Fluorescently tagged polyglutamine expansions expressed in the muscles of a laboratory strain of C .elegans (Bristol N2, left) and a wild strain (DR1350, right). Image source: Gidalevitz et al, BMC Biology, 2013,

C. elegans has already proved useful for studying the cellular toxicity caused by polyglutamine and other disease-associated proteins in the fixed genetic background of a laboratory strain, and the new research extends the utility of the model by showing that numerous natural genetic modifiers of polyglutamine disease  exist in wild worm populations. Previous work has already identified specific genes and pathways that can be manipulated to modify the phenotype caused by protein destabilizing mutations, but amelioration of disease is often achieved at some other cost to the organism. The study establishes C. elegans as a genetically tractable model for identifying modifiers of protein homeostasis among natural variants shaped by selection for the overall fitness of the organism, and could lead to new therapeutic targets.

A worm which as an adult has only about 1,000 cells overall and 301 neurons may seem an unlikely model for studying human neurodegenerative disease, but the cellular pathways governing protein homeostasis are conserved across eukaryotes, and as Matt Kaeberlein from the University of Washington Medical Center, USA, explains in a commentary accompanying the publication of the research article, C. elegans has several features that have contributed to its utility as a model organism, and key aspects of proteotoxic diseases are recapitulated in transgenic worms expressing aggregation-prone proteins.

In the polyQ disease model used by Gidalavitz and Morimoto (one which was originally developed in the Morimoto lab),  a fluorescent-tagged peptide encoding a stretch of 40 glutamines is expressed from a transgene specifically in muscle cells, allowing age-associated aggregation to be monitored in vivo, while assessing toxicity through effects on muscle function and lifespan in the same animals.  In the new study, the researchers introduced the transgene (by introgressive breeding) into three wild strains of C. elegans, finding that genetic background  affected both the age of onset and extent of aggregation, and also which subsets of muscles cells are most likely to form aggregate. They also observed that modifying effects on aggregation and measures of toxicity did not always correlate, with one wild background providing significant protection against muscle paralysis in body wall and reproductive muscle without suppressing aggregation (compared to the original laboratory strain).

The existence of modifiers that can act independently on the aggregation and toxicity phenotypes was further supported by a series of 21 recombinant inbred lines generated between the laboratory strain and the wild strain that showed the greatest enhancement of aggregation, while the existence of multiple modifiers and interactions between them was indicated by transgression – some of the inbred lines had a more extreme aggregation phenotype than either parental line. Overall the results suggest the existence of different and complex modifying pathways, which will be amenable to further dissection and characterization, and may be amenable to therapeutic manipulation.

Understanding the molecular mechanics of the C₄ system has clear implications for future agriculture and food production, yet identifying the regulatory factors involved has proved elusive. In a significant step toward this goal, Shaun Bowman and colleagues in the laboratory of James Berry at the University of Buffalo, USA, have identified a new RNA binding protein that is the first such factor to be implicated in the regulation of an individual photosynthetic gene in a C₄ plant, as published in a recent study in BMC Plant Biology.

Confocal microscopy image of the leaves of the C4 plant maize stained for RLSB (top), Rubisco (middle) and PEPcase (bottom). Image source: Bowman et al, BMC Plant Biology, 2013, 13:138

Both C₃ and C₄ plants contain mesophyll cells, which contain the apparatus needed for photosynthesis. Here, carbon-dioxide assimilating enzymes fix carbon into a usable form, with each enzyme encoded by a specific pattern of gene expression. The primary workhorse of this process in C₃ plants is an enzyme called Rubisco, yet it wastefully also fixes oxygen in the process. C₄ plants overcome this wastefulness by housing Rubisco in a different cellular compartment that is surrounded by the mesophyll – known as the bundle sheath. The bundle sheath cells play host to only the second round of carbon fixation using Rubisco; the primary round of fixation instead being carried out in the mesophyll by the enzyme PEPCase, which is more efficient than Rubisco at fixing carbon.

Although inefficient, Rubisco is nevertheless ubiquitous in plants, and essential to their growth and survival. Given this, comparing the two different photosynthetic processes and identifying what regulates the shared components of their enzymatic pathways could hold the key to understanding how they both operate.

Encoding the larger of Rubisco’s two protein subunits is a chloroplast gene known as rcbL, found in both plant types. Expression of this gene in specific cell-types like the bundle sheath is tightly controlled after transcription via specific stretches of sequences within its messenger RNA (mRNA). The proteins that bind these RNA molecules influence patterns of gene expression, and it was these proteins that Bowman and colleagues sought to find.

In a multi-layered analysis using several C₄ species and the model C₃ plant Arabidopsis, the authors identified a protein called RLSB based on its ability to bind to rcbL mRNA. In both C₃ and C₄ plants, reductions in the levels of RLSB gene expression in RLSB mutant and gene-silenced plants resulted in reduced photosynthetic function, and visibly yellowed leaves.

Confocal/DIC microscopy image of a leaf of the C3 plant Arabidopsis stained for Rubisco. Image source: Bowman et al, BMC Plant Biology, 2013, 13:138

Comparison with other plant species indicates that the RLSB protein is highly conserved across many plant species, and co-localises with Rubisco in all C₃ plant cells. The localisation of this protein specifically to the bundle sheath cells in C₄ plants now adds an extra layer of evidence pointing towards a crucial regulatory role for RLSB in the expression of Rubisco in C₄ plants as well. Taken together, this work suggests that the mechanism of RLSB gene expression observed throughout C₃ plants may have been modified over evolutionary timescales to provide a more localised, specialised, and ultimately similar function in C₄ plants.

C₄ plants currently account for around a quarter of global plant primary productivity, yet few crop species utilise this more efficient form of photosynthesis. Tellingly, it is also the competitive and adaptable grass species that are mostly characterised by this system, with C₄ plants maize and sugarcane among the crop-plant exceptions. Understanding the full scope of this photosynthetic pathway could therefore pave the way toward more highly efficient varieties of non-C₄ crops, and a more efficient answer to future food production.

RHD only affects the Oryctolagus cuniculus (European or common rabbit) species and its symptoms include high fever, internal bleeding and liver disease. It is caused by Rabbit Haemorrhagic Disease virus (RHDV), a single-stranded RNA virus in the Caliciviridae family belonging to the Lagovirus genus. In new research published in Veterinary Research, Ghislaine Le Gall-Reculé from the French Agency for Food, Environmental and Occupational Health and Safety, France and colleagues report a new Lagovirus strain, designated RHDV2, which is related to RHDV but with several key differences.

RHDV has proven to be highly infectious and very resistant, even persisting in frozen conditions. The worldwide prevalence of RHDV, as well as the high lethality of the disease, have resulted in intensive vaccination and epidemiological surveillance programmes. Following reports of RHD in rabbitries and among wild rabbit populations in the north-west of France, the authors used RT-PCR analysis and sandwich ELISA testing to confirm that RHDV2 was the aetiological agent responsible. The virus had killed 25 percent of RHDV-vaccinated does in one particular rabbitry.

RHDV2 differs from RHDV and the previously identified RHDVa strain, in several ways. The authors looked at complete sequences of the protein capsid genes to identify the phylogenetic relationship between the strains. They found that RHDV2, though a member of the same genus as RHDV and RHDVa, is phylogenetically distinct from these strains and forms a new genetic group.

Further molecular analysis and experimental studies carried out by the authors revealed a unique antigenic and symptomatic profile for RHDV2. Though less virulent and with lower observed mortality rates than RHDV and RHDVa, the disease resulting from RHDV2 is longer in duration and will take a chronic or subacute course more often than for RHD caused by the other viral strains.

The identification of RHDV2 and the wide range of molecular, experimental infection and epidemiological data presented by the authors represent a significant contribution to our knowledge of RHD and will likely inform epidemiological surveillance efforts in a number of territories.

Previous work from Métraux’s lab, has shown that aggressive leaf-wounding of the model cress species Arabidopsis thaliana is enough to mount a strong defence against the pathogenic fungus Botrytis cinerea – the causative agent of grey mould – by releasing protective compounds called Reactive Oxygen Species (ROS) in a process independent of the plant’s usual defensive pathways. They questioned, how much of a mechanical insult is needed to induce this protective response that primed the immune system to an attack?

Rather than be forceful with their forceps, the group instead gently rubbed individual leaves between finger and thumb, in what they term soft mechanical stress (SMS). Again, ROS were released in a rapid burst immediately following the treatment, in a coordinated process accompanied by a peak in intracellular calcium levels and activation of genes associated with the touch response. This response also translated into powerful resistance to infection.

Following inoculation of the leaves with spores of B.cinerea, SMS treatment reduced the lesion size of infection in a manner that was strongly dose-dependent – the more times the leaves were rubbed, the greater the reduction in lesion size. However, this effect was transient. After only a few hours, half of this induced resistance was lost, with full susceptibility to infection regained as little as 24 hours after treatment. Could this transient resistance be because the plants sensed the cellular damage, as seen in the Métreaux’s previous work using wounded leaves? Examination of the leaf surface under a powerful electron microscope suggests not.

Although some minor damage was observable to the tiny protrusions on the leaf surface, known as trichomes, very little overt cellular damage was observed under SMS treatment. Indeed, when the researchers replicated these experiments using a mutant Arabidopsis that lacked trichomes, no cellular damage was seen – yet resistance remained. They therefore looked a little closer.

Examining the cellular cuticle for clues, they found that the outer layer of the cell was markedly more permeable than those of untreated leaves, and that this permeability was in turn associated with the leakage of bioactive compounds onto the leaf surface. They speculate that it is these bioactive compounds that may have a key role in preventing the development, and therefore pathogenesis, of mould.

Previous work on wounding in plants has identified several signalling pathways involved in the defence and immune responses – most notably those involving salicylic acid, ethylene, jasmonate, and other hormones. This new research highlights how the wounding response is far more sensitive than previously thought, adding a new layer of complexity to the sensory world of plants and their fight against infection.

MSCs are a group of multipotent stem cells derived from the from the the embryonic germ layer called the mesoblast. They have previously been found to have the capacity to repair damaged tissue. Transplantation of MSCs is considered safe and has been tested in clinical trials for a number of different diseases with encouraging results. Adipose tissue provides an easily obtainable source of MSCs. These human adipose-derived MSCs (hAd-MSCs) secrete a large number of protective cytokines and exhibit other characteristics of MSCs including self-renewal and differentiation into multiple cell lineages.

Lead by Xiangmei Chen of the Chinese PLA General Hospital, Beijing, China, the researchers investigated whether hAd-MSC-conditioned medium (CM) could inhibit podocytic apoptosis induced by high glucose. Using mouse podocyte clone (MPC5) cells, they established an in vitro model of podocytic apoptosis and injury induced by high glucose. The MPC5 cells were then cultured with hAd-MSC-CM, harvested from patients using lipoaspiration.

Unlike the control (CM from a human embryonic lung cell line), hAd-MSC-CM reduced podocytic apoptosis induced by high glucose in a dose-dependent manner, downregulated activated caspase-3, and also prevented the downregulation and rearrangement of synaptopodin, a podocytic cytoskeletal protein. Further experiments showing a similar protective effect of recombinant human epithelial growth factor (EGF) on podocytes, suggest that the beneficial effects of hAd-MSCs are mediated mainly via the secretion of soluble EGF.

These findings suggest a promising role of hAd-MSCs in future therapeutic strategies for the treatment of diabetic nephropathy.

Sørensen and colleagues conducted a cohort study looking at the association between pre-admission metformin use and mortality after ICU admission. Using the unique Danish Civil Registration Number assigned to each citizen, data was collected by individual-level linkage between population based medical registries and databases. All 52,964 adult patients admitted to a medical or surgical ICU in Northern Denmark between January 2005 and December 2011 were identified. Of these patients, 7,404 had type 2 diabetes. Prescription data for anti-diabetic medication, including metformin was collected for each patient. 1,073 were taking metformin alone and 1,335 metformin in combination with other anti-diabetic drugs. Patients were followed up from the date of ICU admission for 30 days (or death if sooner). 30-day mortality was calculated and current metformin use was compared to recent use, former use, and never use.

Current metformin users had a lower 30-day mortality compared to non-users. 30-day mortality in non-users was 25 percent, compared to 17.6 percent for those on metformin monotherapy and 17.9 percent for those on metformin in combination with other anti-diabetics. This reduced mortality persisted after adjustment for pre-admission morbidity and other potential confounders, and was only seen in patients who were currently using metformin. Mortality was not associated with recent or former metformin use. No difference in mortality was observed between those on other anti-diabetic drugs and those who did not use any anti-diabetic drugs.

The reasons for this reduced mortality are not clear. One potential explanation is that, in addition to lowering blood glucose, metformin has other effects that may be beneficial during critical illness. It has anti-inflammatory effects that may modulate the hyper-inflammatory response central to the early phase of sepsis and organ dysfunction, as well as anti-thrombotic effects that could prevent microvascular thrombosis.

Although this study suggests that ICU patients may benefit from pre-admission metformin, the safety and effect of using metformin during critical illness is unclear. Metformin is generally not recommended during hospitalization due to the potential risk of lactic acidosis in patients with severe renal, liver or heart disease, or in patients with shock or post major surgery. It is therefore frequently switched to insulin on admission to hospital. The effect observed in the study is therefore likely due to mediation of the early response to critical illness.

This study suggests that use of metformin, alone or in combination with other anti-diabetic drugs, before admission to an ICU, is associated with reduced 30-day mortality. However, more work is needed to understand and balance the potential risks of lactic acidosis with any potential benefits of metformin in critical illness.

P. aeruginosa typically infects immunocompromised patients, using its quorum sensing system to regulate several important processes during infection. This includes the formation of biofilms – communities of microorganisms that adhere to and cover surfaces – and the production of virulence factor molecules that are responsible for many of the symptoms of infection. These functions make quorum sensing systems a promising target for anti-bacterial drugs that could disrupt these lines of communication. It has also been suggested that such drugs may have the advantage over current antibiotics in being less likely to induce drug resistance.

Helms and colleagues use computational modelling to explore what effects inhibitors of various parts of the quorum sensing system would have on the bacteria P. aeruginosa.

Quorum sensing in P. aeruginosa consists of three interconnected systems, organized in a hierarchy. As many of the details of the connections remain unknown the authors use a simplified, rule-based model of these networks to determine the production of molecules used for signalling between cells and virulence factors.

Tests of the model show that despite its relative simplicity it accurately replicates behaviors of the system reported in the literature. However their findings do suggest that the topology of the molecular network as it is currently understood may need to be revised in order to accurately describe the production of virulence factors.

From this model they show that it is possible to test the effects of mutations or inhibitors that affect various components of the molecular networks. The authors find that blocking the synthesis of signalling molecules is most effective in reducing the production of these networks, and that targeting the receptor molecules is key to reducing virulence factors.

Danielle Drury-Stewart and colleagues at Emory University, USA have previously reported the use of a small molecule based differentiation protocol which reduces the cost and increases efficiency  for the in vitro differentiation of neural precursors and neurons. In their recent study published in Stem Cell Research & Therapy they have further characterised the neural precursors derived from hESCs using this protocol, and demonstrated their use in a mouse model of ischaemic stroke.

hESC-derived precursors expressing neural markers NeuN (red) and neurofilament L (green). Image source: Drury-Stewart et al, Stem Cell Research & Therapy, 2013, 4:93.

Neural differentiation of hESCs in vitro was ascertained using immunocytochemistry and electrophysiological recordings. The hESC-derived precursors expressed the neural markers nestin, PAX1 and SOX1. Following re-plating, the cells differentiated into mature neurons and formed well-connected networks expressing the neuronal markers NeuN and neurofilament L. Whole cell patch-clamp recordings showed that the action potentials and potassium currents of the hESC-derived neurons matured over the four week differentiation period.

In vivo, neural precursors were transplanted into the affected area of the mouse brain seven days after induction of a focal ischemic stroke. Cell survival and neuronal differentiation was verified using immunohistochemistry. Transplantation resulted in an increased proportion of BrdU-positive neurons compared to controls, which may be indicative of cell division associated with increased neurogenesis.

In order to determine whether the mice showed signs of functional recovery, the adhesive removal test was administered. Before stroke induction the mice were trained so that they were consistently removing an adhesive dot from both forepaws within 12 seconds. Time to contact and time to completely remove the dot were recorded. The test was repeated four days after stroke to establish a baseline value for impairment and then repeated at one week intervals for up to four weeks after transplantation.

Although there was no significant difference in the outcome measures between the control and transplantation groups as well as no significant change in time to completely remove the dot, the transplant group did show a significant decreasing trend in time to contact that was not seen in the control group.

Collectively, the results suggest that mice who received transplantation show improved sensory function in the stroke-affected forepaw and also a more consistent recovery compared to the control group.

The demonstration that transplantation of hESC-derived neural precursors into the ischemic brain enhances both neurogenesis and sensory function offers further hope for the development of cell therapy for stroke patients.

In a recent study published in Arthritis Research & Therapy, Anna Blom and colleagues from Lund University, Sweden address the important question of how defective degradation of NETs in SLE patients impacts the clinical phenotype of the disease. Sixty-nine SLE patients with a median age of 39 years were recruited to this prospective study and followed for approximately five years. Recordings of disease activity, infections and other clinically relevant information, as well as serum samples, were taken every two months. Sera from 77 healthy volunteers were used to determine the normal range of ability to degrade NETs.

Significantly, 41 percent of patients exhibited a decreased ability to degrade NETs at least once, with a median of 20 percent of patients when analysing all time points. Decreased degradation was associated with symptoms of glomerulonephritis, low complement levels and increased levels of antibodies against histones and DNA. Patients were also more likely to develop alopecia and fever after an episode of impaired NETs degradation.

This large, well-designed study provides an important insight into disease pathogenesis and highlights a novel correlation between defective clearance of NETs and SLE disease manifestations, though degradation ability may vary for each patient over time. During the course of the study disease activity improved for the majority of patients, and interestingly although no direct efforts were made to restore the ability to degrade NETs, this also improved.

Collectively, the findings  highlight the importance of regular follow-ups with patients to reduce SLE flares, the potential use of NETs as a diagnostic biomarker for disease activity and indicate that therapeutics targeting NET clearance may be of benefit.

The development of antibiotic resistance in bacteria generally has a cost to be paid in terms of their fitness. As such it is often assumed that over generations in the absence of the selective pressure of an antibiotic, resistance is lost due to its detrimental cost on fitness. This situation is complicated by studies now suggesting that the fitness cost of antibiotic resistance may be altered by the development of other resistance mutations. Angst and Hall probed the complexity of this scenario by experimentally evolving antibiotic resistant and antibiotic sensitive strains of E.coli in an antibiotic free environment for 200 generations and looking at the effect on fitness of introducing additional streptomycin resistance alleles into the strains before and after evolution in the antibiotic free medium.

They found that the detrimental effects of antibiotic resistance on fitness were reduced in strains that had evolved in the antibiotic free medium. This suggests that the occurrence of beneficial mutations incorporated during evolution in antibiotic free medium are able to buffer the cost of antibiotic resistance. The history of the evolution of a bacterial strain therefore effects the future evolution of antibiotic resistance and its persistence. This robust study adds strong evidence to previous studies showing the epistatic nature of antibiotic resistance.

The evidence presented in this experimental study has potential relevance when looking at the fitness cost of antibiotic resistance in real populations of disease causing bacteria. It is particularly relevant to chronic infections such as those in cystic fibrosis patients, in which the bacteria have many generations to potentially fix mutations in the absence of antibiotics. If it is the case that these mutations mitigate the cost of antibiotic resistance then research looking at the epistasis between resistance elements and other types of mutations will be important in predicting how likely it is that multi-drug resistant bacteria will remain after different treatments for infection.