Induction,resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.     

Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State

Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N^∗. Fluorescence resonance energy transfer measurements show that N^∗ then expands rapidly but partially to form an early unfolding intermediate I_E. Fluorescence spectral measurements indicate that both N^∗ and I_E have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, I_L, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when I_L forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.     

Effect of Chronic Treatment with D2 Allosteric Modulator PAOPA on the Expression of Cerebral Dopamine Neurotrophic Factor (CDNF) in Select Brain Regions

International Journal of Peptide Research and Therapeutics - Impairment in dopaminergic pathways has been implicated in several detrimental neuropsychiatric and neurodegenerative disorders,...     

Diffusional barrier in the unfolding of a small protein

To determine how the dynamics of the polypeptide chain in a protein molecule are coupled to the bulk solvent viscosity, the unfolding by urea of the small protein barstar was studied in the presence of two viscogens, xylose and glycerol. Thermodynamic studies of unfolding show that both viscogens stabilize barstar by a preferential hydration mechanism, and that viscogen and urea act independently on protein stability. Kinetic studies of unfolding show that while the rate-limiting conformational change during unfolding is dependent on the bulk solvent viscosity, eta, its rate does not show an inverse dependence on eta, as expected by Kramers' theory. Instead, the rate is found to be inversely proportional to an effective viscosity, eta + xi, where xi is an adjustable parameter which needs to be included in the rate equation. xi is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding, at constant urea concentration as well as under isostability conditions. Hence, the unfolding protein chain does not experience the bulk solvent viscosity, but instead an effective solvent viscosity, which is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result is the validation of the isostability assumption, commonly used in protein folding studies but hitherto untested, according to which if a certain concentration of urea can nullify the effect of a certain concentration of viscogen on stability, then the same concentrations of urea and viscogen will also not perturb the free energy of activation of the unfolding of the protein.     

A mathematical model of breast cancer development, local treatment and recurrence

Cancer development is a stepwise process through which normal somatic cells acquire mutations which enable them to escape their normal function in the tissue and become self-sufficient in survival. The number of mutations depends on the patient's age, genetic susceptibility and on the exposure of the patient to carcinogens throughout their life. It is believed that in every malignancy 4-6 crucial similar mutations have to occur on cancer-related genes. These genes are classified as oncogenes and tumour suppressor genes (TSGs) which gain or lose their function respectively, after they have received one mutative hit or both of their alleles have been knocked out. With the acquisition of each of the necessary mutations the transformed cell gains a selective advantage over normal cells, and the mutation will spread throughout the tissue via clonal expansion. We present a simplified model of this mutation and expansion process, in which we assume that the loss of two TSGs is sufficient to give rise to a cancer. Our mathematical model of the stepwise development of breast cancer verifies the idea that the normal mutation rate in genes is only sufficient to give rise to a tumour within a clinically observable time if a high number of breast stem cells and TSGs exist or genetic instability is involved as a driving force of the mutation pathway. Furthermore, our model shows that if a mutation occurred in stem cells pre-puberty, and formed a field of cells with this mutation through clonal formation of the breast, it is most likely that a tumour will arise from within this area. We then apply different treatment strategies, namely surgery and adjuvant external beam radiotherapy and targeted intraoperative radiotherapy (TARGIT) and use the model to identify different sources of local recurrence and analyse their prevention.     

Familial aggregation of nasal conditioning capacity.

In our previous studies on nasal conditioning, we observed a large variability among individuals to condition inspired air. Although we previously investigated various physiological parameters (age, sex, nasal mucosal temperature, heart rate, blood pressure, and nasal volume) that might underlie these differences, we have been unable to explain this variability. Many proteins and molecules, which are under genetic control and could affect nasal conditioning, are involved in water transport,. In this study, we hypothesized that familial factors may contribute to the differences in nasal conditioning capacity (NCC). We performed a prospective study of 47 sibling pairs. Cold dry air was delivered to the nose, and the total water gradient (TWG) was calculated to determine the NCC. We found a highly significant intraclass correlation of 0.53 (P < 0.0001) between sibling pairs for the TWG. These results suggest that there is a familial basis for nasal conditioning and a large enough genetic component to search for genes explaining the observed correlation.     

Optimization of bioreactor using metabolic control analysis approach

A new methodology based on a metabolic control analysis (MCA) approach is developed for the optimization of continuous cascade bioreactor system. A general framework for representation of a cascade bioreactor system consisting of a large number of reactors as a single network is proposed. The kinetic and transport processes occurring in the system are represented as a reaction network with appropriate stoichiometry. Such representation of the bioreactor systems makes it amenable to the direct application of the MCA approach. The process sensitivity information is extracted using MCA methodology in the form of flux and concentration control coefficients. The process sensitivity information is shown to be a useful guide for determining the choice of decision variables for the purpose of optimization. A generalized problem of optimization of the bioreactor is formulated in which the decision variables are the operating conditions and kinetic parameters. The gradient of the objective function to be maximized with respect to all decision variables is obtained in the form of response coefficients. This gradient information can be used in any gradient-based optimization algorithm. The efficiency of the proposed technique is demonstrated with two examples taken from literature: biotransformation of crotonobetaine and alcohol fermentation in cascade bioreactor system.     

Influence of operational variables on properties of piroxicam pellets prepared by extrusion-spheronization: A technical note

Summary and Conclusion  The processing conditions has a pronounced effect on the pellet properties. Drying conditions influenced the mean size and the drug release of the pellets. Because of the shrinking of the pellets upon drying at higher temperatures, the pellets also showed increased densities. Freeze drying almost prevented shrinking and thus led to the highest drug release. With an increase in the temperature of drying, the drug release rate decreased. Both spheronization time and spheronization speed affected the shapes of pellets, and the changes in shapes then affected the pellet flow properties. Within the studied range, the circularity of the pellets was affected more by the spheronization time than by the spheronization speed. Drying conditions influenced pellet friability, which decreased with an increase in drying temperature, indicating the formation of more dense structures at higher temperatures. The same result was obtained with spheronization time. With an increase in spheronization time, the friability decreased, because of the formation of more compact masses at higher spheronization time. Mean size was not affected by spheronization time or spheronization speed. Published: March 9, 2007     

Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO₂ to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco.     

Olfactory Thresholds of the U.S. Population of Home-Dwelling Older Adults: Development and Validation of a Short,Reliable Measure

Current methods of olfactory sensitivity testing are logistically challenging and therefore infeasible for use in in-home surveys and other field settings. We developed a fast, easy and reliable method of assessing olfactory thresholds, and used it in the first study of olfactory sensitivity in a nationally representative sample of U.S. home-dwelling older adults. We validated our method via computer simulation together with a model estimated from 590 normosmics. Simulated subjects were assigned n-butanol thresholds drawn from the estimated normosmic distribution and based on these and the model, we simulated administration of both the staircase and constant stimuli methods. Our results replicate both the correlation between the two methods and their reliability as previously reported by studies using human subjects. Further simulations evaluated the reliability of different constant stimuli protocols, varying both the range of dilutions and number of stimuli (6–16). Six appropriately chosen dilutions were sufficient for good reliability (0.67) in normosmic subjects. Finally, we applied our method to design a 5-minute, in-home assessment of older adults (National Social Life, Health and Aging Project, or NSHAP), which had comparable reliability (0.56), despite many subjects having estimated thresholds above the strongest dilution. Thus, testing with a fast, 6-item constant stimuli protocol is informative, and permits olfactory testing in previously inaccessible research settings.