[Molecular beacon based PNA-FISH method combined with fluorescence scanning for rapid detection of Listeria monocytogenes].

PubMed

Wu, Shan; Zhang, Xiaofeng; Shuai, Jiangbing; Li, Ke; Yu, Huizhen; Jin, Chenchen

2016-07-04

To simplify the PNA-FISH (Peptide nucleic acid-fluorescence in situ hybridization) test, molecular beacon based PNA probe combined with fluorescence scanning detection technology was applied to replace the original microscope observation to detect Listeria monocytogenes The 5′ end and 3′ end of the L. monocytogenes specific PNA probes were labeled with the fluorescent group and the quenching group respectively, to form a molecular beacon based PNA probe. When PNA probe used for fluorescence scanning and N1 treatment as the control, the false positive rate was 11.4%, and the false negative rate was 0; when N2 treatment as the control, the false positive rate decreased to 4.3%, but the false negative rate rose to 18.6%. When beacon based PNA probe used for fluorescence scanning, taken N1 treatment as blank control, the false positive rate was 8.6%, and the false negative rate was 1.4%; taken N2 treatment as blank control, the false positive rate was 5.7%, and the false negative rate was 1.4%. Compared with PNA probe, molecular beacon based PNA probe can effectively reduce false positives and false negatives. The success rates of hybridization of the two PNA probes were 83.3% and 95.2% respectively; and the rates of the two beacon based PNA probes were 91.7% and 90.5% respectively, which indicated that labeling the both ends of the PNA probe dose not decrease the hybridization rate with the target bacteria. The combination of liquid phase PNA-FISH and fluorescence scanning method, can significantly improve the detection efficiency.

Nanoparticles for Site Specific Genome Editing

NASA Astrophysics Data System (ADS)

McNeer, Nicole Ali

Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to translate gene therapies for diseases of the blood and immune system to clinical practice. In addition, we have expanded the use of this technology to an additional nonhematopoietic model system: correction of the human cystic fibrosis transmembrane receptor gene in human bronchial epithelial cells. The work presented here represents (1) the first use of biodegradable nanoparticles for PNA delivery, (2) the first direct in vivo site-specific genome modification in human cells, and (3) the first use of triplex-PNA technology for site-specific genome editing in cystic fibrosis.

Comparative biodistributions and dosimetry of [¹⁷⁷Lu]DOTA-anti-bcl-2-PNA-Tyr³-octreotate and [¹⁷⁷Lu]DOTA-Tyr³-octreotate in a mouse model of B-cell lymphoma/leukemia.

PubMed

Balkin, Ethan R; Liu, Dijie; Jia, Fang; Ruthengael, Varyanna C; Shaffer, Suzanne M; Miller, William H; Lewis, Michael R

2014-01-01

The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene in non-Hodgkin's lymphoma (NHL) is a dominant inhibitor of apoptosis. We developed a (177)Lu-labeled bcl-2 antisense peptide nucleic acid (PNA)-peptide conjugate designed for dual modality NHL therapy, consisting of a radiopharmaceutical capable of simultaneously down-regulating apoptotic resistance and delivering cytotoxic internally emitted radiation. DOTA-anti-bcl-2-Tyr(3)-octreotate was synthesized, labeled with (177)Lu, and purified using RP-HPLC. The PNA-peptide conjugate was evaluated in Mec-1 NHL-bearing mice and compared to [(177)Lu]DOTA-Tyr(3)-octreotate in biodistribution and excretion studies. These data were then used to generate in vivo dosimetry models. The PNA-peptide conjugate was readily prepared and radiolabeled in high yield and radiochemical purity. An in vivo blocking study determined that administration of 50 μg of non-radioactive PNA-peptide was the optimal mass for maximum delivery to the tumor. Based on that result, a dosing regimen of (177)Lu-PNA-peptide, for radiologic effect, followed by the optimal mass of non-radioactive compound, for antisense effect, was designed. Using that dosing regimen, biodistribution of the PNA-peptide showed uptake in the tumor with minimal washout over a 4-day period. Uptakes in receptor-positive normal organs were low and displayed nearly complete washout by 24h. Dosimetry models showed that the tumor absorbed dose of the PNA-peptide conjugate was approximately twice that of the peptide-only conjugate. Biodistribution data showed specific tumor targeting of the (177)Lu-labeled PNA-peptide compound with minimal receptor-positive normal tissue uptake when compared to [(177)Lu]DOTA-Tyr(3)-octreotate. In vivo dosimetry models predicted a more favorable tumor absorbed dose from [(177)Lu]DOTA-anti-bcl-2-Tyr(3)-octreotate. © 2013.

Anionic magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid for DNA base discrimination

NASA Astrophysics Data System (ADS)

Khadsai, Sudarat; Rutnakornpituk, Boonjira; Vilaivan, Tirayut; Nakkuntod, Maliwan; Rutnakornpituk, Metha

2016-09-01

Magnetite nanoparticles (MNPs) were surface modified with anionic poly( N-acryloyl glycine) (PNAG) and streptavidin for specific interaction with biotin-conjugated pyrrolidinyl peptide nucleic acid (PNA). Hydrodynamic size ( D h) of PNAG-grafted MNPs varied from 334 to 496 nm depending on the loading ratio of the MNP to NAG in the reaction. UV-visible and fluorescence spectrophotometries were used to confirm the successful immobilization of streptavidin and PNA on the MNPs. About 291 pmol of the PNA/mg MNP was immobilized on the particle surface. The PNA-functionalized MNPs were effectively used as solid supports to differentiate between fully complementary and non-complementary/single-base mismatch DNA using the PNA probe. These novel anionic MNPs can be efficiently applicable for use as a magnetically guidable support for DNA base discrimination.

Influence of pendant chiral C(γ)-(alkylideneamino/guanidino) cationic side-chains of PNA backbone on hybridization with complementary DNA/RNA and cell permeability.

PubMed

Jain, Deepak R; Anandi V, Libi; Lahiri, Mayurika; Ganesh, Krishna N

2014-10-17

Intrinsically cationic and chiral C(γ)-substituted peptide nucleic acid (PNA) analogues have been synthesized in the form of γ(S)-ethyleneamino (eam)- and γ(S)-ethyleneguanidino (egd)-PNA with two carbon spacers from the backbone. The relative stabilization (ΔTm) of duplexes from modified cationic PNAs as compared to 2-aminoethylglycyl (aeg)-PNA is better with complementary DNA (PNA:DNA) than with complementary RNA (PNA:RNA). Inherently, PNA:RNA duplexes have higher stability than PNA:DNA duplexes, and the guanidino PNAs are superior to amino PNAs. The cationic PNAs were found to be specific toward their complementary DNA target as seen from their significantly lower binding with DNA having single base mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show improved cell permeability.

Strand-invading linear probe combined with unmodified PNA.

PubMed

Asanuma, Hiroyuki; Niwa, Rie; Akahane, Mariko; Murayama, Keiji; Kashida, Hiromu; Kamiya, Yukiko

2016-09-15

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40°C at 100mM NaCl concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes

PubMed Central

Kleiner, Ralph E.; Brudno, Yevgeny; Birnbaum, Michael E.; Liu, David R.

2009-01-01

The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that the polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence-specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry, but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step towards the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities. PMID:18341334

Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics

NASA Astrophysics Data System (ADS)

Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

2017-02-01

Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process.

Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food.

PubMed

Peano, C; Lesignoli, F; Gulli, M; Corradini, R; Samson, M C; Marchelli, R; Marmiroli, N

2005-09-15

In the present study a peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method was developed and applied to the detection of genetically modified organisms (GMO), to test PCR products for band identity and to obtain a semiquantitative evaluation of GMO content. The minimal concentration of PNA necessary to block the PCR was determined by comparing PCRs containing a constant amount of DNA in the presence of increasing concentration of target-specific PNA. The lowest PNA concentration at which specific inhibition took place, by the inhibition of primer extension and/or steric hindrance, was the most efficient condition. Optimization of PCR clamping by PNA was observed by testing five different PNAs with a minimum of 13 bp to a maximum of 15 bp, designed on the target sequence of Roundup Ready soybean. The results obtained on the DNA extracted from Roundup Ready soybean standard flour were verified also on DNA extracted from standard flours of maize GA21, Bt176, Bt11, and MON810. A correlation between the PNA concentration necessary for inducing PCR clamping and the percentage of the GMO target sequence in the sample was found.

Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics.

PubMed

Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

2017-02-17

Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process.

Inducible Alkylation of DNA by a Quinone Methide-Peptide Nucleic Acid Conjugate†

PubMed Central

Liu, Yang; Rokita, Steven E.

2012-01-01

The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alkylation of non-complementary sequences is only possible when a template strand is present to co-localize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self adducts formed between the PNA and its attached QM remained active and reversible over more than eight days in aqueous solution prior to reaction with a chosen target added subsequently. PMID:22243337

Negative ions of p-nitroaniline: Photodetachment, collisions, and ab initio calculations

NASA Astrophysics Data System (ADS)

Smith, Byron H.; Buonaugurio, Angela; Chen, Jing; Collins, Evan; Bowen, Kit H.; Compton, Robert N.; Sommerfeld, Thomas

2013-06-01

The structures of parent anion, M-, and deprotonated molecule, [M-H]-, anions of the highly polar p-nitroaniline (pNA) molecule are studied experimentally and theoretically. Photoelectron spectroscopy (PES) of the parent anion is employed to estimate the adiabatic electron affinity (EAa = 0.75 ± 0.1 eV) and vertical detachment energy (VDE = 1.1 eV). These measured energies are in good agreement with computed values of 0.73 eV for the EAa and the range of 0.85 to 1.0 eV for the VDE at the EOM-CCSD/Aug-cc-pVTZ level. Collision induced dissociation (CID) of deprotonated pNA, [pNA - H]-, with argon yielded [pNA - H - NO]- (i.e., rearrangement to give loss of NO) with a threshold energy of 2.36 eV. Calculations of the energy difference between [pNA - H]- and [pNA - H - NO]- give 1.64 eV, allowing an estimate of a 0.72 eV activation barrier for the rearrangement reaction. Direct dissociation of [pNA - H]- yielding NO_2^ - occurs at a threshold energy of 3.80 eV, in good agreement with theory (between 3.39 eV and 4.30 eV). As a result of the exceedingly large dipole moment for pNA (6.2 Debye measured in acetone), we predict two dipole-bound states, one at ˜110 meV and an excited state at 2 meV. No dipole-bound states are observed in the photodetachment experiments due the pronounced mixing between states with dipole-bound and valence character similar to what has been observed in other nitro systems. For the same reason, dipole-bound states are expected to provide highly efficient "doorway states" for the formation of the pNA- valence anion, and these states should be observable as resonances in the reverse process, that is, in the photodetachment spectrum of pNA- near the photodetachment threshold.

Negative ions of p-nitroaniline: photodetachment, collisions, and ab initio calculations.

PubMed

Smith, Byron H; Buonaugurio, Angela; Chen, Jing; Collins, Evan; Bowen, Kit H; Compton, Robert N; Sommerfeld, Thomas

2013-06-21

The structures of parent anion, M(-), and deprotonated molecule, [M-H](-), anions of the highly polar p-nitroaniline (pNA) molecule are studied experimentally and theoretically. Photoelectron spectroscopy (PES) of the parent anion is employed to estimate the adiabatic electron affinity (EAa = 0.75 ± 0.1 eV) and vertical detachment energy (VDE = 1.1 eV). These measured energies are in good agreement with computed values of 0.73 eV for the EAa and the range of 0.85 to 1.0 eV for the VDE at the EOM-CCSD∕Aug-cc-pVTZ level. Collision induced dissociation (CID) of deprotonated pNA, [pNA - H](-), with argon yielded [pNA - H - NO](-) (i.e., rearrangement to give loss of NO) with a threshold energy of 2.36 eV. Calculations of the energy difference between [pNA - H](-) and [pNA - H - NO](-) give 1.64 eV, allowing an estimate of a 0.72 eV activation barrier for the rearrangement reaction. Direct dissociation of [pNA - H](-) yielding NO2(-) occurs at a threshold energy of 3.80 eV, in good agreement with theory (between 3.39 eV and 4.30 eV). As a result of the exceedingly large dipole moment for pNA (6.2 Debye measured in acetone), we predict two dipole-bound states, one at ~110 meV and an excited state at 2 meV. No dipole-bound states are observed in the photodetachment experiments due the pronounced mixing between states with dipole-bound and valence character similar to what has been observed in other nitro systems. For the same reason, dipole-bound states are expected to provide highly efficient "doorway states" for the formation of the pNA(-) valence anion, and these states should be observable as resonances in the reverse process, that is, in the photodetachment spectrum of pNA(-) near the photodetachment threshold.

Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics

PubMed Central

Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

2017-01-01

Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process. PMID:28211525

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

PubMed Central

2013-01-01

Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina. PMID:23586331

Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs

PubMed Central

Tailhades, Julien; Takizawa, Hotake; Gait, Michael J.; Wellings, Don A.; Wade, John D.; Aoki, Yoshitsugu; Shabanpoor, Fazel

2017-01-01

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2′-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences. PMID:29094037

Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs.

PubMed

Tailhades, Julien; Takizawa, Hotake; Gait, Michael J; Wellings, Don A; Wade, John D; Aoki, Yoshitsugu; Shabanpoor, Fazel

2017-01-01

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2'- O -methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce "on-resin" aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

A combination of immunohistochemistry and molecular approaches improves highly sensitive detection of BRAF mutations in papillary thyroid cancer.

PubMed

Martinuzzi, Claudia; Pastorino, Lorenza; Andreotti, Virginia; Garuti, Anna; Minuto, Michele; Fiocca, Roberto; Bianchi-Scarrà, Giovanna; Ghiorzo, Paola; Grillo, Federica; Mastracci, Luca

2016-09-01

The optimal method for BRAF mutation detection remains to be determined despite advances in molecular detection techniques. The aim of this study was to compare, against classical Sanger sequencing, the diagnostic performance of two of the most recently developed, highly sensitive methods: BRAF V600E immunohistochemistry (IHC) and peptide nucleic-acid (PNA)-clamp qPCR. BRAF exon 15 mutations were searched in formalin-fixed paraffin-embedded tissues from 86 papillary thyroid carcinoma using the three methods. The limits of detection of Sanger sequencing in borderline or discordant cases were quantified by next generation sequencing. BRAF mutations were found in 74.4 % of cases by PNA, in 71 % of cases by IHC, and in 64 % of cases by Sanger sequencing. Complete concordance for the three methods was observed in 80 % of samples. Better concordance was observed with the combination of two methods, particularly PNA and IHC (59/64) (92 %), while the combination of PNA and Sanger was concordant in 55 cases (86 %). Sensitivity of the three methods was 99 % for PNA, 94.2 % for IHC, and 89.5 % for Sanger. Our data show that IHC could be used as a cost-effective, first-line method for BRAF V600E detection in daily practice, followed by PNA analysis in negative or uninterpretable cases, as the most efficient method. PNA-clamp quantitative PCR is highly sensitive and complementary to IHC as it also recognizes other mutations besides V600E and it is suitable for diagnostic purposes.

Solid-phase synthesis of difficult purine-rich PNAs through selective Hmb incorporation: Application to the total synthesis of cell penetrating peptide-PNAs

NASA Astrophysics Data System (ADS)

Tailhades, Julien; Takizawa, Hotake; Gait, Michael J.; Wellings, Don A.; Wade, John D.; Aoki, Yoshitsugu; Shabanpoor, Fazel

2017-10-01

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2’-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

FISH and PNA FISH for the diagnosis of Q fever endocarditis and vascular infections.

PubMed

Prudent, Elsa; Lepidi, Hubert; Angelakis, Emmanouil; Raoult, Didier

2018-06-13

Purpose. Endocarditis and vascular infections are common manifestations of persistent localized infection due to Coxiella burnetii and recently, fluorescence in situ hybridization (FISH) was proposed as an alternative tool for their diagnosis. In this study, we evaluated the efficiency of FISH in a series of valve and vascular samples infected by C. burnetii. Methods. We tested 23 C. burnetii -positive valves and thrombus samples obtained from patients with Q fever endocarditis. Seven aneurysms and thrombus specimens were retrieved from patients with Q fever vascular infection. Samples were analyzed by culture, immunochemistry and FISH with oligonucleotide and PNA probes targeting C. burnetii -specific 16S ribosomal RNA sequences. Results. Immunohistochemical analysis was positive for five (17%) samples with significantly more copies of C. burnetii DNA than the negative ones ( p= 0.02). FISH was positive for 13 (43%) samples and presented 43% and 40% sensitivity compared to qPCR and culture, respectively. PNA FISH detected C. burnetii in 18 (60%) samples and presented 60% and 55% sensitivity compared to qPCR and culture, respectively. Immunohistochemistry had 38% and 28% sensitivity compared to FISH and PNA FISH, respectively. Samples found positive by both immunohistochemistry and PNA FISH contained significantly more copies of C. burnetii DNA than the negative ones ( p= 0.03). Finally, PNA FISH was more sensitive than FISH (60% versus 43%) for the detection of C. burnetii Conclusion. We provide evidence that PNA FISH and FISH are important assays for the diagnosis of C. burnetii endocarditis and vascular infections. Copyright © 2018 American Society for Microbiology.

Applicability of one-stage partial nitritation and anammox in MBBR for anaerobically pre-treated municipal wastewater.

PubMed

Kouba, Vojtech; Widiayuningrum, P; Chovancova, L; Jenicek, P; Bartacek, J

2016-07-01

Energy consumption of municipal wastewater treatment plants can be reduced by the anaerobic pre-treatment of the main wastewater stream. After this pre-treatment, nitrogen can potentially be removed by partial nitritation and anammox (PN/A). Currently, the application of PN/A is limited to nitrogen-rich streams (>500 mg L(-1)) and temperatures 25-35 °C. But, anaerobically pretreated municipal wastewater is characterized by much lower nitrogen concentrations (20-100 mg L(-1)) and lower temperatures (10-25 °C). We operated PN/A under similar conditions: total ammonium nitrogen concentration 50 mg L(-1) and lab temperature (22 °C). PN/A was operated for 342 days in a 4 L moving bed biofilm reactor (MBBR). At 0.4 mg O2 L(-1), nitrogen removal rate 33 g N m(-3) day(-1) and 80 % total nitrogen removal efficiency was achieved. The capacity of the reactor was limited by low AOB activity. We observed significant anammox activity (40 g N m(-3) day(-1)) even at 12 °C, improving the applicability of PN/A for municipal wastewater treatment.

Peptide nucleic acids rather than RNA may have been the first genetic molecule

NASA Technical Reports Server (NTRS)

Nelson, K. E.; Levy, M.; Miller, S. L.

2000-01-01

Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.

Novel Polymeric Nanoparticles for Pulmonary Gene Delivery

NASA Astrophysics Data System (ADS)

Fields, Rachel Jennifer

The lung is an important target for gene and drug therapy of many diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), tubuerculosis (TB) and lung cancer. In fact, the pulmonary route has been employed as a means of delivering drugs for centuries, dating back 4000 years to India where inhaled vapors were used for medicinal purpose. Currently, pulmonary administration of small, hydrophobic drugs leads to rapid local and systemic absorption. However, delivery of large biomacromolecules, such as therapeutic genes, has not yet been accomplished. Here, I test the hypothesis that a rationally engineered nanoparticle (NP) vector can improve delivery of large biomacromolecules. . In this dissertation I tested this hypothesis using a hybrid NP delivery system consisting of a blend of poly(lactic-co-glycolic acid) (PLGA) and a poly(beta-amino ester) (PBAE), a cationic polymer that is particularly useful for delivery of nucleic acids.. PBAE/PLGA nanoparticles (15% PBAE) loaded with plasmid DNA were surface modified with cell-penetrating peptides (CPPs) via a PEGylated phospholipid linker. This optimized NP formulation was able to induce substantial intracellular uptake and transfect lung epithelial cells in vitro while imparting minimal cellular toxicity. In order to determine the most effective method to deliver these NPs to the lung I used fluorescently labeled particles to study the biodistribution of particles after administration to the lung of mice via various administration routes. I determined that the intranasal route was most effective. I further investigated this route and determined that an average of 37.1 +/- 15.1 % of lung cells had NP association after 4hrs. I also investigated the association of particles with different lung cell types like macrophages and alveolar epithelial cells and determined that our best particle formulations associated with approximately 80% of both of these cell types. To demonstrate the ability of the NPs to deliver difficult to gene therapy reagents, such as PNAs, to cells within the lung, I loaded NPs with PNA and DNA and administered them via the intranasal route. Triplex forming peptide nucleic acids (PNAs) are gene therapy reagents that can mediate site-specific homologous recombination with genomic DNA when successfully delivered to the nucleus of cells in combination with donor DNA oligos. Delivery of NPs resulted in EGFP expression in transgenic mice with an aberrant EGFP gene that could be corrected and effectively expressed with nuclear delivery of a PNA/DNA. This work represents the first successful use of PNA/DNA mediated homologous recombination to target cells of the lung.

Size and morphology controlled NiSe nanoparticles as efficient catalyst for the reduction reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subbarao, Udumula; Marakatti, Vijaykumar S.; Amshumali, Mungalimane K.

Facile and efficient ball milling and polyol methods were employed for the synthesis of nickel selenide (NiSe) nanoparticle. The particle size of the NiSe nanoparticle has been controlled mechanically by varying the ball size in the milling process. The role of the surfactants in the formation of various morphologies was studied. The compounds were characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray energy dispersive spectroscopy (EDS). The efficiency of the NiSe nanoparticle as a catalyst was tested for the reduction of para-nitroaniline (PNA) to para-phenyldiamine (PPD) and para-nitrophenol (PNP) to para-aminophenol (PAP)more » using NaBH{sub 4} as the reducing agent. Particle size, morphology and the presence of surfactant played a crucial role in the reduction process. - Graphical abstract: NiSe nanoparticles in different size and morphology were synthesized using facile ball milling and polyol methods. Particle size, morphology and the presence of surfactant in these materials played a crucial role in the hydrogenation of PNA and PNP. - Highlights: • NiSe nanoparticles synthesized using ball milling and solution phase methods. • NiSe nanoparticle is an efficient catalyst for the reduction of PNA and PNP. • NiSe is found to be better than the best reported noble metal catalysts.« less

Influence of Gestational Age and Postnatal Age on Speech Sound Processing in NICU infants

PubMed Central

Key, Alexandra P.F.; Lambert, E. Warren; Aschner, Judy L.; Maitre, Nathalie L.

2012-01-01

The study examined the effect of gestational (GA) and postnatal (PNA) age on speech sound perception in infants. Auditory ERPs were recorded in response to speech sounds (CV syllables) in 50 infant NICU patients (born at 24–40 weeks gestation) prior to discharge. Efficiency of speech perception was quantified as absolute difference in mean amplitudes of ERPs in response to vowel (/a/–/u/) and consonant (/b/–/g/, /d/–/g/) contrasts within 150–250, 250–400, 400–700 ms after stimulus onset. Results indicated that both GA and PNA affected speech sound processing. These effects were more pronounced for consonant than vowel contrasts. Increasing PNA was associated with greater sound discrimination in infants born at or after 30 weeks GA, while minimal PNA-related changes were observed for infants with GA less than 30 weeks. Our findings suggest that a certain level of brain maturity at birth is necessary to benefit from postnatal experience in the first 4 months of life, and both gestational and postnatal ages need to be considered when evaluating infant brain responses. PMID:22332725

Tetrahedral DNA Nanoparticle Vector for Intracellular Delivery of Targeted Peptide Nucleic Acid Antisense Agents to Restore Antibiotic Sensitivity in Cefotaxime-Resistant Escherichia coli.

PubMed

Readman, John Benedict; Dickson, George; Coldham, Nick G

2017-06-01

The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-bla CTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 μM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying bla CTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 μM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.

Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

PubMed

Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

2016-12-01

In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

Effect of arachidonic acid metabolites on thymocyte proliferation.

PubMed

Delebassée, S; Gualde, N

1988-01-01

The influences of prostaglandin E2 (PGE2), 15-hydroxyeicosatetraenoic acid (15-HETE) and leukotrienes (LT) on the proliferative response of mature (PNA-) and immature (PNA+) mouse thymocytes was investigated. Both PNA+ and PNA- thymocytes proliferated when cultured with concanavalin A plus interleukin-2. PGE2 in concentrations of 10(-6) to 10(-9) M caused significant inhibition of proliferation of both PNA+ and PNA- thymocytes in these cultures. In contrast, the lipoxygenase products 15-HETE, LTB4, LTC4 and LTD4 caused marked increases in proliferation of PNA+ thymocytes while having no effect on PNA- cells. Therefore, the effect of leukotrienes on thymocyte proliferation depends upon the level of cell maturation and mainly affects immature PNA+ thymocytes.

Improved Force Fields for Peptide Nucleic Acids with Optimized Backbone Torsion Parameters.

PubMed

Jasiński, Maciej; Feig, Michael; Trylska, Joanna

2018-06-06

Peptide nucleic acids are promising nucleic acid analogs for antisense therapies as they can form stable duplex and triplex structures with DNA and RNA. Computational studies of PNA-containing duplexes and triplexes are an important component for guiding their design, yet existing force fields have not been well validated and parametrized with modern computational capabilities. We present updated CHARMM and Amber force fields for PNA that greatly improve the stability of simulated PNA-containing duplexes and triplexes in comparison with experimental structures and allow such systems to be studied on microsecond time scales. The force field modifications focus on reparametrized PNA backbone torsion angles to match high-level quantum mechanics reference energies for a model compound. The microsecond simulations of PNA-PNA, PNA-DNA, PNA-RNA, and PNA-DNA-PNA complexes also allowed a comprehensive analysis of hydration and ion interactions with such systems.

Maternal peanut exposure during pregnancy and lactation reduces peanut allergy risk in offspring.

PubMed

López-Expósito, Iván; Song, Ying; Järvinen, Kirsi M; Srivastava, Kamal; Li, Xiu-Min

2009-11-01

Maternal allergy is believed to be a risk factor for peanut allergy (PNA) in children. However, there is no direct evidence of maternal transmission of PNA susceptibility, and it is unknown whether maternal peanut exposure affects the development of PNA in offspring. To investigate the influence of maternal PNA on offspring reactions to the first peanut exposure, and whether maternal low-dose peanut exposure during pregnancy and lactation influences these reactions and peanut sensitization in a murine model. Five-week-old offspring of PNA C3H/HeJ mothers (PNA-Ms) were challenged intragastrically with peanut (first exposure), and reactions were determined. In a subset of the experiment, PNA-Ms were fed a low dose of peanut (PNA-M/PN) or not fed peanut (PNA-M/none) during pregnancy and lactation. Their 5-week-old offspring were challenged intragastrically with peanut, and reactions were determined. In another subset of the experiment, offspring of PNA-M/PN or PNA-M/none were sensitized with peanut intragastrically for 6 weeks, and serum peanut-specific antibodies were determined. PNA-M offspring exhibited anaphylactic reactions at first exposure to peanut that were associated with peanut-specific IgG(1) levels and prevented by a platelet activation factor antagonist. In a subset experiment, PNA-M/PN offspring showed significantly reduced first-exposure peanut reactions, increased IgG(2a), and reduced mitogen-stimulated splenocyte cytokine production compared with PNA-M/none offspring. In an additional experiment, PNA-M/PN offspring showed reduction of peanut-specific IgE to active peanut sensitization. We show for the first time maternal transmission of susceptibility to first-exposure peanut reactions and active peanut sensitization. Low-dose peanut exposure during pregnancy and lactation reduced this risk.

Insights into the structural features and stability of peptide nucleic acid with a D-prolyl-2-aminocyclopentane carboxylic acid backbone that binds to DNA and RNA.

PubMed

Poomsuk, Nattawee; Vilaivan, Tirayut; Siriwong, Khatcharin

2018-06-12

Peptide nucleic acid (PNA) is a powerful biomolecule with a wide variety of important applications. In this work, the molecular structures and binding affinity of PNA with a D-prolyl-2-aminocyclopentane carboxylic acid backbone (acpcPNA) that binds to both DNA and RNA were studied using molecular dynamics simulations. The simulated structures of acpcPNA-DNA and acpcPNA-RNA duplexes more closely resembled the typical structures of B-DNA and A-RNA than the corresponding duplexes of aegPNA. The calculated binding free energies are in good agreement with the experimental results that the acpcPNA-DNA duplex is more stable than the acpcPNA-RNA duplex regardless of the base sequences. The results provide further insights in the relationship between structure and stability of this unique PNA system. Copyright © 2018 Elsevier Inc. All rights reserved.

Optofluidic plasmonic onchip nanosensor array for biodetection

NASA Astrophysics Data System (ADS)

Huang, Min

Surface plasmon resonance (SPR) sensing has been demonstrated in the past decade to be the gold standard technique for biochemical interaction analysis, and plays an important role in drug discovery and biomedical research. The technique circumvents the need of fluorescence/radioactive tagging or enzymatic detection, enables ultrasensitive remote sensing, and quantitatively monitors bio-interaction in real time. Although SPR has these attractive features that can satisfy most research/clinic requirements, there still exist problems that limit its applications. First, the reflection geometry of the prism coupling scheme adds limitations for high throughput screening application. Additionally, SPR instrumentations are bulky and not suitable for point-of-care settings. Moreover, the SPR sensor is embedded in conventional micro-fluidic cells, in which the sensor performance is limited by inefficient analyte transport. Suspended plasmonic nanohole array (PNA) offers an opportunity to overcome these limitations. A collinear excitation/collection coupling scheme combined with the small footprint of PNA provides unique platform for multiplexing and system minimization. The suspended nanohole structure also offers a unique configuration to integrate nano-photonics with nano-fluidics. This thesis focuses on developing a lab-on-a-chip PNA platform for point-of-care bio-detection. To achieve this, we first demonstrate that the figure-of-merit of our PNA sensor surpasses that of the prism coupled SPR. We also show that the ultrasensitive label-free PNA sensor is able to directly detect intact viruses from biological media at clinically relevant concentrations with little sample preparation. We then present a plasmonic microarray with over one million PNA sensors on a microscope slide for high throughput screening applications. A dual-color filter imaging method is introduced to increase the accuracy, reliability, and signal-to-noise ratio in a highly multiplexed manner. Finally, we present a nanoplasmonic-nanofluidic platform enabling active delivery of analyte to the sensor. Sensor response time is reduced by an order of magnitude compared to the conventional flow scheme. A dynamic range spanning 5 orders of magnitude from 103 to 107 particles/mL is shown on this platform corresponding to analyte concentration sufficient for clinical applications. The proposed approach opens up opportunities of a lab-on-a-chip bio-detection system for drug screening, disease diagnostic as well as clinic studies.

Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

PubMed Central

Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

2002-01-01

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications. PMID:11842106

Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

PubMed Central

Jankowsky, E; Strunk, G; Schwenzer, B

1997-01-01

Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes. PMID:9207013

Insight into a conformation of the PNA-PNA duplex with (2‧R,4‧R)- and (2‧R,4‧S)-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbones

NASA Astrophysics Data System (ADS)

Maitarad, Amphawan; Poomsuk, Nattawee; Vilaivan, Chotima; Vilaivan, Tirayut; Siriwong, Khatcharin

2018-04-01

Suitable conformations for peptide nucleic acid (PNA) self-hybrids with (2‧R,4‧R)- and (2‧R,4‧S)-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbones (namely, acpcPNA and epi-acpcPNA, respectively) were investigated based on molecular dynamics simulations. The results revealed that hybridization of the acpcPNA was observed only in the parallel direction, with a conformation close to the P-type structure. In contrast, self-hybrids of the epi-acpcPNA were formed in the antiparallel and parallel directions; the antiparallel duplex adopted the B-form conformation, and the parallel duplex was between B- and P-forms. The calculated binding energies and the experimental data indicate that the antiparallel epi-acpcPNA self-hybrid was more stable than the parallel duplex.

Thymic lymphocytes. III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation.

PubMed

Papiernik, M; Jacobson, J B

1986-01-01

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.

Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake.

PubMed

Ellipilli, Satheesh; Ganesh, Krishna N

2015-09-18

Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (apg) backbone (γ-CF2-apg PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard aeg-PNA. Cellular uptake of the fluorinated γ-CF2-apg PNAs in NIH 3T3 and HeLa cells was 2-3-fold higher compared to that of nonfluorinated apg PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents.

Tumor affinity of radiolabeled peanut agglutinin compared with that of Ga-67 citrate in animal models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokoyama, K.; Aburano, T.; Watanabe, N.

1985-05-01

Peanut agglutinin (PNA) binds avidly to the immunodominant group of the tumor associated T antigen. The purpose of this study was to evaluate oncodiagnostic potential of radiolabeled PNA in animal models. PNA was labeled with I-125 or I-131 by Iodogen and also with In-111 by cyclic DTPA anhydride. The biological activity of PNA was examined by a hemaglutination titer with a photometer before and after labeling. Animal tumor models used were Lewis Lung Cancer(LLC), B-16 Melanotic Melanoma(MM), Yoshida Sarcoma(YS), Ehrlich Ascites Tumor(EAT and Hepatoma AH109A(HAH). Inflammatory tissue induced by turpentine oil was used as an abscess model. Serial scintigraphic imagesmore » were obtained following IV injections of 100 ..mu..Ci of I-131 or In-111-DTPA-PNA. The tumor affinity of Ga-67 citrate was studied to compare that of radiolabeled PNA. Tissue biodistribution was studied in EAT bearing mice. All of these tumor models except HAH were clearly visible by radiolabeled PNA without subtraction techniques. In the models of LLC and EAT, PNA showed the better accumulation into the tumor tissue than Ga-67 citrate. In YS and MM, PNA represented almost the same accumulation as Ga-67 citrate. The localization of PNA into abscess tissue wasn't found although Ga-67 citrate markedly accumulated into abscess tissue as well as tumor tissue. The clearance of PNA from tumor was slower than those from any other organs. Tumor to muscle ratio was 5.1 at 48hrs. and tumor to blood ratio increased with time to 2.3 at 96hrs. These results suggested that radiolabeled PNA may have a potential in the detection of tumor.« less

The Crystal Structure of Non-Modified and Bipyridine-Modified PNA Duplexes

PubMed Central

Yeh, Joanne I.; Pohl, Ehmke; Truan, Daphne; He, Wei; Sheldrick, George M.; Du, Shoucheng; Achim, Catalina

2011-01-01

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N-aminoethlyl-glycine backbone. The crystal structure of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATCGG)2 (pdb: 3MBS), and the other containing the same nucleobase pairs and a central pair of bipyridine ligands (pdb: 3MBU), has been solved with a resolution of 1.2 Å and 1.05 Å, respectively. The non-modified PNA duplex adopts a P-type helical structure s i m i l a r t o that of previously characterized PNAs. The atomic-level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. These results support the notion that while PNA typically adopts a P-type helical structure, its flexibility is relatively high. For example, the base pair rise in the bipyridine-containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines are bulged out of the duplex and are aligned parallel to the minor groove of the PNA. In the case of the bipyridine-containing PNA, two bipyridines from adjacent PNA duplexes form a π-stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl-modified DNA duplexes in solution, where the biphenyls are π-stacking with adjacent nucleobase pairs and adopt an intrahelical geometry [Johar et al., Chem. Eur. J., 2008, 14, 2080]. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes. PMID:20859960

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses

PubMed Central

Trubetskoy, Vladimir S.; Griffin, Jacob B.; Nicholas, Anthony L.; Nord, Eric M.; Xu, Zhao; Peterson, Ryan M.; Wooddell, Christine I.; Rozema, David B.; Wakefield, Darren H.; Lewis, David L.

2017-01-01

Abstract The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships. PMID:28180327

Recent Advances in Chemical Modification of Peptide Nucleic Acids

PubMed Central

Rozners, Eriks

2012-01-01

Peptide nucleic acid (PNA) has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification. PMID:22991652

Crystal structure of chiral gammaPNA with complementary DNA strand: insights into the stability and specificity of recognition and conformational preorganization.

PubMed

Yeh, Joanne I; Shivachev, Boris; Rapireddy, Srinivas; Crawford, Matthew J; Gil, Roberto R; Du, Shoucheng; Madrid, Marcela; Ly, Danith H

2010-08-11

We have determined the structure of a PNA-DNA duplex to 1.7 A resolution by multiple-wavelength anomalous diffraction phasing method on a zinc derivative. This structure represents the first high-resolution 3D view of a hybrid duplex containing a contiguous chiral PNA strand with complete gamma-backbone modification ("gammaPNA"). Unlike the achiral counterpart, which adopts a random-fold, this particular gammaPNA is already preorganized into a right-handed helix as a single strand. The new structure illustrates the unique characteristics of this modified PNA, possessing conformational flexibility while maintaining sufficient structural integrity to ultimately adopt the preferred P-helical conformation upon hybridization with DNA. The unusual structural adaptability found in the gammaPNA strand is crucial for enabling the accommodation of backbone modifications while constraining conformational states. In conjunction with NMR analysis characterizing the structures and substructures of the individual building blocks, these results provide unprecedented insights into how this new class of chiral gammaPNA is preorganized and stabilized, before and after hybridization with a cDNA strand. Such knowledge is crucial for the future design and development of PNA for applications in biology, biotechnology, and medicine.

Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays

PubMed Central

Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

2012-01-01

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes. PMID:22772038

Enzymatic Reaction with Unnatural Substrates: DNA Photolyase (Escherichia coli) Recognizes and Reverses Thymine [2+2] Dimers in the DNA Strand of a DNA/PNA Hybrid Duplex

NASA Astrophysics Data System (ADS)

Ramaiah, Danaboyina; Kan, Yongzhi; Koch, Troels; Orum, Henrik; Schuster, Gary B.

1998-10-01

Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson--Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.

Antimicrobial Peptide-PNA Conjugates Selectively Targeting Bacterial Genes

DTIC Science & Technology

2013-07-22

RXR)4XB and (KFF)3K, were previously reported as a potent permeabilizer against E. coli and MRSA cells (Mellbye, 2009). (RW)4D, a small dendrimeric ...lethal concentration (Liu, 2007). Scheme 1. Synthesis of PNA- dendrimer conjugate. (a) (RW)4D-cysteine (b)Free PNA (C) PNA-(RW)4D conjugates

Molecular self-assembly using peptide nucleic acids.

PubMed

Berger, Or; Gazit, Ehud

2017-01-01

Peptide nucleic acids (PNAs) are extensively studied for the control of genetic expression since their design in the 1990s. However, the application of PNAs in nanotechnology is much more recent. PNAs share the specific base-pair recognition characteristic of DNA together with material-like properties of polyamides, both proteins and synthetic polymers, such as Kevlar and Nylon. The first application of PNA was in the form of PNA-amphiphiles, resulting in the formation of either lipid integrated structures, hydrogels or fibrillary assemblies. Heteroduplex DNA-PNA assemblies allow the formation of hybrid structures with higher stability as compared with pure DNA. A systematic screen for minimal PNA building blocks resulted in the identification of guanine-containing di-PNA assemblies and protected guanine-PNA monomer spheres showing unique optical properties. Finally, the co-assembly of PNA with thymine-like three-faced cyanuric acid allowed the assembly of poly-adenine PNA into fibers. In summary, we believe that PNAs represent a new and important family of building blocks which converges the advantages of both DNA- and peptide-nanotechnologies. © 2016 Wiley Periodicals, Inc.

Versatility of peptide nucleic acids (PNAs): role in chemical biology, drug discovery, and origins of life.

PubMed

Sharma, Chiranjeev; Awasthi, Satish Kumar

2017-01-01

This review briefly discussed nomenclature, synthesis, chemistry, and biophysical properties of a plethora of PNA derivatives reported since the discovery of aegPNA. Different synthetic methods and structural analogs of PNA synthesized till date were also discussed. An insight was gained into various chemical, physical, and biological properties of PNA which make it preferable over all other classes of modified nucleic acid analogs. Thereafter, various approaches with special attention to the practical constraints, characteristics, and inherent drawbacks leading to the delay in the development of PNA as gene therapeutic drug were outlined. An explicit account of the successful application of PNA in different areas of research such as antisense and antigene strategies, diagnostics, molecular probes, and so forth was described along with the current status of PNA as gene therapeutic drug. Further, the plausibility of the existence of PNA and its role in primordial chemistry, that is, origin of life was explored in an endeavor to comprehend the mystery and open up its deepest secrets ever engaging and challenging the human intellect. We finally concluded it with a discussion on the future prospects of PNA technology in the field of therapeutics, diagnostics, and origin of life. © 2016 John Wiley & Sons A/S.

Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

PubMed

Dutta, Samrat; Armitage, Bruce A; Lyubchenko, Yuri L

2016-03-15

Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

Incidence and mortality trends of aspiration pneumonia in Parkinson's disease in the United States, 1979-2010.

PubMed

Akbar, Umer; Dham, Bhavpreet; He, Ying; Hack, Nawaz; Wu, Samuel; Troche, Michelle; Tighe, Patrick; Nelson, Eugene; Friedman, Joseph H; Okun, Michael S

2015-09-01

Careful examination of long-term analyses and trends is essential in understanding the medico-economic burden of this common complication. We sought to describe the long-term (32-year) trends of incidence and mortality in PD patients hospitalized with aspiration pneumonia (AsPNA). Incidence and mortality of AsPNA in hospitalized PD versus non-PD patients was assessed by logistic regression analysis applied to a national database between the years 1979 and 2010. Covariates such as age-decennium, gender, year AsPNA occurred, and the interactions with PD diagnosis were investigated. Rate of AsPNA and mortality over the 32-years was trended and compared. AsPNA occurred in 3.6% of PD patients and 1.0% of non-PD patients. The average mortality for PD patients was less (17% vs. 22%). Long-term (32-year) trends revealed a nearly 10-fold increase in incidence of AsPNA in PD (0.4% in 1979, 4.9% in 2010), decreasing mortality overtime, higher likelihood in males, and increasing average age of AsPNA patients (steeper increase in PD). All p-values<0.05. In regression analysis, each successive year had a slight increase in odds of AsPNA (OR 1.03 in PD, OR1.06 in non-PD). Trends over 32 years revealed a 10-fold increase in AsPNA among PD and non-PD patients, and an associated decrease in mortality. Our data suggest that PD patients are living longer, have slightly more AsPNA, but a lower mortality than was seen in past decades. Further research should investigate the causes of AsPNA in PD, and also potential interventions to decrease its occurrence. Copyright © 2015 Elsevier Ltd. All rights reserved.

PNA containing isocytidine nucleobase: synthesis and recognition of double helical RNA

PubMed Central

Zengeya, Thomas; Li, Ming; Rozners, Eriks

2011-01-01

Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Triple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was studied using isothermal titration calorimetry. The iC nucleobase recognized the proposed target, C-G inversion in polypurine tract of RNA, with slightly higher affinity than the natural nucleobases, though the sequence selectivity of recognition was low. Compared to non-modified PNA, PNA1 had lower affinity for its RNA target. PMID:21333533

Study of nucleic acid-gold nanorod interactions and detecting nucleic acid hybridization using gold nanorod solutions in the presence of sodium citrate.

PubMed

Kanjanawarut, Roejarek; Su, Xiaodi

2010-09-01

In this study, the authors report that sodium citrate can aggregate hexadecyl-trimethyl-ammonium ion(+)-coated gold nanorods (AuNRs), and nucleic acids of different charge and structure properties, i.e., single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded peptide nucleic acid (PNA), and PNA-DNA complex, can bind to the AuNRs and therefore retard the sodium citrate-induced aggregation to different extents. The discovery that hybridized dsDNA (and the PNA-DNA complex) has a more pronounced protection effect than ssDNA (and PNA) allows the authors to develop a homogeneous phase AuNRs-based UV-visible (UV-vis) spectral assay for detecting specific sequences of oligonucleotides (20 mer) with a single-base-mismatch selectivity and a limit of detection of 5 nM. This assay involves no tedious bioconjugation and on-particle hybridization. The simple "set and test" format allows for a highly efficient hybridization in a homogeneous phase and a rapid display of the results in less than a minute. By measuring the degree of reduction in AuNR aggregation in the presence of different nucleic acid samples, one can assess how different nucleic acids interact with the AuNRs to complement the knowledge of spherical gold nanoparticles. Besides UV-vis characterization, transmission electron microscopy and zeta potential measurements were conduced to provide visual evidence of the particle aggregation and to support the discussion of the assay principle.

Voluntary Exercise Improves Estrous Cyclicity in Prenatally Androgenized Female Mice Despite Programming Decreased Voluntary Exercise: Implications for Polycystic Ovary Syndrome (PCOS).

PubMed

Homa, Lori D; Burger, Laura L; Cuttitta, Ashley J; Michele, Daniel E; Moenter, Suzanne M

2015-12-01

Prenatal androgen (PNA) exposure in mice produces a phenotype resembling lean polycystic ovary syndrome. We studied effects of voluntary exercise on metabolic and reproductive parameters in PNA vs vehicle (VEH)-treated mice. Mice (8 wk of age) were housed individually and estrous cycles monitored. At 10 weeks of age, mice were divided into groups (PNA, PNA-run, VEH, VEH-run, n = 8-9/group); those in the running groups received wheels allowing voluntary running. Unexpectedly, PNA mice ran less distance than VEH mice; ovariectomy eliminated this difference. In ovary-intact mice, there was no difference in glucose tolerance, lower limb muscle fiber types, weight, or body composition among groups after 16 weeks of running, although some mitochondrial proteins were mildly up-regulated by exercise in PNA mice. Before running, estrous cycles in PNA mice were disrupted with most days in diestrus. There was no change in cycles during weeks 1-6 of running (10-15 wk of age). In contrast, from weeks 11 to 16 of running, cycles in PNA mice improved with more days in proestrus and estrus and fewer in diestrus. PNA programs reduced voluntary exercise, perhaps mediated in part by ovarian secretions. Exercise without weight loss improved estrous cycles, which if translated could be important for fertility in and counseling of lean women with polycystic ovary syndrome.

A Reconfigurable Design and Architecture of the Ethernet and HomePNA3.0 MAC

NASA Astrophysics Data System (ADS)

Khalilydermany, M.; Hosseinghadiry, M.

In this paper a reconfigurable architecture for Ethernet and HomePNA MAC is presented. By using this new architecture, Ethernet and HomePNA reconfigurable network card can be produced. This architecture has been implemented using VHDL language and after that synthesized on a chip. The differences between HomePNA (synchronized and unsynchronized mode) and Ethernet in collision detection mechanism and priority access to media have caused the need to separate architectures for Ethernet and HomePNA, but by using similarities of them, both the Ethernet and the HomePNA can be implemented in a single chip with a little extra hardware. The number of logical elements of the proposed architecture is increased by 19% in compare to when only an Ethernet MAC is implemented

Genome-based microbial ecology of anammox granules in a full-scale wastewater treatment system.

PubMed

Speth, Daan R; In 't Zandt, Michiel H; Guerrero-Cruz, Simon; Dutilh, Bas E; Jetten, Mike S M

2016-03-31

Partial-nitritation anammox (PNA) is a novel wastewater treatment procedure for energy-efficient ammonium removal. Here we use genome-resolved metagenomics to build a genome-based ecological model of the microbial community in a full-scale PNA reactor. Sludge from the bioreactor examined here is used to seed reactors in wastewater treatment plants around the world; however, the role of most of its microbial community in ammonium removal remains unknown. Our analysis yielded 23 near-complete draft genomes that together represent the majority of the microbial community. We assign these genomes to distinct anaerobic and aerobic microbial communities. In the aerobic community, nitrifying organisms and heterotrophs predominate. In the anaerobic community, widespread potential for partial denitrification suggests a nitrite loop increases treatment efficiency. Of our genomes, 19 have no previously cultivated or sequenced close relatives and six belong to bacterial phyla without any cultivated members, including the most complete Omnitrophica (formerly OP3) genome to date.

Genome-based microbial ecology of anammox granules in a full-scale wastewater treatment system

PubMed Central

Speth, Daan R.; in 't Zandt, Michiel H.; Guerrero-Cruz, Simon; Dutilh, Bas E.; Jetten, Mike S. M.

2016-01-01

Partial-nitritation anammox (PNA) is a novel wastewater treatment procedure for energy-efficient ammonium removal. Here we use genome-resolved metagenomics to build a genome-based ecological model of the microbial community in a full-scale PNA reactor. Sludge from the bioreactor examined here is used to seed reactors in wastewater treatment plants around the world; however, the role of most of its microbial community in ammonium removal remains unknown. Our analysis yielded 23 near-complete draft genomes that together represent the majority of the microbial community. We assign these genomes to distinct anaerobic and aerobic microbial communities. In the aerobic community, nitrifying organisms and heterotrophs predominate. In the anaerobic community, widespread potential for partial denitrification suggests a nitrite loop increases treatment efficiency. Of our genomes, 19 have no previously cultivated or sequenced close relatives and six belong to bacterial phyla without any cultivated members, including the most complete Omnitrophica (formerly OP3) genome to date. PMID:27029554

Pacific-North American teleconnection and North Pacific Oscillation: historical simulation and future projection in CMIP5 models

NASA Astrophysics Data System (ADS)

Chen, Zheng; Gan, Bolan; Wu, Lixin; Jia, Fan

2017-09-01

Based on reanalysis datasets and as many as 35 CMIP5 models, this study evaluates the capability of climate models to simulate the spatiotemporal features of Pacific-North American teleconnection (PNA) and North Pacific Oscillation (NPO) in the twentieth century wintertime, and further investigates their responses to greenhouse warming in the twenty-first century. Analysis reveals that while the majority (80%) of models reasonably simulate either the geographical distribution or the amplitude of PNA/NPO pattern, only half of models can well capture both features in space. As for the temporal features, variabilities of PNA and NPO in most models are biased toward higher amplitude. Additionally, most models simulate the interannual variabilities of PNA and NPO, qualitatively consistent with the observation, whereas models generally lack the capability to reproduce the decadal (20-25 years) variability of PNA. As the climate warms under the strongest future warming scenario, the PNA intensity is found to be strengthened, whereas there is no consensus on the direction of change in the NPO intensity among models. The intensification of positive PNA is primarily manifested in the large deepening of the North Pacific trough, which is robust as it is 2.3 times the unforced internal variability. By focusing on the tropical Pacific Ocean, we find that the multidecadal evolution of the North Pacific trough intensity (dominating the PNA intensity evolution) is closely related to that of the analogous trough in the PNA-like teleconnection forced by sea surface temperature anomalies (SSTa) in the tropical central Pacific (CP) rather than the tropical eastern Pacific (EP). Such association is also found to act under greenhouse warming: that is, the strengthening of the PNA-like teleconnection induced by the CP SSTa rather than the EP SSTa is a driving force for the intensification of PNA. This is in part owing to the robust enhancement of the tropical precipitation response to the CP SST variation. Indeed, further inspection suggests that models with stronger intensification of the CP SST variability and its related tropical precipitation response tend to have larger deepening magnitude of the North Pacific trough associated with the PNA variability.

Pacific-North American teleconnection and North Pacific Oscillation: historical simulation and future projection in CMIP5 models

NASA Astrophysics Data System (ADS)

Chen, Zheng; Gan, Bolan; Wu, Lixin; Jia, Fan

2018-06-01

Based on reanalysis datasets and as many as 35 CMIP5 models, this study evaluates the capability of climate models to simulate the spatiotemporal features of Pacific-North American teleconnection (PNA) and North Pacific Oscillation (NPO) in the twentieth century wintertime, and further investigates their responses to greenhouse warming in the twenty-first century. Analysis reveals that while the majority (80%) of models reasonably simulate either the geographical distribution or the amplitude of PNA/NPO pattern, only half of models can well capture both features in space. As for the temporal features, variabilities of PNA and NPO in most models are biased toward higher amplitude. Additionally, most models simulate the interannual variabilities of PNA and NPO, qualitatively consistent with the observation, whereas models generally lack the capability to reproduce the decadal (20-25 years) variability of PNA. As the climate warms under the strongest future warming scenario, the PNA intensity is found to be strengthened, whereas there is no consensus on the direction of change in the NPO intensity among models. The intensification of positive PNA is primarily manifested in the large deepening of the North Pacific trough, which is robust as it is 2.3 times the unforced internal variability. By focusing on the tropical Pacific Ocean, we find that the multidecadal evolution of the North Pacific trough intensity (dominating the PNA intensity evolution) is closely related to that of the analogous trough in the PNA-like teleconnection forced by sea surface temperature anomalies (SSTa) in the tropical central Pacific (CP) rather than the tropical eastern Pacific (EP). Such association is also found to act under greenhouse warming: that is, the strengthening of the PNA-like teleconnection induced by the CP SSTa rather than the EP SSTa is a driving force for the intensification of PNA. This is in part owing to the robust enhancement of the tropical precipitation response to the CP SST variation. Indeed, further inspection suggests that models with stronger intensification of the CP SST variability and its related tropical precipitation response tend to have larger deepening magnitude of the North Pacific trough associated with the PNA variability.

PNA-PEG modified silicon platforms as functional bio-interfaces for applications in DNA microarrays and biosensors.

PubMed

Cattani-Scholz, Anna; Pedone, Daniel; Blobner, Florian; Abstreiter, Gerhard; Schwartz, Jeffrey; Tornow, Marc; Andruzzi, Luisa

2009-03-09

The synthesis and characterization of two types of silicon-based biofunctional interfaces are reported; each interface bonds a dense layer of poly(ethylene glycol) (PEG(n)) and peptide nucleic acid (PNA) probes. Phosphonate self-assembled monolayers were derivatized with PNA using a maleimido-terminated PEG(45). Similarly, siloxane monolayers were functionalized with PNA using a maleimido-terminated PEG(45) spacer and were subsequently modified with a shorter methoxy-terminated PEG(12) ("back-filling"). The long PEG(45) spacer was used to distance the PNA probe from the surface and to minimize undesirable nonspecific adsorption of DNA analyte. The short PEG(12) "back-filler" was used to provide additional passivation of the surface against nonspecific DNA adsorption. X-ray photoelectron spectroscopic (XPS) analysis near the C 1s and N 1s ionization edges was done to characterize chemical groups formed in the near-surface region, which confirmed binding of PEG and PNA to the phosphonate and silane films. XPS also indicated that additional PEG chains were tethered to the surface during the back-filling process. Fluorescence hybridization experiments were carried out with complementary and noncDNA strands; both phosphonate and siloxane biofunctional surfaces were effective for hybridization of cDNA strands and significantly reduced nonspecific adsorption of the analyte. Spatial patterns were prepared by polydimethylsiloxane (PDMS) micromolding on the PNA-functionalized surfaces; selective hybridization of fluorescently labeled DNA was shown at the PNA functionalized regions, and physisorption at the probe-less PEG-functionalized regions was dramatically reduced. These results show that PNA-PEG derivatized phosphonate monolayers hold promise for the smooth integration of device surface chemistry with semiconductor technology for the fabrication of DNA biosensors. In addition, our results confirm that PNA-PEG derivatized self-assembled carboxyalkylsiloxane films are promising substrates for DNA microarray applications.

Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

PubMed Central

Topham, Christopher M.; Smith, Jeremy C.

2007-01-01

Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNA·DNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs. PMID:17071666

Pacific/North American teleconnection controls on precipitation isotope ratios across the contiguous United States

NASA Astrophysics Data System (ADS)

Liu, Zhongfang; Kennedy, Casey D.; Bowen, Gabriel J.

2011-10-01

Large-scale climate teleconnections such as the Pacific/North American (PNA) pattern strongly influence atmospheric processes and continental climate. Here we show that precipitation δ 18O values in the contiguous United States are correlated with an index of the PNA pattern. The δ 18O/PNA relationship varies across the study region and exhibits two prominent modes, with positive correlation in the western USA and negative correlation in the east. This spatial pattern appears not to reflect variation in local climate variables, but rather primarily reflects differences in atmospheric circulation and moisture sources associated with PNA. Our results suggest that strong antiphase variation in paired paleo-water δ 18O proxy records from regions characterized by the two modes of δ 18O/PNA correlation, especially in the Midwest and southwestern USA, may provide a robust basis for reconstruction of past variation in the PNA pattern.

Photodissociation of pernitric acid (HO2NO2) at 248 nm

NASA Technical Reports Server (NTRS)

Macleod, Helene; Smith, Gregory P.; Golden, David M.

1989-01-01

The photodissociation of pernitric acid (PNA) was studied at 248 nm. The quantum yield for production of OH radicals is 34 + or - 16 percent. The yield of OH from PNA was measured relative to that of H2O2. The translational and rotational energy content of the OH photofragment from PNA was characterized. A fluorescent emission was also observed and characterized. It is attributed to electronically excited NO2 produced in the PNA photodissociation. A maximum yield of 30 percent for NO2 production was determined. The intensity of this emission, and a mass spectrometric peak at m/e = 33, were found to be useful means of characterizing the purity of the PNA sample.

Can solar cycle modulate the ENSO effect on the Pacific/North American pattern?

NASA Astrophysics Data System (ADS)

Li, Delin; Xiao, Ziniu

2018-01-01

The ENSO effect on the Pacific/North American pattern (PNA) is well-known robust. Recent studies from observations and model simulations have reported that some important atmospheric circulation systems of extratropics are markedly modulated by the 11-year solar cycle. But less effort has been devoted to revealing the solar influence on the PNA. We thus hypothesize that the instability and uncertainty in the relationship between solar activity and PNA could be due to the ENSO impacts. In this study, solar cycle modulation of the ENSO effect on the PNA has been statistically examined by the observations from NOAA and NCEP/NCAR for the period of 1950-2014. Results indicate that during the high solar activity (HS) years, the PNA has stronger relevance to the ENSO, and the response of tropospheric geopotential height to ENSO variability is broadly similar to the typical positive PNA pattern. However, in the case of low solar activity (LS) years, the correlation between ENSO and PNA decreases relatively and the response has some resemblance to the negative phase of Arctic Oscillation (AO). Also, we find the impacts of solar activity on the middle troposphere are asymmetric during the different solar cycle phases, and the weak PNA-like response to solar activity only presents in the HS years. Closer inspection suggests that the higher solar activity has a much more remarkable modulation on the PNA-like response to the warm ENSO (WE) than that to the cold ENSO (CE), particularly over the Northeast Pacific region. The possible cause of the different responses might be the solar influence on the subtropical westerlies of upper troposphere. When the sea surface temperature (SST) of east-central tropical Pacific is anomalously warm, the upper tropospheric westerlies are significantly modulated by the higher solar activity, resulting in the acceleration and eastward shift of the North Pacific subtropical jet, which favors the propagation of WE signal from the tropical Pacific to the North Pacific, and consequently leading to the development of positive PNA-like pattern during the WE phase. Thus, it seems that the solar cycle can significantly modulate the WE effect on the PNA under the HS background.

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance ▿ †

PubMed Central

Jang, Hyunjung; Kim, Jihyun; Choi, Jae-jin; Son, Yeojin; Park, Heekyung

2010-01-01

The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 102 copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 104 copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms. PMID:20573874

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations

PubMed Central

1992-01-01

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222. PMID:1431803

Probing Lewis Acid-Base Interactions with Born-Oppenheimer Molecular Dynamics: The Electronic Absorption Spectrum of p-Nitroaniline in Supercritical CO2.

PubMed

Cabral, Benedito J Costa; Rivelino, Roberto; Coutinho, Kaline; Canuto, Sylvio

2015-07-02

The structure and dynamics of p-nitroaniline (PNA) in supercritical CO2 (scCO2) at T = 315 K and ρ = 0.81 g cm(-3) are investigated by carrying out Born-Oppenheimer molecular dynamics, and the electronic absorption spectrum in scCO2 is determined by time dependent density functional theory. The structure of the PNA-scCO2 solution illustrates the role played by Lewis acid-base (LA-LB) interactions. In comparison with isolated PNA, the ν(N-O) symmetric and asymmetric stretching modes of PNA in scCO2 are red-shifted by -17 and -29 cm(-1), respectively. The maximum of the charge transfer (CT) absorption band of PNA in scSCO2 is at 3.9 eV, and the predicted red-shift of the π → π* electronic transition relative to the isolated gas-phase PNA molecule reproduces the experimental value of -0.35 eV. An analysis of the relationship between geometry distortions and excitation energies of PNA in scCO2 shows that the π → π* CT transition is very sensitive to changes of the N-O bond distance, strongly indicating a correlation between vibrational and electronic solvatochromism driven by LA-LB interactions. Despite the importance of LA-LB interactions to explain the solvation of PNA in scCO2, the red-shift of the CT band is mainly determined by electrostatic interactions.

Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

PubMed Central

Upert, Gregory; Di Giorgio, Audrey; Upadhyay, Alok; Manvar, Dinesh; Pandey, Nootan; Pandey, Virendra N.; Patino, Nadia

2012-01-01

Human immunodeficiency virus-1 (HIV-1) replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR), to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP) cation-based vectors that efficiently deliver nucleotide analogs (PNAs) into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins. PMID:23029603

Analysis of the present and future winter Pacific-North American teleconnection in the ECHAM5 global and RegCM3 regional climate models

USGS Publications Warehouse

Allan, Andrea M.; Hostetler, Steven W.; Alder, Jay R.

2014-01-01

We use the NCEP/NCAR Reanalysis (NCEP) and the MPI/ECHAM5 general circulation model to drive the RegCM3 regional climate model to assess the ability of the models to reproduce the spatiotemporal aspects of the Pacific-North American teleconnection (PNA) pattern. Composite anomalies of the NCEP-driven RegCM3 simulations for 1982–2000 indicate that the regional model is capable of accurately simulating the key features (500-hPa heights, surface temperature, and precipitation) of the positive and negative phases of the PNA with little loss of information in the downscaling process. The basic structure of the PNA is captured in both the ECHAM5 global and ECHAM5-driven RegCM3 simulations. The 1950–2000 ECHAM5 simulation displays similar temporal and spatial variability in the PNA index as that of NCEP; however, the magnitudes of the positive and negative phases are weaker than those of NCEP. The RegCM3 simulations clearly differentiate the climatology and associated anomalies of snow water equivalent and soil moisture of the positive and negative PNA phases. In the RegCM3 simulations of the future (2050–2100), changes in the location and extent of the Aleutian low and the continental high over North America alter the dominant flow patterns associated with positive and negative PNA modes. The future projections display a shift in the patterns of the relationship between the PNA and surface climate variables, which suggest the potential for changes in the PNA-related surface hydrology of North America.

Identification of canine T lymphocytes by membrane receptor to peanut agglutinin: T-lymphocyte identification in dogs with lupus-like syndrome.

PubMed

Rigal, D; Bendali-Ahcène, S; Monier, J C; Mohana, K; Fournel, C

1983-09-01

Canine T lymphocytes were detected, using fluorescent peanut agglutinin (PNA) as a marker. Using a fluorescent technique and cytofluorometry, 70 +/- 11% and 72.4%, respectively, of peripheral blood lymphocytes were bound to PNA. Of thymocytes, 97 +/- 4.5% were detected by fluorescent PNA, but less than 1% were detected for lymphocytes from bone marrow. The T-lymphocyte depletion and enrichment indicated that PNA was bound to lymphocytes recognized by anti-T-lymphocyte heterologous serum. A T-lymphocyte deficiency was detected among 8 dogs with a lupus-like syndrome.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

PubMed

Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

2015-02-01

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

Prenatal androgenization of female mice programs an increase in firing activity of gonadotropin-releasing hormone (GnRH) neurons that is reversed by metformin treatment in adulthood.

PubMed

Roland, Alison V; Moenter, Suzanne M

2011-02-01

Prenatal androgenization (PNA) of female mice with dihydrotestosterone programs reproductive dysfunction in adulthood, characterized by elevated luteinizing hormone levels, irregular estrous cycles, and central abnormalities. Here, we evaluated activity of GnRH neurons from PNA mice and the effects of in vivo treatment with metformin, an activator of AMP-activated protein kinase (AMPK) that is commonly used to treat the fertility disorder polycystic ovary syndrome. Estrous cycles were monitored in PNA and control mice before and after metformin administration. Before metformin, cycles were longer in PNA mice and percent time in estrus lower; metformin normalized cycles in PNA mice. Extracellular recordings were used to monitor GnRH neuron firing activity in brain slices from diestrous mice. Firing rate was higher and quiescence lower in GnRH neurons from PNA mice, demonstrating increased GnRH neuron activity. Metformin treatment of PNA mice restored firing activity and LH to control levels. To assess whether AMPK activation contributed to the metformin-induced reduction in GnRH neuron activity, the AMPK antagonist compound C was acutely applied to cells. Compound C stimulated cells from metformin-treated, but not untreated, mice, suggesting that AMPK was activated in GnRH neurons, or afferent neurons, in the former group. GnRH neurons from metformin-treated mice also showed a reduced inhibitory response to low glucose. These studies indicate that PNA causes enhanced firing activity of GnRH neurons and elevated LH that are reversible by metformin, raising the possibility that central AMPK activation by metformin may play a role in its restoration of reproductive cycles in polycystic ovary syndrome.

Prepubertal Development of Gonadotropin-Releasing Hormone Neuron Activity Is Altered by Sex, Age, and Prenatal Androgen Exposure.

PubMed

Dulka, Eden A; Moenter, Suzanne M

2017-11-01

Gonadotropin-releasing hormone (GnRH) neurons regulate reproduction though pulsatile hormone release. Disruption of GnRH release as measured via luteinizing hormone (LH) pulses occurs in polycystic ovary syndrome (PCOS), and in young hyperandrogenemic girls. In adult prenatally androgenized (PNA) mice, which exhibit many aspects of PCOS, increased LH is associated with increased GnRH neuron action potential firing. How GnRH neuron activity develops over the prepubertal period and whether this is altered by sex or prenatal androgen treatment are unknown. We hypothesized GnRH neurons are active before puberty and that this activity is sexually differentiated and altered by PNA. Dams were injected with dihydrotestosterone (DHT) on days 16 to 18 post copulation to generate PNA mice. Action potential firing of GFP-identified GnRH neurons in brain slices from 1-, 2-, 3-, and 4-week-old and adult mice was monitored. GnRH neurons were active at all ages tested. In control females, activity increased with age through 3 weeks, then decreased to adult levels. In contrast, activity did not change in PNA females and was reduced at 3 weeks. Activity was higher in control females than males from 2 to 3 weeks. PNA did not affect GnRH neuron firing rate in males at any age. Short-term action potential patterns were also affected by age and PNA treatment. GnRH neurons are thus typically more active during the prepubertal period than adulthood, and PNA reduces prepubertal activity in females. Prepubertal activity may play a role in establishing sexually differentiated neuronal networks upstream of GnRH neurons; androgen-induced changes during this time may contribute to the adult PNA, and possibly PCOS, phenotype. Copyright © 2017 Endocrine Society.

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure.

PubMed

Gade, Chandrasekhar Reddy; Sharma, Nagendra K

2017-12-15

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC 5 ) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

A triphenylamine-functionalized luminescent sensor for efficient p-nitroaniline detection.

PubMed

Ji, Ning-Ning; Shi, Zhi-Qiang; Hu, Hai-Liang; Zheng, He-Gen

2018-05-14

The combination of π-conjugated fluorophores within a hybrid system gives rise to a triphenylamine-functionalized material [Zn(bpba)(NO3)] (1) (Hbpba = 4-(bis(4-(pyridin-4-yl)phenyl)amino)benzoic acid). Compound 1 features a 2D + 2D → 2D parallel polycatenation structure with 63-hcb net. Photophysical studies revealed that the title phase showed superior sensitivity towards p-nitroaniline (p-NA) with a low detection limit (down to ∼0.10 ppm). Specifically, following a new detection route, vapor-sensing experiments using a saturated ethanol solution of nitroaromatic isomers have been established for the first time. Highly sensitive and selective detection of p-NA by the proposed material with a rapid response time (t = 30 s, QE > 90.0%) as compared to that via the control isomers (t = 60s, QE < 6.0%) demonstrates an attractive feasible route and a promising luminescent sensor for nitroaromatic detection.

Jacalin and peanut agglutinin (PNA) bindings in the taste bud cells of the rat: new reliable markers for type IV cells of the rat taste buds.

PubMed

Taniguchi, Ryo; Shi, Lei; Fujii, Masae; Ueda, Katsura; Honma, Shiho; Wakisaka, Satoshi

2005-12-01

Lectin histochemistry of Jacalin (Artocarpus integrifolia) and peanut agglutinin (PNA), specific lectins for galactosyl (beta-1, 3) N-acetylgalactosamine (galactosyl (beta-1, 3) GalNAc), was applied to the gustatory epithelium of the adult rat. In the ordinary lingual epithelium, Jacalin and PNA labeled the cell membrane from the basal to granular cell layer. They also bound membranes of rounded-cells at the basal portion of taste buds, but the number of PNA labeled cells was smaller than that of Jacalin labeled cells. There was no apparent difference in the binding patterns of Jacalin and PNA among the taste buds of the lingual papillae and those of the palatal epithelium. Occasionally, a few spindle-shaped cells were labeled with Jacalin, but not with PNA. Double labeling of Jacalin and alpha-gustducin, a specific marker for type II cells, revealed that Jacalin-labeled spindle-shaped taste cells were immunonegative for alpha-gustducin. Spindle-shaped cells expressing protein gene product 9.5 (PGP 9.5) immunoreactivity lacked Jacalin labeling. During the development of taste buds in circumvallate papillae, the binding pattern of Jacalin became almost identical from postnatal day 5. The present results indicate that rounded cells at the basal portion of the taste buds cells (type IV cells) bind to Jacalin and PNA, and these lectins are specific markers for type IV cells of the rat taste cells.

Kt/V and nPNA in pediatric peritoneal dialysis: a clinical or a mathematical association?

PubMed

Cano, F; Azocar, M; Cavada, G; Delucchi, A; Marin, V; Rodriguez, E

2006-01-01

The relationship between dialysis dose and nutrition is a field of particular interest in chronic pediatric dialysis (PD), and a positive correlation between ureaKt/V and nPNA has been published, suggesting a better nutritional status is associated with higher dialysis doses. However, this relationship has also been criticized as being the result of a mathematical coupling resulting from the same variables. The objective of the study was to establish the relationship between dialysis dose (Kt/V) and nutritional variables: daily protein intake (DPI), protein catabolic rate (PCR), protein equivalent of total nitrogen appearance (PNA) and nitrogen balance (NB) in dialyzed children. A cohort, prospective, observational study was carried out, for which 223 biochemical measurements were performed in 20 patients, ages 1 month to 14.3 years old (13 males), under PD for a 12-month period of follow-up. Monthly residual and total ureaKt/V, DPI, PCR, nPNA and NB were calculated, and the correlation between Kt/V and the nutritional parameters was evaluated. The Borah equation was used to calculate the nPNA. The data are reported as the mean plus or minus the standard error. All statistical comparisons were done with a paired t test, and two-way ANOVA for repeated measures was used to calculate correlations. A P <0.05 was considered significant. Mean total and residual Kt/V was 3.4+/-1.3 and 1.69+/-1.27, respectively; nPNA and PCR were 1.38+/-0.44 and 1.39+/-0.43 g/kg/day, daily protein intake (DPI) was 3.25+/-1.27 g/kg/day, and NB showed a value of 1.86+/-1.25 g/kg/day. A significant positive correlation was found between Kt/V and DPI, PCR, DPC and nPNA (all values P <0.0001), but no correlation was found between total and residual Kt/V vs. nitrogen balance ( P:ns). Total Kt/V showed a significant positive correlation with nPNA, but it did not show any correlation with nitrogen balance, suggesting that the relationship with nPNA is the result of a mathematical association calculated from the same variables.

Organophosphonate-based PNA-functionalization of silicon nanowires for label-free DNA detection.

PubMed

Cattani-Scholz, Anna; Pedone, Daniel; Dubey, Manish; Neppl, Stefan; Nickel, Bert; Feulner, Peter; Schwartz, Jeffrey; Abstreiter, Gerhard; Tornow, Marc

2008-08-01

We investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicate the formation of a dense monolayer on the native silicon oxide that has excellent passivation properties. The monolayer was biofunctionalized with 12 mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-maleimidopropionic-acid-N-hydroxysuccinimidester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with DNA was determined by fluorescence measurements. Finally, the PNA functionalization protocol was translated to 2 microm long, 100 nm wide Si nanowire field effect devices, which were successfully used for label-free DNA/PNA hybridization detection.

Fluorescence Detection of KRAS2 mRNA Hybridization in Lung Cancer Cells with PNA-Peptides Containing an Internal Thiazole Orange

PubMed Central

2015-01-01

We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5′ end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5′ terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5–6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA. PMID:25180641

A First-Principles Approach to the Dynamics and Electronic Properties of p-Nitroaniline in Water.

PubMed

Cabral, Benedito J Costa; Coutinho, Kaline; Canuto, Sylvio

2016-06-09

Born-Oppenheimer molecular dynamics of p-nitroaniline (PNA) in water was carried out and the electronic structure was investigated by time-dependent density functional theory. Hydrogen bonding involving the PNA nitro and amine groups and the water molecules leads to an ∼160 cm(-1) red shift of the ν(N-O) and ν(N-H) stretching frequencies relative to the gas phase species. Our estimate for the peak position of the charge transfer (CT) band in the absorption spectrum of PNA in water (∼3.5 eV) is in good agreement with experimental data (3.3 eV). We have investigated the specific role played by local hydrogen bonding and electrostatic interactions on the electronic absorption spectrum. It is shown that although electrostatic interactions play a major role for explaining the structure of the PNA CT band in water, the theoretical prediction of the observed red shift is improved by the explicit consideration of local hydrogen bonding of PNA to water. For isolated PNA, we predict that the dipole moment of the second excited state (S2) is 9.6 D greater than ground state (S0) dipole, which is in good agreement with experimental information (8.2-9.3 D). Calculation of charge transfer indexes for the two first excitations of PNA in water indicates that despite the feature that a small fraction of S1 states (<5%) may exhibit some CT character, CT states in solution are mainly associated with S2 ← S0 transitions.

Linear and nonlinear winter atmospheric responses to extreme phases of low frequency Pacific sea surface temperature variability

NASA Astrophysics Data System (ADS)

Cao, Dandan; Wu, Qigang; Hu, Aixue; Yao, Yonghong; Liu, Shizuo; Schroeder, Steven R.; Yang, Fucheng

2018-02-01

This study examines Northern Hemisphere winter (DJFM) atmospheric responses to opposite strong phases of interdecadal (low frequency, LF) Pacific sea surface temperature (SST) forcing, which resembles El Niño-Southern Oscillation (ENSO) on a longer time scale, in observations and GFDL and CAM4 model simulations. Over the Pacific-North America (PNA) sector, linear observed responses of 500-hPa height (Z500) anomalies resemble the PNA teleconnection pattern, but show a PNA-like nonlinear response because of a westward Z500 shift in the negative (LF-) relative to the positive LF (LF+) phase. Significant extratropical linear responses include a North Atlantic Oscillation (NAO)-like Z500 anomaly, a dipole-like Z500 anomaly over northern Eurasia associated with warming over mid-high latitude Eurasia, and a Southern Annular anomaly pattern associated with warming in southern land areas. Significant nonlinear Z500 responses also include a NAO-like anomaly pattern. Models forced by LF+ and LF- SST anomalies reproduce many aspects of observed linear and nonlinear responses over the Pacific-North America sector, and linear responses over southern land, but not in the North Atlantic-European sector and Eurasia. Both models simulate PNA-like linear responses in the North Pacific-North America region similar to observed, but show larger PNA-like LF+ responses, resulting in a PNA nonlinear response. The nonlinear PNA responses result from both nonlinear western tropical Pacific rainfall changes and extratropical transient eddy feedbacks. With LF tropical Pacific forcing only (LFTP+ and LFTP-, climatological SST elsewhere), CAM4 simulates a significant NAO response to LFTP-, including a linear negative and nonlinear positive NAO response.

Establishing a reference range for triiodothyronine levels in preterm infants.

PubMed

Oh, Ki Won; Koo, Mi Sung; Park, Hye Won; Chung, Mi Lim; Kim, Min-ho; Lim, Gina

2014-10-01

Thyroid dysfunction affects clinical complications in preterm infants and older children. However, thyroid hormone replacement in preterm infants has no proven benefits, possibly owing to the lack of an appropriate reference range for thyroid hormone levels. We aimed to establish a reference range for triiodothyronine (T3) levels at 1-month postnatal age (PNA) in preterm infants. This retrospective study included preterm infants born at a tertiary referral neonatal center at gestational age (GA)<35 weeks with no apparent thyroid dysfunction, for 6 consecutive years, with follow-up from PNA 2 weeks to 16 weeks. Using thyroid function tests (TFT), the relationships between T3 levels and thyrotropin (TSH) and free thyroxine (fT4) levels, birth weight, GA, postmenstrual age (PMA), and PNA were examined. The conversion trend for fT4 to T3 was analyzed using the T3/fT4 ratio. Overall, 464 TFTs from 266 infants were analyzed, after excluding 65 infants with thyroid dysfunction. T3 levels increased with fT4 levels, birth weight, GA, PMA, and PNA but not with TSH levels. The T3/fT4 ratio also increased with GA, PNA, and PMA. The average T3 level at 1 month PNA was 72.56 ± 27.83 ng/dL, with significant stratifications by GA. Relatively low T3 and fT4 levels in preterm infants were considered normal, with T3 levels and conversion trends increasing with GA, PMA, and PNA. Further studies are required to confirm the role of the present reference range in thyroid hormone replacement therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

PubMed Central

Bergquist, Helen; Rocha, Cristina S. J.; Álvarez-Asencio, Rubén; Nguyen, Chi-Hung; Rutland, Mark. W.; Smith, C. I. Edvard; Good, Liam; Nielsen, Peter E.; Zain, Rula

2016-01-01

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression. PMID:27846236

Design of Organic Nonlinear Optical Materials

DTIC Science & Technology

1990-06-01

Cocrystals 6) "Dinitrourea Polymorphs with SHG Properties" 7) "Symmetry Bias in H-Bonded Crystals" Part II a-c. Attached d. Cocrystallization can be used to...environment of PNA. PNA Cocrystals and Their Crystal Structures I PNA cocrystallizes with the following guest molecules: O-N - C N NO2 NO2 0 PhO=P-Ph...Refereed Journals (and not yet publishe) "Hydrogen-Bond Directed Cocrystallization and Molecular Recognition Properties of Diarylurea," M. C. Etter, Zofia

Gold Nanoparticles Used as Protein Scavengers Enhance Surface Plasmon Resonance Signal

PubMed Central

Ferreira de Macedo, Erenildo; Ducatti Formaggio, Daniela Maria; Salles Santos, Nivia; Batista Tada, Dayane

2017-01-01

Although several researchers had reported on methodologies for surface plasmon resonance (SPR) signal amplification based on the use of nanoparticles (NPs), the majority addressed the sandwich technique and low protein concentration. In this work, a different approach for SPR signal enhancement based on the use of gold NPs was evaluated. The method was used in the detection of two lectins, peanut agglutinin (PNA) and concanavalin A (ConA). Gold NPs were functionalized with antibodies anti-PNA and anti-ConA, and these NPs were used as protein scavengers in a solution. After being incubated with solutions of PNA or ConA, the gold NPs coupled with the collected lectins were injected on the sensor containing the immobilized antibodies. The signal amplification provided by this method was compared to the signal amplification provided by the direct coupling of PNA and ConA to gold NPs. Furthermore, both methods, direct coupling and gold NPs as protein scavengers, were compared to the direct detection of PNA and ConA in solution. Compared to the analysis of free protein, the direct coupling of PNA and ConA to gold NPs resulted in a signal amplification of 10–40-fold and a 13-fold decrease of the limit of detection (LOD), whereas the use of gold NPs as protein scavengers resulted in an SPR signal 40–50-times higher and an LOD 64-times lower. PMID:29186024

Doxorubicin-induced co-assembling nanomedicines with temperature-sensitive acidic polymer and their in-situ-forming hydrogels for intratumoral administration.

PubMed

Wan, Jiangshan; Geng, Shinan; Zhao, Hao; Peng, Xiaole; Zhou, Qing; Li, Han; He, Ming; Zhao, Yanbing; Yang, Xiangliang; Xu, Huibi

2016-08-10

Doxorubicin (DOX)-induced co-assembling nanomedicines (D-PNAx) with temperature-sensitive PNAx triblock polymers have been developed for regional chemotherapy against liver cancer via intratumoral administration in the present work. Owing to the formation of insoluble DOX carboxylate, D-PNAx nanomedicines showed high drug-loading and entrapment efficacy via a simple mixing of doxorubicin hydrochloride and PNAx polymers. The sustained releasing profile of D-PNA100 nanomedicines indicated that only 9.4% of DOX was released within 1day, and 60% was released during 10days. Based on DOX-induced co-assembling behavior and their temperature sensitive in-situ-forming hydrogels, D-PNA100 nanomedicines showed excellent antitumor activity against H22 tumor using intratumoral administration. In contrast to that by free DOX solution (1.13±0.04 times at 9days) and blank PNA100 (2.11±0.34 times), the tumor volume treated by D-PNA100 had been falling to only 0.77±0.13 times of original tumor volume throughout the experimental period. In vivo biodistribution of DOX indicated that D-PNA100 nanomedicines exhibited much stronger DOX retention in tumor tissues than free DOX solution via intratumoral injection. D-PNA100 nanomedicines were hopeful to be developed as new temperature sensitive in-situ-forming hydrogels via i.t. injection for regional chemotherapy. Copyright © 2016. Published by Elsevier B.V.

Promotion of double-duplex invasion of peptide nucleic acids through conjugation with nuclear localization signal peptide.

PubMed

Aiba, Yuichiro; Honda, Yuta; Komiyama, Makoto

2015-03-02

Pseudo-complementary peptide nucleic acid (pcPNA), as one of the most widely used synthetic DNA analogues, invades double-stranded DNA according to Watson-Crick rules to form invasion complexes. This unique mode of DNA recognition induces structural changes at the invasion site and can be used for a range of applications. In this paper, pcPNA is conjugated with a nuclear localization signal (NLS) peptide, and its invading activity is notably promoted both thermodynamically and kinetically. Thus, the double-duplex invasion complex is formed promptly at low pcPNA concentrations under high salt conditions, where the invasion otherwise never occurs. Furthermore, NLS-modified pcPNA is successfully employed for site-selective DNA scission, and the targeted DNA is selectively cleaved under conditions that are not conducive for DNA cutters using unmodified pcPNAs. This strategy of pcPNA modification is expected to be advantageous and promising for a range of in vitro and in vivo applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Vitro Assessment of a Peptide Nucleic Acid (PNA) - Peptide Conjugate Labeled With an Auger-Emitting Radionuclide for Prostate Cell Killing

DTIC Science & Technology

2005-02-01

agents that are designed to improve the survival rates for prostate cancer patients with metastatic disease. Survival rates for prostate cancer drop from...radioiodine is physically located in close proximity to the phosphate backbone of the oligonucleotides, and decay of the radionuclide can cause extensive...reaction, non- radioactive sodium iodide was reacted with chloramine -T to give the iodinated PNA 23. That material was identical to PNA 23 prepared from

Front-End Processing of Cell Lysates for Enhanced Chip-Based Detection

DTIC Science & Technology

2006-07-28

manipulation used in lab-on-a-chip devices. A small unknown sample is first mixed with the PNA surfactants (“PNAA”) to tag the DNA targets, and then the...unknown sample is first mixed with the PNA surfactants (hereafter referred to as “PNA amphiphiles” or “PNAA”) to tag the DNA targets, and then the...prolate ellipsoid, and mixed PNAA/SDS micelles form spherical micelles. On addition of complementary DNA, the PNAA/DNA duplexes do not participate in

Lipid peroxidation in sarcoplasmic reticulum membranes: effect on functional and biophysical properties.

PubMed

Dinis, T C; Almeida, L M; Madeira, V M

1993-03-01

The fluorescent polyunsaturated parinaric acid (PnA) incorporated in sarcoplasmic reticulum membranes (SR) was used to probe the initial stages of membrane lipid peroxidation. The experimental set up of the PnA assay was investigated by means of several peroxidation initiators to ascertain peroxidation conditions. This assay in SR is particularly useful to evaluate the membrane susceptibility to peroxidation and to ascertain suitable conditions (concentration of initiators and cofactors) to challenge peroxidation in each preparation under study. On the basis of the PnA assay, Fe2+/ascorbate was selected among the different initiator systems to assess the effect of lipid peroxidation upon biochemical and biophysical parameters of SR membranes. Under mildly controlled conditions at 25 degrees C, the lipid degradative process, as detected by fatty acid analysis, decreases the Ca2+ uptake (up to about 50% of control) and reduces the Ca2+ pump efficiency (Ca2+/ATP ratio) up to about 58% of control, without inactivation the ATPase enzyme turnover. The effect of lipid peroxidation on the SR bilayer organization is dependent either on the extent of lipid peroxidation or on the depth of the bilayer as probed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and by intramolecular excimerization of 1,3-di(1-pyrenyl)propane. It is concluded that the effect of mild lipid peroxidation on Ca2+ pump activity is partially exerted through the alteration of physical properties in the lipid phase or lipid-protein interfaces.

[Evaluation of PNA-FISH method for direct identification of Candida species in blood culture samples and its potential impact on guidance of antifungal therapy].

PubMed

Doğan, Özlem; İnkaya, Ahmet Çağkan; Gülmez, Dolunay; Uzun, Ömrüm; Akova, Murat; Arıkan Akdağlı, Sevtap

2016-10-01

Early antifungal therapy has a major influence on survival in candidemia. Rapid identification of the species has importance for the treatment, prediction of the species-specific primary resistance and variable antifungal susceptibility. Recently, molecular-based methods attempt to reduce the time between the positive signal of a blood culture and identification of the fungus. PNA-FISH (Peptide nucleic acid fluorescence in situ hybridization) assay distinguishes a number of frequently isolated Candida species in groups following the growth in blood culture. The aim of this study was to investigate the correlation of the species identified by PNA-FISH with conventional identification methods in yeast positive blood cultures and its influence on the selection of antifungal therapy. Specimens of adult patients diagnosed as yeast with Gram stain in signal-positive blood cultures between August to December 2013, were included in the study. The strains were concomitantly cultivated by subculturing from the blood culture bottles onto solid media and identified by conventional methods (germ tube test, ID32C and morphology on cornmeal Tween 80 agar). Rapid species identification was performed by Yeast Traffic Light PNA-FISH, which generates green flourescence for Candida albicans and Candida parapsilosis, yellow for Candida tropicalis, and red for Candida krusei and Candida glabrata. C.tropicalis was identified as a single species whereas the others were identified in pairs. The time points when the yeast positive blood culture bottle was received by the mycology laboratory and reporting of the species identification results by PNA-FISH and the conventional methods were recorded. Seven C.albicans, six C.glabrata, three C.parapsilosis, one C.tropicalis, one C.krusei, one Cryptococcus neoformans, one Saprochaete capitata (Blastoschizomyces capitatus), one C.albicans and Candida dubliniensis, one C.krusei and C.dubliniensis, and one C.glabrata and C.parapsilosis were identified by conventional methods in 23 specimens. Results of PNA-FISH and conventional methods were in full agreement in 19 of the 23 specimens (82.6%). Two specimens were negative by PNA-FISH and yielded S.capitata and C.neoformans which were not included in the test panel. In three specimens that were infected with multiple species, PNA-FISH detected only one of the species. On the other hand and in one specimen, PNA-FISH detected a second species (C.glabrata or C.krusei) that could not be isolated and identified conventionally. Species identification were obtained 72 hours (mean) earlier with PNA-FISH. PNA-FISH provided accurate species identification that were consistent with conventional methods. However and expectedly, it failed to detect species that were not included in the test panel. During the study period, 13 of the 23 patients have passed away. Apart from six patients died prior to blood culture positivity and the one that could not get any antifungal therapy during hospital stay, 16 patients received antifungal treatment. Of sixteen patients who received antifungal therapy, initial antifungal treatment was fluconazole for five and echinocandin for 10 patients. Fluconazole and amphotericin B combination was preferred for one patient. In this study, PNA-FISH result had an influence on the modification of the antifungal treatment of only for one patient in accordance with the clinical findings. We conclude that the utility of PNA-FISH method appeared to be limited in our center since the assay cannot differentiate C.albicans and C.parapsilosis, the two commonly isolated species among our candidemia isolates. However, advantages of the assay might be more pronounced for the centers where C.glabrata is a relatively more frequent species.

Enantiomeric Cross-Inhibition in the Synthesis of Oligonucleotides on a Nonchiral Template

NASA Technical Reports Server (NTRS)

Schmidt, Jurgen G.; Nielsen, Peter E.; Orgel, Leslie E.

1997-01-01

Prebiotic syntheses of chiral monomers always yield racemic mixtures. Living systems, however, utilize L-amino acids and D-nucleotides in their biopolymers. The generation of optical asymmetry by selection and amplification in an autocatalytic process is, therefore, an important element in many theories of the origin of life. Replication of polynucleotides in template-directed syntheses is an obvious candidate for such an amplification step in a pre-'RNA world'. A serious objection to this suggestion is the observation that the efficiency of template-directed syntheses of RNA is limited by enantiomeric cross-inhibition. Peptide Nucleic Acids (PNAs), amide-linked, nonchiral analogues of RNA, have been 'copied' into RNA and constitute an alternative to chiral polynucleotides as an informational replicating system. Here, we use PNA as model for a hypothetical, nonchiral precursor of RNA in experiments re-examining enantiomeric cross-inhibition. We find that enantiomeric cross-inhibition is as serious in the polymerization of nucleotides on a PNA template as it is on a conventional RNA or DNA template.

Antisense Treatments for Biothreat Agents

DTIC Science & Technology

2006-08-01

2001) 19(4):360-364. 82. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using...to PNA enhanced the entry of the antisense molecules and reduced expression of the bacterial target genes both in E coli [81] and Staphylococcus ... aureus [82]. Peptide-tagged PMOs can also efficiently inhibit bacterial growth in pure and infected cultures [75]. In a recent study, we observed that

Thymocyte cyclic AMP and cyclic GMP response to treatment with metabolites issued from the lipoxygenase pathway.

PubMed

Mexmain, S; Cook, J; Aldigier, J C; Gualde, N; Rigaud, M

1985-08-01

Evidence has been presented that cGMP is the second messenger for the lipoxygenase metabolites 15-HETE and LTB4 in the mouse splenocyte and thymocyte. Incubation of splenocytes with 10(-7) to 10(-9) M 15-HETE caused a slight decrease in cAMP levels and an increase in cGMP levels after 10 to 20 min. Mature PNA-, immature PNA+, and whole thymocytes treated with 10(-7) to 10(-10) M 15-HETE and 10(-11) M LTB4 showed an approximately 100% increase in cGMP production. In mixed lymphocyte reactions, 15-HETE- and LTB4-treated PNA+, PNA-, and whole thymocyte populations inhibited thymidine uptake by fresh allostimulated splenocytes. These results demonstrate that the eicosanoid-induced generation of suppressor cells follows a rise in lymphocyte cGMP levels.

Insights into peptide nucleic acid (PNA) structural features: The crystal structure of a d-lysine-based chiral PNA–DNA duplex

PubMed Central

Menchise, Valeria; De Simone, Giuseppina; Tedeschi, Tullia; Corradini, Roberto; Sforza, Stefano; Marchelli, Rosangela; Capasso, Domenica; Saviano, Michele; Pedone, Carlo

2003-01-01

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA–RNA and PNA–DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as d-Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three d-Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH2, in which pn represents a pseudopeptide link and dl represents a d-Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-Å resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. Thed-Lys-based chiral PNA–DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition. PMID:14512516

Antisense imaging of gene expression in the brain in vivo

NASA Astrophysics Data System (ADS)

Shi, Ningya; Boado, Ruben J.; Pardridge, William M.

2000-12-01

Antisense radiopharmaceuticals could be used to image gene expression in the brain in vivo, should these polar molecules be made transportable through the blood-brain barrier. The present studies describe an antisense imaging agent comprised of an iodinated peptide nucleic acid (PNA) conjugated to a monoclonal antibody to the rat transferrin receptor by using avidin-biotin technology. The PNA was a 16-mer antisense to the sequence around the methionine initiation codon of the luciferase mRNA. C6 rat glioma cells were permanently transfected with a luciferase expression plasmid, and C6 experimental brain tumors were developed in adult rats. The expression of the luciferase transgene in the tumors in vivo was confirmed by measurement of luciferase enzyme activity in the tumor extract. The [125I]PNA conjugate was injected intravenously in anesthetized animals with brain tumors and killed 2 h later for frozen sectioning of brain and film autoradiography. No image of the luciferase gene expression was obtained after the administration of either the unconjugated antiluciferase PNA or a PNA conjugate that was antisense to the mRNA of a viral transcript. In contrast, tumors were imaged in all rats administered the [125I]PNA that was antisense to the luciferase sequence and was conjugated to the targeting antibody. In conclusion, these studies demonstrate gene expression in the brain in vivo can be imaged with antisense radiopharmaceuticals that are conjugated to a brain drug-targeting system.

Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

PubMed Central

Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

2002-01-01

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

PubMed Central

Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

2013-01-01

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

PubMed Central

Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

2013-01-01

Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes.

PubMed

Biessen, Erik A L; Sliedregt-Bol, Karen; 'T Hoen, Peter A Chr; Prince, Perry; Van der Bilt, Erica; Valentijn, A Rob P M; Meeuwenoord, Nico J; Princen, Hans; Bijsterbosch, Martin K; Van der Marel, Gijs A; Van Boom, Jacques H; Van Berkel, Theo J C

2002-01-01

In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP.

PNA lectin for purifying mouse acinar cells from the inflamed pancreas.

PubMed

Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z; Gittes, George K

2016-02-17

Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

PNA lectin for purifying mouse acinar cells from the inflamed pancreas

PubMed Central

Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z.; Gittes, George K.

2016-01-01

Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer. PMID:26884345

PNA-encoded chemical libraries.

PubMed

Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

2015-06-01

Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70. Copyright © 2015 Elsevier Ltd. All rights reserved.

Some lumbar sympathetic neurons develop a glutamatergic phenotype after peripheral axotomy with a note on VGLUT2-positive perineuronal baskets

PubMed Central

Brumovsky, Pablo R.; Seroogy, Kim B.; Lundgren, Kerstin H.; Watanabe, Masahiko; Hökfelt, Tomas; Gebhart, G. F.

2011-01-01

Glutamate is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons. However, to date a glutamatergic phenotype of autonomic neurons has not been described. Therefore, we explored the expression of vesicular glutamate transporters (VGLUTs) type 1, 2 and 3 in lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) of naïve BALB/C mice, as well as after pelvic nerve axotomy (PNA), using immunohistochemistry and in situ hybridization. Colocalization with activating transcription factor-3 (ATF-3), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and calcitonin generelated peptide was also examined. Sham-PNA, sciatic nerve axotomy (SNA) or naïve mice were included. In naïve mice, VGLUT2-like immunoreactivity (LI) was only detected in fibers and varicosities in LSC and MPG; no ATF-3-immunoreactive (IR) neurons were visible. In contrast, PNA induced upregulation of VGLUT2 protein and transcript, as well as of ATF-3-LI in subpopulations of LSC neurons. Interestingly, VGLUT2-IR LSC neurons coexpressed ATF-3, and often lacked the noradrenergic marker TH. SNA only increased VGLUT2 protein and transcript in scattered LSC neurons. Neither PNA nor SNA upregulated VGLUT2 in MPG neurons. We also found perineuronal baskets immunoreactive either for VGLUT2 or the acetylcholinergic marker VAChT in non-PNA MPGs, usually around TH-IR neurons. VGLUT1-LI was restricted to some varicosities in MPGs, was absent in LSCs, and remained largely unaffected by PNA or SNA. This was confirmed by the lack of expression of VGLUT1 or VGLUT3 mRNAs in LSCs, even after PNA or SNA. Taken together, axotomy of visceral and non-visceral nerves results in a glutamatergic phenotype of some LSC neurons. In addition, we show previously non-described MPG perineuronal glutamatergic baskets. PMID:21596036

Comparative Effectiveness of Needle Aponeurotomy and Collagenase Injection for Dupuytren's Contracture: A Multicenter Study.

PubMed

Zhou, Chao; Hovius, Steven E R; Pieters, Adriana J; Slijper, Harm P; Feitz, Reinier; Selles, Ruud W

2017-09-01

Although the efficacy of collagenase clostridium histolyticum (CCH) injections has been demonstrated by randomized clinical trials, the relative effectiveness of CCH remains uncertain. Our aim was to compare the outcomes of CCH with those of percutaneous needle aponeurotomy (PNA) in daily clinical practice. We analyzed data from patients undergoing PNA or CCH between 2011 and 2014 at 7 practice sites in the Netherlands. We examined the degree of improvement in contracture and adverse effects at 6-12 weeks after surgery or the last injection. Additionally, we invited patients to complete the Michigan Hand Questionnaire before and at 6-12 months follow-up. To minimize the risk of bias, we used propensity score matching. Among 130 matched patients (93% Tubiana I or II) undergoing PNA (n = 46) and CCH (n = 84), improvement in contracture was similar: 26 degrees (65% improvement from baseline) for PNA versus 31 degrees (71%) for CCH for affected metacarpophalangeal joints ( P = 0.163). This was 16 degrees (50% improvement) versus 17 degrees (42%) for affected proximal interphalangeal joints ( P = 0.395), respectively. No serious adverse effects occurred in either of the 2 treatment groups. Of the mild adverse effects, only skin fissures and sensory disturbances were seen in both groups. Through 1-year follow-up, patients reported similar improvements in the overall Michigan Hand Questionnaire score (PNA 5.3 points versus CCH 4.9 points; P = 0.912). In patients with mild contractures (Tubiana I or II), CCH was as effective as PNA in reducing contractures. Both treatments were safe and improved hand function to a similar extent in daily practice.

Evaluation of dual-color fluorescence in situ hybridization with peptide nucleic acid probes for the detection of Mycobacterium tuberculosis and non-tuberculous mycobacteria in clinical specimens.

PubMed

Kim, Namhee; Lee, Seung Hee; Yi, Jongyoun; Chang, Chulhun L

2015-09-01

Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes. PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain. The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676). Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.

Sensitive immobilization-free electrochemical DNA sensor based on isothermal circular strand displacement polymerization reaction.

PubMed

Xuan, Feng; Luo, Xiaoteng; Hsing, I-Ming

2012-05-15

A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies. Copyright © 2012 Elsevier B.V. All rights reserved.

Zwitterionic polymer functionalization of polysulfone membrane with improved antifouling property and blood compatibility by combination of ATRP and click chemistry.

PubMed

Xiang, Tao; Lu, Ting; Xie, Yi; Zhao, Wei-Feng; Sun, Shu-Dong; Zhao, Chang-Sheng

2016-08-01

The chemical compositions are very important for designing blood-contacting membranes with good antifouling property and blood compatibility. In this study, we propose a method combining ATRP and click chemistry to introduce zwitterionic polymer of poly(sulfobetaine methacrylate) (PSBMA), negatively charged polymers of poly(sodium methacrylate) (PNaMAA) and/or poly(sodium p-styrene sulfonate) (PNaSS), to improve the antifouling property and blood compatibility of polysulfone (PSf) membranes. Attenuated total reflectance-Fourier transform infrared spectra, X-ray photoelectron spectroscopy and water contact angle results confirmed the successful grafting of the functional polymers. The antifouling property and blood compatibility of the modified membranes were systematically investigated. The zwitterionic polymer (PSBMA) grafted membranes showed good resistance to protein adsorption and bacterial adhesion; the negatively charged polymer (PNaSS or PNaMAA) grafted membranes showed improved blood compatibility, especially the anticoagulant property. Moreover, the PSBMA/PNaMAA modified membrane showed both antifouling property and anticoagulant property, and exhibited a synergistic effect in inhibiting blood coagulation. The functionalization of membrane surfaces by a combination of ATRP and click chemistry is demonstrated as an effective route to improve the antifouling property and blood compatibility of membranes in blood-contact. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids.

PubMed

Kerman, Kagan; Saito, Masato; Tamiya, Eiichi

2008-08-01

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at approximately 0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biomaterials for various analytical and pharmaceutical applications.

An evaluation of the proteolytic and lipolytic potential of Penicillium spp. isolated from traditional Greek sausages in submerged fermentation.

PubMed

Papagianni, Maria

2014-01-01

A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.

Peptide-nucleic acids (PNAs) with pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-tetraone (PPT) as a universal base: their synthesis and binding affinity for oligodeoxyribonucleotides.

PubMed

Hirano, Taisuke; Kuroda, Kenji; Kataoka, Masanori; Hayakawa, Yoshihiro

2009-07-21

Peptide-nucleic acids (PNAs) including pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-tetraone (PPT) as a nucleobase were synthesized, and their binding affinity for the complementary oligodeoxyribonucleotides was investigated. We found that PNAs with one or two PPT(s) and natural nucleobases (i.e., adenine, cytosine, guanine, or thymine) have excellent binding affinity for oligodeoxyribonucleotides with complementary bases at the positions facing the natural nucleobases, and with adenine, cytosine, guanine, and thymine at the positions opposite PPT in PNAs. The binding affinity of the PPT-containing PNA is higher than or comparable to that of a PNA consisting of all complementary natural nucleobases, viz. a PNA with a suitable natural nucleobase in place of PPT in the PPT-containing PNA. Consequently, it was concluded that PPT serves as a useful universal base that can recognize all natural nucleobases.

An In Vitro Translation, Selection, and Amplification System for Peptide Nucleic Acids

PubMed Central

Brudno, Yevgeny; Birnbaum, Michael E.; Kleiner, Ralph E.; Liu, David R.

2009-01-01

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of twelve PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory. PMID:20081830

Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution

NASA Technical Reports Server (NTRS)

Eriksson, M.; Christensen, L.; Schmidt, J.; Haaima, G.; Orgel, L.; Nielsen, P. E.

1998-01-01

The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.

Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson-Crick base pairing.

PubMed

Berger, Or; Adler-Abramovich, Lihi; Levy-Sakin, Michal; Grunwald, Assaf; Liebes-Peer, Yael; Bachar, Mor; Buzhansky, Ludmila; Mossou, Estelle; Forsyth, V Trevor; Schwartz, Tal; Ebenstein, Yuval; Frolow, Felix; Shimon, Linda J W; Patolsky, Fernando; Gazit, Ehud

2015-04-01

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

Efficacy, safety and immunological actions of butanol-extracted FAHF-2 on peanut anaphylaxis

PubMed Central

Srivastava, Kamal; Yang, Nan; Chen, Yuming; Lopez-Exposito, Ivan; Song, Ying; Goldfarb, Joseph; Zhan, Jixun; Sampson, Hugh; Li, Xiu-Min

2010-01-01

Background Therapies for peanut allergy (PNA) are urgently needed. Food Allergy Herbal Formula -2 (FAHF-2) has profound therapeutic effects in a murine peanut allergy model and is safe for food allergic adults in clinical trials. However the large FAHF-2 pill-load is not conducive to clinical studies in children. Thus refining FAHF-2 to decrease pill-load is essential for the inclusion of children in clinical trials and to facilitate studying FAHF-2 as a clinically useful botanical drug. Objectives Testing long term efficacy and safety of a butanol-purified extract of FAHF-2 (B-FAHF-2) in a murine model of PNA, and to explore its immunological mechanisms of action. Methods FAHF-2 was purified by butanol extraction. C3H/HeJ mice with established PNA received the 1st course of B-FAHF-2 at 6 mg, twice daily for 7 weeks (PNA/B-FAHF-2) or water (PNA/Sham) and were then challenged immediately after completing the treatment and 6 more times every 1–2 months post treatment up to week 50. Mice then received a second course of B-FAHF-2 treatment at week 52 and were challenged at week 65. In vivo and in vitro immunological effects on T, B and mast cells were also determined. Results Butanol purification reduced the volume of the effective dose ~5 fold. All PNA/B-FAHF-2 mice were completely protected from peanut anaphylaxis until the 5th challenge after the 1st course of treatment, as compared to PNA/sham mice. Partial protection persisted up to 50 weeks. A 2nd treatment course restored complete protection. B-FAHF-2 significantly suppressed Th2 cytokine, IgE and histamine levels in vivo, and showed direct inhibition of Th2, IgE-producing B cells and mast cell activation in vitro. B-FAHF-2 had a high margin of safety. Conclusion and clinical relevance B-FAHF-2 produced long-lasting protection against PN anaphylaxis for approximately half of the murine lifespan without side effects. B-FAHF-2 exhibited direct effects on multiple food allergy effector cells. PMID:21121976

In vivo correction of anaemia in β-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery

PubMed Central

Bahal, Raman; Ali McNeer, Nicole; Quijano, Elias; Liu, Yanfeng; Sulkowski, Parker; Turchick, Audrey; Lu, Yi-Chien; Bhunia, Dinesh C.; Manna, Arunava; Greiner, Dale L.; Brehm, Michael A.; Cheng, Christopher J.; López-Giráldez, Francesc; Ricciardi, Adele; Beloor, Jagadish; Krause, Diane S.; Kumar, Priti; Gallagher, Patrick G.; Braddock, Demetrios T.; Mark Saltzman, W.; Ly, Danith H.; Glazer, Peter M.

2016-01-01

The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% β-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration. PMID:27782131

Psychometric Needs Assessment. Theory and Practice.

ERIC Educational Resources Information Center

Scissons, Edward H.

The Psychometric Needs Assessment (PNA) model was designed to provide a means of describing a target population and various sub-populations contained therein. The specific purpose of such description is to provide a guide to determination of the continuing educational programming needs of professionals. Major attributes of the PNA model are the…

24 CFR 905.300 - Capital fund submission requirements.

Code of Federal Regulations, 2014 CFR

2014-04-01

... PHA must complete a comprehensive physical needs assessment (PNA). (1) Applicability. Small PHAs (PHAs... included; (ii) The information is consistent with the needs identified in the PNA and data available to HUD... Assessment Program (SEMAP) (24 CFR part 985), and/or were identified as noncompliant with section 9(j...

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

PubMed Central

Stender, Henrik; Kurtzman, Cletus; Hyldig-Nielsen, Jens J.; Sørensen, Ditte; Broomer, Adam; Oliveira, Kenneth; Perry-O'Keefe, Heather; Sage, Andrew; Young, Barbara; Coull, James

2001-01-01

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity. PMID:11157265

Enhancement of cell-cell contact by a nonmitogenic lectin increases blastogenic response and IL-2 release by mitogen-stimulated mouse thymocytes.

PubMed

Favero, J; Marti, J; Dornand, J; Bonnafous, J C; Mani, J C

1986-03-01

We have examined the influence of peanut agglutinin (PNA), a lectin which agglutinates but does not stimulate mouse thymocytes, on the responsiveness of these cells to concanavalin A (Con A) or galactose oxidase stimulation. Binding low amounts of PNA on unseparated mouse thymocytes pretreated with neuraminidase highly enhances the mitogenic response and the level of interleukin 2 release in the culture medium upon Con A stimulation. We have shown that PNA present on the cell surface acts as a crosslinking agent which favors intercellular binding between accessory cells (macrophages) and thymocytes, leading through this enhanced cooperation by cell-cell contact to an enhanced blastogenic response.

UV-Vis Spectroscopy and Dynamic Light Scattering Study of Gold Nanorods Aggregation

PubMed Central

Kanjanawarut, Roejarek; Yuan, Bo

2013-01-01

Gold nanorods (AuNRs) were used as spectroscopic sensing elements to detect specific DNA sequences with a single-base mismatch sensitivity. The assay was based on the observation that the stabilizing repulsive forces between CTA+-coated AuNRs can be removed by citrate ions, which causes aggregation among AuNRs; whereas nucleic acids of different structures[ i.e., peptide nucleic acid (PNA), single-stranded DNA (ssDNA), PNA-DNA complex, and double-stranded DNA (dsDNA)] can retard the aggregation. Moreover, the dsDNA PNA-DNA duplexes provide larger retardation than that by unhybridized ssDNA and PNA probe. This assay can differentiate single-base mismatched targets with base substitution at different locations (center and end) with AuNRs of a larger aspect ratio. Besides ultraviolet–visable spectroscopy measurement of particle assembly-induced plasmonic coupling that in turn provides a spectroscopic detection of the specific DNA, dynamic light scattering and transmission electron microscope (TEM) were used to measure smaller degree of aggregation that can reveal sodium citrate– and dsDNA–AuNRs interactions in fine detail. PMID:23902360

UV-vis spectroscopy and dynamic light scattering study of gold nanorods aggregation.

PubMed

Kanjanawarut, Roejarek; Yuan, Bo; XiaoDi, Su

2013-08-01

Gold nanorods (AuNRs) were used as spectroscopic sensing elements to detect specific DNA sequences with a single-base mismatch sensitivity. The assay was based on the observation that the stabilizing repulsive forces between CTA(+)-coated AuNRs can be removed by citrate ions, which causes aggregation among AuNRs; whereas nucleic acids of different structures[ i.e., peptide nucleic acid (PNA), single-stranded DNA (ssDNA), PNA-DNA complex, and double-stranded DNA (dsDNA)] can retard the aggregation. Moreover, the dsDNA PNA-DNA duplexes provide larger retardation than that by unhybridized ssDNA and PNA probe. This assay can differentiate single-base mismatched targets with base substitution at different locations (center and end) with AuNRs of a larger aspect ratio. Besides ultraviolet-visable spectroscopy measurement of particle assembly-induced plasmonic coupling that in turn provides a spectroscopic detection of the specific DNA, dynamic light scattering and transmission electron microscope (TEM) were used to measure smaller degree of aggregation that can reveal sodium citrate- and dsDNA-AuNRs interactions in fine detail.

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycocluster to lectin and tetanus toxin c-fragment

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Minoura, Norihiko

2010-02-01

We have developed a fluorescent ruthenium metalloglycocluster as a powerful molecular probe for evaluating a binding event between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analysis. The fluorescent ruthenium metalloglycoclusters, [Ru(bpy-2Gal)3] and [Ru(bpy-2Glc)3], possess clustered galactose and glucose surrounding the ruthenium center. Changes in FE and FP of these metalloglycoclusters were measured by adding each lectin (Peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), Concanavalin A (ConA), or Wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA, the FE spectrum of [Ru(bpy- 2Gal)3] showed new emission peak and the FP value of [Ru(bpy-2Gal)3] increased. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] showed new emission peak and the FP value increased following the addition of ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were proved by the FE and FP measurement. From nonlinear least-squares fitting, dissociation constants (Kd) of [Ru(bpy-2Gal)3] to PNA was 6.1 μM, while the Kd values of [Ru(bpy)2(bpy-2Gal)] to PNA was ca. 10-4 M. Therefore, the clustered carbohydrates were proved to increase affinity to lectins. Furthermore, the FP measurements proved specific binding of [Ru(bpy-2Gal)3] to TCF.

Synthesis and characterization of metastable, 20 nm-sized Pna2{sub 1}-LiCoPO{sub 4} nanospheres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ludwig, Jennifer; Nordlund, Dennis; Doeff, Marca M.

The majority of research activities on LiCoPO{sub 4} are focused on the phospho-olivine (space group Pnma), which is a promising high-voltage cathode material for Li-ion batteries. In contrast, comparably little is known about its metastable Pna2{sub 1} modification. Herein, we present a comprehensive study on the structure–property relationships of 15–20 nm Pna2{sub 1}-LiCoPO{sub 4} nanospheres prepared by a simple microwave-assisted solvothermal process. Unlike previous reports, the results indicate that the compound is non-stoichiometric and shows cation-mixing with Co ions on the Li sites, which provides an explanation for the poor electrochemical performance. Co L{sub 2,3}-edge X-ray absorption spectroscopic data confirmmore » the local tetrahedral symmetry of Co{sup 2+}. Comprehensive studies on the thermal stability using thermogravimetric analysis, differential scanning calorimetry, and in situ powder X-ray diffraction show an exothermic phase transition to olivine Pnma-LiCoPO{sub 4} at 527 °C. The influence of the atmosphere and the particle size on the thermal stability is also investigated. - Graphical abstract: Blue nano-sized Pna2{sub 1}-LiCoPO{sub 4,} featuring tetrahedrally-coordinated Co{sup 2+}, was synthesized in a rapid one-step microwave-assisted solvothermal process. The phase relation between this metastable and the stable polymorph was analyzed and electrochemical properties are discussed. - Highlights: • Preparation of uniform 15–20 nm nanospheres of metastable Pna2{sub 1}-LiCoPO{sub 4} polymorph. • Structure redetermination shows cation-mixing (Co blocking Li sites). • In situ investigation of phase transformation to olivine Pnma-LiCoPO{sub 4} at 527 °C. • Pna2{sub 1}-LiCoPO{sub 4} reemerges as a stable high-temperature phase above 800 °C. • X-ray absorption spectroscopy confirms local tetrahedral symmetry (T{sub d} Co{sup 2+}).« less

Climatic Data Integration and Analysis - Regional Approaches to Climate Change for Pacific Northwest Agriculture (REACCH PNA)

NASA Astrophysics Data System (ADS)

Seamon, E.; Gessler, P. E.; Flathers, E.; Sheneman, L.; Gollberg, G.

2013-12-01

The Regional Approaches to Climate Change for Pacific Northwest Agriculture (REACCH PNA) is a five-year USDA/NIFA-funded coordinated agriculture project to examine the sustainability of cereal crop production systems in the Pacific Northwest, in relationship to ongoing climate change. As part of this effort, an extensive data management system has been developed to enable researchers, students, and the public, to upload, manage, and analyze various data. The REACCH PNA data management team has developed three core systems to encompass cyberinfrastructure and data management needs: 1) the reacchpna.org portal (https://www.reacchpna.org) is the entry point for all public and secure information, with secure access by REACCH PNA members for data analysis, uploading, and informational review; 2) the REACCH PNA Data Repository is a replicated, redundant database server environment that allows for file and database storage and access to all core data; and 3) the REACCH PNA Libraries which are functional groupings of data for REACCH PNA members and the public, based on their access level. These libraries are accessible thru our https://www.reacchpna.org portal. The developed system is structured in a virtual server environment (data, applications, web) that includes a geospatial database/geospatial web server for web mapping services (ArcGIS Server), use of ESRI's Geoportal Server for data discovery and metadata management (under the ISO 19115-2 standard), Thematic Realtime Environmental Distributed Data Services (THREDDS) for data cataloging, and Interactive Python notebook server (IPython) technology for data analysis. REACCH systems are housed and maintained by the Northwest Knowledge Network project (www.northwestknowledge.net), which provides data management services to support research. Initial project data harvesting and meta-tagging efforts have resulted in the interrogation and loading of over 10 terabytes of climate model output, regional entomological data, agricultural and atmospheric information, as well as imagery, publications, videos, and other soft content. In addition, the outlined data management approach has focused on the integration and interconnection of hard data (raw data output) with associated publications, presentations, or other narrative documentation - through metadata lineage associations. This harvest-and-consume data management methodology could additionally be applied to other research team environments that involve large and divergent data.

DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

PubMed Central

Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

2002-01-01

The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, “real-time” DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated. PMID:12167673

Maternal testosterone exposure increases anxiety-like behavior and impacts the limbic system in the offspring.

PubMed

Hu, Min; Richard, Jennifer Elise; Maliqueo, Manuel; Kokosar, Milana; Fornes, Romina; Benrick, Anna; Jansson, Thomas; Ohlsson, Claes; Wu, Xiaoke; Skibicka, Karolina Patrycja; Stener-Victorin, Elisabet

2015-11-17

During pregnancy, women with polycystic ovary syndrome (PCOS) display high circulating androgen levels that may affect the fetus and increase the risk of mood disorders in offspring. This study investigated whether maternal androgen excess causes anxiety-like behavior in offspring mimicking anxiety disorders in PCOS. The PCOS phenotype was induced in rats following prenatal androgen (PNA) exposure. PNA offspring displayed anxiety-like behavior in the elevated plus maze, which was reversed by flutamide [androgen receptor (AR) blocker] and tamoxifen [selective estrogen receptor (ER) modulator]. Circulating sex steroids did not differ between groups at adult age. The expression of serotonergic and GABAergic genes associated with emotional regulation in the amygdala was consistent with anxiety-like behavior in female, and partly in male PNA offspring. Furthermore, AR expression in amygdala was reduced in female PNA offspring and also in females exposed to testosterone in adult age. To determine whether AR activation in amygdala affects anxiety-like behavior, female rats were given testosterone microinjections into amygdala, which resulted in anxiety-like behavior. Together, these data describe the anxiety-like behavior in PNA offspring and adult females with androgen excess, an impact that seems to occur during fetal life, and is mediated via AR in amygdala, together with changes in ERα, serotonergic, and GABAergic genes in amygdala and hippocampus. The anxiety-like behavior following testosterone microinjections into amygdala demonstrates a key role for AR activation in this brain area. These results suggest that maternal androgen excess may underpin the risk of developing anxiety disorders in daughters and sons of PCOS mothers.

Hypomethylation of specific CpG sites in the promoter region of steroidogeneic genes (GATA6 and StAR) in prenatally androgenized rats.

PubMed

Jahromi, Marziyeh Salehi; Hill, Jennifer W; Tehrani, Fahimeh Ramezani; Zadeh-Vakili, Azita

2018-05-30

The methylation level of promoters is one of the most studied and well-known epigenetic mechanisms that programs the amount of gene expression. Over expression of steroidogenesis genes via epigenetic control can result in hypetandrogenism, which is the main endocrine aspect of polycystic ovarian syndrome (PCOS). In the present study we aimed to determine and compare the promoter methylation levels of three steroidogenic genes, CYP17, GATA6 and StAR, in theca cells of prenatally androgenized (PNA) rats to those of controls. Pregnant Wistar rats in the PNA group received 5 mg free testosterone, dissolved in 500 mL solvent, subcutaneously injected on day 20 of pregnancy, while controls were injected with 500 mL of solvent only. Theca cell samples, taken from the ovaries of eight to ten female offspring of both the PNA and control groups, were measured for promoter methylation levels of the aforementioned genes, using the bisulfite sequence PCR (BSP) method. Although the promoters of all three genes were slightly hypomethylated in the PNA group, the differences observed were not significant compared to the control group. The methylation of -520 and -822 positions, in the GATA6 and the StAR promoter respectively, were significantly decreased in the PNA group. The results of this study suggest that alterations in the steroidogenesis pathway after exposure to excess androgen may be a result of changes in the pattern of the methylation of the relevant genes. Copyright © 2017. Published by Elsevier Inc.

Prenatal androgen excess programs metabolic derangements in pubertal female rats.

PubMed

Yan, Xiaonan; Dai, Xiaonan; Wang, Jing; Zhao, Nannan; Cui, Yugui; Liu, Jiayin

2013-04-01

Owing to the heterogeneity in the clinical symptoms of polycystic ovary syndrome (PCOS), the early pathophysiological mechanisms of PCOS remain unclear. Clinical, experimental, and genetic evidence supports an interaction between genetic susceptibility and the influence of maternal environment in the pathogenesis of PCOS. To determine whether prenatal androgen exposure induced PCOS-related metabolic derangements during pubertal development, we administrated 5α-dihydrotestosterone (DHT) in pregnant rats and observed their female offspring from postnatal 4 to 8 weeks. The prenatally androgenized (PNA) rats exhibited more numerous total follicles, cystic follicles, and atretic follicles than the controls. Fasting glucose, insulin, leptin levels, and homeostatic model assessment for insulin resistance were elevated in the PNA rats at the age of 5-8 weeks. Following intraperitoneal glucose tolerance tests, glucose and insulin levels did not differ between two groups; however, the PNA rats showed significantly higher 30- and 60-min glucose levels than the controls after insulin stimulation during 5-8 weeks. In addition, prenatal DHT treatment significantly decreased insulin-stimulated phosphorylation of AKT in the skeletal muscles of 6-week-old PNA rats. The abundance of IR substrate 1 (IRS1) and IRS2 was decreased in the skeletal muscles and liver after stimulation with insulin in the PNA group, whereas phosphorylation of insulin-signaling proteins was unaltered in the adipose tissue. These findings validate the contribution of prenatal androgen excess to metabolic derangements in pubertal female rats, and the impaired insulin signaling through IRS and AKT may result in the peripheral insulin resistance during pubertal development.

Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.

PubMed

Park, Joonhong; Song, Minsik; Jang, Woori; Chae, Hyojin; Lee, Gun Dong; Kim, KyungTak; Park, Heekyung; Kim, Myungshin; Kim, Yonggoo

2017-02-01

We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Elevated androstenedione in young adult but not early adolescent prenatally androgenized female rats.

PubMed

Shah, Ami B; Nivar, Isaac; Speelman, Diana L

2018-01-01

Elevated testosterone (T) is routinely reported as a marker of hyperandrogenemia in rodent models for polycystic ovary syndrome (PCOS). In women with PCOS, elevated serum androstenedione (A4) is associated with more severe phenotypes, including a positive correlation with serum T, DHEAS, free androgen index (FAI), LH, and LH/FSH ratio. Furthermore, A4, along with calculated free T and FAI, was identified as one of the best predictors of PCOS in adult women of all ages (18 to > 50 y). The objective of this study was to investigate serum A4 levels in early adolescent and young adult prenatally androgenized (PNA) female rats, a model for PCOS. Pregnant rats were injected with 5 mg T daily during gestational days 16-19 (PNA rats, experimental group) or an equal volume of vehicle (control group). Female offspring of both groups had tail vein blood drawn for serum analysis at 8 and 16 weeks of age. ELISAs were used to quantify serum A4 and T levels. Serum A4 and T were elevated in 16-week-old PNA rats compared to controls. There was no significant difference in either hormone at 8 weeks of age. The PNA rats demonstrated elevated serum A4 and T in young adulthood, as has been observed in women with PCOS, further validating this as a model for PCOS and underscoring the importance of serum A4 elevation as a parameter inherent to PCOS and a rodent model for the disorder. Significant A4 elevation develops between early adolescence and early adulthood in this PNA rat model.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector.

PubMed

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis -specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

PubMed Central

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria. PMID:28638803

Maternal testosterone exposure increases anxiety-like behavior and impacts the limbic system in the offspring

PubMed Central

Hu, Min; Richard, Jennifer Elise; Maliqueo, Manuel; Kokosar, Milana; Fornes, Romina; Benrick, Anna; Jansson, Thomas; Ohlsson, Claes; Wu, Xiaoke; Skibicka, Karolina Patrycja; Stener-Victorin, Elisabet

2015-01-01

During pregnancy, women with polycystic ovary syndrome (PCOS) display high circulating androgen levels that may affect the fetus and increase the risk of mood disorders in offspring. This study investigated whether maternal androgen excess causes anxiety-like behavior in offspring mimicking anxiety disorders in PCOS. The PCOS phenotype was induced in rats following prenatal androgen (PNA) exposure. PNA offspring displayed anxiety-like behavior in the elevated plus maze, which was reversed by flutamide [androgen receptor (AR) blocker] and tamoxifen [selective estrogen receptor (ER) modulator]. Circulating sex steroids did not differ between groups at adult age. The expression of serotonergic and GABAergic genes associated with emotional regulation in the amygdala was consistent with anxiety-like behavior in female, and partly in male PNA offspring. Furthermore, AR expression in amygdala was reduced in female PNA offspring and also in females exposed to testosterone in adult age. To determine whether AR activation in amygdala affects anxiety-like behavior, female rats were given testosterone microinjections into amygdala, which resulted in anxiety-like behavior. Together, these data describe the anxiety-like behavior in PNA offspring and adult females with androgen excess, an impact that seems to occur during fetal life, and is mediated via AR in amygdala, together with changes in ERα, serotonergic, and GABAergic genes in amygdala and hippocampus. The anxiety-like behavior following testosterone microinjections into amygdala demonstrates a key role for AR activation in this brain area. These results suggest that maternal androgen excess may underpin the risk of developing anxiety disorders in daughters and sons of PCOS mothers. PMID:26578781

High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

PubMed

Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

2013-07-08

Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

Parasympathetic neural control of canine tracheal smooth muscle.

PubMed

Kobayashi, Ichiro; Kondo, Tetsuri; Hayama, Naoki; Tazaki, Gen

2004-12-01

The middle segment of the trachea is innervated by the recurrent laryngeal and pararecurrent nerves. This study determined the pathway that mediated descending commands to the tracheal smooth muscle. Animals used were seven paralyzed and tracheostomized dogs. Tracheal contraction induced either by apnea, mechanical stimulation of the tracheal bifurcation or hypercapnia was always composed of tonic and rhythmic components. The rhythmic contraction developed in synchrony with rhythmic bursts on phrenic nerve activity (PNA). The respiratory-related bursts were also observed on the recurrent laryngeal nerve activity (RNA) and pararecurrent nerve activity (ParaRNA). During apnea there was no tonic activity neither on RNA or PNA, whereas ParaRNA had both tonic and rhythmic activities. Bursts on RNA preceded to correspondent PNA-bursts by 90+/-13 ms. In contrast, ParaRNA-burst always developed later than PNA-burst and it started at almost the same time as that of tracheal rhythmic contraction. During mechanical stimulation of the trachea or CO2-loading, though RNA did not include tonic component, ParaRNA had tonic activity during tracheal tonic contraction. These findings suggested that rhythmic and tonic contractions of the trachea were mediated through the pararecurrent nerve but not through the recurrent laryngeal nerve.

Principal network analysis: identification of subnetworks representing major dynamics using gene expression data

PubMed Central

Kim, Yongsoo; Kim, Taek-Kyun; Kim, Yungu; Yoo, Jiho; You, Sungyong; Lee, Inyoul; Carlson, George; Hood, Leroy; Choi, Seungjin; Hwang, Daehee

2011-01-01

Motivation: Systems biology attempts to describe complex systems behaviors in terms of dynamic operations of biological networks. However, there is lack of tools that can effectively decode complex network dynamics over multiple conditions. Results: We present principal network analysis (PNA) that can automatically capture major dynamic activation patterns over multiple conditions and then generate protein and metabolic subnetworks for the captured patterns. We first demonstrated the utility of this method by applying it to a synthetic dataset. The results showed that PNA correctly captured the subnetworks representing dynamics in the data. We further applied PNA to two time-course gene expression profiles collected from (i) MCF7 cells after treatments of HRG at multiple doses and (ii) brain samples of four strains of mice infected with two prion strains. The resulting subnetworks and their interactions revealed network dynamics associated with HRG dose-dependent regulation of cell proliferation and differentiation and early PrPSc accumulation during prion infection. Availability: The web-based software is available at: http://sbm.postech.ac.kr/pna. Contact: dhhwang@postech.ac.kr; seungjin@postech.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21193522

Missing-orbital analysis on hyperpolarizability β of organic molecules. Enhancement of the off-diagonal component βzxx using multiple substitutions

NASA Astrophysics Data System (ADS)

Tomonari, Mutsumi; Ookubo, Norio; Takada, Toshikazu

1995-04-01

The first-order hyperpolarizability components, βzzz and βzxx, for C 2v molecules (the z axis being the principal axis) are analyzed after simplified sum-over-states calculations. Compared with p-nitroaniline (PNA), βzxx is three times enhanced by x-extended π conjugation realized by a bulky substrate in 9-amino-10-nitroanthracene (ANA) and by multiple substitutions in 1,5-diamino-2,4-dinitrobenzene (DDB). While ANA shows βzzz unchanged because its z-directed charge transfer (CT) is similar to PNA, DDB has a β zzz1/3 of PNA, which is reduced by two weak CTs introduced by two ortho-positioned donor-acceptor pairs on both sides of the z axis.

Copper-64-labeled anti-bcl-2 PNA-peptide conjugates selectively localize to bcl-2-positive tumors in mouse models of B-cell lymphoma.

PubMed

Jia, Fang; Balaji, Baghavathy S; Gallazzi, Fabio; Lewis, Michael R

2015-11-01

The bcl-2 gene is overexpressed in non-Hodgkin's lymphoma (NHL). We have reported micro-SPECT/CT imaging of Mec-1 human lymphoma xenografts in SCID mice, using [(111)In]DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate. In order to reduce normal organ accumulation and improve imaging contrast, modified monomers with neutral hydrophilic (serine, TS) or negatively charged (aspartic acid, TD) residues were synthesized as substitutes for glycine at T(14) in the PNA sequence. The parent and modified PNA-peptide conjugates were labeled with (64)Cu and evaluated in biodistribution studies and high resolution PET/CT imaging of SCID mice bearing bcl-2-positive Mec-1 xenografts as well as bcl-2-negative Ramos xenografts. Mice were administered the (64)Cu-labeled conjugates for biodistribution and imaging studies. Biodistributions were obtained from 1 to 48 h post-injection. Mice were imaged from 1 to 48 h post-injection. The parent glycine conjugate and two modified conjugates all showed selective tumor uptake in Mec-1 xenografts. The liver uptake of the serine conjugate was significantly reduced compared to the two other PNA conjugates. Its kidney uptake was highest of the three at 47.1% ID/g at 1h and dropped to 20.6% ID/g at 24h. [Copper-64]DOTA-anti-bcl-2-TS-PNA-Tyr(3)-octreotate showed tumor uptake of 1.38% ID/g at 1h and 1.06% ID/g at 24h. The tumor-to-blood ratio was increased by factor of 2 from 1h to 24h. This compound detected Mec-1 tumors by micro-PET/CT as early as 1h post-injection and at time points out to 48 h. However, the negative control Ramos tumor could not be detected. These (64)Cu-labeled, amino acid-modified PNA conjugates showed selective tumor targeting in vivo, and tumor xenografts were detected by micro-PET/CT as early as 1h post-injection, suggesting that bcl-2 expression at the mRNA level can detected by PET in mouse models of NHL. Advances in knowledge and implications for patient care Down-regulating bcl-2, an anti-apoptotic proto-oncogene, is a mechanism to reverse chemotherapy resistance in humans with NHL. Thus, bcl-2 overexpression might be considered a new independent prognostic parameter in NHL, aiding in the identification of patients at risk for treatment failure. We have developed [(64)Cu]DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate conjugates for targeted antisense PET imaging. Our preclinical studies identified an effective combination of antisense and radionuclide imaging, with the goal of future clinical trials in patients. This imaging modality may improve clinical care by identifying patients who might respond better to conventional chemotherapy. Copyright © 2015. Published by Elsevier Inc.

Condition of Si crystal formation by vaporizing Na from NaSi

NASA Astrophysics Data System (ADS)

Morito, Haruhiko; Karahashi, Taiki; Yamane, Hisanori

2012-09-01

NaSi was heated at various Na vapor pressures (pNa 0.1-1.2 atm) and temperatures (973-1173 K) to investigate the condition of Si crystal formation from NaSi by Na evaporation. Silicon single crystals 1-3 mm in diameter were grown by evaporation of Na from Na-Si melt at 1173 K and pNa=0.74 atm.

Side to Side Supercharging Allograft

DTIC Science & Technology

Side-to-side grafting between the PNA and regional in situ nerve trunks may be able to increase the effective critical length of the PNA. Nerve tissue...and provides an effective scaffolding system but depends on in situ Schwann cell migration to support axon regeneration. Though this process appears...loss and retraction can result in segmental gaps requiring some form of grafting. Autologous nerve grafting is associated with potential donor

Computer simulation of the linear and nonlinear optical susceptibilities of p-nitroaniline in cyclohexane, 1,4-dioxane, and tetrahydrofuran in quadrupolar approximation. II. Local field effects and optical susceptibilitities.

PubMed

Reis, H; Papadopoulos, M G; Grzybowski, A

2006-09-21

This is the second part of a study to elucidate the local field effects on the nonlinear optical properties of p-nitroaniline (pNA) in three solvents of different multipolar character, that is, cyclohexane (CH), 1,4-dioxane (DI), and tetrahydrofuran (THF), employing a discrete description of the solutions. By the use of liquid structure information from molecular dynamics simulations and molecular properties computed by high-level ab initio methods, the local field and local field gradients on p-nitroaniline and the solvent molecules are computed in quadrupolar approximation. To validate the simulations and the induction model, static and dynamic (non)linear properties of the pure solvents are also computed. With the exception of the static dielectric constant of pure THF, a good agreement between computed and experimental refractive indices, dielectric constants, and third harmonic generation signals is obtained for the solvents. For the solutions, it is found that multipole moments up to two orders higher than quadrupole have a negligible influence on the local fields on pNA, if a simple distribution model is employed for the electric properties of pNA. Quadrupole effects are found to be nonnegligible in all three solvents but are especially pronounced in the 1,4-dioxane solvent, in which the local fields are similar to those in THF, although the dielectric constant of DI is 2.2 and that of the simulated THF is 5.4. The electric-field-induced second harmonic generation (EFISH) signal and the hyper-Rayleigh scattering signal of pNA in the solutions computed with the local field are in good to fair agreement with available experimental results. This confirms the effect of the "dioxane anomaly" also on nonlinear optical properties. Predictions based on an ellipsoidal Onsager model as applied by experimentalists are in very good agreement with the discrete model predictions. This is in contrast to a recent discrete reaction field calculation of pNA in 1,4-dioxane, which found that the predicted first hyperpolarizability of pNA deviated strongly from the predictions obtained using Onsager-Lorentz local field factors.

Influence of PNA containing 8-aza-7-deazaadenine on structure stability and binding affinity of PNA·DNA duplex: insights from thermodynamics, counter ion, hydration and molecular dynamics analysis.

PubMed

Gupta, Sharad K; Sur, Souvik; Prasad Ojha, Rajendra; Tandon, Vibha

2013-07-01

This paper describes the synthesis of a novel 8-aza-7-deazapurin-2,6-diamine (DPP)-containing peptide nucleic acid (PNA) monomer and Boc protecting group-based oligomerization of PNA, replacing adenine (A) with DPP monomers in the PNA strand. The PNA oligomers were synthesized against the biologically relevant SV40 promoter region (2494-AATTTTTTTTATTTA-2508) of pEGFP-N3 plasmid. The DPP-PNA·DNA duplex showed enhanced stability as compared to normal duplex (A-PNA·DNA). The electronic distribution of DPP monomer suggested that DPP had better electron donor properties over 2,6-diamino purine. UV melting and thermodynamic analysis revealed that the PNA oligomer containing a diaminopyrazolo(3,4-d)pyrimidine moiety (DPP) stabilized the PNA·DNA hybrids compared to A-PNA·DNA. DPP-PNA·DNA duplex showed higher water activity (Δnw = 38.5) in comparison to A-PNA·DNA duplex (Δnw = 14.5). The 50 ns molecular dynamics simulations of PNA·DNA duplex containing DPP or unmodified nucleobase-A showed average H-bond distances in the DPP-dT base pair of 2.90 Å (OH-N bond) and 2.91 Å (NH-N bond), which were comparably shorter than in the A-dT base pair, in which the average distances were 3.18 Å (OH-N bond) and 2.97 Å (NH-N bond), and there was one additional H-bond in the DPP-dT base pair of around 2.98 Å (O2H-N2 bond), supporting the higher stability of DPP-PNA·DNA. The analysis of molecular dynamics simulation data showed that the system binding free energy increased at a rate of approximately -4.5 kcal mol(-1) per DPP base of the PNA·DNA duplex. In summary, increased thermal stability, stronger hydrogen bonding and more stable conformation in the DPP-PNA·DNA duplex make it a better candidate as antisense/antigene therapeutic agents.

Size and morphology controlled NiSe nanoparticles as efficient catalyst for the reduction reactions

NASA Astrophysics Data System (ADS)

Subbarao, Udumula; Marakatti, Vijaykumar S.; Amshumali, Mungalimane K.; Loukya, B.; Singh, Dheeraj Kumar; Datta, Ranjan; Peter, Sebastian C.

2016-12-01

Facile and efficient ball milling and polyol methods were employed for the synthesis of nickel selenide (NiSe) nanoparticle. The particle size of the NiSe nanoparticle has been controlled mechanically by varying the ball size in the milling process. The role of the surfactants in the formation of various morphologies was studied. The compounds were characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray energy dispersive spectroscopy (EDS). The efficiency of the NiSe nanoparticle as a catalyst was tested for the reduction of para-nitroaniline (PNA) to para-phenyldiamine (PPD) and para-nitrophenol (PNP) to para-aminophenol (PAP) using NaBH4 as the reducing agent. Particle size, morphology and the presence of surfactant played a crucial role in the reduction process.

Steroid injection and needle aponeurotomy for Dupuytren contracture: a randomized, controlled study.

PubMed

McMillan, Catherine; Binhammer, Paul

2012-07-01

To compare flexion deformity at 6 months in patients with Dupuytren contracture who had percutaneous needle aponeurotomy (PNA) combined with a series of triamcinolone acetonide (TA) injections to that of patients who had PNA alone. Forty-seven patients with Dupuytren disease who were candidates for PNA (at least 1 contracture of at least 20°) participated in the study. Patients were randomized either to receive TA injections immediately following and 6 weeks and 3 months after the procedure or to receive no injections. Injections were administered into cords. The number of injections and the amount of TA per injection was determined based on the number of digits involved and the cord size. All subjects returned for 3 follow-up visits after the procedure, and contractures were measured using a goniometer. Change in total active extension deficit (TAED) was analyzed using a repeated measures analysis of variance to assess for differences between groups, time points, and interaction between group and time point. Descriptive statistics were calculated for all variables of interest. Continuous measures were summarized using means and standard deviations. There was no significant difference in TAED between groups before cord aponeurotomy. Correction at 6 months was 87% of preoperative TAED for the TA group versus 64% for the control group. This difference was statistically significant. The amount of TA administered did not correlate with TAED improvement. The study group who received TA in combination with PNA experienced a significantly greater degree of correction of flexion deformity at 6 months than those who had PNA alone. Copyright © 2012 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

Prenatal androgen excess enhances stimulation of the GNRH pulse in pubertal female rats.

PubMed

Yan, Xiaonan; Yuan, Chun; Zhao, Nannan; Cui, Yugui; Liu, Jiayin

2014-07-01

In adolescent girls with polycystic ovary syndrome (PCOS), neuroendocrine derangements manifest after the onset of puberty, characterized by rapid LH pulse frequency. The early mechanism underlying the pubertal regulation of the GNRH/LH pulsatile release in adolescents with PCOS remains uncertain. To determine the effects of prenatal androgen exposure on the activation of GNRH neurons and generation of LH pulse at puberty, we administrated 5α-dihydrotestosterone to pregnant rats and observed serum LH levels and expression of hypothalamic genes in female offspring from postnatal 4 to 8 weeks. The 6-week-old prenatally androgenized (PNA) female rats exhibited an increase in LH pulse frequency. The hypothalamic expression of neurokinin B (Nkb (Tac2)) and Lepr mRNA levels in PNA rats increased remarkably before puberty and remained high during puberty, whereas elevated Kiss1 mRNA levels were detected only after the onset of puberty. Exogenous kisspeptin, NK3R agonist, and leptin triggered tonic stimulation of GNRH neurons and increased LH secretion in 6-week-old PNA rats. Leptin upregulated Kiss1 mRNA levels in the hypothalamus of pubertal PNA rats; however, pretreatment with a kisspeptin antagonist failed to suppress the elevated serum LH stimulated by leptin, indicating that the stimulatory effects of leptin may be conveyed indirectly to GNRH neurons via other neural components within the GNRH neuronal network, rather than through the kisspeptin-GPR54 pathway. These findings validate the hypotheses that NKB and leptin play an essential role in the activation of GNRH neurons and initiation of increased LH pulse frequency in PNA female rats at puberty and that kisspeptin may coordinate their stimulatory effects on LH release. © 2014 Society for Endocrinology.

Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

PubMed

Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam

2015-11-11

Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 μM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels. The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse β-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

PubMed

D'Souza, Alicia D; Belotserkovskii, Boris P; Hanawalt, Philip C

2018-02-01

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

Pain Adaptability in Individuals With Chronic Musculoskeletal Pain Is Not Associated With Conditioned Pain Modulation.

PubMed

Wan, Dawn Wong Lit; Arendt-Nielsen, Lars; Wang, Kelun; Xue, Charlie Changli; Wang, Yanyi; Zheng, Zhen

2018-03-27

Healthy humans can be divided into the pain adaptive (PA) and the pain nonadaptive (PNA) groups; PA showed a greater decrease in pain rating to a cold pressor test (CPT) than PNA. This study examined if the dichotomy of pain adaptability existed in individuals with chronic musculoskeletal pain. CPTs at 2°C and 7°C were used to assess the status of pain adaptability in participants with either chronic nonspecific low back pain or knee osteoarthritis. The participants' potency of conditioned pain modulation (CPM) and local inhibition were measured. The strengths of pain adaptability at both CPTs were highly correlated. PA and PNA did not differ in their demographic characteristics, pain thresholds from thermal and pressure stimuli, or potency of local inhibition or CPM. PA reached their maximum pain faster than PNA (t 41 = -2.76, P < .01), and had a gradual reduction of pain unpleasantness over 7 days whereas PNA did not (F 6,246  = 3.01, P = .01). The dichotomy of pain adaptability exists in musculoskeletal pain patients. Consistent with the healthy human study, the strength of pain adaptability and potency of CPM are not related. Pain adaptability could be another form of endogenous pain inhibition of which clinical implication is yet to be understood. The dichotomy of pain adaptability was identified in healthy humans. The current study confirms that this dichotomy also exists in individuals with chronic musculoskeletal pain, and could be reliably assessed with CPTs at 2°C and 7°C. Similar to the healthy human study, pain adaptability is not associated with CPM, and may reflect the temporal aspect of pain inhibition. Copyright © 2018 The American Pain Society. Published by Elsevier Inc. All rights reserved.

Time-resolved energy transfer in DNA sequence detection using water-soluble conjugated polymers: the role of electrostatic and hydrophobic interactions.

PubMed

Xu, Qing-Hua; Gaylord, Brent S; Wang, Shu; Bazan, Guillermo C; Moses, Daniel; Heeger, Alan J

2004-08-10

We have investigated the energy transfer processes in DNA sequence detection by using cationic conjugated polymers and peptide nucleic acid (PNA) probes with ultrafast pump-dump-emission spectroscopy. Pump-dump-emission spectroscopy provides femtosecond temporal resolution and high sensitivity and avoids interference from the solvent response. The energy transfer from donor (the conjugated polymer) to acceptor (a fluorescent molecule attached to a PNA terminus) has been time resolved. The results indicate that both electrostatic and hydrophobic interactions contribute to the formation of cationic conjugated polymers/PNA-C/DNA complexes. The two interactions result in two different binding conformations. This picture is supported by the average donor-acceptor separations as estimated from time-resolved and steady-state measurements. Electrostatic interactions dominate at low concentrations and in mixed solvents.

PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

PubMed

Belotserkovskii, Boris P; Hanawalt, Philip C

2015-11-01

Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Time-resolved energy transfer in DNA sequence detection using water-soluble conjugated polymers: The role of electrostatic and hydrophobic interactions

PubMed Central

Xu, Qing-Hua; Gaylord, Brent S.; Wang, Shu; Bazan, Guillermo C.; Moses, Daniel; Heeger, Alan J.

2004-01-01

We have investigated the energy transfer processes in DNA sequence detection by using cationic conjugated polymers and peptide nucleic acid (PNA) probes with ultrafast pump-dump-emission spectroscopy. Pump-dump-emission spectroscopy provides femtosecond temporal resolution and high sensitivity and avoids interference from the solvent response. The energy transfer from donor (the conjugated polymer) to acceptor (a fluorescent molecule attached to a PNA terminus) has been time resolved. The results indicate that both electrostatic and hydrophobic interactions contribute to the formation of cationic conjugated polymers/PNA-C/DNA complexes. The two interactions result in two different binding conformations. This picture is supported by the average donor–acceptor separations as estimated from time-resolved and steady-state measurements. Electrostatic interactions dominate at low concentrations and in mixed solvents. PMID:15282375

Triple Helical Recognition of Pyrimidine Inversions in Polypurine Tracts of RNA by Nucleobase-modified PNA

PubMed Central

Gupta, Pankaj; Zengeya, Thomas; Rozners, Eriks

2013-01-01

Peptide nucleic acids containing 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E) heterocycles recognized C-G and U-A inversions in a polypurine tract of double helical RNA with high affinity and sequence selectivity at pH 6.25. E-modified PNA bound strongly to bacterial A-site RNA, while no binding was observed to the human A-site RNA. PMID:21909545

Ontogeny and reversal of brain circuit abnormalities in a preclinical model of PCOS.

PubMed

Silva, Mauro Sb; Prescott, Melanie; Campbell, Rebecca E

2018-04-05

Androgen excess is a hallmark of polycystic ovary syndrome (PCOS), a prevalent yet poorly understood endocrine disorder. Evidence from women and preclinical animal models suggests that elevated perinatal androgens can elicit PCOS onset in adulthood, implying androgen actions in both PCOS ontogeny and adult pathophysiology. Prenatally androgenized (PNA) mice exhibit a robust increase of progesterone-sensitive GABAergic inputs to gonadotropin-releasing hormone (GnRH) neurons implicated in the pathogenesis of PCOS. It is unclear when altered GABAergic wiring develops in the brain, and whether these central abnormalities are dependent upon adult androgen excess. Using GnRH-GFP-transgenic mice, we determined that increased GABA input to GnRH neurons occurs prior to androgen excess and the manifestation of reproductive impairments in PNA mice. These data suggest that brain circuit abnormalities precede the postpubertal development of PCOS traits. Despite the apparent developmental programming of circuit abnormalities, long-term blockade of androgen receptor signaling from early adulthood rescued normal GABAergic wiring onto GnRH neurons, improved ovarian morphology, and restored reproductive cycles in PNA mice. Therefore, androgen excess maintains changes in female brain wiring linked to PCOS features and the blockade of androgen receptor signaling reverses both the central and peripheral PNA-induced PCOS phenotype.

Glycoconjugate distribution in early human notochord and axial mesenchyme.

PubMed

Götz, W; Quondamatteo, F

2001-02-01

Glycosylation patterns of cells and tissues give insights into spatially and temporally regulated developmental processes and can be detected histochemically using plant lectins with specific affinities for sugar moieties. The early development of the vertebral column in man is a process which has never been investigated by lectin histochemistry. Therefore, we studied binding of several lectins (AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA) in formaldehyde-fixed sections of the axial mesenchyme of 5 human embryos in Carnegie stages 12-15. During these developmental stages, an unsegmented mesenchyme covers the notochord. Staining patterns did not show striking temporal variations except for SBA which stained the cranial axial mesenchyme only in the early stage 12 embryo and for PNA, of which the staining intensity in the mesenchyme decreased with age. The notochord appeared as a highly glycosylated tissue. Carbohydrates detected may correspond to adhesion molecules or to secreted substances like proteoglycans or proteins which could play an inductive role, for example, for the neural tube. The axial perinotochordal unsegmented mesenchyme showed strong PNA binding. Therefore, its function as a PNA-positive "barrier" tissue is discussed. The endoderm of the primitive gut showed a lectin-binding pattern that was similar to that of the notochord, which may correlate with interactions between these tissues during earlier developmental stages.

Ontogeny and reversal of brain circuit abnormalities in a preclinical model of PCOS

PubMed Central

Silva, Mauro S.B.; Prescott, Melanie; Campbell, Rebecca E.

2018-01-01

Androgen excess is a hallmark of polycystic ovary syndrome (PCOS), a prevalent yet poorly understood endocrine disorder. Evidence from women and preclinical animal models suggests that elevated perinatal androgens can elicit PCOS onset in adulthood, implying androgen actions in both PCOS ontogeny and adult pathophysiology. Prenatally androgenized (PNA) mice exhibit a robust increase of progesterone-sensitive GABAergic inputs to gonadotropin-releasing hormone (GnRH) neurons implicated in the pathogenesis of PCOS. It is unclear when altered GABAergic wiring develops in the brain, and whether these central abnormalities are dependent upon adult androgen excess. Using GnRH-GFP–transgenic mice, we determined that increased GABA input to GnRH neurons occurs prior to androgen excess and the manifestation of reproductive impairments in PNA mice. These data suggest that brain circuit abnormalities precede the postpubertal development of PCOS traits. Despite the apparent developmental programming of circuit abnormalities, long-term blockade of androgen receptor signaling from early adulthood rescued normal GABAergic wiring onto GnRH neurons, improved ovarian morphology, and restored reproductive cycles in PNA mice. Therefore, androgen excess maintains changes in female brain wiring linked to PCOS features and the blockade of androgen receptor signaling reverses both the central and peripheral PNA-induced PCOS phenotype. PMID:29618656

[Identification of hepatitis B virus YMDD point mutation using peptide nucleic acid clamping PCR].

PubMed

Zhang, Yingying; He, Haitang; Yang, Jie; Hou, Jinlin

2013-06-01

To establish a peptide nucleic acid clamping PCR assay for detecting hepatitis B virus (HBV) drug resistance mutation. RtM204I (ATT) mutant, rtM204V (GTG) mutant and rtM204 (ATG) wild-type plasmids mixed at different ratios were detected for mutations by PNA clamping PCR assay and direct sequencing, and the sensitivity and specificity of the two methods were compared. Serum samples from 85 patients with chronic HBV infection were detected for drug resistance using the two methods. The sensitivity of PNA-PCR assay was 0.001% in a 10(5)-fold excess of wild-type HBV DNA with a detection limit of 10(1) copies. The sensitivity of direct sequencing was 10% with a detection limit of 10(4) copies. Mutants were detected in 73 of the 85 serum samples (85.9%), including YIDD in 40 samples, YVDD in 23 samples, and YIDD+YVDD in 10 samples. The agreement of PNA-PCR assay with direct sequencing was only 40% (34/85, YIDD in 21 samples, YVDD in 11 samples, and YIDD+YVDD in 2 samples). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. PNA-PCR assay appears to be a more sensitive and rapid assay for detection of HBV genotypic resistance.

Fluorogenic PNA probes

PubMed Central

2018-01-01

Fluorogenic oligonucleotide probes that can produce a change in fluorescence signal upon binding to specific biomolecular targets, including nucleic acids as well as non-nucleic acid targets, such as proteins and small molecules, have applications in various important areas. These include diagnostics, drug development and as tools for studying biomolecular interactions in situ and in real time. The probes usually consist of a labeled oligonucleotide strand as a recognition element together with a mechanism for signal transduction that can translate the binding event into a measurable signal. While a number of strategies have been developed for the signal transduction, relatively little attention has been paid to the recognition element. Peptide nucleic acids (PNA) are DNA mimics with several favorable properties making them a potential alternative to natural nucleic acids for the development of fluorogenic probes, including their very strong and specific recognition and excellent chemical and biological stabilities in addition to their ability to bind to structured nucleic acid targets. In addition, the uncharged backbone of PNA allows for other unique designs that cannot be performed with oligonucleotides or analogues with negatively-charged backbones. This review aims to introduce the principle, showcase state-of-the-art technologies and update recent developments in the areas of fluorogenic PNA probes during the past 20 years. PMID:29507634

Synthesis and characterisation of hetero-bimetallic organometallic phenylalanine and PNA monomer derivatives.

PubMed

Gasser, Gilles; Brosch, Oliver; Ewers, Alexandra; Weyhermüller, Thomas; Metzler-Nolte, Nils

2009-06-14

The rational, sequential synthesis of two hetero-bimetallic derivatives of the amino acid phenylalanine and one thymine (T) peptide nucleic acid (PNA) monomer is reported. Ferrocene carboxylic acid and (eta-ethene)bis(triphenylphosphine)platinum(0) were successfully reacted with propargylamide amino acid (1a and 1b) or a T PNA monomer derivative (6) to give the expected three bimetallic compounds 4a, 4b and 9 in good yield. An enzymatic route using cross-linked enzyme crystals (CLEC) of subtilopeptidase A in organic solvents gave the ferrocene carboxylate phenylalanine propargylamide precursor (Fc-CO-Phe-NH-CH(2)-CCH, 3a) in comparable yield and purity to the traditional deprotection-peptide coupling sequence. (31)P NMR spectra of these bioorganometallics showed two doublets with (195)Pt satellites corresponding to two chemically different (31)P atoms. Interestingly, in the case of the T PNA monomer derivative 9, these signals were also doubled in a 60 : 40 ratio as a consequence of the existence of two slowly interconverting isomers in solution. Furthermore, the single-crystal X-ray structures of 3a and the hetero-bimetallic phenylalanine derivative 4b were determined, showing the presence of the two organometallics moieties separated by ca. 8.5 A in 4b as well as illustrating the stability of such compounds.

Electrochemical impedance study of the interaction of metal ions with unlabeled PNA.

PubMed

Gao, Lan; Li, Congjuan; Li, Xiaohong; Kraatz, Heinz-Bernhard

2010-09-14

The interactions of the metal ions Mg(2+), Zn(2+), Ni(2+), and Co(2+) with thin films of peptide nucleic acids (PNAs) were studied by electrochemical impedance spectroscopy (EIS), and the results show that Zn(2+), Ni(2+) and Co(2+) interacted favorably with the PNA film involving the backbone and the nucleobases with the exception of Mg(2+) for which the interaction with the backbone appears to be dominant.

Synthesis of Bipartite Tetracysteine PNA Probes for DNA In Situ Fluorescent Labeling.

PubMed

Fang, Ge-Min; Seitz, Oliver

2017-12-24

"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A 1.5 hour procedure for identification of Enterococcus Species directly from blood cultures.

PubMed

Morgan, Margie A; Marlowe, Elizabeth; Novak-Weekly, Susan; Miller, J M; Painter, T M; Salimnia, Hossein; Crystal, Benjamin

2011-02-10

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

PubMed Central

Morgan, Margie A.; Marlowe, Elizabeth; Novak-Weekly, Susan; Miller, J.M.; Painter, T.M.; Salimnia, Hossein; Crystal, Benjamin

2011-01-01

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle. PMID:21339730

In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus.

PubMed

Alcolea, Pedro J; Alonso, Ana; Degayón, María A; Moreno-Paz, Mercedes; Jiménez, Maribel; Molina, Ricardo; Larraga, Vicente

2016-05-20

Leishmania infantum is the protozoan parasite responsible for zoonotic visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been reported in this area. The life cycle of the parasite is digenetic. The promastigote stage develops within the gut of phlebotomine sand flies, whereas amastigotes survive and multiply within phagolysosomes of mammalian host phagocytes. The major vector of L. infantum in Spain is Phlebotomus perniciosus. The axenic culture model of promastigotes is generally used because it is able to mimic the conditions of the natural environment (i.e. the sand fly vector gut). However, infectivity decreases with culture passages and infection of laboratory animals is frequently required. Enrichment of the stationary phase population in highly infective metacyclic promastigotes is achieved by negative selection with peanut agglutinin (PNA), which is possible only in certain Leishmania species such as L. major and L. infantum. In this study, in vitro infectivity and differential gene expression of cultured PNA-negative promastigotes (Pro-PNA(-)) and metacyclic promastigotes isolated from the sand fly anterior thoracic midgut (Pro-Pper) have been compared. In vitro infectivity is about 30 % higher in terms of rate of infected cells and number of amastigotes per infected cell in Pro-Pper than in Pro-PNA(-). This finding is in agreement with up-regulation of a leishmanolysin gene (gp63) and genes involved in biosynthesis of glycosylinositolphospholipids (GIPL), lipophosphoglycan (LPG) and proteophosphoglycan (PPG) in Pro-Pper. In addition, differences between Pro-Pper and Pro-PNA(-) in genes involved in important cellular processes (e.g. signaling and regulation of gene expression) have been found. Pro-Pper are significantly more infective than peanut lectin non-agglutinating ones. Therefore, negative selection with PNA is an appropriate method for isolating metacyclic promastigotes in stationary phase of axenic culture but it does not allow reaching the in vitro infectivity levels of Pro-Pper. Indeed, GIPL, LPG and PPG biosynthetic genes together with a gp63 gene are up-regulated in Pro-Pper and interestingly, the correlation coefficient between both transcriptomes in terms of transcript abundance is R (2) = 0.68. This means that the correlation is sufficiently high to consider that both samples are physiologically comparable (i.e. the experiment was correctly designed and performed) and sufficiently low to conclude that important differences in transcript abundance have been found. Therefore, the implications of axenic culture should be evaluated case-by-case in each experimental design even when the stationary phase population in culture is enriched in metacyclic promastigotes by negative selection with PNA.

The efficacy of oral and subcutaneous antigen-specific immunotherapy in murine cow's milk- and peanut allergy models.

PubMed

Vonk, Marlotte M; Wagenaar, Laura; Pieters, Raymond H H; Knippels, Leon M J; Willemsen, Linette E M; Smit, Joost J; van Esch, Betty C A M; Garssen, Johan

2017-01-01

Antigen-specific immunotherapy (AIT) is a promising therapeutic approach for both cow's milk allergy (CMA) and peanut allergy (PNA), but needs optimization in terms of efficacy and safety. Compare oral immunotherapy (OIT) and subcutaneous immunotherapy (SCIT) in murine models for CMA and PNA and determine the dose of allergen needed to effectively modify parameters of allergy. Female C3H/HeOuJ mice were sensitized intragastrically (i.g.) to whey or peanut extract with cholera toxin. Mice were treated orally (5 times/week) or subcutaneously (3 times/week) for three consecutive weeks. Hereafter, the acute allergic skin response, anaphylactic shock symptoms and body temperature were measured upon intradermal (i.d.) and intraperitoneal (i.p.) challenge, and mast cell degranulation was measured upon i.g. challenge. Allergen-specific IgE, IgG1 and IgG2a were measured in serum at different time points. Single cell suspensions derived from lymph organs were stimulated with allergen to induce cytokine production and T cell phenotypes were assessed using flow cytometry. Both OIT and SCIT decreased clinically related signs upon challenge in the CMA and PNA model. Interestingly, a rise in allergen-specific IgE was observed during immunotherapy, hereafter, treated mice were protected against the increase in IgE caused by allergen challenge. Allergen-specific IgG1 and IgG2a increased due to both types of AIT. In the CMA model, SCIT and OIT reduced the percentage of activated Th2 cells and increased the percentage of activated Th1 cells in the spleen. OIT increased the percentage of regulatory T cells (Tregs) and activated Th2 cells in the MLN. Th2 cytokines IL-5, IL-13 and IL-10 were reduced after OIT, but not after SCIT. In the PNA model, no differences were observed in percentages of T cell subsets. SCIT induced Th2 cytokines IL-5 and IL-10, whereas OIT had no effect. We have shown clinical protection against allergic manifestations after OIT and SCIT in a CMA and PNA model. Although similar allergen-specific antibody patterns were observed, differences in T cell and cytokine responses were shown. Whether these findings are related to a different mechanism of AIT in CMA and PNA needs to be elucidated.

Polynitroxyl albumin and albumin therapy after pediatric asphyxial cardiac arrest: effects on cerebral blood flow and neurologic outcome

PubMed Central

Manole, Mioara D; Kochanek, Patrick M; Foley, Lesley M; Hitchens, T Kevin; Bayır, Hülya; Alexander, Henry; Garman, Robert; Ma, Li; Hsia, Carleton J C; Ho, Chien; Clark, Robert S B

2012-01-01

Postresuscitation cerebral blood flow (CBF) disturbances and generation of reactive oxygen species likely contribute to impaired neurologic outcome after pediatric cardiac arrest (CA). Hence, we determined the effects of the antioxidant colloid polynitroxyl albumin (PNA) versus albumin or normal saline (NS) on CBF and neurologic outcome after asphyxial CA in immature rats. We induced asphyxia for 9 minutes in male and female postnatal day 16 to 18 rats randomized to receive PNA, albumin, or NS at resuscitation from CA or sham surgery. Regional CBF was measured serially from 5 to 150 minutes after resuscitation by arterial spin-labeled magnetic resonance imaging. We assessed motor function (beam balance and inclined plane), spatial memory retention (water maze), and hippocampal neuronal survival. Polynitroxyl albumin reduced early hyperemia seen 5 minutes after CA. In contrast, albumin markedly increased and prolonged hyperemia. In the delayed period after resuscitation (90 to 150 minutes), CBF was comparable among groups. Both PNA- and albumin-treated rats performed better in the water maze versus NS after CA. This benefit was observed only in males. Hippocampal neuron survival was similar between injury groups. Treatment of immature rats with PNA or albumin resulted in divergent acute changes in CBF, but both improved spatial memory retention in males after asphyxial CA. PMID:22126915

Comparative analysis of Gram's stain, PNA-FISH and Sepsityper with MALDI-TOF MS for the identification of yeast direct from positive blood cultures.

PubMed

Gorton, Rebecca L; Ramnarain, P; Barker, K; Stone, N; Rattenbury, S; McHugh, T D; Kibbler, C C

2014-10-01

Fungaemia diagnosis could be improved by reducing the time to identification of yeast from blood cultures. This study aimed to evaluate three rapid methods for the identification of yeast direct from blood cultures; Gram's stain analysis, the AdvanDX Peptide Nucleic Acid in Situ Hybridisation Yeast Traffic Light system (PNA-FISH YTL) and Bruker Sepsityper alongside matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Fifty blood cultures spiked with a known single yeast strain were analysed by blinded operators experienced in each method. Identifications were compared with MALDI-TOF MS CHROMagar Candida culture and ITS rRNA sequence-based identifications. On first attempt, success rates of 96% (48/50) and 76% (36/50) were achieved using PNA-FISH YTL and Gram's stain respectively. MALDI-TOF MS demonstrated a success rate of 56% (28/50) when applying manufacturer's species log score thresholds and 76% (38/50) using in-house parameters, including lowering the species log score threshold to >1.5. In conclusion, PNA-FISH YTL demonstrated a high success rate successfully identifying yeast commonly encountered in fungaemia. Sepsityper(™) with MALDI-TOF MS was accurate but increased sensitivity is required. Due to the misidentification of commonly encountered yeast Gram's stain analysis demonstrated limited utility in this setting. © 2014 Blackwell Verlag GmbH.

Effects of gestational length, gender, postnatal age, and birth order on visual contrast sensitivity in infants.

PubMed

Dobkins, Karen R; Bosworth, Rain G; McCleery, Joseph P

2009-09-30

To investigate effects of visual experience versus preprogrammed mechanisms on visual development, we used multiple regression analysis to determine the extent to which a variety of variables (that differ in the extent to which they are tied to visual experience) predict luminance and chromatic (red/green) contrast sensitivity (CS), which are mediated by the magnocellular (M) and parvocellular (P) subcortical pathways, respectively. Our variables included gestational length (GL), birth weight (BW), gender, postnatal age (PNA), and birth order (BO). Two-month-olds (n = 60) and 6-month-olds (n = 122) were tested. Results revealed that (1) at 2 months, infants with longer GL have higher luminance CS; (2) at both ages, CS significantly increases over a approximately 21-day range of PNA, but this effect is stronger in 2- than 6-month-olds and stronger for chromatic than luminance CS; (3) at 2 months, boys have higher luminance CS than girls; and (4) at 2 months, firstborn infants have higher CS, while at 6 months, non-firstborn infants have higher CS. The results for PNA/GL are consistent with the possibility that P pathway development is more influenced by variables tied to visual experience (PNA), while M pathway development is more influenced by variables unrelated to visual experience (GL). Other variables, including prenatal environment, are also discussed.

Effects of gestational length, gender, postnatal age, and birth order on visual contrast sensitivity in infants

PubMed Central

Dobkins, Karen R.; Bosworth, Rain G.; McCleery, Joseph P.

2010-01-01

To investigate effects of visual experience versus preprogrammed mechanisms on visual development, we used multiple regression analysis to determine the extent to which a variety of variables (that differ in the extent to which they are tied to visual experience) predict luminance and chromatic (red/green) contrast sensitivity (CS), which are mediated by the magnocellular (M) and parvocellular (P) subcortical pathways, respectively. Our variables included gestational length (GL), birth weight (BW), gender, postnatal age (PNA), and birth order (BO). Two-month-olds (n = 60) and 6-month-olds (n = 122) were tested. Results revealed that (1) at 2 months, infants with longer GL have higher luminance CS; (2) at both ages, CS significantly increases over a ~21-day range of PNA, but this effect is stronger in 2- than 6-month-olds and stronger for chromatic than luminance CS; (3) at 2 months, boys have higher luminance CS than girls; and (4) at 2 months, firstborn infants have higher CS, while at 6 months, non-firstborn infants have higher CS. The results for PNA/GL are consistent with the possibility that P pathway development is more influenced by variables tied to visual experience (PNA), while M pathway development is more influenced by variables unrelated to visual experience (GL). Other variables, including prenatal environment, are also discussed. PMID:19810800

Model-based clinical dose optimization for phenobarbital in neonates: An illustration of the importance of data sharing and external validation.

PubMed

Völler, Swantje; Flint, Robert B; Stolk, Leo M; Degraeuwe, Pieter L J; Simons, Sinno H P; Pokorna, Paula; Burger, David M; de Groot, Ronald; Tibboel, Dick; Knibbe, Catherijne A J

2017-11-15

Particularly in the pediatric clinical pharmacology field, data-sharing offers the possibility of making the most of all available data. In this study, we utilize previously collected therapeutic drug monitoring (TDM) data of term and preterm newborns to develop a population pharmacokinetic model for phenobarbital. We externally validate the model using prospective phenobarbital data from an ongoing pharmacokinetic study in preterm neonates. TDM data from 53 neonates (gestational age (GA): 37 (24-42) weeks, bodyweight: 2.7 (0.45-4.5) kg; postnatal age (PNA): 4.5 (0-22) days) contained information on dosage histories, concentration and covariate data (including birth weight, actual weight, post-natal age (PNA), postmenstrual age, GA, sex, liver and kidney function, APGAR-score). Model development was carried out using NONMEM ® 7.3. After assessment of model fit, the model was validated using data of 17 neonates included in the DINO (Drug dosage Improvement in NeOnates)-study. Modelling of 229 plasma concentrations, ranging from 3.2 to 75.2mg/L, resulted in a one compartment model for phenobarbital. Clearance (CL) and volume (V d ) for a child with a birthweight of 2.6kg at PNA day 4.5 was 0.0091L/h (9%) and 2.38L (5%), respectively. Birthweight and PNA were the best predictors for CL maturation, increasing CL by 36.7% per kg birthweight and 5.3% per postnatal day of living, respectively. The best predictor for the increase in V d was actual bodyweight (0.31L/kg). External validation showed that the model can adequately predict the pharmacokinetics in a prospective study. Data-sharing can help to successfully develop and validate population pharmacokinetic models in neonates. From the results it seems that both PNA and bodyweight are required to guide dosing of phenobarbital in term and preterm neonates. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Morphological and glycan features of the camel oviduct epithelium.

PubMed

Accogli, Gianluca; Monaco, Davide; El Bahrawy, Khalid Ahmed; El-Sayed, Ashraf Abd El-Halim; Ciannarella, Francesca; Beneult, Benedicte; Lacalandra, Giovanni Michele; Desantis, Salvatore

2014-07-01

This study describes regional differences in the oviduct of the one-humped camel (Camelus dromedarius) during the growth phase (GP) and the mature phase (MP) of the follicular wave by means of morphometry, scanning electron microscopy (SEM) and glycohistochemistry investigations. Epithelium height significantly increased in the ampulla and decreased in the isthmus passing from the GP to the MP. Under SEM, non-ciliated cells displayed apical blebs (secretory) or short microvilli. Cilia glycocalyx expressed glycans terminating with sialic acid linked α2,6 to Gal/GalNAc (SNA affinity) throughout the oviducts of GP and MP and sialic acid linked α2,3 to Galβ1,3GalNAc (MAL II and KOH-sialidase (K-s)-PNA staining) throughout the MP oviducts. Non-ciliated cells displayed lectin-binding sites from the supra-nuclear cytoplasm to the luminal surface. Ampulla non-ciliated cells showed O-linked (mucin-type) sialoglycans (MAL II and K-s-PNA) during GP and MP and N-linked sialoglycans (SNA) during the MP. Isthmus non-ciliated cells expressed SNA reactivity in GP and MP, also K-s-PNA binders in MP, and MAL II and PNA affinity (Galβ1,3GalNAc) during GP. Galβ1,3GalNAc was sialilated in the non-ciliated cells of GP UTJ. Luminal surface lacked of Galβ1,3GalNAc in GP and MP, whereas it expressed α2,6- and α2,3-linked sialic acids. In GP intraluminal substance reacted with SNA, MAL II, K-s-PNA in ampulla and only with MAL II in the isthmus and UTJ. These results demonstrate that the morphology and the glycan pattern of the camel oviductal epithelium vary during the follicular wave and that could relate to the region-specific functions. Copyright © 2014 Elsevier GmbH. All rights reserved.

Prenatal Androgen Exposure Causes Hypertension and Gut Microbiota Dysbiosis.

PubMed

Sherman, Shermel; Sarsour, Nadeen; Salehi, Marziyeh; Schroering, Allen; Mell, Blair; Joe, Bina; Hill, Jennifer W

2018-02-22

Conditions of excess androgen in women, such as polycystic ovary syndrome (PCOS), often exhibit intergenerational transmission. One way in which the risk for PCOS may be increased in daughters of affected women is through exposure to elevated androgens in utero. Hyperandrogenemic conditions have serious health consequences, including increased risk for hypertension and cardiovascular disease. Recently, gut dysbiosis has been found to induce hypertension in rats, such that blood pressure can be normalized through fecal microbial transplant. Therefore, we hypothesized that the hypertension seen in PCOS has early origins in gut dysbiosis caused by in utero exposure to excess androgen. We investigated this hypothesis with a model of prenatal androgen (PNA) exposure and maternal hyperandrogenemia by single-injection of testosterone cypionate or sesame oil vehicle (VEH) to pregnant dams in late gestation. We then completed a gut microbiota and cardiometabolic profile of the adult female offspring. The metabolic assessment revealed that adult PNA rats had increased body weight and increased mRNA expression of adipokines: adipocyte binding protein 2, adiponectin, and leptin in inguinal white adipose tissue. Radiotelemetry analysis revealed hypertension with decreased heart rate in PNA animals. The fecal microbiota profile of PNA animals contained higher relative abundance of bacteria associated with steroid hormone synthesis, Nocardiaceae and Clostridiaceae, and lower abundance of Akkermansia, Bacteroides, Lactobacillus, Clostridium. The PNA animals also had an increased relative abundance of bacteria associated with biosynthesis and elongation of unsaturated short chain fatty acids (SCFAs). We found that prenatal exposure to excess androgen negatively impacted cardiovascular function by increasing systolic and diastolic blood pressure and decreasing heart rate. Prenatal androgen was also associated with gut microbial dysbiosis and altered abundance of bacteria involved in metabolite production of short chain fatty acids. These results suggest that early-life exposure to hyperandrogenemia in daughters of women with PCOS may lead to long-term alterations in gut microbiota and cardiometabolic function.

Accidentes laborales asociados al desánimo de médicos SERUMS para laborar en el primer nivel de atención de Lima, Perú

PubMed Central

Mejia, Christian R.; Valladares-Garrido, Mario J.; Romero, Brian M.; Valladares-Garrido, Danai; Linares-Reyes, Edgardo

2018-01-01

Introducción la retención laboral es un tema de suma importancia, porque se requiere de profesionales en el primer nivel de atención (PNA). El objetivo fue determinar si los accidentes laborales se asociaron al desánimo de los médicos para trabajar en el PNA de Lima, Perú. Métodos estudio transversal analítico de datos secundarios de una base de datos generada de una encuesta a médicos que realizaron su Servicio Rural y Urbano-Marginal en Salud (SERUMS). Se incluyó solo a los médicos que manifestaron al inicio del SERUMS que podían trabajar en el PNA de Lima. Se definió como cambio de intención de trabajo en el PNA a los que finalizando el SERUMS refirieron que ya no deseaban laborar en Lima. Esto se asoció según si tuvieron un accidente laboral y se ajustó por otras variables. Resultados de los 124 médicos el 63% fueron hombres (78). La mediana de edad fue de 26 años (rango intercuartílico: 25-27 años). Después de su SERUMS, el 12% (15) manifestó que cambió su interés y que deseaba trabajar en la capital. En el análisis multivariado, haber tenido un accidente laboral disminuyó la frecuencia del cambio de intención de trabajo en el PNA (RPa: 0.28, IC 95%: 0.14-0.54, p < 0,001), ajustado por ocho variables. Conclusiones en un estudio previo los accidentes laborales disminuyeron la frecuencia de trabajar en provincias, pero nuestro estudio dice lo contrario, posiblemente por la percepción de que un trabajo en la capital del país permite estar más cerca de los servicios para ser atendido en caso de cualquier emergencia. PMID:29190859

Remote Enantioselection Transmitted by an Achiral Peptide Nucleic Acid Backbone

NASA Technical Reports Server (NTRS)

Kozlov, Igor A.; Orgel, Leslie E.; Nielsen, Peter E.

2000-01-01

short homochiral segment of DNA into a PNA helix could have guaranteed that the next short segment of DNA to be incorporated would have the same handedness as the first. Once two segments of the same handedness were present, the probability that a third segment would have the same handedness would increase, and so on. Evolution could then slowly dilute out the PNA part. This scenario would ultimately allow the formation of a chiral oligonucleotide by processes that are largely resistant to enantiomeric crossinhibition. It is important to note that the ligation of homochiral dinucleotides on a nucleic acid template would probably be at least as enantiospecific as the reaction that we have studied. The disadvantage of using chiral monomers as components of a replicating system arises from the difficulty of generating a first long homochiral template from a racemic mixture of monomers, although results of experiments designed to overcome this difficulty by employing homochiral tetramers have been reported.l l The probability of obtaining a homochiral n-mer from achiral substrates is approximately 1P-I if the nontemplate-directed extension of the primer is not enantioselective. Hence, it would be very hard to get started with a homochiral 40-mer, for example. No such difficulty exists in a scenario that originates with an achiral genetic material and in which the incorporation of very few chiral monomers in this achiral background gradually progresses towards homochirality. It seems possible that some PNA sequences could act as catalysts, analogous to ribozymes, even though PNA lacks clear metal binding sites. Although such catalysts could not be enantioselective, the incorporation of as few as two chiral nucleotides could then impose chiral specificity on the system. Furthermore, such patch chimeras could help to bridge the gap in catalytic potential between PNA and RNA, while guaranteeing enantioselectivity.

Template switching between PNA and RNA oligonucleotides

NASA Technical Reports Server (NTRS)

Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

1995-01-01

The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

Using Electrolyte Free Water Balance to Rationalize and Treat Dysnatremias

PubMed Central

Shah, Sanjeev R.; Bhave, Gautam

2018-01-01

Dysnatremias or abnormalities in plasma [Na+] are often termed disorders of water balance, an unclear physiologic concept often confused with changes in total fluid balance. However, most clinicians clearly recognize that hypertonic or hypotonic gains or losses alter plasma [Na+], while isotonic changes do not modify plasma [Na+]. This concept can be conceptualized as the electrolyte free water balance (EFWB), which defines the non-isotonic components of inputs and outputs to determine their effect on plasma [Na+]. EFWB is mathematically proportional to the rate of change in plasma [Na+] (dPNa/dt) and, therefore, is actively regulated to zero so that plasma [Na+] remains stable at its homeostatic set point. Dysnatremias are, therefore, disorders of EFWB and the relationship between EFWB and dPNa/dt provides a rationale for therapeutic strategies incorporating mass and volume balance. Herein, we leverage dPNa/dt as a desired rate of correction of plasma [Na+] to define a stepwise approach for the treatment of dysnatremias. PMID:29740578

[Abortion in Brazil: a household survey using the ballot box technique].

PubMed

Diniz, Debora; Medeiros, Marcelo

2010-06-01

This study presents the first results of the National Abortion Survey (PNA, Pesquisa Nacional de Aborto), a household random sample survey fielded in 2010 covering urban women in Brazil aged 18 to 39 years. The PNA combined two techniques, interviewer-administered questionnaires and self-administered ballot box questionnaires. The results of PNA show that at the end of their reproductive health one in five women has performed an abortion, with abortions being more frequent in the main reproductive ages, that is, from 18 to 29 years old. No relevant differentiation was observed in the practice of abortion among religious groups, but abortion was found to be more common among people with lower education. The use of medical drugs to induce abortion occurred in half of the abortions, and post-abortion hospitalization was observed among approximately half of the women who aborted. Such results lead to conclude that abortion is a priority in the Brazilian public health agenda.

Impact of In situ Postnitridation Annealing for Successful Fabrication of HfSiON Thin Film

NASA Astrophysics Data System (ADS)

Horii, Sadayoshi; Ishikawa, Dai; Sano, Atsushi; Imai, Yoshinori; Kunii, Yasuo

2007-05-01

For the successful integration of high-k gate dielectrics into advanced complementary metal-oxide-semiconductor (CMOS) processes, it is important to determine the stability of high-k materials during exposure to an ambient atmosphere. In this work, we investigated the effect of exposure to air on the nitrogen concentration in HfSiON films formed by sequentially combining HfSiO chemical vapor deposition (CVD), plasma nitridation, and postnitridation annealing (PNA). We observed that exposure to air after the nitridation step reduces the nitrogen concentration due to a reaction between the HfSiON surface and the constituents of atmospheric air. We also found that exposure to air for even a short time between nitridation and PNA leads to a significant loss of nitrogen concentration, indicating that in situ PNA is critical for achieving precise control of the nitridation. These results confirmed the importance of using clustered multichamber platforms for successful high-k fabrication.

Use of PNA FISH for blood cultures growing Gram-positive cocci in chains without a concomitant antibiotic stewardship intervention does not improve time to appropriate antibiotic therapy.

PubMed

Cosgrove, Sara E; Li, David X; Tamma, Pranita D; Avdic, Edina; Hadhazy, Eric; Wakefield, Teresa; Gherna, Michael; Carroll, Karen C

2016-09-01

Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a rapid diagnostic assay that can identify certain organisms growing in blood cultures 30-90 min from the time of positive Gram-stain. Existing studies have demonstrated a clinical utility with this assay when antibiotic stewardship programs assist clinicians with interpreting the results. However, the benefit of these rapid assays in the absence of concomitant antibiotic stewardship involvement is unclear. In this randomized study of 220 patients with enterococcal or streptococcal bacteremia, we found that PNA FISH, in the absence of concomitant input from an antibiotic stewardship program, had no impact on time to effective or optimal therapy, length of hospital stay, or in-hospital mortality. Our results suggest that in the absence of guidance from an antibiotic stewardship program, the clinical benefits of rapid diagnostic microbiological tools may be reduced. Copyright © 2016 Elsevier Inc. All rights reserved.

Water-Soluble Conjugated Polymers: Self-Assembly and Biosensor Applications

NASA Astrophysics Data System (ADS)

Bazan, Guillermo

2005-03-01

Homogeneous assays can be designed which take advantage of the optical amplification of conjugated polymers and the self-assembly characteristic of aqueous polyelectrolytes. For example, a ssDNA sequence sensor comprises an aqueous solution containing a cationic water soluble conjugated polymer such as poly(9,9-bis(trimethylammonium)-hexyl)-fluorene phenylene) with a peptide nucleic acid (PNA) labeled with a dye (PNA-C*). Signal transduction is controlled by hybridization of the neutral PNA-C* probe and the negative ssDNA target, resulting in favorable electrostatic interactions between the hybrid complex and the cationic polymer. Distance requirements for Förster energy transfer are thus met only when ssDNA of complementary sequence to the PNA-C* probe is present. Signal amplification by the conjugated polymer provides fluorescein emission >25 times higher than that of the directly excited dye. Transduction by electrostatic interactions followed by energy transfer is a general strategy. Examples involving other biomolecular recognition events, such as DNA/DNA, RNA/protein and RNA/RNA, will also be provided. The mechanism of biosensing will be discussed, with special attention to the varying contributions of hydrophobic and electrostatic forces, polymer conformation, charge density, local concentration of C*s and tailored defect sites for aggregation-induced optical changes. Finally, the water solubility of these conjugated polymers opens possibilities for spin casting onto organic materials, without dissolving the underlying layers. This property is useful for fabricating multilayer organic optoelectronic devices by simple solution techniques.

Accelerated Photobleaching of a Cyanine Dye in the Presence of a Ternary Target DNA, PNA Probe, Dye Catalytic Complex: A Molecular Diagnostic

PubMed Central

Wang, M.; Holmes-Davis, R.; Rafinski, Z.; Jedrzejewska, B.; Choi, K. Y.; Zwick, M.; Bupp, C.; Izmailov, A.; Paczkowski, J.; Warner, B.; Koshinsky, H.

2009-01-01

In many settings, molecular testing is needed but unavailable due to complexity and cost. Simple, rapid, and specific DNA detection technologies would provide important alternatives to existing detection methods. Here we report a novel, rapid nucleic acid detection method based on the accelerated photobleaching of the light-sensitive cyanine dye, 3,3′-diethylthiacarbocyanine iodide (DiSC2(3) I−), in the presence of a target genomic DNA and a complementary peptide nucleic acid (PNA) probe. On the basis of the UV–vis, circular dichroism, and fluorescence spectra of DiSC2(3) with PNA–DNA oligomer duplexes and on characterization of a product of photolysis of DiSC2(3) I−, a possible reaction mechanism is proposed. We propose that (1) a novel complex forms between dye, PNA, and DNA, (2) this complex functions as a photosensitizer producing 1O2, and (3) the 1O2 produced promotes photobleaching of dye molecules in the mixture. Similar cyanine dyes (DiSC3(3), DiSC4(3), DiSC5(3), and DiSCpy(3)) interact with preformed PNA–DNA oligomer duplexes but do not demonstrate an equivalent accelerated photobleaching effect in the presence of PNA and target genomic DNA. The feasibility of developing molecular diagnostic assays based on the accelerated photobleaching (the smartDNA assay) that results from the novel complex formed between DiSC2(3) and PNA–DNA is under way. PMID:19231844

Ordered Self-Assembled Monolayers of Peptide Nucleic Acids with DNA Recognition Capability

NASA Astrophysics Data System (ADS)

Briones, C.; Mateo-Marti, E.; Gómez-Navarro, C.; Parro, V.; Román, E.; Martín-Gago, J. A.

2004-11-01

We report on the formation of ordered self-assembled monolayers (SAMs) of single-stranded peptide nucleic acids (ssPNA). In spite of their remarkable length (7nm) thiolated PNAs assemble standing up on gold surfaces similarly to the SAMs of short alkanethiols. SAMs of ssPNA recognize complementary nucleic acids, acting as specific biosensors that discriminate even a point mutation in target ssDNA. These results are obtained by surface characterization techniques that avoid labeling of the target molecule: x-ray photoemission, x-ray absorption and atomic force microscopy.

Interphotoreceptor matrix components in retinal cell transplants.

PubMed

Juliusson, B; Mieziewska, K; Bergström, A; Wilke, K; Van Veen, T; Ehinger, B

1994-05-01

To further investigate the functional potential of retinal transplants we have used immunocytochemistry to study the distribution of four different interphotoreceptor matrix (IPM)-specific components in rabbit retinal transplants. The different components were: interphotoreceptor retinoid-binding protein (IRBP), chondroitin-6-sulfate, F22 antigen and peanut agglutinin (PNA) binding structures. IRBP acts as a retinoid-transport protein between the neural retina and the retinal pigment epithelium. Chondroitin-6-sulfate is a glycosaminoglycan and a part of the insoluble IPM skeleton. The identity and role of the F22 antigen is not known. However, it is a 250 kDa protein localized to specific extracellular compartments such as teh IPM. PNA is a lectin with a high binding affinity for D-galactose-beta (1-3) N-acetyl-D-galactosamine disaccharide linkages and binds to IPM domains surrounding cones, but not rods. The transplants (15-day-old embryonic rabbit retina) were placed between the neural retina and retinal pigment epithelium in adult hosts. The transplants developed the typical rosette formations with photoreceptors toward the center. IRBP labeling was distinct in the IPM in the host retina. However, no IRBP labeling could be detected in the transplants. The chondroitin-6-sulfate and F22 antibodies strongly labeled the IPM in the host retina and corresponding structures in the center of rosettes. A cone-specific labeling with PNA could be seen in the host retina. In the transplants, however, PNA labeling appeared in association with many more photoreceptors than in the host retina. There is no previous study available on the IPM in retinal cell transplants.(ABSTRACT TRUNCATED AT 250 WORDS)

Composite Quality Measures for Common Inpatient Medical Conditions

PubMed Central

Chen, Lena M.; Staiger, Douglas O.; Birkmeyer, John D.; Ryan, Andrew M.; Zhang, Wenying; Dimick, Justin B.

2014-01-01

Background Public reporting on quality aims to help patients select better hospitals. However, individual quality measures are sub-optimal in identifying superior and inferior hospitals based on outcome performance. Objective To combine structure, process, and outcome measures into an empirically-derived composite quality measure for heart failure (HF), acute myocardial infarction (AMI), and pneumonia (PNA). To assess how well the composite measure predicts future high and low performers, and explains variance in future hospital mortality. Research Design Using national Medicare data, we created a cohort of older patients treated at an acute care hospital for HF (n=1,203,595), AMI (n=625,595), or PNA (n=1,234,299). We ranked hospitals based on their July 2005 to June 2008 performance on the composite. We then estimated the odds of future (July to December 2009) 30-day, risk-adjusted mortality at the worst vs. best quintile of hospitals. We repeated this analysis using 2005-2008 performance on existing quality indicators, including mortality. Results The composite (vs. Hospital Compare) explained 68% (vs. 39%) of variation in future AMI mortality rates. In 2009, if an AMI patient had chosen a hospital in the worst vs. best quintile of performance using 2005-2008 composite (vs. Hospital Compare) rankings, he or she would have had 1.61 (vs. 1.39) times the odds of dying in 30 days (p-value for difference < 0.001). Results were similar for HF and PNA. Conclusions Composite measures of quality for HF, AMI, and PNA performed better than existing measures at explaining variation in future mortality and predicting future high and low performers. PMID:23942222

Synthesis and characterization of metastable, 20 nm-sized Pna21-LiCoPO4 nanospheres

NASA Astrophysics Data System (ADS)

Ludwig, Jennifer; Nordlund, Dennis; Doeff, Marca M.; Nilges, Tom

2017-04-01

The majority of research activities on LiCoPO4 are focused on the phospho-olivine (space group Pnma), which is a promising high-voltage cathode material for Li-ion batteries. In contrast, comparably little is known about its metastable Pna21 modification. Herein, we present a comprehensive study on the structure-property relationships of 15-20 nm Pna21-LiCoPO4 nanospheres prepared by a simple microwave-assisted solvothermal process. Unlike previous reports, the results indicate that the compound is non-stoichiometric and shows cation-mixing with Co ions on the Li sites, which provides an explanation for the poor electrochemical performance. Co L2,3-edge X-ray absorption spectroscopic data confirm the local tetrahedral symmetry of Co2+. Comprehensive studies on the thermal stability using thermogravimetric analysis, differential scanning calorimetry, and in situ powder X-ray diffraction show an exothermic phase transition to olivine Pnma-LiCoPO4 at 527 °C. The influence of the atmosphere and the particle size on the thermal stability is also investigated.

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

PubMed Central

Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

2002-01-01

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

An Investigation of the Thermal Decomposition of 1, 4-Butanediammonium Dinitrate and Selected Composites with Ammonium and Potassium Nitrates

DTIC Science & Technology

1989-02-01

Reference 20. (8) PN H dCHCHCHH 2T NO2 + H20 PNA (9) PNA - 2C H2CH 2 CH2 NNO + other products PNSA (10) BDD . 02 NNH(CH2 )4 NHNO 2 + 2H2 0 BONA (11) BDNA ...ONNH(CH2 )4 NHNO + other products BONSA 2H+ (12) BDNA --> ý4-CH2CH2 -N=N=0+] 2 + 2H2 0 (a) (13) (a) --- 4 [(CH2 )4 ]++ + 2N2 0 (b) (14) (b) --- H2 C

Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

PubMed

Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

2016-04-01

Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Testing the hypothesis of neurodegeneracy in respiratory network function with a priori transected arterially perfused brain stem preparation of rat

PubMed Central

Jones, Sarah E.

2016-01-01

Degeneracy of respiratory network function would imply that anatomically discrete aspects of the brain stem are capable of producing respiratory rhythm. To test this theory we a priori transected brain stem preparations before reperfusion and reoxygenation at 4 rostrocaudal levels: 1.5 mm caudal to obex (n = 5), at obex (n = 5), and 1.5 (n = 7) and 3 mm (n = 6) rostral to obex. The respiratory activity of these preparations was assessed via recordings of phrenic and vagal nerves and lumbar spinal expiratory motor output. Preparations with a priori transection at level of the caudal brain stem did not produce stable rhythmic respiratory bursting, even when the arterial chemoreceptors were stimulated with sodium cyanide (NaCN). Reperfusion of brain stems that preserved the pre-Bötzinger complex (pre-BötC) showed spontaneous and sustained rhythmic respiratory bursting at low phrenic nerve activity (PNA) amplitude that occurred simultaneously in all respiratory motor outputs. We refer to this rhythm as the pre-BötC burstlet-type rhythm. Conserving circuitry up to the pontomedullary junction consistently produced robust high-amplitude PNA at lower burst rates, whereas sequential motor patterning across the respiratory motor outputs remained absent. Some of the rostrally transected preparations expressed both burstlet-type and regular PNA amplitude rhythms. Further analysis showed that the burstlet-type rhythm and high-amplitude PNA had 1:2 quantal relation, with burstlets appearing to trigger high-amplitude bursts. We conclude that no degenerate rhythmogenic circuits are located in the caudal medulla oblongata and confirm the pre-BötC as the primary rhythmogenic kernel. The absence of sequential motor patterning in a priori transected preparations suggests that pontine circuits govern respiratory pattern formation. PMID:26888109

Testing the hypothesis of neurodegeneracy in respiratory network function with a priori transected arterially perfused brain stem preparation of rat.

PubMed

Jones, Sarah E; Dutschmann, Mathias

2016-05-01

Degeneracy of respiratory network function would imply that anatomically discrete aspects of the brain stem are capable of producing respiratory rhythm. To test this theory we a priori transected brain stem preparations before reperfusion and reoxygenation at 4 rostrocaudal levels: 1.5 mm caudal to obex (n = 5), at obex (n = 5), and 1.5 (n = 7) and 3 mm (n = 6) rostral to obex. The respiratory activity of these preparations was assessed via recordings of phrenic and vagal nerves and lumbar spinal expiratory motor output. Preparations with a priori transection at level of the caudal brain stem did not produce stable rhythmic respiratory bursting, even when the arterial chemoreceptors were stimulated with sodium cyanide (NaCN). Reperfusion of brain stems that preserved the pre-Bötzinger complex (pre-BötC) showed spontaneous and sustained rhythmic respiratory bursting at low phrenic nerve activity (PNA) amplitude that occurred simultaneously in all respiratory motor outputs. We refer to this rhythm as the pre-BötC burstlet-type rhythm. Conserving circuitry up to the pontomedullary junction consistently produced robust high-amplitude PNA at lower burst rates, whereas sequential motor patterning across the respiratory motor outputs remained absent. Some of the rostrally transected preparations expressed both burstlet-type and regular PNA amplitude rhythms. Further analysis showed that the burstlet-type rhythm and high-amplitude PNA had 1:2 quantal relation, with burstlets appearing to trigger high-amplitude bursts. We conclude that no degenerate rhythmogenic circuits are located in the caudal medulla oblongata and confirm the pre-BötC as the primary rhythmogenic kernel. The absence of sequential motor patterning in a priori transected preparations suggests that pontine circuits govern respiratory pattern formation. Copyright © 2016 the American Physiological Society.

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Defining Subpopulations of Arcuate Nucleus GABA Neurons in Male, Female, and Prenatally Androgenized Female Mice.

PubMed

Marshall, Christopher J; Desroziers, Elodie; McLennan, Timothy; Campbell, Rebecca E

2017-01-01

Arcuate nucleus (ARN) γ-aminobutyric acid (GABA) neurons are implicated in many critical homeostatic mechanisms, from food intake to fertility. To determine the functional relevance of ARN GABA neurons, it is essential to define the neurotransmitters co-expressed with and potentially co-released from ARN GABA neurons. The present study investigated the expression of markers of specific signaling molecules by ARN GABA neurons in brain sections from male, female, and, in some cases, prenatally androgen-treated (PNA) female, vesicular GABA transporter (VGaT)-ires-Cre/tdTomato reporter mice. Immunofluorescence for kisspeptin, β-endorphin, neuropeptide Y (NPY), tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS) was detected by confocal microscopy, and co-localization with tdTomato VGaT reporter expression throughout the ARN was quantified. GABA neurons rarely co-localized with kisspeptin (<2%) or β-endorphin (<1%), and only a small proportion of kisspeptin (∼10%) or β-endorphin (∼3%) neurons co-localized with VGaT in male and female mice. In contrast, one-third of ARN GABA neurons co-localized with NPY, and nearly all NPY neurons (>95%) co-localized with VGaT across groups. Both TH and nNOS labeling was co-localized with ∼10% of ARN GABA neurons. The proportion of TH neurons co-localized with VGaT was significantly greater in males than either control or PNA females, and the proportion of nNOS neurons co-localizing VGaT was higher in control and PNA females compared with males. These data highlight NPY as a significant subpopulation of ARN GABA neurons, demonstrate no significant impact of PNA on signal co-expression, and, for the first time, show sexually dimorphic co-expression patterns of TH and nNOS with ARN GABA neurons. © 2016 S. Karger AG, Basel.

Evaluation of the acrosomal status in Lama glama sperm incubated with acrosome reaction inducers.

PubMed

Carretero, M I; Fumuso, F G; Neild, D M; Giuliano, S M; Cetica, P; Miragaya, M H

2015-09-01

The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP-BSA medium. An aliquot was sonicated to remove the acrosomal content (positive control). The rest of the sample was incubated for 3h at 38 °C with 5% CO2 and 100% humidity. Three aliquots were further incubated 1h with one of the following AR inducers: calcium ionophore, ionomycin or progesterone. One without inducers and the other, incubated with dimethyl sulfoxide (vehicle of the inducing agents). Acrosomes were evaluated at time 0 and after 4h incubation. Calcium ionophore was the most potent agent for inducing the AR (67.2 ± 14.4% live+dead AR sperm) (P < 0.05). These samples showed no motility and viability was very low (0-30%). Both ionomycin and progesterone presented significantly higher (P < 0.05) percentages of total AR sperm than the controls, but had similar percentages of dead reacted sperm to the controls. A positive correlation was observed between the intact acrosome FITC-PNA/PI pattern (live+dead sperm) and the acrosome-present CB pattern (r = 0.64; P = 0.000) in all the evaluated samples. the FITC-PNA/PI technique simultaneously evaluates viability and acrosomal status in llama spermatozoa and calcium ionophore could be used as a control of AR. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of functional variables in epididymal alpaca (Vicugna pacos) sperm using imaging flow cytometry.

PubMed

Santiani, Alexei; Ugarelli, Alejandra; Evangelista-Vargas, Shirley

2016-10-01

Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of group B streptococci in Lim broth by use of group B streptococcus peptide nucleic acid fluorescent in situ hybridization and selective and nonselective agars.

PubMed

Montague, Naomi S; Cleary, Timothy J; Martinez, Octavio V; Procop, Gary W

2008-10-01

The sensitivity, specificity, and positive and negative predictive values for the detection of group B streptococci from Lim enrichment broth with sheep blood agar (SBA), with selective Streptococcus agar (SSA), and by a peptide nucleic acid fluorescent in situ hybridization (PNA FISH) assay were as follows: for culture on SBA, 68.4%, 100%, 100%, and 87.9%, respectively; for culture on SSA, 85.5%, 100%, 100%, and 94.1%, respectively; and for the PNA FISH assay, 97.4%, 98.3%, 96.1%, and 98.9%, respectively.

Detection of Group B Streptococci in Lim Broth by Use of Group B Streptococcus Peptide Nucleic Acid Fluorescent In Situ Hybridization and Selective and Nonselective Agars▿

PubMed Central

Montague, Naomi S.; Cleary, Timothy J.; Martinez, Octavio V.; Procop, Gary W.

2008-01-01

The sensitivity, specificity, and positive and negative predictive values for the detection of group B streptococci from Lim enrichment broth with sheep blood agar (SBA), with selective Streptococcus agar (SSA), and by a peptide nucleic acid fluorescent in situ hybridization (PNA FISH) assay were as follows: for culture on SBA, 68.4%, 100%, 100%, and 87.9%, respectively; for culture on SSA, 85.5%, 100%, 100%, and 94.1%, respectively; and for the PNA FISH assay, 97.4%, 98.3%, 96.1%, and 98.9%, respectively. PMID:18667597

Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.

PubMed

Norregaard, Kamilla; Andersson, Magnus; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B

2014-09-01

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycoclusters to lectins and tetanus toxin C-fragment

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Minoura, Norihiko

2011-03-01

We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)3] shows a new emission peak and the FP value of [Ru(bpy-2Gal)3] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (Kd) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)3] to TCF was confirmed by the FP measurement.

DNA biosensors implemented on PNA-functionalized microstructured optical fibers Bragg gratings

NASA Astrophysics Data System (ADS)

Candiani, A.; Giannetti, S.; Cucinotta, A.; Bertucci, A.; Manicardi, A.; Konstantaki, M.; Margulis, W.; Pissadakis, S.; Corradini, R.; Selleri, S.

2013-05-01

A novel DNA sensing platform based on a Peptide Nucleic Acid - functionalized Microstructured Optical Fibers gratings has been demonstrated. The inner surface of different MOFs has been functionalized using PNA probes, OligoNucleotides mimic that are well suited for specific DNA target sequences detection. The hybrid sensing systems were tested for optical DNA detection of targets of relevance in biomedical application, using the cystic fibrosis gene mutation, and food-analysis, using the genomic DNA from genetic modified organism soy flour. After the solutions of DNA molecules has been infiltrated inside the fibers capillaries and hybridization has occurred, oligonucleotidefunctionalized gold nanoparticles were infiltrated and used to form a sandwich-like system to achieve signal amplification. Spectral measurements of the reflected signal reveal a clear wavelength shift of the reflected modes when the infiltrated complementary DNA matches with the PNA probes placed on the inner fiber surface. Measurements have also been made using the mismatched DNA solution for the c, containing a single nucleotide polymorphism, showing no significant changes in the reflected spectrum. Several experiments have been carried out demonstrating the reproducibility of the results and the high selectivity of the sensors, showing the simplicity and the potential of this approach.

Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA.

PubMed

Machnik, Grzegorz; Skudrzyk, Estera; Bułdak, Łukasz; Ruczyński, Jarosław; Kozłowska, Agnieszka; Mucha, Piotr; Rekowski, Piotr; Szkróbka, Witold; Basiak, Marcin; Bołdys, Aleksandra; Sławska, Helena; Okopień, Bogusław

2018-02-01

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.

The effect of acamprosate on alcohol and food craving in patients with alcohol dependence.

PubMed

Han, Doug Hyun; Lyool, In Kyoon; Sung, Young Hoon; Lee, Sang Hoon; Renshaw, Perry F

2008-03-01

The balance between inhibitory (gamma aminobutyric acid; GABAergic) and excitatory (glutamatergic) neurotransmission is thought to be associated with craving for alcohol and food. The anticraving effect of acamprosate is thought to be mediated through modifying the balance of GABA and glutamate. Recent studies in animals have suggested that acamprosate may have non-selective effects on craving for both alcohol and food. The influence of acamprosate for reducing craving for alcohol and food was assessed in 204 in-patients with alcohol dependence (96 patients treated with acamprosate, PWA; 108 patients were not treated PNA) was assessed at baseline and following 1, 2, and 4 weeks of treatment. There was a significant reduction in craving for alcohol over 4 weeks of treatment in both PWA and PNA groups, but without significant group differences. In contrast, a reduction in food craving was observed only in the PWA group. In addition, there was a significant increase of body mass index (BMI) in the PNA group but not the PWA group over the 4-week period. These results demonstrate acamprosate nonselective effects on craving for drinking and eating in alcoholic patients.

Fischer carbene mediated covalent grafting of a peptide nucleic acid on gold surfaces and IR optical detection of DNA hybridization with a transition metalcarbonyl label

NASA Astrophysics Data System (ADS)

Srivastava, Pratima; Ghasemi, Mahsa; Ray, Namrata; Sarkar, Amitabha; Kocabova, Jana; Lachmanova, Stepanka; Hromadova, Magdalena; Boujday, Souhir; Cauteruccio, Silvia; Thakare, Pramod; Licandro, Emanuela; Fosse, Céline; Salmain, Michèle

2016-11-01

Amine-reactive surfaces comprising N-hydroxysuccinimide ester groups as well as much more unusual Fischer alkoxymetallocarbene groups were generated on gold-coated surfaces via self-assembled monolayers of carboxy- and azido-terminated thiolates, respectively. These functions were further used to immobilize homothymine peptide nucleic acid (PNA) decamer in a covalent fashion involving the primary amine located at its N-terminus. These stepwise processes were monitored by polarization modulation reflection - absorption infrared spectroscopy (PM-RAIRS) that gave useful information on the molecular composition of the organic layers. PNA grafting and hybridization with complementary DNA strand were successfully transduced by quartz crystal microbalance (QCM) measurements. Unfortunately, attempts to transduce the hybridization optically by IR in a label-free fashion were inconclusive. Therefore we undertook to introduce an IR reporter group, namely a transition metalcarbonyl (TMC) entity at the 5‧ terminus of complementary DNA. Evidence for the formation of PNA-DNA heteroduplex was brought by the presence of ν(Ctbnd O) bands in the 2000 cm-1 region of the IR spectrum of the gold surface owing to the metalcarbonyl label.

A safe and reliable neutralization assay based on pseudovirus to measure neutralizing antibody titer against poliovirus.

PubMed

Liu, Shaohua; Song, Dongmei; Bai, Han; Lu, Weiwei; Dai, Xinxian; Hao, Chunsheng; Zhang, Zhongyang; Guo, Huijie; Zhang, Yue; Li, Xiuling

2017-12-01

With the promotion of inactivated poliomyelitis vaccine (IPV) and live attenuated oral poliomyelitis vaccine (OPV), the global reported cases of poliomyelitis have reduced sharply from 0.35 million in 1988 to 74 in 2015. The Polio Eradication & Endgame Strategic Plan published by WHO in 2013 included the strategy of implementation of poliovirus safe handling and containment measures to minimize the risks of facility-associated reintroduction of virus into the polio-free community to prevent the re-import of poliovirus. Toward this strategy, we produced replication-incompetent pseudovirus of poliovirus type 1, 2, 3 attenuated strains by constructing poliovirus capsid expression vectors and poliovirus replicon then transfecting HEK293T cells and developed a pseudovirus-based neutralization assay (pNA) to determine neutralizing antibody titer which is more secure, time-saving and reliable than conventional neutralization assay (cNA). By using anti-poliovirus rat serum, we demonstrated excellent correlation between neutralizing antibody titers measured by cNA and pNA. It was concluded that pNA can be a potential alternative to replace cNA as a safe and time-saving system for titer determination after live poliovirus's safekeeping. © 2017 Wiley Periodicals, Inc.

Growth of binary organic NLO crystals: m.NA-p.NA and m.NA-CNA system

NASA Technical Reports Server (NTRS)

Singh, N. B.; Henningsen, T.; Hopkins, R. H.; Mazelsky, R.

1993-01-01

Experiments were carried out to grow 3.Nitroaniline (m.NA) crystals doped with 4.Nitroaniline (p.NA) and 2.chloro 4.Nitroaniline (CNA). The measured undercooling for m.NA, p.NA, and CNA were 0.21 tm K, 0.23 tm K, and 0.35 tm K respectively, where tm represents the melting temperature of the pure component. Because of the crystals' large heat of fusion and large undercooling, it was not possible to grow good quality crystals with low thermal gradients. In the conventional two-zone Bridgman furnace we had to raise the temperature of the hot zone above the decomposition temperature of CNA, p.NA, and m.NA to achieve the desired thermal gradient. To avoid decomposition, we used an unconventional Bridgman furnace. Two immiscible liquids, silicone oil and ethylene glycol, were used to build a special two-zone Bridgman furnace. A temperature gradient of 18 K/cm was achieved without exceeding the decomposition temperature of the crystal. The binary crystals, m.NA-p.NA and m.NA-CNA, were grown in centimeter size in this furnace. X-ray and optical characterization showed good optical quality.

Functionalization of DNA Nanostructures for Cell Signaling Applications

NASA Astrophysics Data System (ADS)

Pedersen, Ronnie O.

Transforming growth factor beta (TGF-beta) is an important cytokine responsible for a wide range of different cellular functions including extracellular matrix formation, angiogenesis and epithelial-mesenchymal transition. We have sought to use self-assembling DNA nanostructures to influence TGF-beta signaling. The predictable Watson Crick base pairing allows for designing self-assembling nanoscale structures using oligonucleotides. We have used the method of DNA origami to assemble structures functionalized with multiple peptides that bind TGF-beta receptors outside the ligand binding domain. This allows the nanostructures to cluster TGF-beta receptors and lower the energy barrier of ligand binding thus sensitizing the cells to TGF-beta stimulation. To prove efficacy of our nanostructures we have utilized immunofluorescent staining of Smad2/4 in order to monitor TGF-beta mediated translocation of Smad2/4 to the cell nucleus. We have also utilized Smad2/4 responsive luminescence constructs that allows us to quantify TGF-beta stimulation with and without nanostructures. To functionalize our nanostructures we relied on biotin-streptavidin linkages. This introduces a multivalency that is not necessarily desirable in all designs. Therefore we have investigated alternative means of functionalization. The first approach is based on targeting DNA nanostructure by using zinc finger binding proteins. Efficacy of zinc finger binding proteins was assayed by the use of enzyme-linked immunosorbent (ELISA) assay and atomic force microscopy (AFM). While ELISA indicated a relative specificity of zinc finger proteins for target DNA sequences AFM showed a high degree of non-specific binding and insufficient affinity. The second approach is based on using peptide nucleic acid (PNA) incorporated in the nanostructure through base pairing. PNA is a synthetic DNA analog consisting of a backbone of repeating N-(2-aminoethyl)-glycine units to which purine and pyrimidine bases are linked by amide bonds. The solid phase synthesis of PNA allows for convenient extension of the backbone into a peptide segment enabling peptide functionalization of DNA nanostructures. We have investigated how the neutral character of PNA alters the incorporation in DNA based nanostructures compared to a DNA control using biotinylation and AFM. Results indicate that PNA can successfully be used as a way of functionalizing DNA nanostructures. Additionally we have shown that functionalized nanostructures are capable of sensitizing cells to TGF-beta stimulation.

First-principles study of crystal structure, elastic stiffness constants, piezoelectric constants, and spontaneous polarization of orthorhombic Pna21-M2O3 (M = Al, Ga, In, Sc, Y)

NASA Astrophysics Data System (ADS)

Shimada, Kazuhiro

2018-03-01

We perform first-principles calculations to investigate the crystal structure, elastic and piezoelectric properties, and spontaneous polarization of orthorhombic M2O3 (M = Al, Ga, In, Sc, Y) with Pna21 space group based on density functional theory. The lattice parameters, full elastic stiffness constants, piezoelectric stress and strain constants, and spontaneous polarization are successfully predicted. Comparison with available experimental and computational results indicates the validity of our computational results. Detailed analysis of the results clarifies the difference in the bonding character and the origin of the strong piezoelectric response and large spontaneous polarization.

2-Methoxypyridine as a Thymidine Mimic in Watson-Crick Base Pairs of DNA and PNA: Synthesis, Thermal Stability, and NMR Structural Studies.

PubMed

Novosjolova, Irina; Kennedy, Scott D; Rozners, Eriks

2017-11-02

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A-T base pair. The hypothesis was that including protonation in the Watson-Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA-DNA duplexes and ≈20 °C in PNA-DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P-A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In-situ restoration of one-stage partial nitritation-anammox process deteriorated by nitrate build-up via elevated substrate levels

PubMed Central

Wang, Xiaolong; Gao, Dawen

2016-01-01

The one-stage partial nitritation and anammox process (PN/A) has been a promising microbial process to remove ammonia from wastewater especially with low carbon/nitrogen ratio. The main breakdown was the deterioration caused by overgrowth of nitrite oxidizing bacteria (NOB) resulting effluent nitrate build-up in the PN/A process. This study presented an in-situ restoring strategy for suppressing NOB activity in a one-stage granular PN/A system deteriorated over 2 months, using elevated concentrations of substrates (ammonia and nitrite) under limited dissolved oxygen level. The results showed that the NOB activity was successfully suppressed after 56 days of restoration, and finally the ratio of produced nitrate/consumed ammonium was reduced from 36.8% to 7%. On day 66 the nitrogen removal rate obtained as 1.2 kg N/(m3·d). The high FA level (5–40 mg/L) and low dissolved oxygen (<0.13 mg/L) were responsible for NOB suppression. From quantitative PCR (qPCR) analysis, after this restoration, anammox bacteria had a widely growth, and AOB stay stable, but Nitrospira increase and Nitrobacter declined. High amount of NOB was still persistent in the granules, which was not easy to wash-out and threaten the deammonification performance. PMID:27881860

Midcontinental Native American population dynamics and late Holocene hydroclimate extremes

NASA Astrophysics Data System (ADS)

Bird, Broxton W.; Wilson, Jeremy J.; Gilhooly, William P., III; Steinman, Byron A.; Stamps, Lucas

2017-01-01

Climate’s influence on late Pre-Columbian (pre-1492 CE), maize-dependent Native American populations in the midcontinental United States (US) is poorly understood as regional paleoclimate records are sparse and/or provide conflicting perspectives. Here, we reconstruct regional changes in precipitation source and seasonality and local changes in warm-season duration and rainstorm events related to the Pacific North American pattern (PNA) using a 2100-year-long multi-proxy lake-sediment record from the midcontinental US. Wet midcontinental climate reflecting negative PNA-like conditions occurred during the Medieval Climate Anomaly (950-1250 CE) as Native American populations adopted intensive maize agriculture, facilitating population aggregation and the development of urban centers between 1000-1200 CE. Intensifying midcontinental socio-political instability and warfare between 1250-1350 CE corresponded with drier positive PNA-like conditions, culminating in the staggered abandonment of many major Native American river valley settlements and large urban centers between 1350-1450 CE during an especially severe warm-season drought. We hypothesize that this sustained drought interval rendered it difficult to support dense populations and large urban centers in the midcontinental US by destabilizing regional agricultural systems, thereby contributing to the host of socio-political factors that led to population reorganization and migration in the midcontinent and neighboring regions shortly before European contact.

Pollution-Induced Community Tolerance To Diagnose Hazardous Chemicals in Multiple Contaminated Aquatic Systems.

PubMed

Rotter, Stefanie; Gunold, Roman; Mothes, Sibylle; Paschke, Albrecht; Brack, Werner; Altenburger, Rolf; Schmitt-Jansen, Mechthild

2015-08-18

Aquatic ecosystems are often contaminated with large numbers of chemicals, which cannot be sufficiently addressed by chemical target analyses. Effect-directed analysis (EDA) enables the identification of toxicants in complex contaminated environmental samples. This study suggests pollution-induced community tolerance (PICT) as a confirmation tool for EDA to identify contaminants which actually impact on local communities. The effects of three phytotoxic compounds local periphyton communities, cultivated at a reference (R-site) and a polluted site (P-site), were assessed to confirm the findings of a former EDA study on sediments. The sensitivities of R- and P-communities to prometryn, tributyltin (TBT) and N-phenyl-2-naphthylamine (PNA) were quantified in short-term toxicity tests and exposure concentrations were determined. Prometryn and PNA concentrations were significantly higher at the P-site, whereas TBT concentrations were in the same range at both sites. Periphyton communities differed in biomass, but algal class composition and diatom diversity were similar. Community tolerance of P-communities was significantly enhanced for prometryn, but not for PNA and TBT, confirming site-specific effects on local periphyton for prometryn only. Thus, PICT enables in situ effect confirmation of phytotoxic compounds at the community level and seems to be suitable to support confirmation and enhance ecological realism of EDA.

DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity.

PubMed

Usui, Kenji; Okada, Arisa; Sakashita, Shungo; Shimooka, Masayuki; Tsuruoka, Takaaki; Nakano, Shu-Ichi; Miyoshi, Daisuke; Mashima, Tsukasa; Katahira, Masato; Hamada, Yoshio

2017-11-16

The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca 2+ , and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex <--> DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire <--> particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.

Midcontinental Native American population dynamics and late Holocene hydroclimate extremes

PubMed Central

Bird, Broxton W.; Wilson, Jeremy J.; Gilhooly III, William P.; Steinman, Byron A.; Stamps, Lucas

2017-01-01

Climate’s influence on late Pre-Columbian (pre-1492 CE), maize-dependent Native American populations in the midcontinental United States (US) is poorly understood as regional paleoclimate records are sparse and/or provide conflicting perspectives. Here, we reconstruct regional changes in precipitation source and seasonality and local changes in warm-season duration and rainstorm events related to the Pacific North American pattern (PNA) using a 2100-year-long multi-proxy lake-sediment record from the midcontinental US. Wet midcontinental climate reflecting negative PNA-like conditions occurred during the Medieval Climate Anomaly (950–1250 CE) as Native American populations adopted intensive maize agriculture, facilitating population aggregation and the development of urban centers between 1000–1200 CE. Intensifying midcontinental socio-political instability and warfare between 1250–1350 CE corresponded with drier positive PNA-like conditions, culminating in the staggered abandonment of many major Native American river valley settlements and large urban centers between 1350–1450 CE during an especially severe warm-season drought. We hypothesize that this sustained drought interval rendered it difficult to support dense populations and large urban centers in the midcontinental US by destabilizing regional agricultural systems, thereby contributing to the host of socio-political factors that led to population reorganization and migration in the midcontinent and neighboring regions shortly before European contact. PMID:28139698

Soil aggregate stratification of nematodes and ammonia oxidizers affects nitrification in an acid soil.

PubMed

Jiang, Yuji; Jin, Chen; Sun, Bo

2014-10-01

Nitrification plays a central role in global nitrogen cycle, which is affected by interaction between soil microfauna and microorganisms. The impact of synchronized changes in nematodes and ammonia oxidizers within aggregate fractions on nitrification was investigated in an acid soil under 10-year manure application. Nematodes, ammonia oxidizers and potential nitrification activity (PNA) were examined in three soil aggregate fractions under four fertilization regimes. Pyrosequencing data revealed that the dominant bacterial amoA operational taxonomic units (OTUs) were related to Nitrosospira species, while archaeal OTUs were affiliated with Nitrososphaera and Nitrosotalea species. PNA was more strongly correlated with ammonia-oxidizing bacteria (AOB) abundance than ammonia-oxidizing archaea (AOA) abundance, although AOA were dominant in the acid soil. Plant parasites had a negative effect on AOB abundance; however, bacterivores stimulated AOB abundance and contributed more to PNA than plant parasites. Aggregate fractions exerted significant impacts on AOA abundance and AOB community composition. Total carbon content strongly affected the abundance and composition of AOA community, while soil pH primarily affected that of AOB community. Soil variables explained 62.7% and 58.1% variations, and nematode variables explained 11.7% and 19.5% variations in the AOA and AOB community composition respectively. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

In vivo visualization of endogenous miR-21 using hyaluronic acid-coated graphene oxide for targeted cancer therapy.

PubMed

Hwang, Do Won; Kim, Han Young; Li, Fangyuan; Park, Ji Yong; Kim, Dohyun; Park, Jae Hyung; Han, Hwa Seung; Byun, Jung Woo; Lee, Yun-Sang; Jeong, Jae Min; Char, Kookheon; Lee, Dong Soo

2017-03-01

Oncogene-targeted nucleic acid therapy has been spotlighted as a new paradigm for cancer therapeutics. However, in vivo delivery issues and uncertainty of therapeutic antisense drug reactions remain critical hurdles for a successful targeted cancer therapy. In this study, we developed a fluorescence-switchable theranostic nanoplatform using hyaluronic acid (HA)-conjugated graphene oxide (GO), which is capable of both sensing oncogenic miR-21 and inhibiting its tumorigenicity simultaneously. Cy3-labeled antisense miR-21 peptide nucleic acid (PNA) probes loaded onto HA-GO (HGP21) specifically targeted CD44-positive MBA-MB231 cells and showed fluorescence recovery by interacting with endogenous miR-21 in the cytoplasm of the MBA-MB231 cells. Knockdown of endogenous miR-21 by HGP21 led to decreased proliferation and reduced migration of cancer cells, as well as the induction of apoptosis, with enhanced PTEN levels. Interestingly, in vivo fluorescence signals markedly recovered 3 h after the intravenous delivery of HGP21 and displayed signals more than 5-fold higher than those observed in the HGPscr-treated group of tumor-bearing mice. These findings demonstrate the possibility of using the HGP nanoplatform as a cancer theranostic tool in miRNA-targeted therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Climate variability from the Florida Bay sedimentary record: Possible teleconnections to ENSO, PNA and CNP

USGS Publications Warehouse

Cronin, T. M.; Dwyer, Gary S.; Schwede, S.B.; Vann, C.D.; Dowsett, H.

2002-01-01

We analyzed decadal and interannual climate variability in South Florida since 1880 using geochemical and faunal paleosalinity indicators from isotopically dated sediment cores at Russell Bank in Florida Bay (FB). Using the relative abundance of 2 ostracode species and the Mg/Ca ratios in Loxoconcha matagordensis shells to reconstruct paleosalinity, we found evidence for cyclic oscillations in the salinity of central FB. During this time salinity fluctuated from as low as ~18 parts per thousand (ppt) to as high as ~57 ppt. Time series analyses suggest, in addition to a 5.6 yr Mg/Ca based salinity periodicity, there are 3 other modes of variability in paleosalinity indicators: 6-7, 8-9, and 13-14 yr periods which occur in all paleo-proxies. To search for factors that might cause salinity to vary in FB, we compared the Russell Bank paleosalinity record to South Florida winter rainfall, the Southern Oscillation Index (SOI), winter North Atlantic Oscillation (NAO), and the winter Pacific North American (PNA) index, and a surrogate for the PNA in the winter season, the Central North Pacific (CNP) index. SOI and PNA/CNP appear to be associated with South Florida winter precipitation. Time series analyses of SOI and winter rainfall for the period 1910-1999 suggest ~5, 6-7, 8-9 and 13-14 yr cycles. The 6-7 yr and 13-14 yr cycles correspond to those observed in the faunal and geochemical time series from Russell Bank. The main periods of the CNP index are 5-6 and 13-15 yr, which are similar to those observed in FB paleosalinity. Cross-spectral analyses show that winter rainfall and salinity are coherent at 5.6 yr with a salinity lag of ~1.6 mo. These results suggest that regional rainfall variability influences FB salinity over interannual and decadal timescales and that much of this variability may have its origin in climate variability in the Pacific Ocean/atmosphere system.

The efficacy of hypotonic and near-isotonic saline for parenteral fluid therapy given at low maintenance rate in preventing significant change in plasma sodium in post-operative pediatric patients: protocol for a prospective randomized non-blinded study

PubMed Central

2011-01-01

Background Hyponatremia is the most frequent electrolyte abnormality observed in post-operative pediatric patients receiving intravenous maintenance fluid therapy. If plasma sodium concentration (p-Na+) declines to levels below 125 mmol/L in < 48 h, transient or permanent brain damage may occur. There is an intense debate as to whether the administered volume (full rate vs. restricted rate of infusion) and the composition of solutions used for parenteral maintenance fluid therapy (hypotonic vs. isotonic solutions) contribute to the development of hyponatremia. So far, there is no definitive pediatric data to support a particular choice of parenteral fluid for maintenance therapy in post-surgical patients. Methods/Design Our prospective randomized non-blinded study will be conducted in healthy children and adolescents aged 1 to 14 years who have been operated for acute appendicitis. Patients will be randomized either to intravenous hypotonic (0.23% or 0.40% sodium chloride in glucose, respectively) or near-isotonic (0.81% sodium chloride in glucose) solution given at approximately three-fourths of the average maintenance rate. The main outcome of interest from this study is to evaluate 24 h post-operatively whether differences in p-Na+ between treatment groups are large enough to be of clinical relevance. In addition, water and electrolyte balance as well as regulatory hormones will be measured. Discussion This study will provide valuable information on the efficacy of hypotonic and near-isotonic fluid therapy in preventing a significant decrease in p-Na+. Finally, by means of careful electrolyte and water balance and by measuring regulatory hormones our results will also contribute to a better understanding of the physiopathology of post-operative changes in p-Na+ in a population at risk for hyponatremia. Trial registration The protocol for this study is registered with the current controlled trials registry; registry number: ISRCTN43896775. PMID:21729308

Observed and Aogcm Simulated Relationships Between us Wind Speeds and Large Scale Modes of Climate Variability

NASA Astrophysics Data System (ADS)

Schoof, J. T.; Pryor, S. C.; Barthelmie, R. J.

2013-12-01

Previous research has indicated that large-scale modes of climate variability, such as El Niño - Southern Oscillation (ENSO), the Arctic Oscillation (AO) and the Pacific-North American pattern (PNA), influence the inter-annual and intra-annual variability of near-surface and upper-level wind speeds over the United States. For example, we have shown that rawinsonde derived wind speeds indicate that 90th percentile of wind speeds at 700 hPa over the Pacific Northwest and Southwestern USA are significantly higher under the negative phase of the PNA, and the Central Plains experiences higher wind speeds at 850 hPa under positive phase Southern Oscillation index while the Northeast exhibits higher wind speeds at 850 hPa under positive phase NAO. Here, we extend this research by further investigating these relationships using both reanalysis products and output from coupled atmosphere-ocean general circulation models (AOGCMs) developed for the 5th Phase of the Coupled Model Intercomparison Project (CMIP5). The research presented has two specific goals. First, we evaluate the AOGCM simulations in terms of their ability to represent the temporal and spatial representations of ENSO, the AO, and the PNA pattern relative to historical observations. The diagnostics used include calculation of the power spectra (and thus representation of the fundamental frequencies of variability) and Taylor diagrams (for comparative assessment of the spatial patterns and their intensities). Our initial results indicate that most AOGCMs produce modes that are qualitatively similar to those observed, but that differ slightly in terms of the spatial pattern, intensity of specific centers of action, and variance explained. Figure 1 illustrates an example of the analysis of the frequencies of variability of two climate modes for the NCEP-NCAR reanalysis (NNR) and a single AOGCM (BCC CSM1). The results show a high degree of similarity in the power spectra but for this AOGCM the variance of the PNA associated with high frequencies are amplified relative to those in NNR. Second, we quantify the observed and AOGCM-simulated relationships between ENSO, AO, and PNA indices and zonal and meridional wind components at multiple levels for the contiguous United States. The results are presented in form of maps displaying the strength of the relationship at different timescales, from daily to annual, and at multiple atmospheric levels, from 10m to 500 mb. The results of the analysis are used to provide context for regional wind climate projections based on 21st century AOGCM simulations.

Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

NASA Astrophysics Data System (ADS)

Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

2015-01-01

MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression. Electronic supplementary information (ESI) available: Extra figures and tables. See DOI: 10.1039/c4nr05447d

Supramolecular nanofiber of pyrene-lactose conjugates and its two-photon fluorescence imaging.

PubMed

Sun, Qian; Zhu, Hong-Yu; Wang, Jun-Fang; Chen, Xiao; Wang, Ke-Rang; Li, Xiao-Liu

2018-04-23

A lactose modified pyrene derivative (Py-Lac) was synthesized, with which novel twisted supramolecular nanofibers in diameter about 20 nm were constructed by self-assembly. The nanofibers showed solid-state fluorescence between 400 nm and 650 nm with the maximum emission at 495 nm. Furthermore, its recognition reaction with PNA lectin was investigated by fluorescence spectra and turbidity assays. It is interesting found that the supramolecular assembly as multivalent glycocluster exhibited unique and selectively binding interactions with PNA lectin with the binding constant of 5.74 × 10 6  M -1 . Moreover, compound Py-Lac showed two-photon fluorescence imaging with Hep G2 cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Ionizing radiation in the field of hydrogels used for agriculture and medicine

NASA Astrophysics Data System (ADS)

Radoiu, M.; Martin, D.; Oproiu, C.; Toma, M.; Popescu, A. S.; Bestea, V.; Dragusin, M.; Moraru, R.; Calinescu, I.; Manea, A.

1999-01-01

Some hydrogel types, obtained by gamma ray and electron beam irradiation, such as homopolymers of acrylamide (pAA type), co-polymers of acrylamide and sodium acrylate (pAANA type), homo-polymers of sodium acrylate (pNA type) and homo-polymers of 2-hydroxyethylmethacrylate (pHEMA type), are presented. The effects of the solution's chemical composition, swelling medium nature, radiation absorbed dose and radiation absorbed dose rate upon the swelling degree and mechanical strength of pAA, pAANA, pNA types are discussed. For the pHEMA type, which are reinforced in the polyester network, the studies concerning the influence of the irradiation parameters and chemical composition upon the shape stability after swelling and surface's roughness are also discussed.

Winter Precipitation in North America and the Pacific-North America Pattern in GEOS-S2Sv2 Seasonal Hindcast

NASA Technical Reports Server (NTRS)

Li, Zhao; Molod, Andrea; Schubert, Siegfried

2018-01-01

Reliable prediction of precipitation remains one of the most pivotal and complex challenges in seasonal forecasting. Previous studies show that various large-scale climate modes, such as ENSO, PNA and NAO play significant role in winter precipitation variability over the Northern America. The influences are most pronounced in years of strong indices of such climate modes. This study evaluates model bias, predictability and forecast skills of monthly winter precipitation in GEOS5-S2S 2.0 retrospective forecast from 1981 to 2016, with emphasis on the forecast skill of precipitation over North America during the extreme events of ENSO, PNA and NAO by applying EOF and composite analysis.

Efficient systemic DNA delivery to the tumor by self-assembled nanoparticle

NASA Astrophysics Data System (ADS)

Tang, Hailin; Xie, Xinhua; Guo, Jiaoli; Wei, Weidong; Wu, Minqing; Liu, Peng; Kong, Yanan; Yang, Lu; Hung, Mien-Chie; Xie, Xiaoming

2014-01-01

There are few delivery agents that could deliver gene with high efficiency and low toxicity, especially for animal experiments. Therefore, creating vectors with good delivery efficiency and safety profile is a meaningful work. We have developed a self-assembled gene delivery system (XM001), which can more efficiently deliver DNA to multiple cell lines and breast tumor, as compared to commercial delivery agents. In addition, systemically administrated XM001-BikDD (BikDD is a mutant form of proapoptotic gene Bik) significantly inhibited the growth of human breast cancer cells and prolonged the life span in implanted nude mice. This study demonstrates that XM001 is an efficient and widespread transfection agent, which could be a promising tumor delivery vector for cancer targeted therapy.

Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells.

PubMed

Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Kobayashi, Shouhei; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

2016-08-01

Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery. © 2016 Federation of European Biochemical Societies.

Nematicidal Activity of Kojic Acid Produced by Aspergillus oryzae against Meloidogyne incognita.

PubMed

Kim, Tae Yoon; Jang, Ja Yeong; Jeon, Sun Jeong; Lee, Hye Won; Bae, Chang-Hwan; Yeo, Joo Hong; Lee, Hyang Burm; Kim, In Seon; Park, Hae Woong; Kim, Jin-Cheol

2016-08-28

The fungal strain EML-DML3PNa1 isolated from leaf of white dogwood (Cornus alba L.) showed strong nematicidal activity with juvenile mortality of 87.6% at a concentration of 20% fermentation broth filtrate at 3 days after treatment. The active fungal strain was identified as Aspergillus oryzae, which belongs to section Flavi, based on the morphological characteristics and sequence analysis of the ITS rDNA, calmodulin (CaM), and β-tubulin (BenA) genes. The strain reduced the pH value to 5.62 after 7 days of incubation. Organic acid analysis revealed the presence of citric acid (515.0 mg/kg), malic acid (506.6 mg/kg), and fumaric acid (21.7 mg/kg). The three organic acids showed moderate nematicidal activities, but the mixture of citric acid, malic acid, and fumaric acid did not exhibit the full nematicidal activity of the culture filtrate of EML- DML3PNa1. Bioassay-guided fractionation coupled with (1)H- and (13)C-NMR and EI-MS analyses led to identification of kojic acid as the major nematicidal metabolite. Kojic acid exhibited dose-dependent mortality and inhibited the hatchability of M. incognita, showing EC50 values of 195.2 µg/ml and 238.3 µg/ml, respectively, at 72 h postexposure. These results suggest that A. oryzae EML-DML3PNa1 and kojic acid have potential as a biological control agent against M. incognita.

Interannual to multidecadal climate forcings on groundwater resources of the U.S. West Coast

USGS Publications Warehouse

Velasco, Elzie M.; Gurdak, Jason J.; Dickinson, Jesse; Ferré, T.P.A.; Corona, Claudia

2017-01-01

Study regionThe U.S. West Coast, including the Pacific Northwest and California Coastal Basins aquifer systems.Study focusGroundwater response to interannual to multidecadal climate variability has important implications for security within the water–energy–food nexus. Here we use Singular Spectrum Analysis to quantify the teleconnections between AMO, PDO, ENSO, and PNA and precipitation and groundwater level fluctuations. The computer program DAMP was used to provide insight on the influence of soil texture, depth to water, and mean and period of a surface infiltration flux on the damping of climate signals in the vadose zone.New hydrological insights for the regionWe find that PDO, ENSO, and PNA have significant influence on precipitation and groundwater fluctuations across a north-south gradient of the West Coast, but the lower frequency climate modes (PDO) have a greater influence on hydrologic patterns than higher frequency climate modes (ENSO and PNA). Low frequency signals tend to be preserved better in groundwater fluctuations than high frequency signals, which is a function of the degree of damping of surface variable fluxes related to soil texture, depth to water, mean and period of the infiltration flux. The teleconnection patterns that exist in surface hydrologic processes are not necessarily the same as those preserved in subsurface processes, which are affected by damping of some climate variability signals within infiltrating water.

Identification of cell surface glycoprotein markers for glioblastoma-derived stem-like cells using a lectin microarray and LC-MS/MS approach

PubMed Central

He, Jintang; Liu, Yashu; Xie, Xiaolei; Zhu, Thant; Soules, Mary; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within two years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. Lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma. PMID:20235609

Influence of copper surfaces on biofilm formation by Legionella pneumophila in potable water.

PubMed

Gião, M S; Wilks, S A; Keevil, C W

2015-04-01

Legionella pneumophila is a waterborne pathogen that can cause Legionnaires' disease, a fatal pneumonia, or Pontiac fever, a mild form of disease. Copper is an antimicrobial material used for thousands of years. Its incorporation in several surface materials to control the transmission of pathogens has been gaining importance in the past decade. In this work, the ability of copper to control the survival of L. pneumophila in biofilms was studied. For that, the incorporation of L. pneumophila in polymicrobial drinking water biofilms formed on copper, PVC and PEX, and L. pneumophila mono-species biofilms formed on copper and uPVC were studied by comparing cultivable and total numbers (quantified by peptide nucleic acid (PNA) hybridisation). L. pneumophila was never recovered by culture from heterotrophic biofilms; however, PNA-positive numbers were slightly higher in biofilms formed on copper (5.9 × 10(5) cells cm(-2)) than on PVC (2.8 × 10(5) cells cm(-2)) and PEX (1.7 × 10(5) cells cm(-2)). L. pneumophila mono-species biofilms grown on copper gave 6.9 × 10(5) cells cm(-2) for PNA-positive cells and 4.8 × 10(5) CFU cm(-2) for cultivable numbers, showing that copper is not directly effective in killing L. pneumophila. Therefore previous published studies showing inactivation of L. pneumophila by copper surfaces in potable water polymicrobial species biofilms must be carefully interpreted.

[Effect of thyroid hormones on the histotopography of lectin receptors in the rat salivary gland].

PubMed

Lutsik, A D; Iashchenko, A M; Detiuk, E S

1987-04-01

Using lectin-peroxidase technique, the influence of hypo- and hyperthyroidism on histotopography of glycoconjugates has been investigated in rat submandibular gland. The following lectins were used: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Laburnum anagyroides lectin (LAL) and concanavalin A (con A). It has been demonstrated that hyperthyroidism is accompanied by the loss of con A, WGA and LAL receptor sites. Hypothyrodism enhanced con A binding to granular duct cells with a parallel reduction in WGA and LAL binding to these or other duct cells. Hypothyroidism as well as hyperthyroidism markedly enhanced PNA binding to duct epitheliocytes with redistribution of these lectin binding sites from the luminal surface of salivary ducts into the cytoplasm of duct cells. Possible interpretations of the observed phenomena are discussed.

The extracellular matrix architecture relating to myotendinous pattern formation in the distal part of the developing chick limb: an ultrastructural, histochemical and immunocytochemical analysis.

PubMed

Hurle, J M; Hinchliffe, J R; Ros, M A; Critchlow, M A; Genis-Galvez, J M

1989-07-01

In the later developmental stages (Hamburger and Hamilton, 25-34) the distal part of the chick leg possesses a distinctive extracellular matrix (ECM) architecture which relates to myotendinous patterning. There are two components: firstly, a system of dorsoventrally oriented fibrils which link the two ectodermal surfaces through the undifferentiated distal mesenchyme and secondly, a 'mesenchyme lamina' originates at the basement membrane distally, but proximally runs through the mesoderm, subjacent and parallel to the basement membrane. The 'mesenchyme lamina' appears to be a precursor of developing tendons and is spatially related to the distal tips of the myogenic blocks. As developing tendons form on the inner surface of the lamina at its proximal end, it becomes less distinct and disappears. Further dorsoventral fibrils run from the 'mesenchyme lamina' into the developing condensations and chondrogenic elements of the phalanges. The architecture of the ECM was revealed by silver and lectin staining (peanut and Ricinus communis agglutinins, PNA and RCA I), by immunocytochemistry (for fibronectin, tenascin, collagen type I) and by ultrastructural analysis. Both components stain with silver, PNA following neuraminidase digestion, RCA I, tenascin and collagen type I. However, the dorsoventral fibrils are positive for fibronectin and negative for PNA, while conversely the mesenchyme lamina is positive for PNA but much less so for fibronectin. Tenascin has been shown to be a specialized mesenchyme component of tendons and myotendinous junctions (Chiquet and Fambrough, 1984). Such a basement membrane forming a 'mesenchyme lamina' appears to be unique in epithelial-mesenchymal developing systems and points to an ectodermal role in tendon pattern formation within the mesenchyme. We discuss the possible role of mechanical force in converting the dorsoventral tenascin-positive fibrils into the localized pattern of tendon insertions into the proximal parts of the phalanges. Distally the dorsoventral fibrils may shape the digital plate by pulling together the two ectodermal surfaces. A similar ECM architecture is found in corresponding stages in the developing wing.

Northern Hemisphere Autumn and Winter Climate Responses to Realistic Tibetan Plateau and Mongolia Snow Anomalies

NASA Astrophysics Data System (ADS)

Wu, Q.; Yao, Y.; Liu, S.

2017-12-01

The impact of the Eurasian snow cover extent (SCE) on the Northern Hemisphere (NH) circulation is first investigated by applying a lagged maximum covariance analysis (MCA) to monthly satellite-derived SCE and NCEP reanalysis data. Wintertime atmospheric signals significantly correlated with persistently autumn-early winter SCE anomalies are found in the leading two MCA modes. The first MCA mode indicates the effect of Eurasian snow cover anomalies on the Arctic Oscillation/North Atlantic Oscillation (AO/NAO). The second MCA mode links a persistent dipole of autumn and winter SCE anomalies over the Tibetan Plateau (TP) and Mongolia with winter Pacific-North America (PNA)-like atmospheric variations. A modeling study further investigates atmospheric responses to above TP and Mongolia snow forcings using multiple ensemble transient integrations of the CAM4 and CLM4.0 models. Model boundary conditions are based on climatological sea ice extent (SIE) and sea surface temperature (SST), and satellite observations of SCE and snow water equivalent (SWE) over the TP and Mongolia from October to March in 1997/98 (heavy TP and light Mongolia snow) and 1984/85 (light TP and heavy Mongolia snow), with model derived SCE and SWE elsewhere. In various forcing experiments, the ensemble-mean difference between simulations with these two extreme snow states identifies local, distant, concurrent, and delayed climatic responses. The main atmospheric responses to a dipole of high TP and low Mongolia SCE persisting from October to March (versus the opposite extreme) are strong TP surface cooling, warming in the surrounding China and Mongolia region, and a winter positive PNA-like response. The localized response is maintained by persistent diabatic cooling or heating, and the remote PNA response results mainly from the increased horizontal eastward propagation of stationary Rossby wave energy due to persistent TP snow forcing and also a winter transient eddy feedback mechanism. With a less persistent dipole anomaly in autumn or winter only, local responses are similar depending on the specific anomalies, but the winter PNA-like response is nearly absent or noticeably reduced.

Exploration of Using Antisense Peptide Nucleic Acid (PNA)-cell Penetrating Peptide (CPP) as a Novel Bactericide against Fire Blight Pathogen Erwinia amylovora.

PubMed

Patel, Ravi R; Sundin, George W; Yang, Ching-Hong; Wang, Jie; Huntley, Regan B; Yuan, Xiaochen; Zeng, Quan

2017-01-01

Erwinia amylovora is a Gram-negative bacterial plant pathogen in the family Enterobacteriaceae and is the causal agent of fire blight, a devastating disease of apple and pear. Fire blight is traditionally managed by the application of the antibiotic streptomycin during bloom, but this strategy has been challenged by the development and spread of streptomycin resistance. Thus, there is an urgent need for effective, specific, and sustainable control alternatives for fire blight. Antisense antimicrobials are oligomers of nucleic acid homologs with antisense sequence of essential genes in bacteria. The binding of these molecules to the mRNA of essential genes can result in translational repression and antimicrobial effect. Here, we explored the possibility of developing antisense antimicrobials against E. amylovora and using these compounds in fire blight control. We determined that a 10-nucleotide oligomer of peptide nucleic acid (PNA) targeting the start codon region of an essential gene acpP is able to cause complete growth inhibition of E. amylovora . We found that conjugation of cell penetrating peptide (CPP) to PNA is essential for the antimicrobial effect, with CPP1 [(KFF)3K] being the most effective against E. amylovora . The minimal inhibitory concentration (MIC) of anti- acpP -CPP1 (2.5 μM) is comparable to the MIC of streptomycin (2 μM). Examination of the antimicrobial mechanisms demonstrated that anti- acpP -CPP1 caused dose-dependent reduction of acpP mRNA in E. amylovora upon treatment and resulted in cell death (bactericidal effect). Anti- acpP -CPP1 (100 μM) is able to effectively limit the pathogen growth on stigmas of apple flowers, although less effective than streptomycin. Finally, unlike streptomycin that does not display any specificity in inhibiting pathogen growth, anti- acpP -CPP1 has more specific antimicrobial effect against E. amylovora . In summary, we demonstrated that PNA-CPP can cause an effective, specific antimicrobial effect against E. amylovora and may provide the basis for a novel approach for fire blight control.

Characteristics of atmospheric circulation patterns associated with extreme temperatures over North America in observations and climate models

NASA Astrophysics Data System (ADS)

Loikith, Paul C.

Motivated by a desire to understand the physical mechanisms involved in future anthropogenic changes in extreme temperature events, the key atmospheric circulation patterns associated with extreme daily temperatures over North America in the current climate are identified. Several novel metrics are used to systematically identify and describe these patterns for the entire continent. The orientation, physical characteristics, and spatial scale of these circulation patterns vary based on latitude, season, and proximity to important geographic features (i.e., mountains, coastlines). The anomaly patterns associated with extreme cold events tend to be similar to, but opposite in sign of, those associated with extreme warm events, especially within the westerlies, and tend to scale with temperature in the same locations. The influence of the Pacific North American (PNA) pattern, the Northern Annular Mode (NAM), and the El Niño-Southern Oscillation (ENSO) on extreme temperature days and months shows that associations between extreme temperatures and the PNA and NAM are stronger than associations with ENSO. In general, the association with extremes tends to be stronger on monthly than daily time scales. Extreme temperatures are associated with the PNA and NAM in locations typically influenced by these circulation patterns; however many extremes still occur on days when the amplitude and polarity of these patterns do not favor their occurrence. In winter, synoptic-scale, transient weather disturbances are important drivers of extreme temperature days; however these smaller-scale events are often concurrent with amplified PNA or NAM patterns. Associations are weaker in summer when other physical mechanisms affecting the surface energy balance, such as anomalous soil moisture content, are associated with extreme temperatures. Analysis of historical runs from seventeen climate models from the CMIP5 database suggests that most models simulate realistic circulation patterns associated with extreme temperature days in most places. Model-simulated patterns tend to resemble observed patterns better in the winter than the summer and at 500 hPa than at the surface. There is substantial variability among the suite of models analyzed and most models simulate circulation patterns more realistically away from influential features such as large bodies of water and complex topography.

Biological dosimetry of ionizing radiation: Evaluation of the dose with cytogenetic methodologies by the construction of calibration curves

NASA Astrophysics Data System (ADS)

Zafiropoulos, Demetre; Facco, E.; Sarchiapone, Lucia

2016-09-01

In case of a radiation accident, it is well known that in the absence of physical dosimetry biological dosimetry based on cytogenetic methods is a unique tool to estimate individual absorbed dose. Moreover, even when physical dosimetry indicates an overexposure, scoring chromosome aberrations (dicentrics and rings) in human peripheral blood lymphocytes (PBLs) at metaphase is presently the most widely used method to confirm dose assessment. The analysis of dicentrics and rings in PBLs after Giemsa staining of metaphase cells is considered the most valid assay for radiation injury. This work shows that applying the fluorescence in situ hybridization (FISH) technique, using telomeric/centromeric peptide nucleic acid (PNA) probes in metaphase chromosomes for radiation dosimetry, could become a fast scoring, reliable and precise method for biological dosimetry after accidental radiation exposures. In both in vitro methods described above, lymphocyte stimulation is needed, and this limits the application in radiation emergency medicine where speed is considered to be a high priority. Using premature chromosome condensation (PCC), irradiated human PBLs (non-stimulated) were fused with mitotic CHO cells, and the yield of excess PCC fragments in Giemsa stained cells was scored. To score dicentrics and rings under PCC conditions, the necessary centromere and telomere detection of the chromosomes was obtained using FISH and specific PNA probes. Of course, a prerequisite for dose assessment in all cases is a dose-effect calibration curve. This work illustrates the various methods used; dose response calibration curves, with 95% confidence limits used to estimate dose uncertainties, have been constructed for conventional metaphase analysis and FISH. We also compare the dose-response curve constructed after scoring of dicentrics and rings using PCC combined with FISH and PNA probes. Also reported are dose response curves showing scored dicentrics and rings per cell, combining PCC of lymphocytes and CHO cells with FISH using PNA probes after 10 h and 24 h after irradiation, and, finally, calibration data of excess PCC fragments (Giemsa) to be used if human blood is available immediately after irradiation or within 24 h.

Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH).

PubMed

Almeida, Carina; Azevedo, Nuno F; Santos, Sílvio; Keevil, Charles W; Vieira, Maria J

2011-03-29

Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.

Aerosol Drug Delivery During Noninvasive Positive Pressure Ventilation: Effects of Intersubject Variability and Excipient Enhanced Growth

PubMed Central

Walenga, Ross L.; Kaviratna, Anubhav; Hindle, Michael

2017-01-01

Abstract Background: Nebulized aerosol drug delivery during the administration of noninvasive positive pressure ventilation (NPPV) is commonly implemented. While studies have shown improved patient outcomes for this therapeutic approach, aerosol delivery efficiency is reported to be low with high variability in lung-deposited dose. Excipient enhanced growth (EEG) aerosol delivery is a newly proposed technique that may improve drug delivery efficiency and reduce intersubject aerosol delivery variability when coupled with NPPV. Materials and Methods: A combined approach using in vitro experiments and computational fluid dynamics (CFD) was used to characterize aerosol delivery efficiency during NPPV in two new nasal cavity models that include face mask interfaces. Mesh nebulizer and in-line dry powder inhaler (DPI) sources of conventional and EEG aerosols were both considered. Results: Based on validated steady-state CFD predictions, EEG aerosol delivery improved lung penetration fraction (PF) values by factors ranging from 1.3 to 6.4 compared with conventional-sized aerosols. Furthermore, intersubject variability in lung PF was very high for conventional aerosol sizes (relative differences between subjects in the range of 54.5%–134.3%) and was reduced by an order of magnitude with the EEG approach (relative differences between subjects in the range of 5.5%–17.4%). Realistic in vitro experiments of cyclic NPPV demonstrated similar trends in lung delivery to those observed with the steady-state simulations, but with lower lung delivery efficiencies. Reaching the lung delivery efficiencies reported with the steady-state simulations of 80%–90% will require synchronization of aerosol administration during inspiration and reducing the size of the EEG aerosol delivery unit. Conclusions: The EEG approach enabled high-efficiency lung delivery of aerosols administered during NPPV and reduced intersubject aerosol delivery variability by an order of magnitude. Use of an in-line DPI device that connects to the NPPV mask appears to be a convenient method to rapidly administer an EEG aerosol and synchronize the delivery with inspiration. PMID:28075194

Electrogenerated poly(pyrrole-lactosyl) and poly(pyrrole-3'-sialyllactosyl) interfaces: towards the impedimetric detection of lectins

NASA Astrophysics Data System (ADS)

Gondran, Chantal; Dubois, Marie-Pierre; Fort, Sebastien; Cosnier, Serge

2013-07-01

This paper reports on the impedimetric transduction of binding reaction between polymerized saccharides and target lectins. The controlled potential electro-oxidation of pyrrole-lactosyl and pyrrole-3’-sialyllactosyl at 0.95 V vs Ag/AgCl, provides thin and reproducible poly(pyrrole-saccharide) films. The affinity binding of two lectins: Arachis hypogaea, (PNA) and Maackia amurensis (MAA) onto poly(pyrrole-lactosyl) and poly(pyrrole-3’-sialyllactosyl) electrodes, was demonstrated by cyclic voltammetry in presence of ruthenium hexamine and hydroquinone. In addition, rotating disk experiments were carried out to determine the permeability of both polypyrrole films and its evolution after incubating with lectin target. Finally, the possibility of using the poly(pyrrole-lactosyl) or poly(pyrrole-3’-siallyllactosyl) films for the impedimetric transduction of the lectin binding reaction, was investigated with hydroquinone (2×10-3 mol L-1) as a redox probe in phosphate buffer. The resuting impedance spectra were interpreted and modeled as an equivalent circuit indicating that charge transfer resistance (Rct) and relaxation frequency (f°) parameters are sensitive to the lectin binding. Rct increases from 77 to 97 Ω cm2 for PNA binding and from 93 to 131 Ω cm2 for MAA binding. In parallel, f° decreases from 276 to 222 Hz for PNA binding and from 223 to 131 Hz for MAA binding. This evolution of both parameters reflects the steric hindrances generated by the immobilised lectins towards the permeation of the redox probe.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with atmore » least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.« less

Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants.

PubMed

Kaul, Dhananjay K; Liu, Xiao-du; Zhang, Xiaoqin; Ma, Li; Hsia, Carleton J C; Nagel, Ronald L

2006-07-01

In sickle cell anemia (SCA), inflammatory (i.e., intravascular sickling and transient vasoocclusive) events result in chronic endothelial activation. In addition to sickling behavior, sickle (SS) red blood cells exhibit abnormal interaction with the vascular endothelium, which is considered to have an important role in initiation of vasoocclusion. Upregulation of endothelial adhesion molecules caused by oxidants (and cytokines) may lead to increased SS red cell adhesion. We hypothesize that endothelial activation is indispensable in SS red cell adhesion to the endothelium and that antioxidants will have an inhibitory effect on this interaction. We examined the effect of selected antioxidants in ex vivo mesocecum vasculature, a well-established model that allows measurement of hemodynamic parameters and, by intravital microscopy, can allow quantification of adhesion. We tested antioxidant enzymes (SOD and catalase) and an intravascular SOD mimetic, polynitroxyl albumin (PNA), in the presence of platelet-activating factor (PAF); the latter causes endothelial oxidant generation and endothelial activation, which characterize SCA. In ex vivo preparations, PAF not only induced marked endothelial oxidant generation, it also enhanced SS red cell adhesion, resulting in frequent blockage of small-diameter venules. The adhesion, inversely related to venular diameter, and vasoocclusion were markedly inhibited by antioxidants, resulting in improved hemodynamics. PNA, the most effective antioxidant, also abolished SS red cell adhesion in non-PAF-activated preparations. Thus SS red cell adhesion and related vasoocclusion may be ameliorated by antioxidant therapy with a stable and long-acting molecule (e.g., PNA).

Study on the interactions between toxic nitroanilines and lysozyme by spectroscopic approaches and molecular modeling.

PubMed

Gu, Yunlan; Wang, Yanqing; Zhang, Hongmei

2018-05-05

Being exogenous environmental pollutants, nitroanilines (NAs) are highly toxic and have mutagenic and carcinogenic activity. Being lack of studies on interactions between NAs and lysozyme at molecular level, the binding interactions of lysozyme with o-nitroaniline (oNA), m-nitroaniline (mNA) and p-nitroaniline (pNA) were investigated by means of steady-state fluorescence, synchronous fluorescence, UV-vis absorption spectroscopy, as well as molecular modeling. The experimental results revealed that the fluorescence of lysozyme is quenched by oNA and mNA through a static quenching, while the fluorescence quenching triggered by pNA is a combined dynamic and static quenching. The number of binding sites (n) and the binding constant (K b ) corresponding thermodynamic parameters ΔH ⊖ , ΔS ⊖ , ΔG ⊖ at different temperatures were calculated. The reactions between NAs and lysozyme were spontaneous and entropy driven and the binding of NAs to lysozyme induced conformation changes of lysozyme. The difference of the position of -NO 2 group affected the binding and the binding constants K b decreased in the following pattern: K b (pNA) >K b (mNA) >K b (oNA). Molecular docking studies were performed to reveal the most favorable binding sites of NAs on lysozyme. Our recently results could offer mechanistic insights into the nature of the binding interactions between NAs and lysozyme and provide information about the toxicity risk of NAs to human health. Copyright © 2018 Elsevier B.V. All rights reserved.

Plasma Sodium-Osmotic Dissociation and Hormonal Interaction with Drinking-Induced Hypervolemia at 2800 m Altitude

NASA Technical Reports Server (NTRS)

Greenleaf, J. E.; Hinghofer-Szalkay, H.; Roessler, A.; Farrell, P. A.; Loomis, J. L.; Fedele, M. J.; West, J.; Cowell, S. A.; Bowley, Susan M. (Technical Monitor)

2000-01-01

Dissociation between beverage sodium [Na(+)] and osmotic [Osm] concentrations for increasing plasma volume (PV, hypervolemia) appears to refute the high theoretical correlation between plasma [pNa(+)] and [pOsm].

Channels, pumps, and exchangers in the gill and kidney of freshwater fishes: their role in ionic and acid-base regulation.

PubMed

Perry, S F; Shahsavarani, A; Georgalis, T; Bayaa, M; Furimsky, M; Thomas, S L Y

2003-11-01

In freshwater fishes, the gill and kidney are intricately involved in ionic and acid-base regulation owing to the presence of numerous ion channels, pumps, or exchangers. This review summarizes recent developments in branchial and renal ion transport physiology and presents several models that integrate epithelial ion and acid-base movements in freshwater fishes. At the gill, three cell types are potentially involved in ionic uptake: pavement cells, mitochondria-rich (MR) PNA(+) cells, and MR PNA(-) cells. The transfer of acidic or basic equivalents between the fish and its environment is accomplished largely by the gill and is appropriately regulated to correct acid-base imbalances. The kidney, while less important than the gill in overall acid or base excretion, has an essential role in regulating systemic acid-base balance by controlling HCO(3) (-) reabsorption from the filtrate. Copyright 2003 Wiley-Liss, Inc.

Phase-separation induced extraordinary toughening of magnetic hydrogels

NASA Astrophysics Data System (ADS)

Tang, Jingda; Li, Chenghai; Li, Haomin; Lv, Zengyao; Sheng, Hao; Lu, Tongqing; Wang, T. J.

2018-05-01

Phase separation markedly influences the physical properties of hydrogels. Here, we find that poly (N, N-dimethylacrylamide) (PDMA) hydrogels suffer from phase separation in aqueous sodium hydroxide solutions when the concentration is higher than 2 M. The polymer volume fraction and mechanical properties show an abrupt change around the transition point. We utilize this phase separation mechanism to synthesize tough magnetic PDMA hydrogels with the in-situ precipitation method. For comparison, we also prepared magnetic poly (2-acrylamido-2-methyl-propane sulfonic acid sodium) (PNaAMPS) magnetic hydrogels, where no phase separation occurs. The phase-separated magnetic PDMA hydrogels exhibit an extraordinarily high toughness of ˜1000 J m-2; while non-phase-separated magnetic PNaAMPS hydrogels only show a toughness of ˜1 J m-2, three orders of magnitude lower than that of PDMA hydrogels. This phase separation mechanism may become a new approach to prepare tough magnetic hydrogels and inspire more applications.

A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water

PubMed Central

Gogoi, Khirud; Mane, Meenakshi V.; Kunte, Sunita S.; Kumar, Vaijayanti A.

2007-01-01

The specific 1,3 dipolar Hüisgen cycloaddition reaction known as ‘click-reaction’ between azide and alkyne groups is employed for the synthesis of peptide–oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, ∼3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products. PMID:17981837

Health hazard evaluation report HETA 83-248-1515, Arco Philadelphia refinery, Philadelphia, Pennsylvania

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, F.A.; Parrish, G.

1984-10-01

A bulk sample of fractionator residue was analyzed for polynuclear aromatic (PNA) compounds at the catalytic cracking unit of ARCO Philadelphia Refinery (SIC-2911), Philadelphia, Pennsylvania in May, 1983. The study was requested by the Atlantic Independent Union to determine if skin rashes and skin irritation occurring among refinery workers were caused by PNA in the fractionators. The authors conclude that a health hazard from exposure to chemicals at the cracking unit may exist. No specific chemical agent can be identified. Dust from the catalyst and oily residues that could contaminate workers shoes and clothing may have contributed to some ofmore » the dermatitis cases. Recommendations include laundering workers coveralls by dry cleaning to insure the removal of oily residues, providing workers with oil resistant or oil proof work boots, and repairing the ventilator in the sample preparation room adjacent to the block house.« less

Studies on the oligosaccharide heterogeneity of the isoelectric forms of the lower molecular weight acid phosphatase of frog liver.

PubMed

Kubicz, A; Szalewicz, A; Chrambach, A

1991-01-01

1. The lower molecular weight, heterogeneous acid phosphatase (AcPase) from the frog liver (Rana esculenta) containing AcPase I, II, III and IV was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient. 2. The blotted enzyme bands were characterized by their different binding patterns obtained with the lectins concanavalin A, wheat germ agglutinin (WGA), Lens culinaris hemagglutinin (LcH) and peanut agglutinin (PNA). 3. In situ neuraminidase treatment reduced the staining intensity of some WGA-bands and increased that of PNA-bands. 4. The finding that AcPases I, II, III and IV differ in their carbohydrate chain composition, together with previous results showing different bioactivities of AcPases III and IV, indicates a correlation between the glycosylation state of enzyme forms and their physiological action.

Effective and efficient implementation of alternative project delivery : research summary.

DOT National Transportation Integrated Search

2017-05-01

Alternative project delivery (APD) methods such as Design Build (DB) and Construction Manager at Risk (CMAR), are used by state departments of transportation to improve the efficiency and effectiveness of project delivery. The Maryland Department of ...

Efficiency performance of China's health care delivery system.

PubMed

Zhang, Luyu; Cheng, Gang; Song, Suhang; Yuan, Beibei; Zhu, Weiming; He, Li; Ma, Xiaochen; Meng, Qingyue

2017-07-01

Improving efficiency performance of the health care delivery system has been on the agenda for the health system reform that China initiated in 2009. This study examines the changes in efficiency performance and determinants of efficiency after the reform to provide evidence to assess the progress of the reform from the perspective of efficiency. Descriptive analysis, Data Envelopment Analysis, the Malmquist Index, and multilevel regressions are used with data from multiple sources, including the World Bank, the China Health Statistical Yearbook, and routine reports. The results indicate that over the last decade, health outcomes compared with health investment were relatively higher in China than in most other countries worldwide, and the trend was stable. The overall efficiency and total factor productivity increased after the reform, indicating that the reform was likely to have had a positive impact on the efficiency performance of the health care delivery system. However, the health care delivery structure showed low system efficiency, mainly attributed to the weakened primary health care system. Strengthening the primary health care system is central to enhancing the future performance of China's health care delivery system. Copyright © 2017 John Wiley & Sons, Ltd.

Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly "Targeted" Drug Delivery Systems.

PubMed

Maity, Amit Ranjan; Stepensky, David

2016-01-04

Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

How can innovative project delivery systems improve the overall efficiency of GDOT in transportation project delivery?

DOT National Transportation Integrated Search

2013-04-01

The USDOT and Federal Highway Administration (FHWA) recommend the smart use of innovative project : delivery systems, such as design-build, to improve efficiency and effectiveness of developing transportation : projects. Although design-build provide...

The influence of ENSO, PDO and PNA on secular rainfall variations in Hawai`i

NASA Astrophysics Data System (ADS)

Frazier, Abby G.; Elison Timm, Oliver; Giambelluca, Thomas W.; Diaz, Henry F.

2017-11-01

Over the last century, significant declines in rainfall across the state of Hawai`i have been observed, and it is unknown whether these declines are due to natural variations in climate, or manifestations of human-induced climate change. Here, a statistical analysis of the observed rainfall variability was applied as first step towards better understanding causes for these long-term trends. Gridded seasonal rainfall from 1920 to 2012 is used to perform an empirical orthogonal function (EOF) analysis. The leading EOF components are correlated with three indices of natural climate variations (El Niño-Southern Oscillation (ENSO), Pacific Decadal Oscillation (PDO), and Pacific North American (PNA)), and multiple linear regression (MLR) is used to model the leading components with climate indices. PNA is the dominant mode of wet season (November-April) variability, while ENSO is most significant in the dry season (May-October). To assess whether there is an anthropogenic influence on rainfall, two methods are used: a linear trend term is included in the MLR, and pattern correlation coefficients (PCC) are calculated between recent rainfall trends and future changes in rainfall projected by downscaling methods. PCC results indicate that recent observed rainfall trends in the wet season are positively correlated with future expected changes in rainfall, while dry season PCC results do not show a clear pattern. The MLR results, however, show that the trend term adds significantly to model skill only in the dry season. Overall, MLR and PCC results give weak and inconclusive evidence for detection of anthropogenic signals in the observed rainfall trends.

Regionally synchronous fires in interior British Columbia, Canada, driven by interannual climate variability and weakly associated with large-scale climate patterns between AD 1600-1900

NASA Astrophysics Data System (ADS)

Harvey, J. E.; Smith, D. J.

2016-12-01

We investigated the influence of climate variability on forest fire occurrence in west central British Columbia (BC), Canada, between AD 1600 and 1900. Fire history was reconstructed at 8 sites in the Cariboo-Chilcotin region and we identified 46 local (fires that affected 1 site) and 16 moderate (fires that affected 2 sites) fires. Preexisting fire history data collected from nearby sites was incorporated to identify 17 regionally synchronous fire years (fires that affected ³ 3 sites). Interannual and multidecadal relationships between fire occurrence and the Palmer Drought Severity Index (PDSI), El Nino Southern Oscillation (ENSO), Pacific Decadal Oscillation (PDO) and the Pacific North American (PNA) pattern were examined, in addition to the effects of phase interactions between ENSO and PDO. We examined multiple reconstructions of PDO and ENSO and utilized three methodological approaches to characterize climate-fire relationships. We found that the influence of interannual climate expressed as PDSI, increasingly synchronized the occurrence of of fires from local to regional fires. Regional fires were associated with anomalously dry, warm conditions in the year of the fire and in years preceding the fire. We also identified an association between local fires and antecedent moisture conditions, where wetter and cooler conditions persisted 2-3 years prior to fire. This finding suggests that moisture-driven fine fuel development and proximity to grasslands could function as key determinants of local (small-scale) fire history parameters. The relationships we identified between regional fires and ENSO, PDO and PNA suggest that large-scale patterns of climate variability exert a weak and/or inconsistent influence over fire activity in west central BC between AD 1600-1900. The strongest relationships between regional fires and large-scale climate patterns were identified when ENSO and PDO were both in positive phases. We also documented a relationship between regional fires and positive years of the PNA pattern. Our findings suggest that long-term fire planning using predictions of large scale climate patterns may be limited in west central BC, however, the consideration of additive phases of ENSO and PDO, and the PNA pattern, may be effective and has been suggested by others in the inland Pacific Northwest.

Target Oriented Drugs against Leishmania.

DTIC Science & Technology

1980-01-31

present in very significant amounts and there is a possibility of arabinose - normally found only as a component of plant glycoproteins. 6...as does EF. This is also demonstrated by our experiments on promastigote agglutination by PNA (§ 2a). These oligosaccharide substituents, containing

Strong Links Between Teleconnections and Ecosystem Exchange Found at a Pacific Northwest Old-Growth Forest from Flux Tower and MODIS EVI Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wharton, S; Chasmer, L; Falk, M

2009-03-12

Variability in three Pacific teleconnection patterns are examined to see if net carbon exchange at a low-elevation, old-growth forest is affected by climatic changes associated with these periodicities. Examined are the Pacific Decadal Oscillation (PDO), Pacific/North American Oscillation (PNA) and El Nino-Southern Oscillation (ENSO). We use nine years of eddy covariance CO{sub 2}, H{sub 2}O and energy fluxes measured at the Wind River AmeriFlux site, Washington, USA and eight years of tower-pixel remote sensing data from the Moderate Resolution Imaging Spectroradiometer (MODIS) to address this question. We compute a new Composite Climate Index (CCI) based on the three Pacific Oscillationsmore » to divide the measurement period into positive- (2003 and 2005), negative- (1999 and 2000) and neutral-phase climate years (2001, 2002, 2004, 2006 and 2007). The forest transitioned from an annual net carbon sink (NEP = + 217 g C m{sup -2} year{sup -1}, 1999) to a source (NEP = - 100 g C m{sup -2} year{sup -1}, 2003) during two dominant teleconnection patterns. Net ecosystem productivity (NEP), water use efficiency (WUE) and light use efficiency (LUE) were significantly different (P < 0.01) during positive (NEP = -0.27 g C m{sup -2} day{sup -1}, WUE = 4.1 mg C/g H{sub 2}O, LUE = 0.94 g C MJ{sup -1}) and negative (NEP = +0.37 g C m{sup -2} day{sup -1}, WUE = 3.4 mg C/g H{sub 2}O, LUE = 0.83 g C MJ{sup -1}) climate phases. The CCI was linked to variability in the MODIS Enhanced Vegetation Index (EVI) but not to MODIS Fraction of absorbed Photosynthetically Active Radiation (FPAR). EVI was highest during negative climate phases (1999 and 2000) and was positively correlated with NEP and showed potential for using MODIS to estimate teleconnection-driven anomalies in ecosystem CO{sub 2} exchange in old-growth forests. This work suggests that any increase in the strength or frequency of ENSO coinciding with in-phase, low frequency Pacific oscillations (PDO and PNA) will likely increase CO{sub 2} uptake variability in Pacific Northwest conifer forests.« less

Low-Temperature Pd/Zeolite Passive NO x Adsorbers: Structure, Performance, and Adsorption Chemistry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Yang; Kovarik, Libor; Engelhard, Mark H.

Pd/zeolite passive NOx adsorber (PNA) materials were prepared with solution ion-exchange between NH4/zeolites (Beta, ZSM-5 and SSZ-13) and PdCl2 solutions. The nature of Pd (dispersion, distribution and oxidation states) in these materials was characterized with Na+ ion-exchange, TEM imaging, CO titration with FTIR and in situ XPS. The NOx trapping and release properties were tested using feeds with different compositions. It is concluded that multiple Pd species coexist in these materials: atomically dispersed Pd in the cationic sites of zeolites, and PdO2 and PdO particles on the external surfaces. While Pd is largely atomically dispersed in ZSM-5, the small poremore » opening for SSZ-13 inhibits Pd diffusion such that the majority of Pd stays as external surface PdO2 clusters. NOx trapping and release are not simple chemisorption and desorption events, but involve rather complex chemical reactions. In the absence of CO in the feed, cationic Pd(II) sites with oxygen ligands and PdO2 clusters are reduced by NO to Pd(I) and PdO clusters. These reduced sites are the primary NO adsorption sites. In the presence of H2O, the as-formed NO2 desorb immediately. In the presence of CO in the feed, metallic Pd, “naked” Pd2+, and Pd+ sites are responsible for NO adsorption. For Pd adsorption sites with the same oxidation states but in different zeolite frameworks, NO binding energies are not expected to vary greatly. However, NO release temperatures do vary substantially with different zeolite structures. This indicates that NO transport within these materials play an important role in determining release temperatures. Finally, some rational design principles on efficient PNA materials are suggested. The authors gratefully acknowledge the US Department of Energy (DOE), Energy Efficiency and Renewable Energy, Vehicle Technologies Office for the support of this work. The research described in this paper was performed in the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is operated for the US DOE by Battelle. Discussions with Drs. A. Yezerets, J.Y. Luo, K. Kamasamudram and N. Currier Cummins, Inc., and H.Y. Chen from Johnson-Matthey are greatly appreciated.« less

Trends and Variability of the Outdoor Skating Season in Canada during 1951-2005

NASA Astrophysics Data System (ADS)

Damyanov, Nikolay Nikolaev

Climate change affects a range of human activities, including one of Canada's prime sources of entertainment: ice skating. Whether done recreationally or as hockey, its outdoor component is heavily dependent on weather and climate. Based on information obtained from public works officials from various Canadian cities, I have established a meteorological criterion for the initiation of an outdoor skating season (OSS) as the last day in a sequence of the first three consecutive fall/winter days with a maximum temperature below -5 °C. In addition, I derive a proxy of the OSS length, defined as the total number of days with a maximum temperature below -5 °C after the OSS start date and before the start of March. Using these filters, I have extracted the start dates and the lengths of the OSS for each year during the fifty-five year period 1951-2005 from a comprehensive daily temperature dataset (Vincent et al., 2002). For each station, I created time series of both the OSS start dates and OSS lengths, and calculated the magnitude, sign and statistical significance of the slopes of the best-fit lines to each time series. In order to establish a relationship of the OSS with large-scale climate patterns, I grouped stations into six climatic regions. Depending on location, I then tested each region for correlation with the Pacific North-American teleconnection pattern (PNA) or the North Atlantic Oscillation (NAO), using a composite analysis method. Lastly, I removed the signal due to these climate fluctuations from the OSS start date and length trends in order to determine how much of the variability was caused by these interannual climate oscillations. The results of the study indicate that most stations in British Columbia and southwest Alberta, as well as these in the southern Ontario/Quebc region have witnessed a progressively later onset of the OSS over time. The Prairies, northwest Canada, and some Maritime locales show the opposite trend, although the magnitudes of the slopes are smaller. Significance tests on the regression lines show that most of these trends are not significant at the 95% level. However, OSS start dates in western Canada are very well correlated with PNA patterns by happening later on the average whenever PNA is positive and more warm air is channeled towards the west coast; the OSS start dates in eastern Canada show a similar connection with the NAO. The OSS lengths exhibit different trends: five of the six regions show a decrease in OSS length with the only region having experienced a lengthening of the OSS being the Maritimes. The statistical significance of the OSS length slopes is much higher than that of OSS start slopes, and the correlation with the PNA or NAO is similar in both cases. After carrying out the last procedure (removal of the PNA and NAO signals from the OSS start date and length series), I found an increase in the new slopes and their significance for more than half of my geographic regions' OSS start date and length trends.

Dendrimers as Carriers for siRNA Delivery and Gene Silencing: A Review

PubMed Central

Huang, Weizhe; He, Ziying

2013-01-01

RNA interference (RNAi) was first literaturally reported in 1998 and has become rapidly a promising tool for therapeutic applications in gene therapy. In a typical RNAi process, small interfering RNAs (siRNA) are used to specifically downregulate the expression of the targeted gene, known as the term “gene silencing.” One key point for successful gene silencing is to employ a safe and efficient siRNA delivery system. In this context, dendrimers are emerging as potential nonviral vectors to deliver siRNA for RNAi purpose. Dendrimers have attracted intense interest since their emanating research in the 1980s and are extensively studied as efficient DNA delivery vectors in gene transfer applications, due to their unique features based on the well-defined and multivalent structures. Knowing that DNA and RNA possess a similar structure in terms of nucleic acid framework and the electronegative nature, one can also use the excellent DNA delivery properties of dendrimers to develop effective siRNA delivery systems. In this review, the development of dendrimer-based siRNA delivery vectors is summarized, focusing on the vector features (siRNA delivery efficiency, cytotoxicity, etc.) of different types of dendrimers and the related investigations on structure-activity relationship to promote safe and efficient siRNA delivery system. PMID:24288498

Variable Asymmetric Chains in Transition Metal Oxyfluorides: Structure-Second-Harmonic-Generation Property Relationships.

PubMed

Ahmed, Belal; Jo, Hongil; Oh, Seung-Jin; Ok, Kang Min

2018-05-15

Four novel transition metal oxyfluorides, [Zn(pz) 3 ][MoO 2 F 4 ]·0.1H 2 O (1), [Zn(pz) 2 F 2 ][Zn(pz) 3 ] 2 [WO 2 F 4 ] 2 (2), [Cd(pz) 4 ][Cd(pz) 4 (H 2 O)][MoO 2 F 4 ] 2 ·0.625H 2 O (3), and [Zn(mpz) 3 ] 2 [MoO 2 F 4 ] 2 (4) (pz = pyrazole; mpz = 3-methyl pyrazole) have been synthesized. Compounds 1 and 4 contain helical chains. Compound 2 accommodates zigzag chains, and compound 3 has quasi-one-dimensional linear chains. The variable chain structures are found to be attributable to the different structure-directing anionic groups and hydrogen bonding interactions. Compound 4 crystallized in the noncentrosymmetric (NCS) polar space group, Pna2 1 , is nonphase-matchable (Type I), and reveals a moderate second-harmonic-generation (SHG) efficiency (10 × α-SiO 2 ). The observed SHG efficiency of compound 4 is due to the small net polarization occurring from the arrangement of ZnN 3 F 2 trigonal bipyramids. Spectroscopic and thermal characterizations along with calculations for the title materials are reported.

A pliable electroporation patch (ep-Patch) for efficient delivery of nucleic acid molecules into animal tissues with irregular surface shapes.

PubMed

Wei, Zewen; Huang, Yuanyu; Zhao, Deyao; Hu, Zhiyuan; Li, Zhihong; Liang, Zicai

2015-01-05

Delivery of nucleic acids into animal tissues by electroporation is an appealing approach for various types of gene therapy, but efficiency of existing methodsis not satisfactory. Here we present the validation of novel electroporation patch (ep-Patch) for efficient delivery of DNA and siRNA into mouse tissues. Using micromachining technology, closely spaced gold electrodes were made on the pliable parylene substrate to form a patch-like electroporation metrics. It enabled large coverage of the target tissues and close surface contact between the tissues and electrodes, thus providing a uniform electric field to deliver nucleic acids into tissues, even beneath intact skin. Using this ep-Patch for efficiently delivery of both DNA and siRNA, non-invasive electroporation of healthy mouse muscle tissue was successfully achieved. Delivery of these nucleic acids was performed to intact tumors with satisfactory results. Silencing of tumor genes using the ep-Patch was also demonstrated on mice. This pliable electroporation patch method constitutes a novel way of in vivo delivery of siRNA and DNA to certain tissues or organs to circumvent the disadvantages of existing methodologies for in vivo delivery of nucleic acid molecules.

Efficient siRNA delivery system using carboxilated single-wall carbon nanotubes in cancer treatment.

PubMed

Neagoe, Ioana Berindan; Braicu, Cornelia; Matea, Cristian; Bele, Constantin; Florin, Graur; Gabriel, Katona; Veronica, Chedea; Irimie, Alexandru

2012-08-01

Several functionalized carbon nanotubes have been designed and tested for the purpose of nucleic acid delivery. In this study, the capacity of SWNTC-COOH for siRNA deliverey were investigated delivery in parallel with an efficient commercial system. Hep2G cells were reverse-transfected with 50 nM siRNA (p53 siRNA, TNF-alphasiRNA, VEGFsiRNA) using the siPORT NeoFX (Ambion) transfection agent in paralel with SWNTC-COOH, functionalised with siRNA. The highest level of gene inhibition was observed in the cases treated with p53 siRNA gene; in the case of transfection with siPort, the NeoFX value was 33.8%, while in the case of SWNTC-COOH as delivery system for p53 siRNA was 37.5%. The gene silencing capacity for VEGF was 53.7%, respectively for TNF-alpha 56.7% for siPORT NeoFX delivery systems versus 47.7% (VEGF) and 46.5% (TNF-alpha) for SWNTC-COOH delivery system. SWNTC-COOH we have been showed to have to be an efficient carrier system. The results from the inhibition of gene expresion for both transfection systems were confirmed at protein level. Overall, the lowest mRNA expression was confirmed at protein level, especially in the case of p53 siRNA and TNF-alpha siRNA transfection. Less efficient reduction protein expressions were observed in the case of VEGF siRNA, for both transfection systems at 24 h; only at 48 h, there was a statistically significant reduction of VEGF protein expression. SWCNT-COOH determined an efficient delivery of siRNA. SWNTC-COOH, combined with suitable tumor markers like p53 siRNA, TNFalpha siRNA or VEGF siRNA can be used for the efficient delivery of siRNA.

Visualization of melanoma tumor with lectin-conjugated rare-earth doped fluoride nanocrystals

PubMed Central

Dumych, Tetiana; Lutsyk, Maxym; Banski, Mateusz; Yashchenko, Antonina; Sojka, Bartlomiej; Horbay, Rostyslav; Lutsyk, Alexander; Stoika, Rostyslav; Misiewicz, Jan; Podhorodecki, Artur; Bilyy, Rostyslav

2014-01-01

Aim To develop specific fluorescent markers for melanoma tumor visualization, which would provide high selectivity and reversible binding pattern, by the use of carbohydrate-recognizing proteins, lectins, combined with the physical ability for imaging deep in the living tissues by utilizing red and near infrared fluorescent properties of specific rare-earth doped nanocrystals (NC). Methods B10F16 melanoma cells were inoculated to C57BL/6 mice for inducing experimental melanoma tumor. Tumors were removed and analyzed by lectin-histochemistry using LABA, PFA, PNA, HPA, SNA, GNA, and NPL lectins and stained with hematoxylin and eosin. NPL lectin was conjugated to fluorescent NaGdF4:Eu3+-COOH nanoparticles (5 nm) via zero length cross-linking reaction, and the conjugates were purified from unbound substances and then used for further visualization of histological samples. Fluorescent microscopy was used to visualize NPL-NaGdF4:Eu3+ with the fluorescent emission at 600-720 nm range. Results NPL lectin selectively recognized regions of undifferentiated melanoblasts surrounding neoangiogenic foci inside melanoma tumor, PNA lectin recognized differentiated melanoblasts, and LCA and WGA were bound to tumor stroma regions. NPL-NaGdF4:Eu3+ conjugated NC were efficiently detecting newly formed regions of melanoma tumor, confirmed by fluorescent microscopy in visible and near infrared mode. These conjugates possessed high photostability and were compatible with convenient xylene-based mounting systems and preserved intensive fluorescent signal at samples storage for at least 6 months. Conclusion NPL lectin-NaGdF4:Eu3+ conjugated NC permitted distinct identification of contours of the melanoma tissue on histological sections using red excitation at 590-610 nm and near infrared emission of 700-720 nm. These data are of potential practical significance for development of glycans-conjugated nanoparticles to be used for in vivo visualization of melanoma tumor. PMID:24891277

Molecular Gut Content Profiling to Investigate the In Situ Grazing and Selectivity of Dolioletta gegenbauri in Summer Continental Shelf Intrusion Waters of the South Atlantic Bight, USA

NASA Astrophysics Data System (ADS)

Walters, T. L.; Frazier, L.; Gibson, D. M.; Paffenhofer, G. A.; Frischer, M. E.

2016-02-01

Gelatinous metazooplankton play a crucial role in marine planktonic food webs and it has been suggested that they may become increasingly important in the Future Ocean. However, largely due to methodological challenges and reliance on laboratory cultivation approaches, the in situ diet of zooplankton with complex life histories and diverse prey choices remains poorly investigated. This is particularly true for the gelatinous zooplankton including the pelagic tunicate, Dolioletta gegenbauri that form large blooms in productive subtropical continental shelf environments. To investigate the diet of D. gegenbauri we developed a molecular gut profiling approach based on the use of a Peptide Nucleic Acid (PNA) PCR blocker. Using a doliolid-specific PNA blocker, it was possible to enrich the amplification of prey and parasite DNA from whole animal DNA extracts of doliolids. Gut contents from the water column, wild and captive-fed doliolids were profiled after PNA-PCR by denaturing HPLC (dHPLC), clone library and next generation sequencing (NGS) approaches. Studies were conducted during 5 summer cruises in the mid-shelf of the South Atlantic Bight. Comparison of gut profiles to available prey in the water column revealed evidence of prey selection towards larger prey species, including diatoms, dinoflagelletes and also metazoan prey that were likely captured as larvae and eggs. Wild-caught doliolids contained significantly more metazoan sequences than did the captive-fed doliolids. Ingestion of metazoan prey suggests that metazoans may contribute both the nutrition of doliolids and the potential role of doliolids as trophic cascade agents in continental shelf pelagic food webs.

Rapid single nucleotide polymorphism detection for personalized medicine applications using planar waveguide fluorescence sensors

NASA Astrophysics Data System (ADS)

Herron, James N.; Tolley, Samuel E.; Smith, Richard; Christensen, Douglas A.

2006-02-01

Personalized medicine is an emerging field in which clinical diagnostics information about a patient's genotype or phenotype is used to optimize his/her pharmacotherapy. This article evaluates whether planar waveguide fluorescent sensors are suitable for determining such information from patient testing in point-of-care (POC) settings. The model system was Long QT Syndrome, a congenital disease associated with single nucleotide polymorphisms (SNPs) in genes encoding for cardiac ion channels. Three different SNP assay formats were examined: DNA/DNA hybridization, DNA/PNA hybridization (PNA: "peptide nucleic acid"), and single base extension (SBEX). Although DNA/DNA hybridization produced a strong intensity-time response for both wildtype and SNP analytes in a 5-min assay at 32°C, their hybridization rates differed by only 32.7%, which was insufficient for clinical decision-making. Much better differentiation of the two rates was observed at 53°C, where the wildtype's hybridization rate was two-thirds of its maximum value, while that of the SNP was essentially zero. Such all-or-nothing resolution would be adequate for clinical decision-making; however, the elevated temperature and precise temperature control would be hard to achieve in a POC setting. Results from DNA/PNA hybridization studies were more promising. Nearly 20-fold discrimination between wildtype and SNP hybridization rates was observed in a 5-min assay at 30°C, although the low ionic strength conditions required necessitated a de-salting step between sample preparation and SNP detection. SBEX was the most promising of the three, determining the absolute identity of the suspected polymorphism in a 5-min assay at 40°C.

Organization of extratropical transients during El Nino

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoerling, M.P.; Ting, Mingfang

Four observed El Nino-Southern Oscillation (ENSO) events are studied to determine the mechanisms responsible for the anomalous extratropical atmospheric circulation during northern winter. A parallel analysis of a GCM's response to El Nino is performed in order to assess if similar mechanisms are operative in the model atmosphere. The observed stationary wave anomalies over the Pacific/North American (PNA) region are found to be similar during the four winters despite appreciable differences in sea surface temperatures. The anomalous transient vorticity fluxes are remarkably robust over the North Pacific during each even, with an eastward extension of the climatological storm track leadingmore » to strong cyclonic forcing near 40[degrees]N, 150[degrees]W. This forcing is in phase with the seasonal mean Aleutian trough anomaly suggesting the important of eddy-mean flow interaction. By comparison, the intersample variability of the GCM response over the PNA region is found to exceed the observed inter-El Nino variability. This stems primarily from a large variability in the model's anomalous transients over the North Pacific. Further analysis reveals that extratropical vorticity transients are the primary mechanism maintaining the stationary wave anomalies over the PNA region during all four observed ENSO winters. In the case of the GCM, the organization of transient eddies is ill defined over the North Pacific, a behavior indicative of model error. A physical model is proposed to explain the robustness of the tropical controlling influence of the extratropical transients in nature. A simple equatorial Pacific heat source directly forces a tropical anticyclone whose phase relative to the climatological tropical anticyclone leads to an eastward extension of the subtropical jet stream. This mechanism appears to be equally effective for a heat source located either in the central or eastern Pacific basin. 36 refs., 14 figs.« less

Ex vivo gene editing of the dystrophin gene in muscle stem cells mediated by peptide nucleic acid single stranded oligodeoxynucleotides induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy.

PubMed

Nik-Ahd, Farnoosh; Bertoni, Carmen

2014-07-01

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNA-ssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients. © 2014 AlphaMed Press.

Relationship between hospital financial performance and publicly reported outcomes.

PubMed

Nguyen, Oanh Kieu; Halm, Ethan A; Makam, Anil N

2016-07-01

Hospitals that have robust financial performance may have improved publicly reported outcomes. To assess the relationship between hospital financial performance and publicly reported outcomes of care, and to assess whether improved outcome metrics affect subsequent hospital financial performance. Observational cohort study. Hospital financial data from the Office of Statewide Health Planning and Development in California in 2008 and 2012 were linked to data from the Centers for Medicare and Medicaid Services Hospital Compare website. Hospital financial performance was measured by net revenue by operations, operating margin, and total margin. Outcomes were 30-day risk-standardized mortality and readmission rates for acute myocardial infarction (AMI), congestive heart failure (CHF), and pneumonia (PNA). Among 279 hospitals, there was no consistent relationship between measures of financial performance in 2008 and publicly reported outcomes from 2008 to 2011 for AMI and PNA. However, improved hospital financial performance (by any of the 3 measures) was associated with a modest increase in CHF mortality rates (ie, 0.26% increase in CHF mortality rate for every 10% increase in operating margin [95% confidence interval: 0.07%-0.45%]). Conversely, there were no significant associations between outcomes from 2008 to 2011 and subsequent financial performance in 2012 (P > 0.05 for all). Robust financial performance is not associated with improved publicly reported outcomes for AMI, CHF, and PNA. Financial incentives in addition to public reporting, such as readmissions penalties, may help motivate hospitals with robust financial performance to further improve publicly reported outcomes. Reassuringly, improved mortality and readmission rates do not necessarily lead to loss of revenue. Journal of Hospital Medicine 2016;11:481-488. © 2016 Society of Hospital Medicine. © 2016 Society of Hospital Medicine.

In situ characterization of glycans in the urothelium of donkey bladder: evidence of secretion of sialomucins.

PubMed

Desantis, Salvatore; Accogli, Gianluca; Zizza, Sara; Arrighi, Silvana

2013-09-01

The glycoprotein pattern was investigated by lectin histochemistry in the urothelium lining the urinary bladder of the donkey Equus asinus. Tissue sections were stained with a panel of twelve lectins, in combination with saponification and sialidase digestion (K-s). The urinary bladder urothelium has three distinct layers from the basal zone to the lumen consisting of basal, intermediate and superficial cells (umbrella cells). Cytoplasm of basal cells reacted with SNA, PNA, K-s-PNA, GSA I-B4 and Con A showing glycans ending with Neu5Acα2,6Gal/GalNAc, Neu5AcGalβ1,3GalNAc, αGal and with terminal/internal αMan. The cytoplasm of umbrella cells displayed an increase of Neu5AcGalβ1,3GalNAc and the appearance of Neu5AcGalβ1,3GalNAc, Neu5acα2,3Galβ1,4GlcNAc and Neu5AcGalNAc residues (MAL II, K-s-SBA and K-s-HPA staining). Scattered umbrella cells were characterized by glycans terminating with GalNAc binding DBA, SBA and HPA. The mucosa forms folds with a crypt-like appearance where the urothelium shows a different pattern of glycans. The bladder luminal surface stained with K-s-PNA, K-s-DBA, KOH-s-SBA, and K-s-HPA displaying a coating of sialoglycoproteins belonging to O-linked glycans (typical secretory moieties). These findings show that different glycosylation patterns exist along the donkey bladder urothelium, and different sub-populations of umbrella cells are present secreting the sialoglycans which constitute the protective gel layer lining the bladder. Copyright © 2013 Elsevier GmbH. All rights reserved.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: The effect of pH, dextran sulfate and probe concentration.

PubMed

Rocha, Rui; Santos, Rita S; Madureira, Pedro; Almeida, Carina; Azevedo, Nuno F

2016-05-20

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

Improving IMRT delivery efficiency using intensity limits during inverse planning.

PubMed

Coselmon, Martha M; Moran, Jean M; Radawski, Jeffrey D; Fraass, Benedick A

2005-05-01

Inverse planned intensity modulated radiotherapy (IMRT) fields can be highly modulated due to the large number of degrees of freedom involved in the inverse planning process. Additional modulation typically results in a more optimal plan, although the clinical rewards may be small or offset by additional delivery complexity and/or increased dose from transmission and leakage. Increasing modulation decreases delivery efficiency, and may lead to plans that are more sensitive to geometrical uncertainties. The purpose of this work is to assess the use of maximum intensity limits in inverse IMRT planning as a simple way to increase delivery efficiency without significantly affecting plan quality. Nine clinical cases (three each for brain, prostate, and head/neck) were used to evaluate advantages and disadvantages of limiting maximum intensity to increase delivery efficiency. IMRT plans were generated using in-house protocol-based constraints and objectives for the brain and head/neck, and RTOG 9406 dose volume objectives in the prostate. Each case was optimized at a series of maximum intensity ratios (the product of the maximum intensity and the number of beams divided by the prescribed dose to the target volume), and evaluated in terms of clinical metrics, dose-volume histograms, monitor units (MU) required per fraction (SMLC and DMLC delivery), and intensity map variation (a measure of the beam modulation). In each site tested, it was possible to reduce total monitor units by constraining the maximum allowed intensity without compromising the clinical acceptability of the plan. Monitor unit reductions up to 38% were observed for SMLC delivery, while reductions up to 29% were achieved for DMLC delivery. In general, complicated geometries saw a smaller reduction in monitor units for both delivery types, although DMLC delivery required significantly more monitor units in all cases. Constraining the maximum intensity in an inverse IMRT plan is a simple way to improve delivery efficiency without compromising plan objectives.

Effect of Surface Properties on Liposomal siRNA Delivery

PubMed Central

Xia, Yuqiong; Tian, Jie; Chen, Xiaoyuan

2015-01-01

Liposomes are one of the most widely investigated carriers for siRNA delivery. The surface properties of liposomal carriers, including the surface charge, PEGylation, and ligand modification can significantly affect the gene silencing efficiency. Three barriers of systemic siRNA delivery (long blood circulation, efficient tumor penetration and efficient cellular uptake/endosomal escape) are analyzed on liposomal carriers with different surface charges, PEGylations and ligand modifications. Cationic formulations dominate siRNA delivery and neutral formulations also have good performance while anionic formulations are generally not proper for siRNA delivery. The PEG dilemma (prolonged blood circulation vs. reduced cellular uptake/endosomal escape) and the side effect of repeated PEGylated formulation (accelerated blood clearance) were discussed. Effects of ligand modification on cationic and neutral formulations were analyzed. Finally, we summarized the achievements in liposomal siRNA delivery, outlined existing problems and provided some future perspectives. PMID:26695117

Effects of local structural transformation of lipid-like compounds on delivery of messenger RNA

NASA Astrophysics Data System (ADS)

Li, Bin; Luo, Xiao; Deng, Binbin; Giancola, Jolynn B.; McComb, David W.; Schmittgen, Thomas D.; Dong, Yizhou

2016-02-01

Lipid-like nanoparticles (LLNs) have shown great potential for RNA delivery. Lipid-like compounds are key components in LLNs. In this study, we investigated the effects of local structural transformation of lipid-like compounds on delivery of messenger RNA. Our results showed that position change of functional groups on lipid-like compounds can dramatically improve delivery efficiency. We then optimized formulation ratios of TNT-b10 LLNs, a lead material, increasing delivery efficiency over 2-fold. More importantly, pegylated TNT-b10 LLNs is stable for over four weeks and is over 10-fold more efficient than that of its counterpart TNT-a10 LLNs. Additionally, the optimal formulation O-TNT-b10 LLNs is capable of delivering mRNA encoding luciferase in vivo. These results provide useful insights into the design of next generation LLNs for mRNA delivery.

ASSOCIATIONS BETWEEN NAO VARIBILITY AND U.S. MID-ATLANTIC REGION HYDROCLIMATOLOGY

EPA Science Inventory

Variability in the climate of the US Mid-Atlantic Region is associated with larger scale variability in the El Nino-Southern Oscillation (ENSO), the Pacific North American (PNA) teleconnection pattern, and the North Atlantic Oscillation (NAO). Collectively, these three large-scal...

24 CFR 905.108 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-04-01

... F of this part. Physical Needs Assessment (PNA). A systematic review of all the major physical... capital costs required to meet the replacement need. PIH Information Center (PIC). PIH's current system.... Public Housing Assessment System (PHAS). The assessment system under 24 CFR part 902 for measuring the...

Detection of unamplified genomic DNA by a PNA-based microstructured optical fiber (MOF) Bragg-grating optofluidic system.

PubMed

Bertucci, Alessandro; Manicardi, Alex; Candiani, Alessandro; Giannetti, Sara; Cucinotta, Annamaria; Spoto, Giuseppe; Konstantaki, Maria; Pissadakis, Stavros; Selleri, Stefano; Corradini, Roberto

2015-01-15

Microstructured optical fibers containing microchannels and Bragg grating inscribed were internally functionalized with a peptide nucleic acid (PNA) probe specific for a gene tract of the genetically modified Roundup Ready soy. These fibers were used as an optofluidic device for the detection of DNA by measuring the shift in the wavelength of the reflected IR light. Enhancement of optical read-out was obtained using streptavidin coated gold-nanoparticles interacting with the genomic DNA captured in the fiber channels (0%, 0.1%, 1% and 10% RR-Soy), enabling to achieve statistically significant, label-free, and amplification-free detection of target DNA in low concentrations, low percentages, and very low sample volumes. Computer simulations of the fiber optics based on the finite element method (FEM) were consistent with the formation of a layer of organic material with an average thickness of 39 nm for the highest percentage (10% RR soy) analysed. Copyright © 2014 Elsevier B.V. All rights reserved.

A route to possible civil engineering materials: the case of high-pressure phases of lime

NASA Astrophysics Data System (ADS)

Bouibes, A.; Zaoui, A.

2015-07-01

Lime system has a chemical composition CaO, which is known as thermodynamically stable. The purpose here is to explore further possible phases under pressure, by means of variable-composition ab initio evolutionary algorithm. The present investigation shows surprisingly new stable compounds of lime. At ambient pressure we predict, in addition to CaO, CaO2 as new thermodynamically stable compound. The latter goes through two phases transition from C2/c space group structure to Pna21 at 1.5 GPa, and Pna21 space group structure to I4/mcm at 23.4 GPa. Under increasing pressure, further compounds such as CaO3 become the most stable and stabilize in P-421m space group structure above 65 GPa. For the necessary knowledge of the new predicted compounds, we have computed their mechanical and electronic properties in order to show and to explain the main reasons leading to the structural changes.

Activation of respiratory muscles during respiratory muscle training.

PubMed

Walterspacher, Stephan; Pietsch, Fabian; Walker, David Johannes; Röcker, Kai; Kabitz, Hans-Joachim

2018-01-01

It is unknown which respiratory muscles are mainly activated by respiratory muscle training. This study evaluated Inspiratory Pressure Threshold Loading (IPTL), Inspiratory Flow Resistive Loading (IFRL) and Voluntary Isocapnic Hyperpnea (VIH) with regard to electromyographic (EMG) activation of the sternocleidomastoid muscle (SCM), parasternal muscles (PARA) and the diaphragm (DIA) in randomized order. Surface EMG were analyzed at the end of each training session and normalized using the peak EMG recorded during maximum inspiratory maneuvers (Sniff nasal pressure: SnPna, maximal inspiratory mouth occlusion pressure: PImax). 41 healthy participants were included. Maximal activation was achieved for SCM by SnPna; the PImax activated predominantly PARA and DIA. Activations of SCM and PARA were higher in IPTL and VIH than for IFRL (p<0.05). DIA was higher applying IPTL compared to IFRL or VIH (p<0.05). IPTL, IFRL and VIH differ in activation of inspiratory respiratory muscles. Whereas all methods mainly stimulate accessory respiratory muscles, diaphragm activation was predominant in IPTL. Copyright © 2017 Elsevier B.V. All rights reserved.

A route to possible civil engineering materials: the case of high-pressure phases of lime.

PubMed

Bouibes, A; Zaoui, A

2015-07-23

Lime system has a chemical composition CaO, which is known as thermodynamically stable. The purpose here is to explore further possible phases under pressure, by means of variable-composition ab initio evolutionary algorithm. The present investigation shows surprisingly new stable compounds of lime. At ambient pressure we predict, in addition to CaO, CaO2 as new thermodynamically stable compound. The latter goes through two phases transition from C2/c space group structure to Pna21 at 1.5 GPa, and Pna21 space group structure to I4/mcm at 23.4 GPa. Under increasing pressure, further compounds such as CaO3 become the most stable and stabilize in P-421m space group structure above 65 GPa. For the necessary knowledge of the new predicted compounds, we have computed their mechanical and electronic properties in order to show and to explain the main reasons leading to the structural changes.

Noncovalent interaction-assisted drug delivery system with highly efficient uptake and release of paclitaxel for anticancer therapy.

PubMed

Wei, Yuping; Ma, Liang; Zhang, Liang; Xu, Xia

2017-01-01

An effective drug delivery system requires efficient drug uptake and release inside cancer cells. Here, we report a novel drug delivery system, in which paclitaxel (PTX) interacts with a novel cell penetrating peptide (CPP) through noncovalent interaction designed based on molecular simulations. This CPP/PTX complex confers high efficiency in delivering PTX into cancer cells not by endocytosis but by an energy-independent pathway. Once inside cells, the noncovalent interaction between PTX and the CPP may allow fast release of PTX within cells due to the direct translocation of CPP/PTX. This drug delivery system exhibits strong capacity for inhibition of tumor growth and offers a new avenue for the development of advanced drug delivery systems for anticancer therapy.

The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone.

PubMed

Li, D; Kong, Y; Yu, H; Lehtinen, A; Huang, H; Shen, F; Min, L; Zhou, J; Tang, G; Wang, Q

2008-04-01

A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity. The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including (1)H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments. The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15.0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa. These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.

2-Aminoimidazole facilitates efficient gene delivery in a low molecular weight poly(amidoamine) dendrimer.

PubMed

Wang, Jing; Hu, Xuefeng; Wang, Dongli; Xie, Cao; Lu, Weiyue; Song, Jie; Wang, Ruifeng; Gao, Chunli; Liu, Min

2018-06-20

Functional groups have shown great potential in gene delivery. However, a number of the reported functional groups can only overcome one certain physiological barrier, resulting in limited transfection efficiencies. Based on the structure-activity relationships of both imidazolyl and guanidyl, we designed a novel multifunctional group, 2-aminoimidazole (AM), for gene delivery. On modifying with the AM group, the transfection efficiency of low molecular weight poly(amidoamine) (G2) was 200 times greater than the parent dendrimer in vitro. In contrast, the transfection efficiency of G2 showed a decreasing trend when it was grafted with imidazole. Assays revealed that the AM group played multiple roles in gene delivery, including condensing DNA into monodisperse nanoparticles of 80-90 nm in diameter, achieving nearly ten times higher cellular-uptake efficacy, and enhancing the abilities of endosome/lysosome escape and nuclear localization. What's more, AM showed low toxicity. These results demonstrate that the AM group could be a promising tool in non-viral gene delivery.

A RNA nanotechnology platform for a simultaneous two-in-one siRNA delivery and its application in synergistic RNAi therapy

PubMed Central

Jang, Mihue; Han, Hee Dong; Ahn, Hyung Jun

2016-01-01

Incorporating multiple copies of two RNAi molecules into a single nanostructure in a precisely controlled manner can provide an efficient delivery tool to regulate multiple gene pathways in the relation of mutual dependence. Here, we show a RNA nanotechnology platform for a two-in-one RNAi delivery system to contain polymeric two RNAi molecules within the same RNA nanoparticles, without the aid of polyelectrolyte condensation reagents. As our RNA nanoparticles lead to the simultaneous silencing of two targeted mRNAs, of which biological functions are highly interdependent, combination therapy for multi-drug resistance cancer cells, which was studied as a specific application of our two-in-one RNAi delivery system, demonstrates the efficient synergistic effects for cancer therapy. Therefore, this RNA nanoparticles approach has an efficient tool for a simultaneous co-delivery of RNAi molecules in the RNAi-based biomedical applications, and our current studies present an efficient strategy to overcome multi-drug resistance caused by malfunction of genes in chemotherapy. PMID:27562435

Efficient intradermal delivery of superoxide dismutase using a combination of liposomes and iontophoresis for protection against UV-induced skin damage.

PubMed

Kigasawa, Kaoru; Miyashita, Moeko; Kajimoto, Kazuaki; Kanamura, Kiyoshi; Harashima, Hideyoshi; Kogure, Kentaro

2012-01-01

Superoxide dismutase (SOD) is a potent antioxidant agent that protects against UV-induced skin damage. However, its high molecular weight is a significant obstacle for efficient delivery into the skin through the stratum corneum and development of antioxidant activity. Recently, we developed a non-invasive transfollicular delivery system for macromolecules using a combination of liposomes and iontophoresis, that represents promising technology for enhancing transdermal administration of charged drugs (IJP, 403, 2011, Kajimoto et al.). In this study, in rats we attempted to apply this system to intradermal delivery of SOD for preventing UV-induced skin injury. SOD encapsulating in cationic liposomes was subjected to anodal iontophoresis. After iontophoretic treatment, the liposomes were diffused widely in the viable skin layer around hair follicles. In contrast, passive diffusion failed to transport liposomes efficiently into the skin. Iontophoretic delivery of liposomes encapsulating SOD caused a marked decrease in the production of oxidative products, such as malondialdehyde, hexanoyl lysine, and 8-hydroxi-2-deoxyguanosine, in UV-irradiated skin. These findings suggested that functional SOD can be delivered into the skin using a combination of iontophoresis and a liposomal system. In conclusion, we succeeded in developing an efficient intradermal SOD delivery system, that would be useful for delivery of other macromolecules.

Galactose Derivative-Modified Nanoparticles for Efficient siRNA Delivery to Hepatocellular Carcinoma.

PubMed

Huang, Kuan-Wei; Lai, Yu-Tsung; Chern, Guann-Jen; Huang, Shao-Feng; Tsai, Chia-Lung; Sung, Yun-Chieh; Chiang, Cheng-Chin; Hwang, Pi-Bei; Ho, Ting-Lun; Huang, Rui-Lin; Shiue, Ting-Yun; Chen, Yunching; Wang, Sheng-Kai

2018-05-29

Successful siRNA therapy requires suitable delivery systems with targeting moieties such as small molecules, peptides, antibodies, or aptamers. Galactose (Gal) residues recognized by the asialoglycoprotein receptor (ASGPR) can serve as potent targeting moieties for hepatocellular carcinoma (HCC) cells. However, efficient targeting to HCC via galactose moieties rather than normal liver tissues in HCC patients remains a challenge. To achieve more efficient siRNA delivery in HCC, we synthesized various galactoside derivatives and investigated the siRNA delivery capability of nanoparticles modified with those galactoside derivatives. In this study, we assembled lipid/calcium/phosphate nanoparticles (LCP NPs) conjugated with eight types of galactoside derivatives and demonstrated that phenyl β-d-galactoside-decorated LCP NPs (L4-LCP NPs) exhibited a superior siRNA delivery into HCC cells compared to normal hepatocytes. VEGF siRNAs delivered by L4-LCP NPs downregulated VEGF expression in HCC in vitro and in vivo and led to a potent antiangiogenic effect in the tumor microenvironment of a murine orthotopic HCC model. The efficient delivery of VEGF siRNA by L4-LCP NPs that resulted in significant tumor regression indicates that phenyl galactoside could be a promising HCC-targeting ligand for therapeutic siRNA delivery to treat liver cancer.

43 CFR 418.28 - Conditions of delivery.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Enforcement § 418.28 Conditions of delivery. There are four basic elements for enforcement with all necessary... efficiency. To the extent that the actual District efficiency determined for an irrigation season is greater...

Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

PubMed

Zheng, Hao; Tang, Cui; Yin, Chunhua

2015-06-01

Present study aimed at exploring advantages/disadvantages of amino acid modified trimethylated chitosan in conquering multiple gene delivery obstacles and thus providing comprehensive understandings for improved transfection efficiency. Arginine, cysteine, and histidine modified trimethyl chitosan were synthesized and employed to self-assemble with plasmid DNA (pDNA) to form nanocomplexes, namely TRNC, TCNC, and THNC, respectively. They were assessed by structural stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. Besides, sodium tripolyphosphate (TPP) was added into TRNC to compromise certain disadvantageous attributes for pDNA delivery. Optimal endosomal escape ability failed to bring in satisfactory transfection efficiency of THNC due to drawbacks in structural stability, cellular uptake, pDNA liberation, and nuclear distribution. TCNC evoked the most potent gene expression owing to multiple advantages including sufficient stability, preferable uptake, efficient pDNA release, and high nucleic accumulation. Undesirable stability and insufficient pDNA release adversely affected TRNC-mediated gene transfer. However, incorporation of TPP could improve such disadvantages and consequently resulted in enhanced transfection efficiencies. Coordination of multiple contributing effects to conquer all delivery obstacles was necessitated for improved transfection efficiency, which would provide insights into rational design of gene delivery vehicles.

How to achieve optimal organization of primary care service delivery at system level: lessons from Europe.

PubMed

Pelone, Ferruccio; Kringos, Dionne S; Spreeuwenberg, Peter; De Belvis, Antonio G; Groenewegen, Peter P

2013-09-01

To measure the relative efficiency of primary care (PC) in turning their structures into services delivery and turning their services delivery into quality outcomes. Cross-sectional study based on the dataset of the Primary Healthcare Activity Monitor for Europe project. Two Data Envelopment models were run to compare the relative technical efficiency. A sensitivity analysis of the resulting efficiency scores was performed. PC systems in 22 European countries in 2009/2010. Model 1 included data on PC governance, workforce development and economic conditions as inputs and access, coordination, continuity and comprehensiveness of care as outputs. Model 2 included the previous process dimensions as inputs and quality indicators as outputs. There is relatively reasonable efficiency in all countries at delivering as many as possible PC processes at a given level of PC structure. It is particularly important to invest in economic conditions to achieve an efficient structure-process balance. Only five countries have fully efficient PC systems in turning their services delivery into high quality outcomes, using a similar combination of access, continuity and comprehensiveness, although they differ on the adoption of coordination of services. There is a large variation in efficiency levels obtained by countries with inefficient PC in turning their services delivery into quality outcomes. Maximizing the individual functions of PC without taking into account the coherence within the health-care system is not sufficient from a policymaker's point of view when aiming to achieve efficiency.

One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring Opening and Screening for Gene Delivery.

PubMed

Molla, Mijanur R; Böser, Alexander; Rana, Akshita; Schwarz, Karina; Levkin, Pavel A

2018-04-18

Efficient delivery of nucleic acids into cells is of great interest in the field of cell biology and gene therapy. Despite a lot of research, transfection efficiency and structural diversity of gene-delivery vectors are still limited. A better understanding of the structure-function relationship of gene delivery vectors is also essential for the design of novel and intelligent delivery vectors, efficient in "difficult-to-transfect" cells and in vivo clinical applications. Most of the existing strategies for the synthesis of gene-delivery vectors require multiple steps and lengthy procedures. Here, we demonstrate a facile, three-component one-pot synthesis of a combinatorial library of 288 structurally diverse lipid-like molecules termed "lipidoids" via a thiolactone ring opening reaction. This strategy introduces the possibility to synthesize lipidoids with hydrophobic tails containing both unsaturated bonds and reducible disulfide groups. The whole synthesis and purification are convenient, extremely fast, and can be accomplished within a few hours. Screening of the produced lipidoids using HEK293T cells without addition of helper lipids resulted in identification of highly stable liposomes demonstrating ∼95% transfection efficiency with low toxicity.

Use of plan quality degradation to evaluate tradeoffs in delivery efficiency and clinical plan metrics arising from IMRT optimizer and sequencer compromises

PubMed Central

Wilkie, Joel R.; Matuszak, Martha M.; Feng, Mary; Moran, Jean M.; Fraass, Benedick A.

2013-01-01

Purpose: Plan degradation resulting from compromises made to enhance delivery efficiency is an important consideration for intensity modulated radiation therapy (IMRT) treatment plans. IMRT optimization and/or multileaf collimator (MLC) sequencing schemes can be modified to generate more efficient treatment delivery, but the effect those modifications have on plan quality is often difficult to quantify. In this work, the authors present a method for quantitative assessment of overall plan quality degradation due to tradeoffs between delivery efficiency and treatment plan quality, illustrated using comparisons between plans developed allowing different numbers of intensity levels in IMRT optimization and/or MLC sequencing for static segmental MLC IMRT plans. Methods: A plan quality degradation method to evaluate delivery efficiency and plan quality tradeoffs was developed and used to assess planning for 14 prostate and 12 head and neck patients treated with static IMRT. Plan quality was evaluated using a physician's predetermined “quality degradation” factors for relevant clinical plan metrics associated with the plan optimization strategy. Delivery efficiency and plan quality were assessed for a range of optimization and sequencing limitations. The “optimal” (baseline) plan for each case was derived using a clinical cost function with an unlimited number of intensity levels. These plans were sequenced with a clinical MLC leaf sequencer which uses >100 segments, assuring delivered intensities to be within 1% of the optimized intensity pattern. Each patient's optimal plan was also sequenced limiting the number of intensity levels (20, 10, and 5), and then separately optimized with these same numbers of intensity levels. Delivery time was measured for all plans, and direct evaluation of the tradeoffs between delivery time and plan degradation was performed. Results: When considering tradeoffs, the optimal number of intensity levels depends on the treatment site and on the stage in the process at which the levels are limited. The cost of improved delivery efficiency, in terms of plan quality degradation, increased as the number of intensity levels in the sequencer or optimizer decreased. The degradation was more substantial for the head and neck cases relative to the prostate cases, particularly when fewer than 20 intensity levels were used. Plan quality degradation was less severe when the number of intensity levels was limited in the optimizer rather than the sequencer. Conclusions: Analysis of plan quality degradation allows for a quantitative assessment of the compromises in clinical plan quality as delivery efficiency is improved, in order to determine the optimal delivery settings. The technique is based on physician-determined quality degradation factors and can be extended to other clinical situations where investigation of various tradeoffs is warranted. PMID:23822412

Gene delivery through the use of a hyaluronate-associated intracellularly degradable cross-linked polyethyleneimine

PubMed Central

Xu, Peisheng; Quick, Griffin; Yeo, Yoon

2009-01-01

For a non-viral gene delivery system to be clinically effective, it should be non-toxic, compatible with biological components, and highly efficient in gene transfection. With this goal in mind, we investigated the gene delivery efficiency of a ternary complex consisting of DNA, an intracellularly degradable polycation, and sodium hyaluronate (DPH complex). Here, we report that the DPH ternary complex achieved significantly higher transfection efficiency than other polymer systems, especially in the presence of serum. The high transfection efficiency and serum tolerance of DPH are attributed to a unique interplay between CLPEI and HA, which leads to (i) the improved stability of DNA in the extracellular environment and at the early stage of intracellular trafficking and (ii) timely dissociation of the DNA-polymer complex. This study reinforces findings of earlier studies that emphasized each step as a bottleneck for efficient gene delivery; yet, it is the first to show that it is possible to overcome these obstacles simultaneously by taking advantage of two distinctive approaches. PMID:19631979

Liposome-chaperoned cell-free synthesis for the design of proteoliposomes: Implications for therapeutic delivery.

PubMed

Lu, Mei; Zhao, Xiaoyun; Xing, Haonan; Xun, Zhe; Yang, Tianzhi; Cai, Cuifang; Wang, Dongkai; Ding, Pingtian

2018-04-03

Cell-free (CF) protein synthesis has emerged as a powerful technique platform for efficient protein production in vitro. Liposomes have been widely studied as therapeutic carriers due to their biocompatibility, biodegradability, low toxicity, flexible surface manipulation, easy preparation, and higher cargo encapsulation capability. However, rapid immune clearance, insufficient targeting capacity, and poor cytoplasmic delivery efficiency substantially restrict their clinical application. The incorporation of functional membrane proteins (MPs) or peptides allows the transfer of biological properties to liposomes and imparts them with improved circulation, increased targeting, and efficient intracellular delivery. Liposome-chaperoned CF synthesis enables production of proteoliposomes in one-step reaction, which not only substantially simplifies the production procedure but also keeps protein functionality intact. Building off these observations, proteoliposomes with integrated MPs represent an excellent candidate for therapeutic delivery. In this review, we describe recent advances in CF synthesis with emphasis on detailing key factors for improving CF expression efficiency. Furthermore, we provide insights into strategies for rational design of proteoliposomal nanodelivery systems via CF synthesis. Liposome-chaperoned CF synthesis has emerged as a powerful approach for the design of recombinant proteoliposomes in one-step reaction. The incorporation of bioactive MPs or peptides into liposomes via CF synthesis can facilitate the development of proteoliposomal nanodelivery systems with improved circulation, increased targeting, and enhanced cellular delivery capacity. Moreover, by adapting lessons learned from natural delivery vehicles, novel bio-inspired proteoliposomes with enhanced delivery properties could be produced in CF systems. In this review, we first give an overview of CF synthesis with focus on enhancing protein expression in liposome-chaperoned CF systems. Furthermore, we intend to provide insight into harnessing CF-synthesized proteoliposomes for efficient therapeutic delivery. Copyright © 2018. Published by Elsevier Ltd.

Recent Advances in Skin Penetration Enhancers for Transdermal Gene and Drug Delivery.

PubMed

Amjadi, Morteza; Mostaghaci, Babak; Sitti, Metin

2017-01-01

There is a growing interest in transdermal delivery systems because of their noninvasive, targeted, and on-demand delivery of gene and drugs. However, efficient penetration of therapeutic compounds into the skin is still challenging largely due to the impermeability of the outermost layer of the skin, known as stratum corneum. Recently, there have been major research activities to enhance the skin penetration depth of pharmacological agents. This article reviews recent advances in the development of various strategies for skin penetration enhancement. We show that approaches such as ultrasound waves, laser, and microneedle patches have successfully been employed to physically disrupt the stratum corneum structure for enhanced transdermal delivery. Rather than physical approaches, several non-physical route have also been utilized for efficient transdermal delivery across the skin barrier. Finally, we discuss some clinical applications of transdermal delivery systems for gene and drug delivery. This paper shows that transdermal delivery devices can potentially function for diverse healthcare and medical applications while further investigations are still necessary for more efficient skin penetration of gene and drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

TREATMENT OF A PENTACHLOROPHENOL AND CREOSOTE-CONTAMINATED SOIL USING THE LIGNIN-DEGRADING FUNGUS PHANERO- CHAETE SORDIDA: A FIELD DEMONSTRATION

EPA Science Inventory

The feasibility of large-scale fungal bioaugmentation was evaluated by assessing the ability of the lignin-degrading fungus Phanerochaete sordida to decrease the soil concentrations of pentachlorophenol (PCP) and 13 priority pollutant polynuclear aromatic (PNA) creosote component...

Solubilities of Peroxyacetyl Nitrate and Peroxynitrate in Water and Aqueous H(sub 2)SO(sub 4)

NASA Technical Reports Server (NTRS)

Leu, M. T.; Zhang, R.

1998-01-01

In addition, the effective Henry's law constant and the associative enthalpy change of solvation of PNA in water are determined to be 39.95 mol kg^-1 atm^-1 and -69.84 kJ mol^-1 at 298.15K, respectively.

Simulation study of electric-guided delivery of 0.4µm monodisperse and polydisperse aerosols to the ostiomeatal complex.

PubMed

Xi, Jinxiang; Yuan, Jiayao Eddie; Si, Xiuhua April

2016-05-01

Despite the high prevalence of rhinosinusitis, current inhalation therapy shows limited efficacy due to extremely low drug delivery efficiency to the paranasal sinuses. Novel intranasal delivery systems are needed to enhance targeted delivery to the sinus with therapeutic dosages. An optimization framework for intranasal drug delivery was developed to target polydisperse charged aerosols to the ostiomeatal complex (OMC) with electric guidance. The delivery efficiency of a group of charged aerosols recently reported in the literature was numerically assessed and optimized in an anatomically accurate nose-sinus model. Key design variables included particle charge number, particle size and distribution, electrode strength, and inhalation velocity. Both monodisperse and polydisperse aerosol profiles were considered. Results showed that the OMC delivery efficiency was highly sensitive to the applied electric field and electrostatic charges carried by the particles. Through the synthesis of electric-guidance and point drug release, focused deposition with significantly enhanced dosage in the OMC can be achieved. For 0.4 µm charged aerosols, an OMC delivery efficiency of 51.6% was predicted for monodisperse aerosols and 34.4% for polydisperse aerosols. This difference suggested that the aerosol profile exerted a notable effect on intranasal deliveries. Sensitivity analysis indicated that the OMC deposition fraction was highly sensitive to the charge and size of particles and was less sensitive to the inhalation velocity considered in this study. Experimental studies are needed to validate the numerically optimized designs. Further studies are warranted to investigate the targeted OMC delivery with both electric and acoustics controls, the latter of which has the potential to further deliver the drug particles into the sinus cavity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery.

PubMed

Wang, Xu-Li; Ramusovic, Sergej; Nguyen, Thanh; Lu, Zheng-Rong

2007-01-01

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence disease-related genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal-lysosomal escape of siRNA delivery systems at the endosomal-lysosomal pH. The surfactants formed compact nanoparticles (160-260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40-88% silencing of luciferase expression with 100 nM siRNA and 35-75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.

Exploring the role of peptides in polymer-based gene delivery.

PubMed

Sun, Yanping; Yang, Zhen; Wang, Chunxi; Yang, Tianzhi; Cai, Cuifang; Zhao, Xiaoyun; Yang, Li; Ding, Pingtian

2017-09-15

Polymers are widely studied as non-viral gene vectors because of their strong DNA binding ability, capacity to carry large payload, flexibility of chemical modifications, low immunogenicity, and facile processes for manufacturing. However, high cytotoxicity and low transfection efficiency substantially restrict their application in clinical trials. Incorporating functional peptides is a promising approach to address these issues. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we systematically summarize the role of peptides in polymer-based gene delivery, and elaborate how to rationally design polymer-peptide based gene delivery vectors. Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Spray-Dried Nanoparticle-in-Microparticle Delivery Systems (NiMDS) for Gene Delivery, Comprising Polyethylenimine (PEI)-Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix.

PubMed

Schulze, Jan; Kuhn, Stephanie; Hendrikx, Stephan; Schulz-Siegmund, Michaela; Polte, Tobias; Aigner, Achim

2018-03-01

Nucleic acid-based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid-based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready-to-use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine-based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray-dried nanoparticle-in-microparticle delivery system is presented for the encapsulation of polyethylenimine-based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy-to-handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine-based nanoparticles from the nanoparticle-in-microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle-in-microparticle delivery system is demonstrated as ready-to-use dry powder to be an efficient device for the inhalative delivery of polyethylenimine-based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biophysical and biological contributions of polyamine-coated carbon nanotubes and bidimensional buckypapers in the delivery of miRNAs to human cells.

PubMed

Celluzzi, Antonella; Paolini, Alessandro; D'Oria, Valentina; Risoluti, Roberta; Materazzi, Stefano; Pezzullo, Marco; Casciardi, Stefano; Sennato, Simona; Bordi, Federico; Masotti, Andrea

2018-01-01

Recent findings in nanomedicine have revealed that carbon nanotubes (CNTs) can be used as potential drug carriers, therapeutic agents and diagnostics tools. Moreover, due to their ability to cross cellular membranes, their nanosize dimension, high surface area and relatively good biocompatibility, CNTs have also been employed as a novel gene delivery vector system. In our previous work, we functionalized CNTs with two polyamine polymers, polyethyleneimine (PEI) and polyamidoamine dendrimer (PAMAM). These compounds have low cytotoxicity, ability to conjugate microRNAs (such as miR-503) and, at the same time, transfect efficiently endothelial cells. The parameters contributing to the good efficiency of transfection that we observed were not investigated in detail. In fact, the diameter and length of CNTs are important parameters to be taken into account when evaluating the effects on drug delivery efficiency. In order to investigate the biophysical and biological contributions of polymer-coated CNTs in delivery of miRNAs to human cells, we decided to investigate three different preparations, characterized by different dimensions and aspect ratios. In particular, we took into account very small CNTs, a suspension of CNTs starting from the commercial product and a 2D material based on CNTs (ie, buckypapers [BPs]) to examine the transfection efficiency of a rigid scaffold. In conclusion, we extensively investigated the biophysical and biological contributions of polyamine-coated CNTs and bidimensional BPs in the delivery of miRNAs to human cells, in order to optimize the transfection efficiency of these compounds to be employed as efficient drug delivery vectors in biomedical applications.

Highly efficient local delivery of endothelial progenitor cells significantly potentiates angiogenesis and full-thickness wound healing.

PubMed

Wang, Chenggui; Wang, Qingqing; Gao, Wendong; Zhang, Zengjie; Lou, Yiting; Jin, Haiming; Chen, Xiaofeng; Lei, Bo; Xu, Huazi; Mao, Cong

2018-03-15

Wound therapy with a rapid healing performance remains a critical clinical challenge. Cellular delivery is considered to be a promising approach to improve the efficiency of healing, yet problems such as compromised cell viability and functionality arise due to the inefficient delivery. Here, we report the efficient delivery of endothelial progenitor cells (EPCs) with a bioactive nanofibrous scaffold (composed of collagen and polycaprolactone and bioactive glass nanoparticles, CPB) for enhancing wound healing. Under the stimulation of CPB nanofibrous system, the viability and angiogenic ability of EPCs were significantly enhanced through the activation of Hif-1α/VEGF/SDF-1α signaling. In vivo, CPB/EPC constructs significantly enhanced the formation of high-density blood vessels by greatly upregulating the expressions of Hif-1α, VEGF, and SDF-1α. Moreover, owing to the increased local delivery of cells and fast neovascularization within the wound site, cell proliferative activity, granulation tissue formation, and collagen synthesis and deposition were greatly promoted by CPB/EPC constructs resulting in rapid re-epithelialization and regeneration of skin appendages. As a result, the synergistic enhancement of wound healing was observed from CPB/EPC constructs, which suggests the highly efficient delivery of EPCs. CPB/EPC constructs may become highly competitive cell-based therapeutic products for efficient impaired wound healing application. This study may also provide a novel strategy to develop bioactive cell therapy constructs for angiogenesis-related regenerative medicine. This paper reported a highly efficient local delivery of EPCs using bioactive glass-based CPB nanofibrous scaffold for enhancing angiogenesis and wound regeneration. In vitro study showed that CPB can promote the proliferation, migration, and tube formation of EPCs through upregulation of the Hif-1α/VEGF/SDF-1α signaling pathway, indicating that the bioactivity and angiogenic ability of EPCs can be highly maintained and promoted by the CPB scaffold. Moreover, CPB/EPC constructs effectively stimulated the regeneration of diabetic wounds with satisfactory vascularization and better healing outcomes in a full-thickness wound model, suggesting that the highly efficient delivery of EPCs to wound site facilitates angiogenesis and further leads to wound healing. The high angiogenic capacity and excellent healing ability make CPB/EPC constructs highly competitive in cell-based therapeutic products for efficient wound repair application. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Recent developments in anticancer drug delivery using cell penetrating and tumor targeting peptides.

PubMed

Dissanayake, Shama; Denny, William A; Gamage, Swarna; Sarojini, Vijayalekshmi

2017-03-28

Efficient intracellular trafficking and targeted delivery to the site of action are essential to overcome the current drawbacks of cancer therapeutics. Cell Penetrating Peptides (CPPs) offer the possibility of efficient intracellular trafficking, and, therefore the development of drug delivery systems using CPPs as cargo carriers is an attractive strategy to address the current drawbacks of cancer therapeutics. Additionally, the possibility of incorporating Tumor Targeting Peptides (TTPs) into the delivery system provides the necessary drug targeting effect. Therefore the conjugation of CPPs and/or TTPs with therapeutics provides a potentially efficient method of improving intracellular drug delivery mechanisms. Peptides used as cargo carriers in DDS have been shown to enhance the cellular uptake of drugs and thereby provide an efficient therapeutic benefit over the drug on its own. After providing a brief overview of various drug targeting approaches, this review focusses on peptides as carriers and targeting moieties in drug-peptide covalent conjugates and summarizes the most recent literature examples where CPPs on their own or CPPs together with TTPs have been conjugated to anticancer drugs such as Doxorubicin, Methotrexate, Paclitaxel, Chlorambucil etc. A short section on CPPs used in multicomponent drug delivery systems is also included. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient packet transportation on complex networks with nonuniform node capacity distribution

NASA Astrophysics Data System (ADS)

He, Xuan; Niu, Kai; He, Zhiqiang; Lin, Jiaru; Jiang, Zhong-Yuan

2015-03-01

Provided that node delivery capacity may be not uniformly distributed in many realistic networks, we present a node delivery capacity distribution in which each node capacity is composed of uniform fraction and degree related proportion. Based on the node delivery capacity distribution, we construct a novel routing mechanism called efficient weighted routing (EWR) strategy to enhance network traffic capacity and transportation efficiency. Compared with the shortest path routing and the efficient routing strategies, the EWR achieves the highest traffic capacity. After investigating average path length, network diameter, maximum efficient betweenness, average efficient betweenness, average travel time and average traffic load under extensive simulations, it indicates that the EWR appears to be a very effective routing method. The idea of this routing mechanism gives us a good insight into network science research. The practical use of this work is prospective in some real complex systems such as the Internet.

Nanomaterials in cancer-therapy drug delivery system.

PubMed

Zhang, Gen; Zeng, Xin; Li, Ping

2013-05-01

Nanomaterials can enhance the delivery and treatment efficiency of anti-cancer drugs, and the mechanisms of the tumor-reducing activity of nanomaterials with cancer drug have been investigated. The task for drug to reach pathological areas has facilitated rapid advances in nanomedicine. Herein, we summarize promising findings with respect to cancer therapeutics based on nano-drug delivery vectors. Relatively high toxicity of uncoated nanoparticles restricts the use of these materials in humans. In order to reduce toxicity, many approaches have focused on the encapsulation of nanoparticles with biocompatible materials. Efficient delivery systems have been developed that utilized nanoparticles loaded with high dose of cancer drug in the presence of bilayer molecules. Well-established nanotechnologies have been designed for drug delivery with specific bonding. Surface-modified nanoparticles as vehicles for drug delivery system that contains multiple nano-components, each specially designed to achieve aimed task for the emerging application delivery of therapeutics. Drug-coated polymer nanoparticles could efficiently increase the intracellular accumulation of anti-cancer drugs. This review also introduces the nanomaterials with drug on the induction of apoptosis in cancer cells in vitro and in vivo. Direct interactions between the particles and cellular molecules to cause adverse biological responses are also discussed.

Superparamagnetic Iron Oxide Nanoparticle-Based Delivery Systems for Biotherapeutics

PubMed Central

Mok, Hyejung; Zhang, Miqin

2014-01-01

Introduction Superparamagnetic iron oxide nanoparticle (SPION)-based carrier systems have many advantages over other nanoparticle-based systems. They are biocompatible, biodegradable, facilely tunable, and superparamagnetic and thus controllable by an external magnetic field. These attributes enable their broad biomedical applications. In particular, magnetically-driven carriers are drawing considerable interest as an emerging therapeutic delivery system because of their superior delivery efficiency. Area covered This article reviews the recent advances in use of SPION-based carrier systems to improve the delivery efficiency and target specificity of biotherapeutics. We examine various formulations of SPION-based delivery systems, including SPION micelles, clusters, hydrogels, liposomes, and micro/nanospheres, as well as their specific applications in delivery of biotherapeutics. Expert opinion Recently, biotherapeutics including therapeutic cells, proteins and genes have been studied as alternative treatments to various diseases. Despite the advantages of high target specificity and low adverse effects, clinical translation of biotherapeutics has been hindered by the poor stability and low delivery efficiency compared to chemical drugs. Accordingly, biotherapeutic delivery systems that can overcome these limitations are actively pursued. SPION-based materials can be ideal candidates for developing such delivery systems because of their excellent biocompatibility and superparamagnetism that enables long-term accumulation/retention at target sites by utilization of a suitable magnet. In addition, synthesis technologies for production of finely-tuned, homogeneous SPIONs have been well developed, which may promise their rapid clinical translation. PMID:23199200

Development of "College CHEF," a Campus-Based Culinary Nutrition Program

ERIC Educational Resources Information Center

McMullen, Jennifer; Ickes, Melinda; Noland, Melody; Erwin, Heather; Helme, Don

2017-01-01

Background: More than one third of college students are obese, with many lacking the knowledge and skills necessary to cook healthfully. Purpose: The purpose of this study was to describe the PRECEDE-PROCEED model-driven primary and secondary needs assessments (PNA, SNA), which contributed to the development of the College CHEF. Methods: Staff…

Changes to Tensile Strength and Electromagnetic Shielding Effectiveness in Neutron Irradiated Carbon Nanocomposites

DTIC Science & Technology

2013-03-01

3 II. Background ...Agilent Technologies E8362B PNA Series Microwave Network Analyzer. The waveguide is secured by hex cap screws and nuts tightened to the torque...believed to result in a reduction in tensile strength of the composite. 5 II. Background Chapter Overview This chapter describes the

DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles.

PubMed

Mejia-Ariza, Raquel; Rosselli, Jessica; Breukers, Christian; Manicardi, Alex; Terstappen, Leon W M M; Corradini, Roberto; Huskens, Jurriaan

2017-03-23

A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal-organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or non-covalent interactions, resulting in alkyne-modified and biotin-modified MIL-88A, respectively. Covalent surface coupling of an azide-dye and the alkyne-MIL-88A was achieved by means of a click reaction. Non-covalent streptavidin-biotin interactions were employed to link biotin-PNA to biotin-MIL-88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single-base mismatched, and randomized DNA targets. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

GCM simulations of intraseasonal variability in the Pacific/North American region

NASA Technical Reports Server (NTRS)

Schubert, Siegfried; Suarez, Max; Park, Chung-Kyu; Moorthi, Shrinivas

1993-01-01

General circulation model (GCM) simulations of low-frequency variability with time scales of 20 to 70 days are analyzed for the Pacific sector during boreal winter. The GCM's leading mode in the upper-tropospheric zonal wind is associated with fluctuations of the East Asian jet; this mode resembles, in both structure and amplitude, the Pacific/North American (PNA) pattern found in the observations on these time scales. In both the model and observations, the PNA anomaly is characterized by: (1) a linear balance in the upper-tropospheric vorticity budget with no significant Rossby wave source in the tropics, (2) a barotropic conversion of kinetic energy from the time mean Pacific jet, and (3) a north/south displacement of the Pacific storm track. In the GCM, the latter is associated with synoptic eddy heat flux and latent heat anomalies that appear to contribute to a strong lower-tropospheric source of wave activity over the North Pacific. This is in contrast to the observations, which show only a weak source of wave activity in this region.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

PubMed

Quirin, Kayla A; Kwon, Jason J; Alioufi, Arafat; Factora, Tricia; Temm, Constance J; Jacobsen, Max; Sandusky, George E; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Mendell, Joshua T; Korc, Murray; Kota, Janaiah

2018-03-16

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10 12 viral genomes (vg). Intraductal delivery of 1 × 10 11 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10 11 vg. In a Kras G12D -driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Polyethyleneimine Coating Enhances the Cellular Uptake of Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA Constructs

PubMed Central

Xia, Tian; Kovochich, Michael; Liong, Monty; Meng, Huan; Kabehie, Sanaz; Zink, Jeffrey I.; Nel, Andre E.

2014-01-01

Surface-functionalized mesoporous silica nanoparticles (MSNP) can be used as an efficient and safe carrier for bioactive molecules. In order to make the MSNP a more efficient delivery system, we modified the surface of the particles by a functional group that enhances cellular uptake and allows nucleic acid delivery in addition to traditional drug delivery. Non-covalent attachment of polyethyleneimine (PEI) polymers to the surface not only increases MSNP cellular uptake, but also generates a cationic surface to which DNA and siRNA constructs could be attached. While efficient for intracellular delivery of these nucleic acids, the 25 KD PEI polymer unfortunately changes the safety profile of the MSNP that is otherwise very safe. By experimenting with several different polymer molecular weights, it was possible to retain high cellular uptake and transfection efficiency while reducing or even eliminating cationic MSNP cytotoxicity. The particles coated with the 10 KD PEI polymer was particularly efficient for transducing HEPA-1 cells with a siRNA construct that was capable of knocking down GFP expression. Similarly, transfection of a GFP plasmid induced effective expression of the fluorescent protein in > 70% cells in the population. These outcomes were quantitatively assessed by confocal microscopy and flow cytometry. We also demonstrated that the enhanced cellular uptake of the non-toxic cationic MSNP enhance the delivery of the hydrophobic anticancer drug, paclitaxel, to pancreatic cancer cells. In summary, we demonstrate that by a careful selection of PEI size, it is possible to construct cationic MSNP that are capable of nucleotide and enhanced drug delivery with minimal or no cytotoxicity. This novel use of a cationic MSNP extends its therapeutic use potential. PMID:19739605

Rapidly separating microneedles for transdermal drug delivery.

PubMed

Zhu, Dan Dan; Wang, Qi Lei; Liu, Xu Bo; Guo, Xin Dong

2016-09-01

The applications of polymer microneedles (MNs) into human skin emerged as an alternative of the conventional hypodermic needles. However, dissolving MNs require many minutes to be dissolved in the skin and typically have difficulty being fully inserted into the skin, which may lead to the low drug delivery efficiency. To address these issues, we introduce rapidly separating MNs that can rapidly deliver drugs into the skin in a minimally invasive way. For the rapidly separating MNs, drug loaded dissolving MNs are mounted on the top of solid MNs, which are made of biodegradable polylactic acid which eliminate the biohazardous waste. These MNs have sufficient mechanical strength to be inserted into the skin with the drug loaded tips fully embedded for subsequent dissolution. Compared with the traditional MNs, rapidly separating MNs achieve over 90% of drug delivery efficiency in 30s while the traditional MNs needs 2min to achieve the same efficiency. With the in vivo test in mice, the micro-holes caused by rapidly separating MNs can heal in 1h, indicating that the rapidly separating MNs are safe for future applications. These results indicate that the design of rapidly separating dissolvable MNs can offer a quick, high efficient, convenient, safe and potentially self-administered method of drug delivery. Polymer microneedles offer an attractive, painless and minimally invasive approach for transdermal drug delivery. However, dissolving microneedles require many minutes to be dissolved in the skin and typically have difficulty being fully inserted into the skin due to the skin deformation, which may lead to the low drug delivery efficiency. In this work we proposed rapidly separating microneedles which can deliver over 90% of drug into the skin in 30s. The in vitro and in vivo results indicate that the new design of these microneedles can offer a quick, high efficient, convenient and safe method for transdermal drug delivery. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Poly(ester amine) Composed of Polyethylenimine and Pluronic Enhance Delivery of Antisense Oligonucleotides In Vitro and in Dystrophic mdx Mice

PubMed Central

Wang, Mingxing; Wu, Bo; Tucker, Jason D; Bollinger, Lauren E; Lu, Peijuan; Lu, Qilong

2016-01-01

A series of poly(esteramine)s (PEAs) constructed from low molecular weight polyethyleneimine (LPEI) and Pluronic were evaluated for the delivery of antisense oligonuclotides (AOs), 2′-O-methyl phosphorothioate RNA (2′-OMePS) and phosphorodiamidate morpholino oligomer (PMO) in cell culture and dystrophic mdx mice. Improved exon-skipping efficiency of both 2′-OMePS and PMO was observed in the C2C12E50 cell line with all PEA polymers compared with PEI 25k or LF-2k. The degree of efficiency was found in the order of PEA 01, PEA 04 > PEA 05 > others. The in vivo study in mdx mice demonstrated enhanced exon-skipping of 2′-OMePS with the order of PEA 06 > PEA 04, PEA 07 > PEA 03 > PEA 01 > others, and much higher than PEI 25k formulated 2′-OMePS. Exon-skipping efficiency of PMO in formulation with the PEAs were significantly enhanced in the order of PEA 02 > PEA 10 > PEA 01, PEA 03 > PEA 05, PEA 07, PEA 08 > others, with PEA 02 reaching fourfold of Endo-porter formulated PMO. PEAs improve PMO delivery more effectively than 2′-OMePS delivery in vivo, and the systemic delivery evaluation further highlight the efficiency of PEA for PMO delivery in all skeletal muscle. The results suggest that the flexibility of PEA polymers could be explored for delivery of different AO chemistries, especially for antisense therapy. PMID:27483024

Characterisation of glycoconjugate sugar residues in the vomeronasal organ of the armadillo Chaetophractus villosus (Mammalia, xenarthra)

PubMed Central

CARMANCHAHI, P. D.; FERRARI, C. C.; ALDANA MARCOS, H. J.; AFFANNI, J. M.; SONEZ, C. A.; PAZ, D. A.

2000-01-01

Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus. The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA120. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA120, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL-I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA120 and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA120 stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes. PMID:10853958

Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment.

PubMed

Pavlekovic, Marko; Schmid, Markus C; Schmider-Poignee, Nadja; Spring, Stefan; Pilhofer, Martin; Gaul, Tobias; Fiandaca, Mark; Löffler, Frank E; Jetten, Mike; Schleifer, K-H; Lee, Natuschka M

2009-08-01

Fluorescence in situ hybridization (FISH) using fluorochrome-labeled DNA oligonucleotide probes has been successfully applied for in situ detection of anaerobic ammonium oxidizing (anammox) bacteria. However, application of the standard FISH protocols to visualize anammox bacteria in biofilms from a laboratory-scale wastewater reactor produced only weak signals. Increased signal intensity was achieved either by modifying the standard FISH protocol, using peptide nucleic acid probes (PNA FISH), or applying horse radish peroxidase- (HRP-) labeled probes and subsequent catalyzed reporter deposition (CARD-FISH). A comparative analysis using anammox biofilm samples and suspended anammox biomass from different laboratory wastewater bioreactors revealed that the modified standard FISH protocol and the PNA FISH probes produced equally strong fluorescence signals on suspended biomass, but only weak signals were obtained with the biofilm samples. The probe signal intensities in the biofilm samples could be enhanced by enzymatic pre-treatment of fixed cells, and by increasing the hybridization time of the PNA FISH protocol. CARD-FISH always produced up to four-fold stronger fluorescent signals but unspecific fluorescence signals, likely caused by endogenous peroxidases as reported in several previous studies, compromised the results. Interference of the development of fluorescence intensity with endogenous peroxidases was also observed in cells of aerobic ammonium oxidizers like Nitrosomonas europea, and sulfate-reducers like Desulfobacter postgatei. Interestingly, no interference was observed with other peroxidase-positive microorganisms, suggesting that CARD-FISH is not only compromised by the mere presence of peroxidases. Pre-treatment of cells to inactivate peroxidase with HCl or autoclavation/pasteurization failed to inactive peroxidases, but H(2)O(2) significantly reduced endogenous peroxidase activity. However, for optimal inactivation, different H(2)O(2) concentrations and incubation time may be needed, depending on nature of sample and should therefore always be individually determined for each study.

A base-modified PNA-graphene oxide platform as a turn-on fluorescence sensor for the detection of human telomeric repeats

NASA Astrophysics Data System (ADS)

Sabale, Pramod M.; George, Jerrin Thomas; Srivatsan, Seergazhi G.

2014-08-01

Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures.Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures. Electronic supplementary information (ESI) available. Figures, tables, experimental procedures and NMR spectra. See DOI: 10.1039/c4nr00878b

[Dialysis dose, nutrition and growth among pediatric patients on peritoneal dialysis].

PubMed

Cano, Francisco; Azócar, Marta; Marín, Verónica; Rodríguez, Eugenio; Delucchi, Angela; Ratner, Rinat; Cavada, Gabriel

2005-12-01

Stunting is common among pediatric patients on peritoneal dialysis. To establish the best profile for urea kinetic variables associated to growth in children on chronic peritoneal dialysis (PD). Twenty patients, aged 1 month to 14 years, 13 males, were followed for 6-12 months, with monthly measurements of weight/age and height/age Z score; plasma creatinine, BUN, protein and albumin and urine and dialysate urea nitrogen, creatinine, protein and albumin. Minimum total Kt/V was 2.1. Dialysis dose (Kt/V), Protein Equivalent of Urea Nitrogen Appearance (PNA), Protein Catabolic Rate (PCR) and Nitrogen Balance (NB) were calculated. To identify the variable(s) associated to growth, the Tree Classification Model (CART) Enterprise Miner 8.1 was applied. Mean total/residual Kt/V: 3.4+/-1.3/1.69+/-1.27; Daily Protein Intake (DPI) was 3.25+/-1.27 g/kg/day. nPNA, PCR and NB were 1.37+/-0.44, 0.84+/-0.33 and 1.86+/-1.25 g/kg/day, respectively. Mean height/age Z score was -2.3+/-1.19. Eleven patients showed a positive height/age delta Z (mean 0.55+/-0.38) and nine showed a negative growth (mean -0.50+/-0.42). The main variable explaining the positive growth was a Nitrogen Balance between 0.54 and 2.37 g/kg/day, mean 1.55+/-0.21 (p <0.001). The second associated variable to growth was a residual Kt/V between 0.43 and 4.6 (2.02+/-0.49) (p <0.05). Kt/V and nPNA showed a significant correlation, but no correlation could be found between Kt/V and NB. Nitrogen Balance was the main variable associated to growth in pediatric PD, with values between 0.53 to 2.38 g/kg/day. The second variable was a residual Kt/V between 0.43 and 4.6. Therapy should be reassessed with NB values less than 0.54 or above 2.37 g/kg/day.

Carbon nanotubes: a potential concept for drug delivery applications.

PubMed

Kumar, Rakesh; Dhanawat, Meenakshi; Kumar, Sudhir; Singh, Brahma N; Pandit, Jayant K; Sinha, Vivek R

2014-04-01

The unique properties of carbon nanotubes (CNTs) make them a highly interesting and demandable nanocarrier in the field of nanoscience. CNTs facilitate efficient delivery of therapeutics like drugs, proteins, genes, nucleic acids, vitamins and lot more. Even though highly beneficial, the biocompatibility of CNTs is a major issue in their questioning their potential application in targeting drug delivery. Studies confirmed subdued toxicity of CNTs following slight modifications like functionalization, controlled dimensions, purification etc. A well-established mechanism for cellular internalization is an insistent need to attain a more efficient and targeted delivery. Recent patents have been thoroughly discussed in the text below.

Integration mechanisms and hospital efficiency in integrated health care delivery systems.

PubMed

Wan, Thomas T H; Lin, Blossom Yen-Ju; Ma, Allen

2002-04-01

This study analyzes integration mechanisms that affect system performances measured by indicators of efficiency in integrated delivery systems (IDSs) in the United States. The research question is, do integration mechanisms improve IDSs' efficiency in hospital care? American Hospital Association's Annual Survey (1998) and Dorenfest's Survey on Information Systems in Integrated Healthcare Delivery Systems (1998) were used to conduct the study, using IDS as the unit of analysis. A covariance structure equation model of the effects of system integration mechanisms on IDS performance was formulated and validated by an empirical examination of IDSs. The study sample includes 973 hospital-based integrated health care delivery systems operating in the United States, carried in the list of Dorenfests Survey on Information Systems in Integrated Health care Delivery Systems. The measurement indicators of system integration mechanisms are categorized into six related domains: informatic integration, case management, hybrid physician-hospital integration, forward integration, backward integration, and high tech medical services. The multivariate analysis reveals that integration mechanisms in system operation are positively correlated and positively affect IDSs' efficiency. The six domains of integration mechanisms account for 58.9% of the total variance in hospital performance. The service differentiation strategy such as having more high tech medical services have much stronger influences on efficiency than other integration mechanisms do. The beneficial effects of integration mechanisms have been realized in IDS performance. High efficiency in hospital care can be achieved by employing proper integration strategies in operations.

In Vivo Tumor Gene Delivery Using Novel Peptideticles: pH-Responsive and Ligand Targeted Core-Shell Nanoassembly.

PubMed

Alipour, Mohsen; Majidi, Asia; Molaabasi, Fatemeh; Sheikhnejad, Reza; Hosseinkhani, Saman

2018-04-30

Modulating cancer causing genes with nucleic acid based-molecules as cutting-edge approaches need efficient delivery systems to succeed in clinic. Herein, we report design and fabrication of a novel tissue penetrating Peptideticle with charge-structure switching in tumor microenvironment for an effective gene delivery. The comparative in vitro studies indicate that peptideticles identify and bind to tumor endothelial cells and efficiently penetrate into multicellular tumor spheroid. In addition, negatively charged peptideticle at pH 7.4, prevent unwanted interaction while it's sharp charge-structure switching at pH 6.2-6.9 (e.g.in tumor tissue) facilitates malignant cells penetration. More importantly, upon systemic administration into tumor bearing mice, peptideticles effectively localized in tumor tissue and delivered luciferase gene with a 200-fold higher efficiency compared to their non-pH-responsive counterparts. In conclusion, this study presents a robust nanoassembly of safe materials for high efficient tumor gene delivery. This article is protected by copyright. All rights reserved. © 2018 UICC.

Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles12

PubMed Central

Shahzad, Mian MK; Mangala, Lingegowda S; Han, Hee Dong; Lu, Chunhua; Bottsford-Miller, Justin; Nishimura, Masato; Mora, Edna M; Lee, Jeong-Won; Stone, Rebecca L; Pecot, Chad V; Thanapprapasr, Duangmani; Roh, Ju-Won; Gaur, Puja; Nair, Maya P; Park, Yun-Yong; Sabnis, Nirupama; Deavers, Michael T; Lee, Ju-Seog; Ellis, Lee M; Lopez-Berestein, Gabriel; McConathy, Walter J; Prokai, Laszlo; Lacko, Andras G; Sood, Anil K

2011-01-01

RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches. PMID:21472135

Injectable supramolecular hydrogel formed from α-cyclodextrin and PEGylated arginine-functionalized poly(l-lysine) dendron for sustained MMP-9 shRNA plasmid delivery.

PubMed

Lin, Qianming; Yang, Yumeng; Hu, Qian; Guo, Zhong; Liu, Tao; Xu, Jiake; Wu, Jianping; Kirk, Thomas Brett; Ma, Dong; Xue, Wei

2017-02-01

Hydrogels have attracted much attention in cancer therapy and tissue engineering due to their sustained gene delivery ability. To obtain an injectable and high-efficiency gene delivery hydrogel, methoxypolyethylene glycol (MPEG) was used to conjugate with the arginine-functionalized poly(l-lysine) dendron (PLLD-Arg) by click reaction, and then the synthesized MPEG-PLLD-Arg interacted with α-cyclodextrin (α-CD) to form the supramolecular hydrogel by the host-guest interaction. The gelation dynamics, hydrogel strength and shear viscosity could be modulated by α-CD content in the hydrogel. MPEG-PLLD-Arg was confirmed to bind and deliver gene effectively, and its gene transfection efficiency was significantly higher than PEI-25k under its optimized condition. After gelation, MMP-9 shRNA plasmid (pMMP-9) could be encapsulated into the hydrogel matrix in situ and be released from the hydrogels sustainedly, as the release rate was dependent on α-CD content. The released MPEG-PLLD-Arg/pMMP-9 complex still showed better transfection efficiency than PEI-25k and induced sustained tumor cell apoptosis. Also, in vivo assays indicated that this pMMP-9-loaded supramolecular hydrogel could result in the sustained tumor growth inhibition meanwhile showed good biocompatibility. As an injectable, sustained and high-efficiency gene delivery system, this supramolecular hydrogel is a promising candidate for long-term gene therapy. To realize the sustained gene delivery for gene therapy, a supramolecular hydrogel with high-efficiency gene delivery ability was prepared through the host-guest interaction between α-cyclodextrin and PEGylated arginine-functionalized poly(l-lysine) dendron. The obtained hydrogel was injectable and biocompatible with adjustable physicochemical property. More importantly, the hydrogel showed the high-efficiency and sustained gene transfection to our used cells, better than PEI-25k. The supramolecular hydrogel resulted in the sustained tumor growth inhibition meanwhile keep good biocompatibility. As an injectable, sustained and high-efficiency gene delivery system, this supramolecular hydrogel is a promising candidate in long-term gene therapy and tissue engineering. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

PMO Delivery System Using Bubble Liposomes and Ultrasound Exposure for Duchenne Muscular Dystrophy Treatment.

PubMed

Negishi, Yoichi; Ishii, Yuko; Nirasawa, Kei; Sasaki, Eri; Endo-Takahashi, Yoko; Suzuki, Ryo; Maruyama, Kazuo

2018-01-01

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, "Bubble liposomes" (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.

Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery

PubMed Central

Zhou, Jiangbing; Liu, Jie; Cheng, Christopher J.; Patel, Toral R.; Weller, Caroline E.; Piepmeier, Joseph M.; Jiang, Zhaozhong; Saltzman, W. Mark

2014-01-01

Many synthetic polycationic vectors for non-viral gene delivery show high efficiency in vitro, but their usually excessive charge density makes them toxic for in vivo applications. Here we describe the synthesis of a series of high molecular weight terpolymers with low charge density, and show that they exhibit efficient gene delivery, some surpassing the efficiency of the commercial transfection reagents Polyethylenimine and Lipofectamine 2000. The terpolymers were synthesized via enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol, and their hydrophobicity adjusted by varying the lactone content and by selecting a lactone comonomer of specific ring size. Targeted delivery of the pro-apoptotic TRAIL gene to tumour xenografts by one of the terpolymers results in significant inhibition of tumour growth, with minimal toxicity both in vitro and in vivo. Our findings suggest that the gene delivery ability of the terpolymers stems from their high molecular weight and increased hydrophobicity, which compensates for their low charge density. PMID:22138789

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.

PubMed

Wang, Ming; Zuris, John A; Meng, Fantao; Rees, Holly; Sun, Shuo; Deng, Pu; Han, Yong; Gao, Xue; Pouli, Dimitra; Wu, Qi; Georgakoudi, Irene; Liu, David R; Xu, Qiaobing

2016-03-15

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

Intracellular trafficking of hybrid gene delivery vectors.

PubMed

Keswani, Rahul K; Lazebnik, Mihael; Pack, Daniel W

2015-06-10

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles. Copyright © 2015 Elsevier B.V. All rights reserved.

Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets: vaporization efficiency dictates large molecular delivery

NASA Astrophysics Data System (ADS)

Wu, Shih-Ying; Fix, Samantha M.; Arena, Christopher B.; Chen, Cherry C.; Zheng, Wenlan; Olumolade, Oluyemi O.; Papadopoulou, Virginie; Novell, Anthony; Dayton, Paul A.; Konofagou, Elisa E.

2018-02-01

Focused ultrasound with nanodroplets could facilitate localized drug delivery after vaporization with potentially improved in vivo stability, drug payload, and minimal interference outside of the focal zone compared with microbubbles. While the feasibility of blood-brain barrier (BBB) opening using nanodroplets has been previously reported, characterization of the associated delivery has not been achieved. It was hypothesized that the outcome of drug delivery was associated with the droplet’s sensitivity to acoustic energy, and can be modulated with the boiling point of the liquid core. Therefore, in this study, octafluoropropane (OFP) and decafluorobutane (DFB) nanodroplets were used both in vitro for assessing their relative vaporization efficiency with high-speed microscopy, and in vivo for delivering molecules with a size relevant to proteins (40 kDa dextran) to the murine brain. It was found that at low pressures (300-450 kPa), OFP droplets vaporized into a greater number of microbubbles compared to DFB droplets at higher pressures (750-900 kPa) in the in vitro study. In the in vivo study, successful delivery was achieved with OFP droplets at 300 kPa and 450 kPa without evidence of cavitation damage using ¼ dosage, compared to DFB droplets at 900 kPa where histology indicated tissue damage due to inertial cavitation. In conclusion, the vaporization efficiency of nanodroplets positively impacted the amount of molecules delivered to the brain. The OFP droplets due to the higher vaporization efficiency served as better acoustic agents to deliver large molecules efficiently to the brain compared with the DFB droplets.

Reducing Bottlenecks to Improve the Efficiency of the Lung Cancer Care Delivery Process: A Process Engineering Modeling Approach to Patient-Centered Care.

PubMed

Ju, Feng; Lee, Hyo Kyung; Yu, Xinhua; Faris, Nicholas R; Rugless, Fedoria; Jiang, Shan; Li, Jingshan; Osarogiagbon, Raymond U

2017-12-01

The process of lung cancer care from initial lesion detection to treatment is complex, involving multiple steps, each introducing the potential for substantial delays. Identifying the steps with the greatest delays enables a focused effort to improve the timeliness of care-delivery, without sacrificing quality. We retrospectively reviewed clinical events from initial detection, through histologic diagnosis, radiologic and invasive staging, and medical clearance, to surgery for all patients who had an attempted resection of a suspected lung cancer in a community healthcare system. We used a computer process modeling approach to evaluate delays in care delivery, in order to identify potential 'bottlenecks' in waiting time, the reduction of which could produce greater care efficiency. We also conducted 'what-if' analyses to predict the relative impact of simulated changes in the care delivery process to determine the most efficient pathways to surgery. The waiting time between radiologic lesion detection and diagnostic biopsy, and the waiting time from radiologic staging to surgery were the two most critical bottlenecks impeding efficient care delivery (more than 3 times larger compared to reducing other waiting times). Additionally, instituting surgical consultation prior to cardiac consultation for medical clearance and decreasing the waiting time between CT scans and diagnostic biopsies, were potentially the most impactful measures to reduce care delays before surgery. Rigorous computer simulation modeling, using clinical data, can provide useful information to identify areas for improving the efficiency of care delivery by process engineering, for patients who receive surgery for lung cancer.

Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

PubMed

Gao, Ding; Lin, Xiu-Ping; Zhang, Zhi-Ping; Li, Wei; Men, Dong; Zhang, Xian-En; Cui, Zong-Qiang

2016-02-01

Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

A method for the 32P labeling of peptides or peptide nucleic acid oligomers

NASA Technical Reports Server (NTRS)

Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

1998-01-01

A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

43 CFR 418.28 - Conditions of delivery.

Code of Federal Regulations, 2010 CFR

2010-10-01

... particulars including the known or estimated location and amounts; (3) The amount will not be included as a valid headgate delivery for purposes of computing the Project efficiency and resultant incentive credit... treated directly as a debit to Lahontan storage in the same manner as an efficiency debit. (b) District...

Effects of the microbubble shell physicochemical properties on ultrasound-mediated drug delivery to the brain.

PubMed

Wu, Shih-Ying; Chen, Cherry C; Tung, Yao-Sheng; Olumolade, Oluyemi O; Konofagou, Elisa E

2015-08-28

Lipid-shelled microbubbles have been used in ultrasound-mediated drug delivery. The physicochemical properties of the microbubble shell could affect the delivery efficiency since they determine the microbubble mechanical properties, circulation persistence, and dissolution behavior during cavitation. Therefore, the aim of this study was to investigate the shell effects on drug delivery efficiency in the brain via blood-brain barrier (BBB) opening in vivo using monodisperse microbubbles with different phospholipid shell components. The physicochemical properties of the monolayer were varied by using phospholipids with different hydrophobic chain lengths (C16, C18, and C24). The dependence on the molecular size and acoustic energy (both pressure and pulse length) were investigated. Our results showed that a relatively small increase in the microbubble shell rigidity resulted in a significant increase in the delivery of 40-kDa dextran, especially at higher pressures. Smaller (3kDa) dextran did not show significant difference in the delivery amount, suggesting that the observed shell effect was molecular size-dependent. In studying the impact of acoustic energy on the shell effects, it was found that they occurred most significantly at pressures causing microbubble destruction (450kPa and 600kPa); by increasing the pulse length to deliver the 40-kDa dextran, the difference between C16 and C18 disappeared while C24 still achieved the highest delivery efficiency. These indicated that the acoustic energy could be used to modulate the shell effects. The acoustic cavitation emission revealed the physical mechanisms associated with different shells. Overall, lipid-shelled microbubbles with long hydrophobic chain length could achieve high delivery efficiency for larger molecules especially with high acoustic energy. Our study, for the first time, offered evidence directly linking the microbubble monolayer shell with their efficacy for drug delivery in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Elastic liposomes as novel carriers: recent advances in drug delivery

PubMed Central

Hussain, Afzal; Singh, Sima; Sharma, Dinesh; Webster, Thomas J; Shafaat, Kausar; Faruk, Abdul

2017-01-01

Elastic liposomes (EL) are some of the most versatile deformable vesicular carriers that comprise physiologically biocompatible lipids and surfactants for the delivery of numerous challenging molecules and have marked advantages over other colloidal systems. They have been investigated for a wide range of applications in pharmaceutical technology through topical, transdermal, nasal, and oral routes for efficient and effective drug delivery. Increased drug encapsulation efficiency, enhanced drug permeation and penetration into or across the skin, and ultradeformability have led to widespread interest in ELs to modulate drug release, permeation, and drug action more efficiently than conventional drug-release vehicles. This review provides insights into the versatile role that ELs play in the delivery of numerous drugs and biomolecules by improving drug release, permeation, and penetration across the skin as well as stability. Furthermore, it provides future directions that should ensure the widespread use of ELs across all medical fields. PMID:28761343

Last Mile Towards Efficient Healthcare Delivery in Switzerland: eHealth Enabled Applications Could Speed Up the Care Process.

PubMed

Deng, Yihan; Bürkle, Thomas; Holm, Jürgen; Zetz, Erwin; Denecke, Kerstin

2018-01-01

A precise and timely care delivery depends on an efficient triage performed by primary care providers and smooth collaboration with other medical specialities. In recent years telemedicine gained increasing importance for efficient care delivery. It's use, however, has been limited by legal issues, missing digital infrastructures, restricted support from health insurances and the digital divide in the population. A new era towards eHealth and telemedicine starts with the establishment of national eHealth regulations and laws. In Switzerland, a nation-wide digital infrastructure and electronic health record will be established. But appropriate healthcare apps to improve patient care based on this infrastructure remain rare. In this paper, we present two applications (self-anamnesis and eMedication assistant) for eHealth enabled care delivery which have the potential to speed up diagnosis and treatment.

Targeted chimera delivery to ovarian cancer cells by heterogeneous gold magnetic nanoparticle

NASA Astrophysics Data System (ADS)

Chen, Yao; Xu, Mengjiao; Guo, Yi; Tu, Keyao; Wu, Weimin; Wang, Jianjun; Tong, Xiaowen; Wu, Wenjuan; Qi, Lifeng; Shi, Donglu

2017-01-01

Efficient delivery of small interfering RNAs (siRNAs) to the targeted cells has remained a significant challenge in clinical applications. In the present study, we developed a novel aptamer-siRNA chimera delivery system mediated by cationic Au-Fe3O4 nanoparticles (NPs). The chimera constructed by VEGF RNA aptamer and Notch3 siRNA was bonded with heterogeneous Au-Fe3O4 nanoparticles by electrostatic interaction. The obtained complex exhibited much higher silencing efficiency against Notch3 gene compared with chimera alone and lipofectamine-siRNA complex, and improved the antitumor effects of the loaded chimera. Moreover, the efficient delivery of the chimera by Au-Fe3O4 NPs could reverse multi-drug resistance (MDR) of ovarian cancer cells against the chemotherapeutic drug cisplatin, indicating its potential capability for future targeted cancer therapy while overcoming MDR.

Elastic liposomes as novel carriers: recent advances in drug delivery.

PubMed

Hussain, Afzal; Singh, Sima; Sharma, Dinesh; Webster, Thomas J; Shafaat, Kausar; Faruk, Abdul

2017-01-01

Elastic liposomes (EL) are some of the most versatile deformable vesicular carriers that comprise physiologically biocompatible lipids and surfactants for the delivery of numerous challenging molecules and have marked advantages over other colloidal systems. They have been investigated for a wide range of applications in pharmaceutical technology through topical, transdermal, nasal, and oral routes for efficient and effective drug delivery. Increased drug encapsulation efficiency, enhanced drug permeation and penetration into or across the skin, and ultradeformability have led to widespread interest in ELs to modulate drug release, permeation, and drug action more efficiently than conventional drug-release vehicles. This review provides insights into the versatile role that ELs play in the delivery of numerous drugs and biomolecules by improving drug release, permeation, and penetration across the skin as well as stability. Furthermore, it provides future directions that should ensure the widespread use of ELs across all medical fields.

Special problems in aerosol delivery: neonatal and pediatric considerations.

PubMed

Cole, C H

2000-06-01

Identification of the determinants of efficient aerosol delivery and the specific challenges of aerosol delivery to infants and children can facilitate a systematic approach to optimize aerosol delivery to this population. There are inherent anatomical, physiologic, pathophysiologic, and technical limitations of aerosol efficiency in infants and young children. Nevertheless, one can enhance aerosol efficiency through application of sound principles of aerosol delivery and by exerting control over factors that are amenable to intervention. Improvements in aerosol formulations and delivery systems are being made that will enhance efficiency, decrease risk, and reduce waste and cost. Attention to aerosol particle size (1-3 microm mass median aerodynamic diameter and geometric standard deviation < 2 microm), and the concentration of this respirable particle fraction produced by an aerosol system may enhance delivery through endotracheal tubes and to the lower respiratory tract in infants and children with low V(T) and low inspiratory rates. Attention to the choice of delivery system and to details of proper MDI technique (shaking, priming, immediate actuation, and avoiding multiple actuations prior to inhalation), choice of the aerosol spacer and patient interface (type of face mask, endotracheal tube, mouthpiece), spacer cleaning, and consideration of the medicine to be aerosolized (solution or suspension, viscosity) permit adjustment of the aerosol regimen to optimize delivery. All the patient-related, system-related, and operator-dependent considerations combined can greatly impact aerosol delivery efficacy and improve therapeutic response. Therefore, education and motivation of medical personnel, parents and caregivers, and patients regarding factors that influence aerosol efficiency and teaching of proper technique must be prioritized in order to improve aerosol delivery. Aerosol therapy to all patients, especially infants and young children, would be well served if we had a clear understanding of the efficiency and functional differences among the various drugs and devices. These are substantive issues with daily therapeutic impact that have received increasingly outspoken concern over the past decade by aerosol scientists and clinicians. These issues must be given due attention by drug and device manufacturers as well as by regulatory agencies. The medication, the device, and the conditions under which they are tested must be considered together and studied as thoroughly as the medications themselves with respect to total output and particle size distribution. As noted by Bisgaard, medication dose recommendations are useless unless the device and technique used are specified. Medication dose recommendation could be facilitated by setting equivalent standards for generic and brand-name medications and devices. In addition, standardization of in vitro models with better replicas of infants' and children's anatomy (oropharynx, upper airways), and better in vitro lung models, plus utilization of realistic breathing patterns of infants and children will improve in vitro prediction of the in vivo dose delivered to lower airways. This would greatly facilitate selection of delivery systems under specific circumstances for infants and children of various ages). Safety profile, therapeutic efficacy, and efficiency of aerosolized medications delivered to infants and children need to be rigorously studied. This is particularly true for medications with potentially great benefit but possible adverse effects, such as inhaled glucocorticoid therapy in extremely premature infants. Common sense, ethics, and due respect for the same high standard of approval requirements of adults and older children should motivate further research in understanding and improving aerosol delivery in infants and young children.

Biomimetics in drug delivery systems: A critical review.

PubMed

Sheikhpour, Mojgan; Barani, Leila; Kasaeian, Alibakhsh

2017-05-10

Today, the advanced drug delivery systems have been focused on targeted drug delivery fields. The novel drug delivery is involved with the improvement of the capacity of drug loading in drug carriers, cellular uptake of drug carriers, and the sustained release of drugs within target cells. In this review, six groups of therapeutic drug carriers including biomimetic hydrogels, biomimetic micelles, biomimetic liposomes, biomimetic dendrimers, biomimetic polymeric carriers and biomimetic nanostructures, are studied. The subject takes advantage of the biomimetic methods of productions or the biomimetic techniques for the surface modifications, similar to what accrues in natural cells. Moreover, the effects of these biomimetic approaches for promoting the drug efficiency in targeted drug delivery are visible. The study demonstrates that the fabrication of biomimetic nanocomposite drug carriers could noticeably promote the efficiency of drugs in targeted drug delivery systems. Copyright © 2017 Elsevier B.V. All rights reserved.

State-of-the-art in design rules for drug delivery platforms: lessons learned from FDA-approved nanomedicines.

PubMed

Dawidczyk, Charlene M; Kim, Chloe; Park, Jea Ho; Russell, Luisa M; Lee, Kwan Hyi; Pomper, Martin G; Searson, Peter C

2014-08-10

The ability to efficiently deliver a drug to a tumor site is dependent on a wide range of physiologically imposed design constraints. Nanotechnology provides the possibility of creating delivery vehicles where these design constraints can be decoupled, allowing new approaches for reducing the unwanted side effects of systemic delivery, increasing targeting efficiency and efficacy. Here we review the design strategies of the two FDA-approved antibody-drug conjugates (Brentuximab vedotin and Trastuzumab emtansine) and the four FDA-approved nanoparticle-based drug delivery platforms (Doxil, DaunoXome, Marqibo, and Abraxane) in the context of the challenges associated with systemic targeted delivery of a drug to a solid tumor. The lessons learned from these nanomedicines provide an important insight into the key challenges associated with the development of new platforms for systemic delivery of anti-cancer drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

Reduction in the size of layered double hydroxide nanoparticles enhances the efficiency of siRNA delivery.

PubMed

Chen, Min; Cooper, Helen M; Zhou, Ji Zhi; Bartlett, Perry F; Xu, Zhi Ping

2013-01-15

Small interfering RNAs (siRNAs) are a potentially powerful new class of pharmaceutical drugs for many disease. However, the delivery of unprotected siRNAs is ineffective due to their susceptibility to degradation by ubiquitous nucleases under physiological conditions. Layered double hydroxide nanoparticles (LDHs) have been found to be efficient carriers of anionic drugs and nucleic acids. Our previous research has shown that LDHs (with the Z-average particle size of approximately 110 nm) can mediate siRNA delivery in mammalian cells, resulting in gene silencing. However, short double-stranded nucleic acids are mostly adsorbed onto the external surface and not well protected by LDHs. In order to enhance the intercalation of siRNA into the LDH interlayer and the efficiency of subsequent siRNA delivery, we prepared smaller LDHs (with the Z-average particle size of approximately 45 nm) with an engineered non-aqueous method. We demonstrate here that dsDNA/siRNA is more effectively intercalated into these small LDH nanoparticles, more dsDNA/siRNA is transfected into HEK 293T cells, and more efficient silencing of the target gene is achieved using smaller LDHs. Thus, smaller LDH particles have greater potential as a delivery system for the application of RNA interference. Copyright © 2012 Elsevier Inc. All rights reserved.

Hybrid inorganic-organic capsules for efficient intracellular delivery of novel siRNAs against influenza A (H1N1) virus infection.

PubMed

Timin, Alexander S; Muslimov, Albert R; Petrova, Aleksandra V; Lepik, Kirill V; Okilova, Maria V; Vasin, Andrey V; Afanasyev, Boris V; Sukhorukov, Gleb B

2017-03-07

The implementation of RNAi technology into the clinical practice has been significantly postponing due to the issues regarding to the delivery of naked siRNA predominantly to target cells. Here we report the approach to enhance the efficiency of siRNA delivery by encapsulating the siRNA into new carrier systems which are obtained via the combination of widely used layer-by-layer technique and in situ modification by sol-gel chemistry. We used three types of siRNAs (NP-717, NP-1155 and NP-1496) in encapsulated form as new therapeutic agents against H1N1 influenza virus infection. By employing the hybrid microcontainers for the siRNA encapsulation we demonstrate the reduction of viral nucleoprotein (NP) level and inhibition of influenza virus production in infected cell lines (MDCK and A549). The obtained hybrid carriers based on assembled biodegradable polyelectrolytes and sol-gel coating possess several advantages such as a high cell uptake efficiency, low toxicity, efficient intracellular delivery of siRNAs and the protection of siRNAs from premature degradation before reaching the target cells. These findings underpin a great potential of versatile microencapsulation technology for the development of anti-viral RNAi delivery systems against influenza virus infection.

Waste-efficient materials procurement for construction projects: A structural equation modelling of critical success factors.

PubMed

Ajayi, Saheed O; Oyedele, Lukumon O

2018-05-01

Albeit the understanding that construction waste is caused by activities ranging from all stages of project delivery process, research efforts have been concentrated on design and construction stages, while the possibility of reducing waste through materials procurement process is widely neglected. This study aims at exploring and confirming strategies for achieving waste-efficient materials procurement in construction activities. The study employs sequential exploratory mixed method approach as its methodological framework, using focus group discussion, statistical analysis and structural equation modelling. The study suggests that for materials procurement to enhance waste minimisation in construction projects, the procurement process would be characterised by four features. These include suppliers' commitment to low waste measures, low waste purchase management, effective materials delivery management and waste-efficient Bill of Quantity, all of which have significant impacts on waste minimisation. This implies that commitment of materials suppliers to such measures as take back scheme and flexibility in supplying small materials quantity, among others, are expected of materials procurement. While low waste purchase management stipulates the need for such measures as reduced packaging and consideration of pre-assembled/pre-cut materials, efficient delivery management entails effective delivery and storage system as well as adequate protection of materials during the delivery process, among others. Waste-efficient specification and bill of quantity, on the other hand, requires accurate materials take-off and ordering of materials based on accurately prepared design documents and bill of quantity. Findings of this study could assist in understanding a set of measures that should be taken during materials procurement process, thereby corroborating waste management practices at other stages of project delivery process. Copyright © 2018. Published by Elsevier Ltd.

An efficient delivery of DAMPs on the cell surface by the unconventional secretion pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Haiyan; Wang, Lan; Ruan, Yuanyuan

2011-01-21

Research highlights: {yields} Hsp60 transported to cell surface through the classical secretory pathway was modified with N-glycosylation. {yields} HSAPB-N18 could efficiently deliver Hsp60 to the cell surface via the unconventional secretory pathway. {yields} Cell surface Hsp60 delivered by HASPB-N18 has a proper conformation. {yields} HASPB-N18 is an efficient delivery signal for other DAMP molecules such as Hsp70 and HMGB1. -- Abstract: Damage-associated molecular patterns (DAMPs) are signals released from dying cells evoking the immune system response in several inflammatory disorders. In normal situations, many of DAMPs are nuclear or cytosolic proteins with defined intracellular function, but they could be foundmore » on the cell surface following tissue injury. The biological function of the translocated DAMPs is still not well known and an efficient delivery of these molecules on the cell surface is required to clarify their biological effects. In this study, we demonstrated that an unclassical secretory signal peptide, N-terminal 18 amino acids of HASPB (HASPB-N18), could efficiently deliver Hsp60, Hsp70, and HMGB1 on the cell surface. Furthermore, the delivery of these molecules on the cell surface by HASPB-N18 is not limited to a special cell line because several cell lines could use this delivery signal to deliver these molecules on the cell surface. Moreover, we demonstrated that Hsp60 on the cell surface delivered by HASPB-N18 could be recognized by a soluble form of LOX-1, which implies that DAMPs on the cell surface delivered by HASPB-N18 have a proper conformation during transport. Therefore, delivery of DAMPs by HASPB-N18 is a reliable model to further understand the biological significance of DAMPs on the cell surface.« less

Sonophoresis Using Ultrasound Contrast Agents: Dependence on Concentration.

PubMed

Park, Donghee; Song, Gillsoo; Jo, Yongjun; Won, Jongho; Son, Taeyoon; Cha, Ohrum; Kim, Jinho; Jung, Byungjo; Park, Hyunjin; Kim, Chul-Woo; Seo, Jongbum

2016-01-01

Sonophoresis can increase skin permeability to various drugs in transdermal drug delivery. Cavitation is recognized as the predominant mechanism of sonophoresis. Recently, a new logical approach to enhance the efficiency of transdermal drug delivery was tried. It is to utilize the engineered microbubble and its resonant frequency for increase of cavitation activity. Actively-induced cavitation with low-intensity ultrasound (less than ~1 MPa) causes disordering of the lipid bilayers and the formation of aqueous channels by stable cavitation which indicates a continuous oscillation of bubbles. Furthermore, the mutual interactions of microbubble determined by concentration of added bubble are also thought to be an important factor for activity of stable cavitation, even in different characteristics of drug. In the present study, we addressed the dependence of ultrasound contrast agent concentration using two types of drug on the efficiency of transdermal drug delivery. Two types of experiment were designed to quantitatively evaluate the efficiency of transdermal drug delivery according to ultrasound contrast agent concentration. First, an experiment of optical clearing using a tissue optical clearing agent was designed to assess the efficiency of sonophoresis with ultrasound contrast agents. Second, a Franz diffusion cell with ferulic acid was used to quantitatively determine the amount of drug delivered to the skin sample by sonophoresis with ultrasound contrast agents. The maximum enhancement ratio of sonophoresis with a concentration of 1:1,000 was approximately 3.1 times greater than that in the ultrasound group without ultrasound contrast agent and approximately 7.5 times greater than that in the control group. These results support our hypothesis that sonophoresis becomes more effective in transdermal drug delivery due to the presence of engineered bubbles, and that the efficiency of transdermal drug delivery using sonophoresis with microbubbles depends on the concentration of microbubbles in case stable cavitation is predominant.

Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells

PubMed Central

Hindriksen, Sanne; Bramer, Arne J.; Truong, My Anh; Vromans, Martijn J. M.; Post, Jasmin B.; Verlaan-Klink, Ingrid; Snippert, Hugo J.; Lens, Susanne M. A.

2017-01-01

The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B. PMID:28640891

Efficient delivery of recombinant human bone morphogenetic protein (rhBMP-2) with dextran sulfate-chitosan microspheres.

PubMed

Xia, Yuan-Jun; Xia, Hong; Chen, Ling; Ying, Qing-Shui; Yu, Xiang; Li, Li-Hua; Wang, Jian-Hua; Zhang, Ying

2018-04-01

Bone morphogenetic protein-2 (BMP-2) serves an important role in the development of bone and cartilage. However, administration of BMP-2 protein alone by intravenous delivery is not very effective. Sustained delivery of stabilized BMP-2 by carriers has been proven necessary to improve the osteogenesis effect of BMP-2. The present study constructed a novel drug delivery system using dextran sulfate (DS)-chitosan (CS) microspheres and investigated the efficiency of the delivery system on recombinant human bone morphogenetic protein (rhBMP-2). The microsphere morphology, optimal ratio of DS/CS/rhBMP-2, and drug loading rate and entrapment efficiency of rhBMP-2 CS nanoparticles were determined. L929 cells were used to evaluate the cytotoxicity and effect of DS/CS/rhBMP-2 microspheres on cell proliferation. Differentiation study was conducted using bone marrow mesenchymal stem cells (BMSCs-C57) cells treated with DS/CS/rhBMP-2 microspheres or the control microspheres. The DS/CS/rhBMP-2 microspheres delivery system was successfully established. Subsequent complexation of rhBMP-2-bound DS with polycations afforded well defined microspheres with a diameter of ~250 nm. High protein entrapment efficiency (85.6%) and loading ratio (47.245) µg/mg were achieved. Release of rhBMP-2 from resultant microspheres persisted for over 20 days as determined by ELISA assay. The bioactivity of rhBMP-2 encapsulated in the CS/DS microsphere was observed to be well preserved as evidenced by the alkaline phosphatase activity assay and calcium nodule formation of BMSCs-C57 incubated with rhBMP-2-loaded microspheres. The results demonstrated that microspheres based on CS-DS polyion complexes were a highly efficient vehicle for delivery of rhBMP-2 protein. The present study may provide novel orientation for bone tissue engineering for repairing and regenerating bone defects.

Erythrocyte membrane based cationic polymer-mcDNA complexes as an efficient gene delivery system.

PubMed

Huang, Ping; Zhao, Jing; Wei, Chiju; Hou, Xiaohu; Chen, Pingzhang; Tan, Yan; He, Cheng-Yi; Wang, Zhiyong; Chen, Zhi-Ying

2016-12-20

Gene therapy has great promise for the treatment of obtained and inherited serious diseases. However, the lack of safe and efficient gene delivery systems remains a barrier for their clinical application. Here, we reported a potential gene delivery vehicle composed of the erythrocyte membrane and cationic polymers, for example the XtremeGENE from Roche and the ε-caprolactone modified polyethylenimine. In addition to high efficiency, this system showed negligible cytotoxicity compared to the two cationic polymers alone in various cell lines, including human embryonic kidney cells (293T), human liver cancer cells (Huh7 and HepG2), murine dendritic cells (DC2.4) and human umbilical cord mesenchymal stem cells (Hu-MSCs). Moreover, the results of confocal laser scanning microscopy and flow cytometry suggested that the cell uptake of this gene vector was improved and might be introduced by the fusion interaction between the erythrocyte membrane and targeted cells.Thus, all the results revealed that the erythrocyte membrane based gene delivery system might be able to serve as an excellent gene delivery system.

Evaluation of Ocular Irritation and Bioavailability of Voriconazole Loaded Microemulsion.

PubMed

Kumar, Rakesh; Sinha, Vivek Ranjan

2017-01-01

Voriconazole (VCZ), a second-generation antifungal with excellent attributes like, broad-spectrum activity, targeted delivery, and tolerability. VCZ loaded microemulsion could be an effective strategy for efficient ocular delivery of the drug. To perform corneal irritation studies and in vivo delivery of VCZ microemulsion to establish its potential as an efficient ocular delivery system. Ocular irritancy was performed by HETCAM (Hen's Egg Test Chorio Allantoic Membrane) assay, corneal histopathology and Draize test. Ex vivo and in vivo studies were performed to determine permeation efficiency of VCZ microemulsion. The irritation studies suggested the non-irritant nature of the microemulsion. The ex vivo studies performed on excised cornea displayed significant enhancement in drug permeation/penetration from microemulsion in contrast to the drug suspension. Further, the in vivo study confirmed the higher availability of VCZ (from microemulsion) in aqueous humor with minimal nasolacrimal drainage (lower plasma drug content) when compared with the drug suspension. The non-irritant nature and high corneal permeation of VCZ encourages the role of microemulsion as a potential ocular delivery system. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study

PubMed Central

Oliveira, Ana V.; Silva, Andreia P.; Bitoque, Diogo B.; Silva, Gabriela A.; Rosa da Costa, Ana M.

2013-01-01

OBJECTIVE: Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently. MATERIALS AND METHODS: Chitosan and thiolated chitosan nanoparticles (NPs) were prepared in order to obtain a NH3+:PO4− ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells. RESULTS: In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency. CONCLUSION: The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery. PMID:23833516

Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing.

PubMed

Suresh, Bharathi; Ramakrishna, Suresh; Kim, Hyongbum

2017-01-01

The clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system represents an efficient tool for genome editing. It consists of two components: the Cas9 protein and a guide RNA. To date, delivery of these two components has been achieved using either plasmid or viral vectors or direct delivery of protein and RNA. Plasmid- and virus-free direct delivery of Cas9 protein and guide RNA has several advantages over the conventional plasmid-mediated approach. Direct delivery results in shorter exposure time at the cellular level, which in turn leads to lower toxicity and fewer off-target mutations with reduced host immune responses, whereas plasmid- or viral vector-mediated delivery can result in uncontrolled integration of the vector sequence into the host genome and unwanted immune responses. Cell-penetrating peptide (CPP), a peptide that has an intrinsic ability to translocate across cell membranes, has been adopted as a means of achieving efficient Cas9 protein and guide RNA delivery. We developed a method for treating human cell lines with CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs that leads to endogenous gene disruption. Here we describe a protocol for preparing an efficient CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs, as well as treatment methods to achieve safe genome editing in human cell lines.

Improved and targeted delivery of bioactive molecules to cells with magnetic layer-by-layer assembled microcapsules

NASA Astrophysics Data System (ADS)

Pavlov, Anton M.; Gabriel, Samantha A.; Sukhorukov, Gleb B.; Gould, David J.

2015-05-01

Despite our increasing knowledge of cell biology and the recognition of an increasing repertoire of druggable intracellular therapeutic targets, there remain a limited number of approaches to deliver bioactive molecules to cells and even fewer that enable targeted delivery. Layer-by-layer (LbL) microcapsules are assembled using alternate layers of oppositely charged molecules and are potential cell delivery vehicles for applications in nanomedicine. There are a wide variety of charged molecules that can be included in the microcapsule structure including metal nanoparticles that introduce physical attributes. Delivery of bioactive molecules to cells with LbL microcapsules has recently been demonstrated, so in this study we explore the delivery of bioactive molecules (luciferase enzyme and plasmid DNA) to cells using biodegradable microcapsules containing a layer of magnetite nanoparticles. Interestingly, significantly improved intracellular luciferase enzyme activity (25 fold) and increased transfection efficiency with plasmid DNA (3.4 fold) was observed with magnetic microcapsules. The use of a neodymium magnet enabled efficient targeting of magnetic microcapsules which further improved the delivery efficiency of the cargoes as a consequence of increased microcapsule concentration at the magnetic site. Microcapsules were well tolerated by cells in these experiments and only displayed signs of toxicity at a capsule : cell ratio of 100 : 1 and with extended exposure. These studies illustrate how multi-functionalization of LbL microcapsules can improve and target delivery of bioactive molecules to cells.

THE APPLICATION OF PEPTIDE NUCLEIC ACID PROBES FOR RAPID DETECTION AND ENUMERATION OF EUBACTERIA, STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA IN RECREATIONAL BEACHES OF S. FLORIDA. (R828830)

EPA Science Inventory

A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, S...