G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

High-throughput clone screening followed by protein expression cross-check: A visual assay platform.

In high-throughput biotechnology and structural biology, molecular cloning is an essential prerequisite for attaining high yields of recombinant protein. However, a rapid, cost-effective, easy clone screening protocol is still required to identify colonies with desired insert along with a cross check method to certify the expression of the desired protein as the end product. We report an easy, fast, sensitive and cheap visual clone screening and protein expression cross check protocol employing gold nanoparticle based plasmonic detection phenomenon. This is a non-gel, non-PCR based visual detection technique, which can be used as simultaneous high throughput clone screening followed by the determination of expression of desired protein.

Visual assessment of parasitic burden in infected macrophage by plasmonic detection of leishmania specific marker RNA.

Visceral leishmaniasis is a neglected tropical disease and may prove fatal if not diagnosed and treated early. The amastigotes of Leishmania donovani nest in the macrophage of human host and thus, determination of parasitic burden in the infected macrophages has been the most crucial step in diagnosis, dose determination and medical management of relapse cases of this fatal disease. Microscopic count following Giemsa staining and other morphological analysis are the classical ways vastly used in the resource stringent endemic areas. The current method introduced a high throughput, rapid, cheap, non-gel, non-PCR and nonculture based visual detection platform employing salt triggered aggregation of gold nanoparticle in presence of extracted total RNA from infected macrophages and leishmania specific oligo-nucleotide probe to determine the parasite burden in macrophages. Amastigote's small subunit ribosomal RNA (SSU rRNA, PMID 1565128) was used as the leishmania specific marker and its abundance in the total RNA extracts of infected macrophages were determined by this visual colorimetric assay.

Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients.

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients' sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients' group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients' group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients' group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients' groups, to the contrary high glycan branching was observed in HCC patients' group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients' group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.

Visual detection of serum asialohaptoglobin by plasmonic sandwich ELLSA--a new platform for cirrhosis diagnosis.

The cirrhotic condition of the liver has long been acknowledged as the preface to liver cancer. The desialylation status of the serum acute phase protein, haptoglobin, has been introduced as a new diagnostic analyte for liver cirrhosis. The reliability of this new diagnostic molecule has been evaluated in 30 liver cirrhosis patients having a history of earlier viral hepatitis C (HCV-LC). A novel enzyme linked lectinosorbent assay has been developed coupled with the plasmon mechanism of gold nanoparticle aggregation as the colorimetric read out which can visually distinguish the cirrhotic liver patients from the normal healthy and hepatitis C controls. The assay can be useful for rapid point-of-care detection, and even an untrained person can execute it without a specialized instrument. This method employs Sambucus nigra agglutinin (SNA) to detect the extent of α-2,6 sialylation of serum haptoglobin, the new diagnostic molecule for liver cirrhosis.

Synthesis and evaluation of antineurotoxicity properties of an amyloid-ß peptide targeting ligand containing a triamino acid.

Peptide-like compounds containing an arginine have been shown to bind and stabilize the central helix of the Alzheimer's disease related amyloid-ß peptide (Aß) in an α-helical conformation, thereby delaying its aggregation into cytotoxic species. Here we study a novel Aß targeting ligand AEDabDab containing the triamino acid, N(γ)-(2-aminoethyl)-2,4-diaminobutanoic (AEDab) acid. The new AEDab triamino acid carries an extra positive charge in the side chain and is designed to be incorporated into a ligand AEDabDab where the AEDab replaces an arginine moiety in a previously developed ligand Pep1b. This is done in order to increase the Aß-ligand interaction, and molecular dynamics (MD) simulation of the stability of the Aß central helix in the presence of the AEDabDab ligand shows further stabilization of the helical conformation of Aß compared to the previously reported Pep1b as well as compared to the AEOrnDab ligand containing an N(δ)-(2-aminoethyl)-2,5-diaminopentanoic acid unit which has an additional methylene group. To evaluate the effect of the AEDabDab ligand on the Aß neurotoxicity the AEDab triamino acid building block is synthesized by reductive alkylation of N-protected-glycinal with α-amino-protected diaminobutanoic acid, and the Aß targeting ligand AEDabDab is prepared by solid-phase synthesis starting with attachment of glutarate to the Wang support. Replacement of the arginine residue by the AEDab triamino acid resulted in an improved capability of the ligand to prevent the Aß1-42 induced reduction of gamma (γ) oscillations in hippocampal slice preparation.

Influence of sequence variation on the RNA cleavage activity of Zn2+-dimethyl-dppz-PNA-based artificial enzymes.

The development of Zn2+-dependent dimethyl-dppz-PNA conjugates (PNAzymes) as efficient site-specific artificial ribonucleases enables rapid sequence-specific degradation of clinically relevant RNA target sequences, but the significance of the RNA/PNAzyme sequence and structural demands for the identification of novel RNA targets are not fully understood. In the present study, we investigated the influence of sequence variation in the recognition arms of the RNA/PNAzyme complex on the RNA cleavage activity of the artificial enzymes. The base pairs closing the 3-nucleotide bulge region on both sides of the bulge as well as the neighbouring nucleobases were shown to significantly influence the RNA cleavage activity. Elongation of the RNA/PNAzyme complex was shown to be tolerated, although potentially prohibitive for catalytic turnover. The specificity of PNAzyme action was clearly demonstrated by the significantly reduced or absent cleavage activity in complexes containing mismatches. Further investigation into 2- and 4-nucleotide RNA bulges indicated that formation of 3-nucleotide bulges in the target RNA gives the optimal cleavage rates, while some potential off-target cleavage of formed 4-nucleotide bulges of select sequences should be considered.

Zn2+-Dependent peptide nucleic acid-based artificial ribonucleases with unprecedented efficiency and specificity.

We present Zn2+-dependent dimethyl-dipyridophenazine PNA conjugates as efficient RNA cleaving artificial enzymes. These PNAzymes display site-specific RNA cleavage with 10 minute half-lives and cleave clinically relevant RNA models.

In vitro ADMET and physicochemical investigations of poly-N-methylated peptides designed to inhibit Abeta aggregation.

N-Methylation is a common strategy for improving oral bioavailability of peptide-based lead structures. Herein, we present a detailed study on how the degree of N-methylation affects the absorption-distribution-metabolism-excretion-toxicity (ADMET) properties such as solubility, membrane transport, proteolytic stability, and general cell toxicity of the investigated peptides. As representative structures we chose hexapeptides 1-8. These peptides, corresponding to N-methylated analogues of residues 16-21 and 32-37 of the Abeta-peptide, pathological hallmark of Alzheimer's disease (AD), have previously been shown to inhibit aggregation of Abeta fibrils in vitro. This study suggests that poly-N-methylated peptides are non-toxic and have enhanced proteolytic stability over their non-methylated analogues. Furthermore, solubility in aqueous solution is seen to increase with increased degree of N-methylation, while membrane transport was found to be low for all investigated hexapeptides. The present results, together with those reported in the literature, suggest that poly-N-methylated peptides, especially shorter or equal to six residues, can be suitable candidates for drug design.

Macrophage ghost entrapped amphotericin B: a novel delivery strategy towards experimental visceral leishmaniasis.

Leishmania donovani, an intracellular parasite, poses many challenges against attempted chemotherapy. After the resistance towards the first-line antileishmanial drug, amphotericin B has become the treatment of choice against visceral leishmaniasis, a fatal tropical disease. However, unfavorable toxicity profile, severe side effects, and prolonged parenteral administration limit its use. Lack of available specific delivery system also makes this drug unsafe for long-term use. In the current study, a "ghost cell" strategy based on macrophage membrane-derived nanovesicle has been introduced as a specific carriage for amphotericin B. Membrane proteins of macrophage ghost play a crucial role in the dissemination of infection in host by communicating between infected neutrophil-macrophage system and non-infected macrophages. These membrane proteins are the basis of specificity of the drug delivery to the infected tissues in this current macrophage ghost cell carrier. This cheap and biocompatible delivery vehicle has significantly improved the toxicity profile and lowered LD50 value of the drug compared to traditional way of its direct administration and widely accepted antileishmanial therapy, AmBisome.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA.

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

Decoration of Au and Ag nanoparticles on self-assembling pseudopeptide-based nanofiber by using a short peptide as capping agent for metal nanoparticles.

The surface of a nanofiber that is formed from a self-assembling pseudopeptide has been decorated by gold and silver nanoparticles that are stabilized by a dipeptide. Transmission electron microscopic images make the decoration visible. In this paper, a new strategy of mineralizing a pseudopeptide based nanofiber by gold and silver nanoparticles with use of a two-component nanografting method is described.

Two Cases of Esophageal Injury Following Anterior Cervical Discectomy and Fusion: One Overt and One Covert.

Esophageal perforation is a dreaded complication of anterior cervical spinal surgery. A 52-year-old diabetic man had undergone a surgery for a C6-C7 disc prolapse and developed spiking fever with chills and rigor on the 7th postoperative day. No cause could be found out but a CT scan of thorax done in the course of investigations revealed pneumomediastimum. The patient succumbed on the 10th day after surgery. Autopsy revealed the cause of death to be mediastinitis following iatrogenic esophageal perforation. A second patient, 53 years of age, following surgery for C5-C6 disc prolapse and developed intractable dysphagia. Later, fever and purulent discharge from the wound prompted an MRI showing prevertebral collection extending to the superior mediastinum. Presuming only wound infection, debridement and implant removal was done. However persistent serous discharge from the wound revealed an esophageal injury. Late diagnosis precluded primary repair. With conservative treatment, the fistula finally closed after 42 days. Postoperative dysphagia, a common complaint following surgery, may not always be present in cases of esophageal injury. A high index of suspicion is required for diagnosing and initiating treatment for esophageal perforation before complications set in.

Formation of triple helical nanofibers using self-assembling chiral benzene-1,3,5-tricarboxamides and reversal of the nanostructure's handedness using mirror image building blocks.

Intertwining triple helical nanofibers with an overall handedness have been formed from self-assembling chiral benzene-1,3,5-tricarboxamides , and , whereas the achiral benzene-1,3,5-tricarboxamide upon self-association gives rise to straight nanofibers without any twist and transmission electron microscopy images of chiral compounds clearly demonstrate that the handedness of the triple helical nanofibers can be reversed by using the enantiomeric benzene-1,3,5-tricarboxamide building blocks.

Hemoglobin guided nanocarrier for specific delivery of amphotericin B to Leishmania infected macrophage.

Leishmania donovani being an intracellular parasite poses many challenges against the attempted chemotherapy. After the resistance towards the first line of antileishmanial drug, Amphotericin B has been the treatment of choice against visceral leishmaniasis, a fatal tropical disease. However, unfavorable toxicity profile, severe side effects, prolonged parenteral administration procedure limits the use of Amphotericin B. Lack of available specific delivery system also makes this drug unsafe for the prolonged use. In this current study, a chitosan-chondroitin sulfate based nanodelivery vehicle has been introduced. Hemoglobin has been attached on the surface of the delivery system for specifically targeting the leishmania infected macrophage taking the advantage of Leishmania being highly auxotrophic for heme. This cheap and biodegradable delivery vehicle has improved the toxicity profile and lowered LD50 value of the drug significantly compared to traditional way of its direct administration.

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations.

BACKGROUND: Lectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied. METHODS: Purification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed. RESULTS: The purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (Ka=4.9 (±0.2)×104 M-1), which indicated static quenching (Stern-Volmer (SV) constant Ksv=4.6 (±0.2)×104 M-1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa. CONCLUSIONS: A novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored. GENERAL SIGNIFICANCE: The newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.

Concurrent visual diagnosis and susceptibility profiling of the first line drug against visceral leishmaniasis by plasmonic detection of PCR amplified genetic biomarker.

Visceral form of leishmaniasis (also known as Kala-azar) is a fatal neglected tropical disease affecting 95 countries worldwide. Recently, substantial proportion of resistance related treatment failure cases have been reported against its first line drug, sodium-antimony gluconate (SAG). We report an easy, fast, sensitive and cheap visual diagnosis and SAG susceptibility profiling for this disease based on recently recognized genetic biomarker and gold nanoparticle based plasmonic detection phenomenon. This is a non-gel, non-culture based detection technique, which can be used as simultaneous high throughput detection and SAG-susceptibility profiling in Leishmania endemic resource stringent countries.

Antiproliferative effect of T/Tn specific Artocarpus lakoocha agglutinin (ALA) on human leukemic cells (Jurkat, U937, K562) and their imaging by QD-ALA nanoconjugate.

T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19-21, 24-27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative effect on leukemic cells, with the highest effect toward Jurkat cells (IC(50) 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat, U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells, where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient fluorescent marker for identification of leukemic cell lines that gives rise to high quality images.