Heart rate dynamics in the prediction of coronary artery disease and myocardial infarction using artificial neural network and support vector machine.

BACKGROUND: Atherosclerosis leads to coronary artery disease (CAD) and myocardial infarction (MI), a major cause of morbidity and mortality worldwide. The computer-aided prognosis of atherosclerotic events with the electrocardiogram (ECG) derived heart rate variability (HRV) can be a robust method in the prognosis of atherosclerosis events. METHODS: A total of 70 male subjects aged 55 ± 5 years participated in the study. The lead-II ECG was recorded and sampled at 200 Hz. The tachogram was obtained from the ECG signal and used to extract twenty-five HRV features. The one-way Analysis of variance (ANOVA) test was performed to find the significant differences between the CAD, MI, and control subjects. Features were used in the training and testing of a two-class artificial neural network (ANN) and support vector machine (SVM). RESULTS: The obtained results revealed depressed HRV under atherosclerosis. Accuracy of 100% was obtained in classifying CAD and MI subjects from the controls using ANN. Accuracy was 99.6% with SVM, and in the classification of CAD from MI subjects using SVM and ANN, 99.3% and 99.0% accuracy was obtained respectively. CONCLUSIONS: Depressed HRV has been suggested to be a marker in the identification of atherosclerotic events. The good accuracy observed in classification between control, CAD, and MI subjects, revealed it to be a non-invasive cost-effective approach in the prognosis of atherosclerotic events.

Simulating cellular galectin networks by mixing galectins in vitro reveals synergistic activity.

BACKGROUND: Even though members of the family of adhesion/growth-regulatory galectins are increasingly detected to be co-expressed, they are still being routinely tested separately. The recent discovery of heterodimer formation among galectins-1, -3, and -7 in mixtures prompts further study of their functional activities in mixtures. METHODS: Cell agglutination, galectin binding to cells, as well as effects on cell proliferation, onset of apoptosis and migration were determined in assays using various cell types and mixtures of galectins-1, -3, and -7. RESULTS: Evidence for a more than additive increases of experimental parameters was consistently obtained. CONCLUSION: Testing galectins in mixtures simulates the situation of co-expression in situ and reveals unsuspected over-additive activities. This new insight is relevant for analyzing galectin functionality in (patho)physiological conditions.

PD-1 derived CA-170 is an oral immune checkpoint inhibitor that exhibits preclinical anti-tumor efficacy.

Small molecule immune checkpoint inhibitors targeting PD-1 and other pathways may offer advantages including ease of dosing, ability to manage immune-related adverse events (irAEs) due to their shorter pharmacokinetic exposure and opportunity to target more than one pathway for improving efficacy. Here we describe the identification and characterization of CA-170, an amino acid inspired small molecule inhibitor of PD-L1 and VISTA derived from the interface of PD-1 and PD-L1. CA-170 exhibited potent rescue of proliferation and effector functions of T cells inhibited by PD-L1/L2 and VISTA with selectivity over other immune checkpoint proteins as well as a broad panel of receptors and enzymes. Observed blocking of PD-L1 signaling and binding to PD-L1 in the cellular context without preventing the assembly of PD-1:PD-L1 complex support the formation of a defective ternary complex as the mechanism of action of CA-170. Oral administration of CA-170 resulted in increased proliferation and activation of T cells in the tumor, and significant anti-tumor efficacy in a number of immunocompetent mouse tumor models either as a single agent or in combination with approved therapeutics. These results prompted the advancement of CA-170 to human clinical trials.

Reversible re-programing of cell-cell interactions.

The ability to engineer and re-program the surfaces of cells would provide an enabling synthetic biological method for the design of cell- and tissue-based therapies. A new cell surface-engineering strategy is described that uses lipid-chemically self-assembled nanorings (lipid-CSANs) that can be used for the stable and reversible modification of any cell surface with a molecular reporter or targeting ligand. In the presence of a non-toxic FDA-approved drug, the nanorings were quickly disassembled and the cell-cell interactions reversed. Similar to T-cells genetically engineered to express chimeric antigen receptors (CARS), when activated peripheral blood mononuclear cells (PBMCs) were functionalized with the anti-EpCAM-lipid-CSANs, they were shown to selectively kill antigen-positive cancer cells. Taken together, these results demonstrate that lipid-CSANs have the potential to be a rapid, stable, and general method for the reversible engineering of cell surfaces and cell-cell interactions.

Protein lysine-Nζ alkylation and O-phosphorylation mediated by DTT-generated reactive oxygen species.

Reactive oxygen species (ROS) play crucial roles in physiology and pathology. In this report, we use NMR spectroscopy and mass spectrometry (MS) to demonstrate that proteins (galectin-1, ubiquitin, RNase, cytochrome c, myoglobin, and lysozyme) under reducing conditions with dithiothreitol (DTT) become alkylated at lysine-N(ζ) groups and O-phosphorylated at serine and threonine residues. These adduction reactions only occur in the presence of monophosphate, potassium, trace metals Fe/Cu, and oxygen, and are promoted by reactive oxygen species (ROS) generated via DTT oxidation. Superoxide mediates the chemistry, because superoxide dismutase inhibits the reaction, and hydroxyl and phosphoryl radicals are also likely involved. While lysine alkylation accounts for most of the adduction, low levels of phosphorylation are also observed at some serine and threonine residues, as determined by western blotting and MS fingerprinting. The adducted alkyl group is found to be a fragment of DTT that forms a Schiff base at lysine N(ζ) groups. Although its exact chemical structure remains unknown, the DTT fragment includes a SH group and a --CHOH--CH2-- group. Chemical adduction appears to be promoted in the context of a well-folded protein, because some adducted sites in the proteins studied are considerably more reactive than others and the reaction occurs to a lesser extent with shorter, unfolded peptides and not at all with small organic molecules. A structural signature involving clusters of positively charged and other polar groups appears to facilitate the reaction. Overall, our findings demonstrate a novel reaction for DTT-mediated ROS chemistry with proteins.

Structure-based optimization of angiostatic agent 6DBF7, an allosteric antagonist of galectin-1.

Galectin-1 (gal-1), which binds ß-galactoside groups on various cell surface receptors, is crucial to cell adhesion and migration, and is found to be elevated in several cancers. Previously, we reported on 6DBF7, a dibenzofuran (DBF)-based peptidomimetic of the gal-1 antagonist anginex. In the present study, we used a structure-based approach to optimize 6DBF7. Initial NMR studies showed that 6DBF7 binds to gal-1 on one side of the ß-sandwich away from the lectin's carbohydrate binding site. Although an alanine scan of 6DBF7 showed that the two cationic groups (lysines) in the partial peptide are crucial to its angiostatic activity, it is the hydrophobic face of the amphipath that appears to interact directly with the surface of gal-1. Based on this structural information, we designed and tested additional DBF analogs. In particular, substitution of the C-terminal Asp for alanine and branched alkyl side chains (Val, Leu, Ile) for linear ones (Nle, Nva) rendered the greatest improvements in activity. Flow cytometry with gal-1(-/-) splenocytes showed that 6DBF7 and two of its more potent analogs (DB16 and DB21) can fully inhibit fluorescein isothiocyanate-gal-1 binding. Moreover, heteronuclear single-quantum coherence NMR titrations showed that the presence of DB16 decreases gal-1 affinity for lactose, indicating that the peptidomimetic targets gal-1 as a noncompetitive, allosteric inhibitor of glycan binding. Using tumor mouse models (B16F10 melanoma, LS174 lung, and MA148 ovarian), we found that DB21 inhibits tumor angiogenesis and tumor growth significantly better than 6DBF7, DB16, or anginex. DB21 is currently being developed further and holds promise for the management of human cancer in the clinic.

Antitumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding.

Calix[4]arene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide Anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, (15)N-(1)H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin's carbohydrate binding site and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of antitumor calixarene compound 0118.

Determination of an unusual secondary structural element in the immunostimulating tetrapeptide rigin in aqueous environments: insights via MD simulations, (1)H NMR and CD spectroscopic studies.

An immunomodulating tetrapeptide, rigin (H-Gly-Gln-Pro-Arg-OH), has been examined for its secondary structure preferences through combined use of high-temperature unrestrained MD simulations in implicit water and 1D and 2D 1H NMR spectroscopy.The distribution of backbone torsion angles revealed the predominance of trans conformers across the Xaa-Pro peptide bond. The results of MD simulations revealed that of the five predicted families A-E, the predominant families, family A (92 structures), family C (63 structures) and family D (31 structures), could be complemented by extensive 1D and 2D 1H NMR parameters acquired in aqueous PBS solution. A survey of specific inter- and intraresidue NOEs substantiated the predominance of an unusual type VII beta-turn structure, defined by two torsion angles, i.e. psiGln approximately 155 degrees and psiPro approximately -65 degrees across the Gln-Pro segment. The proposed semi-folded kinked topology precluded formation of any intramolecular interaction, i.e. hydrogen bond or electrostatic interaction. Far-UV CD spectral characteristics of rigin in aqueous PBS solution and non-aqueous structure promoting organic solvents, TFE and TMP, revealed its strong solvent dependence. However, in aqueous PBS solution, the presence of a weak negative shoulder at approximately 234 nm could be ascribed to a small population with ordered, semi-folded topology.We propose that the plausible structural attributes may be exploited for design and rigidification of the bioactive conformation of this immunomodulator toward improved immunopharmacological properties.

Crystallographically observed folded topology of an unsubstituted γ-aminobutyric acid incorporated in a model peptide: importance of a C--H···O interaction.

To validate the existing hypothesis put forward by Navarro et al., we performed single crystal X-ray diffraction structural analysis of a designed model peptide incorporating an unsubstituted achiral γ-aminobutyric acid: Boc-Pro-γ-Abu-OH (1) lacking C-terminal amide group. The analysis established existence of an overall unusual tightly folded topology stabilized by a conventional N(i)···H--N(i + 1) and an unconventional C(i)--H···O(i) type intramolecular hydrogen bonding interactions, encompassing a five-membered and a six-membered ring motifs, respectively. Moreover, in conjunction with Fourier transform infrared (FT-IR) absorption study in solid KBr, the results provided evidence that two conventional and one unconventional noncovalent intermolecular interaction stabilize a right-handed helical architecture generated via molecular self-assembly by translating the symmetry related molecules along the crystallographic b axis. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 927-931, 2010.

Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis

A reliable method for characterizing microbial communities on the basis of their differences in the 16S ribosomal RNA (rRNA) gene sequences in the hot arid zone sandy soils has been optimized. A desert plant (Calligonum polygonoides) was chosen to provide the rhizospheric soil samples, collected from three different agro-ecological locations. Total community DNA was efficiently extracted at small-scale level using direct lysis with hot sodium dodecyl sulphate (SDS), glass bead beating and finally subjecting the sandy soil to liquid nitrogen freeze-thaw cycles. To amplify V3 region of bacterial 16S rRNA gene, universal conserved primers were used. Second round polymerase chain reaction (PCR) was attempted to increase product concentration and to minimize the effect of inhibitory substances. To enhance the detection sensitivity of the denaturing gradient gel electrophoresis (DGGE), the effect of change in template DNA concentration was studied. The separation of bands were greatly enhanced in the fingerprints obtained after the second round of PCR representing low abundant species which were not differentiated at single optimized concentration of DNA.