Mathematical modelling of tumour response in primary breast cancer.

Although breast cancer is perceived to be relatively chemosensitive, cytotoxic drug therapy only leads to cure in the adjuvant setting. In advanced disease, primary resistance and inadequate cell kill may be important in determining the lack of a durable response to cytotoxics, but for an individual patient's tumour there is no consistent way of determining the importance of these two factors. An adaptation of Skipper's log cell kill model of tumour response to chemotherapy was applied to serial tumour measurements of 46 locally advanced primary breast carcinomas undergoing neoadjuvant chemotherapy. Assuming a log-normal distribution of errors in the clinically measured volumes, the model produced, for each tumour separately, in vivo estimates of proportional cell kill, initial resistance and tumour doubling times during therapy. After 4 weeks' treatment, these data could then be used to predict subsequent tumour volumes with good accuracy. In addition, for the 13 tumours that became operable after the neoadjuvant chemotherapy, there was a significant association between the final volume as predicted by the model and the final pathological volume (P < 0.05). This approach could be usefully employed to determine those tumours that are primarily resistant to the treatment regimen, permitting changes of therapy to more effective drugs at a time when the tumour is clinically responding but destined to progress

Analysis of the role of PCNA-DNA contacts during clamp loading

<p>Abstract</p> <p>Background</p> <p>Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.</p> <p>Results</p> <p>We built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 Å resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.</p> <p>Conclusion</p> <p>Mutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.</p

Basic linker of macroH2A stabilizes DNA at the entry/exit site of the nucleosome

PMC3458575MacroH2A is a histone H2A variant that is typically found in heterochromatic regions of the genome. A positively charged linker that connects the histone-fold with the macro-domain was suggested to have DNA-binding properties, and has been shown to promote oligomerization of chromatin fibers. Here we examine the influence of this basic linker on DNA of mononucleosomes. We find that the macro-linker reduces accessibility to extranucleosomal DNA, and appears to increase compaction of the nucleosome. These properties arise from interactions between the H1-like basic linker region and DNA around the entry/exit site, which increases protection of nucleosomal DNA from exonuclease III digestion by 10 bp. By stabilizing the wrapping of DNA around the histone core, this basic linker of macroH2A may alter the distribution of nucleosome-associated factors, and potentially contribute to the more compacted nature of heterochromatin.JH Libraries Open Access Fun

Microarray-based resequencing of multiple Bacillus anthracis isolates

We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication (discrepancy rate of 7.4 × 10(-7)) and by comparison to independently generated shotgun sequence (discrepancy rate < 2.5 × 10(-6)). Population genomics studies of microbial pathogens using rapid resequencing technologies such as resequencing arrays are critical for recognizing newly emerging or genetically engineered strains

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer\u27s disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein

Rapid, energy-efficient synthesis of the layered carbide, Al₄C₃

The phase-pure binary aluminium carbide, Al4C3 can be synthesised in vacuo from the elements in 30 minutes via microwave heating in a multimode cavity reactor. The success of the reaction is dependent on the use of finely divided aluminium and graphite starting materials, both of which couple effectively to the microwave field. The yellow-brown powder product was characterised by powder X-ray diffraction, scanning electron microscopy/energy dispersive X-ray spectroscopy thermogravimetric-differential thermal analysis and Raman spectroscopy. Powders were composed of hexagonal single crystallites tens of microns in diameter (rhombohedral space group R[3 with combining macron]m; Z = 3; a = 3.33813(5) Å, c = 25.0021(4) Å) and were stable to 1000 °C in air, argon and nitrogen. Equivalent microwave reactions of the elements in air led to the formation of the oxycarbide phases Al2OC and Al4O4C

Naturally occurring genetic variants in the oxytocin receptor alter receptor signaling profiles

The hormone oxytocin is commonly administered during childbirth to initiate and strengthen uterine contractions and prevent postpartum hemorrhage. However, patients have wide variation in the oxytocin dose required for a clinical response. To begin to uncover the mechanisms underlying this variability, we screened the 11 most prevalent missense genetic variants in the oxytocin receptor