Thermodynamics and Kinetics of Single-Chain Monellin Folding with Structural Insights into Specific Collapse in the Denatured State Ensemble

Proteins, which behave as random coils in high denaturant concentrations undergo collapse transition similar to polymers on denaturant dilution. We study collapse in the denatured ensemble of single-chain monellin (MNEI) using a coarse-grained protein model and molecular dynamics simulations. The model is validated by quantitatively comparing the computed guanidinium chloride and pH-dependent thermodynamic properties of MNEI folding with the experiments. The computed properties such as the fraction of the protein in the folded state and radius of gyration (R_g) as function of [GuHCl] are in good agreement with the experiments. The folded state of MNEI is destabilized with an increase in pH due to the deprotonation of the residues Glu24 and Cys42. On decreasing [GuHCl], the protein in the unfolded ensemble showed specific compaction. The R_g of the protein decreased steadily with [GuHCl] dilution due to increase in the number of native contacts in all the secondary structural elements present in the protein. MNEI folding kinetics is complex with multiple folding pathways and transiently stable intermediates are populated in these pathways. In strong stabilizing conditions, the protein in the unfolded ensemble showed transition to a more compact unfolded state where R_g decreased by ≈ 17% due to the formation of specific native contacts in the protein. The intermediate populated in the dominant MNEI folding pathway satisfies the structural features of the dry molten globule inferred from experiments.     

A 16-Gene Signature Distinguishes Anaplastic Astrocytoma from Glioblastoma

Anaplastic astrocytoma (AA; Grade III) and glioblastoma (GBM; Grade IV) are diffusely infiltrating tumors and are called malignant astrocytomas. The treatment regimen and prognosis are distinctly different between anaplastic astrocytoma and glioblastoma patients. Although histopathology based current grading system is well accepted and largely reproducible, intratumoral histologic variations often lead to difficulties in classification of malignant astrocytoma samples. In order to obtain a more robust molecular classifier, we analysed RT-qPCR expression data of 175 differentially regulated genes across astrocytoma using Prediction Analysis of Microarrays (PAM) and found the most discriminatory 16-gene expression signature for the classification of anaplastic astrocytoma and glioblastoma. The 16-gene signature obtained in the training set was validated in the test set with diagnostic accuracy of 89%. Additionally, validation of the 16-gene signature in multiple independent cohorts revealed that the signature predicted anaplastic astrocytoma and glioblastoma samples with accuracy rates of 99%, 88%, and 92% in TCGA, {"type":"entrez-geo","attrs":{"text":"GSE1993","term_id":"1993"}}GSE1993 and {"type":"entrez-geo","attrs":{"text":"GSE4422","term_id":"4422"}}GSE4422 datasets, respectively. The protein-protein interaction network and pathway analysis suggested that the 16-genes of the signature identified epithelial-mesenchymal transition (EMT) pathway as the most differentially regulated pathway in glioblastoma compared to anaplastic astrocytoma. In addition to identifying 16 gene classification signature, we also demonstrated that genes involved in epithelial-mesenchymal transition may play an important role in distinguishing glioblastoma from anaplastic astrocytoma.     

Effects of ionizing radiation on cell cycle progression

Irradiation of normal eukaryotic cells results in delayed progression through the G1, S, and G2 phases of the cell cycle. The G1 arrest is regulated by the p53 tumor suppressor gene product. Irradiation results in increased expression of p53, which in turn induces a 21 kDa protein, WAF 1/Cip 1, that inhibits cyclin CDK kinases. S-phase delay is observed after relatively high doses of radiation. This delay has both radiosensitive and radioresistant components, corresponding to inhibition of DNA replicon initiation and DNA chain elongation, respectively. The mechanism for this delay is as yet undefined, but the extent of the delay appears to be under genetic control and is sensitive to the kinase inhibitor staurosporine. A delay in G2 has been demonstrated in virtually all eukaryotic cells examined in response to irradiation. Our studies have focused on the mechanisms responsible for this delay. Cyclin B1 and p34^cdc2 are cell cycle control proteins that together form a kinase complex required for passage through G2 and mitosis [22]. Control of radiation-induced G2 delay is likely therefore to involve modulation of cyclin B1/p34^cdc2 activity. We have shown in HeLa cells that cyclin B1 expression is decreased in a dose-dependent manner following irradiation. This decrease is controlled at both the level of mRNA and protein accumulation. We have also shown that radiation-sensitive rat embryo fibroblast lines (REF) immortalized with v- or c-myc display a minimal G2 delay when compared to radiation resistant cells transformed with v-myc + H-ras. These REF lines respond to irradiation with a decrease in cyclin B mRNA, which parallels the extent of their respective G2 delays. The duration of the G2 delay in radiation-resistant REF can be shortened by treatment with low doses of the kinase inhibitor staurosporine. We have also been able to markedly reduce the radiation-induced G2 delay in HeLa cells using either staurosporine or caffeine. Attenuation of the G2 delay is accompanied by reversal of the radiation-induced inhibition of cyclin B mRNA accumulation. The results of these studies are consistent with the hypothesis that reduced expression of cyclin B in response to radiation is in part responsible for the G2 delay. The duration of the G2 delay may also be influenced by the activation state of the cyclin B/p34_cdc2 complex.Invited paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Efficient Semiparametric Marginal Estimation for the Partially Linear Additive Model for Longitudinal/Clustered Data

We consider the efficient estimation of a regression parameter in a partially linear additive nonparametric regression model from repeated measures data when the covariates are multivariate. To date, while there is some literature in the scalar covariate case, the problem has not been addressed in the multivariate additive model case. Ours represents a first contribution in this direction. As part of this work, we first describe the behavior of nonparametric estimators for additive models with repeated measures when the underlying model is not additive. These results are critical when one considers variants of the basic additive model. We apply them to the partially linear additive repeated-measures model, deriving an explicit consistent estimator of the parametric component; if the errors are in addition Gaussian, the estimator is semiparametric efficient. We also apply our basic methods to a unique testing problem that arises in genetic epidemiology; in combination with a projection argument we develop an efficient and easily computed testing scheme. Simulations and an empirical example from nutritional epidemiology illustrate our methods.     

Effect of envenomation on enzymes of brain of albino rats

Effects of high doses of cobra venom, (150 micrograms/120 +/- 20 g body wt) and viper venom (300 micrograms/120 +/- 20 g body wt) on aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), acetylcholinesterase (ACh) and alkaline phosphatase (ALP) of brain of albino rats were studied. While AST, LDH, ACh and ALP activities increased in both viper and cobra venom treated rats, ALT decreased in both groups compared to control.     

The disappearing seasonality of autism conceptions in california

Background

Autism incidence and prevalence have increased dramatically in the last two decades. The autism caseload in California increased 600% between 1992 and 2006, yet there is little consensus as to the cause. Studying the seasonality of conceptions of children later diagnosed with autism may yield clues to potential etiological drivers.

Objective

To assess if the conceptions of children later diagnosed with autism cluster temporally in a systematic manner and whether any pattern of temporal clustering persists over time.

Method

We searched for seasonality in conceptions of children later diagnosed with autism by applying a one-dimensional scan statistic with adaptive temporal windows on case and control population data from California for 1992 through 2000. We tested for potential confounding effects from known risk factors using logistic regression models.

Results

There is a consistent but decreasing seasonal pattern in the risk of conceiving a child later diagnosed with autism in November for the first half of the study period. Temporal clustering of autism conceptions is not an artifact of composition with respect to known risk factors for autism such as socio-economic status.

Conclusion

There is some evidence of seasonality in the risk of conceiving a child later diagnosed with autism. Searches for environmental factors related to autism should allow for the possibility of risk factors or etiological drivers that are seasonally patterned and that appear and remain salient for a discrete number of years.     

Mutational and Structural Analysis of Conserved Residues in Ribose-5-Phosphate Isomerase B from Leishmania donovani: Role in Substrate Recognition and Conformational Stability

Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB.     

Coherent Motion of Monolayer Sheets under Confinement and Its Pathological Implications

Coherent angular rotation of epithelial cells is thought to contribute to many vital physiological processes including tissue morphogenesis and glandular formation. However, factors regulating this motion, and the implications of this motion if perturbed, remain incompletely understood. In the current study, we address these questions using a cell-center based model in which cells are polarized, motile, and interact with the neighboring cells via harmonic forces. We demonstrate that, a simple evolution rule in which the polarization of any cell tends to orient with its velocity vector can induce coherent motion in geometrically confined environments. In addition to recapitulating coherent rotational motion observed in experiments, our results also show the presence of radial movements and tissue behavior that can vary between solid-like and fluid-like. We show that the pattern of coherent motion is dictated by the combination of different physical parameters including number density, cell motility, system size, bulk cell stiffness and stiffness of cell-cell adhesions. We further observe that perturbations in the form of cell division can induce a reversal in the direction of motion when cell division occurs synchronously. Moreover, when the confinement is removed, we see that the existing coherent motion leads to cell scattering, with bulk cell stiffness and stiffness of cell-cell contacts dictating the invasion pattern. In summary, our study provides an in-depth understanding of the origin of coherent rotation in confined tissues, and extracts useful insights into the influence of various physical parameters on the pattern of such movements.     

VEGF: a modifier of the del22q11 (DiGeorge) syndrome?

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.