Refolding of a membrane protein in a microfluidics reactor

Membrane protein production for structural studies is often hindered by the formation of non-specific aggregates from which the protein has to be denatured and then refolded to a functional state. We developed a new approach, which uses microfluidics channels, to refold protein correctly in quantities sufficient for structural studies. Green fluorescent protein (GFP), a soluble protein, and bacteriorhodopsin (BR), a transmembrane protein, were used to demonstrate the efficiency of the process. Urea-denatured GFP refolded as the urea diffused away from the protein, forming in the channel a uniform fluorescent band when observed by confocal microscopy. Sodium dodecyl sulphate-denatured BR refolded within the channel on mixing with detergent–lipid mixed micelles. The refolding, monitored by absorbance spectroscopy, was found to be flow rate dependent. This potential of microfluidic reactors for screening protein-folding conditions and producing protein would be particularly amenable for high-throughput applications required in structural genomics. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Exogenous Application of Melatonin Induces Tolerance to Salt Stress by Improving the Photosynthetic Efficiency and Antioxidant Defense System of Maize Seedling

Journal of Plant Growth Regulation - Melatonin is a ubiquitous signaling plant hormone that plays a crucial role in regulating the growth and development of plants under stress conditions. Since a...     

Solitary Renal Fossa Recurrence of Renal Cell Carcinoma After Nephrectomy

Renal cell carcinoma without metastasis responds well to surgical excision but is known to recur postnephrectomy. In a small but significant number of patients this recurrence is not accompanied by metastasis, which is important as these people benefit from further surgery. We examined 20 articles from the current literature to ascertain how best to treat this condition. Surgical management renders better results than conservative or medical therapies. Readily available investigations such as blood tests and computed tomography can help determine the right patients for surgery in an evidence-based fashion. Current findings have allowed us to suggest a protocol for the treatment of solitary renal fossa recurrence of postnephrectomy renal cell carcinoma. There are further opportunities for study in validating our protocol, and in novel renal cell carcinoma treatment strategies that have not been tested on solitary renal fossa recurrences.Key words: Renal cancer, Recurrence, Nephrectomy, Complications, ManagementKidney cancers represent 2% of cancers worldwide; the most common type is renal cell carcinoma. Curative treatment of localized disease is a nephrectomy. Following surgery, recurrence can happen locally with an incidence of 1.61%.^1–5 A solitary renal fossa local recurrence is rare but important to distinguish from local recurrence with metastasis, which would not benefit from surgical resection. The 5-year survival postresection of local recurrence for those without metastasis compared with those with metastasis was 62% compared with 0%.⁴ The kidneys are bordered by the colon, spleen, liver, stomach, and associated neurovascular structures, all of which may be invaded in this form of recurrence; specific morbidity is related to the invasion and subsequent resection of these organs. General morbidity is caused by the surgery itself, with pain, infection, and hemorrhage being major contributors (Figure 1). This article explains predictive factors in recurrence, useful diagnostic modalities, and management, and provides recommendations and highlights opportunities for further study.Open in a separate windowFigure 1Computed tomography image of a patient with renal fossa recurrence of renal cancer after nephrectomy. Of note is the large mass identifiable in the spleen.     

Stochastic model for tumor control probability: effects of cell cycle and (a)symmetric proliferation

Background

Estimating the required dose in radiotherapy is of crucial importance since the administrated dose should be sufficient to eradicate the tumor and at the same time should inflict minimal damage on normal cells. The probability that a given dose and schedule of ionizing radiation eradicates all the tumor cells in a given tissue is called the tumor control probability (TCP), and is often used to compare various treatment strategies used in radiation therapy.

Method

In this paper, we aim to investigate the effects of including cell-cycle phase on the TCP by analyzing a stochastic model of a tumor comprised of actively dividing cells and quiescent cells with different radiation sensitivities. Moreover, we use a novel numerical approach based on the method of characteristics for partial differential equations, validated by the Gillespie algorithm, to compute the TCP as a function of time.

Results

We derive an exact phase-diagram for the steady-state TCP of the model and show that at high, clinically-relevant doses of radiation, the distinction between active and quiescent tumor cells (i.e. accounting for cell-cycle effects) becomes of negligible importance in terms of its effect on the TCP curve. However, for very low doses of radiation, these proportions become significant determinants of the TCP. We also present the results of TCP as a function of time for different values of asymmetric division factor.

Conclusion

We observe that our results differ from the results in the literature using similar existing models, even though similar parameters values are used, and the reasons for this are discussed.

     

Effect of exercise on blood flow through the aortic valve: a combined clinical and numerical study

The aim of this study was to measure the cardiac output and stroke volume for a healthy subject by coupling an echocardiogram Doppler (echo-Doppler) method with a fluid–structure interaction (FSI) simulation at rest and during exercise. Blood flow through aortic valve was measured by Doppler flow echocardiography. Aortic valve geometry was calculated by echocardiographic imaging. An FSI simulation was performed, using an arbitrary Lagrangian–Eulerian mesh. Boundary conditions were defined by pressure loads on ventricular and aortic sides. Pressure loads applied brachial pressures with (stage 1) and without (stage 2) differences between brachial, central and left ventricular pressures. FSI results for cardiac output were 15.4% lower than Doppler results for stage 1 (r = 0.999). This difference increased to 22.3% for stage 2. FSI results for stroke volume were undervalued by 15.3% when compared to Doppler results at stage 1 and 26.2% at stage 2 (r = 0.94). The predicted mean backflow of blood was 4.6%. Our results show that numerical methods can be combined with clinical measurements to provide good estimates of patient-specific cardiac output and stroke volume at different heart rates.     

Bovine and human lactoferricin peptides: chimeras and new cyclic analogs

Lactoferrin (LF) is an important antimicrobial and immune regulatory protein present in neutrophils and most exocrine secretions of mammals. The antimicrobial activity of LF has been related to the presence of an antimicrobial peptide sequence, called lactoferricin (LFcin), located in the N-terminal region of the protein. The antimicrobial activity of bovine LFcin is considerably stronger than the human version. In this work, chimera peptides combining segments of bovine and human LFcin were generated in order to study their antimicrobial activity and mechanism of action. In addition, the relevance of the conserved disulfide bridge and the resulting cyclic structure of both LFcins were analyzed by using “click chemistry” and sortase A-catalyzed cyclization of the peptides. The N-terminal region of bovine LFcin (residues 17–25 of bovine LF) proved to be very important for the antimicrobial activity of the chimera peptides against E. coli, when combined with the C-terminal region of human LFcin. Similarly the cyclic bovine LFcin analogs generated by “click chemistry” and sortase A preserved the antimicrobial activity of the original peptide, showing the significance of these two techniques in the design of cyclic antimicrobial peptides. The mechanism of action of bovine LFcin and its active derived peptides was strongly correlated with membrane leakage in E. coli and up to some extent with the ability to induce vesicle aggregation. This mechanism was also preserved under conditions of high ionic strength (150 mM NaCl) illustrating the importance of these peptides in a more physiologically relevant system.     

Intracellular Locations of Replication Proteins and the Origin of Replication during Chromosome Duplication in the Slowly Growing Human Pathogen Helicobacter pylori

We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.