Historically, inorganic nitrate was believed to be an inert by‐product of nitric oxide (NO) metabolism that was readily excreted by the body. Studies utilising doses of nitrate far in excess of dietary and physiological sources reported potentially toxic and carcinogenic effects of the anion. However, nitrate is a significant component of our diets, with the majority of the anion coming from green leafy vegetables, which have been consistently shown to offer protection against obesity, type 2 diabetes and metabolic diseases. The discovery of a metabolic pathway in mammals, in which nitrate is reduced to NO, via nitrite, has warranted a re‐examination of the physiological role of this small molecule. Obesity, type 2 diabetes and the metabolic syndrome are associated with a decrease in NO bioavailability. Recent research suggests that the nitrate‐nitrite‐NO pathway may be harnessed as a therapeutic to supplement circulating NO concentrations, with both anti‐obesity and anti‐diabetic effects, as well as improving vascular function. In this review, we examine the key studies that have led to the re‐evaluation of the physiological function of inorganic nitrate, from toxic and carcinogenic metabolite, to a potentially important and beneficial agent in the treatment of metabolic disease. BATbrown adipose tissuecGMPcyclic guanosine mononucleotide phosphateCVDcardiovascular diseaseeNOSendothelial nitric oxide synthaseiNOSinducible nitric oxide synthaseNADPHnicotinamide adenosine dinucleotide phosphateNOnitric oxideNOSnitric oxide synthaseT2DMtype 2 diabetes mellitusUCP‐1uncoupling protein 1WATwhite adipose tissueXORxanthine oxidoreductase1N‐nitroso compounds in the presence of nitrosatable compounds. The conclusions of these reports were based on epidemiological studies Until recently dietary inorganic nitrate was thought to be hazardous to human health. In 2006, the International Agency for Research on Cancer (IARC) concluded that it was probable ingested nitrate was carcinogenic in conditions that promoted endogenous nitrosation Nitrate is an important constituent of our diet. Sources of dietary nitrate intake include processed meats, fruits and vegetables. In addition, nitrates and nitrites are often added as a preservative to meats, including sausages, hot dogs and bacon, and were initially suggested to be contributing to the risk to human health posed by preserved and processed meats Since the concentration of nitrate and nitrite in vegetables is so much higher than processed meats, it appears unlikely that nitrates and nitrites are the active agents in the risk to health associated with processed meats. In fact, the World Health Organisation acceptable daily intake for nitrate is 0–3.7 mg/kg. This figure is vastly exceeded (approximately 550%) by a diet relatively rich in fruit and vegetables high in nitrate Here, we discuss the therapeutic potential of inorganic nitrate, as a means of providing bioavailable NO, to treat obesity, diabetes and the metabolic syndrome.2l‐arginine to l‐citrulline NO is a highly bio‐active molecule, signalling via soluble guanylyl cyclase and cyclic guanosine mononucleotide phosphate (cGMP) to bring about a host of physiological functions Following the description of the non‐enzymatic physiological reduction of nitrate through nitrite to NO, work conducted by Jansson et al. identified an enzymatic process functioning to reduce nitrate to NO in mammalian tissue, with xanthine oxidoreductase (XOR) postulated as the enzyme responsible 1 couple (complex III), as essential in the reduction of nitrite to NO by mitochondria A number of additional physiological sources of nitrate reduction have been identified. Cosby et al. demonstrated that infusions of nitrite into the forearm brachial artery resulted in rapid formation of erythrocyte iron‐nitrosylated haemoglobin NO has a short half‐life (0.05–1.18 ms in human blood), meaning that it can only act locally in an autocrine and paracrine manner to bring about physiological changes l‐arginine‐NO pathway is impaired in hypoxic conditions While on the surface it appears that the nitrate‐nitrite‐NO pathway is a redundant mechanism for generating NO, the recycling of nitrate and nitrite in fact provides an important method of producing NO in conditions where NOS enzyme activity is compromised. The NOS enzymes require oxygen to produce NO, therefore NO production via the canonical However, the enzymatic reduction of nitrate has been observed to occur in both hypoxic and normoxic conditions, implying a role beyond that of a compensatory mechanism for compromised eNOS activity 3The metabolic syndrome is a cluster of risk factors for cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM). These factors include obesity, dyslipidaemia, hypertension and insulin resistance In recent years, the perturbation of NO synthesis and signalling has emerged as a potential modulator of both cardiovascular morbidity and metabolic dysfunction in both rodent models and humans Following the characterisation of eNOS‐null phenotypes, Carlstrom et al. sought to restore NO signalling in eNOS‐null mice via dietary nitrate supplementation and subsequent metabolism through the nitrate‐nitrite‐NO pathway The work of Carlstrom et al. provided functional justification for the use of nitrate to treat impaired NO synthesis and highlighted the effects of dietary nitrate on, not only one, but multiple risk factors underlying the metabolic syndrome. However, while this study showed the potential of dietary nitrate as an anti‐obesity and anti‐diabetic agent on a phenotypic level, the mechanisms driving these effects were yet to be elucidated.4Obesity is characterised by inappropriate accumulation of white adipose tissue (WAT) and an increase in adipocyte size. WAT stores energy as lipid which can then be released via lipolysis when required In contrast, brown adipose tissue (BAT) is more metabolically active and utilises lipids as an energy source in a process termed non‐shivering thermogenesis Interestingly, a small number of cells within WAT were found to express UCP‐1 upon cold exposure Numerous activators of the browning response have been identified, including cardiac natriuretic peptides, fibroblast growth factor 21, irisin and β‐aminoisobutyric acid Dietary nitrate is known to increase the circulating concentration of cGMP in humans Roberts et al. demonstrated that inorganic nitrate increased brown adipocyte‐specific gene expression, including UCP‐1, in WAT both in vitro and in vivo As previously mentioned, the nitrate‐nitrite‐NO pathway acts to complement the oxygen‐dependent NO synthases and utilises nitrate as a substrate in hypoxic conditions, thus providing NO in situations in which NO synthases are compromised Carlstrom et al.’s work highlighted how dietary nitrate can bring about a reduction in body weight and dyslipidaemia, while increasing glucose tolerance, on a phenotypic level 5Dietary nitrate has a profound effect on hypertension, a major risk factor for CVD and another strand of the metabolic syndrome Endothelial dysfunction is a significant determinant of hypertension A number of recent studies have sought to identify the mechanisms underpinning the effect of dietary nitrate on hypertension. Wilcox postulated that during hypertension renal oxidative stress decreases NO bioavailability through the production of reactive oxygen species, and suggested that the kidney may mediate the effects of dietary nitrate on blood pressure 6l‐arginine concentrations in the heart. It is postulated that this l‐arginine could then be redirected from the arginase‐dependent production of ornithine and urea to the production of NO. Arginase activity is associated with numerous cardiac pathologies As previously mentioned, nitrate is likely a key factor behind the cardioprotective effects of green leafy vegetables , 18, 1177Obesity has long been associated with inflammation in insulin target tissues, especially adipose tissue depots, which is suggested to contribute to the pathology of the metabolic syndrome 8Having initially been considered an inactive, then toxic and carcinogenic molecule, dietary inorganic nitrate is gradually being identified as a modulator of numerous aspects of the metabolic syndrome, with therapeutic potential against obesity, diabetes and hypertension. In the wake of recent work identifying the ability of nitrate to induce the browning response in adipose tissue and alter energy balance, lower blood pressure and decrease the haematocrit, the means by which nitrate protects against the metabolic syndrome are slowly being elucidated.However, mechanistically, the picture remains incomplete. The identification of the nitrate‐nitrite‐NO pathway appeared to clarify that the bioactive effects of dietary nitrate were brought about by increasing bioavailable NO and downstream cGMP signalling. However, the recent discoveries that nitrate can inhibit the activity of both NADPH oxidase and arginase suggests the activity of nitrate is far more complex.Furthermore, much of the in vivo work on dietary nitrate has been conducted in rodents, and there is a clear need to confirm these studies in humans to determine the therapeutic potential of dietary nitrate within the population. Nevertheless, it is evident that the use of dietary inorganic nitrate as a tool to tackle the metabolic syndrome has great potential. Dietary adjustment, with a focus on food groups high in nitrate, such as green leafy vegetables, provides an important starting point, but perhaps it is also time to reassess the attitudes to, and strict controls on, nitrate in our diets.The authors have declared no conflict of interest.


In 2010, a tissue‐engineered trachea was transplanted into a 10‐year‐old child using a decellularized deceased donor trachea repopulated with the recipient's respiratory epithelium and mesenchymal stromal cells. We report the child's clinical progress, tracheal epithelialization and costs over the 4 years. A chronology of events was derived from clinical notes and costs determined using reference costs per procedure. Serial tracheoscopy images, lung function tests and anti‐HLA blood samples were compared. Epithelial morphology and T cell, Ki67 and cleaved caspase 3 activity were examined. Computational fluid dynamic simulations determined flow, velocity and airway pressure drops. After the first year following transplantation, the number of interventions fell and the child is currently clinically well and continues in education. Endoscopy demonstrated a complete mucosal lining at 15 months, despite retention of a stent. Histocytology indicates a differentiated respiratory layer and no abnormal immune activity. Computational fluid dynamic analysis demonstrated increased velocity and pressure drops around a distal tracheal narrowing. Cross‐sectional area analysis showed restriction of growth within an area of in‐stent stenosis. This report demonstrates the long‐term viability of a decellularized tissue‐engineered trachea within a child. Further research is needed to develop bioengineered pediatric tracheal replacements with lower morbidity, better biomechanics and lower costs. This case presents a 4‐year follow‐up of a tissue‐engineered pediatric tracheal transplant and reports on the longterm viability of this approach and areas for further research. GMPgood manufacturing practiceGOSHGreat Ormond Street HospitalICUintensive care unitThe application of tissue engineering to restore a segment of airway in those with long‐segment tracheal stenosis resistant to treatment is now realistic. Such therapies remain highly controversial, however, due to uncertainty regarding long‐term benefit, quality of life and comparative cost. A 5‐year follow‐up of the first tissue‐engineered airway in an adult reported normal lung function and a good quality of life long‐term albeit with the need for serial insertions of airway stents In 2012, we reported a 2‐year follow‐up of the first tissue‐engineered tracheal transplant in a child using a decellularized deceased donor trachea repopulated with the patient's own respiratory epithelial cells and mesenchymal stromal cells Clinical data were collated from the patient notes documenting his long‐term care at Great Ormond Street Hospital (GOSH). The Peds QL parent‐proxy assessment tool was used as a validated method of analyzing health‐related quality of life in children with chronic disease and associated learning disabilities ® Innovation Suite . Cross‐sectional area of the trachea was calculated following methods previously described Neck and chest computed tomography scans at 3 months (June 2010), 42 months (September 2013) and 49 months (April 2014) following transplantation were compared. 3D reconstructions of the tracheal lumen were performed using MimicsEndoscopy images of the transplanted trachea were obtained during microlaryngobronchoscopy performed at 15 days, 6, 15 and 42 months following transplantation using a 15‐mm zero degree Hopkins Rod and image capture .Biopsy samples were routinely processed following formalin fixation, followed by paraffin wax embedding and cutting of 4‐μm sections onto glass slides for hematoxylin and eosin (H&E) staining and immunostaining using an automated immunostainer . Samples of cilia were obtained during routine flexible bronchoscopy by brushing the trachea with a 2‐mm cytology brush  The costing for the clinical items were obtained using a combination of NHS reference costs (2010–2011 period) per procedure appropriate for a male child and typical costs for complex tracheal patients provided by GOSH. The laboratory costs were estimated from data gathered from the good manufacturing practice (GMP) laboratories where further grafts were constructed to treat two later patients (data not shown).Following transplantation at GOSH in March 2010, the child returned to the intensive care unit  where he remained an inpatient for 8 days, before being discharged from critical care The child was discharged home in August 2010, but required a number of return visits to GOSH to address retained secretions, granulation and a malacic segment in the distal transplanted trachea, necessitating the insertion of further stents, one bioabsorbable and two self‐expanding nitinol. There were two further admissions to ICU during the first year follow up period and one ICU admission during the second year follow up period.By 6 months, the airway was sufficiently stable to allow the child to return to school and the need for repeated interventions declined up to late 2013 when an infection and stenosis within the tracheal stents and un‐transplanted left main bronchus necessitated intervention. Following balloon dilatation of the trachea and left main bronchus, the child made a good recovery and has now returned to full‐time education.A Peds QL 4 · 0 parent proxy quality of life assessment was completed in May 2014. Scores were 50, 60, 55 and 35 for physical, emotional, social and school subscales, respectively, with a total score of 50 out of 100. Since transplantation, the child has grown 15 cm in height to 168 cm and gained 21 kg in weight to 58 kg.Figure Microlaryngoscopy 15 days after transplantation showed a dense web covering the stent and partially occluding the lumen Figure A. At 42 A section of the excised homograft trachea exhibited normal ciliated respiratory type epithelium Figure A. A biopBrushings on routine endoscopy in April 2014 demonstrated ciliated epithelial cells. Ciliary beat frequency was within the normal range at 10.7 Hz  with a normal ciliary beat pattern. On electron microscopy, structurally normal ciliary axonemes were observed.The total clinical cost of the transplant and follow‐up care to March 2014 was calculated to be US$565 414 (£/$ exchange rate 1:1.56) Table . The labThe use of a tissue‐engineered tracheal transplant is currently a potential treatment of last resort. In the reported case, all conventional therapies had failed and the technique was used on compassionate grounds in an urgent setting. This intervention has not only preserved life for more than 4 years, but has enabled the child to mature, continue education and be free of medical intervention for long periods. Indeed, the timing and frequency of interventions between the 1st and 4th years following transplantation compare favorably to children suffering from recurrent tracheal stenosis or those with metallic tracheal stents treated in our institution.The Peds QL score is consistent with a child suffering from a complex chronic disease in vivo in humans prohibits observations of the fate of such epithelium. A number of preclinical studies have indicated re‐epithelialization occurs from migration of cells from the wound edge following tracheal transplantation The emergent nature of our case necessitated the use of intraoperative native airway epithelial patches to re‐epithelialize the graft Alternative protocols for epithelizing tracheal grafts use cadaveric tracheal cartilage prevascularized within radial forearm fascia and lined with autologous buccal mucosa A cross‐sectional centerline analysis suggested growth at the proximal and distal portions of the trachea over time. Cross‐sectional area is restricted at 15–45 mm below the first tracheal ring in all scans corresponding to an area of in‐stent stenosis. It is not possible to determine whether the transplanted trachea would have grown had in‐stent stenosis been avoided. Whether the transplanted section includes viable tracheal cartilage can also not be determined with routine investigations. However, a complete mucosal layer, dynamic movement of the airway and demonstrable tracheal rings on endoscopy indicate the transplanted trachea is functioning as a cartilaginous frame. Of note, recurrent stenosis has been reported in the other example of a decellularized airway transplant We also report, for the first time to our knowledge, the use of computational fluid dynamic simulations within a reconstructed trachea. This validated technique provides detailed information on how distinct geometrical features correlate with airflow distribution and provides information on pressure The total cost of this tissue‐engineered tracheal transplant is greater than conventional forms of airway management; however, our approach is only indicated for a small subset of children with airway stenosis and the calculated costs herein are unlikely to be prohibitive within a modern healthcare system. Our observations and interpretations of the problems the child faced in this early period have fed back into substantially improved tissue‐engineering protocols, and an advanced GMP process which will lead to substantial, iterative, reductions in hospital stay, complications and, thus, costs.To our knowledge, this is the first long‐term follow‐up report of a child receiving a tissue‐engineered trachea. Further research is required to develop bioengineered pediatric tracheal replacements with lower morbidity, better biomechanics and at a cost acceptable to healthcare providers. Future clinical trials of such constructs should include comprehensive assessments of airway physiology, biology, quality of life, and health economics.American Journal of Transplantation.The authors of this manuscript have no conflicts of interest to disclose as described by the Supplementary Materials and MethodsClick here for additional data file.


Tanzania and Vietnam using pyrosequencing of 16S rRNA genes, and correlated with a number of intrinsic environmental factors such as pH, temperature, organic matter content/composition and geographical factors. Clear differences were observed at the operational taxonomic unit, family and phylum level in terms of richness and community composition between latrines in Tanzania and Vietnam. The results also clearly show that environmental variables, particularly substrate type and availability, can exert a strong structuring influence on bacterial communities in latrines from both countries. The origins and significance of these environmental differences are discussed. This work describes the bacterial ecology of pit latrines in combination with inherent latrine characteristics at an unprecedented level of detail. As such, it provides useful baseline information for future studies that aim to understand the factors that affect decomposition rates in pit latrines.Improving the rate and extent of faecal decomposition in basic forms of sanitation such as pit latrines would benefit around 1.7 billion users worldwide, but to do so requires a major advance in our understanding of the biology of these systems. As a critical first step, bacterial diversity and composition was studied in 30 latrines in  Annually, 2.4 million deaths could be prevented through improved hygiene, drinking water and sanitation  on pit latrine community composition has yet to be determined. The work presented in this paper investigates the diversity and composition of bacterial communities found in pit latrines from different geographical regions, in tandem with physical and bio‐chemical characteristics  of pit latrine material, in order to better understand the decomposition process.et al., The observed diversities in the individual latrine samples were relatively high compared with the human gut  (Table P < 0.01) although it was not observed for the total diversity estimates suggesting that the Tanzanian latrines contain a longer tail of low abundance or rare OTUs. We also observed lower phyla and family numbers in the Tanzanian latrines per sample compared with Vietnamese latrines  and  respectively.To account for the impact of sequencing depth on richness, we used parametric methods to extrapolate to total diversities. These values Table  and the Methods section characterizing the latrines . From this figure, we observed that total solids (TS) and pH are positively correlated with each other but are negatively related to all the other environmental variables, which are then mostly positively correlated with each other.We also explored correlations between the different intrinsic environmental variables . More detailed descriptions are given in the The correlations between these variables, and the rarefied 3% OTU richness are shown in Fig. Firmicutes, followed by the Bacteroidetes, Proteobacteria and Actinobacteria in order of decreasing abundance. There are clear differences between the proportional abundances of phyla between the two countries, and these differences are found to be highly significant at the whole community level from the permutational multivariate analysis of variance (P < 0.001), explaining 31% in the variance of the community structures. From fitting a Dirichlet‐multinomial model to the two countries separately (Supplementary Table S1), we can show that Proteobacteria, Actinobacteria, Deinococcus‐Thermus and Verrucomicrobia are all significantly more proportionally abundant in the Vietnamese latrines, whereas the Firmicutes, Synergistetes and Spirochaetes are more prominent in Tanzania.The bar plot in Fig. P < 0.001). The Dirichlet‐multinomial model finds a large number of families that differ significantly in abundance between the countries (Supplementary Table S2). These results are summarized in Fig. Synergistaceae, and the Firmicutes lineages Clostridiaceae, Ruminococcaceae, Incertae Sedis XI and Erysipelotrichaceae were associated with Tanzanian pits, while Xanthomonadaceae, Actinomycetales, Flavobacteriaceae and Trueperaceae were more likely to be found in Vietnamese pits. The impact of the country where the latrine was sampled on latrine community composition is also detected at the 3% OTU level, our proxy for species, and 11% of the variation in 3% OTU community structure is explained by the country of origin (P < 0.001).These differences continue at the family level. The country that the latrine was derived from explains 14% of the variation in family‐level community structure . Depth was significant (P = 0.009) but only explained 2% of the variance. The same was found for the 3% OTU compositions (Supplementary Table S6), with explained variances of 15%, 48% and 2% respectively. Figure In addition to the impact of geographical location described above, to explore the impact of both latrine identity  and the depth within latrines that the samples were taken from (intra‐latrine variability), on the community composition we performed a multivariate analysis of variance at the phylum, family and 3% OTU levels. At the phylum level, latrine identity had a further significant impact on community composition but depth did not (Supplementary Table S4). Similarly, at the family level (Supplementary Table S5), country of origin explained 22% of the variance but including latrine identity explained a further 47%, and both of these variables had highly significant These analyses establish that both latrine identity and country of origin have a major impact on community structure at all taxonomic levels, while depth within a given latrine has a lesser, but still significant, impact.We next attempted to determine the extent to which this is due to differences in the local intrinsic environment of the latrines and the extent to which it may depend on other factors, such as the gut microbiota or diets of the individuals using the latrines, or indeed the local soil microbiota by including the measured intrinsic environmental variables in the multivariate analysis of variance. The results are shown for family composition in Supplementary Table S7. Many of the environmental variables are shown to be significantly correlated with the latrine community structure. In fact, only those variables associated with COD fail to have a significant impact , suggesting that the amount of resources available is less important than the type of resources (e.g. % carbohydrate) or abiotic environment (pH and temperature) in structuring the community. After incorporating the intrinsic environmental data, latrine identity still explains a large amount of variation (33%), whereas the country, while still significant, is less important (6%). Depth is no longer a significant driver of community structure. This implies that all the effect of depth was due to correlation with the environment and that most of the difference between Tanzania and Vietnam is driven by the intrinsic environmental factors. However, the remaining highly significant impact of latrine identity implies a role for local factors beyond the intrinsic environment or for environmental factors that we did not measure.There are clearly large differences between the latrine microbiota of the two countries. To investigate whether intrinsic environment controls microbiota within a country, we separated the samples by country of origin and performed permutational multivariate analysis of variance on the family level community composition for each separately. The results are given for Vietnam and Tanzania in Supplementary Tables S8 and S9 respectively. In Vietnam more significant results were found with TS, volatile solids (VS), volatile fatty acids (VFA) and protein all impacting the pit latrine microbiota, whereas only TS and %CODs/CODt were significant in Tanzanian latrines.Actinomycetales and Rhodobacteraceae) to gut associated organisms . The nature of this gradient is given in more detail, complete with estimated contours, in Supplementary Fig. S3.In order to explore the relationships within Vietnam in more detail, we generated a NMDS plots for both the samples and all the families with an average abundance of greater than 1% Fig. . Onto thMicrobial populations within pit latrines are likely derived from a combination of species originating from human faeces and microbes from the environment, which might enter the pit latrine through the addition of household waste, or from surrounding soil or groundwater.et al., et al., When faecal bacterial communities leave the gut, their survival and persistence will depend on their ability to respond to changing environmental surroundings. This ability is governed by cellular physiology, stress response mechanisms and the physical properties of the bacterium. Examples of these include adaptations to varying availability of nutrients e.g. low iron, and variation in atmospheric oxygen percentage, and the ability to survive by changing physical properties such as spore formation , and latrine maintenance approaches like different emptying patterns that can potentially explain the differences in pit latrine microbiota composition observed between the two countries and between each latrine. Tanzanian users have a predominantly vegetarian diet compared with the Vietnamese population, which may explain why in Tanzania we found bacteria groups associated with cellulose degradation.between latrines are far greater than differences within latrines. One potential explanation for this is that the rate of growth and change in the latrine microbiota may slow as resources become depleted. One limitation of 16S rRNA gene sequencing is that it is unable to distinguish between live and dead/inactive microbes, potentially limiting our ability to detect the level of growth and activity of the microbial communities present at different depths within each latrine. Nonetheless, latrine identity and country of origin were both shown to have a major impact on community structure at all taxonomic levels. However, the local intrinsic environment of the latrines has a greater influence on the community structure. Only variables associated with COD failed to have a significant impact, suggesting that total available resources are less important than the type of resources or abiotic environment in structuring the community. Previous work . Tanzania has a tropical climate; typically it experiences a short rainy season from November to December and longer heavier rains from April to May. Average daily temperatures range from 19°C to 32°C.In Vietnam, the neighbouring communes of Hoang Tay and Nhat Tan, Ha Nam, province  were selected for this study. The area is predominantly agricultural with intensive rice cultivation and animal husbandry . Pits in Vietnam are raised in order to allow excreta collection for use in agriculture, and pit volume is much smaller than that typically found in Tanzania, with the vaults 1 metre deep at maximum . Many households separate urine from faeces and paper is used for anal cleansing. The use of ash or lime to reduce odours is widely practised. Diet in Vietnam is diverse though tends to consist of rice, noodles, a variety of green vegetables and meat or poultry. Average daily temperatures range from 3°C to 38°C.A total of 30 latrines were selected for this study. Latrines were selected based on the number of users, design characteristics, such as presence or absence of a roof, and materials used for construction. Eight latrines were selected in Tanzania, while 22 were selected in Vietnam. Characteristics of selected latrines from both countries are presented in Table In‐situ temperature and pH measurements were taken with a hand‐held meter . Pit latrine samples were collected in sterile containers and transported in a cool box for further analysis on the same day as collection. Samples were analysed for TS and moisture content as indicators of the amount of inorganic and water content in a sample respectively. Total and soluble COD and VS were measured to give a gross and indirect indication of the organic matter content present in each sample, and measurements of carbohydrate and protein content provided information about the type of organic substrates present in each sample. Volatile fatty acids are important intermediates and metabolites in biological processes, and their presence in a sample matrix is often indicative of bacterial activity. Ammonia and total phosphate were also measured in each sample as their concentrations are determined by microbiological processes. All these tests were selected to describe the prevailing latrine environmental conditions, which we hereafter refer to as intrinsic environmental factors.Approximately 200 g of material was collected at 20 cm intervals from top to bottom in each pit latrine. Latrines in Tanzania were much deeper and as a result more samples could be collected from each latrine. Depending on the consistency of the material in the pit, samples were collected with a standard soil auger , or with a sterile 150 ml plastic container attached to the soil auger. In cases where the liquid layer was deeper than 20 cm, samples were collected with a sampler that was specifically designed for this project, which allowed us to collect samples from deep layers. The sampler consisted of a sampling tube that covers a gate with a spring‐loaded fin device . The sampler was inserted into the latrine and, once the required layer was reached, the device opened and a lever was used for collecting a small amount of sample. ddH2O. After homogenization and dilution, the mixture was passed through a 0.45 μm filter. Samples were analysed using HACH‐Lange test kits and methods  following which 1 g was diluted in 20 ml of ds HACH, ; for totds HACH, , with saSamples for DNA sequence analysis were kept at −80°C until DNA extraction was performed. Deoxyribonucleic acid was extracted from the samples following the kit protocol for the FastDNA SPIN kit for soil  in conjunction with a FastPrep‐24 bead‐beading instrument .CCATCTCATCCCTGCGTGTCTCCGACTCAG‐barcode‐CCGTCAATTCMTTTRAGT‐3′. The 338F primer contained the 454 Lib‐L kit ‘B’ adaptor (bold) followed by the 16S rRNA gene primer: 338F – 5′‐CCTATCCCCTGTGTGCCTTGGCAGTCTCAGACTCCTACGGGAGGCAGCAG‐3′. Polymerase chain reaction (PCR) amplification of 16S rRNA genes from the extracted DNA involved initial denaturation at 94°C for 2 min; 20 cycles of denaturation (30 s at 94°C), annealing (30 s at 53°C), extension (68°C for 2 min). Amplification was performed using Accuprime Taq DNA polymerase from Life Technologies  using manufacturer's instructions. Polymerase chain reaction amplicons were generated in quadruplicate for each sample and then pooled. Amplicon concentrations were checked for each sample using a Qubit from Life Technologies  and then added in equimolar amounts to a mastermix for sequencing. Polymerase chain reaction products were cleaned using the Wizard PCR product purification kit  and were then pyrosequenced at the Wellcome Trust Sanger Institute using the Lib‐L kit on the 454 GS FLX Titanium System .Primers 338F and 926R, targeted towards variable regions 3 to 5 of the bacterial 16S rRNA gene, were used to generate amplicons from each sample. Each 926R primer contained a unique 12‐mer barcode sequence, plus the standard 454 Lib‐L kit ‘A’ adaptor (shown in bold), in the following configuration: 926R primers 5′‐et al., et al., et al., et al., The sequence data was processed using the AmpliconNoise pipeline  allowing the variance in a multivariate data set to be partitioned between both continuous and categorical variables .Fig. S3. Non‐metric multidimensional scaling of family compositions of Vietnamese latrine samples with bubble plots and gradients for the four environmental variables judged significant in Supplementary Table S7  plotted using the envirosurf function of vegan. The size of the bubble indicates the value of the environmental variable.Fig. S4. Sampler device used to sample deep latrines with more liquid consistency material. (https://www.youtube.com/watch?v=q5JDu0emYxk).Table S1. Percentage relative abundance of the 30 phyla in the Dirichlet means in the 55 Vietnamese samples and 24 Tanzanian samples. The upper and lower 95% credible intervals are also given. These are calculated as the maximum posterior estimate (MPE) minus/plus two standard deviations as calculated from the inverse Hessian. Phyla are ranked in order of their contribution to the total mean difference between the two means. This is the second from last column in the table. The total difference was 37%. The cumulative fraction of this difference accounted for by each family is given in the last column of the table.*Indicates those phyla that differ significantly between groups in that their confidence intervals do not overlap. Phyla that were more proportionally abundant in Tanzanian latrines are highlighted in blue; those that were more proportionally abundant in Vietnamese latrines are highlighted in red.Table S2. Percentage relative abundance of the first 30 out of 180 families in the Dirichlet means in the 55 Vietnamese samples and 24 Tanzanian samples. The upper and lower 95% credible intervals are also given. These are calculated as the maximum posterior estimate (MPE) minus/plus two standard deviations as calculated from the inverse Hessian. Families are ranked in order of their contribution to the total mean difference between the two means. This is the second from last column in the table. The total difference was 37%. The cumulative fraction of this difference accounted for by each family is given in the last column of the table.*Indicates those families that differ significantly between groups in that their confidence intervals do not overlap. Families that were more proportionally abundant in Tanzanian latrines are highlighted in blue; those that were more proportionally abundant in Vietnamese latrines are highlighted in red.Table S3. Percentage relative abundance of the first 10 out of 12,335 3% OTUs in the Dirichlet means in the 55 Vietnamese samples and 24 Tanzanian samples. The upper and lower 95% credible intervals are also given. These are calculated as the maximum posterior estimate (MPE) minus/plus two standard deviations as calculated from the inverse Hessian. OTUs are ranked in order of their contribution to the total mean difference between the two means. This is the second from last column in the table. The total difference was 129%. The cumulative fraction of this difference accounted for by each family is given in the last column of the table.*Indicates those families that differ significantly between groups in that their confidence intervals do not overlap. OTUs that were more proportionally abundant in Tanzanian latrines are highlighted in blue; those that were more proportionally abundant in Vietnamese latrines are highlighted in red.Table S4. Permutational multivariate analysis of variance using Bray‐Curtis distances for the phylum latrine composition data as a function of country of origin and latrine identity (adonis function vegan – Oksanen et al., Table S5. Permutational multivariate analysis of variance using Bray–Curtis distances for the family latrine composition data as a function of country of origin and latrine identity (adonis function vegan – Oksanen et al., Table S6. Permutational multivariate analysis of variance using Bray‐Curtis distances for the 3% OTU latrine composition data as a function of country of origin and latrine identity (adonis function vegan – Oksanen et al., Table S7. Permutational multivariate analysis of variance using Bray‐Curtis distances for latrine composition family data as a function of intrinsic environmental variables, country of origin and latrine identity (adonis function vegan – Oksanen et al., Table S8. Permutational multivariate analysis of variance using Bray–Curtis distances for Vietnam latrine microbial composition family data as a function of intrinsic environmental variables (adonis function vegan – Oksanen et al., Table S9. Permutational multivariate analysis of variance using Bray–Curtis distances for Tanzanian latrine microbial composition family data as a function of intrinsic environmental variables (adonis function vegan – Oksanen et al., Click here for additional data file.


After temporarily implementing stricter emissions standards and shutting down nearby industrial polluters, pollutant measurements between June and October 2008 showed reductions of 18–59% compared with pre-Olympics levels. But once the Games were over, air pollution began to rise again.The current study included two month-by-month analyses. The authors included only term births (37–41 weeks) and births in which the infant was neither large nor small for gestational age.In the first analysis, the researchers assessed records for 71,803 births at Beijing Obstetrics and Gynecology Hospital to determine which month of each pregnancy fell during the Games (8 August–24 September 2008). They compared the birth weights with those in which the same month of pregnancy occurred in the same calendar period either one year earlier or one year later. They found the strongest association when the eighth month fell during the Games, with babies born in 2008 weighing an average of 23 g (0.8 oz) more than the babies born in 2007 or 2009.2.5, CO, NO2 and SO2 during each month of pregnancy. They then looked at 32,506 birth records in which women were pregnant for at least one full month during the Games. They found that interquartile increases in the pollutants during the eighth month of pregnancy were associated with reductions of 17–34 g in birth weight, depending on the pollutant. They found no such associations for increased air pollution during other months of pregnancy.In the second analysis, the team used data on air pollution, temperature, and humidity gathered at a site in central Beijing to calculate average monthly concentrations of PMLead author David Rich, a public health sciences professor at the University of Rochester Medical Center, says it will take further study to figure out why the eighth month may be a critical time for pregnant women and air pollution exposure. “It could be that the early months are important—if pollution levels went down in the first or second month, it could have a beneficial effect on the baby’s growth—but then the pollution levels went back up after the Olympic Games, so any improvement in birth weight may be ‘washed away.’” He says another possibility is that since late pregnancy is when the fetus grows most rapidly, air pollutant exposure during this time period may affect some biologic mechanism—perhaps the placenta’s ability to deliver nutrients—that impedes fetal growth.10 and PM2.5 across the span of a pregnancy were associated with increases in the risk of low birth weight and decreased birth weight among term births.A 2014 meta-analysis of previous studies showed that increased concentrations of PMDavid Savitz, a professor of epidemiology at Brown University School of Public Health, says he found the new results more persuasive than previous studies looking at the impact of air pollution in reducing birth weights. “Some people are already convinced that it’s a known fact that pollution [lowers birth weights],” he says. “I’m not among them. I’m pretty skeptical about it.” But while this study is far from the final word on the issue, he says it “really adds a significant amount of weight to the evidence in support of an effect on birth weight.” Savitz was not involved in the study.Lyndsey Darrow, an assistant professor of epidemiology at Emory University who also was not involved in the study, says she would like to see a similar study but with a longer duration of reduced air pollution. “Ideally, future complementary studies could take advantage of other natural experiments to investigate the cumulative impact of reducing air pollution over the full course of pregnancy,” she says, adding, “Of course, such natural experiments are hard to come by.”


Following the previous two Educational Research Symposiums in 2012 and 2013 which have received overwhelming support from vocational education and training (VET) practitioners within the Vocational Training Council (VTC) in Hong Kong, the scope of this Symposium was expanded and renamed as Practical Social and Industrial Research (PSIR) symposium. The aim of this Symposium was to provide a platform for researchers and practitioners in VET to share and exchange their knowledge and experience with one another. This year’s theme “Advancing VET Practice” incorporated both empirical and practice-based researches that addressed VET issues.The symposium included 3 keynote speeches entitled “On Translating Technology Research into Industrial Applications” presented by Prof Stephen Wong , “Assisted Technology for Daily Living” presented by Prof Raymond Tong , “TVET as an Important Factor in Country’s Economic Development” presented by Dr. Margarita Pavlova . After the keynote speech, a total of 3 poster and 9 oral presentations were held covering the sub-themes of “Students and Teachers in VET” and “Teaching and Learning in VET”. Not only the latest research, ideas and innovations that could help to strengthen VET were showcased, evidence-based practices in VET were also shared to inspire practitioners and researchers. The event provided participants with a great opportunity to network with each other and fostered partnership that would support future cooperation.The PSIR symposium is a regular annual event organised by the Vocational Training Council in Hong Kong. Potential focus areas in 2015 will include vocational education and training, applied research in social innovation or related issues, and environment and sustainability.


Increased neuronal spontaneous firing rates have been observed throughout the central auditory system after trauma to the cochlea and this hyperactivity is believed to be associated with the phantom perception of tinnitus. Previously, we have shown in an animal model of hearing loss, that an acute injection with furosemide can significantly decrease hyperactivity after cochlear trauma and eliminate behavioral evidence of tinnitus of early onset. However, furosemide also has the potential to affect cochlear thresholds. In this paper, we measured the effects of a chronic (daily injections for 7 days) furosemide treatment on the spontaneous firing rate of inferior colliculus neurons and on cochlear thresholds in order to establish whether a beneficial effect on hyperactivity can be obtained without causing additional hearing loss. Guinea pigs were exposed to a 10–kHz, 124 dB, 2 h acoustic trauma, and after 5 days of recovery, were given daily i.p. injections of 80 mg/kg furosemide or an equivalent amount of saline. The activity of single IC neurons was recorded 24 h following the last injection. The furosemide treatment had no effect on cochlear thresholds compared to saline injections but did result in significant reductions in spontaneous firing rates recorded in inferior colliculus. These results that suggest a long-term beneficial effect of furosemide on hyperactivity after cochlear trauma may be achievable without detrimental effects on hearing, which is important when considering therapeutic potential. Tinnitus, a phantom auditory perception, is generally thought to be the results of abnormal activity along the central auditory pathways that is often triggered by damage to the auditory receptor, the cochlea . One forHyperactivity is thought to be the result of an increased central gain following peripheral denervation  and avaiIf indeed hyperactivity plays a role in the generation of tinnitus, these data suggest that tinnitus of recent onset may be sensitive to treatments affecting the activity of the auditory nerve. This was confirmed in a recent paper in which it was reported that an acute injection with furosemide, at a dose that caused a reduction of the spontaneous activity of the auditory nerve and a reduction of central hyperactivity, also caused an elimination of the early behavioral signs of tinnitus in guinea pigs exposed to cochlear trauma . These dThe effects of acute furosemide administration on hyperactivity and tinnitus suggest a therapeutic potential for furosemide in treating early onset tinnitus, although, it is likely that chronic treatment would be needed to achieve ongoing suppression. However, it is also known that furosemide can affect cochlear thresholds, causing hearing loss. Therefore, the present study investigated whether chronic administration of furosemide could decrease hyperactivity without deleterious effects on cochlear thresholds. The effects of a chronic (administered daily for 7 days) furosemide treatment on central hyperactivity and cochlear thresholds were studied in our guinea pig model of hearing loss and hyperactivity after acoustic trauma.Nine adult pigmented guinea pigs of either sex were used. Animals weighed between 290 and 500 g at the time of acoustic trauma. The experimental protocols conformed to the Code of Practice of the National Health and Medical Research Council of Australia, and were approved by the Animal Ethics Committee of The University of Western Australia.Animals received a sub-cutaneous injection of 0.1 ml atropine sulfate (0.6 mg/ml) as pre-medication, followed by an intraperitoneal injection of diazepam (5 mg/kg) and an intramuscular injection of Hypnorm . The surgery commenced when there was an absence of the foot withdrawal reflex indicating deep anesthesia. Animals were placed on a heating pad in a sound proof room and positioned in hollow ear bars. Eyes were protected from drying out by application of an eye-gel. A small incision was made behind the left ear and the bulla was opened to visualize the cochlea in order to place an insulated silver wire electrode on the cochlear round window. This enabled recording a compound action potential (CAP) audiogram  for the All sound stimuli were presented in a closed sound system through a 1/2″ condenser microphone driven in reverse as a speaker . Pure tone stimuli were synthesized by a computer equipped with a DIGI 96 soundcard connected to an analog/digital interface . Sample rate was 96 kHz. The interface was driven by a custom-made computer program , which was also used to collect single neuron data during the final experiments. CAP signals were amplified, filtered (100–3 kHz bandpass) and recorded with a second data acquisition system .When cochlear sensitivity was within the normal range , animalsn = 5) or a saline control group (n = 4). Intraperitoneal injections of 80 mg/kg furosemide  were administered daily for 7 days (day 5 till day 11 post-trauma). Single neuron recordings in CNIC and CAP threshold measurements (as described above) were taken 1 day after the final furosemide or saline treatment (day 12 post-trauma).Animals were allowed to recover for 5 days after acoustic trauma and were then assigned randomly to either a furosemide group (2 and 5% CO2). The electrocardiogram was continuously monitored and heart rate never increased over pre-paralysis levels at any stage of the experiments. After the animals were mounted in hollow ear bars, the left and right cochleae were exposed and CAP audiograms were recorded on both sides with a silver wire placed on the round window as described for the recovery procedures. Paralysis was then induced with 0.1 ml pancuronium bromide (2 mg/ml intramuscularly).Animals received a sub-cutaneous injection with 0.1 ml atropine followed by an intraperitoneal injection of Nembutal  and a 0.15 ml intramuscular injection of Hypnorm. Anesthesia was maintained with full Hypnorm doses every hour and half doses of Nembutal every 2 h. When deep anesthesia was obtained as determined by the absence of the foot withdrawal reflex, the areas of incision were shaved and animals were placed on a heating blanket in a sound proof room and artificially ventilated on carbogen  a small craniotomy overlying the visual cortex was performed and a glass-insulated tungsten microelectrode  was advaHearing loss can result in changes to the tuning curves of neurons, so that determination of CF based on the lowest threshold of tuning curves in the damaged region can be erroneous . We therTo identify statistically significant differences in spontaneous firing rates between chronic intraperitoneal injections with furosemide or saline, the Mann–Whitney test was used when comparing all neurons in each group with each other and the Kruskall–Wallis test and Dunn’s multiple comparison post-test when comparing the neurons sorted according to CF range. Statistical analysis of CAP threshold changes at each frequency was performed using a one-way ANOVA.Figure p < 0.0001).In total, data were obtained from 362 neurons in the CNIC of saline treated animals and 398 neurons from the CNIC of furosemide treated animals. Spontaneous firing rates in saline treated animals varied between 0 and 77.6 spikes/s with a mean spontaneous firing rate of 6.5 ± 0.65 median 1.5) spikes/s and in furosemide treated animals between 0 and 52.5 spikes/s with an average rate of 3.45 ± 0.36 spikes/s (median 0.4) . Statistical analysis showed a significant decrease after furosemide in both CF groups (p < 0.01 for CFs <12 kHz and p < 0.0001 for CFs >12 kHz). The largest effect of furosemide was seen in the frequency region that showed the most prominent hyperactivity.In order to investigate whether furosemide was equally affecting the spontaneous firing rate of all neurons throughout the tonotopic map of the IC, neurons were also sorted according to CF. The spontaneous firing rates in saline and furosemide treated animals were compared for neurons with a CF < 12 kHz (non-hearing loss region) and a CF > 12 kHz (hearing loss region). Results from this analysis are shown in Figure The results presented in this paper show that 1 day after a 7-day course of furosemide central hyperactivity in inferior colliculus is reduced without effects on peripheral auditory thresholds. These results are in agreement with the results of single acute furosemide injections, which also result in suppression of hyperactivity and they show that chronic administration can have beneficial effects centrally without causing further hearing loss. Further experiments are required to investigate the duration of the benefit from repeated administration and whether this is accompanied by a concomitant reduction of tinnitus.Importantly, chronic administration of furosemide did not affect the magnitude of the hearing loss following acoustic trauma compared to saline injections. This indicates that furosemide did not have long-lasting effects on cochlear thresholds, even though studies do suggest a possible short-term effect on sound-evoked responses in the cochlea –20. FuroOverall spontaneous firing rates were significantly reduced after the chronic treatment with furosemide as compared to saline. This is the same effect as observed after acute administration of furosemide at the same dosage . In addiWhat is the cause of the reduction in spontaneous firing rates in IC? Firstly, the level of hyperactivity is known to be affected by the magnitude of hearing loss ; howeverIn view of the fact of our previous study showing that acute administration of furosemide lowers the hyperactivity in IC and eliminates the behavioral signs of tinnitus , the preThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scientific Reports6: Article number: 1814010.1038/srep18140; published online: 01052016; updated: 03082016The original version of this Article contained an error in the Abstract.“These considerations give a new perspective on previous results perspective, and suggest that studies of biotic effects on GHG emissions from dung pats on a global scale are a priority for current research.”now reads:“These considerations give a new perspective on previous results, and suggest that studies of biotic effects on GHG emissions from dung pats on a global scale are a priority for current research.”This has now been corrected in the PDF and HTML versions of the Article.


Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision‐making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrin‐associated adhesion complex composition.Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literature‐based reference set of cell adhesion‐related components and using ontological and protein–protein interaction network analyses.Adhesion complex‐specific proteins in MSCs were identified that comprised predominantly cell adhesion‐related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain‐containing proteins in MSC adhesion complexes were highlighted, which may act as force‐sensing components.These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention. ECMextracellular matrixFNfibronectinLIM domainLIN‐11, Isl1, and MEC‐3 domainMSCmesenchymal stem cellPDLd‐Lysinepoly‐The use of mesenchymal stem cells (MSCs) for tissue engineering and regenerative medicine applications is attractive, in part due to their ability to differentiate into multiple cell types and their ease of expansion in vitro. MSC growth and differentiation are influenced by the extracellular environment, which is sensed by integrin cell‐surface receptors binding to extracellular matrix (ECM) components. This binding allows the formation of intracellular protein complexes that signal to determine specific cellular outcomes in response to different environmental cues. A more detailed knowledge of how MSCs sense the mechanical, compositional and topological features of the ECM via integrins and their associated proteins will aid our understanding of the regulation of MSC growth and differentiation, and in turn will benefit clinical applications of these cells.Multipotent mesenchymal stem cells (MSCs) have the capacity to differentiate into multiple mesenchymal lineages Integrins are a family of cell‐surface ECM receptors that mediate signalling across the plasma membrane from the extracellular environment to the actin cytoskeleton d‐Lysine (PDL) were used as a control substrate that allowed cell spreading but did not support engagement of integrin and the formation of integrin‐associated adhesion complexes  spread on fibronectin (FN) was confirmed by immunofluorescence staining for the well‐defined adhesion complex components vinculin, integrin‐linked kinase and active integrin β1  For proteomic analysis of integrin‐associated complexes, MSCs were spread on FN and the control ligand PDL and the isolation procedure performed in triplicate. Samples were analysed by LC‐MS/MS. In total, from all conditions, MS analyses identified 1352 proteins with an FDR < 0.01% and a minimum of two unique peptides per protein (Supporting Information). Using spectral counts as a measure of protein abundance, and a subtractive approach utilising control PDL isolations, 475 proteins were classified as specifically enriched to protein complexes isolated from cells spread on FN over PDL . Further analysis of the dataset highlighted 86 proteins as statistically enriched to FN compared to PDL (FDR<1%), including the integrin α5 and β1 subunits along with several well‐characterised adhesion complex components (Supporting Information). The current literature‐curated adhesome inventory reported 232 proteins To provide a simplified view of the molecular organisation of the isolated adhesion complexes in the context of known adhesion complex protein interactions, a network was constructed using the identified MSC adhesome components and organised according to their defined roles Fig.  19. ThisPrevious MS analyses of adhesion complexes isolated from fibroblasts have established that the LIN‐11, Isl1 and MEC‐3 (LIM) domain‐containing proteins are recruited to adhesion complexes via forces generated by the myosin II contractile machinery In summary, we have enriched and isolated adhesion complex structures from MSCs spread on FN and catalogued their composition by LC‐MS/MS, revealing a subset of adhesome proteins found in MSCs that contained many potentially mechanoresponsive LIM domain‐containing proteins. Application of MSCs to cell therapy or tissue engineering requires an understanding of how to maintain and differentiate MSCs both in vitro and in vivo As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supplementary data: General Sample and Data Analysis StatisticsClick here for additional data file.


Autism Res2016, 9: 471–479. © 2015 The Authors Autism Research published by Wiley Periodicals, Inc. on behalf of International Society for Autism ResearchA deficit in empathy has been suggested to underlie social behavioural atypicalities in autism. A parallel theoretical account proposes that reduced social motivation  can account for the said atypicalities. Recent evidence suggests that autistic traits modulate the link between reward and proxy metrics related to empathy. Using an evaluative conditioning paradigm to associate high and low rewards with faces, a previous study has shown that individuals high in autistic traits show reduced spontaneous facial mimicry of faces associated with high vs. low reward. This observation raises the possibility that autistic traits modulate the magnitude of evaluative conditioning. To test this, we investigated (a) if autistic traits could modulate the ability to implicitly associate a reward value to a social stimulus ; (b) if the learned association could modulate participants’ prosocial behaviour ; (c) if the strength of this modulation was influenced by autistic traits. In 43 neurotypical participants, we found that autistic traits moderated the relationship of social reward learning on prosocial behaviour but not reward learning itself. This evidence suggests that while autistic traits do not directly influence social reward learning, they modulate the relationship of social rewards with prosocial behaviour.  The social motivation hypothesis posits that atypical social behaviour in autism spectrum condition (ASC) could be caused by a failure to assign reward values to social stimuli and interactions  participants had to use one key to identify pictures of PosC identity or losses and the other for NegC identity or wins. Each participant was administered two versions of the cIAT, one in which the congruent blocks appeared before the incongruent (cIAT‐A) and one with the opposite pattern (cIAT‐B). The sequence of administration of the two versions was counterbalanced across participants.The cIAT was used to test whether participants implicitly learned the association between wins and PosC face and between losses and NegC face. The cIAT consisted of a seven‐block design  involving five practice blocks and two test blocks. Participants were instructed to use a right (P) or left (Q) key to categorize possible pictures belonging to one of the four categories  that appeared in the centre of the screen. In three blocks or 20 trials each  participants had to respond according to two categories: either PosC vs. NegC identity or wins vs. losses. In the remaining blocks participants had to sort the pictures into four categories. Specifically, in two blocks  participants had to use one key to identify pictures that belonged either to the PosC identity The stimuli consisted of the PosC and NegC pictures used during the conditioning paradigm, plus three pictures representing the concept of wins and three pictures representing the concept of loss .M = 20.02 SD = 7.10). No participant scored above 32 on the full AQ, which has been found to be a reliable threshold score for a potential clinical diagnosis of ASC.Participants filled the Autism Spectrum Quotient (AQ) score online after they took part in the study. Four participants did not complete the questionnaire, thus, they were excluded from the AQ analysis; thus the total number of participants for these analyses was 39. Scores on the AQ ranged between 5 and 31  = 18.59, P < 0.001 ηp2 = 0.31 showing that RTs for incongruent blocks were significantly higher than RTs for congruent blocks. The interaction between Congruency and Version showed to be significant F = 55.62, P < 0.001 ηp2 = 0.57 revealing that incongruent RTs were higher than congruent RTs only for the cIAT‐A version  (Table Ps > 0.05). Thus, irrespective of the order of presentation (A–B vs. B–A), the cIAT‐A version (where the congruent block was presented before the incongruent), was the only version that reflected the conditioning. Consequently, only the cIAT‐A version was used for the subsequent analysis.In order to control for the order of presentation, reaction times (RTs) of the cIAT were submitted to a 2 × 2 × 2 analysis of variance (ANOVA) with Congruency (congruent vs. incongruent) and Version (cIAT‐A vs. cIAT‐B) as within‐subject factors and Order of Presentation (A–B vs. B–A) as between‐subject factor. This ANOVA revealed a main effect of Congruency F,41 = 18.t(42) = 0.87, P = 0.38). However, the number of balls tossed to the PosC identity significantly correlated with cIAT measure  , and Congruency, AQ and Congruency*AQ as fixed effects of our design. Only the Congruency predictor showed to be significant  was transferred to prosocial behaviour , moderated multiple regression analysis was used. This analysis allows to test if the effect of the independent variable (Conditioning: Incongruent RTs of cIAT‐A) on the dependent variable (Tosses to PosC) is moderated by a moderator variable (AQ). The interaction is tested by entering in the regression model the two predictor variables and the product of the two (the interaction term) , t = 3.06, P = 0.004; when the AQ were on average or high the relationship failed to reach the significance . This results show that only when AQ traits are low the learned conditioning is transferred to a social behaviour  was found to be significant, showing that the extent to which the conditioning affected the dependent variable  was moderated by participants’ autistic traits To control for a possible confound due to the gender of participants, all analyses were rerun using gender as a covariate. All effects reported above remained unchanged.The central aim of this study was to test if autistic traits influenced the extent of reward learning or/and the extent to which the learned association translated into prosocial behaviour. We found three key results. First, the strength of conditioning was correlated to the number of ball tosses directed to the PosC face. Evaluative conditioning has already been seen to influence different proxy metrics related to empathy such as spontaneous mimicry of happy faces [Sims et al., Second, while there was no evidence for any influence of autistic traits on the reward learning performance, we found that autistic traits moderated the extent to which reward learning for social stimuli was transferred to prosocial behaviour toward those faces. The first of these results is consistent with previous reports that demonstrated comparable reward learning behaviour in adults with and without Asperger Syndrome [Johnson, Yechiam, Murphy, Queller, & Stout, Crucially, we found that autistic traits moderated the extent to which the learnt reward value of the face translated to prosocial behaviour. Specifically, only participants with low autistic traits showed to transform the learned association into prosocial behaviour. This result is in line with the previous findings that showed that autistic traits modulate frontostriatal connectivity [Sims et al., Interestingly, the effect of the evaluative conditioning paradigm was reflected in the cIAT‐A, but not in the cIAT‐B. It has already been documented [Greenwald et al., By introducing independent measures of reward conditioning  and social behaviour (CT) this study is able to parse the role that autistic traits might have on: (a) implicitly learning the stimulus‐reward association; (b) transferring the acquired reward value to prosocial behaviour. Our result expand upon the previous findings that showed link between reward and behavioural and neural markers related to empathy [Haffey et al.,


Purkinje cell axons are the sole output of the cerebellar cortex, understanding pathologic processes within these cells is crucial to develop strategies to prevent their loss and thus reduce ataxia. We studied pathologic changes occurring within Purkinje cells of the cerebellum. Using immunohistochemic techniques, we found changes in neurofilament phosphorylation states within Purkinje cells, including loss of dephosphorylated neurofilament and increased phosphorylated and hyperphosphorylated neurofilament. We also found Purkinje axonal spheroids and Purkinje cell loss, both of which occurred predominantly within areas of leucocortical demyelination within the cerebellar cortex. These changes have important implications for the study of cerebellar involvement in multiple sclerosis and may help design therapies to reduce the burden of ataxia in the condition.Cerebellar ataxia commonly occurs in multiple sclerosis, particularly in chronic progressive disease. Previous reports have highlighted both white matter and grey matter pathological changes within the cerebellum; and demyelination and inflammatory cell infiltrates appear commonly. As  The cerebellum and its efferent and afferent pathways are commonly affected in multiple sclerosis (MS). In patients with established MS, ataxia occurs in about 80% with symptoms and is particularly prevalent in those with progressive disease v1.8 sensory neuron‐specific sodium channel is normally expressed at very low levels in Purkinje cells, but its expression is markedly up‐regulated in MS together with annexin light chain (p11), which facilitates the functional expression of this sodium channel Cerebellar white matter lesions are commonly found and are often apparent in magnetic resonance imaging (MRI) scans of patients with MS. Recent observations concerning grey matter demyelination in cerebral cortex have led to studies evaluating grey matter disease in the cerebellum Purkinje cells represent the sole output neuron of the cerebellar cortex and thus changes in their function have significant impact on the function of the cerebellum as a whole. The aims of this study were to further characterize Purkinje cell pathology in MS cerebellum particularly with respect to neurofilament phosphorylation states, in light of descriptions of neurofilament abnormalities within white and grey matter of the cerebral hemispheres in MS Post‐mortem cerebellar samples from five control cases and six patients with MS were obtained from the UK Multiple Sclerosis Tissue Bank at the Imperial College London, UK as previously described DAB  staining for myelin basic protein (MBP)  and the macrophage/microglial markers   were performed in cerebellar sections of MS and control tissue, according to previously published reports from our laboratory ® 488 and 555 conjugated secondary antibodies  were used to visualize primary antibody staining. DAPI  Vectasheild™  was used for nuclear identification. Sections were visualized using a Leica (DMI 6000) fluorescence light microscope with a Leica LAS‐AF software and also a Leica TCS SP5‐AOBS confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope to obtain three‐dimensional images.Paraffin‐embedded sections were double‐stained as previously described The total number of Purkinje cells was counted in MS and control section, scanning the entire length of the Purkinje cell layer. Purkinje cells were identified based on Calbindin positivity and their specific location. To account for the difference in section sizes and plane of cutting, the length of the Purkinje cell layer was traced and measured in each section using Image J software to obtain the percentage of Purkinje cells per standardized unit length. For these sections, the length of Purkinje cell layer was measured and the number of Purkinje cells in each area counted. Additionally, MS sections were subdivided into non‐demyelinated Figure D blue, lThe total number of spheroids, Purkinje cells bodies positive for SMI‐31, Purkinje cells bodies positive for SMI‐34 and Purkinje cells bodies negative for SMI‐32 were counted in MS and control sections, scanning the entire Purkinje cell layer. As above, MS sections were subdivided into non‐demyelinated, leucocortical lesion, intracortical lesion regions and the frequency of spheroids and abnormal Purkinje cell body staining (SMI‐31/SMI‐34/SMI‐32) were counted and divided by the total number of Purkinje cells in the specific region. Furthermore, regions with leucocortical lesions were subdivided based on presence/absence of inflammatory cell infiltrates using the markers HLA‐DP, ‐DQ and ‐DR . Data were analyzed using one‐way analysis of variance (ANOVA) with post hoc Bonferroni's test between groups where appropriate and unpaired To investigate whether abnormal neurofilament phosphorylation was present in Purkinje cell bodies of MS patients, cerebellar sections from control and MS cases were double immunostained. Antibodies used were the Purkinje cell marker Calbindin with SMI‐31 (anti‐phosphorylated neurofilament), SMI‐34 (anti‐hyperphosphorylated neurofilament) or SMI‐32 (anti‐non‐phosphorylated neurofilament).In control sections, Purkinje cell bodies were negative for SMI‐31  in control cerebellum revealed that almost all Purkinje cells bodies were positive for this marker  spheroids were observed ; and demyelination restricted to cerebellar cortex and not expending to white matter . MS sections were subdivided into non‐demyelinated regions Figure D blue, lP < 0.05). The Purkinje cell number within intracortical lesions was slightly reduced compared with non‐demyelinated areas of MS and control cases, but this reduction was not significant  and Purkinje cells bodies positive for SMI‐31  and SMI‐34  in MS patients compared with control cases. SMI‐32 cell body staining was decreased in MS cases, with a significantly higher number of Purkinje cells bodies negative for SMI‐32 in MS compared with controls . No spheroids were positive for SMI‐32. MS sections were subdivided into non‐demyelinated regions, leucocortical lesions and intracortical lesions. Low magnification of the double‐immunostaining for calbindin and SMI‐31  and Purkinje cell bodies positive for SMI‐31  and SMI‐34  were higher in the leucocortical lesions compared with non‐demyelinated areas of MS cases. In accordance, in areas with leucocortical lesions, a significant number of Purkinje cells were negative for SMI‐32 cell body staining compared with non‐demyelinated areas . In intracortical lesions of MS cases, Purkinje cell bodies staining for SMI31/34 and the frequency of spheroids were increased compared with non‐demyelinated areas although not significant  and SMI‐34  were present with a high frequency in leucocortical lesions positive for HLA compared with lesions without inflammation, suggesting that spheroid formation may be driven by inflammation. No significant differences were present in the frequency of Purkinje cell body staining for SMI‐31  that perform many diverse cellular functions, but are predominantly involved in providing structural stability The current study has revealed neurofilament phosphorylation changes in Purkinje cells, namely reduced numbers of cells staining for SMI‐32 (non‐phosphorylated NF) and increased numbers of cells staining for SMI‐31 and SMI‐34 (phosphorylated and hyper‐phosphorylated NF). Reduced staining for SMI‐32 in ventral spinal neurons has previously been documented in MS samples We have shown that Purkinje cell bodies positive for SMI‐34 displayed often an abnormal morphology, with a less flask‐like shape. Accumulation of hyperphosphorylated neurofilaments in Purkinje cells of the cerebellar cortex in MS may represent an important neurodegenerative mechanism. Although the precise relationship between hyperphosphorylated neurofilament accumulation and neuronal damage remains unknown, aberrant perikaryal accumulation may contribute to neuronal dysfunction and eventually to cell death.Axonal spheroids (torpedoes) are found extensively within MS white matter lesions Regions with leucocortical lesions were subdivided based on presence/absence of inflammatory cell infiltrates using the markers HLA‐DP, ‐DQ and –DR. This subdivision was not made for intracortical lesions as it was harder to define the inflammation within this lesion. This study has shown that spheroid formation in the cerebellar cortex appear most marked in leucocortical lesions with high levels of inflammatory cell infiltration. This suggests a possible link between axonal transection/spheroid formation and inflammation. In MS white matter, acute axonal injury (measured by amyloid precursor protein accumulation) is elevated in acute lesions and correlates to the degree of inflammation v1.8 sensory neuron‐specific sodium channel is normally expressed at very low levels in Purkinje cells, but its expression is markedly up‐regulated in MS v1.8 in Purkinje cells causes significant abnormalities of firing patterns of these cells Other changes in Purkinje cell phenotype have been documented in MS, notably changes in ion channel expression and receptor profiles. The NaOptimal Purkinje cell function is vital to the overall function of the cerebellum, as Purkinje cell axons represent the sole output of the cerebellar cortex. Loss of Purkinje cells is a common finding in a range of neurologic conditions characterized by ataxia, and in all likelihood, is irreversible. Thus, designing therapies to protect Purkinje cells in MS and related conditions is vital to preserve cerebellar function. Recent reports of bi‐nucleate Purkinje cells and heterokaryon formation in experimental models and in human disease have suggested a potential intrinsic repair mechanism of Purkinje cells, whereby nuclear material is donated to an injured cell


DNA after they completed their function. Metabolic genes in particular are dynamically regulated and continuously adjusted to cellular requirements. Transcription factors controlling metabolism are therefore under constant surveillance by the ubiquitin–proteasome system, which can degrade DNA‐bound proteins in a site‐specific manner. Several of these metabolic transcription factors are critical to cancer cells, as they promote uncontrolled growth and proliferation. This review highlights recent findings in the emerging field of nuclear proteolysis and outlines novel paradigms for cancer treatment, with an emphasis on multiple myeloma.The recruitment of transcription factors to promoters and enhancers is a critical step in gene regulation. Many of these proteins are quickly removed from  AR androgen receptorChIP chromatin immunoprecipitationCREB cAMP response element‐binding proteinEBPδ enhancer‐binding protein deltaGR glucocorticoid receptorHIF‐1α hypoxia‐inducible factor‐1 αIKZF1 Ikaros family zinc finger protein 1IKZF3 Ikaros family zinc finger protein 3MM multiple myelomaNCoR1 nuclear receptor corepressor 1NRF‐2 nuclear respiratory factor‐2pVHL von Hippel–Lindau tumor suppressor proteinROS vreactive oxygen speciesT‐ALL T‐cell acute lymphoblastic leukemiaTGF‐β transforming growth factor‐βUPS ubiquitin–proteasome systemOver the past decade, extensive work has confirmed the existence of a link between cancer cell‐intrinsic metabolism and tumor signaling pathways. It is now clear that dysregulation of oncogenic signal transduction pathways alters metabolic flux; conversely, mutations in enzymes regulating metabolic flux can initiate cellular transformation and contribute to tumor maintenance and progression Recent evidence has emerged that a direct cross‐talk between metabolism and epigenetics is a key regulatory mechanism in cancer development. Specifically, proteome modifications provide a fast mechanism to respond to environmental changes and to adjust homeostasis. Regulated protein catabolism via the ubiquitin–proteasome system (UPS) controls a wide variety of cellular functions, from transcriptional regulation and stress response to cell cycle regulation.This review focuses on the mechanisms by which the turnover of transcription factors regulates gene activity and metabolism, and how these functions are interconnected Fig. . After aThe proteasome and the upstream ubiquitination pathway are an evolutionary conserved and highly regulated proteolytic system that precisely controls selective protein degradation in order to tightly maintain proper protein content and quality. The UPS acts as a disposal pathway and optimizes cellular functions by removing misshaped proteins. The UPS also has a ‘surgical’ role in controlling cell function by adjusting the abundance of regulatory proteins involved in numerous biological pathways, such as signal transduction, stress response, immunity, cell cycle regulation, and transcriptional regulation.Protein degradation through the UPS pathway is a complex process, involving multiple steps and enzyme machineries that begins when proteins are targeted for degradation by sequential conjugation, usually at lysine residues, to isopeptide‐linked ubiquitin molecules. Ubiquitination is achieved through the coordinated actions of E1 ubiquitin‐activating enzymes, E2 ubiquitin‐conjugating enzymes, and E3 ubiquitin ligases. The interaction between the E3 ubiquitin ligase and its target protein provides a high level of specificity in this dynamic process Once at least four ubiquitin molecules have been conjugated to a substrate in a particular chain formation, the proteasome recognizes the poly‐ubiquitinated proteins as substrates and degrades them in vivo with a half‐life of 20–30 min, and c‐Myc proteolysis is mediated very rapidly by the ubiquitin–proteasome pathway The expression of a gene is not only controlled by the type of transcription factor that binds to its promoter but also by the duration of this interaction. Many transcription factors, particularly those involved in the regulation of cell proliferation, are unstable regulators. Upon completion of their tasks, for instance the recruitment of additional enzymes or cofactors, they are quickly catabolized by the UPS in order to vacate the promoters and allow new regulatory proteins to bind. The stability of the c‐Myc protein, for example, is actively regulated Interestingly, a global study of the budding yeast proteome categorized half‐lives of proteins based on their function. Based on the integration of proteomic data with previous measurements of mRNA levels and translation rate, two functional clusters of coregulated genes can be distinguished: a first group (‘production cluster’) encodes for abundant and generally stable proteins that appear to be optimized for maximum protein production and maintenance efficiency . The second group, called ‘regulatory cluster’, is enriched in proteins produced in smaller quantities and with short half‐lives, whose elimination is regulated by the UPS. This second cluster seems to be optimized for regulatory flexibility and therefore mainly consists of cell cycle proteins and transcriptional regulators Saccharomyces cerevisiae, diverse subsets of genes were identified in connection with their enrichment in particular biological and functional processes. Constitutive cellular processes like translation , and central metabolism (glycolysis) were mainly regulated by a first subset of genes with stable proteins, whereas a second group of genes including many transcriptional regulators, signal transduction genes, chromatin modifying enzymes, as well as genes involved in mitosis or cell cycle, encodes short‐lived proteins The first quantitative genome‐scale prediction of protein abundance and turnover in mammalian cells confirmed a link between protein stability and function The UPS influences transcription through proteolytic and nonproteolytic activities, including regulation of transcription factors and RNA polymerase activity, as well as epigenetic histone ubiquitination The concept that the spatio‐temporal regulation of gene expression is influenced by the binding as well as the removal of transcriptional regulators is well established. Ubiquitination of transcription factors and their proteasome‐mediated turnover are crucial steps in this process. A review from the Tansey laboratory suggests two distinct strategies in which cells control the nuclear abundance of transcription factors by ubiquitin‐mediated proteolysis. The first mechanism takes place off chromatin and represents a constitutive turnover in which the transcription factor is maintained in a constitutively unstable form and in restricted abundance by the UPS in situ, and allows a ‘fresh’ pool of transcription factors to bind to the cleared promoter region and catalyze a new round of transcription The second proteolytic method occurs on chromatin at promoter and enhancer sites and during the process of transcriptional activation et al. show that the ability of a transcription factor to function can be associated with its ubiquitin‐dependent degradation. With a half‐life of only 5 min, MATα2 is a short‐lived protein known to be rapidly ubiquitinated by at least two distinct E3 ubiquitin ligases Saccharomyces cerevisiae. The authors reported that several mutations within the second degradation target site and homeodomain of MATα2 result in stabilization of this protein, but impair the ability of MATα2 to interact with DNA Saccharomyces cerevisiae requires the F‐box protein Dsg1/Mdm30 that targets Gal4 for UPS‐mediated proteolysis in cells grown under activating conditions . Dsg1/Mdm30‐mediated Gal4 turnover surprisingly stimulates the expression of GAL genes, while in Dsg1/Mdm30‐deficient cells, Gal4 stabilization prevents the transcriptional activation of GAL genes. The authors concluded that the proteolytic degradation of Gal4 under activating conditions is essential for its activator function Growing evidence supports the concept that the function of a particular transcription factor is more strongly linked to its binding dynamics than to static gene occupancy. It is well established that transcriptional activators are fine‐tuned by the UPS and ultimately turned off after their activation et al. in vivo. Another example of how the UPS closely impacts transcription is the direct protein–protein interaction of the yeast Asr1 ubiquitin ligase with the carboxy‐terminal domain of RNA polymerase II. Ubiquitination by Asr1 alters the subunit composition of the enzymatic complex and is associated with inactivation of polymerase function in vitroUbiquitin‐mediated proteolysis of activators is a critical component of transcription regulation, as evidenced by the deep physical connections between the transcription machinery and the UPS. First, ubiquitin ligases have been demonstrated to interact with genomic sites of activator function. Von der Lehr GAL1 promoter by the Gal4 transactivator upon induction with galactose in vivo, and promotes the association of a specific coactivator/histone acetyl transferase in order to facilitate the recruitment of TFIID for transcriptional initiation GAL10 gene in a similar temporal and spatial manner Saccharomyces cerevisiae, ChIP combined with global transcriptome analyses identified genomic regions regulated by the proteasome, providing evidence that proteasome association correlates with a vast array of highly transcribed genes Furthermore, the proteasome is intimately involved in the regulation of gene expression. This notion is supported by chromatin immunoprecipitation (ChIP) studies in yeast showing that proteasome subunits actually bind to chromatin Even though proteolysis and transcription are functionally interconnected and might be key determinants of cellular metabolism, the genome‐wide degradation pattern of transcription factors is still poorly understood. Chromatin immunoprecipitation assays are widely used to produce a static snapshot of transcription factor occupancy at specific DNA regulatory sequences, but they fail to reflect the transcription factor‐binding dynamics across the genome Proteolytic activities of the proteasome occur in the immediate vicinity of DNA and participate in accessing, modifying, or controlling chromatin in order to regulate transcription. Different models for recruitment of proteasome subunits to chromatin have been proposed. Active proteasomes can be connected to the chromatin by direct contact with activators; indirectly through their interactions with intermediate proteins, or they can also bind to chromatin in response to the presence of ubiquitinated substrates at specific loci −/− mice exhibit enhanced muscle mass and oxidative capacity of muscle fibers, which present a higher mitochondrial content and activity Like transcriptional coactivators, transcriptional repressors and corepressors are also restricted by the UPS and failure to eliminate repressors results in aberrant transcriptional silencing. When mapping DNA‐associated protein turnover on a genome‐wide scale, we found that promoter‐linked proteolysis has a positive effect on the transcription of a significant number of genes, one of the most prevalent groups being the nuclear‐encoded mitochondrial genes Mitochondria play a central role in the metabolism of most eukaryotic cells. Mitochondrial dysfunction has been implicated in the development of a variety of pathological conditions and illnesses, including not only age‐related neurodegenerative diseases such as Alzheimer's dementia, Parkinson's disease, or amyotrophic lateral sclerosis, but also type 2 diabetes and cancer The majority of genes encoding mitochondrial proteins is located in the nucleus C. elegans demonstrated that dysregulation of mitochondrial respiratory complexes induces elevated levels of mitochondria‐derived ROS The UPS is tightly linked to mitochondrial function. Degradation events control the fusion and fission cycle of these organelles, trigger mitophagy, and also eliminate misfolded or mislocalized mitochondrial proteins Among other key signaling events triggered by dysfunctional mitochondria is the altered production of metabolites that have an effect on the epigenetic code. Components such as α‐ketoglutarate, fumarate, or succinate can modify the methylation status of histones and DNA, and alter the stability of transcription factors through prolyl‐hydroxylases The E3 ubiquitin ligase seven in absentia 2 (Siah2), together with proteins of the NCoR1 corepressor complex, such as transducin β‐like 1 (TBL1) and TBL‐related 1 (TBLR1) cis‐sequences and allows replacement by p300‐bound active ARs. This example highlights the complexity of nuclear degradation, which is not only protein‐specific but also specific in terms of the chromatin location at which it occurs.In addition, a recent study identified a role of Siah2 in the development and progression of castration‐resistant prostate cancer via its role in cell cycle regulation and DNA repair pathways. Bhaskara et al. Given the importance of Siah2 in the regulation of mitochondrial metabolism and its tumor‐promoting role, it is worth mentioning that the NCoR1/HDAC3 repressor complex also controls cell proliferation Given its fundamental and ubiquitous role in maintaining cellular homeostasis, it is perhaps surprising that pharmalogical inhibition of the proteasome can be effective in the treatment of cancer, with relatively limited general side effects. Historically, proteasome‐targeted therapy has first shown positive results in the treatment of the hematological malignancy multiple myeloma (MM) Carfilzomib, the second FDA‐approved proteasome inhibitor for treating relapsed and resistant MM previously treated with Bortezomib, has improved proteasome‐targeting selectivity and efficacy, with a lower toxicity profile However, one of the major remaining questions is how proteasome inhibitors actually kill MM cells. Knowing which pathway disruption is critical to MM treatment would help us devise more specific modes of intervention that target a defined E3 ligase rather than use ‘blunt’ proteasome inhibition. Originally, the proposed mode of action of Velcade was to block inflammatory signals that are critical for MM cells by stabilizing the NF‐κB inhibitor IκB. However, Velcade actually seems to stimulate this pathway Diffuse large B‐cell lymphoma (DLBCL) is a malignancy that can be differentiated based on its metabolic profile into a ‘respiratory’ and a ‘nonrespiratory’ subgroup Gene expression profiling and correlation with outcome in clinical trials for Velcade in MM patients shows that many of the pathways associated with progressive disease and response to treatment regulate protein biosynthesis and mitochondrial functions. Results of Velcade chemotherapy in solid tumors, including lung, breast, and prostate cancers, have been somewhat disappointing. However, as the uncontrolled synthesis of antibodies and the associated high energy demand increases their dependence on mitochondrial activity, MM cells may be more sensitive to metabolic treatments Beyond its impact on basic molecular and biological processes, site‐selective transcription factor degradation also holds great medical promise. Transcription factors are generally deemed ‘undruggable’. The ability to modify transcription factor‐specific E3 ubiquitin ligases with small molecules would allow us to facilitate or block the elimination of DNA‐binding regulatory proteins. Because the process of ubiquitination is located upstream of the proteasome and because E3 ubiquitin ligases mediate the specificity of substrate ubiquitination, small molecule modifiers of these enzymes represent more specific and valuable drug candidates than therapies based on proteasome inhibition. Small molecules that inhibit or stimulate specific E3 ubiquitin ligases represent the most appealing target for drug development and are currently being tested for utility in hematological malignancies Another example of a promising therapeutic strategy is the targeting of the E3 enzyme Mdm2. Inhibitors of the ubiquitin ligase are being tested, as the enzyme plays an essential role in regulating p53 turnover. Not only does Mdm2 promote proteasomal degradation of p53, but it also inhibits the transactivation domain of the tumor suppressor protein Recently, new roles have emerged for FBXW7, a substrate‐targeting subunit of the SCF (Skp1‐Cul1‐Fbox) ubiquitin ligase complex, in leukemia. Indeed, a comprehensive screen of FBXW7 mutations in various human malignancies detected that more than 30% of T‐cell acute lymphoblastic leukemia (T‐ALL) patients carry FBXW7 mutations. As FBXW7 mediates the ubiquitin‐dependent proteolysis of several oncoproteins including c‐Myc, cyclin E1, c‐Jun, and Notch, FBXW7 is considered a tumor suppressor. In particular, FBXW7 plays a pivotal role in maintaining quiescence in the small pool of leukemia‐initiating cells by mediating c‐Myc degradation If NCoR1 is indeed a critical substrate of the proteasome and a determinant of the sensitivity to proteasome inhibitors, it would be desirable to have drugs that specifically block its degradation. Given the importance of the E3 ubiquitin ligase Siah2 in the regulation of NCoR1, it is reasonable to consider Siah2 as a target of interest in MM In conclusion, these observations define E3 ubiquitin ligases as the most attractive druggable components of the UPS. It is possible to identify drugs that either stimulate or inhibit specific E3 ligases and thereby degrade or stabilize target transcription factors. Given the selective sensitivity of transcription factors to degradation at certain target genes, one would expect such drugs to interfere with the expression of defined gene sets. These drugs would likely be more effective and feature less side effects than proteasome inhibitors and moreover also have the potential capacity to increase degradation as shown with Thalidomide.Transcription factors are short‐lived proteins under constant surveillance by the UPS. As such, protein turnover is a critical component of genome regulation. Metabolic genes are continuously being adjusted and it is no surprise that these two highly connected systems, degradation and transcription, converge in the control of mitochondrial genes Fig. . The comLM and AC co‐wrote the manuscript.


Since the discovery of nerve growth factor (NGF) more than a half century ago  designed to block the brain-specific PKC isoform protein kinase Mζ (PKMζ) were used to demonstrate this effect  family in the context of synaptic development , tumor necrosis factor-α (TNF-α), all of which were first identified for their roles other than synaptogenesis (Poon et al., The field of neuronal growth factors is continuing to grow, and new discoveries, some which are highlighted in this volume, will prompt new questions. For example, do these growth factors work together, competitively or separately? Is there cell-type specificity for each factor? Do they play a deterministic or a modulatory role in synaptic specificity during development ? Advances in genomics and proteomics will help us understand not only single cascade but also multiple signaling pathways as network. Various genetic tools allow spacial and temporal controls of gene expression and neuronal activities (Boyden et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.