Mcl-1 and Bcl-x sequestration of Bak confers differential resistance to BH3-only proteins.

The prosurvival Bcl-2 family proteins Mcl-1 and Bcl-x inhibit apoptosis by sequestering BH3-only proteins such as Bid and Bim (MODE 1) or the effector proteins Bak and Bax (MODE 2). To better understand the contributions of MODE 1 and MODE 2 in blocking cell death, and thus how to bypass resistance to cell death, we examined prescribed mixtures of Bcl-2 family proteins. In a Bim and Bak mixture, Bcl-x and Mcl-1 each sequestered not only Bim but also Bak as it became activated by Bim.

BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove.

BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear.

Epigenetic control of mitochondrial cell death through PACS1-mediated regulation of BAX/BAK oligomerization.

PCAF and ADA3 associate within the same macromolecular complexes to control the transcription of many genes, including some that regulate apoptosis. Here we show that PCAF and ADA3 regulate the expression of PACS1, whose protein product is a key component of the machinery that sorts proteins among the trans-Golgi network and the endosomal compartment. We describe a novel role for PACS1 as a regulator of the intrinsic pathway of apoptosis and mitochondrial outer membrane permeabilization.

Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies.

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2-α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1-α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release.

Rapid, Multiplexed Phosphoprotein Profiling Using Silicon Photonic Sensor Arrays.

Extracellular signaling is commonly mediated through post-translational protein modifications that propagate messages from membrane-bound receptors to ultimately regulate gene expression. Signaling cascades are ubiquitously intertwined, and a full understanding of function can only be gleaned by observing dynamics across multiple key signaling nodes. Importantly, targets within signaling cascades often represent opportunities for therapeutic development or can serve as diagnostic biomarkers.

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis.

During apoptosis, Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes. Here, we characterize those changes using 11 antibodies that were epitope mapped using peptide arrays and mutagenesis. After Bak activation by Bid, epitopes throughout the α1 helix are exposed indicating complete dissociation of α1 from α2 in the core and from α6-α8 in the latch.

Photopatterning of vascular endothelial growth factor within collagen-glycosaminoglycan scaffolds can induce a spatially confined response in human umbilical vein endothelial cells.

Biomolecular signals within the native extracellular matrix are complex, with bioactive factors found in both soluble and sequestered states. In the design of biomaterials for tissue engineering applications it is increasingly clear that new approaches are required to locally tailor the biomolecular environment surrounding cells within the matrix. One area of particular focus is strategies to improve the speed or quality of vascular ingrowth and remodeling.

Collagen scaffold arrays for combinatorial screening of biophysical and biochemical regulators of cell behavior.

Arrays of 3D macroporous collagen scaffolds with orthogonal gradations of structural and biomolecular cues are described. Gradient maker technology is applied to create linear biomolecular gradients within microstructurally distinct sections of a single CG scaffold array. The array set up is used to explore cell behaviors including proliferation and regulation of stem cell fate.

Fas ligand-mediated immune surveillance by T cells is essential for the control of spontaneous B cell lymphomas.

Loss of function of the tumor suppressor gene PRDM1 (also known as BLIMP1) or deregulated expression of the oncogene BCL6 occurs in a large proportion of diffuse large B cell lymphoma (DLBCL) cases. However, targeted mutation of either gene in mice leads to only slow and infrequent development of malignant lymphoma, and despite frequent mutation of BCL6 in activated B cells of healthy individuals, lymphoma development is rare. Here we show that T cells prevent the development of overt lymphoma in mice caused by Blimp1 deficiency or overexpression of Bcl6 in the B cell lineage.

Reconstruction of the ancestral marsupial karyotype from comparative gene maps.

Background: The increasing number of assembled mammalian genomes makes it possible to compare genome organisation across mammalian lineages and reconstruct chromosomes of the ancestral marsupial and therian (marsupial and eutherian) mammals. However, the reconstruction of ancestral genomes requires genome assemblies to be anchored to chromosomes. The recently sequenced tammar wallaby (Macropus eugenii) genome was assembled into over 300,000 contigs.

Cyclin E facilitates dysplastic hepatocytes to bypass G1/S checkpoint in hepatocarcinogenesis.

Background And Aim: By array-comparative genomic hybridization, we demonstrated cyclin E as one of seven genes associated with hepatocellular carcinoma (HCC) development in Ku70 DNA repair-deficient mice. We therefore explored the hypothesis that during hepatocarcinogenesis, cyclin E kinase can overcome the inhibitory effects of p53 and establish whether abnormal miRNA(mi-R)-34, a co-regulator of cyclin E and p53, can account for their interactions as "drivers" of HCC.

Methods: Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3-12 months.

Bak apoptotic function is not directly regulated by phosphorylation.

During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation.

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii).

Background: The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species.

Deciphering the molecular events necessary for synergistic tumor cell apoptosis mediated by the histone deacetylase inhibitor vorinostat and the BH3 mimetic ABT-737.

The concept of personalized anticancer therapy is based on the use of targeted therapeutics through in-depth knowledge of the molecular mechanisms of action of these agents when used alone and in combination. We have identified the apoptotic proteins and pathways necessary for synergistic tumor cell apoptosis and in vivo antitumor responses seen when the HDAC inhibitor vorinostat is combined with the BH3-mimetic ABT-737 in lymphomas overexpressing Bcl-2. Vorinostat "primes" tumors overexpressing Bcl-2 for rapid ABT-737-mediated apoptosis by inducing expression of the BH3-only gene bmf.

Collaborative working in end-of-life care: developing a guide for health and social care professionals.

This article describes a project to develop collaborative working between palliative care nurse specialists and community matrons for patients with a non-cancer diagnosis. Pathways to clarify decision-making in end-of-life care were created as part of the project and were subsequently developed into a guide for use by health or social care professionals caring for any patient, irrespective of diagnosis. The guide is designed to facilitate best practice in end-of-life care, by identifying the key questions which need to be addressed and the appropriate responses at different stages of the patient journey.

Defining the target specificity of ABT-737 and synergistic antitumor activities in combination with histone deacetylase inhibitors.

The apoptotic and therapeutic activities of the histone deacetylase inhibitor (HDACi) vorinostat are blocked by overexpression of Bcl-2 or Bcl-X(L). Herein, we used the small molecule inhibitor ABT-737 to restore sensitivity of Emu-myc lymphomas overexpressing Bcl-2 or Bcl-X(L) to vorinostat and valproic acid (VPA). Combining low-dose ABT-737 with vorinostat or VPA resulted in synergistic apoptosis of these cells.

Defensins and the convergent evolution of platypus and reptile venom genes.

When the platypus (Ornithorhynchus anatinus) was first discovered, it was thought to be a taxidermist's hoax, as it has a blend of mammalian and reptilian features. It is a most remarkable mammal, not only because it lays eggs but also because it is venomous. Rather than delivering venom through a bite, as do snakes and shrews, male platypuses have venomous spurs on each hind leg.

Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes.

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination.

Homozygous deletions may be markers of nearby heterozygous mutations: The complex deletion at FRA16D in the HCT116 colon cancer cell line removes exons of WWOX.

Homozygous deletions in cancer cells have been thought to harbor tumor suppressor genes. We show that the 25 and 50 kb homozygous deletions in WWOX in the colon cancer cell line HCT116 result from a complex set of heterozygous deletions, some of which overlap to give homozygous loss. One of the heterozygous deletions has removed exons 6-8 of one allele of WWOX, and there is also a third copy of the distal region of WWOX in an unbalanced translocation.

Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus).

Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes.