Rapid and sensitive detection of an intracellular pathogen in human peripheral leukocytes with hybridizing magnetic relaxation nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample's magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen.     

Molecular dynamics modeling of the sub-THz vibrational absorption of thioredoxin from <Emphasis Type="Italic">E. coli</Emphasis>

Sub-terahertz (THz) vibrational modes of the protein thioredoxin in a water environment were simulated using molecular dynamics (MD) in order to find the conditions needed for simulation convergence, improve the correlation between experimental and simulated absorption frequencies, and ultimately enhance the predictive capabilities of computational modeling. Thioredoxin from E. coli was used as a model molecule for protocol development and to optimize the simulation parameters. The empirically parameterized software packages Amber 8 and 10 were used in this work. Using atomic trajectories from the constant energy and volume MD simulations, thioredoxin’s sub-THz vibrational spectra and absorption coefficients were calculated in a quasi-harmonic approximation. An optimal production run length ∼100 ps was found, in agreement with experimental data on thioredoxin relaxation dynamics. At the same time, a new procedure was developed for averaging correlation matrices of atomic coordinates in MD simulations. In particular, the open source package ptraj was edited to improve a matrix-analyzing function. Averaging only six matrices gave much more consistent results, with absorption peak intensities exceeding those from the individual spectra and a rather good correlation between simulated vibrational frequencies and experimental data.     

The role of scale explants in the growth of regenerating lily bulblets in vitro

Plant Cell, Tissue and Organ Culture (PCTOC) - Lily scale-explants cultured in vitro regenerate adventitious bulblets at their base. Large scale-explants (6?×?18&nbsp;mm; the...     

Calprotectin Pegylation Enhanced Its Physical and Structural Properties

Calprotectin is member of the S-100 protein family with a wide plethora of intra-and extracellular functions. Anticancer activities, antimicrobial effects and being a qualified disease marker are among the compelling features of this protein to be used as a pharmaceutical agent. However, there are several impediments to applications of protein pharmaceuticals including: proteolytic degradation, short circulating half-life, low solubility and immunogenicity. Pegylation is a common bioconjugation polymer capable of overcoming these drawbacks. Recombinant expression and purification of calprotectin along with its pegylation would result in enhanced pharmaco-dynamic and pharmacokinetic properties. Our florescence spectroscopy and far Ultraviolet-optical density results indicate that pegylation altered the physical and structural properties of the calprotectin to become in a more stable and functionally active state. Due to enhanced pharmacodynamic and pharmacokinetic properties of the calprotectin via pegylation, this study would pave the way for better in vitro and in vivo validations of calprotectin applications in medical practice.     

Development of Acid-Resistant Alginate/Trimethyl Chitosan Nanoparticles Containing Cationic β-Cyclodextrin Polymers for Insulin Oral Delivery

In this study, the use of trimethylchitosan (TMC), by higher solubility in comparison with chitosan, in alginate/chitosan nanoparticles containing cationic β-cyclodextrin polymers (CPβCDs) has been studied, with the aim of increasing insulin uptake by nanoparticles. Firstly, TMCs were synthesized by iodomethane, and CPβCDs were synthesized within a one-step polycondensation reaction using choline chloride (CC) and epichlorohydrine (EP). Insulin–CβCDPs complex was prepared by mixing 1:1 portion of insulin and CPβCDs solutions. Then, nanoparticles prepared in a three-step procedure based on the iono-tropic pregelation method. Nanoparticles screened using experimental design and Placket Burman methodology to obtain minimum size and polydispercity index (pdI) and the highest entrapment efficiency (EE). CPβCDs and TMC solution concentration and pH and alginate and calcium chloride solution concentrations are found as the significant parameters on size, PdI, and EE. The nanoparticles with proper physicochemical properties were obtained; the size, PdI, and EE% of optimized nanoparticles were reported as 150.82 ± 21 nm, 0.362 ± 0.036, and 93.2% ± 4.1, respectively. The cumulative insulin release in intestinal condition achieved was 50.2% during 6 h. By SEM imaging, separate, spherical, and nonaggregated nanoparticles were found. In the cytotoxicity studies on Caco-2 cell culture, no significant cytotoxicity was observed in 5 h of incubation, but after 24 h of incubation, viability was decreased to 50% in 0.5 mμ of TMC concentration. Permeability studies across Caco-2 cells had been carried out, and permeability achieved in 240 min was 8.41 ± 0.39%, which shows noticeable increase in comparison with chitosan nanoparticles. Thus, according to the results, the optimized nanoparticles can be used as a new insulin oral delivery system.KEY WORDS: alginate, cationic β-cyclodextrin, insulin nanoparticle, oral delivery, trimethyl chitosan     

Comparison of the adsorbed conformation of barley lipid transfer protein at the decane-water and vacuum-water interface: a molecular dynamics simulation

Molecular dynamics simulation is used to model the adsorption of the barley lipid transfer protein (LTP) at the decane-water and vacuum-water interfaces. Adsorption at both surfaces is driven by displacement of water molecules from the interfacial region. LTP adsorbed at the decane surface exhibits significant changes in its tertiary structure, and penetrates a considerable distance into the decane phase. At the vacuum-water interface LTP shows small conformational changes away from its native structure and does not penetrate into the vacuum space. Modification of the conformational stability of LTP by reduction of its four disulphide bonds leads to an increase in conformational entropy of the molecules, which reduces the driving force for adsorption. Evidence for changes in the secondary structure are also observed for native LTP at the decane-water interface and reduced LTP at the vacuum-water interface. In particular, intermittent formation of short (six-residue) regions of beta-sheet is found in these two systems. Formation of interfacial beta-sheet in adsorbed proteins has been observed experimentally, notably in the globular milk protein beta-lactoglobulin and lysozyme.     

Troponin elevation in patients with various tachycardias and normal epicardial coronaries

Troponin elevation is usually synonymous with acute coronary syndrome (ACS). Although sensitive for ACS, the elevation of serum troponin, in the absence of clinical evidence of ischemia, should prompt a search for other etiologies of myocardial necrosis. In fact, elevated values of troponin are correlated with myocardial necrosis even though it does not discriminate the mechanism involved. We report a series of seven patients (age range 18-67 years), who presented with complaints of chest discomfort and were found to have regular supraventricular tachycardia (5 patients) and one patient each with atrial fibrillation and ventricular tachycardia. All these patients had elevated troponin I and underwent coronary angiography that revealed normal epicardial coronary arteries. This is first case series in which all patients underwent coronary angiography and none of the patients was hemodynamically unstable at the time of presentation. Patients with elevated troponin due to conditions other than ACS can receive inappropriate and delayed definitive diagnosis and treatment.     

Metal/metalloid stress tolerance in plants: role of ascorbate,its redox couple,and associated enzymes

The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfully metabolized in order to maintain plant growth, development, and productivity. Ascorbate (AsA) is a highly abundant metabolite and a water-soluble antioxidant, which besides positively influencing various aspects in plants acts also as an enigmatic component of plant defense armory. As a significant component of the ascorbate-glutathione (AsA-GSH) pathway, it performs multiple vital functions in plants including growth and development by either directly or indirectly metabolizing ROS and its products. Enzymes such as monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintain the reduced form of AsA pool besides metabolically controlling the ratio of AsA with its oxidized form (dehydroascorbate, DHA). Ascorbate peroxidase (APX, EC 1.11.1.11) utilizes the reduced AsA pool as the specific electron donor during ROS metabolism. Thus, AsA, its redox couple (AsA/DHA), and related enzymes (MDHAR, DHAR, and APX) cumulatively form an AsA redox system to efficiently protect plants particularly against potential anomalies caused by ROS and its products. Here we present a critical assessment of the recent research reports available on metal/metalloid-accrued modulation of reduced AsA pool, AsA/DHA redox couple and AsA-related major enzymes, and the cumulative significance of these antioxidant system components in plant metal/metalloid stress tolerance.