Differences in individual responses to drug treatment are generally assumed to have a strong genetic component but identifying individuals who may be at higher risk of adverse reactions from studies conducted entirely in people is fraught with difficulty. Looking at variations in susceptibility to paracetamol (acetaminophen)-induced hepatotoxicity, a team led by researchers at North Carolina State University has now shown how inbred mouse strains can be used to model genetic diversity in human populations.

Paracetamol is a widely available over-the-counter treatment for pain and fevers. Although generally considered safe at recommended doses, paracetamol has a narrow therapeutic index and overdoses can cause potentially fatal liver failure. For a significant number of people, even the recommended dose can cause serious liver damage and recent studies have shown that relatively short term use of paracetamol leads to increased levels of alanine transaminase (ALT) in about a third of healthy individuals. These individuals may be at increased risk of liver injury from high doses of paracetamol and the team have now identified a genetic marker linked to the risk of paracetamol-induced liver damage. Using 36 different strains of mice with well characterised genetic differences, the team were able to link specific genes to liver damage following paracetamol treatment. When they sequenced the corresponding genes in people who showed an increase in ALT after taking paracetamol, they found that a variation in one of the candidate genes, CD44, was significantly associated with elevated ALT levels.

Hepatotoxicity following paracetamol overdose is attributed not to the drug itself but to a minor alkylating metabolite, N-acetyl-p-benzoquinone imine (NARQI). NARQI is formed primarily by the action of cytochrome P450 enzymes, and differences in susceptibility to paracetamol poisoning have previously been linked to polymorphisms in P450 genes. CD44 is a cell surface glycoprotein involved in cell/cell and cell/matrix interactions and, although its role in liver toxicity is not yet understood, it could serve as a useful marker to identify people at high risk of paracetamol-induced liver damage. The team believe that routine use of genetic differences in early safety testing will provide more accurate predictions of clinical responses. The study is published in full in the journal Genome Research.

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Researchers from University of Rochester Medical Center have shown that eliminating the gene for cyclophilin A completely protects mice from developing abdominal aortic aneurysms, a late stage complication of atherosclerosis. An aortic aneurysm is a thin, weakened section of the aorta which can rupture, leading to massive internal blood loss and death. Aneurysms occur most frequently in the abdominal section of the aorta and cause around 15,000 deaths a year, most in older men. In abdominal aortic aneurysm, angiotensin II is known to stimulate oxidative stress in blood vessels leading to increased activity of matrix metalloproteinases which, in turn, degrade the matrix structure of the vessel wall. Increased activity of matrix metalloproteinases also plays a role in atherosclerosis, allowing smooth muscle cells from the blood vessel walls to contribute to the development of plaques. In both abdominal aortic aneurysm and atherosclerosis, angiotensin II also contributes to local inflammation by recruiting immune cells to the blood vessel wall.

Using genetically modified mice, cyclophilin A was found to promote all three events involved in angiotensin II mediated damage to blood vessels – oxidative stress, matrix degradation and inflammation. Cyclophilin A is highly expressed in vascular smooth muscle cells and studies showed that both intracellular and extracellular cyclophilin A are required for generation of reactive oxygen species and activation of matrix metalloproteinases. The team is hoping to develop anti-cyclophilin A drugs that will reduce the processes underlying cardiovascular diseases such as abdominal aortic aneurysm, atherosclerosis and hypertension. The study is published in full in the journal Nature Medicine.

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Although many studies have shown the potential for gene silencing using short interfering RNA (siRNA), a major hurdle to the therapeutic use of the technique has been the lack of effective delivery systems. Writing in the journal Nature, researchers at the University of Massachusetts Medical School now report a method of delivering siRNA to specific cell types following oral administration. The researchers exploited a characteristic of macrophages – the ability to engulf yeast particles – to deliver the siRNA. Macrophages are attractive targets for RNA interference therapy since they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Yeast particles were treated to remove components that would elicit an immune response to give β-1,3-D glucan, a polysaccharide formed from D-glucose, as the delivery vehicle. Oral delivery of β-1,3-D glucan-encapsulated siRNA particles containing as little as 20 µg kg^-1 siRNA directed against tumour necrosis factor α (TNF-α) depleted messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum TNF-α levels. The technique was also used to identify the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) as a previously unknown mediator of cytokine expression. Silencing Map4k4 in macrophages protected mice from lipopolysaccharide-induced lethality by inhibiting TNF-α and interleukin-1β production. The siRNA-carrying particles were engulfed by macrophages in the gut and, over time, a large proportion of macrophages exhibited gene silencing, resulting in systemic immune suppression.

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Histones are basic proteins that interact with negatively charged phosphate groups on DNA, compacting and protecting the DNA, and controlling gene expression. Histones are subject to a number of post-translational modifications, including methylation and acetylation. The balance between acetylation by histone acetyltransferases (HAT) and deacylation by histone deacetylases (HDAC) alters the strength of DNA interactions and plays a key role in regulating gene expression.

Collaborators led by scientists at the Picower Institute for Learning and Memory have identified a promising target that could enable the development of therapeutics to improve memory and learning in patients with neurodegenerative disorders such as Alzheimer’s. The team demonstrated that treatment with HDAC inhibitors enhanced memory and learning ability in normal mice and mouse models of neurodegeneration.

Histone and DNA are the major components of chromatin, the complex that packages genetic information into the chromosomes and inhibitors of the HDAC family have received much attention in recent years as potential treatments for various cancers. Chromatin modification, particularly via deacetylation, has also been implicated in memory formation.

Although HDACs are a family of 11 members, the team has shown that neuron-specific overexpression of HDAC2, but not HDAC1, in mice decreased synaptic plasticity, synapse number and memory formation. This effect was ameliorated by treatment with HDAC inhibitors. Conversely, Hdac2 deficient mice displayed enhancement of synapse number and memory facilitation.

The results, published in full in the journal Nature, suggest exploration of selective HDAC2 inhibitors for treatment of human neurodegenerative diseases involving memory impairment.

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Since the mid 1990s, more and more companies have turned to fragment-based lead discovery as an alternative to high-throughput screening to identify new lead compounds. The method aims to identify very small, low molecular weight, molecules that bind to the target protein with low affinity (high µM to mM). When the target protein has a well-defined binding site, growing the fragment can be an effective strategy to gain greater potency but when the target protein has neighboring shallow pockets, linking two or more weakly binding fragments may be a better approach. In the latter situation, the best possible outcome is that the binding affinity of individual fragments will not be compromised by the linker and that overall affinity will be improved by reducing rotational and translation entropy.

Often, however, the linked fragments bind differently to the individual fragments, and a study published in the journal Nature Chemical Biology has now shown that the nature of the tether may be as important as the individual fragments in determining overall affinity. The authors explored the energetic and structural effects of rigid and flexible linkers on the binding of a fragment-based inhibitor of human uracil DNA glycosylase and found that the flexibility and strain of a given linker can have a significant impact on binding affinity, even when the individual fragments are optimally positioned. The study explored the effect of variable length amine and oxime linkages between a uracil substrate fragment and random library fragments and provided unambiguous evidence that the tether is not just a passive agent for presenting the individual fragments. The linkers do not interact directly with the enzyme and differences in binding affinities were attributed largely to the conformational preferences of a given linker and how well it presents each fragment to the appropriate binding site. The observed effects were not apparent from an inspection of the structures and emphasize the importance of linker optimisation in fragment-based lead discovery.

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Green fluorescent protein (GFP), originally isolated from the jellyfish, Aequorea victoria, fluoresces green when exposed to blue light. Because the protein is easily detected, its gene has become widely used as a reporter gene in biological experiments. The gene is fused with the gene for the protein of interest and, when this gene is switched on, GFP is also produced by the cell and production of the target protein can be monitored by measuring the green fluorescence.

Although the protein has proved so useful to biologists, the natural function of GFP is not well understood. Writing in the journal Nature Chemical Biology, scientists from the Shemiakin-Ovchinnikov Institute have now shown that, when exposed to light, GFP can act as an electron donor. It was already known that under low (<1%) oxygen conditions, GFP undergoes a photoconversion into a red fluorescent state, a phenomenon the authors call ‘redding’. Although the GFP red state is stable for many hours in the absence of oxygen, its structure and mechanism of formation were not understood. The authors found that redding could also be brought about by treatment with electron acceptors under both aerobic and anaerobic conditions. In the presence of electron acceptors such as nicotinamide adenine dinucleotide (NAD) or cytochrome c, the green glow reddened – the same effect as seen under low-oxygen conditions –suggesting that transfer of electrons could be changing the structure of the chromophore. The authors were also able to demonstrate redding in living cells, although there was a high variability of redding rate between individual cells within a number of different cell lines.

Rather than just passive light absorbers/emitters, the study points to a new role for GFPs in light-induced electron transfer – a role that should be kept in mind when designing experiments using these proteins.

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Autism, the best known of the autism spectrum disorders (ASDs), is a relatively common condition affecting around 1 in 150 children in the US, with about four times more boys than girls affected. People with autism spectrum disorders struggle with social communication and interactions, and have difficulty relating to other people and their emotions. A number of factors – both genetic and environmental – have been suggested to be linked to autism and two recent studies have now provided evidence of associations with genetic variations.

In the first study, which is published in the journal Nature, variations in a region close to the genes for two neuronal cell-adhesion molecules, cadherin 9 (CDH9) and cadherin 10 (CDH10) were found to occur more frequently in children with ASDs than in unaffected children. These cadherin molecules, which are expressed on the surface of neurons, mediate calcium-dependent cell-cell adhesion and are important in shaping the physical structure of the developing brain as well as the functional connections between different areas of the brain. The researchers propose that these gene variants are new susceptibility factors for ASDs and estimate that they may contribute to up to 15% of cases.

The second study, also published in the journal Nature, identified copy number variations – deletions or duplications of DNA – in genes belonging to two biological pathways. Interestly, one pathway involved the same neuronal cell-adhesion molecule gene family that was identified in the first study, whilst the other involved genes in the ubiquitin degradation pathway. The role of ubiquitin, which tags proteins – including the neuronal cell-adhesion molecules – for proteasome-mediated degradation, presents a mechanism that links the two gene pathways. The new data support previous evidence from functional magnetic resonance imaging studies showing that children with ASDs may have reduced connectivity among neural cells, and with anatomy studies that have found abnormal development in the frontal lobes in autistic patients.

Although the new information does not fully explain why some children develop ASDs and cannot immediately be used to provide clinical treatments, it should provide ideas for further experiments that may eventually lead to strategies for the prevention or early treatment of ASDs.

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Although it is ten years since the anti-cancer properties of (+)-11,11′-dideoxyverticillin A were first described, it is only now that the first total synthesis has been reported. The compound was originally isolated from the mycelium of a marine-derived fungus of the genus Penicillium and has since been found in several other fungal species including Shiraia bambusicola, a parasitic fungus which grows on some species of bamboo.

With ten rings and eight stereogenic centres, (+)-11,11′-dideoxyverticillin A is one of the most complex of a family of dimeric epidithiodiketopiperazine natural products. Writing in the journal Science, chemists at the Massachusetts Institute of Technology have now described an enantioselective 11-step synthesis, starting from the commercially available amino acids, tryptophan and alanine. The synthesis was designed to mimic a plausible biosynthetic pathway and, as well as providing ready access to (+)-11,11′-dideoxyverticillin A itself, should provide access to analogues which may have enhanced pharmacological activity. The elegant synthesis used a minimum of protecting groups and, by taking full advantage of the inherent reactivity of intermediates, offers insights into the natural biosynthetic pathways. (+)-11,11′-Dideoxyverticillin A inhibits the tyrosine kinase activity of the epidermal growth factor receptor with an IC₅₀ of 0.14nM, shows anti-angiogenic activity, and prevents the growth of several cancer cell lines.

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Despite enormous efforts worldwide, there is still no effective way to prevent transmission of HIV-1. Some non-human primates produce θ-defensins (retrocyclins), which act as HIV-1 entry inhibitors by inhibiting fusion of HIV-1 Env. In humans, however, θ-defensin genes have a premature stop codon that blocks translation. Defensins are small cationic, tri-disulfide bonded peptides expressed by leukocytes and epithelial cells that contribute to immune defenses against bacterial and viral infections. In primates, there are three sub-families of defensins, α-, β-, and θ-, classified on the basis of cysteine position and disulphide bonding pattern. In rhesus macaques, formation of θ-defensins, which are 18-residue cyclic peptides and the only known cyclic peptides in mammals, involves post-translational head-to-tail ligation of two nonapeptides. It was not known whether this process could occur in human cells, but researchers at the University of Central Florida, working with scientists from the University of California and the Centers for Disease Control, have now demonstrated that human cells retain the ability to produce retrocyclins and have further used the aminoglycoside antibiotics, gentamicin and tobramycin, to read-through the premature stop codon found in the mRNA transcripts and induce synthesis of bioactive retrocyclins. Although much more work needs to be done to demonstate the safety and effectiveness of this approach, the study provides hope that topical aminoglycoside-based microbicides – or other compounds that allow read-through of the stop codon –could one day be used topically to prevent sexual transmission of HIV-1.

The study is published in full in PLoS Biology.

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