Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.     

Rapid Prototyping of a High Sensitivity Graphene Based Glucose Sensor Strip

A rapid prototyping of an inexpensive, disposable graphene and copper nanocomposite sensor strip using polymeric flexible substrate for highly sensitive and selective nonenzymatic glucose detection has been developed and tested for direct oxidization of glucose. The CuNPs were electrochemically deposited on to the graphene sheets to improve electron transfer rates and to enhance electrocatalytic activity toward glucose. The graphene based electrode with CuNPs demonstrated a high degree of sensitivity (1101.3±56 μA/mM.cm²), excellent selectivity (without an interference with Ascorbic Acid, Uric Acid, Dopamine, and Acetaminophen), good stability with a linear response to glucose ranging from 0.1 mM to 0.6 mM concentration, and detection limits of 0.025 mM to 0.9 mM. Characterization of the electrodes was performed by scanning electron microscopy (FESEM and SEM). The electrochemical properties of the modified graphene electrodes were inspected by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry.     

A Molecular Dynamics Simulation Study of Nanomechanical Properties of Asymmetric Lipid Bilayer

A very important part of the living cells of biological systems is the lipid membrane. The mechanical properties of this membrane play an important role in biophysical studies. Investigation as to how the insertion of additional phospholipids in one leaflet of a bilayer affects the physical properties of the obtained asymmetric lipid membrane is of recent practical interest. In this work a coarse-grained molecular dynamics simulation was carried out in order to compute the pressure tensor, the lateral pressure, the surface tension and the first moment of lateral pressure in each leaflet of such a bilayer. Our simulations indicate that adding more phospholipids into one monolayer results in asymmetrical changes in the lateral pressure of the individual bilayer leaflets. Interestingly, it has been observed that a change in phospholipid density in one leaflet affects the physical properties of unperturbed leaflet as well. The asymmetric behavior of the physical properties of the two leaflets as a result of a change in the contribution of the various intermolecular forces in the presence of additional phospholipids may be expressed formally.     

New immunological investigations on Helicobacter pylori‐induced gastric ulcer in patients

Although Helicobacter pylori (Hp) plays an important role in the pathogenesis of chronic gastritis and gastric ulcer, little is known about the probable mechanisms of these types of gastrointestinal damage. To determine the precise mechanisms involved in ulcer formation, immune responses in patients with gastric ulcer (GUP) caused by Hp infection (Hp⁺) were compared with those of other gastritis patients (GP). The sensitivity and proliferation of peripheral blood mononuclear cells (PBMNCs) obtained from patients were evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay against exposure with complex Hp crude antigen (HPCA) and mitogen (phytohemagglutinin, PHA). Production of inflammatory cytokines, including interleukin (IL)‐1β and IL‐8, in serum and supernatants of PBMNCs were then measured by ELISA. It was found that, after stimulation with PHA, both IL‐8 and IL‐1β concentrations in sera and supernatants as well as proliferation and sensitivity were statistically greater in GUP Hp⁺ than GP Hp^?. Furthermore, HPCA inhibited the proliferation of PBMNCs dose‐dependently; however, it stimulated IL‐8 and IL‐1β production in supernatants of mononuclear cells. Therefore, the up‐regulated concentrations of IL‐8 and IL‐1β may have been caused by increase in the size of mononuclear cell subpopulations or in their cytokine secretory activity, indicating the greatest cell responsiveness in GUP Hp⁺ patients. These results suggest that tissue damage and ulcers occur in patients who produce more IL‐8 and IL‐1β than patients who do not develop ulcers; the former consequently have more activated immune cells at the site of infection. Therefore, both host responses and Hp virulence factors may be involved in the development of gastric ulcers.

     

Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology

An experimental design was used to optimize plasmid purification from an alkaline lysate of Escherichia coli cells using PEG-sodium citrate aqueous two-phase systems (ATPS), and to evaluate the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology (RMS) with an orthogonal rotatable central composite design was defined based on the conditions found for the highest purification by preliminary tests. The adequacy of the calculated models for the plasmid recovery and remaining RNA were confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, PEG molecular weight, tie line length, and cell lysate load for three different phase volume ratios revealed different effects of these five factors on the studied parameters. Plasmid recovery of 99% was predicted for a system with PEG 400, pH 6.9, tie line length of 38.7%, phase volume ratio of 1.5, and lysate load of 10% (v/v). Under these conditions the predicted RNA removal was 68%.     

Synthesis and biological evaluation of 1,3-diphenylprop-2-en-1-ones possessing a methanesulfonamido or an azido pharmacophore as cyclooxygenase-1/-2 inhibitors

A group of (E)-1,3-diphenylprop-2-en-1-one derivatives (chalcones) possessing a MeSO(2)NH, or N(3), COX-2 pharmacophore at the para-position of the C-1 phenyl ring were synthesized using a facile stereoselective Claisen-Schmidt condensation reaction. In vitro COX-1/COX-2 structure-activity relationships were determined by varying the substituents on the C-3 phenyl ring (4-H, 4-Me, 4-F, and 4-OMe). Among the 1,3-diphenylprop-2-en-1-ones possessing a C-1 para-MeSO(2)NH COX-2 pharmacophore, (E)-1-(4-methanesulfonamidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7b) was identified as a selective COX-2 inhibitor (COX-2 IC(50)=1.0 microM; selectivity index >100) that was less potent than the reference drug rofecoxib (COX-2 IC(50)=0.50 microM; SI>200). The corresponding 1,3-diphenylprop-2-en-1-one analogue possessing a C-1 para-N(3) COX-2 pharmacophore, (E)-1-(4-azidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7f), exhibited potent and selective COX-2 inhibition (COX-1 IC(50)=22.2 microM; COX-2 IC(50)=0.3 microM; SI=60). A molecular modeling study where 7b and 7f were docked in the binding site of COX-2 showed that the p-MeSO(2)NH and N(3) substituents on the C-1 phenyl ring are oriented in the vicinity of the COX-2 secondary pocket (His90, Arg513, Phe518, and Val523). The structure-activity data acquired indicate that the propenone moiety constitutes a suitable scaffold to design new acyclic 1,3-diphenylprop-2-en-1-ones with selective COX-1 or COX-2 inhibitory activity.     

LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Two‐dimensional blue native/SDS‐PAGE is widely applied to investigate native protein–protein interactions, particularly those within membrane multi‐protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we investigated the potential of using LC‐MS/MS as an alternative for SDS‐PAGE in blue native (BN) analysis of protein complexes. By subjecting equal slices from BN gel lanes to label‐free semi‐quantitative LC‐MS/MS, we determined an abundance profile for each protein across the BN gel, and used these profiles to identify potentially interacting proteins by protein correlation profiling. We demonstrate the feasibility of this approach by considering the oxidative phosphorylation complexes I–V in the native human embryonic kidney 293 mitochondrial fraction, showing that the method is capable of detecting both the fully assembled complexes as well as assembly/turnover intermediates of complex I (NADH:ubiquinone oxidoreductase). Using protein correlation profiling with a profile for subunits NDUFS2, 3, 7 and 8 we identified multiple proteins possibly involved in the biogenesis of complex I, including the recently implicated chaperone C6ORF66 and a novel candidate, C3ORF60.     

Strain energy density as a rupture criterion for the kidney: impact tests on porcine organs, finite element simulation, and a baseline comparison between human and porcine tissues

High-velocity (up to 25 m/s) impact tests were performed on pig kidneys to characterize failure behavior at deformation rates associated with traumatic injury. Cylindrical tissue samples (n = 45) and whole perfused organs (n = 34) were impacted using both falling weights and a high-velocity pneumatic projectile impactor. Impact energy was incrementally increased until visible rupture occurred. The strain energy density failure threshold fell between 25 and 60 kJ/m3 for excised porcine tissue samples, and between 15 and 30 kJ/m3 for whole, perfused organs. The relationship between localized failure in whole organ impacts and tissue level failure thresholds observed in cylindrical tissue samples was explored using a detailed finite element model of the human kidney. The model showed good correlation between experimentally observed injury patterns and predicted strain energy density distributions within the renal parenchyma. Finally, to facilitate interpretation of the porcine renal impact results with regard to human trauma, quasi-static compression test results of freshly excised human kidney cortex samples (n = 30) were compared against similar tests on pig kidneys. Human tissues failed at Lagrange strain levels similar to porcine tissue (63+/-6.3%), but at 52% lower Lagrange stress (116+/-28 kPa), and 35% lower strain energy density (17.1+/-4.4 kJ/m3). Thus conservative interpretation of porcine test results is recommended.     

Safety testing for neurovirulence of novel live, attenuated flavivirus vaccines: infant mice provide an accurate surrogate for the test in monkeys.

Current requirements for control of live viral vaccines, including yellow fever 17D, produced from potentially neurotropic wild-type viruses include tests for neurovirulence in nonhuman primates. We have used yellow fever 17D virus as a live vector for novel flavivirus vaccines (designated ChimeriVax) against dengue, Japanese encephalitis (JE), and West Nile (WN) viruses. For control of these vaccines, it would be preferable to substitute a test in mice for the test in a higher species (monkeys). In this study, we compare the neurovirulence of ChimeriVax vaccine candidates in suckling mice inoculated by the intracerebral (IC) route with graded doses of the test article or yellow fever 17D vaccine as a reference control. Mortality ratio and survival distribution are the outcome measures. The monkey safety test is performed as described for control of yellow fever vaccines. In both mice and monkeys, all chimeric vaccines were significantly less neurovirulent than yellow fever 17D vaccine. The test in suckling mice discriminated between strains of two different vaccines (ChimeriVax-JE and ChimeriVax-DEN1) differing by a single amino acid change, and was more sensitive for detecting virulence differences than the test in monkeys. The results indicate that the suckling mouse test is simple to perform, highly sensitive and, with appropriate validation, could complement or possibly even replace the neurovirulence component of the monkey safety test. The test in infant mice is particularly useful as a means of demonstrating biological consistency across seed virus and vaccine lots.     

Long-term outcome of posterior spinal fusion for the correction of adolescent idiopathic scoliosis

Background

Adolescent idiopathic scoliosis (AIS) is the most common form of idiopathic scoliosis, and surgery is considered as one of the therapeutic options. However, it is associated with a variety of irreversible complications, in spite of the benefits it provides. Here, we evaluated the long-term outcome of posterior spinal fusion (PSF) of AIS to shed more light on the consequences of this surgery.

Methods

In a cross-sectional study, a total of 42 AIS patients who underwent PSF surgery were radiographically and clinically inspected for the potential post-operative complications. Radiographic assessments included the device failure, union status, and vertebral tilt below the site of fusion. Clinical outcomes were evaluated using the Oswestry disability index (ODI) and visual analogue scale (VAS).

Results

The mean age of the surgery was 14.4?±?5.1 years. The mean follow-up of the patients was 5.6?±?3.2 years. Complete union was observed in all patients, and no device failure was noticed. Pre- and post-operative vertebral tilt below the site of fusion were 11.12°?±?7.92° and 6.21°?±?5.73°, respectively (p?<?0.001). The mean post-operative ODI was 16.7?±?9.8. The mean post-operative VAS was 2.1?±?0.7. ODI value was positively correlated with follow-up periods (p?=?0.04, r?=?0.471). New degenerative disc disease (DDD) was observed in 6 out of 37 (16%) patients.

Conclusion

In spite of the efficacy and safety of PSF surgery of AIS, it might result in irreversible complications such as DDD. Moreover, the amount of post-operative disability might increase over the time and should be discussed with the patients.