Biomolecular Binding under Confinement: Statistical Predictions of Steric Influence in Absence of Long-Distance Interactions

We propose a theoretical model for the influence of confinement on biomolecular binding at the single-molecule scale at equilibrium, based on the change of the number of microstates (localization and orientation) upon reaction. Three cases are discussed: DNA sequences shorter and longer than the single strain DNA Kuhn length and spherical proteins, confined into a spherical container (liposome, droplet, etc.). The influence of confinement is found to be highly dependent on the molecular structure and significant for large molecules (relative to container size)

Impact of the Coverage of Aptamers on a Nanoparticle on the Binding Equilibrium and Kinetics between Aptamer and Protein

Knowledge of the interaction between aptamer and protein is integral to the design and development of aptamer-based biosensors. Nanoparticles functionalized with aptamers are commonly used in these kinds of sensors. As such, studies into how the number of aptamers on the nanoparticle surface influence both kinetics and thermodynamics of the binding interaction are required. In this study, aptamers specific for interferon gamma (IFN-γ) were immobilized on the surface of gold nanoparticles (AuNPs), and the effect of surface coverage of aptamer on the binding interaction with its target was investigated using fluorescence spectroscopy. The number of aptamers were adjusted from an average of 9.6 to 258 per particle. The binding isotherm between AuNPs-aptamer conjugate and protein was modeled with the Hill-Langmuir equation, and the determined equilibrium dissociation constant (K′D) decreased 10-fold when increasing the coverage of aptamer. The kinetics of the reaction as a function of coverage of aptamer were also investigated, including the association rate constant (kon) and the dissociation rate constant (koff). The AuNPs-aptamer conjugate with 258 aptamers per particle had the highest kon, while the koff was similar for AuNPs-aptamer conjugates with different surface coverages. Therefore, the surface coverage of aptamers on AuNPs affects both the thermodynamics and the kinetics of the binding. The AuNPs-aptamer conjugate with the highest surface coverage is the most favorable in biosensors considering the limit of detection, sensitivity, and response time of the assay. These findings deepen our understanding of the interaction between aptamer and target protein on the particle surface, which is important to both improve the scientific design and increase the application of aptamer-nanoparticle based biosensor

Locked nucleic acid molecular beacon for multiplex detection of loop mediated isothermal amplification

Loop mediated isothermal amplification (LAMP) holds incredible promise for point - of - care molecular diagnostics because of its high sensitivity and isothermal amplification behaviour. The issues related to the spurious non-specific amplification caused by the template independent amplification of primers itself causes false positive detection. This can be exacerbated by the common indirect methods used for detection of LAMP. Developing robust and specific detection methods for LAMP is a challenge due to the complex nature of the LAMP amplicons. To see wider adaptation of LAMP, we employ locked nucleic acid bases in molecular beacon to provide the structural stability to the hairpin probes that enable specific and multiplex detection of LAMP. Locked nucleic acid (LNA) modification provides ultra - high thermal stability to the molecular beacons resulting in negligible background fluorescence in the closed state. In this study, various combinations of LNA modifications in the stem and loop region were used and characterized for their thermal stability and influence on hybridization efficiency. The sequence specificity and ultra - high thermal stability of the LNA bases was exploited to develop a multiplex LAMP assay for detection of clinically important antibiotic resistance in S.aureus in 30 min. Multiplex approaches hold a significant advancement in LAMP and would find widespread applications in molecular diagnostics

Two sittings of Autologous Bone Marrow Stem Cells within two years in a case of Ischemic Cardiomyopathy

A 66yrs old Diabetic and Hypertensive female, who had Anterior Wall MI 5yrs ago and had undergone PTCA with Stent to LAD, was admitted for refractory CHF with Severe LVD 2yrs ago and the LVEF then was 25%. Coronary Angiogram was done which showed Total Occlusion of LAD and 50% Stenosis of RCA. Method: 100ml of her bone marrow was harvested from posterior iliac crest and the BMMNCs were isolated as per cGMP protocols at NCRM, Chennai and 325X106 cells with a CD34+ count of 0.84% were injected the next day by transfemoral catheter into the coronary arteries. Post treatment she had clinical improvement. EF increased by 5%. She was in Class-II for 1 year. After 1 yr, she was admitted with severe CHF and EF had deteriorated to 20%. This time BMMNCs isolated from the bone marrow were subjected to in vitro expansion by which the initial 0.15% CD34+ cells increased by nearly 30 fold to 4.62%. Totally 315X106 cells were injected into the coronaries. Post treatment there is clinical as well as Echo evidence of improvement and BNP level has come down by 30%. Conclusion:　 Isolated and expanded CD34+ cells from bone marrow mononuclear cells of autologous origin, administered into the coronaries in an Ischemic Cardiomyopathy patient has been proven to be safe. The clinical and Echo cardiographic improvement that has sustained for long-term, proves the feasibility and efficacy of two consecutive autologous bone marrow stem cell applications, one isolated and the second ex vivo expanded. More case studies may be undertaken to further evaluate the results

Comparison of peritoneal transport characteristics at the second week and at six months of peritoneal dialysis commencement

Peritoneal equilibration test (PET) is an important tool for managing peritoneal dialysis (PD) prescription. The Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines suggest that the first PET be performed 4-8 weeks after PD commencement. The main reason for this delay is because of the peritoneal membrane might change its character once it is exposed to the glucose based dialysate. In this study, we compared PET 2 weeks after PD commencement to PET after 6 months to evaluate the changes in the peritoneal membrane character with time. This study included 126 patients who underwent PD initiation between March 2007 and December 2011. The PET was performed as per the standard protocol at 2 nd week and 6 th month after PD initiation. Transport status was categorized as low, low average, high average, and high as per the standard definition. There was no change in transport character in 115 patients (91.2%) between the two PET measurements. When the Early PET at 2 nd week and 6 th month PET data were analyzed, no significant changes were observed in measured D/P creatinine (0.59 ± 0.14 vs. 0.62 ± 0.14 respectively P = 0.26) and D/D0 Glucose (0.46 ± 0.12 vs. 0.46 ± 0.11, P = 0.65). Using the Bland-Altman analysis the repeatability coefficients were 0.27 and 0.25 for creatinine and glucose values respectively. In our study, the PET performed at the 2 nd week are similar to that of the 6 th month PET in 91.2% of our patients and the test did not significantly change with time. In conclusion, we could do PET early at 2 nd week to assess the peritoneal membrane character and this would help in proper dialysis prescription to the patients