Position effects at the FGF8 locus are associated with femoral hypoplasia

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals’ cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.M.S. and A.S.-S. were supported by the Polish National Science Centre (UMO-2016/23/N/NZ2/02362 to M.S. and UMO-2016/21/D/NZ5/00064 to A.S.-S.). A.S.-S. was also supported by the Polish National Science Centre scholarship for PhD students (UMO-2013/08/T/NZ2/00027). C.L. is supported by postdoctoral Beatriu de Pinós from Secretaria d’Universitats I Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and by the Marie Sklodowska-Curie COFUND program from H2020 (2018-BP-00055). A.J. was supported by the Polish National Science Centre (UMO-2016/22/E/NZ5/00270) as well as the Polish National Centre for Research and Development (LIDER/008/431/L-4/12/NCBR/2013). M.S. is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SP1532/3-1, SP1532/4-1, and SP1532/5-1), the Max Planck Foundation, and the Deutsches Zentrum für Luft- und Raumfahrt (DLR 01GM1925)

Targeting somatostatin receptors using in situ-bioconjugated fluorescent nanoparticles

Aim: The author's group report, for the first time, on the development of a quantum dot (QD)-based fluorescent somatostatin (somatotropin release-inhibiting factor [SRIF]) probe that enables specific targeting of somatostatin receptors. Receptor-mediated endocytosis of SRIF was imaged using this probe. Materials & methods: Biotinylated SRIF-analog (SRIF-B) and streptavidin (Sav)-coated QDs were used for the probe synthesis. A dye-labeled streptavidin complex was used to evaluate the effect of Sav binding on the activity of SRIF-B. Results: A preconjugated probe of the form SRIF-B:Sav-QD, was inactive and unable to undergo receptor-mediated endocytosis. An alternative in situ bioconjugation strategy, where SRIF-B and Sav-QD were added in two consecutive steps, enabled visualization of the receptor-mediated endocytosis. The process of Sav binding appeared to be responsible for the inactivity in the first case. Conclusion: The in situ two-step bioconjugation strategy allowed QDs to be targeted to somatostatin receptors. This strategy should enable flexible fluorescent tagging of SRIF for the investigation of molecular trafficking in cells and targeted delivery in live animals.10 page(s

Feasibility study of the optical imaging of a breast cancer lesion labeled with upconversion nanoparticle biocomplexes

Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The feasibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-antibodies were grafted onto the UCNPs via a high-affinity molecular linker barstar: barnase (Bs:Bn) to allow their specific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10:1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a monolayer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model predicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth showing considerable potential for diagnostic and image-guided surgery applications.10 page(s

Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-β (Aβ) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aβ40 and Aβ42 remain predominantly monomeric up to 3 μm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aβ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers