Clinical validation of droplet digital PCR assays in detecting BRAF-mutant circulating tumour DNA as a prognostic biomarker in patients with resected stage III melanoma receiving adjuvant therapy (COMBI-AD): a biomarker analysis from a double-blind, randomised phase 3 trial.

Background: Cell-free, circulating tumour DNA (ctDNA) is an established measure of minimal residual disease; however, it is not utilised in melanoma management. We investigated whether ctDNA measurements could predict survival outcomes during adjuvant targeted therapy or placebo treatment in stage III melanoma, thereby identifying patients at high risk and low risk of recurrence.

Methods: Analytically validated mutation-specific droplet digital PCR assays were used to measure BRAF or BRAF ctDNA in patients aged 18 years or older who were enrolled in the COMBI-AD trial, which was a double-blind, randomised, phase 3 study of oral dabrafenib (150 mg twice daily) plus oral trametinib (2 mg once daily) combination therapy versus two matched placebos in resected BRAF-mutant stage III melanoma.

Use of Microcomputed Tomography to Measure the Relaxin-induced Expansion of Intrapubic Ligaments in Mice.

Relaxin is a 6-kDa peptide in the insulin superfamily of hormones. In addition to its effects on reproductive and musculoskeletal ligaments, relaxin has demonstrated beneficial effects on cardiac, renal, and vascular systems in preclinical models. The mouse intrapubic ligament ex vivo bioassay is the current standard for measuring in vivo relaxin bioactivity.

Relaxin and Matrix Metalloproteinase-9 in Angiotensin II-Induced Abdominal Aortic Aneurysms.

Background: This study determined whether relaxin or matrix metalloproteinase (MMP)-9 influences angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA).

Methods and results: Male C57BL/6 or apolipoprotein Emice were infused with AngII with or without relaxin. Relaxin did not influence AngII-induced AAA in either mouse strain.

Chronic Inhibition of Renal Outer Medullary Potassium Channel Not Only Prevented but Also Reversed Development of Hypertension and End-Organ Damage in Dahl Salt-Sensitive Rats.

The renal outer medullary potassium (ROMK) channel mediates potassium recycling and facilitates sodium reabsorption through the Na/K/2Cl cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Evidence from the phenotype of humans and rodents with functional ROMK deficiency supports the contention that selective ROMK inhibitors (ROMKi) will represent a novel diuretic with potential of therapeutic benefit for hypertension. ROMKi have recently been synthesized by Merck & Co, Inc.

Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure.

The renal outer medullary potassium channel (ROMK, KCNJ1) mediates potassium recycling and facilitates sodium reabsorption through the Na(+)/K(+)/2Cl(-) cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome.

Prevention of experimental colitis by a selective inhibitor of the immunoproteasome.

The proteasome, a multicatalytic protease, is responsible for the degradation of intracellular proteins. Stimulation of cells with inflammatory cytokines, such as IFN-gamma, leads to the replacement of the constitutive catalytic proteasome subunits by the inducible subunits low molecular mass polypeptide (LMP)2 (beta1i), multicatalytic endopeptidase complex-like-1 (beta2i), and LMP7 (beta5i), which are required for the production of certain MHC class I-restricted T cell epitopes. In this study, we investigated the effect of immunoproteasomes on the development of dextran sulfate sodium-induced colitis.

Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047).

Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents.

Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome.

Clinical studies with bortezomib have validated the proteasome as a therapeutic target for the treatment of multiple myeloma and non-Hodgkin's lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing. Here we describe the antitumor activity of PR-171, a novel epoxyketone-based irreversible proteasome inhibitor that is currently in clinical development.

Non-viral delivery of nuclear factor-kappaB decoy ameliorates murine inflammatory bowel disease and restores tissue homeostasis.

Background: Nuclear factor-kappaB (NF-kappaB) is a key transcriptional regulator of inflammatory bowel disease (IBD).

Aim: To investigate the therapeutic potential of a locally administered "non-viral" nuclear factor-kappaB decoy (NFkappaBD) in multiple experimental models of IBD.

Methods: A fully phosphorothioated decoy oligonucleotide with improved stability that specifically binds NF-kappaB and blocks inflammatory mediators regulated by this transcription factor without the help of viral envelope-assisted delivery was developed.

Blockade of experimental atopic dermatitis via topical NF-kappaB decoy oligonucleotide.

Atopic dermatitis (AD) is a common chronic skin inflammatory disease. Long-term use of topical corticosteroids in skin inflammation poses risks of systemic and local side effects. The NF-kappaB transcription factor family plays a central role in the progression and maintenance of AD.

Method for analyzing signaling networks in complex cellular systems.

Now that the human genome has been sequenced, the challenge of assigning function to human genes has become acute. Existing approaches using microarrays or proteomics frequently generate very large volumes of data not directly related to biological function, making interpretation difficult. Here, we describe a technique for integrative systems biology in which: (i) primary cells are cultured under biologically meaningful conditions; (ii) a limited number of biologically meaningful readouts are measured; and (iii) the results obtained under several different conditions are combined for analysis.

NF-kappaB blockade and oncogenic Ras trigger invasive human epidermal neoplasia.

The nuclear factor NF-kappaB and oncogenic Ras can alter proliferation in epidermis, the most common site of human cancer. These proteins are implicated in epidermal squamous cell carcinoma in mice, however, the potential effects of altering their function are uncertain. Whereas inhibition of NF-kappaB enhances apoptosis in certain tumours, blockade of NF-kappaB predisposes murine skin to squamous cell carcinoma.

Inducible activation of Ras and Raf in adult epidermis.

Ras effects vary with developmental setting, with oncogenic RAS activation implicated in epithelial carcinogenesis. In epidermal cells, previous studies described conflicting Ras impacts on growth and differentiation, with the only in vivo studies relying on constitutive alterations of Ras function throughout development. To study Ras effects in developmentally mature adult epidermis, we expressed a 4-hydroxytamoxifen (4OHT)-regulated Ras fusion in transgenic mice using the keratin 14 promoter.

CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis.

Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase.

Epidermal Ras blockade demonstrates spatially localized Ras promotion of proliferation and inhibition of differentiation.

While important in carcinogenesis, the role of Ras in normal self-renewing tissues such as epidermis is unclear. To address this, we altered Ras function in undifferentiated and differentiating epidermal layers. Ras blockade within undifferentiated basal epidermal cells leads to decreased integrin expression, diminished growth capacity and induction of differentiation.