The identification and the elimination of clashes in the structure of an early-stage intermediate in the protein folding process

Correlation between Protein Stability Cores and Protein Folding Kinetics: A Case Study on Pseudomonas aeruginosa Apo-Azurin

Protein folding is the process that a protein molecule transforms from the linear polymer of peptides to a three-dimensional native structure with specific biological function. By now, the protein folding problem has been studied for more than 50 years and already became a broad and active research field. To answer the 58th question raised by Science in 2005, in this article we briefly reviewed the background and research history of the protein folding problem, and introduced the progresses of protein folding prediction research from four aspects: the protein folding process prediction (protein folding simulation), the folding process related parameter prediction, the protein folding result prediction (protein structure prediction), and the folding result related parameter prediction. The studies on the protein folding problem began in the 60s of 20th century, with the efforts to seek a solution to the paradox that a protein can actually form a native 3D structure in only several seconds but the time scale estimated by a thermodynamic ergodic hypothesis would be longer than the age of universe. Computer simulation is an important approach for protein folding study. The protein models can be classified into 3 categories: lattice model, off-lattice model and all-atom model. The current knowledge about protein folding mechanism is based on the concept of folding funnel on a free-energy landscape, and the current opinion is that the protein folding mechanism is not unique for the whole protein universe and that there may exist a continuum between the two extreme ends of hierarchical folding and nucleation folding scenarios. The hardware for protein folding simulation was becoming more powerful; distributed systems (e.g, Folding@home), special-purpose machines (e.g, ANTON), and GPU-based platforms have been developed for protein folding simulation. Meanwhile, the folding simulation software was continuously enhanced. An important issue in protein folding simulation is to overcome the local energy barrier to find the global energy minimum; several approaches such as replica-exchange, multi-scale modeling and Modeling Employing Limited Data (MELD) were developed to tackle this issue; human intelligence involvement (e.g, “Foldit” Game) is another interesting effort. During the past two decades, the ability of protein folding simulation was continuously rising. For now, the folding simulation for the proteins with dozens of amino acids can reach a time scale of millisecond, while the protein size able to do effective folding simulation is around 100 amino acids. The targets of protein folding simulation have been largely expanded and now include both the in vitro and the in vivo folding such as co-translational folding, chaperone-assistant folding, small-molecule- induced folding and metal-coupled folding. Folding rate and folding type are two important parameters related with the protein folding process and now they can be predicted by statistical and machine-learning approaches based on different levels of structural features such as the topological properties of tertiary structure, the contents of secondary structure and the amino acid frequencies of primary structure. The result of a protein folding process is the formation of a protein structure. According to the hierarchy of structural organization, the protein structure prediction problem includes secondary structure prediction, tertiary structure prediction and quaternary structure prediction. By now, the secondary structure prediction algorithm has experienced five generations and the current accuracy is about 80% for 3-classes prediction. The tertiary structure prediction approaches mainly include two categories: template-based modeling and free modeling, with the former having higher accuracy and the latter having larger application scope. The quaternary structure prediction includes the prediction of complex structure and the prediction of the possibility of protein-protein interaction, and these predictions can be performed based on protein 3D structure or merely amino acid sequence. Structure related parameter prediction also attracted research interests, including the predictions of protein structural classes, secondary structure contents, disordered regions, solvent accessible surface region and the amino acid contacting pairs in the interface of protein-protein interaction. In the end, some possible development directions worth noticing in the future of protein folding research were suggested and they are: the coupling between protein folding and binding, the fusion of protein folding research with systems biology and the application of deep-learning techniques in the field of protein folding prediction.

4013 Background: Intensive postoperative surveillance is associated with improved survival and recommended for patients with late stage (stage IIB & III) colon cancer. We hypothesized that stage I & IIA colon cancer patients would experience similar benefits. Methods: Secondary analysis of data from the Clinical Outcomes of Surgical Therapy trial was performed by analyzing results according to TNM stage; early (stage I & IIA; 537 patients) and late (stage IIB & III; 254 patients) stage disease. Five-year recurrence rates were higher in patients with late (35.7%) versus early stage disease (9.5%). Early and late stage salvage rates, recurrence patterns and methods of first detection were compared by χ2 test. Results: Salvage rates for early and late stage disease patients with recurrence were the same (35.9% versus 37%, p=0.9 respectively). Median survival following second surgery after recurrence was 35.8 months late stage and 51.2 months for early stage patients respectively. Sites of first recurrence did not significantly differ between late and early stage disease: liver (37.4% vs. 27.2%); lung (29.7% vs.23.6%); intraabdominal (24.2% vs.10.9%); and locoregional (12.1% vs.10.9%). Methods of first detection of recurrence were also not significantly different: CEA (37.4% vs. 29.1%); CT scan (26.4% vs. 23.6%); chest X-ray (12.1% vs. 7.3%); and colonoscopy (8.8% vs. 12.7%), for late versus early stage disease, respectively. Conclusions: Patients with early stage colon cancer have similar sites of recurrence, and receive similar benefit from post-recurrence therapy as later stage patients; implementation of existing surveillance guidelines for early stage patients is appropriate. No significant financial relationships to disclose.

Molecular Mechanism of Protein Folding in the Cell

Chapter 4 - Nuclear magnetic resonance spectroscopy for protein structure, folding, and dynamics

The expression of proinflammatory (IL-1β, IFN-γ, TNF-α) and regulatory (IL-10, TGF-β, IL-4) cytokines, as well as the transcription factor FoxP3, was quantified in the liver and hepatic lymph node (HLN) of sheep primoinfected and reinfected with Fasciola hepatica at early (4, 8 and 16 days post-infection [dpi]) and late (100 dpi) stages. The liver exerted a Th2 immune response at very early stages after the primoinfection with F. hepatica that induced the downregulation of IFN-γ, followed by a Th1/Th2/Treg response although the late stages were characterised by the expression of Th1/Th2 immune mediators. Contrarily, in reinfected sheep a robust mixed Th1/Th2/Treg immune response was found at very early stages meanwhile at late stages we observed a Th2/Treg immune response overcoming the expression of Th1 immune mediators. However, the HLN displayed a completely different Th1/Th2/Treg expression profile compared to the liver. Primoinfections with F. hepatica in HLN induced a mixed Th1/Th2/Treg environment from early stages, establishing a Th2 immune response at a late stage. However, the reinfected sheep exerted a Th2 immune response at early stages led by the IL-4 expression in opposition to the Th1/Th2/Treg found in the liver, meanwhile at late stages the HLN of reinfected sheep exerted a mixed Th1/Th2/Treg immune response. This is the first work publishing the expression of immune mediators in the liver and HLN from reinfected sheep with F. hepatica. The study of the immune responses exerted by the natural host in the target organs directly implied in the development of F. hepatica are crucial to better understand the immunopathogenesis of the fasciolosis being a key factor to develop effective vaccines.

Fluid evolution of the Paleoproterozoic Hujiayu copper deposit in the Zhongtiaoshan region: Evidence from fluid inclusions and carbon–oxygen isotopes

e13570 Background: The treatment of metastatic colorectal cancer has been improved by combining cytotoxic chemotherapy with bevacizumab. Newer anti-angiogenic agents are required to improve survival rates as response rates with the current therapies are modest. The preclinical development of such drugs is time-consuming and new methods are required to test the efficacy of lead drugs which represent the entire tumor micro-environment. Methods: Chemical screens were performed in zebrafish larvae to identify hits that affected intersegmental angiogenesis. Five lead drugs were then tested for cytotoxicity using the crystal violet assay. These drugs were tested using ex vivo colorectal tumor explants to determine their effect on secretion of IL-1β, TNF, IL-6 and VEGF. Human explants from 20 patients (Dukes A-D) were cultured for 72 hours and the levels of the above factors measured by ELISA. Comparisons were made between early (Dukes A&B) and late stages (Dukes C&D). Results: One thousand drugs were screened and 5 were found to reduce intersegmental angiogenesis in zebrafish larvae: AM1, AM2, AM3, AM4 and AM5. The drugs did not affect cell growth levels at 10 µM concentration. From the explant studies: AM1 decreased secretion of VEGF in late stages (p=0.005) and IL-6 in early stages (p=0.009). AM2 decreased VEGF in early (p=0.004) and late stages (p=0.02). AM3 decreased TNF secretion in late stages (p=0.02) and IL-6 secretion in early (p=0.009) and late stages (p<0.0001). AM4 decreased VEGF secretion in early stages (p=0.001), IL-6 in early stages (p<0.001) and late stages (p=0.03) and IL-1β in early stages (p=0.002). AM5 decreased IL-6 secretion in the early stages (p=0.03) and IL-1β in the early stages (p=0.001).Overall, IL-1β secretion was reduced by AM4, AM3 and AM5 (p<0.05). IL-6 secretion was reduced by AM1, AM3 and AM4 (p=0.001). TNF secretion was reduced by AM3 (p=0.02) and VEGF secretion was reduced by AM1, AM2 and AM4 (p<0.005). Conclusions: These studies show these drugs have stage-specific effects in colorectal tumor explants and may have the potential as new anti-angiogenic agents in colorectal cancer.

Protein folding has always been a key problem in the field of computational biology. Several models have been proposed for protein folding in the past. The sequence via which a protein folds has a strong impact on the presence or absence of a disease in an organism. Given their significance to the health of an organism, in this paper, we present a Probabilistic Model Checking (PMC) based approach which provides statistical answers to questions that one may pose about the protein folding process. In such an approach, the protein folding model is represented as a probabilistic state transition system, and the questions are expressed formally in form of temporal properties. The PMC engine provides the probability of satisfying the property, which provides an insight into the protein folding process. Such properties are of several kinds - liveness properties, safety properties and fairness properties. We verify the properties on a probabilistic model checker. In this paper, we present our approach, and demonstrate its applicability by using the protein pro-insulin as our test case. We first express the dynamics of this protein using a probabilistic state transition system. Based on the state transition model we express several liveness, fairness and safety properties which target the folding steps/transitions. The probabilistic model checker tool (Prism [29-32]) is invoked to provide the probability of satisfying each of these queries. We have analyzed the change in the property satisfaction probability as a function of the change in the concentration level of the oxidation and reduction reagents. To the best of the authors’ knowledge, this is the first paper to model protein folding dynamics probabilistically, using Probabilistic Model Checking, a powerful technique from the domain of formal verification of state transition systems.

CT features in the early and late stages of chronic total coronary occlusions

Introduction: Endometrial cancer (EC) is the most common gynecologic malignancy in the United States. It is generally divided into one of two categories: Type I, which present as low grade, early stage tumors with good prognosis and Type II which are high grade and usually advanced stage with poor outcomes, despite chemotherapy, even in early stage disease. Despite the well-documented clinico-pathologic differences between the two types, little is known about the genetic etiology of the discrepancies in behavior and outcomes between the two subtypes. The goal of this study was to identify distinctly expressed genes (DEG) in early stages vs. late stages in each of type I and II tumors as well as DEGs in ‘good’ vs. ‘poor’ tumor outcomes in each of the two types. Materials and Methods: A total of 24 EC tissue samples were collected (type I =12, type II =12). Six cases of each type were early stages and six were late stage. The cases were further categorized as having either ‘good’ prognosis, defined as the patient being alive with no evidence of disease at follow-up or ‘poor’ prognosis, with the patient having either disease progression, persistence, recurrence or death from disease at follow-up. RNA was extracted and human genome wide illumine bead microarrays carrying 48,000 genes was performed. Statistical analysis using the limma program in the R-based bioconductor package was used to calculate the level of gene differential expression of each comparison conducted. Results: Four separate comparisons based on patient characteristics were performed including: type I (late vs. early stage), type II (late vs. early), type I prognosis (‘good’ vs. ‘poor’), and type II (‘good’ vs. ‘poor’). Using the expression value of the identified DEGs we were able to accurately cluster patients into their respective clino-pathologic groups (late vs. early stages and ‘good’ vs. ‘poor’ prognosis). For type I tumors (late vs. early), 111 DEGs were identified (p<0.01). For type II tumors (late vs. early) 274 DEGs were identified (p<0.01). For prognosis (good vs. poor) there were 112 DEGs for type I and 135 for type II tumors (p<0.01 each). Furthermore, there was little overlap in the DEG sets between stages in type I and type II tumors (only 4 shared DEGs among the 111 and 274 DEGs, respectively). Furthermore, there was an insignificant overlap in DEG between good and poor prognosis in type I and type II (only 1 shared DEG among the 112 and 135, respectively). Remarkably, there was no overlap between the stage-derived DEGs and the prognosis-derived DEGs for each of type I and type II tumors. Conclusion: These data suggest that type I and type II tumors have very distinct gene expression signatures. We have shown the involvement of very distinct groups of genes in tumor progression (early vs. late stage) and that they are different from those involved in tumor outcome in type I and type II tumors. These genes might be important in tumor staging and predicting outcome. The evaluation of mRNA expression of some of these genes by RT-PCR affymetrix technique is in progress in our laboratory to identify which ones have value in determining pathogenesis and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-124.

Aim: Loss of balance can be seen in idiopathic Parkinson's disease (IPD) There are only a few studies in the literature in which brainstem involvement in IPD has been researched with neurophysiological tests such as vestibular evoked myogenic potential (VEMP). In this study, it was investigated whether there is a difference in the results of VEMP testing in early or late stage of IPD. Material and method: The IPD cases were classified as early stage and late stage according to the Hoehn-Yahr scale. The presence of a positive wave with a latency of P13 and a negative wave with a latency of N23 was investigated as the first reflex response The latencies of these potentials and the absolute amplitude of the P13-N23 component were measured. The VEMP results of the patients with early and late stage IPD were compared with those of the control group. Results: The right P13 latency mean value in the late stage patient group was significantly prolonged than in the early stage patient group and the control group. The right P13-N23 amplitude mean value of the late and early stage patient groups was significantly smaller than that of the control group (p < 0.002 and p < 0.001, respectively). Among the patients with IPD, the P13 latency was statistically increased in those with a fall history than in those without a fall history. In conclusion, this study indicates that the VEMP pathway is affected over time especially in patients with late stage Parkinson’s disease.

Yanshanian magmatisms are intensive in the southern Anhui Province and can be divided into early (152–137 Ma) and late (136–122 Ma) stages. A Yanshanian granitic zone was found to crop out along Qingshan to Changgai areas in the Tunxi district in Field investigation which has a genetic link with molybdenum multiple metal mineralization. To be a representative syenitic granite in the southern Anhui Province, the Huangshan pluton has not been found so far to have any genetic link with mineralization. Zircon LA-ICP-MS dating indicate that the four granitic bodies from the Qingshan-Changgai zone have concurrent formed ages from 140±4 to 141±2 Ma, belonging to the Yanshanian early stage magmatism. However, the Huangshan granite is dated to be 129±2 Ma, belonging to the Yanshanian late stage magmatism. The Qingshan-Changgai granites show high SiO2 and K2O contents, low P2O5 contents and middle Al2O3 contents and are high-K calc-alkaline series metaluminum I-type granite. These rocks are characterized by enrichments in the large ion lithophile elements and light rare earth elements (REE), depletions in the high field-strength elements, and middle degree negative anomalies of Eu, geochemical features of arc or continent crustal derived magma affinities. These rocks have 87Sr/86Sr( t ) ratios from 0.7120 to 0.7125, e Nd( t ) values from ‒7.24 to ‒4.38 and zircon e Hf( t ) values of ‒4.4 to 6.7, similar to that of the coeval ore-bearing granodiorites in the southern Anhui Province. Integrated geochemical studies indicate that the Yanshanian ore-bearing granodiorites were formed by partial melting of the Meso-Neoproterozoic accreted thickened low crust. Meanwhile, the Qingshan-Changgai granites were formed through a AFC process of plagioclase+amphibole+Shangxi Group of magmas that formed the ore-bearing granodiorites. The Huangshan granites are characterized by high SiO2 and K2O contents, moderate Al2O3 contents, seagull shape REE distributed pattern and distinct Eu negative abnormities. Comparing with the Qingshan-Changgai granites, the Huangshan granites show more Ba, Sr, P, and Ti negative abnormities with no Nb and Ta depletions and are high-K calc-alkaline series metaluminum A-type granite. e Hf( t ) values of the Huangshan granites are from ‒6.6 to ‒1.2, similar to that of the early stage ore-bearing granodiorites, indicating that they were also formed by anatexis of the Meso-Neoproterozoic accreted crust, but their magma sources might be residual granulitic crust which ever underwent Yanshanian early stage I-type intermediate-acid magma extraction. Comparing studies on the two stages granites indicate that the early stage granites derived from a relative thickened low crust under a lower temperature condition. Their magma sources were Meso-Neoproterozoic accreted crust which enriched in ore-forming materials and further became more enriched through processes of magma AFC evolution. However, the late stage A-type granites originated from relative shallow crust under a higher temperature condition. Their magma source was depleted in ore-forming materials due to the early stage magma extraction and thus had weak ore-forming capacity. From early to late stage, the magmatisms tectonic setting translated from post-orogenic to anorogenic and the later corresponded to a back-arc extensional setting as increase of the slab subducted angle of the Paleo-Pacific plate.

Introduction: The protein folding process is very sensitive to environmental conditions. Many possibilities in the form of numerous pathways for this process can-if an incorrect one is chosen-lead to the creation of forms described as misfolded. The aqueous environment is the natural one for the protein folding process. Nonetheless, other factors such as the cell membrane and the presence of specific molecules (chaperones) affect this process, ensuring the correct expected structural form to guarantee biological activity. All these factors can be considered components of the external force field for this process. Methods: The fuzzy oil drop-modified (FOD-M) model makes possible the quantitative evaluation of the modification of the external field, treating the aqueous environment as a reference. The FOD-M model (tested on membrane proteins) includes the component modifying the water environment, allowing the assessment of the external force field generated by prefoldin. Results: In this work, prefoldin was treated as the provider of a specific external force field for actin and tubulin. The discussed model can be applied to any folding process simulation, taking into account the changed external conditions. Hence, it can help simulate the in silico protein folding process under defined external conditions determined by the respective external force field. In this work, the structures of prefoldin and protein folded with the participation of prefoldin were analyzed. Discussion: Thus, the role of prefoldin can be treated as a provider of an external field comparable to other environmental factors affecting the protein folding process.

Investigated the contractile activity of 34 myocardium strips of right ventricular of 17 nonpregnant rats, including with estrogen (9) and progesterone (8) background, and 17 pregnant rats — at the early (5–10 days; n = 7), middle (11–18 days; n = 7) and late (19–21 days; n = 3) stages. Contractions caused by electrostimuls (5 ms, 20 B, 1 Hz), and their registration was performed using the force transducer. Adrenoreactivity of strips were evaluated by the pattern of inotropic and batmotropic effects of adrenaline (10-9–10-5 g/ml). It has been established that contractility of myocardium is not changed during the estrous cycle. In pregnancy, it does not increase, and even decreases, which is typical for middle and late stages. Adrenoreactivity of myocardium, according to the inotropic effect of adrenaline, did not depend on the phase of the estrous cycle (PhEC), but depended on the presence of pregnancy — in early stages it remained relatively high, and in the middle and late stages — decreased. Excitability of intactmyocardium did not depend on PhEC and the presence of pregnancy. Adrenaline showed positive batmotropic effect. Its expression was dependent on the PhEC (it decreases with the dominance of progesterone background) and the presence of pregnancy (in early stage it increases, and in the middle and late stages — it decreases). It is assumed that changes of adrenoreactivity of the myocardium of the rat right ventricle during pregnancy are due by increasing of the expression of alpha1-, beta2- and beta3-adrenoceptors. Refs 58. Figs 5. Table 1.