Quantifying the Extent of Calcification of a Coccolithophore Using a Coulter Counter.

Although, in principle, the Coulter Counter technique yields an absolute measure of particle volume, in practice, calibration is near-universally employed. For regularly shaped and non-biological samples, the use of latex beads for calibration can provide sufficient accuracy. However, this is not the case with particles encased in biogenically formed calcite. To date, there has been no effective route by which a Coulter Counter can be calibrated to enable the calcification of coccolithophores─single cells encrusted with biogenic calcite─to be quantified. Consequently, herein, we seek to answer the following question: to what extent can a Coulter Counter be used to provide accurate information regarding the calcite content of a single-species coccolithophore population? Through the development of a new calibration methodology, based on the measurement and dynamic tracking of the acid-driven calcite dissolution reaction, a route by which the cellular calcite content can be determined is presented. This new method allows, for the first time, a Coulter Counter to be used to yield an absolute measurement of the amount of calcite per cell.

Construction of a Targeting Nanoparticle of 3',3″-Bis-Peptide-siRNA Conjugate/Mixed Lipid with Postinserted DSPE-PEG2000-cRGD.

The cyclic Arg-Gly-Asp (cRGD) peptides are widely used as tumor-targeting ligands due to their specific binding ability to integrin αvß3, which is overexpressed on the surface of various cancer cells and the endothelial cells of new blood vessels within tumor tissues. In this paper, the postinsertion strategy of DSPE-PEG2000-cRGD has been applied to the nanoparticles of 3',3″-bis-peptide-siRNA (pp-siRNA) encapsulated by gemini-like cationic lipid (CLD) and neutral cytosin-1-yl lipid (DNCA) from our lab. It was confirmed that the nanoparticles of pp-siRNA/CLD/DNCA/DSPE-PEG2000-cRGD (PCNR) were able to specifically target tumor cells with highly expressed integrin αvß3; moreover, it efficiently downregulated the levels of BRAF mRNA and the BRAF protein and inhibited cell proliferation in A375 cells, in comparison with the nontargeted nanocomplex of pp-siRNA/CLD/DNCA/cRAD (PCNA). The uptake pathways of PCNR are mostly dependent on CvME-mediated endocytosis and macropinocytosis in A375 cells, which could bypass lysosome or quickly lead to the lysosomal escape to reduce siRNA degradation. Finally, the biodistribution study showed that PCNR exhibited a high ability to accumulate in tumor tissues. These results suggest that the nanocomplex of PCNR is promising to be highly effective in the treatment of melanomas including their mutation.

Chemical modification improves the stability of the DNA aptamer GBI-10 and its affinity towards tenascin-C.

Aptamers are useful tools in molecular imaging due to their numerous attractive properties, such as excellent affinity and selectivity to diverse types of target molecules and biocompatibility. We carried out structure-activity relationship studies with the tenascin-C (TN-C) binding aptamer GBI-10, which is a promising candidate in tumor imaging. To increase the tumor targeting ability and nuclease resistance under physiological conditions, systematic modifications of GBI-10 with single and multiple 2'-deoxyinosine (2'-dI) or d-/l-isonucleoside (d-/l-isoNA) were performed. Results indicated that sector 3 of the proposed secondary structure is the most important region for specific binding with TN-C. By correlating the affinity of eighty-four GBI-10 derivatives with their predicted secondary structure by Zuker Mfold, we first validated the preferred secondary structure at 37 °C. We found that d-/l-isoNA modified GBI-10 derivatives exhibited improved affinity to the target as well as plasma stability. Affinity measurement and confocal imaging analysis highlighted one potent compound: 4AL/26TL/32TL, which possessed a significantly increased targeting ability to tumor cells. These results revealed the types of modified nucleotides, and the position and number of substituents in GBI-10 that were critical to the TN-C binding ability. Stabilized TN-C-binding DNA aptamers were prepared and they could be further developed for tumor imaging. Our strategy to introduce 2'-dI and d-/l-isoNA modifications after the selection process is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding ability.

Biological Properties of a 3',3″-Bis-Peptide-siRNA Conjugate in Vitro and in Vivo.

This study proposes an effective melanoma small interfering RNA (siRNA), named siMB3, which targets the mRNA of mutant BRAF protein. We found that Bis-pep-siMB3, with peptide KALLAL-conjugated siMB3 at the 3'-termini of both strands, inhibited translation of the target genes and expression of the related protein as effectively as siMB3, but for substantially longer, and the conjugates could alleviate off-target effects. Further studies on the mechanisms of action showed that the stability of Bis-pep-siMB3 in fetal bovine serum improved and the half-life period of Bis-pep-siMB3 was increased 21-fold over that of siMB3. Peptide conjugation could improve the combination of siRNA and cationic lipid vectors. Bis-pep-siMB3 is likely to reach the lysosome earlier and stay longer, and appears to increase the release of siRNA from the endosome. At the animal level, application of Bis-pep-siMB3 showed good therapeutic potential, inhibiting the growth of xenograft tumors in athymic mice slightly better than siMB3 and greatly prolonging the circulating time in vivo. Moreover, it distributed widely in mice. These results show the promising potential of Bis-pep-siRNA conjugates as therapeutic siRNAs for cancer treatment.

Stability and bioactivity of thrombin binding aptamers modified with D-/L-isothymidine in the loop regions.

Thrombin binding aptamer (TBA) is a 15-mer single-strand DNA that was identified by SELEX screening technology. It adopts a chair-type antiparallel G-quadruplex and can specifically interact with thrombin, thus inhibiting blood coagulation. Isonucleoside (isoNA) is a type of nucleoside isomer in which the base is shifted to 2'-positions of the glycosyl group, endowed with the ability to modulate local conformation of nucleotides, and L-isoNA could alter the conformation more due to the inversion of glycosyl configuration. Incorporation of L-isothymidine (L-isoT) at T3, T9, T12 positions and D-isoT at the T7 position in TBA's loop regions promoted the formation of G-quadruplex, resulting in enhanced affinity with thrombin and an increased anticoagulant effect. Computer simulation indicated that TBA-12L showed the strongest binding with thrombin, which was consistent with experimental results. The bioactivity of double isoNA incorporated TBA with D-IsoT at T7 and L-IsoT at T12 was comparable to that of TBA-12L, suggesting that the T12 of TBA was very important in interaction with thrombin. Our study also suggested that TBA might interact with two thrombin molecules through the TT loops (T3T4, T12T13) and TGT loop, but the second bonding did not show additional biological effects.

Single Calcite Particle Dissolution Kinetics: Revealing the Influence of Mass Transport.

Calcite dissolution kinetics at the single particle scale are determined. It is demonstrated that at high undersaturation and in the absence of inhibitors the particulate mineral dissolution rate is controlled by a saturated calcite surface in local equilibrium with dissolved Ca2+ and CO3 2- coupled with rate determining diffusive transport of the ions away from the surface. Previous work is revisited and inconsistencies arising from the assumption of a surface-controlled reaction are highlighted. The data have implications for ocean modeling of climate change.

The Bioactivity of D-/L-Isonucleoside- and 2'-Deoxyinosine-Incorporated Aptamer AS1411s Including DNA Replication/MicroRNA Expression.

In this study, chemical modification of 2'-deoxyinosine (2'-dI) and D-/L-isothymidine (D-/L-isoT) was performed on AS1411. They could promote the nucleotide-protein interaction by changing the local conformation. Twenty modified sequences were obtained, FCL-I and FCL-II showed the most noticeable activity improvement. They stabilized the G-quadruplex, remained highly resistant to serum degradation and specificity for nucleolin, further inhibited tumor cell growth, exhibited a stronger ability to influence the different phases of the tumor cell cycle, induced S-phase arrest, promoted the inhibition of DNA replication, and suppressed the unwound function of a large T antigen as powerful as AS1411. The microarray analysis and TaqMan PCR results showed that FCL-II can upregulate the expression of four breast-cancer-related, lowly expressed miRNAs and downregulate the expression of three breast-cancer-related, highly expressed miRNAs (>2.5-fold). FCL-II resulted in enhanced treatment effects greater than AS1411 in animal experiments (p < 0.01). The computational results further proved that FCL-II exhibits more structural advantages than AS1411 for binding to the target protein nucleolin, indicating its great potential in antitumor therapy.

Bioactivity of 2'-deoxyinosine-incorporated aptamer AS1411.

Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2'-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2'-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2'-deoxyinosine at positions 12 and 24 (FAN-1224dI), 13 and 24 (FAN-1324dI), and 15 and 24 (FAN-1524dI) promoted G-quartet formation, as well as inhibition of DNA replication and tumor cell growth, and induced S-phase cell cycle arrest. In further animal experiments, FAN-1224dI, FAN-1324dI and FAN-1524dI resulted in enhanced treatment effects than AS1411 alone. These results suggested that the position and number of modification substituents in AS1411 are critical parameters to improve the diagnostic and therapeutic function of the aptamer. Structural investigations of the FAN-1524dI/nucleolin complex structure, using molecular dynamics simulation, revealed the critical interactions involving nucleolin and 2'-dI incorporated AS1411 compared with AS1411 alone. These findings augment understanding of the role of 2'-deoxyinosine moieties in interactive binding processes.