Pdgfra and Pdgfrb genetically interact during craniofacial development.

BACKGROUND: One of the most prevalent congenital birth defects is cleft palate. The palatal skeleton is derived from the cranial neural crest and platelet-derived growth factors (Pdgf) are critical in palatogenesis. Of the two Pdgf receptors, pdgfra is required for neural crest migration and palatogenesis. However, the role pdgfrb plays in the neural crest, or whether pdgfra and pdgfrb interact during palatogenesis is unclear. RESULTS: We find that pdgfrb is dispensable for craniofacial development in zebrafish. However, the palatal defect in pdgfra;pdgfrb double mutants is significantly more severe than in pdgfra single mutants. Data in mouse suggest this interaction is conserved and that neural crest requires both genes. In zebrafish, pdgfra and pdgfrb are both expressed by neural crest within the pharyngeal arches, and pharmacological analyses demonstrate Pdgf signaling is required at these times. While neither proliferation nor cell death appears affected, time-lapsed confocal analysis of pdgfra;pdgfrb mutants shows a failure of proper neural crest condensation during palatogenesis. CONCLUSIONS: We provide data showing that pdgfra and pdgfrb interact during palatogenesis in both zebrafish and mouse. In zebrafish, this interaction affects proper condensation of maxillary neural crest cells, revealing a previously unknown interaction between Pdgfra and Pdgfrb during palate formation. Developmental Dynamics 245:641-652, 2016. © 2016 Wiley Periodicals, Inc.

Preliminary Development of a DNA Aptamer-Magnetic Bead Capture Electrochemiluminescence Sandwich Assay for Brain Natriuretic Peptide.

Fifty-two candidate DNA aptamer sequences were selected for binding to the cardiovascular biomarker B-type or brain natriuretic peptide (BNP). Candidate aptamers were screened to rank their relative affinities against BNP by an aptamer-based ELISA-like aptamer microplate assay (ELASA). The highest affinity aptamers from ELASA screening were also paired in all possible combinations and screened for electrochemiluminescence (ECL) assay potential in capture aptamer-magnetic bead and ruthenium trisbipyridine (Ru(bpy)32+)-reporter aptamer sandwich formats. The top ECL sandwich combinations utilized the same aptamer pair in either capture or reporting roles with nanogram to low picogram per mL levels of detection even in 50% human serum. ECL assay sensitivity and linearity even in 50% human serum suggest that the aptamer-based assay is at least comparable to other reported immunoassays for BNP.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid leishmania detection in sandflies.

A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (∼ 100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.

Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses.

BACKGROUND: Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF), dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications. RESULTS: Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA) was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA) emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow chromatographic assays and a fluorescent sandwich assay on the surface of magnetic microbeads is also demonstrated. CONCLUSIONS: This article catalogues numerous DNA aptamer sequences which can bind various important pathogenic arboviruses and have, in some cases, already demonstrated diagnostic potential. These aptamer sequences are proprietary, patent-pending, and partially characterized. Therefore, they are offered to the scientific community for potential research use in diagnostic assays, biosensor applications or for possible passive immunity and prophylaxis against pathogenic viruses.

An aptamer beacon responsive to botulinum toxins.

Sixty candidate DNA aptamers were developed against botulinum neurotoxin (BoNT) type A light chain (LC) from ten rounds of selection, resulting in several identical sequences. Secondary structures of the identical aptamers were compared to structures of previously reported BoNT A DNA aptamers. A series of ten candidate loop structures were selected from this comparison as potential binding pockets and aptamer beacons. These candidate beacons were synthesized with 5'-TYE 665 and 3'-Iowa Black quencher labels for comparison of fluorescence levels as a function of BoNT A LC concentration. Only three of the ten candidates exhibited any fluorescence response to increasing levels of BoNT A LC. However, of the two most responsive candidates, one represented a subset loop of the larger more intensely fluorescent double-looped structure, designated Beacon 10. This beacon yielded a lower limit of detection of 1 ng/mL in buffer using a spectrofluorometer and a portable handheld fluorometer, but also responded substantially to BoNT A, B, E holotoxins and heavy or light chain components even in a dilute soil suspension, but not in 50% human serum. Beacon 10 did not respond strongly to a variety of other divergent peptides, suggesting that it is relatively specific to the level of botulinum toxins and is only useful for environmental testing. Beacon 10 also shared short sequence segments with other published BoNT aptamer DNA sequences, suggesting that these may be points of physical contact between the aptamers and BoNTs.

Cooperation between the PDGF receptors in cardiac neural crest cell migration.

Neural crest cells (NCCs) are essential components of the sympathetic nervous system, skin, craniofacial skeleton, and aortic arch. It has been known for many years that perturbation of migration, proliferation, and/or differentiation of these cells leads to birth defects such as cleft palate and persistent truncus arteriosus (PTA). Previously, we had shown that disruption of the platelet-derived growth factor receptor (PDGFR) alpha in NCCs resulted in defects in craniofacial and aortic arch development, the latter with variable penetrance. Because we observed ventricular septal defects in embryos that are null for the PDGFRbeta, we hypothesized that both PDGF receptors are involved in NCC formation. Here, we show that both receptors are expressed in cardiac NCCs and that the combined loss of the PDGFRalpha and PDGFRbeta in NCCs resulted in NCC-related heart abnormalities, including PTA and a ventricular septal defect (VSD). Using NCC lineage tracing, we observed that loss of PDGF receptor signaling resulted in reduced NCCs in the conotruncus region, leading to defects in aortic arch septation. These results indicate that while PDGFRalpha plays a predominant role in NCC development, the PDGFRbeta is expressed by and functions in cardiac NCCs. Combined PDGF receptor signaling is required for sufficient recruitment of cardiac NCCs into the conotruncal region and for formation of the aortico-pulmonary and ventricular septum.