Molecular mechanisms of congenital limb malformations

Congenital limb malformations occur in 1 in 500 to 1 in 1000 human live births and are diverse in their epidemiology, aetiology and anatomy. The molecular analysis of disturbed gene function in inherited limb malformations provides essential information for the understanding of physiological and pathophysiological limb development in humans as well as in other vertebrates. The following Ph.D thesis focussed on the identification and molecular characterisation of disease causing genes and their pathophysiological mechanism for selected human limb defects such as Cenani-Lenz syndrome (CLS), Werner mesomelic syndrome (WMS), Bardet-Biedl syndrome (BBS), Split hand/ foot malformation (SHFM) and Temtamy preaxial brachydactyly syndrome (TPBS). In this context, we were able to identify novel limb specific genes and causative mutations in different components of evolutionary highly conserved pathways and, furthermore, to elucidate their role in physiological as well as in pathophysiological limb development. In detail, we found (i) alterations in the low-density-lipoprotein-related protein 4 (LRP4), an antagonistic receptor of Wnt signalling, causing the rare autosomal recessive CLS, (ii) specific mutations in the cis-acting limb-specific enhancer of the sonic hedgehog (SHH) gene being causative for WMS, and (iii) mutations in CHSY1 to be responsible for TPBS. Furthermore, we could show that mutations in the ciliary protein BBS12 can cause a very mild BBS phenotype. Moreover, we used in vitro studies to obtain insights into the molecular pathogenesis of these limb malformations. We studied the effect of five LRP4 mutants on the transduction and activation of canonical Wnt signalling by using a Dual-Luciferase Reporter Assay and showed that co-expression of each of the five missense mutations with LRP6 and WNT1 abolish the known antagonistic effect of LRP4 on LRP6-mediated activation of Wnt/ß-catenin signalling and thus conclude that homozygous LRP4 mutations in CLS cause a loss of protein function. Additionally, we functionally characterized the first autosomal recessive p.R332W mutation in the WNT10b gene causing SHFM6 and rise evidence that p.R332W causes loss of function of Lrp6-mediated Wnt signalling. In this regard we examined the role of the SHFM3 candidate gene Fgf8 in altering Wnt signalling and demonstrated that Fgf8 is a novel putative Wnt signalling antagonist which functions by direct interaction with Wnt10b. Hence, we present the first direct cross-talk between Fgf and Wnt signalling pathways and, therefore, physically link two important signalling pathways involved in limb initiation and outgrowth

Energy transfer study on Tb3+/Eu3+ Co-activated sol-gel glass-ceramic materials containing MF3 (M = Y, La) nanocrystals for NUV optoelectronic devices

In the present work, the Tb3+/Eu3+ co-activated sol-gel glass-ceramic materials (GCs) containing MF3 (M = Y, La) nanocrystals were fabricated during controlled heat-treatment of silicate xerogels at 350 C. The studies of Tb3+ ! Eu3+ energy transfer process (ET) were performed by excitation and emission spectra along with luminescence decay analysis. The co-activated xerogels and GCs exhibit multicolor emission originated from 4fn–4fn optical transitions of Tb3+ (5D4 !7FJ, J = 6–3) as well as Eu3+ ions (5D0 ! 7FJ, J = 0–4). Based on recorded decay curves, it was found that there is a significant prolongation in luminescence lifetimes of the 5D4 (Tb3+) and the 5D0 (Eu3+) levels after the controlled heat-treatment of xerogels. Moreover, for both types of prepared GCs, an increase in ET e ciency was also observed (from ET 16% for xerogels up to ET = 37.3% for SiO2-YF3 GCs and ET = 60.8% for SiO2-LaF3 GCs). The changes in photoluminescence behavior of rare-earth (RE3+) dopants clearly evidenced their partial segregation inside low-phonon energy fluoride environment. The obtained results suggest that prepared SiO2-MF3:Tb3+, Eu3+ GC materials could be considered for use as optical elements in RGB-lighting optoelectronic devices operating under near-ultraviolet (NUV) excitation

Hydrocarbon degradation potential and plant growth-promoting activity of culturable endophytic bacteria of Lotus corniculatus and Oenothera biennis from a long-term polluted site

Many endophytic bacteria exert beneficial effects on their host, but still little is known about the bacteria associated with plants growing in areas heavily polluted by hydrocarbons. The aim of the study was characterization of culturable hydrocarbon-degrading endophytic bacteria associated with Lotus corniculatus L. and Oenothera biennis L. collected in long-term petroleum hydrocarbon-polluted site using culture-dependent and molecular approaches. A total of 26 hydrocarbon-degrading endophytes from these plants were isolated. Phylogenetic analyses classified the isolates into the phyla Proteobacteria and Actinobacteria. The majority of strains belonged to the genera Rhizobium, Pseudomonas, Stenotrophomonas, and Rhodococcus. More than 90% of the isolates could grow on medium with diesel oil, approximately 20% could use n-hexadecane as a sole carbon and energy source. PCR analysis revealed that 40% of the isolates possessed the P450 gene encoding for cytochrome P450-type alkane hydroxylase (CYP153). In in vitro tests, all endophytic strains demonstrated a wide range of plant growth-promoting traits such as production of indole-3-acetic acid, hydrogen cyanide, siderophores, and phosphate solubilization. More than 40% of the bacteria carried the gene encoding for the 1-aminocyclopropane-1-carboxylic acid deaminase (acdS). Our study shows that the diversity of endophytic bacterial communities in tested plants was different. The results revealed also that the investigated plants were colonized by endophytic bacteria possessing plant growth-promoting features and a clear potential to degrade hydrocarbons. The properties of isolated endophytes indicate that they have the high potential to improve phytoremediation of petroleum hydrocarbon-polluted soils

Reddish-Orange Luminescence from BaF2:Eu3+ Fluoride Nanocrystals Dispersed in Sol-Gel Materials

Nanocrystalline transparent BaF2:Eu3+ glass-ceramic materials emitting reddish-orange light were fabricated using a low-temperature sol-gel method. Several experimental techniques were used to verify structural transformation from precursor xerogels to sol-gel glass-ceramic materials containing fluoride nanocrystals. Thermal degradation of xerogels was analyzed by thermogravimetric analysis (TG) and di erential scanning calorimetry method (DSC). The presence of BaF2 nanocrystals dispersed in sol-gel materials was confirmed by the X-ray di raction (XRD) analysis and transmission electron microscopy (TEM). In order to detect structural changes in silica network during annealing process, the infrared spectroscopy (IR-ATR) was carried out. In particular, luminescence spectra of Eu3+ and their decays were examined in detail. Some spectroscopic parameters of Eu3+ ions in glass-ceramics containing BaF2 nanocrystals were determined and compared to the values obtained for precursor xerogels. It was observed, that the intensities of two main red and orange emission bands corresponding to the 5D0!7F2 electric-dipole transition (ED) and the 5D0!7F1 magnetic-dipole (MD) transition are changed significantly during transformation from xerogels to nanocrystalline BaF2:Eu3+ glass-ceramic materials. The luminescence decay analysis clearly indicates that the measured lifetime 5D0 (Eu3+) considerably enhanced in nanocrystalline BaF2:Eu3+ glass-ceramic materials compared to precursor xerogels. The evident changes in luminescence spectra and their decays suggest the successful migration of Eu3+ ions from amorphous silica network to low-phonon BaF2 nanocrystals

The expansion of CD4(+)CD28(- )T cells in patients with rheumatoid arthritis

Clonal expansion of CD4(+)CD28(- )T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4(+ )clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4(+)CD28(- )T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4(+)CD28(- )T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4(+)CD28(- )T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4(+)CD28(- )T cells may be a marker correlating with extra-articular manifestations and joint involvement

Cutaneous Adverse Events of Systemic Melanoma Treatments: A Retrospective Single-Center Analysis

Recent progress in the treatment of advanced melanoma has led to the improved survival of affected patients. However, novel treatments also lead to considerable and distinct skin toxicity. To further characterize cutaneous adverse events (AE) of systemic treatments, we conducted a single-center retrospective study of biopsy-proven cutaneous adverse events of melanoma treatment over a period of 10 years at the University Hospital of Zurich, Switzerland. In 102 identified patients, 135 individual skin AEs developed. Immune checkpoint blockade (ICB) was causal for 81 skin AEs, and 54 were related to targeted therapies (TT). Recorded types of skin AEs included lichenoid, maculopapular, acneiform, urticarial, panniculitis, folliculitis, psoriasiform, granulomatous, eczematous, and others. The incidence of skin AEs was higher with TT (18.54%) than with ICB (9.64%, p = 0.0029). Most AEs were low-grade, although 19.21% of AEs were common terminology criteria for adverse events (CTCAE) Grades 3 or 4. A large spectrum of skin AEs was documented during treatment of advanced melanoma, and distinct phenotypes were observed, depending on treatment classes. AEs occurred earlier during treatment with TT than with ICB, and distinct types of skin AEs were associated with respective treatment classes. This study comprehensively describes skin AEs occurring during systemic treatment for melanoma at a single center

Nanoparticles for Directed Immunomodulation: Mannose-Functionalized Glycodendrimers Induce Interleukin-8 in Myeloid Cell Lines

New therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles. © 2021 The Authors. Published by American Chemical Society

Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-κB Pathway in a Myeloid Cell Line

Purpose: Fourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors. Methods: To estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4. Results: We found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 μM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25–100 μM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected. Conclusion: We conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines)

Sol-Gel Glass-Ceramic Materials Containing CaF2:Eu3+ Fluoride Nanocrystals for Reddish-Orange Photoluminescence Applications

CaF2:Eu3+ glass-ceramic sol-gel materials have been examined for reddish-orange photoluminescence applications. The transformation from precursor xerogels to glass-ceramic materials with dispersed fluoride nanocrystals was verified using several experimental methods: di erential scanning calorimetry (DSC), thermogravimetric analysis (TG), X-ray di raction (XRD), transmission electron microscopy (TEM), infrared spectroscopy (IR-ATR), energy dispersive X-ray spectroscopy (EDS) and photoluminescence measurements. Based on luminescence spectra and their decays, the optical behavior of Eu3+ ions in fabricated glass-ceramics were characterized and compared to those of precursor xerogels. In particular, the determined luminescence lifetime of the 5D0 excited state of Eu3+ ions in nanocrystalline CaF2:Eu3+ glass-ceramic materials is significantly prolonged in comparison with prepared xerogels. The integrated intensities of emission bands associated to the 5D0 ! 7F2 electric-dipole transition (ED) and the 5D0 !7F1 magnetic-dipole transition (MD) are changed drastically during controlled ceramization process of xerogels. This implies the e cient migration of Eu3+ ions from amorphous silicate sol-gel network into low-phonon energy CaF2 nanocrystals