores in matrix.