Soon after birth, the human body is colonised by bacteria. Trillions of bacteria take up residence in the gut and perform a range of useful functions such as helping with digestion and absorption of nutrients, producing vitamins, preventing growth of pathogenic bacteria, and developing the immune system. In 2006, it was shown that the proportion of Bacteroidetes relative to Firmicutes was reduced in the guts of obese people compared with lean individuals and also in the guts of genetically obese mice compared with lean littermates. Researchers at Emory University have now shown that mice engineered to lack toll-like receptor 5 (TLR5) – a component of the innate immune system that is expressed in the gut mucosa and that helps defend against infection – are 20% heavier than normal mice and have elevated triglycerides, cholesterol and blood pressure as well as slightly elevated blood sugar and a decreased response to insulin. TLR5-deficient mice consume about 10% more food than wild type mice and, although they lose weight when food is restricted, they still show insulin resistance. On a high fat diet, TLR5-deficient mice gain more weight than normal mice and develop full-blown diabetes and fatty liver disease, mimicking “metabolic syndrome” which increases the risk of developing heart disease and diabetes in humans.

Treating TLR5-deficient mice with antibiotics to kill most of the bacteria in the intestine reduced their metabolic abnormalities and, conversely, transfer of intestinal bacteria from TLR5-deficient mice to germ-free wild type mice transferred many of the characteristics of metabolic syndrome, including increased appetite, obesity, elevated blood sugar, and insulin resistance. Although earlier studies had shown that greater numbers of Firmicutes bacteria lead to more calories being extracted from the diet, the TLR5-deficient mice had normal proportions of Firmicutes and Bacteroidetes but differed in the composition of bacterial species in the two families. The new study shows that, as well as influencing how well energy is absorbed from food, gut flora can also influence appetite and may contribute to human obesity and metabolic disease.

The study is published in Science Express.

The dopamine reuptake inhibitor, Ritalin® (methylphenidate) has been used for almost 50 years to treat children with attention-deficit hyperactivity disorder (ADHD) and, more recently – and controversially, has been used by students to enhance academic performance and as a recreational drug. Although Ritalin® has been prescribed for millions of children, the mechanisms by which it modifies behavioural performance remain poorly understood. Researchers at the University of California, San Francisco have now shown, in animals at least, that Ritalin® improves ability to focus on tasks and directly enhances speed of learning by distinct dopamine receptor-mediated mechanisms.

By co-administering Ritalin® with either the dopamine D₁ receptor antagonist, SCH-23390, or the dopamine D₂ receptor antagonist, raclopride, the team were able to show the well-known benefit of improved focus was mediated through D₂ receptor-dependent mechanisms whereas learning efficiency was enhanced through D₁ receptor-dependent mechanisms. The study also established that Ritalin® strengthens synapses and enhances neuroplasticity. A better understanding of the way that Ritalin® improves focus and enhances learning could lead to the development of more targeted drugs for ADHD and learning enhancement.

The study is published in Nature Neuroscience.

Systemic inflammatory response syndrome (SIRS) is an exaggerated host inflammatory response to infection (sepsis) or to physical insults such as trauma. SIRS can lead to multiple organ dysfunction syndrome (MODS) and, amongst the under-35s, trauma is the leading cause of death in the United States. Although the pathways leading from infection to sepsis are relatively well understood, it has been much less clear why physical insults lead to SIRS. A study led by researchers at Beth Israel Deaconess Medical Center has now suggested a link between sepsis and SIRS that is caused by trauma. The team propose that mitochondria are released into the bloodstream after physical injury and, because mitochondria closely resemble the symbiotic bacteria from which they are believed to originate, they elicit a sepsis-like response.

Pathogen-associated molecular patterns (PAMPs) are molecules such as bacterial endotoxins which are recognised by pattern recognition receptors (PRRs) as non-self, and so trigger an innate immune response. Injured or necrotic tissue generates molecules known as damage-associated molecular pattern molecules (DAMPs) that can also initiate and perpetuate an immune response. Many DAMPs are molecules that are usually found exclusively within cells and, when released into the bloodstream, are not recognised as self and trigger an immune response. The team found blood samples from patients who had suffered multiple trauma contained high levels of mitochondrial DNA – often thousands-of-fold higher than normal levels – and that this DNA activates immune cells via toll-like receptor 9 which normally recognises bacterial or viral DNA. Mitochondrial peptides were also found to elicit an immune response via the formyl peptide receptor 1 (FPR1) which also plays a role in the immune response to bacterial infections. Mitochondrial DDA and peptides were found to act synergistically to activate neutrophils via downstream kinase pathways. In further experiments, injection of mitochondria into rats caused peritonitis and reproduced the pulmonary and hepatic inflammation typical of traumatic SIRS.

The study, which is published in the journal Nature, shows that trauma can initiate innate immune pathways identical to those activated in sepsis and may lead to new strategies for treating trauma patients as well as re-evaluation of patients believed to be suffering from sepsis.

Post-translational modifications modulate the function of most eukaryote proteins by altering their activity state, localization, turnover, and interactions with other proteins. Many important proteins, including antibodies, hormones and receptors, are glycosylated but the development of chemical and enzymatic methods for the synthesis of homogeneous glycoproteins has proved a considerable challenge.

A paper published in Science in 2004 was later retracted when it proved impossible to repeat the results but researchers at the University of Maryland School of Medicine and ETH, Zurich have now described a new process for preparing homogeneous eukaryotic glycoproteins. The method, which involves engineering and functionally transferring the glycosylation machinery from Campylobacter jejuni into Escherichia coli, provides a means of expressing glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans can then be trimmed and remodelled in vitro by enzymatic transglycosylation to provide N-glycosylated eukaryotic proteins. The authors believe that the new methodology, which is published in the journal Nature Biotechnology, should lead to a general platform for producing eukaryotic N-glycoproteins.

Gene expression profiling is used to guide treatment options for women with breast cancer. Endocrine therapies – tamoxifen or aromatase inhibitors – are offered to women whose cancer is oestrogen receptor (ER) positive whilst the monoclonal antibody, trastuzumab (Herceptin®) and the small molecule, lapatinib (Tykerb®) are used to treat women whose cancer overexpresses the HER2 receptor. About 15% of breast cancers – the so-called triple negative breast cancers that don’t have receptors for oestrogen, progesterone or HER2 – don’t respond to hormone therapy or to HER2 blockers and the prognosis for women with these cancers is relatively poor.

Researchers at Washington University University School of Medicine in St. Louis have now identified a gene that is overexpressed mainly in ER-negative, HER2-negative and triple negative breast cancers, leading to the possibility of a new clinical biomarker and potential treatments. Upregulation of Wnt signalling coreceptor, LRP6 (low-density lipoprotein receptor-related protein 6), was found in about a quarter of the breast cancer samples that the researchers examined. Previous studies had shown that the protein Mesd (mesoderm development) blocks LRP6 and was able to slow the growth of cultured breast cancer cells. Mesd also inhibits the development of mammary tumours in mice, without producing known pathway-dependent side-effects such as bone lesions, skin disorders or intestinal malfunctions. A smaller fragment of Mesd was found to be as effective as full length Mesd and to have improved stability.

The study is published in the Proceedings of the National Academy of Sciences.

Although the study offers the prospect of targeted therapy for women with breast cancer that is currently difficult to treat, both screening and prescribing practices need to improve for such discoveries to realise their full potential. A recent news feature in Nature Biotechnology highlights differing views on testing as well as the problems associated with diagnostic tests for HER2 – both of which may be compromising women’s access to appropriate and effective treatment.

Spinal muscular atrophy (SMA), a muscle-wasting disease caused by a deficiency in survival motor neuron protein (SMN), is a leading genetic cause of death in infants. There is currently no cure for the disease and treatment consists of managing the symptoms and preventing complications. The protein deficiency, which is restricted to motor neurones, is caused by loss of or mutation in the SMN 1 gene and a team led by researchers at Ohio State University have shown that, in mice at least, the disease can be reversed by intravenous, virally mediated gene delivery.

SMA mice, treated at one day old, showed increased levels of SMN in the brain, spinal cord and muscles within ten days. Although levels remained lower than in normal mice, the increase appeared to be sufficient to reverse the effects of the disease and the team believe that the same would be true in children with the disease. Dramatic improvements were seen only in mice treated within the first two days of life and the potential treatment window for children is not yet clear. The researchers hope to be able to progress to human clinical trials and, as a first step, have shown that the virus is also able to cross the blood-brain barrier and penetrate motor neurones in a one day old macaque. One difficulty in translating the research into a treatment option for children with SMA is that symptoms are typically absent in very young infants and the only available screening method for newborns is considered to be prohibitively expensive.

The study is published in Nature Biotechnology.

In back-to-back papers published in the Proceedings of the National Academy of Sciences, researchers describe retro-inverted peptide mimetics targeting the vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) pathways. The team begin by screening a phage display library to identify peptides that bind to the target receptor and refining the hits by structural and functional analysis. Since natural peptides are often unstable to proteolysis, the amino acid retro-inversion method (ie substitution of D-amino acids for L-amino acids in conjunction with reversal of chain direction) was used to produce molecules with greater resistance to proteolysis. The side chain topology of the resulting peptide mimetics is similar to that of the original peptide – albeit with the amide bonds and terminal charges reversed.

The formation of new blood vessels has been associated with a number of pathologies including cancer and diseases of the eye such as diabetic retinopathy and age-related macular degeneration. VEGF comprises a family of five growth factors that promote angiogenesis by binding to, and selectively activating, several membrane-bound tyrosine kinase receptors (VEGFR-1, -2, and -3) and neurophilins (NRP-1 and -2). The minimal structural requirement for binding of a small peptide fragment to VEGFR-1 and NRP-1 was determined to be the tripeptide, RPL. The retro-inverted tripeptidomimetic, D-LPR was found to be resistant to proteolysis and to bind effectively to VEGFR-1 and NRP-1. In competition binding experiments, the IC₅₀ values of RPL for VEGFR-1 and NRP-1 were 30 nM and 4 pM and the IC₅₀ values of D-LPR for VEGFR-1 and NRP-1 were 2 pM and 2 pM. D-LPR was shown to be effective in three animal models of angiogenesis, including a mouse model of retinopathy. When administered topically, D-LPR led to a significant (ca 50%) reduction in angiogenesis, suggesting that the peptide mimetic can penetrate the vitreous humour and may provide a lead for the development of soluble and permeable small drug molecules that can be administered in eye drops.

EGFR, a tyrosine kinase receptor, is abnormally activated in many types of epithelial tumours, including including lung, colon, and head and neck cancers. Although, theoretically, the EGFR pathway could be blocked by three drug classes – antibodies, kinase inhibitors or soluble ligand receptor traps/decoys – so far no EGFR decoy has been developed. In the second paper, the team describe small molecules that mimick EGFR and behave as soluble decoys for EGFR ligands, EGF and TGFα. The cysteine-bridged pentapeptide, CVRAC, was found to bind specifically to EGFR ligands and both CVRAC and the retro-inverted derivative, D-CARVC, markedly reduced proliferation of tumour cell lines. In immunocompetent female mice bearing mammary tumours, mice treated with D-CARVC had significantly smaller tumour volumes than control mice.

Although very different at a molecular level, hepatitis viruses B and C (HBV and HCV) both infect only humans and chimpanzees which means that there is a lack of suitable small animal models for studying viral lifecycles and for testing new drugs. One alternative would be to study the viruses and test new compounds in liver cells grown in vitro, but human hepatocytes are very difficult to grow and maintain in culture.

A team of researchers led by scientists at the Salk Institute has now provided a solution to the problem by generating a mouse with a liver that is almost completely ‘humanised’. The team had previously generated a mouse with a partially humanised liver but wanted to achieve more complete transformation. Around 95% of the liver cells of the new mice are human in origin and the animals are susceptible to infection by both HBV and HCV. Mice infected with HCV were shown to respond to drugs such as pegylated interferon α2a and ribavirin that are used to treat human patients. Adefovir dipivoxil, used to treat HBV patients, was found to lower viral titres in mice infected with HBV.

The mice were generated by using genetic and pharmacological pressures to lead to a growth disadvantage for mouse hepatocytes and positive selection for transplanted human hepatocytes. The mice provide a new way to study pathogens that target the human liver and to test drugs to treat human hepatitis. In the future, the mice could also be used to study other hepatotrophic pathogens such as malaria, as well as cirrhosis and liver cancer.

The study is published in the Journal of Clinical Investigation.

Ion channels, proteins that regulate the transfer of ions across the cell membrane, can be broadly classified according to ‘gating mechanism’ – what makes the channel open and close. Voltage-gated channels respond to a voltage gradient across the plasma membrane whereas ligand-gated channels are activated by binding of extracellular ligands or intracellular second messengers. Recent detailed studies of ion channels are showing, however, that things are not quite so simple.

Researchers in the US and Germany have now shown that they can confer significant voltage dependence to the inwardly rectifying K⁺ channel, K_ir6.2, by introducing a point mutation, L157E. K_ir6.2 is a ligand-gated channel that lacks a canonical voltage-sensing domain (VSD). In classical models of voltage-dependent gating, the VSD strongly influences opening and closing of the pore-forming domain so that the channel open probability is reduced to virtually zero at sufficiently negative voltages and increased to near unity upon depolarization. Previous observations have shown that such ‘tight coupling’ between the VSD and the pore does not apply to all channel types and the new study shows that substitution of charged residues at pore-lining positions can affect channel gating in very unexpected ways. Comparing K_ir6.2[L157E] with wild type K_ir6.2, the team found that the probability of opening under conditions of low intracellular K⁺ was much greater for the mutant channel. The presence of a natural ligand, phosphatidyl inositol bis-phosphate (PIP₂), removed the voltage dependence of the mutant channel. Both voltage- and ligand-dependent gating of K_ir6.2[L157E] were highly sensitive to intracellular [K⁺], indicating an interaction between ion permeation and gating.

The authors of the study, which is published in PLoS Biology, propose that ions flowing through the ion channel pore can significantly affect channel activity and that the mechanisms of voltage-gating and ligand-gating may be more closely linked than previously supposed. They further suggest that such interactions are likely to be a general, if latent, feature of the superfamily of cation channels.

Some 200 million people worldwide are estimated to be infected with the hepatitis C virus (HCV) which can eventually lead to cirrhosis, liver cancer, and in some cases, death. The current standard-of-care treatment for persistent infection – a combination of pegylated interferon-α and ribavirin – is able to clear the infection in around 50% of patients. In some patients, however, treatment is associated with haemolytic anaemia which may be severe enough to need dosage reduction or even discontinuation of treatment.

A team led by scientists at Duke University’s Institute for Genome Sciences & Policy (IGSP) have now discovered that loss of function mutations in the gene ITPA, which encodes the enzyme inosine triphosphatase, protect against the development of anaemia. Previous studies had identified the genetic variants with enzyme deficiency and, through a genome-wide association study, the Duke team were able to show that they were also protective against anaemia caused by ribavirin. The finding may offer new treatment opportunities for HCV patients with coronary artery disease or kidney disease who are often not treated with ribavirin because of fears that anaemia could exacerbate their condition.

Inosine triphosphatase deficiency was first recognised over 30 years ago and is not thought to be clinically important. A diagnostic test that could predict deficiency, and hence reduced susceptibility to ribavirin-associated anaemia, would allow broader treatment options for HCV patients.

The study is published in the journal Nature.