Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.     

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.     

Biomimetic nanoparticles for siRNA delivery in the treatment of leukaemia

Leukaemia is a bone marrow cancer occurring in acute and chronic subtypes. Acute leukaemia is a rapidly fatal cancer potentially causing death within a few weeks, if untreated. Leukaemia arises as a result of disruption to haematopoietic precursors, caused either by acquired gene fusions, gene mutations or inappropriate expression of the relevant oncogenes. Current treatment options have made significant progress, but the 5 year survival for acute leukaemia remains under 10% in elderly patients, and less than 50% for some types of acute leukaemia in younger adults. For chronic leukaemias longer survival is generally expected and for chronic myeloid leukaemia patients on tyrosine kinase inhibitors the median survival is not yet reached and is expected to exceed 10 years. Chemotherapy and haematopoietic stem cell transplantation (HSCT) for acute leukaemia provide the mainstay of therapy for patients under 65 and both carry significant morbidity and mortality. Alternative and superior therapeutic strategies for acute leukaemias are urgently required. Recent molecular-based knowledge of recurring chromosome rearrangements, in particular translocations and inversions, has resulted in significant advances in understanding the molecular pathogenesis of leukaemia. Identification of a number of unique fusion genes has facilitated the development of highly specific small interfering RNAs (siRNA). Although delivery of siRNA using multifunctional nanoparticles has been investigated to treat solid cancers, the application of this approach to blood cancers is at an early stage. This review describes current treatments for leukaemia and highlights the potential of leukaemic fusion genes as therapeutic targets for RNA interference (RNAi). In addition, the design of biomimetic nanoparticles which are capable of responding to the physiological environment of leukaemia and their potential to advance RNAi therapeutics to the clinic will be critically evaluated.     

Divide and Conquer: Sub-Grouping of ASD Improves ASD Detection Based on Brain Morphometry

Low success (<60%) in autism spectrum disorder (ASD) classification using brain morphometry from the large multi-site ABIDE dataset and inconsistent findings on brain morphometric abnormalities in ASD can be attributed to the ASD heterogeneity. In this study, we show that ASD brain morphometry is highly heterogeneous, and demonstrate that the heterogeneity can be mitigated and classification improved if autism severity (AS), verbal IQ (VIQ) and age are used with morphometric features. Morphometric features from structural MRIs (sMRIs) of 734 males (ASD: 361, controls: 373) of ABIDE were derived using FreeSurfer. Applying the Random Forest classifier, an AUC of 0.61 was achieved. Adding VIQ and age to morphometric features, AUC improved to 0.68. Sub-grouping the subjects by AS, VIQ and age improved the classification with the highest AUC of 0.8 in the moderate-AS sub-group (AS = 7–8). Matching subjects on age and/or VIQ in each sub-group further improved the classification with the highest AUC of 0.92 in the low AS sub-group (AS = 4–5). AUC decreased with AS and VIQ, and was the lowest in the mid-age sub-group (13–18 years). The important features were mainly from the frontal, temporal, ventricular, right hippocampal and left amygdala regions. However, they highly varied with AS, VIQ and age. The curvature and folding index features from frontal, temporal, lingual and insular regions were dominant in younger subjects suggesting their importance for early detection. When the experiments were repeated using the Gradient Boosting classifier similar results were obtained. Our findings suggest that identifying brain biomarkers in sub-groups of ASD can yield more robust and insightful results than searching across the whole spectrum. Further, it may allow identification of sub-group specific brain biomarkers that are optimized for early detection and monitoring, increasing the utility of sMRI as an important tool for early detection of ASD.     

Desiccation as a mitigation tool to manage biofouling risks: trials on temperate taxa to elucidate factors influencing mortality rates

The desiccation tolerance of biofouling taxa (adults and early life-stages) was determined under both controlled and ‘realistic’ field conditions. Adults of the ascidian Ciona spp. died within 24 h. Mortality in the adult blue mussel Mytilus galloprovincialis occurred within 11 d under controlled conditions, compared with 7 d when held outside. The Pacific oyster Crassostrea gigas was the most desiccation-tolerant taxon tested (up to 34 d under controlled conditions). Biofouling orientated to direct sunlight showed faster mortality rates for all the taxa tested. Mortality in Mytilus juveniles took up to 24 h, compared with 8 h for Ciona, with greater survival at the higher temperature (18.5°C) and humidity (~95% RH) treatment combination. This study demonstrated that desiccation can be an effective mitigation method for a broad range of fouling taxa, especially their early life-stages. Further work is necessary to assess risks from other high-risk species such as algae and cyst forming species.     

Specification, annotation, visualization and simulation of a large rule-based model for ERBB receptor signaling

ABSTRACT: BACKGROUND: Mathematical/computational models are needed to understand cell signaling networks, which are complex. Signaling proteins contain multiple functional components and multiple sites of post-translational modification. The multiplicity of components and sites of modification ensures that interactions among signaling proteins have the potential to generate myriad protein complexes and post-translational modification states. As a result, the number of chemical species that can be populated in a cell signaling network, and hence the number of equations in an ordinary differential equation model required to capture the dynamics of these species, is prohibitively large. To overcome this problem, the rule-based modeling approach has been developed for representing interactions within signaling networks efficiently and compactly through coarse-graining of the chemical kinetics of molecular interactions. RESULTS: Here, we provide a demonstration that the rule-based modeling approach can be used to specify and simulate a large model for ERBB receptor signaling that accounts for site-specific details of protein-protein interactions. The model is considered large because it corresponds to a reaction network containing more reactions than can be practically enumerated. The model encompasses activation of ERK and Akt, and it can be simulated using a network-free simulator, such as NFsim, to generate time courses of phosphorylation for 55 individual serine, threonine, and tyrosine residues. The model is annotated and visualized in the form of an extended contact map. CONCLUSIONS: With the development of software that implements novel computational methods for calculating the dynamics of large-scale rule-based representations of cellular signaling networks, it is now possible to build and analyze models that include a significant fraction of the protein interactions that comprise a signaling network, with incorporation of the site-specific details of the interactions. Modeling at this level of detail is important for understanding cellular signaling.