Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

A novel method for human hematopoietic stem/progenitor cell isolation from umbilical cord blood based on immunoaffinity aqueous two-phase partitioning

A novel cell separation process based on immunoaffinity aqueous two phase systems is presented to isolate and purify CD34⁺ stem/progenitor cells directly from the whole umbilical cord blood (UCB). A system, composed of polyethylene glycol and dextran, was evaluated for the selective recovery of CD34⁺ cells from UCB. A monoclonal antibody against the CD34 surface antigen was used for the direct partitioning of CD34⁺ cells in UCB to the PEG-rich phase. The initial population of CD34⁺ cells (0.2% of the initial sample) was enriched to values up to 42% in a single partitioning step, while the majority of contaminant cells were partitioned to the dextran-rich phase (1.37 × 10⁻² < K_P < 2.76 × 10⁻²). This novel selection method allowed a recovery yield of 95% of CD34⁺ cells with a purification factor of 245 and is expected to pave a new way to purify hematopoietic stem/progenitor cells for use in a variety of clinical settings.     

Diatoms and Other Epibionts Associated with Olive Ridley (Lepidochelys olivacea) Sea Turtles from the Pacific Coast of Costa Rica

Although the sea turtles have long been familiar and even iconic to marine biologists, many aspects of their ecology remain unaddressed. The present study is the first of the epizoic diatom community covering the olive ridley turtle’s (Lepidochelys olivacea) carapace and the first describing diatoms living on sea turtles in general, with the primary objective of providing detailed information on turtle epibiotic associations. Samples of turtle carapace including the associated diatom biofilm and epizoic macro-fauna were collected from Ostional beach (9° 59´ 23.7´´ N 85° 41´ 52.6´´ W), Costa Rica, during the arribada event in October 2013. A complex diatom community was present in every sample. In total, 11 macro-faunal and 21 diatom taxa were recorded. Amongst diatoms, the most numerous were erect (Achnanthes spp., Tripterion spp.) and motile (Haslea sp., Navicula spp., Nitzschia spp., Proschkinia sp.) forms, followed by adnate Amphora spp., while the most common macro-faunal species was Stomatolepas elegans (Cirripedia). Diatom densities ranged from 8179 ± 750 to 27685 ± 4885 cells mm^-2. Epizoic microalgae were either partly immersed or entirely encapsulated within an exopolymeric coat. The relatively low diatom species number, stable species composition and low inter-sample dissimilarities (14.4% on average) may indicate a mutualistic relationship between the epibiont and the basibiont. Dispersal of sea turtle diatoms is probably highly restricted and similar studies will help to understand both diatom diversity, evolution and biogeography, and sea turtle ecology and foraging strategies.     

ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase

The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.     

Quantitative trait loci for plant height in Maresi × CamB barley population and their associations with yield-related traits under different water regimes

High-yielding capacity of the modern barley varieties is mostly dependent on the sources of semi-dwarfness associated with the sdw1/denso locus. The objective of the study was to identify quantitative trait loci (QTLs) associated with the plant height and yield potential of barley recombinant inbred lines (RILs) grown under various soil moisture regimes. The plant material was developed from a hybrid between the Maresi (European cv.) and CamB (Syrian cv.). A total of 103 QTLs affecting analysed traits were detected and 36 of them showed stable effects over environments. In total, ten QTLs were found to be significant only under water shortage conditions. Nine QTLs affecting the length of main stem were detected on 2H-6H chromosomes. In four of the detected QTLs, alleles contributed by Maresi had negative effects on that trait, the most significant being the QLSt-3H.1-1 in the 3H.1 linkage group. The close linkage between QTLs identified around the sdw1/denso locus, with positive alleles contributed by Maresi, indicates that the semi-dwarf cv. Maresi could serve as a donor of favourable traits resulting in grain yield improvement, also under water scarcity. Molecular analyses revealed that the Syrian cv. also contributed alleles which increased the yield potential. Available barley resources of genomic annotations were employed to the biological interpretation of detected QTLs. This approach revealed 26 over-represented Gene Ontology terms. In the projected support intervals of QGWS_l-5H.3-2 and QLSt-5H.3 on the chromosome 5H, four genes annotated to ‘response to stress’ were found. It suggests that these QTL-regions may be involved in a response of plant to a wide range of environmental disturbances.     

The Impact of Model Peptides on Structural and Dynamic Properties of Egg Yolk Lecithin Liposomes – Experimental and DFT Studies

Electron spin resonance (ESR), ¹H‐NMR, voltage and resistance experiments were performed to explore structural and dynamic changes of Egg Yolk Lecithin (EYL) bilayer upon addition of model peptides. Two of them are phenylalanine (Phe) derivatives, Ac‐Phe‐NHMe ( 1 ) and Ac‐Phe‐NMe₂ ( 2 ), and the third one, Ac‐(Z)‐ΔPhe‐NMe₂ ( 3 ), is a derivative of (Z)‐α,β‐dehydrophenylalanine. The ESR results revealed that all compounds reduced the fluidity of liposome's membrane, and the highest activity was observed for compound 2 with N‐methylated C‐terminal amide bond (Ac‐Phe‐NMe₂). This compound, being the most hydrophobic, penetrates easily through biological membranes. This was also observed in voltage and resistance studies. ¹H‐NMR studies provided a sound evidence on H‐bond interactions between the studied diamides and lecithin polar head. The most significant changes in H‐atom chemical shifts and spin‐lattice relaxation times T₁ were observed for compound 1 . Our experimental studies were supported by theoretical calculations. Complexes EYL? Ac‐Phe‐NMe₂ and EYL? Ac‐(Z)‐ΔPhe‐NMe₂, stabilized by NH???O or/and CH???O H‐bonds were created and optimized at M06‐2X/6‐31G(d) level of theory in vacuo and in H₂O environment. According to our molecular‐modeling studies, the most probable lecithin site of H‐bond interaction with studied diamides is the negatively charged O‐atom in phosphate group which acts as H‐atom acceptor. Moreover, the highest binding energy to hydrocarbon chains were observed in the case of Ac‐Phe‐NMe₂ ( 2 ).