Over the past 15 years, computational models have had a considerable impact on basal-ganglia research. Most of these models implement multiple distinct basal-ganglia pathways and assume them to fulfill different functions. As there is now a multitude of different models, it has become complex to keep track of their various, sometimes just marginally different assumptions on pathway functions. Moreover, it has become a challenge to oversee to what extent individual assumptions are corroborated or challenged by empirical data. Focusing on computational, but also considering non-computational models, we review influential concepts of pathway functions and show to what extent they are compatible with or contradict each other. Moreover, we outline how empirical evidence favors or challenges specific model assumptions and propose experiments that allow testing assumptions against each other. Basal ganglia (BG) contain a variety of both glutamatergic and GABAergic fiber tracts. Why is BG organization that complex? Two influential theories, published more than 20 years back  and contains an additional glutamatergic synapse  and substantia nigra reticulata (SNr), two largely analog BG output nuclei that tonically inhibit the thalamus Figure . The dire Figure . Corticae Figure  are eithIn the last decade, the rise of computational simulation techniques has boosted model development. Today, there is a multitude of different models, none of which yet accounts for all relevant empirical findings (section 9). Most of these models assume a clear anatomical separation between the different pathways. Although this is likely a simplification  implementation. Such mathematical implementations offer important advantages, including, but not limited to the following: they allow computing the effects of non-linear interactions between simulated neurons that would be impossible to compute mentally. Moreover, they are innately precise, thus preventing fuzzy assumptions; if some of a model's various assumptions contradict each other or do not interact well, the model will fail to produce meaningful output. Finally, computational models produce predictions that do not immediately originate from their assumptions. Such predictions might, for instance, relate to model performance during specific behavioral tasks. As a note of caution, however, computational models are often hard to grasp intuitively: a set of mathematical formulas does not innately reveal what function a model serves. Rather, extensive and often iterative simulations are required to reveal these functions. To report and review computational models, thus, verbal and graphical descriptions of model assumptions and outputs are required as well. These, however, may suffer from lack of precision and in any case simplify a model's “real” computational details.In the context of BG functioning, computational modeling has been particularly fruitful in recent years. The complexity of BG anatomy and physiology, in light of their substantial interactions with cortex, thalamus and other sub-cortical nuclei makes them a good target for computational modeling.The striatum (which is part of direct and indirect pathways) receives topographically organized inputs both from intratelencephalically-projecting cortical cells and from axon collaterals of cortical pyramidal-tract neurons Figure . CorticoNext to its MSNs, striatum contains cholinergic and several types of GABAergic interneurons Tepper, . While GSTN (which gives rise to the hyperdirect pathway) receives topographically organized inputs from frontal and motor cortices , including internal cognitive and emotional states, based upon activation of any other representation , they have been hypothesized to establish and execute sequences of motor processes by linking each single response of a sequence to its respective predecessor  information by reducing dimensionality of cortical information . This hypothesis therefore directly contradicts the global-facilitation hypothesis outlined in section 4.1. Recent computational models mostly follow Mink's  may serve as stimuli for stimulus-response associations as well. In an even broader generalization, the direct pathway may interlink any two cortical representations  result in reversal-learning deficits in non-human primates . In the model by Schroll et al. , therefoIn consideration of the long indirect pathway's relatively slow conduction velocity (section 2.2), this pathway has been hypothesized to provide termination signals for motor execution Nambu, . In lineThe global-response-termination hypothesis is well compatible with the hypothesis that the long indirect pathway globally inhibits responses (section 5.1). Response termination, however, requires a delay in suppression such that responses can be initiated first.We do not know of any direct functional evidence for a termination function of the long indirect pathway.According to Brown et al. , the shoBoth indirect pathways have been hypothesized to establish a surround inhibition of unwanted motor programs during motor responding Mink, . Mink 1 proposedChallenging the subdivision of BG fiber tracts into direct, indirect and hyperdirect pathways, BG have been proposed to consist of a selection pathway, containing what is referred to as direct and hyperdirect pathways in this review, and of a control pathway, vaguely consisting of what is termed long and short indirect pathways here , while synaptic plasticity in this pathway is not yet well understood (section 2.4).Based on its fast and relatively global effects on GPi, the hyperdirect pathway has been proposed to globally prevent premature responses until response selection has been completed Frank, . Along tBeing instructed to maximize rewards, subjects are supposed to learn to choose the stimulus of each pair that provides better average outcomes . In subsequent test trials, pairs are shuffled such that high-conflict pairs  and low conflict pairs  may be presented. Using such a task, human patients ON deep brain stimulation (DBS) of the STN , the hyperdirect pathway has been hypothesized to establish surround-inhibition of unwanted motor programs during responding . We don't know of any empirical study investigating the strict surround-inhibition hypothesis against the more unspecific surround-inhibition hypothesis.The hyperdirect pathway has been hypothesized to clear information from working-memory and to thus allow for updating of working-memory content  representations Nambu, . As outlCenter-surround collaboration has been proposed for direct and indirect pathways as well (section 5.5). Mink  assumed facilitating activation of cortical representations.According to a different set of hypotheses, global inhibition of cortical representations via the hyperdirect pathway is powerful enough to overrule any specific activation caused by the direct pathway Figure . WheneveAn again different set of hypotheses (section 5.6) suggests that the long indirect pathway modulates the direct pathway's effects. Suri et al.  proposedConflicting hypotheses on pathway functions may be empirically tested against each other. Critically, such tests will have to link brain processes to overt behavior and will thus have to be performed in awake animals or humans. In the following sub-sections 8.1 to 8.3, we suggest a few such experiments.Models are relatively unanimous about the direct pathway's functional contribution to motor responding. With regard to its potential involvement in working-memory processes, however, two relatively incompatible hypotheses have been proposed (section 4.5): according to the first, the direct pathway takes part in gating working-memory content (Gruber et al., training of working-memory tasks, since this might more consistently involve BG participation than an already automatized task (cf. Antzoulatos and Miller, To test the maintenance against the updating hypothesis, an experimenter could inactivate direct-pathway MSNs in genetically modified mice (cf. Hikida et al., inactivating the direct pathway, however, natural variances in direct-pathway gene expression could be related to working-memory performance (cf. Heinz et al., Related studies could also be performed with human subjects: rather than It has been proposed that the short indirect pathway learns response inhibition based on aversive events (Frank et al., A, show an increase in activity during competitive response B and that there are other neurons that behave vice versa. If the long indirect pathway is responsible for such a surround inhibition, lesions of STN and GPe should each eliminate the phasic increase in GPI activity. If, however, the short indirect pathway is involved, only lesions of GPe should eliminate it. If the hyperdirect pathway is critical, only STN lesions should abolish the phasic increase in firing.While some authors hypothesize surround-inhibition of unwanted motor programs to be implemented via long or short indirect pathways (section 5.5; Mink, Although existing models of BG pathways account for a variety of anatomical, physiological and biochemical data, some major findings have not yet been implemented at all. Table It may be noted that Table In the Introduction we posed the question, why BG contain such a multitude of nuclei and fiber tracts. By reviewing influential hypotheses on the functions of BG pathways, we hope to have outlined potential functional advantages of such complexity. It has to be admitted, however, that all of the interpretations reviewed have been developed from a reverse-engineering standpoint, asking why BG are complex, given that this is the case. They do not answer why complexities evolved in the first place or if there might have been simpler solutions that would have guaranteed equivalent functionality.Most theorists assume that BG nuclei and fiber tracts give rise to separate pathways and that these pathways fulfill distinct functions. While they mostly agree that the direct BG pathway activates specific cortical representations, the functions of indirect and hyperdirect pathways are under intense debate. We have outlined various hypotheses on these pathways' functions and suggested that they may be grouped according to these pathways' hypothesized interactions with the direct pathway. Specifically, we have identified push-and-pull opposition, center-surround cooperation, global blocking of direct-pathway effects and modulation of direct-pathway effects as major proposed interaction patterns.We hope to have motivated stringent empirical tests of hypotheses on pathway functions, believing that theory-based research promises exciting advances in the understanding of BG complexity.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.


Alcohol consumption within elite sport has been continually reported both anecdotally within the media and quantitatively in the literature. The detrimental effects of alcohol on human physiology have been well documented, adversely influencing neural function, metabolism, cardiovascular physiology, thermoregulation and skeletal muscle myopathy. Remarkably, the downstream effects of alcohol consumption on exercise performance and recovery, has received less attention and as such is not well understood. The focus of this review is to identify the acute effects of alcohol on exercise performance and give a brief insight into explanatory factors. Athletes, like the rest of the population, consume alcohol. Sporting clubs and associations are frequently reported in the media to place bans or restrictions on the availability and consumption of alcohol by contracted athletes. Yet the same media organizations also report on alcohol-fuelled violence or misdemeanors perpetrated by these same athletes, suggesting anecdotally that athletes consume alcohol, occasionally to excess. This is quantitatively supported by dietary surveys of athletic populations that demonstrate self-reported alcohol intake constitutes up to 5% of the total daily energy intake in elite athletes . HoweverThe detrimental effects of alcohol on human physiology have been well documented with acute alcohol ingestion affecting many aspects of metabolism, neural function, cardiovascular physiology, thermoregulation and skeletal muscle myopathy ,8,9. YetAlcohol consumption has a deleterious effect on a multitude of systems within the body and an in-depth analysis of each is beyond the scope of this review. However a brief insight into common symptoms linked to acute alcohol misuse will guide the following discussion on how alcohol influences human performance and recovery.2+ transients into the myocyte by inhibiting sarcolemmal Ca2+ channel actions. This action is reported in isolated human myotubes and rodent muscle tubes in-vitro [in-vitro evidence [Multiple detrimental actions of alcohol within skeletal muscle are likely. Firstly, alcohol inhibits Cain-vitro ,11,12. Cevidence . Secondlevidence . Indeed,evidence  and in hevidence ,16,17,18evidence ; howeverThe effects of alcohol on hydration and its diuretic function are historically well recognised. The identification of alcohol as a potent diuretic date back to 1948, where a 10 mL excess urine production was evident following each gram of ethanol consumed . The mecAlcohol has further been shown to act as a peripheral vasodilator. This presents several complications. Primarily this increases fluid loss through evaporation which further exacerbates the dehydration that is potentially already present. There is further an interference of central thermoregulatory mechanisms consequently resulting in a reduction in core body temperature ,22. ThusIn additional to being a readily accessible source of energy (29kJ per gram) , alcoholAlcohol is a well-known depressant and thus acts to reduce central nervous system (CNS) excitability and cerebral activity , as demoThe effect of alcohol on neurological function is likely to be caused by a myriad of factors. The aforementioned effect of alcohol on glucose metabolism could affect cerebral functioning leading to symptoms of alcohol intoxication . AlternaGiven the numerous and complex mechanisms by which ethanol impacts on physiological systems it can be strongly hypothesized that elevated blood alcohol concentrations at the time of exercise will impair performance. Remarkably there are relatively few clinical trials that address this question.Earlier studies found no significant consequence of alcohol on a sub-maximal endurance performance and a 5-mile treadmill time trial respectively ,40. ContDespite the long list of skeletal muscle and neurological symptomatology associated with alcohol consumption, the majority of literature has been unable to establish a significant cause-effect relationship between alcohol and anaerobic performance. To the reviewers knowledge McNaughton and Pierce  have conMost of the studies examining alcohol and athlete recovery have focused predominately on functional measures of muscle performance and blood borne markers of cellular tissue damage. To date, these studies have produced inconclusive results that fail to demonstrate a dose-dependency or critical threshold above which muscular recovery is compromised. Creatine kinase (CK) is an intra-muscular enzyme which when present in the peripheral circulation is widely used as a measure of muscle damage. Despite the clinical association between chronic alcohol abuse and skeletal muscle myopathy, acute ingestion appears to have little impact on exercise-mediated muscular damage ,16,18. TSimilarly to the analysis of markers of muscle damage, the intra-muscular consequences of acute alcohol ingestion on aspects metabolic pathways of recovery are also ambiguous in humans. Alcohol ingestion immediately following prolonged cycling exercise has a modest impact to impair glycogen re-synthesis . This acOf particular relevance to the recovery of strength athletes is the enhanced protein synthesis that occurs post-exercise to facilitate repair and adaptive hypertrophy . Acute aFunctionally, the consumption of moderate amounts of alcohol augments the loss of force associated strenuous eccentric exercise ,56. To tBoth the affects of alcohol on human physiology and the parameters that determine athletic performance are multi-factorial and extremely complicated. A significant body of literature has established an array of adverse symptoms caused by acute alcohol ingestion. However the notion that alcohol consumption effects performance has not received enough consistent validation to advance beyond being anecdotal. Nevertheless, just because alcohol is not yet comprehensively shown to have a negative influence on performance, does not imply this review advocates its use prior to, or following competition. Indeed, the data demonstrates a severe lack of analysis on the possible detrimental action of alcohol in the recovering athlete. However, based on the available experimental evidence in cellular and rodent-models, athletes should remain wary of ingesting alcohol following intense exercise, focusing instead on effective dietary strategies proven to enhance recovery.


The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented. Current structure-based research has used high-resolution MX and NMR-derived structures to guide hypothesis-driven research. This has been effective for well-folded, compact enzymes, and has enabled atomic-level dissection of an enzyme’s active site. Nevertheless, estimates suggest that over 50 % of eukaryotic proteins contain significant functional unstructured regions , the presence of high experimental noise, or by non-ideal buffer subtraction  functions derived from them  versus q frequently does not  models, where amino-acid residues are presented as spherical beads centered at corresponding Cα atom positions  (Rochel et al. Although exhaustive conformational sampling significantly increases the number of realistic models to be used for modeling experimental SAXS data, a single best-fit conformation may be incapable of explaining the observed SAXS profile. The lack of convergence of a single best-fit conformation has been shown to correlate with conformational disorder rather than a limitation of the search space algorithm (Pelikan et al. Structural biology now recognizes that partially populated states are crucial to biological function. The single conformation description of a macromolecule is only a snapshot of a macromolecular ensemble. We have seen that integrative methods that utilize NMR and MX with SAXS are proving to be essential for providing a larger description of the macromolecular ensemble. Using SAXS data as a source of experimental restraints for modeling macromolecular flexibility is an exciting and relatively underdeveloped discipline. SAXS data can provide important experimental feedback, and can be extended to include dynamic conformational changes characterized by time-resolved experiments. Time-resolved measurements require very high X-ray flux and fast detectors designed for rapid electronic shuttering. Both are now available, and SAXS, unlike traditional NMR and fluorescence experiments, is not affected by molecular rotation times, so time-resolved SAXS can be performed in an equivalent manner to the traditional static experiments. The development of the approaches for characterizing highly fluctuating conformational equilibria on the basis of traditional static experiments are becoming essential in the description of intrinsic dynamic biomolecular systems (Bernado and Blackledge


One of the major challenges in addiction treatment is relapse prevention, as rates of relapse following treatment remain very high across the main classes of drugs of abuse. Relapse prevention could be improved by a better understanding of the factors that influence treatment outcomes, including better predictors of risk of relapse following treatment. Recent developments in cognitive neuroscience point to neurocognitive measures  as potential predictors of relapse. These might even be better predictors than self-report measures, such as craving. We first give an overview of the current state of the field, and then discuss the outstanding challenges and future directions in this area of research. Substance-dependent individuals often relapse, despite their efforts to stay abstinent . SubstanOver the years, various kinds of predictors have been studied in relation to substance-use relapse such as demographic characteristics and other variables such as drug use severity, medical problems, and psychopathology . In Neurocognitive measures, including neurophysiological measures such as functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) during cognitive-task performance, arguably overcome some of the limitations of self-report measures. During neurocognitive assessments, participants are often unaware of the purpose of the assessment. Automatic, fast cognitive processes that are unavailable to conscious introspection can influence behavior . These Various recent theories of addiction suggest an imbalance in motivational and cognitive control processes in substance-dependent individuals –27. MoreA wide range of behavioral studies have confirmed the presence of an attentional bias to substance cues in dependent individuals [for review see Ref. ] and itsOther theories suggest that the ability to control drug use behaviors is reduced in drug-dependent individuals, particularly in conditions that deplete cognitive recourses, like craving or cue-exposure , 39. SevIn sum, both drug-related motivational processes as well as dysfunctions in cognitive control may contribute to compulsive drug use behavior and this might explain why substance-dependent individuals cannot control their drug use and often relapse after a period of abstinence , 38, 39.Well-established research on the role of cognitive and neurobiological processes in addiction has resulted in an increased focus on neurocognitive measures as predictors of treatment outcome and relapse. On the behavioral level, results have mainly showed an association between attentional bias and treatment outcome in substance dependency . To theOverall, the cue-reactivity studies show that enhanced brain-activity during substance cue-exposure in prefrontal, sensory, motor, and limbic (sub)cortical areas is associated with substance relapse . OnMost of the abovementioned studies indicate that cognitive and motivational processes are associated with relapse vulnerability. This is only a first step toward clinical implementation, and current findings need to be interpreted with caution since group results are not necessarily valid on individual results. Different methodologies and designs hamper a direct comparison between these studies. Further, it is not known whether the results found in one substance-dependent group  can be generalized to other substances. In addition, the treatment settings are quite diverse. However, if these findings will stand after replication and provide more accurate information on individual-level prediction, they might eventually help to identify substance-dependent patients that are at risk of relapse into substance use. Obviously, this is not only the case for neurocognitive predictors, but for all predictors of relapse, including demographical, self-report, or behavioral measures.It is clear that neurocognitive measures such as fMRI and – to a lesser degree – EEG are relative expensive and time consuming, limiting their daily use in clinical practice. Therefore, in order to advocate the use of neurocognitive measures in clinical practice, there should be a clear advantage compared to inexpensive and more feasible self-report predictors of relapse such as self-reported craving and substance-use severity. One hypothetical advantage could be that neurocognitive measures explain relapse better than self-report measures – or at least explain additional variance in predicting relapse over and above self-report measures.Current relapse prediction studies addressing motivational and cognitive control processes provide some preliminary indications for this. Some of the studies addressing motivational aspects found that brain-activity during substance cue-exposure was associated with relapse, whereas self-reported craving and substance-use severity were not associated with relapse –46. MarhHence, some relapse prediction studies indicate that neurocognitive measures might make a unique contribution to the prediction of relapse and may even be able to better predict outcomes than self-report measures such as craving. In addition, regarding the specific role of subjective craving, results suggest that the relationship between neurocognitive measures and relapse is not accounted for or mediated by craving . SpeculNeurocognitive methods provide us with crucial information of how brain responses are related to clinical outcomes. Although there are some indications that neurocognitive measures could be relevant in clinical practice there are some issues that need further research before these measures can be applied in a clinical setting. Obviously, one of the biggest challenges is moving from group-level associations with treatment outcomes/relapse to individual-level prediction of such outcomes, for example by using receiver operating characteristic analyses , 54. ThiIn the long run, neurocognitive techniques such as fMRI could be used to investigate individual risk profiles for example with the use of machine-learning approaches . HoweverAnother limitation is that all studies use different measures of treatment outcome/relapse  which makes it difficult to compare the present results. Using the same (multiple) outcome measures across studies would certainly advance the field, also beyond neurocognitive studies.Also, current prediction studies using neurocognitive measures have not addressed the role of response inhibition, which is another important index of cognitive control. Some evidence comes from cross-sectional studies showing that ex-substance users have increased brain-activity during inhibitory control (assessed with Go-Nogo) compared to current users, suggesting that response inhibition might underlie recovery from substance dependence [smokers: ; cocaineFinally, it is of theoretical as well as clinical importance that studies investigating the predictive value of neurocognitive processes of addiction include self-report and behavioral measures in prediction models. It is important to test whether the unique contribution of these relatively “new” measures is high enough in addition to well-established, more easily administered measures. Ideally, relapse prediction models should be multifactorial and should include (socio)demographic, psychological, physiological, and cognitive variables. Of course, large sample sizes are needed to accomplish sufficient power .Neuroimaging research has yielded important information on neurocognitive mechanisms of substance dependence in relation to treatment processes and outcome. Results have shown that neurocognitive measures can provide information on relapse vulnerability over and above the information gained from self-report measures such as craving on a group level. However, regarding clinical utility it is important that all prediction studies report the sensitivity and specificity of neurocognitive relapse predictors. This will allow gaining more knowledge on the suitability of neurocognitive measures for individual risk taxation, necessary for implementation in clinical settings.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.


Mycobacterium tuberculosis (Mtb) has evolved into a highly successful human pathogen. It deftly subverts the bactericidal mechanisms of alveolar macrophages, ultimately inducing granuloma formation and establishing long-term residence in the host. These hallmarks of Mtb infection are facilitated by the metabolic adaptation of the pathogen to its surrounding environment and the biosynthesis of molecules that mediate its interactions with host immune cells. The sulfate assimilation pathway of Mtb produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival. This pathway is regulated by diverse environmental cues and regulatory proteins that mediate sulfur transactions in the cell. Here, we discuss the transcriptional and biochemical mechanisms of sulfur metabolism regulation in Mtb and potential small molecule regulators of the sulfate assimilation pathway that are collectively poised to aid this intracellular pathogen in its expert manipulation of the host. From this global analysis, we have identified a subset of sulfur-metabolizing enzymes that are sensitive to multiple regulatory cues and may be strong candidates for therapeutic intervention. Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis in humans, infects roughly 2 billion people worldwide Mtb show signs of active disease Mtb must withstand phagocytosis by alveolar macrophages, the host's primary line of defense against airborne pathogens Mtb is able to replicate and eventually induce granuloma formation Mtb persists in the hostile phagosomal environment and orchestrates the transition to latency are ill defined. Elucidating the molecular machinery and metabolic pathways that facilitate these pivotal events is crucial to identifying new therapeutic targets for this global human pathogen.In order to mount a latent infection, Mtb pathogenesis Mtb have been intimately linked to bacterial virulence  and nutrient starvation , conditions that also induce the ATP sulfurylase genes cysD and cysN, as well as the cysC gene that encodes APS kinase  following diamide treatment Streptomyces coelicolor, a related actinomycete, is also induced by diamide and regulated by the redox-sensitive anti-sigma factor RsrA  regulon that is positively regulated by the transcription factor CysB In E. coli as a blueprint for those of Mtb. For example, the reductive branch of the Mtb sulfate assimilation pathway emanates from APS, whereas E. coli initiates the biosynthesis of reduced sulfur metabolites from PAPS Mtb also encodes four eukaryotic-like sulfotransferases , which are absent from E. coliMtb encodes two independent pathways for cysteine biosynthesis: the canonical pathway, which utilizes L-serine (as in E. coli), and a novel route that utilizes O-phospho-L-serine Mtb, they do suggest that the transcriptional regulation of mycobacterial sulfur-metabolizing enzymes may diverge considerably from that of E. coli. Additional studies are needed to elucidate the transcriptional machinery mediating the expression of these enzymes in Mtb. Notably, a functional analog of CysB was recently identified in Corynebacterium glutamicum, a gram-positive bacterium belonging to the same suborder as Mtb, which will likely facilitate the search for related proteins in mycobacteria Though the enzymes involved in sulfur metabolism are largely conserved between these two prokaryotes, there are some notable differences in the organization of their respective sulfate assimilation pathways that caution against using the transcriptional regulatory networks of Mtb is an ABC transporter encoded by the subI-cysTWA1 operon cysA1 gene in M. bovis completely inhibits sulfate uptake in vitro and renders the mutant auxotrophic for methionine, yet does not impair bacterial survival in mice Mtb encodes two additional putative sulfate transporters, Rv1739c and Rv1707, whose genes are induced 24 h postinfection of activated macrophages E. coli has been shown to enhance sulfate uptake, though complementation of the M. bovis cysA1 mutant with the Rv1739c gene is not sufficient to restore sulfate prototrophy Rv1707 gene product. Assuming all three of these transporters are functional, it is possible they modulate sulfate uptake in response to discrete environmental signals.In addition to the various environmental conditions that modulate sulfur metabolism at the transcriptional level, several proteins regulate the metabolic pipeline by mediating the availability of sulfate and its flux through the sulfate assimilation pathway. Most notable are the sulfate transporters, which enable the uptake of extracellular sulfate. The primary sulfate permease of Mtb genome encodes six type I sulfatases, yet little is known about their biochemistry Mtb exhibits residual sulfatase activity that may be attributed to FGE-independent type II and type III sulfatases The sulfatases are a second class of proteins that likely contribute to the intracellular concentration of free sulfate. These enzymes catalyze the hydrolysis of sulfate esters from sulfated proteins, peptides, and small molecules  [55]. TyMtb sulfur metabolism is CysQ, a 3′-phosphoadenosine-5′-phosphatase. Unlike sulfate permeases and sulfatases, whose activity directly influences the availability of free sulfate, CysQ has the ability to modulate the levels of intermediates that may affect flux through the sulfate assimilation pathway. Specifically, CysQ dephosphorylates 3′-phosphoadenosine 5′-phosphate (PAP), a byproduct of sulfotransferase reactions, and its sulfated counterpart, PAPS Mtb sulfotransferase Streptococcus mutans confers resistance to superoxide stress Another regulator of Mtb, several paradigms have emerged from other bacteria that may prove functional in mycobacteria. While it is important to consider genus- and species-specific variations in sulfur metabolism when drawing such parallels MtbSmall molecule regulation of sulfur metabolism is another important mechanism directing the biosynthesis of sulfur-containing metabolites in bacteria. Though this phenomenon has not been thoroughly investigated in S. typhimurium, cysteine, sulfide, and thiosulfate have all been shown to inhibit sulfate assimilation O-acetylserine, the biosynthetic precursor of cysteine. In contrast, sulfide and thiosulfate bind the CysB transcriptional regulator, repressing the transcription of genes that facilitate sulfide biosynthesis or in the case of thiosulfate, its uptake from outside the cell. In E. coli, APS has been shown to inhibit Cbl-mediated transcription, preventing the induction of SSI genes that facilitate sulfur metabolism in the absence of inorganic sulfate Mtb, they are likely to play an important role in regulating sulfur metabolism. Consistent with this hypothesis, transcription of the cysDNC operon of Mtb increases in response to sulfur limitation and decreases following treatment with exogenous cysteine In Mtb. As mentioned previously, the alternative sigma factor SigR from S. coelicolor has been shown to modulate the biosynthesis of MSH in response to the intracellular MSH concentration MSH is also likely to trigger transcriptional regulation of sulfur-metabolizing enzymes in A global assessment of the transcriptional, biochemical, and molecular mechanisms of sulfur metabolism regulation described herein suggests nodes of regulatory convergence, or enzymes that are sensitive to multiple regulatory cues. These enzymes are apparently poised to tune flux through the sulfate assimilation pathway in response to substrate availability and a broad range of environmental signals. While many sulfur-metabolizing enzymes are promising drug targets  of Mtb, which converts free sulfate into PAPS cysDNC transcription Mtb sulfur metabolism.ATP sulfurylase and APS kinase, encoded by the Mtb sulfate assimilation pathway cysN with cysC) distinguish ATP sulfurylase from its human counterpart, which may facilitate the discovery of selective inhibitors Notably, ATP sulfurylase, a heterodimeric enzyme with both GTPase (CysN) and sulfurylase (CysD) domains, possesses several unique features that make it a particularly compelling drug target cysK2, cysM, and the subI-cysTWA1 operon. Both cysK2 and cysM are annotated as putative O-acetyl-L-serine sulfhydrylases (OAS), enzymes that catalyze the PLP-dependent biosynthesis of L-cysteine from O-acetyl-L-serine and a sulfur donor such as sulfide cysK1, encodes the true OAS of MtbO-phosphoserine as a substrate cysK2 and cysM are transcriptionally regulated by multiple environmental cues (subI-cysTWA1 operon, which encodes the primary sulfate transport system of Mtb, is sensitive to diverse stress conditions Mtb sulfur metabolism.Additional regulatory nodes suggested by the combined transcriptional data  are cysKtal cues  [21], [2Mtb is responsible for the biosynthesis of sulfur-containing metabolites that influence bacterial pathogenesis. The transcriptional regulation of this pathway in response to multifarious environmental cues, including those typically encountered in the phagosome, likely facilitates adaptation to host immune cells. In addition, several proteins that mediate the flux of sulfate through the pathway, such as sulfate permeases, sulfatases, and the phosphatase CysQ, modulate the biosynthesis of sulfur-containing compounds in response to the evolving metabolic demands of the cell. Finally, the potential for small molecule regulation of Mtb sulfur metabolism abounds; the metabolites produced by the sulfate assimilation pathway are themselves candidate regulators of this network, as evidenced by cysteine and MSH in other bacteria. Realizing the extent of this regulation remains an outstanding challenge in the field, as does identifying the transcriptional proteins that orchestrate this pathway's response to the bacterial environment. Characterization of CysK2 activity and the design of selective inhibitors targeting it and other nodes of regulatory convergence, particularly ATP sulfurylase, also warrant exploration. Addressing these aims will not only advance our understanding of sulfur metabolism in Mtb, but may also reveal a molecular linchpin whose inhibition could dismantle an essential metabolic pathway in this ubiquitous human pathogen.The sulfate assimilation pathway of


In recent years, we came to realize that obesity, broadly defined as increased body mass index or increased total body fat, is not necessarily associated with metabolic dysfunction and greater risk for cardiometabolic disease. In fact, there are several obese persons who are “metabolically healthy,” as there are nonobese persons who are “metabolically abnormal.” Although the reason(s) underlying this phenomenon are still not entirely clear, a number of studies conducted over the past several years indicate that the anatomical location of excess fat is more important than total body adiposity in determining metabolic outcomes. Ectopic fat accumulation, particularly in the liver, is frequently observed in obese persons and is strongly associated with metabolic dysfunction, including multiorgan insulin resistance and dyslipidemia. Intrahepatic fat, possibly more than visceral or intramyocellular fat, may thus be a prominent factor modifying the metabolic risk associated with increasing whole-body adiposity. However, cause-and-effect relationships have not yet been established, and it is also possible that intrahepatic triglyceride content is not a determinant but merely a marker of metabolic health. Understanding the regulation of fat accumulation in the liver will thus have important implications in both research and clinical practice. Little is known regarding the specific effects of lifestyle factors such as diet and exercise in regulating the accumulation of fat in the liver and its depletion thereof. In this special issue, we have invited a few papers in an attempt to partly fill this gap in our knowledge. Putative factors that may modulate the effect of exercise on liver fat: insights from animal studies,” several studies in animals are reviewed in order to highlight putative factors that may modulate the effect of exercise on liver fat. This includes the fat content of the diet (exercise appears to be more effective under high-fat feeding), the role of concurrent exercise-induced loss of body weight or visceral fat, sex , prandial status (fasted versus fed), and the duration of training, as well as the time elapsed from the last bout of exercise. The potential importance of these factors in modifying the exercise-induced changes in liver fat has not yet been formally tested in man, thereby providing a wide array of opportunities for future research. The second paper of this issue, “Nafld, estrogens, and physical exercise: the animal model,” focuses on the effects of exercise on liver fat in relation to estrogen availability. Estrogen deficiency, such as that occurring naturally after menopause in women, is strongly associated with fatty liver in animals. Exercise training exerts an estrogenic-like effect on the expression of genes involved in hepatic lipid metabolism and is a powerful means for preventing liver fat accumulation in estrogen-deficient animals. The third paper of this special issue, “Dietary conjugated linoleic acid and hepatic steatosis: species specific effects on liver and adipose lipid metabolism and gene expression,” reviews the effects of dietary conjugated linoleic acid on liver fat content and hepatic and adipose tissue fatty acid metabolism in animals. Conjugated linoleic acids, particularly the trans-10, cis-12, lead to hepatic steatosis owing to increased de novo lipogenesis and increased hepatic fatty acid uptake, at rates far exceeding the rates of disposal of intrahepatic fatty acids towards oxidation, esterification, and triglyceride export. In the first paper of this issue, “Effects of exercise training on molecular markers of lipogenesis and lipid partitioning in fructose-induced liver fat accumulation,” examines the effects of exercise training on liver fat in starved and subsequently fructose-refed animals. Fructose, a simple sugar, is a potent dietary trigger for liver fat accretion. Exercise training in this model is not able to reverse the fructose-induced changes in lipogenic enzymes and does not reduce intrahepatic fat content. Thus, contrary to the large body of evidence demonstrating that exercise is effective in alleviating hepatic steatosis induced by high-fat feeding, exercise is not able to reverse the changes induced by fructose feeding. The final paper of this special issue, “Exercise and omega-3 polyunsaturated fatty acid supplementation for the treatment of hepatic steatosis in hyperphagic OLETF rats,” evaluates the effects of exercise on a hyperphagic model of obesity, with or without concurrent omega-3 polyunsaturated fatty acid supplementation. Exercise training in this animal model alleviates hepatic steatosis even under low-fat feeding conditions, predominantly by increasing hepatic fatty acid oxidation, whereas supplementation with omega-3 fatty acids slightly increases liver-fat content and attenuates the liver-fat-depleting effect of exercise. It is noteworthy that omega-3 fatty acid supplementation in this study accounted for only 3% of total dietary energy, whereas in several previous studies showing that omega-3 fatty acids reduce liver fat the supplement was administered at much greater doses. The fourth paper of this issue, “Research presented and reviewed in this special issue not only highlights the independent effects of exercise and diet on liver fat accumulation but also, more importantly, raises the intriguing possibility of interactive effects between exercise and diet on the mechanisms regulating liver fat accretion and depletion. It seems that several dietary factors are able to either augment or attenuate the intrahepatic triglyceride-depleting effect of exercise. Faidon MagkosFaidon MagkosJean-Marc LavoieJean-Marc LavoieKonstantinos KantartzisKonstantinos KantartzisAmalia GastaldelliAmalia Gastaldelli


Despite significant improvement in modalities for treatment of cancer that led to a longer survival period, the death rate of patients with solid tumors has not changed during the last decades. Emerging studies have identified several physical barriers that limit the therapeutic efficacy of cancer therapeutic agents such as monoclonal antibodies, chemotherapeutic agents, anti-tumor immune cells, and gene therapeutics. Most solid tumors are of epithelial origin and, although malignant cells are de-differentiated, they maintain intercellular junctions, a key feature of epithelial cells, both in the primary tumor as well as in metastatic lesions. Furthermore, nests of malignant epithelial tumor cells are shielded by layers of extracellular matrix (ECM) proteins  whereby tumor vasculature rarely penetrates into the tumor nests. In this chapter, we will review potential strategies to modulate the ECM and epithelial junctions to enhance the intratumoral diffusion and/or to remove physical masking of target receptors on malignant cells. We will focus on peptides that bind to the junction protein desmoglein 2 and trigger intracellular signaling, resulting in the transient opening of intercellular junctions. Intravenous injection of these junction openers increased the efficacy and safety of therapies with monoclonal antibodies, chemotherapeutics, and T cells in mouse tumor models and was safe in non-human primates. Furthermore, we will summarize approaches to transiently degrade ECM proteins or downregulate their expression. Among these approaches is the intratumoral expression of relaxin or decorin after adenovirus- or stem cell-mediated gene transfer. We will provide examples that relaxin-based approaches increase the anti-tumor efficacy of oncolytic viruses, monoclonal antibodies, and T cells. This tumor stroma is basically composed of the non-malignant cells  of the tumor such as cancer-associated fibroblasts (CAFs), immune cells [tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs)], and mesenchymal stem cells as well as the extracellular matrix (ECM) consisting of fibrous structural proteins (collagen and elastin), fibrous adhesive proteins (fibronectin and laminin), and proteoglycans –3 FigurA. In mosIt is well established that stroma that is associated with normal tissue development and homeostasis is strikingly distinct from that associated with carcinomas . SpecifiThe major contributors of abnormal ECM in solid tumors are stromal cells such as cancer-associated fibroblasts (CAFs), TAMs, and TANs , 13, 31.Cancer-associated fibroblasts are the predominant cell type in the tumor-associated stroma. Their numbers are elevated in tumor stroma compared to stroma found in healthy tissue , 35. In regs and generation of an immune suppressive microenvironment . Under hypoxic condition, tumors produce a number of chemokines that attract and differentiate CAFs, TAMs, and TANs. Approaches that reduced intratumoral hypoxia therefore block pro-tumoral functions of these cells, including the production of stroma proteins. It is therefore thought that hypoxia targeting strategies improve the intratumoral penetration of drugs .About 90% of solid tumors are of epithelial origin, often featuring a stratified epithelium characterized by multilayered cells with three-dimensional intercellular junctions. This is in contrast to monolayered epithelial cells lining epithelial tracts , epithelial ducts , or cavities , which possess an apical-basal polarization of their cell membranes and cytoskeleton. The epithelial phenotype is generally defined by tight and adherence junctions that seal the paracellular space between adjacent cells and thereby providing a barrier that restricts passing of ions and macromolecules Figure A 80)..80).Tight junctions play a key role in the formation of epithelial sheets. Strictly linked to tight junctions is a barrier function within a sheet of cells that restricts ions and small molecules to pass through the paracellular space between two adjacent epithelial cells . Additio2+-dependent manner. Their cytoplasmic domain binds cytosolic catenins that link the cadherin/catenin complex to the actin cytoskeleton. The formation of adherens junctions consequently leads to the assembly of tight junctions, but E-cadherin is dispensable for tight junction maintenance . Members of this protein superfamily promote homophilic intercellular adhesion in a Cantenance . A centrntenance , 89. Thentenance , whereasntenance . Similarntenance , 93.Desmosomes are molecular complexes of cell adhesion proteins and linking proteins that attach the cell surface adhesion proteins to intracellular keratin cytoskeletal filaments. The cell adhesion proteins of the desmosome, desmoglein, and desmocollin, are members of the cadherin family of cell adhesion molecules. They are transmembrane proteins that bridge the space between adjacent epithelial cells by way of homophilic binding of their extracellular domains to other desmosomal cadherins on the adjacent cell. Both have five extracellular domains, and have calcium-binding motifs. One of these junction proteins, DSG2, is upregulated in malignant cells , 95. DSGOne of the key features of epithelial tumors is the presence of intercellular junctions, which link cells to one another, and act as barriers to the penetration of molecules with a molecular weight of>400 Da –100. Mosneu, the receptor for the widely used monoclonal antibody Herceptin (trastuzumab) co-stained with the tight junction proteins claudin 7  are important mechanisms that drive tumor progression and therapy resistance, and indirectly affect intratumoral drug penetration. EMT and MET have been accredited important roles in embryogenic development, tissue regeneration, cancer progression, and recently also the induction and maintenance of stem cell properties . ImportaDuring progression toward metastatic disease carcinoma cells engage EMT. The EMT program can be activated by a multitude of factors secreted by tumor stroma cells, which triggers a complex signaling network including TGF-β, Wnt, HGF, EGF, and PDGF pathways . The morSuccessful EMT induction ultimately enables cancer cells to leave the primary tumor, enter the bloodstream, and attach to distant organ sites in order to build metastases. The endpoint of this process however, involves the reversed process (MET), where cells that underwent EMT regain epithelial properties and form tumors that histopathologically resemble the primary cancer , 134. Alin vitro and undergo MET to form tumors containing epithelial cells when injected into immunodeficient mice , compared to naïve T cell treatment alone (0%). Better survival of relaxin-expressing mice was due to a higher number of neu-specific T cells inside the tumor.Access of anti-tumor immune cells and their intratumoral infiltration is limited by tumor stroma . More spTumor ECM as well as tumor cell density can inhibit diffusional transport of monoclonal antibody therapeutics in tumor tissues. In a study of the monoclonal antibody trastuzumab (Herceptin) penetration, Beyer et al. observed extensive tumor ECM and intercellular junctions in breast cancer patients and xenograft models . Therefoin vivo prostate xenograft tumor growth and angiogenesis  is produced by Vibrio cholerae strains and possesses the ability to reversibly modify intestinal epithelial tight junctions, granting the passage of macromolecules through mucosal barriers  currently containing 57 serotypes. Wang et al. recently reported that a group of human Ads uses DSG2 as a receptor for infection . Among D Figures B,C and imacaques , 181.in vitro and in xenograft models have shown that the DSG2 ECD is cleaved upon binding of JO-1, which results in DSG2 internalization , paclitaxel (Taxol®), nanoparticle albumin bound paclitaxel (Abraxane®), and irinotecan (Camptosar®) in tumor xenograft models of breast, lung, and prostate cancer . ECM production and epithelial junctions can also be targeted through influencing signaling pathways in tumor cells, specifically pathways involved in the regulation of EMT/MET and hypoxia. In conclusion, there is an increasing arsenal of approaches that can be used to enhance the efficacy of more classical cancer therapeutics and overcome treatment resistance.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.


Objectives: Sex hormones strongly influence skin physiology and evidences suggest wound healing as well. Estrogens establish a prohealing setting, and androgens play an antagonist role. Methods and Results: We present the case of a young woman in whom pregnancy with its associated hormonal shifts allowed the spontaneous healing of a chronic wound on the left leg with bone exposure, which had followed a car accident and had resisted medical and surgical treatments for 5 years. Discussion: Systemic or topical estrogens may offer alternative options in the therapy of chronic wounds, in particular in the elderly adults who have physiologically reduced estrogens levels. Chronic skin ulcers are a major social burden in an increasingly elderly population, which hamper patients’ life and represent high costs for health care systems. Incapacity to heal is determined by a multitude of local and systemic factors, such as peripheral arteriopathy, venous insufficiency, diabetes, nutritional deficits, and infection. Alone or, more often, in combination, these exacerbate inflammatory phenomena and frustrate the proliferative phase of wound healing by an ultimate action on microenvironment and cells. The contribution of hormonal unbalances is often less appreciated. However, hormones deeply influence cells activity. Glucocorticoids are powerful inhibitors on almost all cell types, while a lack of thyroid hormones impairs fibroblasts function.A 30-year-old woman consulted us for a chronic skin ulcer with extensive soft tissues loss and tibial exposure at the left leg, without fever or other signs of acute infection . She had,9The influence of sex hormones on wound healing only recently has been gaining higher attention. Chronic wounds mostly occur in the elderly adults,5Pregnancy is a peculiar condition in which the sex-hormone balance is strongly altered. A sudden shift toward estrogens and progesterone occurs, estradiol increasing 5-fold in the first trimester and 32-fold by week 40, while androgens remain substantially stable and the free androgen index markedly decreases (5-fold between week 5 and week 21).Other pregnancy-associated elements may also have played a role in the induction of healing seen in our patient, for example, changes in blood flow, liquids retention, or availability of metabolites affecting the wound environment, as well as parallel alternative signal pathways.On the contrary, given the cumulating evidences, the role of sex hormones in tissue repair seems to merit further investigation; our clinical observation suggests a possible major impact of the introduction of hormonal therapy as skin lesions medical treatment.


Most medical schools list self-awareness, self-care and personal growth as key educational competencies. Yet, studies have reported that traits such as altruism and empathy tend to decline throughout medical school training. To foster medical student self-awareness and mindfulness, which may improve their emotional intelligence (EI), Georgetown University School of Medicine (GUSOM) offers an experiential course to medical students in mind-body medicine skills. The purpose is to expose students to a variety of mind-body approaches , as well as group sharing that involves listening generously and without judgment.The aim of this study was to assess the impact of the Mind Body Medicine Skills (MBS) course on several behavioral measures such as perceived stress (PSS), mindfulness (Freiberg Mindfulness Inventory – FMI), positive and negative affect (PANAS) and elements of EI .Data were obtained from 72 first year medical students , who completed the survey instruments before and after participating in the course. A significant decline was seen in perceived stress among all participants , while mindfulness increased significantly . Both males and females showed a significant increase in positive affect, but only females had a decrease in negative affect . Female students reported greater increases in attention to feelings and perspective taking, and a decrease in the personal distress in response to distress in others. However, both genders reported significant increases in empathic concern.These findings suggest that participation in a one-semester MBS course during the first year of medical school is effective in enhancing important traits such as mindfulness and empathic concern, while reducing students’ perceived stress.


The interactions of nucleic acids with lipid membranes are of great importance for biological mechanisms as well as for biotechnological applications in gene delivery and drug carriers. The optimization of liposomal vectors for clinical use is absolutely dependent upon the formation mechanisms, the morphology, and the molecular organization of the lipoplexes, that is, the complexes of lipid membranes with DNA. Differential scanning calorimetry (DSC) has emerged as an efficient and relatively easy-to-operate experimental technique that can straightforwardly provide data related to the thermodynamics and the kinetics of the DNA—lipid complexation and especially to the lipid organization and phase transitions within the membrane. In this review, we summarize DSC studies considering nucleic acid—membrane systems, accentuating DSC capabilities, and data analysis. Published work involving cationic, anionic, and zwitterionic lipids as well as lipid mixtures interacting with RNA and DNA of different sizes and conformations are included. It is shown that despite limitations, issues such as DNA- or RNA-induced phase separation and microdomain lipid segregation, liposomal aggregation and fusion, alterations of the lipid long-range molecular order, as well as membrane-induced structural changes of the nucleic acids can be efficiently treated by systematic high-sensitivity DSC studies. Important biological mechanisms like chromatin function, as well as biomedical applications involving liposomal carriers for DNA delivery for clinical use in gene therapy, are based upon the incorporation of nucleic acids within lipid membranes.–4 LipoplThe enormous possibilities presented by liposomes, exhibiting versatility with respect to their size, fluidity, and surface charge, have over the last 20 years paved the way for systematic research in the direction of developing stable liposomal complexes with proteins, peptides, metabolites, and drugs. A large The negative charge density of the DNA molecule makes the direct electrostatic interaction with cationic liposomes possible. The addition of DNA to liposomes brings about substantial changes related to the molecular order and the structure of the lipid membrane. These changes heavily depend upon the fluidity of the membrane, the surface charge, and often the size of the preformed cationic liposomes. In this review, we summarize the nucleic acids–membrane interactions studied via DSC. These interactions depend upon parameters such as electrostatic interactions, lipid structure and membrane composition, entropic contributions, mesoscale conformations of membranes, and nucleic acids properties. DSC has definite limitations for studying thermal processes even more so for extracting detailed information about molecular order and structure. Despite these limitations, DSC is a sound technique for exploring membrane–biomolecules interactions through changes demonstrated in the calorimetric trace of lipid phase transitions. By employing thermodynamic analysis, high-sensitivity DSC has become a systematic method for investigating DNA or RNA interactions with lipid bilayers, shedding light into phase separation, lipid segregation, liposomal aggregation and fusion, as well as membrane-induced structural changes of the nucleic acids.Lipid molecules, when dispersed in aqueous media, self-organize spontaneously to a great variety of supramolecular structures depending upon the physicochemical properties of the lipids and the dispersant. This amazing polymorphism of lipids mainly includes lamellar, micellar, hexagonal, cubic normal, and inverted phases.21 These 22DSC is the experimental technique of choice to systematically characterize the thermodynamics of phase transitions and conformational changes of biological macromolecules and supramolecular structures. DSC measures isobaric changes in specific heat capacity as a function of temperature.–26 ThermDNA and RNA of a great variety in size and origin have been used for interaction studies . Synthetic lipid membranes have been tested either as multilamellar vesicles (MLVs) or as large unilamellar vesicles (LUVs). Lipid compositions assayed varied from pure zwitterionic phosphatidylcholine (PC) membranes to three lipid systems containing cationic compounds, fusogenic-helper lipids, or anionic lipids. Lipid phases involved were mainly lamellar  as well as inverted hexagonal or cubic. DSC coulet al. dimethylammonium bromide (BPDAB), bis[2- (11-butyloxyundecanoate)ethyl] dimethylammonium bromide (BBDAB), and N-hexadecyl-N-{10-[O-(4-acetoxy)-phenylundecanoate] ethyl} dimethylammonium bromide (HADAB). All cationic lipids (CLs) bear a dimethylammonium bromide headgroup (DAB) as the DNA-binding moiety yet different hydrophobic parts.-carbamoyl}cholesterol) while for helper lipids DOPE, DOPC, or cholesterol where chosen.It must be mentioned at this point that DSC has been employed in several cases to characterize the thermal denaturation of DNA in relation to membrane binding, where increasing melting temperature indicated enhanced stability for the formed lipoplex.57–59 Lobo 122 °C. This effo 122 °C.6061 CatiDSC can provide information regarding thermally induced phase transitions and with that as a probe information about DNA and membranes interactions can be extracted. DSC cannot give direct lipid–DNA structural information and must be combined with other experimental techniques in order to interpret the acquired calorimetric data with greater confidence.et al., lipid phases were assessed relatively to their efficiency for DNA delivery.[O-phosphatidylcholine (EDLPC-EDOPC), DOTAP-DOPE, and their lipoplexes were assessed. Anionic lipids – phosphatidylglycerol, phosphatidylserine, and cardiolipin – were mixed and characterized in connection with DNA dissociation and delivery. The molecular structure of the lipids and the consequential apolar-polar interface curvature regulated the final structural conformation. Lipoplexes of EDLPC-EDOPC exhibited lamellar conformation; EDLPC-EDOPC-cardiolipin exhibited inverted micellar cubic – the highest interfacial curvature – while the less-effective mixtures EDLPC-EDOPC-cardiolipin exhibited inverted hexagonal, cubic bilayered, and/or coexisting lamellar phases. The most widely studied mixture, DOTAP-DOPE, at low temperature appears to be, while at higher temperatures exhibits an inverted hexagonal phase. Early SAXS structural studies by Lasic et al. on DODAB-cholesterol-DNA complexes revealed a bilayered packing with dehydrated DNA ordered in a 2D layer between the cationic lipid bilayers.[As it was mentioned earlier, in the study by Koynova delivery. In yet adelivery. Structurbilayers.Very few studies employ only DSC for the investigation of membrane–DNA interactions. Most of 303147634346The power of DSC as an experimental tool for characterizing nucleic acids–lipid membrane interactions is projected throughout all the studies reviewed here. DSC peaks associated with phase transitions or with the thermal denaturation of nucleic acids are used as the principal experimental evidence in all the studies. Straightforward analysis provides information on the enthalpy, the transition temperatures, and the cooperativity of the lipoplex phases. Most of the studies conducted involved binary lipid mixtures. It is essential, as a first step, to study the miscibility of the two lipids by DSC and characterize the phase transitions for safe interpretation of the experimental data from the DNA or RNA impact. In most cases and at low DNA concentration, surface-bound DNA will stabilize the lipid membranes through electrostatic interactions with the polar headgroup, though always depending on the membrane fluidity. When DNA penetrates the lipid membrane, the results are severe; the interactions can be slow and the fluidity of the lipid phase is almost always enhanced whilst lipid interdigitation is most surely abolished as is the lipid pretransition. In the binary lipid mixtures, one of the two components is the DNA-binding partner, while the other plays either a stabilizing or a destabilizing role for the long-range molecular organization. Thus, DNA is anticipated to demix lipids. Indeed, lipid demixing and segregation take place in most charged-zwitterionic binary systems resulting in phase-separated domains. Another issue accentuated is the concentration effect of nucleic acids; at low concentration, DNA can induce long-range order, while at high concentration it induces disorder. Information about the kinetics and the nature of the nucleic acids–membranes interactions can also be extracted with DSC. For very weak or slow-kinetics interactions, the establishment of a stable state can be monitored and demonstrated by successive heating and cooling scans and the repeatability of the so-obtained DSC thermograms. In several studies, the steady state is established after long incubation times and a large number of heating and cooling cycles. Since nucleic acid–membrane interactions also involve apolar interactions, several passes from order to disorder of the lipid bilayer will enhance apolar–apolar interactions and assist the establishment of a new phase for the complex. Recent developments in gene delivery involve novel cationic delivery reagents as amidines, polyethylenimines (PEI), and commercially available reagents  as well and exhibit increased transfection efficiencies.–68 An ex69DSC is a valuable technique for studying DNA–lipid complexes, which exhibits many advantages such as the relatively easy-to-use experimental setups and the straightforward data analysis. As a technique, DSC is efficient and relatively of low cost, reliable as an ensemble technique, and most importantly it can assess systems in physiological conditions. DSC should be used in combination with other techniques like structure analysis techniques or binding assays. Once the thermal behavior of a system is correlated with structure facts, then DSC can be used independently. Both qualitative and quantitative analysis can be carried out with DSC, contributing to the elucidation of biological mechanisms as well as to the development of improved gene carriers.


Previous research suggests overlap between brain regions that show task-induced deactivations and those activated during the performance of social-cognitive tasks. Here, we present results of quantitative meta-analyses of neuroimaging studies, which confirm a statistical convergence in the neural correlates of social and resting state cognition. Based on the idea that both social and unconstrained cognition might be characterized by introspective processes, which are also thought to be highly relevant for emotional experiences, a third meta-analysis was performed investigating studies on emotional processing. By using conjunction analyses across all three sets of studies, we can demonstrate significant overlap of task-related signal change in dorso-medial prefrontal and medial parietal cortex, brain regions that have, indeed, recently been linked to introspective abilities. Our findings, therefore, provide evidence for the existence of a core neural network, which shows task-related signal change during socio-emotional tasks and during resting states. Previous research suggests overlap between those brain regions that show task-induced deactivations across a wide range of different fMRI studies , resting state (hereafter: DMN) and emotional processing (hereafter: EMO) rely on a common core network of brain regions we took the following approach: we used the activation likelihood estimation (ALE) approach, which allows to statistically investigate the convergence of published neuroimaging findings on activations or deactivations to delineate brain regions, that are consistently activated by social-cognitive and emotional tasks, as well as those that consistently deactivate following the engagement in a wide range of other experimental tasks In order to address whether social-cognitive  were analyzed separately. In a second step, conjunction analyses were performed to reveal brain areas, which show consistent task-related signal change across two sets of studies. Given the phenomenal and conceptual similarities of social-cognitive and emotional processes and their allegedly comparable importance for the understanding of other minds e.g. , we usedinclusion criteria comprised considering only data from neurotypicals , exclusive consideration of studies that report coordinates provided in a standard anatomical reference space  and reliance on full-brain coverage  or functional localizers). The exclusion criteria consisted in non-consideration of neuroimaging experiments that investigate age, gender, patient groups or drug effects. For retrieval of the relevant experiments, the BrainMap database was filtered based on the metadata of the archived experiments that describe the experimental paradigm used in the respective experiment as well as the specific mental process isolated by a given statistical contrast. Behavioural domains (BD) include the main categories of cognition, action, perception, emotion, interoception, as well as their related subcategories. The respective paradigm classes (PC) classify the specific task employed (a complete list of BDs and PCs can be found at http://brainmap.org/scribe/). The BDs and PCs together constitute a complete cognitive taxonomy for the conceptual distinction of mental processes conceived by leaders of the neuroscientific community Functional imaging experiments for the meta-analyses were obtained from the BrainMap database In the current analysis of the DMN, we included all experiments that reported deactivations relative to a low-level (resting) baseline, i.e. all experiments providing coordinates for regions that decreased their activity when subjects were engaged in an externally structured task (DMN: n = 533), while excluding those studies, which were part of the other individual meta-analyses. That is, the individual meta-analyses were based on independent pools of experiments. Furthermore, we profited from the BrainMap taxonomy, to identify the sub-pools of the databased neuroimaging experiments that have been labelled as being closely related to emotional and social processing, respectively. Here, the considered studies on emotional processing are defined as pertaining to “the mental faculty of experiencing an affective state of consciousness such as joy, sorrow, fear, hate”. In particular, facial emotion is thought to be an evolutionarily shaped adaption that allows for the efficient transmission of affective states Meta-analyses were carried out using the revised version The probabilities of all activation foci in a given experiment were combined for each voxel, resulting in a modelled activation map (MA map). Taking the union across these MA maps yields voxel-wise ALE scores describing the convergence of results at each particular location. Since neurophysiologically, neuronal effects should predominantly be localized within the grey matter, all analyses were restricted to those voxels where a probability of at least 10% for grey matter could be assumed based on the International Consortium of Brain Mapping (ICBM) tissue probability maps. To distinguish ‘true’ convergence between studies from random convergence, i.e., noise, ALE scores were compared to an empirical null-distribution reflecting a random spatial association between experiments. Hereby, a random-effects inference is invoked, focusing on inference on the above-chance convergence between studies, not clustering of foci within a particular study. Computationally, deriving this null-hypothesis involved sampling a voxel at random from each of the MA maps and taking the union of these values in the same manner as done for the  voxels in the true analysis. The p-value of a ‘true’ ALE was then given by the proportion of equal or higher values obtained under the null-distribution. The resulting non-parametric p-values for each meta-analysis were thresholded at a cluster-level family wise error (FWE) corrected threshold of p<0.05 and transformed into Z-scores for display.Conjunction analyses between the separate meta-analyses were carried out by calculating a voxel-wise minimum statistic In order to formally test whether the core ‘introspection network’ - represented by the results of our triple conjunction analysis (SOC ∩ EMO ∩ DMN) – shows anatomical overlap with the findings from the study by Fleming and colleagues Consistent activation across the social-cognitive tasks was found in left dorso-medial prefrontal cortex (DMPFC), the left precuneus, bilateral middle temporal gyrus, anterior temporal cortex (ATC) and the temporo-parietal junction (TPJ) area as well as the left superior frontal gyrus , Table 1Consistent activation across the emotional processing tasks was found in the amygdala (AMY) bilaterally, the ventral (VS) and dorsal striatum (DS) bilaterally, middle and superior temporal gyrus and insular cortex, anterior cingulate cortex (ACC), posterior cingulate cortex (PCC) and the precuneus, area V5 and fusiform gyrus (FG) , Table 2Consistent task-induced deactivation was found in PCC extending into the precuneus, ACC, ventro- and dorso-medial prefrontal cortex, bilateral supramarginal gyrus, bilateral TPJ, left superior and right middle temporal gyrus, left middle occipital gyrus and left middle frontal gyrus , Table 3Brain regions, which showed consistent activation across both the emotional and social-cognitive tasks (SOC ∩ EMO), comprise DMPFC, precuneus, middle and anterior superior temporal gyrus and VS , Table 4Brain regions, which showed consistent involvement both in the social- cognitive tasks and those studies that investigated the DMN (SOC ∩ DMN), comprise DMPFC, precuneus and the TPJ bilaterally , Table 5Using a triple conjunction analysis, we formally tested for brain regions, which show consistent involvement across all different types of studies, i.e. social-cognitive, emotional processing and resting state experiments (SOC ∩ EMO ∩ DMN). To assess whether these are the same regions as the ones reported to underlie introspective abilities , we calculated a minimum conjunction analysis across the thresholded maps from the triple conjunction analysis and results from the morphometric study by Fleming and colleagues (2010). This additional analysis, indeed, demonstrates involvement of DMPFC  and precuneus , thereby confirming anatomical overlap between their results and the triple-conjunction of meta-analyses.Using ALE-based meta-analyses of neuroimaging studies from the BrainMap database, we were able to demonstrate consistent patterns of activations and deactivations across different types of experimental paradigms. Providing such a quantitative assessment of convergence across studies crucially goes beyond what qualitative reviews of studies investigating social cognition and emotional processing can provide. Furthermore, our approach allows to formally investigate commonalities in the neural networks subserving different types of tasks by applying conjunction analyses, which test for convergence across different pairs and across all three individual meta-analyses. By using the latter type of analysis, we can demonstrate overlap of task-related signal change in dorso-medial prefrontal and medial parietal cortex, brain regions that have recently been linked to introspective abilities.The meta-analysis targeting consistent activations across studies exploring the neural correlates of social cognition revealed activations in the DMPFC, the precuneus, the TPJ and anterior temporal cortex , which iMore specifically, DMPFC has commonly been related to mental state attribution or ‘mentalizing’ as e.g. classical false-belief tasks and stories, which require (cognitively) taking someone else's perspective, have been shown to result in a differential increase of neural activity in this area Our analysis of consistent brain activations for emotional processing revealed a highly significant, bilateral pattern of activation, which comprises the AMY, the VS and DS, the ACC/DMPFC and PCC/precuneus, area V5 and insular cortex .Co-activations of ACC and anterior insular cortex bilaterally have commonly been described as a “salience network” believed to be involved in the attribution of salience to both internal events and extrapersonal stimuli, thereby guiding behavior Additionally, our analysis revealed involvement of the bilateral amygdala, a brain region whose primary function appears to reside in signaling what is important and to then modulate appropriate (response) processes to deal with the challenges and opportunities present in a given situation Finally, our meta-analysis also provides evidence for the recruitment of the ventral and dorsal striatum during emotional tasks: while the ventral striatum has been described as a core node of the neural network of reward, especially involved in the anticipation and experience of the hedonic aspects of rewards The meta-analysis that explored consistent, task-induced deactivations across a large number of neuroimaging studies from different task domains demonstrated involvement of ACC/DMPFC and PCC/precuneus, VMPFC and TPJ bilaterally . These r1), which has been described as a core element of the functional neuroanatomy of reward and is thought to be involved in the anticipation and experience of the hedonic aspects of rewards  demonstrates involvement of DMPFC, the precuneus, middle and anterior superior temporal gyrus and the ventral striatum . Consistrds e.g. . The curResults of the conjunction analysis between social cognition and the task-related deactivations (SOC ∩ DMN), demonstrate involvement of DMPFC, precuneus and the TPJ bilaterally . Based oAs the key finding of our study, the conjunction analysis performed by including the results of all individual ALE meta-analyses (SOC ∩ EMO ∩ DMN) provides compelling empirical evidence for a shared neural network, which comprises the precuneus and DMPFC . This anFindings, which suggest that introspection can be enhanced via social interaction Taken together, or results suggest that on the neural level, introspective, social and affective processes converge and rely upon recruitment of a network that comprises dorso-medial prefrontal and medial parietal cortex.activations during social cognition and emotional processing and deactivations across a wide range of experimental tasks. By applying conjunction analyses, we demonstrated a close convergence of the brain regions involved. These results provide robust empirical evidence for a shared neural network that underlies emotional processing, social and unconstrained cognition, which localizes to the precuneus and anterior medial prefrontal cortex. These two regions are known to be critical hubs in the neurofunctional architecture of the human brain In this study, we took a meta-analytic approach to delineating brain regions, which consistently show Future research into the functional and effective connectivity of the different nodes of this shared neural network and its susceptibility to intervention and plasticity during social interactions are likely to be informative concerning the exact functional roles of the different brain regions. One important question our findings raise is whether social cognition, emotional processing, and resting state cognition simply recruit brain structures that subtend conscious introspection or meta-cognitive abilities, or whether they are in fact instrumental in the shaping of such introspective abilities. One possibility is that in social or emotional contexts, not only adequate behavior is important, but that the successful prediction and monitoring of the outcomes of this behavior is equally essential. Such re-description of one's behavior could turn out to be exactly the mechanism that underlies introspection


Although much has been learned concerning the mechanisms of secretory vesicle formation and fusion at donor and acceptor membrane compartments, relatively little attention has been paid toward understanding how cells maintain a homeostatic membrane balance through vesicular trafficking. In neurons and neuroendocrine cells, release of neurotransmitters, neuropeptides, and hormones occurs through calcium-regulated exocytosis at the plasma membrane. To allow recycling of secretory vesicle components and to preserve organelles integrity, cells must initiate and regulate compensatory membrane uptake. This review relates the fate of secretory granule membranes after full fusion exocytosis in neuroendocrine cells. In particular, we focus on the potential role of lipids in preserving and sorting secretory granule membranes after exocytosis and we discuss the potential mechanisms of membrane retrieval. Mammalian cells exhibit complex and dynamic patterns of intracellular membrane traffic between various organelles. Although much has been learned concerning the mechanisms of vesicle transport and vesicle fusion at donor and acceptor compartments, relatively little attention has been paid to understanding how organelle homeostasis is maintained. This aspect is particularly important in neurosecretory cells in which intense membrane trafficking and mixing occurs between secretory vesicles and the plasma membrane during secretion of various transmitters, peptides, and hormones. Calcium-regulated exocytosis, i.e., fusion of secretory vesicles with the plasma membrane results in the merging of these two membrane compartments, hence triggering an increase in plasma membrane surface and loss of identity. As a consequence, exocytosis must be coupled to a compensatory endocytotic process allowing the plasma membrane to recover its integrity and the granule membrane to be recycled. In neurons, molecular mechanisms of synaptic vesicle recycling and coupling with exocytosis have been intensively studied, but is still debated [for reviews see –3]. HoweIn neuroendocrine cells, secretion can occur through different modes of exocytosis depending on the physiological demand . Some omponents . In neurmponents –23. As aOf particular interest, the formation of membrane domains corresponding to exocytotic sites is regulated by annexin-A2 , 24, a c2] has been largely described as an important regulator in exocytosis P2 (2 concentration regulates the membrane binding and deformation capacity of proteins containing Bin/amphiphysin/Rvs (BAR) domains including endophilin, syndapin, and amphiphysin, three membrane-deforming scaffold proteins that have been implicated in endocytotic processes Pocytosis , 29 but IP2 . Local Processes , 32. Intesses  and phosphatidylethanolamine (PE) reside in the inner cytoplasmic leaflet while phosphatidylcholine and sphingomyelin are located in the outer leaflet . In non-To date no current evidence is available to explain the mechanism by which PS externalization is linked to compensatory endocytosis. However, two scenarios are possible. Firstly, as loss of phospholipid asymmetry can modify the mechanical stability of membranes , this miHowever, whether externalization of PS simply reflects the loss of PS asymmetry or reveals more profound lipid reorganization is basically unknown and requires further investigation.in vitro clathrin baskets assemble with a mean diameter of 100 nm or even less (80 nm) in the presence of adaptor proteins (In chromaffin cells, we have found that clathrin is rapidly recruited to the granule membrane right after merging with the plasma membrane and that knocking-down clathrin expression drastically blocks compensatory endocytosis . The claproteins . In INS-proteins . In mousproteins . Accordiproteins . Altogetproteins .The intracellular route followed by the post-exocytotic internalized granules has not been fully characterized. The main difference with synaptic vesicle recycling is that, to be reused, large dense core granules need to be reloaded with matrix proteins, which most likely implies a re-maturation process involving the Golgi apparatus. The transient accessibility of granule-bound proteins at the cell surface during full fusion exocytosis has been exploited to label and follow post-fusion granules with specific antibodies or biotinylation. This approach was widely used in the 80s in order to demonstrate that granule-bound proteins transit through the Golgi region before being recycled in newly mature granules , 50, 51.New evidence is now emerging to support the idea that, in neuroendocrine cells but also in neurons, vesicle/granule membranes do not intermix with the plasma membrane following full fusion exocytosis , 57. ComThe 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.