The use of nucleic acid derived aptamers has rapidly expanded since the introduction of SELEX in 1990. Nucleic acid aptamers have demonstrated their ability to target a broad range of molecules in ways that rival antibodies, but advances have been very uneven for different biochemical classes of targets, and clinical applications have been slow to emerge. What sets different aptamers apart from each other and from rivaling molecular recognition platforms, specifically proteins? What advantages do aptamers as a reagent class offer, and how do the chemical properties and selection procedures of aptamers influence their function? Do the building blocks of nucleic acid aptamers dictate inherent limitations in the nature of molecular targets, and do existing aptamers give us insight in how these challenges might be overcome? This review is written as an introduction for potential endusers of aptamer technology who are evaluating the advantages of aptamers as a versatile, affordable, yet highly expandable platform to target a broad range of biological processes or interactions. Aptamers can rival antibodies in affinity and specificity, providing low nanomolar or even picomolar affinity, yet they are also uniquely distinct from proteins in their mode of binding. New applications of aptamers are literally emerging every day, as are improved methods of selection. The current review will instead introduce, or reintroduce, some basic questions with respect to the fundamental molecular recognition properties of aptamers and their relation to the basic building blocks used for aptamer creation. This overview can by its very nature not be exhaustive. While some areas, such as aptamer based in vitro diagnostics or technical aspects of selection and minimization, are covered in great abundance in the literature, it can be much harder for a putative enduser to find at least an entry level discussion of other questions. Can aptamers target carbohydrate targets with antibody-like affinity? Is it possible to target a basic protein or peptide and still retain specificity? Can targets with negative net charge be effectively selected against while retaining specificity? The purpose of this review is to raise some of these questions and to initiate a discussion of these questions for potential users. In raising these questions, we are particularly mindful of the large divide that often seems to exist between the perceptions about aptamers in the scientific community at large and the views among established aptamer users. We also regard the relative simplicity of conventional SELEX as one of its major advantages that opens up aptamer selection to a broad audience of users. Our discussion is therefore geared more towards classic SELEX methodology that most laboratories with a primary focus on questions of biochemistry, molecular recognition or cell biology would be able to implement in a relatively cost effective manner.Aptamers, small peptides or nucleic acid sequences with unique and usually selected functional properties, have rapidly taken an important place in the available repertoire of pharmacological or diagnostic reagents. The term “aptamer”  may in its general sense apply to peptides, nucleic acids or chimera. However, it is most commonly associated with nucleic acid aptamers which are uniquely suited for SELEX . Initially employed for the study of regulatory mRNA in phage  and the Once non-essential sequence components are removed, aptamers tend to be approximately 1/10 the size of the average IgG antibody, another rapidly expanding class of therapeutics. This size of 7–15 kD and the phosphodiester backbone places aptamers in many ways in a niche between small molecule drugs and antibody therapeutics. Unlike most small molecule drugs, aptamers cannot readily penetrate cells unless equipped with specific uptake enhancing features that may facilitate cellular uptake. Compared to antibodies, their smaller size gives aptamers enhanced tissue penetration properties but also faster renal clearance. Modifications such as cholesterol or PEG attachment are therefore often applied to enhance serum half-life . For aptin vitro applications. This complexity stands in contrast to antibodies. For therapeutic antibodies, reagent class specific questions on safety, purity, or production have been addressed. This allows evaluations to focus more rapidly on target specific studies. However, with the introduction of improved coupling methods and decreasing production costs for the most common forms of aptamers [Arguably some of the strongest aspects of aptamers are the relative ease and low cost of classic SELEX, the ease of transition from selection to synthetic production, and the versatile repertoire of defined chemical modifications for the synthetic end product. These aspects have been the driving forces for the extremely innovative research in a large number of relatively small research labs. While this has resulted in a broad spectrum of analytical applications or early studies of therapeutic usages, it stands in contrast to the relatively modest number of established clinical applications. The therapeutic application of aptamers has in part been held back by the challenges involved in the cost effective upscaling of aptamer synthesis from the laboratory scale to one that meets the reagent requirements of clinical trials. Few roadmaps existed for the large scale GMP grade synthesis and purification of nucleic acid based biopolymers. New chemical modifications continue to improve basic features of aptamers, but each new modification creates additional questions that have to be addressed in preparation for preclinical and clinical tests. As a consequence, existing therapeutic aptamers do not reflect the diversity of chemical platforms that is represented in aptamers , we are At present, neither peptide based approaches nor aptamers can compete with most small molecule drugs when it comes to cellular uptake. Some success has been obtained through the fusion of entry mediating peptides  or the tChimeric aptamers that combine distinct selected functions may initially encounter ambiguous folding as well. However, if the problematic sequence components cannot be readily identified the required optimization of fused properties can be addressed directly by SELEX itself. This concept was demonstrated early by Burke and Willis who optimized fused aptamers against very divergent cytoplasmic small molecule targets . Anotherin vitro diagnostics, but greatly enhances the utility and evaluation of therapeutic aptamers through the introduction of fluorescent probes in mechanistic cell culture studies or the introduction of tracers in pharmacokinetic and imaging applications.The analysis of aptamer properties in biological systems is in most cases a more time consuming and costly undertaking than the initial selection. The ability to retroactively enhance the functionality of aptamers already under investigation is therefore of considerable advantage. In contrast to small molecule drug optimization, ongoing improvements in stabilization, pharmacokinetics or label attachment are largely substance class specific and will not have to be investigated exhaustively by each end user of aptamer SELEX methodology. Aptamers are generally considered non-immunogenic. Despite the ability of short stretches of double stranded RNA to activate Toll-like receptors, aptamers are poor activators of an innate immune response, and this response is further reduced by chemically modified RNAs ,20,21,22A survey of aptamer selections over the years reveals a strong initial bias towards ribonucleotides as building blocks despite the inherent enzymatic and chemical instability of unmodified RNA. This initial preference for RNA over DNA largely reflects the believe that the expanded ability of RNA to form complex three dimensional structures is more likely to mimic the structural repertoire of proteins. Due to the subsequent introduction of modified and more nuclease RNA, current RNA derived aptamers exceed DNA in serum stability. However, DNA aptamers have become more popular in recent years, largely due to a selection procedure that does not require an intermediate reverse transcription step, the relatively high intrinsic serum stability of DNA without the need for modified deoxyribonucleotides, and the low cost and high yield in the synthetic production of final products. In addition, the documented ability of select DNA aptamers to rival their RNA counterparts in affinity demonstrates that the lower structural complexity of DNA may be much less of an inherent limitation than was initially believed. At present, existing DNA aptamers exhibit a strong bias towards quadruplex structures or three-way junctions. Whether the apparently more restricted structural space of DNA aptamers will ultimately translate into a more limited molecular recognition space remains to be seen. So far, this has not emerged as an obvious limitation.O-methyl modified ribonucleotides  in E. coli [O-methyl modification to achieve serum stabilization. The drawback of this approach is the significantly higher cost and technical challenge involved in the chemical synthesis of the 2'-fluoro-substituted RNA end product. The latter is no significant impediment for 2'-O-methyl modified RNA. T7 polymerase mutants have been described that produce 2'-O-methyl RNA [O-methyl modified aptamer against VEGF [O-Me accepting polymerase reaction has until recently remained a limiting aspect to its widespread use.A subset of nucleases with exceptionally high ability to digest even modified RNAs is found in various strains of mycoplasm unless 2'-leotides . In retr E. coli , and the E. coli . Interesthyl RNA  as has tnst VEGF . HoweverRNA or DNA Spiegelmers are an alternative solution to the problem of aptamer stabilization, conceptually akin to anti-idiotype antibodies. Spiegelmers (mirror images) are the synthetic enantiomers  of conventional aptamers that were initially selected against the mirror image of the final target. Spiegelmers against L-arginine were an early examples . Since mde novo identification of biomarkers on live cells [in vivo optimized reagents need to be tackled.In addition to targeting well established biomarkers such as PSMA for cargo delivery ,33, aptave cells ,34,35, ove cells . The latve cells , BrdU [1ve cells  or thio-ve cells . These avice versa. In addition, the biophysical properties of the enzymatically incorporated 2'-fluoro and 2'-O-methyl modified aptamers are fundamentally different on various functionally relevant levels, maybe most importantly their hybridization energy. Average melting temperatures of homo duplexes increase in the order DNA < RNA < 2'-O-methyl-RNA < 2' F-RNA [Whether or not functionality in a cellular setting is ultimately desired has important consequences in the choice of the selection platform. The introduction of 2'-ribose modifications does alter folding, such that aptamers selected with standard nucleotides cannot be readily translated into their corresponding counterparts post selection, or 2' F-RNA ,39. This2' F-RNA . On the 2' F-RNA . The polHence, additional means of fine tuning hybridization could give aptamers a broader range of conformational dynamics, much as proteins can evolve to accommodate a broad range of demands on their intrinsic flexibility depending on their operating environment. However, the functional impact of these modifications on aptamer functions other than nuclease resistance is at present rarely exploited in a systematic manner, largely due the difficulties involved in optimizing the enzymatic incorporation of modified building blocks during classic, amplification dependent SELEX.in vitro diagnostics, most aptamers used in a biological setting represent or incorporate targeting modalities aimed at non-nucleic acid ligands. Small molecule binding aptamers and the control of mRNA processing are combined when the aptamer is introduced into the UTR region of mRNA. In such chimeric constructs, the aptamer can directly regulate translation [ds of 100 nM and 600 nM but discriminated against the closely related caffeine 104-fold. More importantly, this represented a more than a 10-fold improvement in discriminating power compared to antibodies [The challenges aptamers face in molecular recognition are obviously heavily dependent on the reagent class to be targeted. Many applications of aptamers make use of their intrinsic ability to base pair to nucleic acid targets. The rules governing specificity in these cases are relatively well understood. For the previously mentioned use of aptamers as tools in siRNA delivery, sequence components providing siRNA functionality must strike a balance between stability and processing by the cellular machinery. However, while hybridization driven functionality is a very prominent feature for aptamers, especially for aptamers used for nslation  or can cnslation , reviewenslation ). More rnslation  or tetranslation  induced nslation ). One oftibodies . While tin vivo setting, aptamers have also been selected against both alkaloids and steroids. The latter are largely variations of the well studied and conformational switch capable cocaine binding DNA aptamers, an example of a three way junction. In its most basic form, three way junctions provide a hydrophobic binding pocket at the central branching point (D approx. 10−15 M) which bind biotin in a buried binding site through a channel that maximizes interactions with the hydrophobic linker.While proteins have a series of side chains at their disposal for contacts with hydrophobic targets, nucleic acids are more limited in their repertoire of available interactions. A therefore somewhat surprising example for the formation of an exclusively hydrophobic ligand binding pocket was found in an aptamer that binds the acyl component of the phosphoglycolipid antibiotic moenomycin A with high (400 nM) affinity . While fng point C, left [ng point C right. ng point B through−6 and 10−4 M. These include challenging proof of principle targets such as the selective binding of various monosacharides [D of 10−7 M. While most affinities remain relatively modest compared to other aptamer targets, selectivity is in most cases very high providing good potential for catalytic aptamers or multivalent homo or bifunctional aptamers that may bind complex carbohydrate polymers with high affinity. Low micromolar binding affinities also have to be viewed in the context of relevant biological concentrations of metabolites and the affinities against single carbohydrate units generated by proteins. Glucose binding protein, first found in the periplasmic space of Pseudomonas aeruginosa, is considered to be a very sensitive and selective sensor of glucose in the extracellular milieu. It binds glucose with a KD of 0.35 μM and withstands challenges by access competing carbohydrates in 10-fold but not 100-molar excess [D = 4.9 μM) and provided an important proof of principle for expanded SELEX with charge modified nucleotides [SELEX has generated various examples of selective binding to carbohydrates (reviewed in ). As wascharides  or cellucharides  for whicr excess . Given tleotides .A 2'-fluoropyrimidine modified RNA aptamer (NX1838) against VEGF ,68, intrin vivo [Factors that control blood clotting are of great importance for the acute treatment of a large number of cases of thrombosis and have been a significant emphasis of aptamer research over the years, providing several in depth comparative studies. High affinity (20 nM) thrombin aptamers, reported in 1992 , were amin vivo .The initial selection against thrombin capitalized on the presentation of positively charged epitopes representing the sites binding of fibrinogen and heparin respectively. Since many RNA binding proteins utilize arginine or lysine residues to interact with their respective targets, it is tempting to extrapolate from our knowledgebase of nucleic acid binding proteins to predict the mode of binding and limitations in specificity for aptamers. However, a structural analysis of arginine directed aptamers surprisingly reveals an avoidance of charge based interaction and instead shows a reliance on hydrogen bonding in a complex with a fully engulfed arginine side chain. This mode of recognition is in stark contrast to the reverse interaction exhibited by RNA binding proteins. It has been suggested that this reflects the intrinsically different selection pressure for aptamers that allows exclusive emphasis on selectivity and affinity without the more complex functional requirements imposed on proteins . RegardlD = 3.5 nM) with specificity in an in vitro and cellular setting [The selection of aptamers against transcription factors or other nucleic acid binding proteins can be a challenge for target regions other than the nucleic acid binding portion. Positively charged surfaces can steer aptamers towards an epitope during selection, even if the final binding mode does not directly capitalize on charge interactions. But does such a favorable attraction undermine specificity? A direct evaluation of this question in 2011 revealed that aptamers can readily discriminate a panel of basic proteins , thereby setting . While NWhile this represents one viable approach to overcome the challenge imposed by very pronounced negative surface charges, an alternative and potentially more universal approach was outlined in principle by the earlier mentioned selection of modified aptamers against Sialyllactose. A similar selection using arginyl dUTP during SELEX yielded high affinity DNA aptamers against glutamic acid  as well A particularly interesting challenge of molecular recognition by aptamers and their ability to rival proteins in functionality involves the quest for nucleic acid GFP equivalents. A unique feature of fluorescent proteins is the spontaneous oxidation and cyclization chemistry of several core amino acids. In the case of GFP the final fluorophore resulting from this intramolecular reaction is 4-hydroxybenzlidene imidazolinone, shielded in the core of the GFP beta barrel. The limited chemical diversity of aptamers restricts options for similar chemistry, but fluorophores and other modifiers are readily attached to synthetic aptamers carrying amino or thiol functionalities. The enzymatic site directed incorporation of intrinsically fluorescent nucleotide analogs has been achieved using T7 polymerase and DNA templates in which modified bases flank the site of insertion . While t40 or 1024 possible sequences is already beyond the maximum experimentally achievable complexity, which is more often around 1013 molecules per screen. Some recent protocols operate with randomized sequences of around 20 nucleotides and thus can at least in principle be fully represented in the experimentally accessible population of sequences carrying 420 or 1012 permutations. In addition, the need for reamplification adds non randomized flanks that can serve as both primer sites for amplification and unique tags that can be highly beneficial if multiple SELEX procedures are being carried out in parallel. Thus, initial aptamers derived from SELEX range frequently from 50–80 nucleotides. An important first step in further analysis is the identification of the minimum size needed for function. Since the non-randomized flanking reagents can take part in the overall base pairing, it is not necessarily clear whether the randomized core does indeed represent the functional unit, and a variety of tools have emerged over the years to stream line this process of minimization .While classic approaches to aptamer minimization relied mainly on the reiterative use of biochemical methods and algorithms that evaluate the energetics of alternative two dimensional folding options, more advanced approaches include direct predictions of three dimensional folds ,90,91. HBeyond minimization approaches, one of the more profound shifts in the day to day business of aptamer selection has serendipitously arisen from the rapidly dropping costs of high throughput sequencing. This makes it for the first time possible to follow aptamer “enrichment” throughout the selection cycle on a sequence level. Data derived from such efforts are readily processed by an array of parsimony based methods, initially designed to deconstruct protein and gene evolution. As more studies are emerging, it becomes clear that many previously held believes about the underlying driving forces of selection and readouts of selection progress may have to be revisited. In addition, such studies have demonstrated the contribution of the amplification methodology to drifts in the aptamer population . Such drifts occurs in the complete absence of biochemical enrichment or partitioning steps . One exaet al. [The ability to follow the early rounds of selection directly for the emergence of sequence clusters instead of driving selection towards complete convergence has also coincided with technical advances in enrichment and detection methodology that bypass reamplification steps. While in principle already represented in the before mentioned direct selection of anti-thrombin DNA aptamers from defibrotide in 1994, a systematic exploitation of consecutive rounds of enrichment without reamplification was formally introduced by Berezovski et al. . This “net al.  while onWhat does the future hold for aptamers? Traditionally, the role of nucleic acids in therapeutics has been relegated to the coding of delivered genes, possibly supplemented by the enhanced delivery of fused aptamers. An area of rapidly increasing importance is the delivery of nucleic acids as regulatory entities. In the near future, aptamers are likely to capitalize on the advances made in stabilization and targeting to deliver highly potent regulatory cargo with functions such as siRNAs in ways that are unique to nucleic acid carriers. In the more distant future, aptamers with improved catalytic functions may provide new paradigms for enzyme replacement therapy by not only enhancing delivery but by directly representing the catalytic entity itself. The question whether nucleic acids ultimately remain dominant as the basic building block in aptamer creation is directly linked to advances in enrichment, detection, characterization, and cost effective multiplexing in non-SELEX approaches. In the meantime, the classic SELEX procedure has undergone major improvements over the years but it continues to remain readily accessible to most research labs. In addition, nucleic acid aptamers are likely to profit, much like the field of antibody therapeutics did, from the fact that many critical issues, such as the fine tuning of serum stability, pharmacokinetics, or the evaluation of chemical modifications, can be evaluated for aptamers as a compound class. Hence, the current rapid growth in the use of nucleic acid aptamers is unlikely to slow down in the near future.


Background: Chronic obstructive pulmonary disease (COPD) is a respiratory disease associated with a systemic inflammatory response. Peripheral muscle dysfunction has been well characterized in individuals with COPD and results from a complex interaction between systemic and local factors.Objective: In this narrative review, we will describe muscle wasting in people with COPD, the associated structural changes, muscle regenerative capacity and possible mechanisms for muscle wasting. We will also discuss how structural changes relate to impaired muscle function and mobility in people with COPD.Key Observations: Approximately 30–40% of individuals with COPD experience muscle mass depletion. Furthermore, muscle atrophy is a predictor of physical function and mortality in this population. Associated structural changes include a decreased proportion and size of type-I fibers, reduced oxidative capacity and mitochondrial density mainly in the quadriceps. Observations related to impaired muscle regenerative capacity in individuals with COPD include a lower proportion of central nuclei in the presence or absence of muscle atrophy and decreased maximal telomere length, which has been correlated with reduced muscle cross-sectional area. Potential mechanisms for muscle wasting in COPD may include excessive production of reactive oxygen species (ROS), altered amino acid metabolism and lower expression of peroxisome proliferator-activated receptors-gamma-coactivator 1-alpha mRNA. Despite a moderate relationship between muscle atrophy and function, impairments in oxidative metabolism only seems weakly related to muscle function.Conclusion: This review article demonstrates the cellular modifications in the peripheral muscle of people with COPD and describes the evidence of its relationship to muscle function. Future research will focus on rehabilitation strategies to improve muscle wasting and maximize function. Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and presents a major burden of disease in middle and high-income countries  in a large cohort of patients with moderate to severe COPD. Similarly Schols et al.  in COPD patients what suggests a compensatory response to reduced O2 availability to augment ATP production  activity in the human VL (Alonso et al., Although cardiac output and ventilatory limitation have been observed during exercise in COPD patients (Cuttica et al., There are several interactive mechanisms that may contribute to the underlying development of muscle wasting in people with COPD. For a thorough review of the pathways leading to muscle atrophy, the reader to referred to the review by Langen et al. . We presMuscle apoptosis was hypothesized to be one factor underlying the development of muscle atrophy in COPD, however there are limited data in this area. Agusti et al.  found thThere are some data to suggest that muscle regenerative capacity may be impaired in people with COPD. Theriault et al.  describe1; and the degree of lipidation of the LCB3 protein was associated with low thigh muscle cross-sectional area. Further studies are needed to understand the contributions of the ubiquitin-proteasome pathway and autophagy-lysosome pathway in muscle wasting in COPD.Muscle mass is regulated through a balance of protein synthesis and degradation. In COPD, it is not clear whether protein synthesis is downregulated, protein degradation is upregulated or whether muscle mass depletion is a result of both processes. One of the proteolytic systems which has been studied in COPD is the ubiquitin-protease system. There is evidence for increased activation of this system in people with COPD, which may contribute to muscle wasting (Fermoselle et al., In terms of protein synthesis, the IGF-1-Akt pathway has been studied in people with COPD, however the results are conflicting. Circulating levels of IGF-1 have been shown to be similar between people with COPD and controls across disease severities (Piehl-Aulin et al., Reduced exercise capacity, poor quality of life, difficulty with activities of daily living and recurrent acute exacerbation are not simply the consequence of pulmonary impairment, but also impacted by peripheral muscle dysfunction.1 (Bernard et al., Although a causal relationship has not been established, significant association have been observed between measures of muscle function, lung function and exercise performance. Lower quadriceps strength is correlated with lower FEVReduced limb muscle strength also contributes to increased dyspnea, poor quality of life and health status (Shoup et al., Although lower extremity strength has received most of the attention, upper extremity strength is also compromised in individuals with COPD. The force-generating capacity of upper limb muscles are reduced in patients with COPD compared to healthy control (Gosselink et al., Finally, there is increasing evidence of impaired postural control in patients with COPD (Butcher et al., Impaired exercise capacity and peripheral muscle dysfunction contribute to increased mortality and reduced health status, even when accounting for age and lung function status (Marquis et al., Despite COPD being primarily a respiratory disease, these individuals present with important secondary dysfunction in their peripheral muscles characterized by muscle atrophy alterations in muscle fiber type, fiber composition as well as reduction in oxidative enzymatic activity, capillarity and mitochondrial dysfunction. These muscle changes have important clinical consequences such as impaired exercise tolerance, low physical activity and quality of life in people with COPD.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.


The wide range of disease pathologies seen in multiple organ sites associated with human cytomegalovirus (HCMV) infection results from the systemic hematogenous dissemination of the virus, which is mediated predominately by infected monocytes. In addition to their role in viral spread, infected monocytes are also known to play a key role in viral latency and life-long persistence. However, in order to utilize infected monocytes for viral spread and persistence, HCMV must overcome a number of monocyte biological hurdles, including their naturally short lifespan and their inability to support viral gene expression and replication. Our laboratory has shown that HCMV is able to manipulate the biology of infected monocytes in order to overcome these biological hurdles by inducing the survival and differentiation of infected monocytes into long-lived macrophages capable of supporting viral gene expression and replication. In this current review, we describe the unique aspects of how HCMV promotes monocyte survival and differentiation by inducing a “finely-tuned” macrophage cell type following infection. Specifically, we describe the induction of a uniquely polarized macrophage subset from infected monocytes, which we argue is the ideal cellular environment for the initiation of viral gene expression and replication and, ultimately, viral spread and persistence within the infected host. Human cytomegalovirus (HCMV), a ubiquitous betaherpesvirus that infects 60%–90% of the population worldwide, establishes a lifelong infection of the host . The disde novo viral gene expression, as monocytes are initially non-permissive for viral gene expression and replication and only become permissive upon their differentiation into macrophages . Fu. Fu24]. Although the precise mechanisms for how HCMV directs the polarization of infected monocytes/macrophages remain undetermined, our data clearly indicate that HCMV serves as a unique ligand that induces the differentiation and polarization of infected monocytes/macrophages towards a distinct phenotype that shares common characteristics with both M1 and M2 polarized macrophages ,79 Figu. In this


Mutual interactions in the form of symbioses can increase the fitness of organisms and provide them with the capacity to occupy new ecological niches. The formation of obligate symbioses allows for rapid evolution of new life forms including multitrophic consortia. Microbes are important components of many known endosymbioses and their short generation times and strong potential for genetic exchange may be important drivers of speciation. Hosts provide endo- and ectosymbionts with stable, nutrient-rich environments, and protection from grazers. This is of particular importance in aquatic ecosystems, which are often highly variable, harsh, and nutrient-deficient habitats. It is therefore not surprising that symbioses are widespread in both marine and freshwater environments. Symbioses in aquatic ciliates are good model systems for exploring symbiont-host interactions. Many ciliate species are globally distributed and have been intensively studied in the context of plastid evolution. Their relatively large cell size offers an ideal habitat for numerous microorganisms with different functional traits including commensalism and parasitism. Phagocytosis facilitates the formation of symbiotic relationships, particularly since some ingested microorganisms can escape the digestion. For example, photoautotrophic algae and methanogens represent endosymbionts that greatly extend the biogeochemical functions of their hosts. Consequently, symbiotic relationships between protists and prokaryotes are widespread and often result in new ecological functions of the symbiotic communities. This enables ciliates to thrive under a wide range of environmental conditions including ultraoligotrophic or anoxic habitats. We summarize the current understanding of this exciting research topic to identify the many areas in which knowledge is lacking and to stimulate future research by providing an overview on new methodologies and by formulating a number of emerging questions in this field. Ciliates are an extraordinarily widespread group of protists that occur in almost all aquatic environments. These include coastal waters, hydrothermal vents, anoxic sediments, hyporheic zones, and oxic as well as anoxic parts of the water column. Ciliates are very abundant phagotrophs in the biosphere and are capable of forming extensive blooms. They are important grazers of algae, bacteria, and other microorganisms , dinoflagellates and cryptophytes in marine ciliates , and kleptochloroplasts in both marine and freshwater ecosystems . Lithoautotrophic symbionts studied include methanogens and sulfur oxidizers in anaerobic or microaerobic ciliates, and heterotrophic symbionts include Paramecium and its parasite Holospora, Caedibacter , and Euplotes with Polynucleobacter necessarius—a stable and inseparable symbiosis. These well-studied interactions appear to be highly specific and to result from long-term co-evolution, suggesting stable and highly conserved systems. Nevertheless, insights into interactions between the single symbiont species and different hosts, and comparison of sequence data from free-living with symbiotic microorganisms indicate that ciliate symbioses represent rather open and highly dynamic entities rather than closed systems , chemosynthetic, and heterotrophic. Studied photoautotrophic groups include zoochlorellae in freshwater ciliates  is a particularly interesting case. Although almost completely phototrophic, it is an obligate mixotrophic ciliate. Long included with zooplankton in plankton analyses  Caedibacter, and (3) Polynucleobacter these interactions are poorly understood and studied.Symbionts providing defense for the host have only been identified for ciliates so far , which protect their host against the predator Litonotus lamella by committing suicide  belonging to the Syntrophomonadaceae (Firmicutes). This suggests that this symbiont is specifically associated to strains of Trimyema  and phylogenetic analysis  allow very detailed structural analysis of host-symbiont interactions they are limited in the study of physiological and functional aspects. As the majority of symbionts do not grow in pure culture most studies today rely on indirect approaches to investigate symbiosis in ciliates , physiological, biochemical as well as on a molecular level  to minimize false interpretations of the data. One option is to label dead cells with propidium monoazide (“live-dead staining”) followed by flow cytometry to separate dead bacteria from living symbionts  can be used to generate enough material from a few fg of template DNA for subsequent studies Lasken, . Howeveromics approaches  to explore host-symbiont interactions . In microbial ecology FISH is traditionally being used to label microbes with oligonucleotide probes against the small-subunit ribosomal RNA to distinguish between different phylogenetic lineages  could provide increased resolution in the study of bacterial diversity. Standard methods of determining taxon composition using16S rRNA OTUs are known to provide only “coarse” estimates of functional diversity or evolutionarily distinct populations. Improved methods of “species” delineation in bacteria that use DNA sequences will be provided by alternative gene regions  hold promise, we feel that new conceptual approaches are needed to unravel the hidden secrets of bacteria-ciliate interactions. We highlight a number of topics below.The independent and repeated establishment of different symbioses between aquatic ciliates and microorganisms suggests that environmental conditions at least partly regulate incorporation, maintenance, and termination of these symbioses. Unfortunately, the function of many symbionts is unknown and hence it is still unclear whether there is a directed establishment of symbioses for required functional traits. For example, it seems likely that many aquatic ciliates incorporate and maintain nitrogen-fixing bacteria under nitrogen-limited conditions. If proven to be correct, this would also imply a highly dynamic establishment of symbioses with only loosely linked microbial partners. Alternatively, potentially beneficial symbionts may be retained in the ciliate in low numbers and with reduced activity also when not needed. This might be of particular advantage in unstable and fluctuating environments.Our review shows that ciliate symbiosis has arisen across a broad range of taxa and environments; nonetheless, little is known about the evolutionary basis of the symbiosis. One important question is whether symbionts are species-specific or can affect a range of hosts? If species-specific symbiosis is common, then it may be that host-symbiont co-speciation has given rise to ciliate diversity. If a few small lineages form most symbioses, it suggests only one or a few genomic or genetic mechanisms may be involved, whereas if many unrelated lineages can form symbioses there may be functional redundancy. These and similar questions require broad taxonomic sampling that is increasingly available with large-scale sequencing methods (above) and can employ sequence-based delineation methods , but also of communication . How does the host recognize which symbiont is beneficial at a given time? How can it allocate nutrients to, e.g., a phototrophic symbiont, but not to a symbiont promoting digestion of the host's food? Which mechanisms are involved in the host-symbiont communication?Some closely related ciliates occur in marine, brackish, and freshwaters. Thus, for these species different mechanisms in the establishment and maintenance of symbioses are unlikely. However, marine and freshwater ecosystems differ besides salinity in many parameters such as limiting nutrients. This may result in the acquisition of specific microbial functions. For example, most marine systems are limited by nitrogen compounds, thus, acquiring bacteria with the capability of fixing nitrogen is highly advantageous. For these bacteria the host must provide an anoxic or microoxic environment to fix gaseous nitrogen. In freshwater systems on the other hand, phosphate is mainly the limiting factor which may result in symbionts capable of storing phosphate under favorable conditions. Thus, the host might need to move to zones rich in phosphate and establish behavior to circulate this phosphate rich water through its cell.If yes, what can we learn for biogeochemical cycling and evolution ? Containing methanogens seems to be restricted to ciliates living in anoxic or microoxic environments. This symbiosis includes tight coupling between the host's hydrogenosomes and the symbionts, thus even the evolution of hydrogenosomes may represent adaptation to symbiotic lifestyle and a unique niche for symbionts. Another special environment is the bridge between oxic and anoxic conditions where ciliates may favor their symbionts' growth by actively moving between these two zones. On the other hand, ciliates in the oxic zone may provide niches for anaerobic or microaerophilic bacteria in some of their cell compartments.Hereby, it is also interesting if this genetic exchange happens predominately between symbiont and host, symbiont and symbiont or even between symbiont and prey. Additionally the exchange and change of the symbiont's genomic signature after genetic depletion is highly interesting. Which evolutionary principles can be dissected from microbe-ciliate symbioses ?Many ciliates contain microorganisms such as algae, Bacteria, and Archaea Table  and inteEcosystem functioning is an important and often little understood parameter in biodiversity research. Unfortunately, symbionts are hardly taken into account although their capability of photosynthesis, sulfur transformation, methanogenesis and their demand on different elements including iron and magnesium is well known. To calculate their importance for regional and global matter cycling will be an interesting and challenging task in upcoming research by also determining their impact for other element cycling such as nitrogen and phosphorus.Our review shows how diverse and complex symbioses between aquatic ciliates and associated microbes can be and how many gaps in our knowledge still exist. In particular, gaining more information on how symbioses are established and maintained not only extends our scientific knowledge but also may give new insights into species evolution and material cycling and may underline that teamwork can outcompete the individual.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.


Venous aneuysmal dilatation or flebectasia (venous ectasia) or venous congenital cyst is a benign pathology which represents an abnormal dilatation of a vein and it is frequently seen with the improvements in non-invasive diagnostic methods of laryngeal and cervical diseases.Our case was a 68 year-old male. On the coronary angiography, which was performed with the complaint of chest pain, three-vessel disease was detected and the patient was hospitalized for surgery. Routine bilateral carotid colored duplex ultrasonography revealed 70% stenosis in the proximal segment of right internal carotid artery (ICA). Provided that, he was undertaken to carotid angiography and 75% stenosis was seen in the right ICA.In the light of these findings, right carotid endarterectomy was planned electively and simultaneously with coronary by-pass grafting (CABG) operation under general anesthesia. As right sternocleidomastoid muscle was carefully dissected for exploration of common carotid artery (CCA), huge fusiform aneurysmal internal jugular vein was confronted. CCA and its’ branches were carefully explored, and then carotid endarterectomy and CABG were performed simultaneously. He was taken to intensive care unit then to service follow-up without any complications.Venous aneurismal dilatation or flebectasia was thought to be congenital as seen rarely in adulthood, and as it doesn’t cause any symptom diagnosed coincidentally thus. Conservative clinical follow-up is required in this situation. Rupture of dilated vein is a hypothetic complication. Surgery may be needed in case of symptomatic patients, cosmetic reasons, thrombotic occasions and rupture of the vein.


Neuroimaging technology has afforded advances in our understanding of normal and pathological brain function and development in children and adolescents. However, noncompliance involving the inability to remain in the magnetic resonance imaging (MRI) scanner to complete tasks is one common and significant problem. Task noncompliance is an especially significant problem in pediatric functional magnetic resonance imaging (fMRI) research because increases in noncompliance produces a greater risk that a study sample will not be representative of the study population.In this preliminary investigation, we describe the development and application of an approach for increasing the number of fMRI tasks children complete during neuroimaging. Twenty-eight healthy children ages 9-13 years participated. Generalization of the approach was examined in additional fMRI and event-related potential investigations with children at risk for depression, children with anxiety and children with depression (N = 120). Essential features of the approach include a preference assessment for identifying multiple individualized rewards, increasing reinforcement rates during imaging by pairing tasks with chosen rewards and presenting a visual 'road map' listing tasks, rewards and current progress.Our results showing a higher percentage of fMRI task completion by healthy children provides proof of concept data for the recommended tactics. Additional support was provided by results showing our approach generalized to several additional fMRI and event-related potential investigations and clinical populations.We proposed that some forms of task noncompliance may emerge from less than optimal reward protocols. While our findings may not directly support the effectiveness of the multiple reward compliance protocol, increased attention to how rewards are selected and delivered may aid cooperation with completing fMRI tasksThe proposed approach contributes to the pediatric neuroimaging literature by providing a useful way to conceptualize and measure task noncompliance and by providing simple cost effective tactics for improving the effectiveness of common reward-based protocols. Functional magnetic resonance imaging is increasingly being used to advance our understanding of normal and pathological brain function and development in children and adolescents. However, there are a number of challenges that clinicians and researchers encounter. Noncompliance involving an inability to remain in a scanner to complete fMRI tasks is one common and significant problem. This paper describes the development and application of an approach we believe may improve the effectiveness of conventional reward-based approaches used to encourage task compliance. We discuss some issues surrounding task noncompliance and offer that integrating tactics derived from learning based behavior therapies into conventional reward-based protocols may help encourage compliance. Using a case study design, preliminary results show improvements in the number of study tasks completed in healthy children and those with clinical disorders, providing proof of concept data for the recommended tactics. While the present discussion focuses primarily on task compliance during functional neuroimaging investigations, some of the problems discussed and recommendations may generalize to other forms of neuroimaging .Noncompliance has been recognized as a central issue within pediatric fMRI research -5, as weOur understanding of the prevalence of task noncompliance within the pediatric neuroimaging literature is another area of weakness. Several functional neuroimaging studies have reported that task compliance improves with age and is higher in typically developing children relative to variety of clinical populations ,5,14. WhIn all pediatric functional neuroimaging studies, researchers and clinicians use rewards to encourage and maintain task compliance. This highlights an important tie to reinforcement learning theories. The widespread application of reward protocols also highlights recognition of the relationship between task compliance and a sufficiently rewarding neuroimaging environment. Accordingly, conventional reward-based protocols often employ multiple sources and different kinds of rewards to encourage motivation and task completion. One source is the monetary compensation provided for participation . Task cReward-based approaches clearly help to create a positive, encouraging, and supportive environment necessary for successful pediatric neuroimaging research. It seems important to note, however, that for a significant number of children, especially young children and sensitive clinical populations, reward-based approaches will not be enough to promote task compliance. Nevertheless, there still may be ways of improving or strengthening existing conventional reward-based approaches. Results of developmental studies on reinforcement processes, tactics used in behavior therapy for children and head motion training preparations that tap reinforcement as a change agent offer some important insights into why a reward-based protocol may fail and how to improve its effectiveness.One of the ways a researcher-identified reward, such as sticker or trinket, may fail to maintain task compliance is that the reward does not have the capability to function as a reinforcer, which strengthens or makes a behavior more likely to occur . Thus, while a subject may report that they 'like' or 'want' a preselected reward, it may simply not encourage or maintain a target behavior. In fact, finding appropriate rewards that work as reinforcers is a major component of effective sticker charts -23. A seIn this paper, we describe how integrating tactics derived from learning based behavior therapies into conventional reward-based protocols may help to improve or maintain task compliance. These tactics include (1) using preference assessments to identify multiple subject-specific rewards, (2) increasing reinforcement rates during imaging by providing a reward for each task, and (3) presenting a visual 'road map' during imaging that lists tasks, associated rewards and progress. For brevity, this collection of tactics will be referred to as the multiple reward compliance protocol. In what follows, we describe the development and application of our approach in several groups of children that participated in an fMRI investigation. Our results showing increases in the percentage of fMRI tasks completed in several groups of children provide proof of concept data for the multiple reward compliance protocol. Additional support is provided by results showing our approach generalized to several additional fMRI and event-related potential investigations and clinical populations .Twenty-eight healthy children ages 9-13 years  participated. Participation required completing a battery of clinical assessments and a 2 hr 3T functional neuroimaging session. Exclusion criteria for the study included: (a) symptoms suggestive of an Axis I psychiatric disorder based on parent report on the Child or Adolescent Symptom Inventory-4 ,27, (b) The twenty-eight subjects recruited for the study were subdivided into three groups based upon their order of recruitment. Table Table For the primary study groups, seven tasks were presented during neuroimaging in a randomized order, each separated by rest periods that lasted up to 5 minutes. The tasks used assessed attention, reward sensitivity, threat processing and discriminated avoidance and approach . CompliaThe general approach we employed to encourage and maintain compliance followed many conventional practices -5,17 witAs noted above, conventional reward-based protocols can fail to maintain task compliance for a variety of reasons---researcher-selected rewards may be inappropriate, discounted, devalued or uncompetitive. Fortunately, basic and clinical research studies on reinforcement processes and tactics used in learning based behavior therapies for children suggest some tactics for enhancing conventional reward-based protocols. Below we describe the rationale behind the development of a multiple reward compliance protocol. Critical elements of the protocol include (1) using preference assessments prior to imaging to identify multiple subject-specific rewards, (2) increasing reinforcement rates during imaging by providing a reward for each task, and (3) presenting a visual 'road map' during imaging that lists tasks, associated rewards and current progress.One approach to adverting task noncompliance issues related to selection of an ineffective reward(s) is to let subjects identify their own. Clinicians developing behavioral treatments for typically developing children and those with cognitive dysfunction commonly employ 'preference assessments' to identify preferred stimuli that may serve as subsequent reinforcers . AccordiOne approach to combating decreases in reward effectiveness caused by delays or cost/effort is by reducing delays and increasing the number of rewards earned per unit time. While increasing the frequency of verbal praise, encouragement and feedback can be effective in facilitating task compliance, a potentially more effective approach is to use preference assessment results to pair selected rewards with fMRI tasks. Accordingly, the second element of the multiple reward compliance protocol involved pairing subject-selected rewards highlighted in the preference assessment with imaging tasks and informing subjects they will earn one of their chosen rewards after completing each task. Providing multiple rewards during imaging effectively insulates rewards against the negative effects of temporal delays and cost/effort by increasing the local rate of reinforcement . In effect, the multiple reward compliance protocol approach models "catch them being good" clinical approaches that stress providing high rates of reinforcement for appropriate behavior -23.®, Powerpoint® or programmed using Eprime®. The format presents information about the pairing of tasks with subject-identified rewards and current progress. Each task is listed (under 'Jobs') and separated by imaging run, such that tasks requiring multiple imaging runs are listed multiple times. In the center of the display is a progress bar the researcher can advance downward toward a 'finish line' after each task is completed. Presentation of the visual display during breaks enables subjects to quickly observe their progress, tasks, earned rewards and upcoming rewards. The information provided about upcoming tasks may also insulate against the development of negative affective responses associated with the uncertainty of future events and provide an increased sense of control. At least one published protocol has employed a version of this approach with a "virtual sticker chart" that highlights progress [Considerable developmental research shows age-related improvements in memory and information processing speed and reductions in susceptibility to interference ,31. Withprogress . One strTable p < 0.05), and the difference of percentage has a 95% CI of . As a result, about 41.52% of subjects would be predicted to finish all seven tasks in the Reward group, while 74.6% of subjects would be predicted to finish all seven tasks in the MRCP combined group. Lastly, panel C in Figure p > 0.05), than ratings for subjects in the Reward group .To explore the level of compliance improvement, we used a censored geometric distribution model to estimate the probability of task compliance. The analysis revealed that 11.8% fewer participants were estimated as likely to complete each consecutive task in the Reward group compared to only 4.1% for the MRCP combined group. Bootstrap analysis  suggested the percentage of subjects that end participation is significantly higher in Reward group than the MRCP combined group  investigations that employed different research assistants and pediatric populations . For all studies, task compliance was again defined as the percentage of total scheduled tasks completed. Table Noncompliance is a common phenomenon in pediatric neuroimaging ,2. RecogOne contribution of our approach to pediatric neuroimaging was our focus on task noncompliance as an issue worth addressing apart from other forms noncompliance. Within the pediatric imaging literature, the term noncompliance encompasses a wide range of problems encountered by researchers and clinicians. These may include failures to enter a scanner, remain in the scanner, complete tasks, follow task instructions accurately and remain motionless. While all forms of noncompliance are significant barriers, it seems reasonably well established that maintaining participation involves addressing many problem areas with different types of tactics and doing so at different times. For example, some approaches encourage cooperation early in the process by having subjects watch a video of a child completing a routine fMRI study in a scanner . AnotherAnother contribution of our approach is that it highlights the idea that task noncompliance may result from less optimally designed reward-based protocols. This approach emphasizes the importance of creating an environment conducive to completing study tasks which differs in focus from other perspectives that might emphasize subject-related factors such as fear, anxiety or boredom as contributors to task noncompliance. In our case, task compliance was viewed as emerging from an imaging environment that provides adequate and sufficient reward. The end result was an enhanced reward protocol that included (1) a preference assessment to identify multiple subject-specific rewards, (2) increasing reinforcement rates during imaging by providing a reward for each task, and (3) presenting a visual 'road map' during imaging that lists tasks, associated rewards and progress. It is equally important to recognize that while our findings may not directly support the effectiveness of the multiple reward compliance protocol, our rationale is supported by the behavior therapy literature and basic behavioral research on learning processes. All of this is not to say that fear or anxiety are irrelevant to our understanding of noncompliance or that our tactics should replace using relaxation techniques or exposure training. It is merely to point out that improving reward protocols can contribute to enhancing task compliance.There were several limitations of the present investigation that restrict the conclusions that can be drawn about the multiple reward compliance protocol. The principal limitation was our inability to use a randomized control trial during the course of our investigation, which leaves results open to biases and experimenter expectancies normally controlled for with randomized control trial. Nevertheless, we submit that our results showing a higher percentage of fMRI task completion by healthy children provides proof of concept data for the recommended tactics. Additional support was also provided by results showing our approach generalized to several additional fMRI and event-related potential investigations and clinical populations . This level of generalization suggests that the multiple reward compliance protocol may extend to populations with significant cognitive impairments, such as children with developmental disabilities who are at increased risk of hypoxia when sedation approaches are used . AnotherFunctional magnetic resonance imaging is increasingly being used to advance our understanding of normal and pathological brain function and development in children and adolescents. Noncompliance involving an inability to remain in a scanner to complete fMRI tasks is one common and significant problem. Consequently, researchers and clinicians devote considerable effort to developing a supportive, positive environment. We proposed that some forms of task noncompliance may emerge from less than optimal reward protocols. Our findings suggest that increasing our attention to how rewards are selected and delivered may aid cooperation with completing fMRI tasks. The approach presented and preliminary findings contribute to the pediatric neuroimaging literature by providing a useful way to conceptualize and measure task noncompliance and a set of cost effective tactics for improving the effectiveness of common reward-based protocols.MRCP: Multiple reward compliance protocolThe authors declare that they have no competing interests.Design: MS, MC, GS. Data collection: MS, AM, JS, CL, GS. Analysis: MS, GS, IS.Writing: MS, GS, MC, CL, JS, EF, RD and NR All authors read and approved the final manuscript.


Dichloroacetate (DCA) is known to environmental scientists as a by-product of water chlorination and as a metabolite of industrial solvents, including Superfund chemicals. In contrast, DCA is studied by clinical investigators for its therapeutic potential in several life-threatening conditions, including genetic mitochondrial diseases, pulmonary arterial hypertension, and cancer. Thus, DCA holds an almost unique position at the interface between environmental science and allopathic medicine.I critically reviewed laboratory and clinical pharmacological research on DCA to address questions about the current and future status of DCA in relation to human health.Recent information on the clinical toxicogenetics of DCA is interpreted particularly in light of its use as an investigational drug. Adverse effects from chronic DCA exposure have been identified in several target organs in animals. However, in humans, toxicity has so far been limited to reversible effects on the nervous system and liver. DCA is primarily biotransformed to glyoxylate by the bifunctional enzyme glutathione transferase zeta1 and maleylacetoacetate isomerase (GSTz1/MAAI), which also catalyzes the penultimate step in the phenylalanine and tyrosine catabolic pathway. DCA is a suicide inhibitor of GSTz1/MAAI, which can result in delayed plasma clearance of DCA and the accumulation of potentially toxic tyrosine intermediates. Age and GSTz1/MAAI haplotype can markedly affect the toxicokinetics of DCA in humans and rodents.I have defined new potential avenues of research that focus on discrete human populations that may be at increased health risk or that may receive increased health benefit from chronic exposure to DCA at both environmentally and clinically relevant concentrations. To many readers of this journal, dichloroacetate (DCA) has a bad reputation that is mainly the result of its association with Superfund chemicals and other halogenated hydrocarbons of ill repute , the finThe DCA dilemma has been framed by three broad fields of science: epidemiological studies of populations exposed to environmental levels of measured by-products of water disinfection or Superfund chemicals that are precursors of DCA; mechanism-based toxicological experiments in which inbred rodent strains receive doses of DCA usually vastly higher than those to which most humans are exposed in the environment; and research in which subjects are administered DCA as a drug at doses similar to those used in animal toxicological investigations. Together, the data generated by such research address three questions that are at the heart of the DCA dilemma: Is DCA a significant environmental health hazard? Are certain populations at increased health risk or benefit? What are the important areas of future research that will help confirm, modify, or reject current opinions about DCA’s impact on human health?Is DCA a significant environmental health hazard? Human populations have been exposed to DCA for generations, primarily by ingesting chlorinated drinking water that may contain up to 160 μg/L . Thus, cNevertheless, considerable weight has been given to assigning causality to the oxidative metabolites of TCE  in evaluating the toxicity of DCA. Data from population-based pharmacokinetic modeling of the toxicokinetics and risk assessment of TCE and its metabolites  indicateAre certain populations at increased health risk or benefit? As a medicinal agent, DCA has been administered as the sodium salt in doses ranging from 10 to > 100 mg/kg/day by intravenous and oral routes for a few days to > 10 years . These clinical studies have reported occasional asymptomatic increases in serum concentrations of alanine aminotransferase and aspartate aminotransferase that seldom exceeded two to three times the pretreatment values of these enzymes and that returned to pretreatment levels within a few weeks after the DCA dose was reduced or stopped . A more One possible explanation for the hepatic and neurological toxicity of DCA relates to the unusual consequences of its biotransformation. DCA is dehalogenated to glyoxylate by the cytosolic zeta-1 family isoform of glutathione transferase (GSTz1), which is identical to maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway . DCA causes a mechanism-based inhibition of GSTz1/MAAI , an effeGenetic or DCA-induced ablation of GSTz1/MAAI causes accumulation of the MAAI substrates maleylacetone and, presumably, maleylacetoacetate (MAA) , althougKi = 0.3 μM; Another important consequence of tyrosinemia type 1 is the diversion of carbon precursors to succinylacetone, high urinary levels of which are diagnostic for the disease. Succinylacetone is a competitive inhibitor of δ-aminolevulinate dehydratase  with congenital forms of lactic acidosis, Recently, age and GSTz1/MAAI gene contains four nonsynonymous single-nucleotide polymorphisms, with products that show different activities toward DCA and variable frequencies among ethnic and racial groups . Those who metabolized DCA slowly showed markedly delayed plasma clearance and biotransformation, increased excretion of unmetabolized drug , and increased urinary accumulation of maleylacetone and δ-aminolevulinate. Atomic modeling of the effect of GSTz1/MAAI polymorphism showed that variants associated with slow DCA metabolism induced structural changes in the enzyme homodimer leading to protein instability or abnormal protein–protein interactions. These data suggest that GSTz1/MAAI haplotype may predict, at least in part, the toxicogenetics of DCA. Moreover, such information may be used prospectively to adjust dosing and mitigate the risk of adverse events from DCA.Age alone does not account entirely for variability in the kinetics and biotransformation of DCA. The human Ki = 0.2 mM)  complex , a gatek 0.2 mM)  toward tin vitro to a variety of cultured human tumor cells or to human tumors implanted into immunocompromised rodents  condition in which mitochondrial dysfunction and hyperproliferation of cells lining the pulmonary vasculature lead to increased pulmonary vascular resistance, failure of the right ventricle of the heart, and premature death. In animal models of primary arterial hypertension, DCA has been shown to prevent or reverse mitochondrial damage and cellular proliferation, improve lung function, and increase survival by its ability to stimulate oxidative phosphorylation and induce apoptosis in proliferating cells . ResultsGSTz1/MAAI and further impair DCA metabolism.What are important areas of future research? Few chemicals have integrated the disciplines of environmental science and clinical research as effectively as DCA. Recent studies investigating the toxicogenetics of this compound when administered as a drug have important implications for human populations chronically exposed to environmentally relevant concentrations. The findings are particularly germane to persons in contact with water sources contaminated with TCE or other industrial solvents or who are administered drugs that may be biotransformed to DCA , has a worldwide annual incidence that varies widely from approximately 3 to > 300 per million births . AnotherGSTz1/MAAI haplotype. Finally, careful consideration must be given to age and genotype when conducting future treatment-oriented research with DCA, particularly cancer trials involving adults.Likewise, humans who metabolize DCA slowly may also be genetically predisposed to exhibit reduced flux through the phenylalanine and tyrosine catabolic pathway. Chronic consumption of a high-protein diet by such individuals could lead to an accumulation of toxic tyrosine intermediates, with adverse consequences for hepatic, neurological, and other organ system failure. Testing this postulate could involve studies of stable isotope amino acid kinetics in relation to GSTz1/MAAI genotype. We continue to move incrementally, but steadily, from puzzled observations about the pharmacology of DCA to established conclusions about its effect on human health.DCA has so far been proven to be neither a major environmental health hazard nor a panacea for human ills. Future clinical and environmental research on this unusual molecule should be focused increasingly on understanding its impact on biological processes in discrete subpopulations of humans with known


Short linear motifs (SLiMs) are functional stretches of protein sequence that are of crucial importance for numerous biological processes by mediating protein–protein interactions. These motifs often comprise peptides of less than 10 amino acids that modulate protein–protein interactions. While well-characterized in eukaryotic intracellular signaling, their role in prokaryotic signaling is less well-understood. We surveyed the distribution of known motifs in prokaryotic extracellular and virulence proteins across a range of bacterial species and conducted searches for novel motifs in virulence proteins. Many known motifs in virulence effector proteins mimic eukaryotic motifs and enable the pathogen to control the intracellular processes of their hosts. Novel motifs were detected by finding those that had evolved independently in three or more unrelated virulence proteins. The search returned several significantly over-represented linear motifs of which some were known motifs and others are novel candidates with potential roles in bacterial pathogenesis. A putative C-terminal G[AG].$ motif found in type IV secretion system proteins was among the most significant detected. A KK$ motif that has been previously identified in a plasminogen-binding protein, was demonstrated to be enriched across a number of adhesion and lipoproteins. While there is some potential to develop peptide drugs against bacterial infection based on bacterial peptides that mimic host components, this could have unwanted effects on host signaling. Thus, novel SLiMs in virulence factors that do not mimic host components but are crucial for bacterial pathogenesis, such as the type IV secretion system, may be more useful to develop as leads for anti-microbial peptides or drugs. Short linear motifs (SLiMs) are functional microdomains in proteins that play a critical role in many distinct biological processes such as cell signaling and regulation, post-translational modifications, proteolytic cleavage, and protein trafficking  which presents a severe threat to public health.Since SLiMs are used in a plethora of cellular processes in eukaryotes and are utilized by both pathogenic bacteria and viruses, discovering and characterizing new linear motifs is of great importance. As well as shedding light on the mechanisms of fundamental cellular processes they also hold promise as future therapeutic targets. There is an urgent need for new classes of antimicrobial therapeutics that are effective against multidrug resistant bacteria. Conventional antibiotics are becoming increasingly ineffective against pathogenic bacteria, such as methicillin resistant We were interested in whether SLiMs may be valuable when developing new antimicrobial peptides or drugs. Compared with recombinant proteins, the smaller size of peptides makes them easier to manufacture and deliver. The use of chemically synthesized peptides in pharmacological and clinical applications is relatively limited by their low systemic stability and high clearance, poor membrane permeability, negligible activity when administered orally and their high cost of manufacture in comparison to small chemical compounds. However, to date more than 100 peptide-based drugs have already reached the market and of these, the majority are at the smaller end of the size spectrum at 8–10 amino acids , which integrates DNA and protein sequence information from Tox-Prot, SCORPION, the PRINTS database of virulence factors, VFDB, TVFac, Islander, ARGO, CONUS, KNOTTIN, a subset of VIDA and sequences derived by means of literature searches  value returned in the SLiMSearch results. The N_UPC for an individual motif in a specific organism was normalized by dividing the value by the total amount of UPCs (Unrelated Protein Clusters) in the specific organism and the average N_UPCs of a motif across all 60 organisms. For motifs where there were no incidences in a specific organism the frequency was set to an arbitrary value lower than the minimum actual observed value.Datasets comprised of protein sequences obtained from UniProtKB that are predicted to be effector proteins from a selection of 60 organisms represented in the MvirDB were used to assess the distribution of prokaryotic protein motifs. The presence of both known and novel motifs in these datasets was investigated using the predictive computational tool SLiMSearch which can be used to determine the occurrences of predefined motifs in protein sequences . The “phenogram” taxonomic tree (*.phy format) obtained from the NCBI server was fed into Drawgram tree drawing program of Phylip package (version 3.695). Branches were colored according to the following scheme: Purple, High GC Gram+ bacteria; Blue, Firmicutes; Yellow, a-proteobacteria; Light Brown, b-proteobacteria; Dark Brown, e-proteobacteria; Green, g-proteobacteria (non-enterobacteria); Red, g-proteobacteria (enterobacteria); Black, others (CFB).The organisms in Figures p-value (Sig) less than 0.05. Bonferroni correction for significance with 12 search datasets would suggest that motifs with a Sig value of less than 0.004 are significant. Since pathogenesis proteins from bacteria often interact with host protein components, we examined whether any of the identified motifs showed similarity to known eukaryotic linear motifs, using the Comparimotif tool. However, we did not find any convincing similarities, in spite of the known occurrence of eukaryotic motifs in bacterial effector proteins. We also investigated if any of the motifs were known prokaryotic motifs identified in the literature.Our objective was to discover novel SLiMs in non-homologous bacterial proteins with similar roles in virulence that may have functional importance in pathogenesis, and thus have potential to be developed into antimicrobial peptides or drugs. Our analysis returned both previously characterized and novel motifs in several different functional categories indicating the suitability of SLiMFinder for the analysis of bacterial sequence data as well as eukaryotic data. We focused on 12 groups of bacterial proteins with predefined roles in pathogenicity (Table N-acyl-S-diacylglyceryl-Cys) has been found to be an essential, ubiquitous, and unique bacterial post-translational modification. Such a modification allows anchoring of even highly hydrophilic proteins to the membrane surface leaving the rest of the protein to carry out a variety of relevant functions in the aqueous or aqueous-membrane interface  that are integral membrane proteins embedded within the cytoplasmic membrane. These glucosyl transferases contribute to the altering of the structure of the bacterial surface lipopolysaccharide (LPS) O-antigen along with O-acetyltransferase  found in adherence and lipoprotein datasets Table . This mop-value 0.0003) discovered is a C-terminal G[AG].$ motif in the type IV secretion system dataset. The full list of unrelated proteins containing the G[AG].$ motif is represented in Table Agrobacterium tumefaciens: VirB4, VirB8, VirB11, and VirB7 .PY and ∧..I[ST] are found among the effector proteins of many species, but are absent in those of many other species, including those with a reasonable number of annotated effector proteins (Figure We visualized the cross-species distribution of the SLiMFinder identified novel motifs (see Table s Figure .We also looked at the distribution of known motifs (see Tables It can be seen that strains of a species often have very similar motif distributions Figures –4. ThereWe believe that SLiMs are one potential class of new antimicrobial substances for the development of antimicrobial peptides and drugs. While they may lack the potency of antimicrobial peptides that damage the bacterial membrane, they may have other benefits. In particular, those that mimic peptide components of uniquely prokaryotic motifs are likely to have less off-target effects. The value of developing such therapeutic approaches depends on the range of species likely to be affected by the peptide therapeutic. While targeting eukaryotic peptides mimicked by prokaryote effector proteins provides a potential line of attack, the evolutionary plasticity of such motifs in both bacteria Figure  and in hThe shortlist of predicted motifs that we have generated provides a resource for researchers interested in the mechanisms of action of virulence factor proteins across a diverse range of bacterial species. The limitations of the list are well-illustrated by the fact that the motif discovery failed to rediscover the many mimicked eukaryotic motifs. This reflects not only the fact that some motifs have not evolved multiple times in unrelated proteins, but also the limitations in the datasets provided to the SLiMFinder approach. Ideally, datasets should have less than a 100 proteins which have clearly identified similar functions. The challenge is to group proteins according to function efficiently, since the annotation of protein function is highly variable, and frequently relies on computational predictions arising from homology rather than from direct experimentation. The bigger challenge is how to test and manipulate these motifs to provide insights into the mechanisms of action and to determine potential means of interrupting pathogenic processes. While mutagenesis studies can identify the key features of motif function, targeting of a motif may also be progressed by experimental use of bioactive peptides. However, identification of more potent peptidomimetic compounds that resemble such motifs will ideally need 3D models of the peptide regions in complex with their target interactors.What, then, is the contribution that computational screening of novel motifs may play in the discovery of novel antimicrobial peptides? Firstly, it clearly will not identify all known motifs, since patterns of recurrent evolution or of strong sequence conservation are not seen for all antimicrobial peptides. Computational screens will also have some “false positives” in two senses: firstly, statistical false positives where the motif arose simply by chance; and secondly, biological false positives where the motif that functions effectively within its biological context of a larger protein and that protein's complexes, but it will not function as a stand-alone synthetic peptide. This could reflect a lack of strong affinity for its targets or it could reflect an inability to be delivered to the appropriate context in the first place. Nevertheless, computational screens have the advantage that they can be performed on high throughput sequencing of organisms about which little else is known and for which biological screening by mutagenesis is painstaking or impossible. The advantage of computational prioritization is that it identifies a subset of peptides which are enriched for biologically active peptides. Clearly, the strategy we adopted here is only detecting a small fraction of known motifs, in part because of the stringent correction for statistical mismatches that could be false positives, but also because many motifs do not recur in known unrelated proteins that fall into the same functional class. Discovery for bioactive peptides could follow other strategies, including searches for evolutionary conservation (Davey 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.