Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage. Cellular genome holds the blueprint of life; hence, a fundamental evolutionary goal of organisms is to preserve the integrity of their genomes. Throughout the lifespan of an organism, their genome is constantly under threat from exogenous agents, endogenous insults and cellular processes that have the capability to inflict damage and severely compromise genome integrity. In this regard, cells have developed evolutionarily conserved defense mechanisms to counteract the detrimental effects of these events and safeguard the genetic information. An important mechanism is the DNA damage response (DDR), which is a complex signal transduction pathway that has the ability to sense DNA damage and transduce this information to influence cellular responses to DNA damage and promote repair ,2. In hiImportantly, the complexity and diversity in chromatin organization influences genome stability at several levels. This includes the suppression of endogenous damage by promoting fine-tuned DNA templated processes such as DNA replication and transcription in a spatio-temporal manner during various cell cycle phases. However, chromatin structure also poses a significant obstacle to detection and repair of DNA lesions. In this regard, plasticity of chromatin structure plays a central role in facilitating and regulating cellular response to DNA damage ,5. MoreoThe key determinant of nuclear organization is evolutionarily conserved packaging of genome into chromatin, a hierarchically organized complex of DNA, histones and non-histone proteins . At the A defining genomic characteristic of heterochromatin is that it is highly enriched in repetitive sequences and concentrated in pericentromeric and telomeric regions in most eukaryotes . These rFurthermore, transposable elements owing to their intrinsic property of being excised and subsequently inserted elsewhere in the genome have the potential to remodel gene structure and expression by being directly inserted into either the coding sequence or the regulatory elements of genes ,23. CompAberrant overexpression of pericentromeric satellite repeats have been observed in lung cancer while decompaction and transcriptional activation of pericentromeric heterochromatin has been reported in several genetic disorders ,27. SimiIn order to ensure genome stability and to prevent illicit recombination between the vast amounts of repetitive DNA, heterochromatic structures must be faithfully replicated. In this regard, several chromatin-remodeling enzymes, such as SMARCA5 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5) ensure efficient replication of heterochromatic late replicating domains most likely by facilitating chromatin remodeling to allow the progression of the replication fork . FurtherHuman cells are subjected to a daily load of approximately 70,000 genomic lesions , most ofSince chromatin is the physiological template for DNA repair machinery, the structural context of chromatin environment may facilitate or alternatively make it more challenging for repair factors to maintain and restore genome integrity ,38. DepeNotably, the arrangement of the genome into heterochromatin- and euchromatin-like domains may substantially influence regional variations in genome instability in human somatic cells . This vaIn fact, numerous studies over the years lend support to a central role for chromatin plasticity in DNA repair, where repair of lesions follows a set of orchestrated events aptly described as the “access-repair-restore” model . EvidencSince the initial discovery of chromatin structure as a repeating array of nucleosomes, it has been speculated that it plays much more diverse roles than mere packaging and compaction of genomic DNA. This is substantiated by observations that presence of nucleosomes blocks transcription in-vitro  as well 9 nucleotides in every cell cycle, replicating DNA with high fidelity is a challenging task and prone to errors and disruptions. Consequently, cells have evolved to instruct the DNA replication program in spatially and temporally regulated manner . . 100]. IIn addition to DNA damage induced histone PTMs, the chromatin landscape also changes through mobilization of bound proteins to make the DNA strands more accessible to repair factors. For instance, HP1-β, a member of the HP1 family, seen bound to H3K9me is mobilized in response to DNA breaks. The interaction between HP1-β and H3K9me is transiently disrupted upon phosphorylation by casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair . Thus, dThe influence of chromatin compaction on the induction of DNA breaks after ionizing radiation has been studied both in-vivo and in-vitro. The organization of genomic DNA into a chromatin fiber both at the very basic nucleosomal level and also the higher-order chromatin structures provide significant protection against break induction ,106,107.Within euchromatin, a complex of the heterochromatin-associated proteins KAP1 (KRAB-associated protein 1) and HP1 together with the H3K9 methyltransferase SUV39H1 is loaded onto DSBs rapidly following damage where it facilitates H3K9 tri-methylation . This inThe choice between either a homology-directed repair (HR) pathway or non-homologous end joining (NHEJ) for DSBR is dependent on several factors including cell cycle status wherein HR is active in the S/G2 phases; and NHEJ is the single DSBR pathway active in all cell cycle phases . AdditioOne of the factors that affect regulated chromatin expansion and mobility is the nuclear oncogene SET that is recruited to DSBs to limit uncontrolled DNA end resection and HR . SET intThus, chromatin relaxation can promote both HR and NHEJ, which could be cell cycle or genome location specific. However, a contributing aspect could also be limited availability of techniques to accurately measure NHEJ frequency within the context of chromatin. It is challenging to assess repair through the NHEJ pathway as the resulting ligated DNA strands if repaired precisely are not detectable in most assays, hence, several cycles of cleavage and repair may be missed leading to an erroneous estimate of NHEJ vs. HR events . To studFurther to providing access to lesion sites, changes in chromatin structure also play a role in DDR through initiating a DNA damage-signaling cascade. In this regard, it has been suggested that it is in fact a change in the chromatin structure resulting from IR-induced DNA strand breaks that serves as an initiating signal to activate ATM . MoreoveReduction in the expression levels of linker histone H1 is associated with a more “open” chromatin structure in embryonic stem (ES) cells  and thisWhereas, chromatin decompaction can initiate DDR signaling, Burgess et al. suggested that forced chromatin compaction also induced DDR signaling albeit only the upstream ATM- and ATR-dependent factors were activated in a break-independent manner . HoweverThe plasticity of chromatin structure enables access to cellular machineries to perform DNA-based processes as well as protects the underlying sequence information contained in the genome. For instance, deregulation of the replication-timing program by changes in chromatin domain organization may lead to a disruption in segregation of the early and late replicating domains. However, it is not known if changes in replication timing program lead to loss of genome integrity. Therefore, it would be very interesting to study the effect of chromatin structural organization in this context.The increased DNA DSBs and erroneous recombination events arise in repetitive DNA sequences primarily due to their transcriptional and transpositional activities, giving rise to genome instability. Therefore, heterochromatinization of repetitive elements appears as an evolutionarily conserved mechanism to prevent unnecessary transcription and transposition of these elements in the genome and in turn safeguard genome stability. In this manner, chromatin structural organization functions to repress genomic instability arising from unstable elements within the genome by imposing structural restraints to prevent their activation. However, the transcriptionally dynamic nature of repetitive elements begs the question if all repeats are the same in terms of their capacity to cause genome instability. The complexity and diversity in chromatin organization as evident by the presence of “secondary” and “tertiary” chromatin structures may also pose a significant obstacle to repair of DNA lesions. Importantly, endogenous damage leads to higher number of mutations in more compact chromatin structure mainly due to restricted accessibility and DDR signaling. Thus, the dynamic nature of chromatin compaction is crucial to genome integrity through structurally restricting unstable repetitive elements on the one hand while allowing access to repair factors upon damage on the other hand. In this regard, a thorough mechanistic understanding of factors and modifications involved in altering chromatin structure in response to both endogenous and exogenous damage is still lacking.Studies so far argue that chromatin structure also influences the repair choice for DSBR and thus it would be interesting to test whether cancers deficient in one of the repair pathways could be targeted using a synthetic lethality approach, whereby the choice between HR or NHEJ is forced through chromatin structure alteration. Finally, it is generally thought that more open chromatin structure promotes repair efficiency, however, transcriptionally active open chromatin regions are also prone to chromosomal rearrangements as compared to more compact, transcriptionally inactive heterochromatic regions. Moreover, altering chromatin structure would also deregulate other DNA-templated processes such as transcription and replication, which could lead to genomic catastrophe. Hence, further studies are vital to obtain a deeper understanding of chromatin-based regulation of DNA processes in order to ascertain the role of chromatin structural organization in maintaining genome stability.


In neutrophils , different inflammatory stimuli induce histone deimination, chromatin decondensation, and NET formation. These web-like structures that trap and kill microbes contain DNA, cationic granule proteins, and antimicrobial peptides, but the most abundant proteins are core histones. Histones contained in NETs have been deiminated, and arginines are converted in citrullines. While deimination is a physiological process amplified in inflammatory conditions, only individuals carrying genetic predisposition to develop rheumatoid arthritis (RA) make antibodies to deiminated proteins. These antibodies, collectively identified as anti-citrullinated proteins/peptides antibodies (ACPA), react with different deiminated proteins and display partially overlapping specificities. In this paper, we will summarize current evidence supporting the role of NETosis as critical mechanism in the breach of tolerance to self-antigens and in supporting expansion and differentiation of autoreactive cells. In fact, several lines of evidence connect NETosis with RA: RA unstimulated synovial fluid neutrophils display enhanced NETosis; sera from RA patients with Felty’s syndrome bind deiminated H3 and NETs; a high number of RA sera bind deiminated H4 contained in NETs; human monoclonal antibodies generated from RA synovial B cells decorate NETs and bind deiminated histones. In RA, NETs represent on one side an important source of autoantigens bearing posttranslational modifications and fueling the production of ACPA. On the other side, NETs deliver signals that maintain an inflammatory milieu and contribute to the expansion and differentiation of ACPA-producing B cells. NETosis was discovered as a new function of neutrophils and thoroughly investigated as an important mechanism in the protection against bacterial, fungal, and parasitic infections . When thStaphylococcus aureus stimulus), acts by a NADPH oxidase-independent pathway, and leads to the release of NET after nuclear envelope blebbing and vesicle formation, thereby preserving plasma membrane integrity. During this vital NETosis, cells are still able of some typical functions, such as chemotaxis and phagocytosis.Recent data however challenge the prevalent view of NETosis as a cellular suicide. An early NETosis has been described, which occurs rapidly after exposure to microbial specific molecular patterns  deficiency , an immuConversely, a subset of neutrophils, identified for their lower density on gradients, is more abundantly represented in systemic lupus erythematosus (SLE) patients and is more prone to NETosis.Netting neutrophils have not only been identified in nephritic kidneys in systemic lupus but also in ANCA-associated vasculitides (AAV), suggesting that NET constituents may be involved in the induction of severe manifestations of these systemic inflammatory disorders.NET may also contribute to the pathogenesis of human diseases in a more subtle way, making potential autoantigens accessible to the immune system and creating the milieu where an autoimmune response may be triggered and fueled.In this review, we shall summarize the current knowledge accumulated in recent years that point toward an important contribution of NET to the breach of immunological tolerance and the maintenance of autoimmunity and chronic inflammation in rheumatoid arthritis (RA).Neutrophils are the most abundant cells in the synovial fluid of RA patients although they appear a less important component of the chronic synovial inflammatory infiltrate where neutrophils are believed to only transiently populate the synovial tissue. In RA, circulating but especially tissue-infiltrating and synovial fluid neutrophils have all the features of activated cells, characterized by a prolonged survival and by the ability to secrete a wide range of inflammatory mediators including chemokines and cytokines . NeutropA number of autoantibodies have so far been described in RA, but only anti-citrullinated proteins/peptides antibodies (ACPA) can be considered specific disease markers with sufficient specificity and sensitivity to be used as diagnostic tests . ACPA arPorhyromonas gingivalis-induced periodontitis or in the lung of smokers  as well as exogenous antigens . All these proteins become target of ACPA after deimination (or citrullination), a posttranslational modification (PTM) catalyzed by the calcium-dependent enzyme peptidyl arginine deiminase (PAD). PADs are inactive at intracellular calcium concentrations but can be activated by Ca2+ influx due to different stimuli: ionophore-induced macrophage apoptosis   or lipopFurthermore, PADs can be released from the cell and become activated, as a result of the extracellular Ca2+ concentration .Citrullination is a physiological process that regulates the homeostasis of several organs but is strongly amplified during inflammation. In RA, multiple proteins are citrullinated, especially in target organs of the disease, primarily the synovium but also in the lungs  and in mNeutrophils contribute to protein citrullination in RA in several ways. Cells contained in synovial fluid (mainly neutrophils and monocytes) are characterized by the citrullination of a wide variety of proteins. Neutrophil exposed to a variety of stimuli  contain deiminated histones . MoreoveAlthough a large spectrum of citrullinated proteins is produced by neutrophils in RA joints, the immune response detected in RA sera is relatively restricted Figure .A major contribution to the generation of citrullinated proteins comes from the propensity of RA neutrophils from peripheral blood or synovial fluid to form NET, either spontaneously or after LPS stimulation . IndirecIn synovial fluid, netting neutrophils release enzymatically active PAD2 and PAD4 that under the local conditions of inflamed joints may citrullinate extracellular proteins. Both soluble and NET-associated PAD can be detected, thus suggesting that NET may act as a molecular scaffold for protein citrullination .Several recent works have evaluated the mechanisms behind NET regulation by PAD4. A single nucleotide polymorphism (SNP) at position 1858 (C1858T) in the DNA encoding a protein tyrosine phospatase (PTPN22), which results in the conversion of an arginine (R620) to a tryptophan (W620), has risen interest due to its strong connection with RA . InteresNET from RA patients is also able to activate synoviocytes, upregulating the production of pro-inflammatory cytokines and amplifying joint inflammation.Thus, neutrophils have an active role in the inflammatory process of RA not only regulating the function of other immune or structural cells  but alsoAn increased NETosis has also been observed in SLE. A peculiar subset of neutrophils, identified by density gradients as low density granulocytes (LDGs) and more frequently detectable in active SLE, has a pro-inflammatory phenotype  and formIncreased NETosis has not only been described in type 1 diabetes, correlated with autoantibody titers and beta cell damage , but alsMore recently, an increased number of netting neutrophils has been reported in type 1 and type 2 diabetes, and their direct role in retarding wound healing has been demonstrated , 45.The core nucleosome, comprising an H3–H4 tetramer and two H2A/H2B dimers is not a static DNA packaging structure, but on the contrary is a dynamic complex, and the modulation of its structure is an important component of transcriptional regulation.Modifications in the conformation of histones, highly conserved in eukaryotic cells, from yeast to humans, are widely used in the dynamic modulation of chromatin structure and function. Indeed, evolutionary PTMs are more useful than amino acid substitutions.So far, 20 types of histone PTMs have been described, which are able to modulate chromatin function by either altering the amino acid charge and consequently the inter-nucleosomal interactions or by enabling/inhibiting interactions with specific binding proteins external to nucleosomes but nonetheless essential for DNA regulation. Among all, one of the latest described is the deimination of arginine.The first description of histone deimination was reported by the Yamada’s group . They obIn 2004, Cuthbert et al.  and WangIn particular, the group of Koutzarides  showed tWhen PAD4 activity is inhibited by Cl-amidine, an increase in the expression of p53 and p53-related genes is observed as described by the group of Coonrod .PAD4 is not the only PAD isoform involved in chromatin regulation. Indeed, Zhang et al.  suggesteImportantly, deimination may involve arginine but also methylarginine on H4 and H3 induced by PRMT1 and CARM1, respectively, thus dubbing PAD4 as a demethylating enzyme, thereby reverting the epigenetic modification of arginine methylation.Moreover, deimination of the H2A/H2B dimer, probably involving three arginines (given the mass increase of 2.7 Da) stabilizes the dimer, making it less susceptible to harsh conditions than the native complex, as demonstrated by mass spectrometry analysis with increasing concentration of ammonium acetate .Like core histones, extranucleosomal linker histones can also be the target of PAD activity. Christophorou et al.  recentlyNevertheless the topic of histone deimination is still a tangled issue, given the high number of PTM co-expressed on histones and the limitations of the chemical and biological methods presently available for citrulline detection, which are not fully citrulline specific .For instance, the antibody-detecting citrulline after chemical modification with antipyrine and 2,3-butanedione, the so called “Senshuo reagent,” also recognizes carbamylated proteins .This lack of citrulline complete specificity is also a characteristic of some anti-unmodified citrulline antibodies commercially available.To overcome this problem, recently Bicker et al.  suggesteBesides technologies like mass spectrometry that allow determination of site-specific citrulline on proteins with high sensitivity but at high costs, the development of simple but nonetheless reliable and specific chemical or biological tools is a field still open to innovation.Taken together, these results show that histone citrullination is a key regulatory mechanism for cell life, but in particular cells  histone deimination may lead to decondensation of the entire cell chromatin, thus affecting in an irreversible way the cell life and leading to ETosis (extracellular traps formation).Core histones are the most abundant proteins in NET , and sevWe have recently shown that RA sera, tested by immunoblot on acid-extracted proteins from calcium ionophore-stimulated neutrophils, frequently react with a band of 11 kDa. Its identity with deiminated H4 has been suggested by specific antibody recognition and demonstrated by MALDI/TOF analysis. The recognition of deiminated H4 has been confirmed by ELISA using either the entire molecule or citrullinated peptides corresponding to H4 sequences. When RA sera are tested with proteins contained in NET, the reactivity with a band identified as H4 is again detected. Moreover, by derivatization of citrulline residues, it has been shown that H4 contained in NET and recognized by RA sera is deiminated on arginine 23.14–34 and HCP2 – H431–50) are present in 67 and 63% of established RA F1-hbcl-2-transgenic mice , and anyApproximately 50% of patients with RA are characterized by the presence of clusters of infiltrating lymphomononuclear cells in the RA joint synovium forming ectopic lymphoid structures (ELS). Synovial ELS not only resemble secondary lymphoid organs (SLO) but they can also support a germinal center (GC) response. In particular, ELS are characterized by segregation of T and B lymphocytes, differentiation of high endothelial venules (HEVs), and networks of stromal follicular dendritic cells (FDC) , 67. MorOf relevance, we have recently demonstrated that up to 40% of recombinant monoclonal antibodies derived from single CD19+ synovial tissue cells (RA-syn-rmAbs) obtained from ACPA+ RA patients with functional ectopic GC display reactivity toward citrullinated histones . In partAs discussed above, NETs formation is critically dependent on histone citrullination, and citrullinated histones comprise around 70% of all NETs proteins. A proteomic analysis of NETs derived from healthy control neutrophils has identified at least 25 different proteins that decorate these chromatin structures, such as citrullinated vimentin and α-enolase, which are also targets of ACPA, as discussed above . TherefoP. gingivalis)  , 77 and givalis)  can brinormation , 80.However, periodontitis is also characterized by an increased production of antibodies toward the non-citrullinated form of RA-associated antigens, suggesting that in some patients periodontal disease could break the tolerance to non-modified autoantigens in the preclinical phase of RA .In periodontal disease , 83, NETNETs expose citrullinated proteins together with danger signals like cathelicidin or HMGB1 that promote the activation and maturation of professional antigen-presenting cells. NETs uptake and processing may allow the expansion of T cells able to support the affinity maturation and clonal diversification of B cells, leading to ACPA production.Thus, the current hypothesis is that the local release of citrullinated histones and probably other citrullinated and non-citrullinated antigens during an excessive NETosis can sustain the antigen-driven generation of high affinity anti-NETs antibodies within ELS with functional ectopic GC Figure .All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Research investigating Whiplash Associated Disorder (WAD) has largely focused on the cervical spine yet symptoms can be widespread. Thoracic spine pain prevalence is reported ~66%; perhaps unsurprising given the forceful stretch/eccentric loading of posterior structures of the spine, and the thoracic spine’s contribution to neck mobility/function. Approximately 50% WAD patients develop chronic pain and disability resulting in high levels of societal and healthcare costs. It is time to look beyond the cervical spine to fully understand anatomical dysfunction in WAD and provide new directions for clinical practice and research.To evaluate the scope and nature of dysfunction in the thoracic region in patients with WAD.CRD42015026983) and published protocol. All forms of observational study were included. A sensitive topic-based search strategy was designed from inception to 1/06/16. Databases, grey literature and registers were searched using a study population terms and key words derived from scoping search. Two reviewers independently searched information sources, assessed studies for inclusion, extracted data and assessed risk of bias. A third reviewer checked for consistency and clarity. Extracted data included summary data: sample size and characteristics, outcomes, and timescales to reflect disorder state. Risk of bias was assessed using the Newcastle-Ottawa Scale. Data were tabulated to allow enabling a semi-qualitative comparison and grouped by outcome across studies. Strength of the overall body of evidence was assessed using a modified GRADE.A systematic review and data synthesis was conducted according to a pre-defined, registered  which were conducted across a range of countries were included. Few authors responded to requests for further data (5 of 9 contacted). Results were reported in the context of overall quality and were presented for measures of pain or dysfunction and presented, where possible, according to WAD severity and time point post injury. Key findings include: 1) high prevalence of thoracic pain (>60%); higher for those with more severe presentations and in the acute stage, 2) low prevalence of chest pain (<22%), 3) evidence of thoracic outlet syndrome, with some association to and involvement of the brachial plexus, 4) muscle dysfunction in the form of heightened activity of the sternocleidomastoid or delayed onset of action of the serratus anterior, 5) high prevalence of myofascial pain and trigger points in the scalene muscles, sternocleidomastoid and mid/lower fibres of trapezius muscle (48–65%), and 6) inconclusive evidence of altered thoracic posture or mobility.Considerable evidence supports thoracic pain and dysfunction in patients with WAD, involving primarily nerves and muscles. Notwithstanding the low/very low level of evidence from this review, our findings do support a more extensive clinical evaluation of patients presenting with WAD. Additional high quality research is required to further characterise dysfunction across other structures in the thoracic region, including but not limited to the thoracic spine (mobility and posture) and thoracic muscles . In turn this may inform the design of clinical trials targeting such dysfunction. The cumulative incidence of patients seeking healthcare post-whiplash from a road traffic accident has increased over the last 30 years to an annual incidence of between 3 and 6/1000 inhabitants in North America and Western Europe . The sevWhilst research has identified risk factors for poor prognosis , 5, and Although current research into WAD has focused on the primary complaint of neck pain , symptomWith reduced cervical mobility being characteristic of chronic WAD and evidence that the thoracic spine contributes up to 33% and 21% of the movement occurring during neck flexion and rotation respectively  perhaps Nevertheless, a systematic review of the current evidence has never been conducted to examine the scope and nature of dysfunction/impairment in the thoracic spine region following whiplash injury and in WAD. Knowledge of such dysfunction may be used to inform clinical practice and examination of patients with WAD, but also future clinical trials of novel interventions targeting thoracic impairments in WAD.The primary objective is to evaluate the scope and nature of dysfunction in the thoracic spine region in patients with WAD. A secondary objective is to explore the scope and nature of such changes based on severity using the Quebec Task Force classification (I-III) and stage post injury (acute/sub-acute less than 3 months or chronic 3+ months). Thirdly, we wish to make evidence based recommendations for clinical practice and future research.PRISMA)  included: ‘whiplash’, ‘whiplash associated disorder’, ‘WAD’, ‘whiplash injury’, ‘motor vehicle accident OR collision’, ‘road traffic accident’, ‘cervical strain’ and ‘thoracic spine’, ‘dorsal spine’, ‘mid-spine’, ‘thoracic injuries’, limiting to adults >19 years and diagnosis to achieve the best balance of sensitivity and specificity. An example a search from Medline is included Two reviewers  independently searched information sources and assessed identified studies for inclusion, facilitated by grading each eligibility criterion as eligible/not eligible/might be eligible . Full teUsing a standardised form, the two reviewers extracted data independently . A furthData were extracted from each study, including: study design, sample characteristics including age, gender, severity of WAD using the QTF Classification if reported, time point post injury and patient reported or performance based measures of thoracic dysfunction.Risk of bias for each included study was independently assessed by the same initial reviewers. The third reviewer mediated in situations of disagreement. All tools and processes were piloted prior to use. Risk of bias was only assessed for cohort and case-control studies using the Newcastle-Ottawa Scale ; that inSummary measures of patient reported or performance based measures of thoracic dysfunction are presented in the form of prevalence data and confidence intervals where provided, for thoracic pain, chest pain, thoracic outlet syndrome (TOS), myofascial pain and trigger points, dysfunction involving the brachial plexus, thoracic spine posture or mobility. Results are presented where possible, according to severity (QTF) and stage of WAD i.e. acute/sub-acute (< 3 months) or chronic (> 3 months).In accordance with the protocol  meta-anaupgraded where a large dose response was evident, or the effects could not be accounted for by bias [very low’ where concerns were identified from the body of studies relating to precision, consistency, directness, precision or potentially other additional domains relating to strength of association e.g. magnitude of effect [Quality of evidence, including risk of bias across studies was evaluated using GRADE  for indi by bias . Likewisf effect .A total of 38 studies met the eligibility criteria, including 19 cohort studies, 16 case control studies and 3 single case studies/reports. The process of selection is detailed in etc. An overview of the types of dysfunction is provided in Study characteristics are detailed in Agreement of risk of bias following discussion was excellent with studies ranging considerably in quality from 1/9 to 7/9. Key methodological flaws included poor definition of cases, representativeness of cases, lack of non-exposed cohort, lack of blinding, controlling for risk factors beyond age and gender and clarity of follow up time point in cohort studies. There were many instances where information was missing and email requests for additional data or clarification was unforthcoming, resulting in ratings being ‘unclear’. See From this review we identified evidence of the following dysfunction,: thoracic spine pain in acute/sub-acute/chronic WAD ranging from minor injuries to more severe (WADIII) presentations , 58, 62;Synthesis of findings from cohort and case control studies across outcomes of dysfunction are provided in Tables Despite the very low quality of included studies, there is evidence of thoracic spine pain in a sub-acute WAD population n = 11,576) [,576 11,, 52, 60,etc.), time points post injury, sample heterogeneity and the overall methodological low quality of research. This may also be a related to the focus of this review, being to those with mild to moderate presentations of WAD where studies including WADIV or fractures were excluded.Prevalence of chest pain in acute/sub-acute was reported to range 0.0–19% , 17, 52,Evidence of thoracic postural dysfunction is inconclusive given conflicting findings from a small number of studies of low methodological quality evidence where postural assessment was not a primary focus , 61. FutThere is a notable gap in the evidence exploring thoracic and chest mobility in WAD, with just two studies (n = 54) of very low quality suggesting a trend for reduced chest mobility (p>0.05) and flexion hypomobility at the cervico-thoracic junction in women. In terms of thoracic mobility, evidence from one study (n = 7)  suggestsFive studies found, a prevalence of 31–74+% , 47, 51 Acute/sub-acute WADNotwithstanding that evidence is drawn principally from one research group, there is evidence of thoracic dysfunction in relation to the brachial plexus provocation test (BPPT) in WADI-III. From the research by Sterling et al , 57, 59,Chronic WADFor participants with CWADII and/or III, evidence indicates dysfunction detected during the BPPT, with pain levels and restriction in elbow extension almost double those found in asymptomatic controls , 55. FurThere are limited and very variable findings of thoracic muscle dysfunction (activation) in WAD with studies investigating a relatively small number of muscles: sternocleidomastoid , 49, 56;Myofascial trigger points are highly prevalent in WAD with estimates ranging between 48–64% , 37, 40.This is the first methodologically rigorous systematic review investigating thoracic dysfunction in whiplash associated disorders. From a comprehensive search, 38 studies were included and evaluated as part of the review. Many studies were at risk of bias, primarily due to poor reporting with most studies published prior to the introduction of the STROBE reporting guidelines for observational studies. Notwithstanding the low quality of the evidence, there is unequivocal evidence of thoracic dysfunction in WAD.Although there is a high prevalence of acute and chronic pain experienced in the thoracic spine region following injury, distinguishing the interplay of pain mechanisms is however challenging. Whilst we know peripheral and central sensitisation begin immediately following injury , it is pWith limited very low quality evidence and relative to the cervical spine, there is a paucity of research investigating thoracic posture and mobility in WAD , 48, 61.etc. [Although this review suggests a relatively high prevalence of thoracic outlet syndrome, the quality of evidence is very low and derived from relatively older studies, including two studies from the same group of authors , 36. Whietc. . Future There is considerable evidence of brachial plexus dysfunction in both acute/sub-acute and chronic WAD from research investigating the brachial plexus provocation test. Although coming from a relatively small group of researchers, the evidence supports further investigation. Future research could usefully consider the relationship of brachial plexus dysfunction to other musculoskeletal changes in the cervical and thoracic spine following a whiplash injury, but also approaches to managing this; a notable gap within the whiplash management evidence , 10, 66.There is unequivocal evidence of altered function of muscles following whiplash injury , howeverThis review has identified that myofascial trigger points are highly prevalent in WAD , 37, 40,This review provides unequivocal evidence of thoracic dysfunction in WAD, albeit from evidence of low/very low quality. The findings do support a more extensive clinical evaluation of patients following a whiplash injury and the need for more methodologically robust observational studies to further characterise thoracic dysfunction in WAD across stages of the condition and levels of severity. Knowledge and understanding of thoracic dysfunction, where anatomical and biomechanical relationships with the cervical spine exist, offers novel directions for research into management of this disabling condition.etc. targeting the cervical spine [Research into WAD management has been, and continues to be primarily focused on managing cervical spine dysfunction, with interventions such as manual therapy, exercise al spine , and taral spine . With inal spine  it is peetc.[Whilst exercise as part of multimodal packages of care is recommended in the management of acute and chronic WAD , 68, theetc.. The autetc., 10 and etc. further It has not until recent years that the thoracic spine, coined the ‘Cinderella’ of the spine , has staThis review is rigorous and original, with a design and focus on the thoracic spine region using a pre-defined rigorous and published protocol with subject and methodological experts contributing to the evaluation. The key limitation of the review is lack of high quality evidence and compounding this was that few authors responded to requests for additional information or data.This first and rigorous systematic review found considerable evidence of thoracic pain and dysfunction in patients at all stages following whiplash injury. Notwithstanding the low/very low level of evidence, our findings do support a more extensive clinical evaluation of patients presenting with WAD. Key findings include 1) a high prevalence of thoracic spine pain, with the highest levels of pain immediately following injury and in more severe presentations 2) evidence of muscle dysfunction (delayed onset or heightened levels of activity) in a limited number of muscles 3) evidence of thoracic outlet syndrome and brachial plexus involvement 4) inconclusive/limited evidence of postural changes and effect on thoracic spinal mobility. Additional high quality research is required to further characterise dysfunction across other structures in the thoracic region, including but not limited to the thoracic spine (mobility and posture) and thoracic muscles . In turn this may inform the design of clinical trials targeting such dysfunction.S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Approximately one-third of individuals diagnosed with colorectal cancer have a family history of cancer, suggesting that CRCs may result from a heritable component. Despite the availability of current gene-identification techniques, only 5% of all CRCs emerge from well-identifiable inherited causes for predisposition, including polyposis and nonpolyposis syndromes. Hereditary nonpolyposis colorectal cancer represents a large proportion of cases, and robustly affected patients are at increased risk for early onset, synchronous, and metachronous colorectal malignancies and extracolonic malignancies. HNPCC encompasses several cancer syndromes, such as Lynch syndrome, Lynch-like syndrome, and familial colorectal cancer type X, which have remarkable clinical presentations and overlapping genetic profiles that make clinical diagnosis a challenging task. Therefore, distinguishing between the HNPCC disorders is crucial for physicians as an approach to tailor different recommendations for patients and their at-risk family members according to the risks for colonic and extracolonic cancer associated with each syndrome. Identification of these potential patients through epidemiological characteristics and new genetic testing can estimate the individual risk, which informs appropriate cancer screening, surveillance, and/or treatment strategies. In the past three years, many appealing and important advances have been made in our understanding of the relationship between HNPCC and CRC-associated syndromes. The knowledge from the genetic profile of cancer syndromes and unique genotype-phenotype profiles in the different syndromes has changed our cognition. Therefore, this review presents and discusses HNPCC and several common nonpolyposis syndromes with respect to molecular phenotype, histopathologic features, and clinical presentation. Colorectal cancers (CRCs) are one of the most common malignancies and represent the third most common cancer in men and the second in women worldwide. It is estimated that one-third of individuals diagnosed with colorectal cancer have a family history of cancer . HoweverThe hereditary CRCs syndromes are broadly divided into nonpolyposis and polyposis syndromes. In 1966, Lynch first defined familial CRC type I for families with CRC only and type II for families with both CRC and gynecological cancer –9, and HAs such, understanding and distinguishing the various syndromes are useful and clinically meaningful as the approach to diagnosing and surveilling patients and their at-risk family members. Previously, testing of patient tumors for microsatellite instability (MSI) and mutation of DNA MMR genes is an effective strategy to discriminate patients at risk for Lynch syndrome. Cancer-free individuals whose family history indicates suspicion for a hereditary cancer syndrome should undertake clinical genetic evaluation and receive genetic counseling. Even if a mutation is not detected, people may benefit from the genetic evaluation in other interventions to reduce future cancer risk.This paper provides a comprehensive literature review on the most common HNPCC and HNPCC-associated cancer syndromes  that present distinguishing molecular phenotypes, histopathologic features, and clinical presentations among different subtypes .The Amsterdam I clinical criteria for HNPCC, which focus on the number and ages of family members with colorectal cancer, were published in 1990 to standardize the inclusion criteria for clinical research studies . SimilarRegardless, the Amsterdam criteria fail to identify approximately 50% of cases, and Bethesda guidelines fail to identify at least 30% of cases , which hOne of the first hereditary nonpolyposis cancer-associated syndromes to be identified, Lynch syndrome, is also the most relevant to HNPCC. It is estimated that LS accounts for approximately 3% of all CRC cases, and its prevalence in the general population is one in 440 . Lynch sLynch syndrome is an autosomal dominant condition caused by a malfunctioning MMR system resulting from the pathological mutation in at least one of the MMR genes. Germline testing for germline mutations in the MMR genes—mutS homologue 2 (MSH2), mutL homologue (MLH1), mutS homologue 6 (MSH6), and postmeiotic segregation increased 2 (PMS2)—can discriminate Lynch syndrome from Lynch kindreds. During cellular proliferation and differentiation, DNA randomly produces errors that may include single base mismatch, insertions, misincorporation, and deletion of bases. The function of the MMR gene system is to recognize and maintain the fidelity of DNA by correcting the structure of DNA replication errors . ApproxiRecent studies have focused on exploring the novel somatic mutations in the Lynch syndrome. For instance, RNF43, one of the E3 ubiquitin ligases, acts as a negative-regulatory factor of the WNT pathway via reducing membrane surface expression . RNF43 hSince Lynch syndrome has been extensively characterized, however, the incidence of Lynch syndrome varies by the endemicity of all diagnosed CRC patients in different populations. Similar to the case of European individuals with Lynch syndrome , recent Very few studies have compared the epidemiological characteristics and clinicopathological differences between Lynch syndrome patients with other disease and those with Lynch syndrome only. The MSH6 and PMS2 mutations indicate a decreased risk of cancer incidence when compared with MLH1 and MSH2 mutations ; howeverScreening by colonoscopy enables the early detection and removal of preinvasive neoplasia or adenomas before the presence of symptoms and is the main strategy of secondary prevention in Lynch syndrome patients , 48. AltFor treatment, MSI in a tumor usually causes frameshift mutations in the DNA, resulting in immunogenic neoantigens that induce an immune reaction against the tumor. The approval of pembrolizumab use for solid tumors with high-level microsatellite instability or mismatch repair deficiency by the US Food and Drug Administration highlights the promise of precision immuno-oncology. Immunotherapy strategies are based on immunopathology. Hence, patients would benefit from tumor immunopathology, especially for some immune checkpoints, such as PD1 (programmed cell death 1) and PDL1 (programmed cell death 1 ligand 1).As many as 60–70% patients who fulfill the AC criteria in the clinical and MMR deficiency in the tumor but for whom germline testing lacks a detectable germline mutation are defined as having “Lynch-like syndrome” . Due to The mechanism between Lynch syndrome and Lynch-like syndrome for causing the generation of MSI within Lynch-like syndrome patients is appealing and elusive. Three hypotheses have been proposed to explain why Lynch-like syndrome patients show MSI in the tumors but no DNA MMR germline mutation: (1) there exist unknown gene mutations other than the DNA MMR genes in the germline that can drive MSI. Lynch syndrome and Lynch-like syndrome CRC frequently harbor not only activating ERBB2 mutations but also specific mutational patterns in PIK3CA and KRAS. Furthermore, ERBB2-mutated MSI CRC is susceptible to irreversible pan-HER blockade . RecentlOther possible causes of Lynch-like syndrome-associated cancers could include false-positive results showing MSI. Each of these possibilities may be part of the reason for Lynch-like syndrome because they are not mechanisms that conflict with each other, and given the standardized incidence ratios of Lynch-like syndrome between the ratios for Lynch syndrome and sporadic colorectal cancer, Lynch-like syndrome may be a heterogeneous condition between these two extremes. In exploring the three hypotheses, the first regarding an unknown germline gene driving MSI is the most remote. The DNA MMR apparatus is well studied, and most associated components are known, with no other reports of germline mutations outside of these genes, other than EPCAM as described. Mensenkamp et al. made significant headway in identifying that somatic mutations in MLH1 and MSH2 are a frequent cause for inactivating DNA MMR function and subsequent MSI generation within Lynch-like syndrome cancers .MUTYH encodes a base excision repair DNA glycosylase. Mutations in this gene are found in MUTYH-associated polyposis (MAP) syndrome, an autosomal inherited condition commonly featured by the presence of a few to hundreds of colonic adenomatous polyps and an increased CRC risk at a young age . BiallelHNPCC is a clinically heterogeneous disease of which approximately 4% of cases are associated with Lynch syndrome, which may not conform to AC, <1% of cases are correlated to a Lynch-like syndrome, and 2–4% of cases are diagnosed as FCCX . FamiliaThe genomic profiles of FCCX cancers show similarities to sporadic MMR-proficient CRC since the clinical performance and histopathological features resemble sporadic CRC. Chromosomal instability (CIN) is a type of genomic instability; as a result, the chromosomal structures are unstable and hence facilitate carcinogenesis. Recently, high-resolution genomic profiling elaborated a CIN-like profile that discriminated 65% of colorectal cancers associated with FCCX from LS based on the gain of chromosomal region 20q and loss of 18 . The genMoreover, previous studies suggest that mutations of the GALNT12 gene might elucidate the unknown etiology of familial CRC. However, investigators found that GALNT12 is not a major gene modulator in the predisposition to FCCX, when delineating the relevance of GALNT12 mutations in the etiology of FCCX . BRCA2 iRASL10B encodes a small GTPase with antitumor properties, and epigenetic silencing of this gene has been attributed to hepatocellular carcinoma cells and breast cancer , 79. RASFXXC presents a special clinically different phenotype compared to that of Lynch syndrome families as follows : (1) lowObviously, FCCX is clearly easily differentiated from Lynch syndrome . The ideTo our limited knowledge, hereditary CRC syndromes are a few causes of CRC in the general population. Different cancer syndromes encompass a spectrum of similar clinical presentations and genetic profiles. These overlapping concepts and terms make early diagnosis in hereditary CRC cases clinically challenging. Therefore, an awareness and understanding of these unique syndromes may help early diagnosis and preventative interventions in individual patients and their family members. On the other hand, patients with cancer syndromes and their high-risk family members also expect early detection and intense surveillance to prevent and manage several life-threatening malignancies. Moreover, screening, genetic testing, and counseling of at-risk kindred can translate into a significant benefit across multiple generations, demonstrating the tremendous importance of understanding the genetic profile and clinicopathological features of each syndrome. We have herein provided a comprehensive overview of several common hereditary colorectal nonpolyposis and cancer syndromes for clinicians to successfully handle while engaged in a busy clinical practice. Physicians should therefore stay abreast of these discoveries.There still remain some problems. Interactions with environmental factors make the identification and validation of genetic deleterious mutations or genes complicated. First, the human genome harbors a mass of rare variants, most of which may not be clearly or directly associated with disease phenotypes, and second, some of these alterations may interplay with other genetic and/or environmental factors, which may in turn influence their expression.In the era of gene sequencing and other new molecular technologies, a number of new genes/mutations are being identified for the prevention and management of many cancers. These new methods will contribute to the investigation of unique genotype-phenotype profiles and may provide the treatment strategies based on individual risk in precision medicine.


Melusin is a chaperone protein selectively expressed in heart and skeletal muscles. Melusin expression levels correlate with cardiac function in pre-clinical models and in human patients with aortic stenosis. Indeed, previous studies in several animal models indicated that Melusin plays a broad cardioprotective role in different pathological conditions. Chaperone proteins, besides playing a role in protein folding, are also able to facilitate supramolecular complex formation and conformational changes due to activation/deactivation of signaling molecules. This role sets chaperone proteins as crucial regulators of intracellular signal transduction pathways. In particular Melusin activates AKT and ERK1/2 signaling, protects cardiomyocytes from apoptosis and induces a compensatory hypertrophic response in several pathological conditions. Therefore, selective delivery of the Melusin gene in heart via cardiotropic adenoviral associated virus serotype 9 (AAV9), may represent a new promising gene-therapy approach for different cardiac pathologies. Proteins are synthesized as linear amino acid chains that must fold in a specific three-dimensional structure and maintain their functional conformation to carry out their biological functions , controlling their activity and degradation. It is thus conceivable that Hsp90 plays multiple important roles in sustaining heart function upon stress adaptation, by inducing protein refolding, directing unfolded proteins to proteasome degradation, and assisting conformational changes in signal transduction molecules  family, is devoid of ATPase activity and characterized by an α-crystallin domain responsible for their oligomerization. The association between sHsp oligomers and client proteins is required for their anti-aggregation activity , a CS domain, shared by CHORD proteins and by the co-chaperone protein Sgt1  mutations are very rare within the population, with no significant relevance in the epidemiology of cardiomyopathy. On the other hand, pre-clinical studies indicate that the regulation of Melusin expression may play a key role in improving the ability of the myocardium to cope with different stressors.A link between Melusin expression and cardiac functional parameters has also been observed in humans. In a cohort of 17 patients with aortic stenosis evolved to severe heart failure, Melusin expression positively correlates with left ventricle ejection fraction (Brokat et al., in vivo (Sbroggiò et al., Mechanical stress induces in the heart the activation of specific signal transduction pathways and the release of neurohumoral mediators acting on cardiomyocytes, fibroblasts and endothelial cells, regulating the heart's response to stress. The balance between these signals may direct the overall cardiac response to a compensatory or to a maladaptive remodeling (Tarone and Lembo, These data suggest that Melusin, along with Hsp90, mediates the assembly of a signalosome organized on the scaffold protein IQGAP1 to activate and integrate beneficial ERK1/2 signaling (Tarone et al., Melusin, indeed, potentiates ERK1/2 and AKT phosphorylation in transgenic mice subjected to transverse aortic constriction, preventing the evolution to dilated cardiomyopathy (Brancaccio et al., Melusin is a chaperone protein selectively expressed in cardiac and skeletal muscles, indicating a tissue specific function for this protein. Accordingly, its expression is upregulated in response to mechanical overload in the heart during the hypertrophic phase (De Acetis et al., MS wrote the manuscript and drew the figures. MB wrote the manuscript.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.


DEPDC5) gene, and lately in the genes encoding Nitrogen Permease Regulator Like 2 and 3 proteins (NPRL2 and NPRL3), have been revealed in families with inherited focal epilepsies [An increasingly recognized role of genes encoding components of the mammalian Target Of Rapamycin (mTOR) signal transduction pathway has recently emerged among familial focal epilepsies. Loss-of-function mutations in the Dishevelled, Egl-10 and Pleckstrin Domain Containing protein 5  and hemimegalencephaly . Distinepilepsy . The diset al. reported the first animal model of the pathology: a Depdc5 knockout rat [Depdc5-knockout rats exhibited several features of rodent models of mTORopathy, demonstrating that the pathogenic mechanism of DEPDC5-related epilepsies is clearly associated to an upregulation of the mTORC1 pathway.Lately, Marsan kout rat . NoteworTSC1 and TSC2) encoding proteins of the Tuberous Sclerosis Complex (TSC), another negative regulator of mTORC1 [DEPDC5 mutations. Second, since mTORC1 is known to regulate gene transcription and protein translation impaired mTORC1 activity could consequently lead to abnormal gene expression profiles and aberrant protein synthesis. Thus, an interesting line from this point of view would be to explore divergent transcriptional profiles in patient resected brain tissue, with an emphasis on ion channels and neurotransmitters levels of expressions, in order to link mTORopathies to disturbed neuronal activity.One of the upcoming challenges will be to elucidate why mutations in a gene encoding a ubiquitously expressed protein lead to purely neurological symptoms with focal seizures and/or lesions. In sharp contrast, tuberous sclerosis, a well-characterized multisystem disease with abnormal brain development and intractable seizures, is due to mutations in either of the two genes (f mTORC1 . Future DEPDC5 mutations are the most frequent cause in familial focal epilepsies, and differ from any other mutations in ion channel encoding genes by the presence of cortical malformations. The generation of relevant in vivo and in vitro models is thus critical to better characterize the molecular mechanisms involved. At last, efforts will be directed towards the identification of new therapeutic approaches and drug selection to improve drug-resistant patient prognosis.Overall,


SERCA plays a fundamental role for cell calcium homeostasis and signaling in muscle cells and also in cells of other tissues. Because of its prominent role in many physiological processes, SERCA dysfunction is associated to diseases displaying various degrees of severity. SERCA transport activity can be inhibited by a variety of compounds with different chemical structures. Specific SERCA inhibitors were identified which have been instrumental in studies of the SERCA catalytic and transport mechanism. It has been proposed that SERCA inhibition may represent a novel therapeutic strategy to cure certain diseases by targeting SERCA activity in pathogens, parasites and cancer cells. Recently, novel small molecules have been developed that are able to stimulate SERCA activity. Such SERCA activators may also offer an innovative and promising therapeutic approach to treat diseases, such as heart failure, diabetes and metabolic disorders. In the present review the effects of pharmacologically relevant compounds on SERCA transport activity are presented. In particular, we will discuss the interaction of SERCA with specific inhibitors and activators that are potential therapeutic agents for different diseases.The sarco(endo)plasmic reticulum Ca P-type ATPases are membrane transporters that couple the energy provided by ATP hydrolysis to the active transport of various ions or phospholipids. These enzymes generate and maintain crucial electrochemical potential gradients across biological membranes plasmic reticulum (SERCA), belonging to the PIIA-ATPase subfamily, is an intracellular membrane-associated protein of approximately 110 KDa which is involved in cell calcium signaling and homeostasis   is the final reaction step, which allows the enzyme to undergo a new transport cycle. High resolution crystal structures of various conformational states in the SERCA transport cycle were obtained, as described in detailed reviews , N (nucleotide binding), and P (phosphorylation) domains, and a transmembrane region of 10 helical segments (TM1–TM10) including the two CaIn this short review we will focus our attention on the interaction of SERCA with specific inhibitors and activators that may represent potential therapeutic agents for different diseases. To investigate the effects of pharmacologically relevant compounds on SERCA transport activity, we employ an electrophysiological technique, which is discussed in the next section.+,K+-ATPase, SERCA, and Cu+-ATPases (ATP7A and ATP7B)  is able to analyze 96 SSM sensors in a fully parallel mode allowing determination of the dose dependence of 100 compounds in <30 min , preventing cytoplasmic calcium binding and catalytic activation , a sesquiterpene lactone derived from the plant 2-TITU), another SERCA inhibitor .The inhibitory effects of TG, CPA, DBHQ, and1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene  than of Na+,K+-ATPase (IC50 of 11.1 μM). We propose that cisplatin inhibition of SERCA and Na+,K+-ATPase activities may be relevant to the molecular mechanisms that underlie the various adverse effects of cisplatin and other platinum-containing anticancer drugs.In the context of antitumor agents, SSM-based electrophysiology was employed to analyze the interaction of metal-based anticancer drugs with P-type ATPases, i.e., SERCA, Nad Figure   from the SERCA2a/PLN complex, thereby removing the inhibitory effect of PLN on this complex. Displacement of PLN from SERCA2a may favor the SERCA2a conformational transition E2 to E1, thus accelerating Ca2+ cycling. Istaroxime with its unique mode of action may provide a new small-molecule therapeutics for the treatment of both acute and chronic heart failure.Molecules that are able to stimulate SERCA activity have been recently identified Table . A remarIn vitro and in vivo experiments have demonstrated that the pyridone derivative stimulates the Ca2+-dependent ATPase activity of cardiac SR vesicles, increases Ca2+ transients of isolated adult rat cardiomyocytes and accelerates contraction and relaxation of isolated perfused rat hearts. It was concluded that the pyridone derivative behaves like a SERCA2a activator, which binds and inhibits PLN, and enhances systolic and diastolic functions of the heart. The pyridone derivative is thus proposed as a novel lead compound for therapeutic applications in heart failure.Very recently, a pyridone derivative was reported to activate the SERCA2a isoform by attenuating the inhibitory effect of PLN , suggesting that SERCA activators may represent promising pharmacological agents to treat diabetes and metabolic dysfunction. Finally, since defective SERCA is a significant cause of ER stress and neuron loss in Parkinson's disease, direct activation of SERCA by small-molecule drugs is currently explored as a viable strategy to develop a novel therapeutic approach to cure Parkinson's disease  stress. ER stress is associated with a variety of common diseases (Oyadomari and Mori, se Dahl, .P. falciparum may serve as innovative and effective antimalarial agents. In addition, studies on novel SERCA activators indicate that pharmacological activation of SERCA may constitute a promising strategy for the treatment of heart failure and diabetes. Research effort is devoted to the synthesis of highly selective and potent drugs, that can target SERCA in a tissue-specific manner. As most tissue/cell types can express more than one SERCA isoform, it would be highly desirable, although very challenging, to develop isoform-specific drugs that can interact selectively with one SERCA isoform, leaving the other protein molecules intact (Yatime et al., SERCA plays an essential role in the maintenance of cellular calcium homeostasis and calcium pump malfunction is associated to severe disorders and several pathophysiological conditions. Therefore, the SERCA enzyme represents an important target for the development of new drugs. SERCA inhibitors have been identified as potential drug candidates against various diseases. As discussed above, inhibition of SERCA activity in cancer cells provides an alternative therapeutic approach to cure different types of cancer. Moreover, specific inhibitors targeting the SERCA ortholog PfATP6 in the parasite FT-B: Wrote the manuscript and revised it critically for important intellectual content; SS, RG, and MM: Edited the manuscript and revised it critically for important intellectual content; SS: Helped FT-B in preparing the figures. All authors have read and agreed with the final version of the manuscript.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.


Plants confront multifarious environmental stresses widely divided into abiotic and biotic stresses, of which heavy metal stress represents one of the most damaging abiotic stresses. Heavy metals cause toxicity by targeting crucial molecules and vital processes in the plant cell. One of the approaches by which heavy metals act in plants is by over production of reactive oxygen species (ROS) either directly or indirectly. Plants act against such overdose of metal in the environment by boosting the defense responses like metal chelation, sequestration into vacuole, regulation of metal intake by transporters, and intensification of antioxidative mechanisms. This response shown by plants is the result of intricate signaling networks functioning in the cell in order to transmit the extracellular stimuli into an intracellular response. The crucial signaling components involved are calcium signaling, hormone signaling, and mitogen activated protein kinase (MAPK) signaling that are discussed in this review. Apart from signaling components other regulators like microRNAs and transcription factors also have a major contribution in regulating heavy metal stress. This review demonstrates the key role of MAPKs in synchronously controlling the other signaling components and regulators in metal stress. Further, attempts have been made to focus on metal transporters and chelators that are regulated by MAPK signaling. III), arsenate (AsV), cadmium (Cd), chromium (Cr), lead (Pb), copper (Cu), mercury (Hg), aluminum (Al), etc. These metals have not only known to perturb animal kingdom but also plant kingdom. Their damaging impact on our agriculture has also been very well-documented  that bind to Ca2+ and trigger different downstream signaling pathways , arsenite , heavy metal transport ATPase (CPx- and P1B-ATPase), natural resistant associated macrophage protein (NRAMP), cation diffusion facilitator (CDF), and ATP-binding cassette (ABC) transporters, present at plasma membrane and on tonoplast membrane of cell  are the metal binding SOD and require Fe3+ metal ion as cofactor  as corroborated by different studies acts as a universal secondary messenger in the normal functioning of plants as well as in response to various environmental stresses  that bind to Ca2+ and trigger different downstream signaling pathways  by blocking the Ca2+ channels  of vacuolar and Golgi membrane in coping with Cd toxicity. This is achieved through cooperation with a Glutathione-conjugated transporter Ycf1p whose activity is controlled by phosphorylation once again insinuating an interface between different signaling pathways in response to environmental stresses   exhibit higher levels of tolerance to Ni toxicity. Contrastingly, the same plants were found to be hypersensitive to Pb2+, depicting an exclusion of Ni2+ and augmented accumulation of Pb2+ as compared to wild type plants , hydrogen sulfide was found to interact with Ca2+ signaling in imparting improved tolerance to Chromium (Cr VIIV)-mediated heavy metal stress. It has also been discerned that Ca2+ provides tolerance against Cr stress by enhancing the activity of antioxidant enzymes  dose and acid rain strength. The adverse effects on the roots caused by acid rain could be alleviated by low concentrations of LaIII and synergistic effects on the roots were observed upon combined exposures at higher concentrations of La(III) and acid rain  resulted in increased IAA concentration in Arabidopsis roots promoting lateral root growth, thus protecting roots from Cd  are N6-prenylated adenine derivatives involved in the regulation of plant growth and development and in biotic and abiotic stresses  is a gaseous plant hormone regulating various important growth aspects. It is biosynthesized by ACC synthase (ACCS) that convert AdoMet to ACC, while ACC oxidase (ACCO) catalyzes the conversion of ACC to ethylene. ACCS and ACCO are encoded by multigene families and regulated by many biotic and abiotic factors Kende, . SeveralERF1, ERF2, and ERF5  is being considered as a major limiting factor for plant development interfering with cellular redox equilibrium. Similar to other metals, Al stress also downregulates most of the miRNAs in rice such as miR156, miR395, miR398, miR159 except miR399, miR166, miR168 which showed upregulation , bZIP (basic region leucine ZIPper), AP2 (Activator Protein 2), ERF (ethylene-responsive factor), and DREB (dehydration responsive element-binding protein), have been identified as potential downstream targets of MAPKs  that regulates As-associated transporters genes. He found that OsARM1 binds to the conserved MYB binding sites in the promoters of OsLsi1, OsLsi2, and OsLsi6, which encode key As transporters and affects their expression. Several studies suggest MYB TFs to be the downstream targets of MAPKs. Most recently, Li et al.  plant has showed that it positively regulates heavy metal stress tolerance by improving root growth upon overexpression , ZAT12 expression modulated upon short-term exposure to Cu while no such influence was observed upon long-term Cd exposure , NRAMP, CDF, and ABC transporters  by MEK1 (MAPKK) Table . This isApart from the transporters activating MAPKs or vice versa, there are also studies suggesting the activation of MAPKs by metal chelators. The important metal chelators are PCs and metallothioneins and these are small cystein rich peptides with metal binding capacity (Singh S. et al., From this review it is implied that metal exerts tremendous effect on plant by modulating its functioning at various levels. Metal stress activates several signaling pathways, known to have important role in imparting resistance against environmental stresses. Of these, an important signaling pathway contributing majorly in managing stress response is MAPK signaling pathway. Activation of signaling pathways magnifies the activation and functioning of various downstream components like transcription factors and other cytosolic protein thereby altering the expression of genes. In this review, the impact of heavy metals on activation of MAPK, calcium and hormone signaling along with other regulators like transcription factors and miRNAs is certainly reported. Several studies performed throughout different scientific groups suggest important role of MAPK signaling in heavy metal stress. However, a detailed evaluation of complete MAPK cascade working in combating heavy metal stress is required. Further, this review compiles the results revealing interplay of MAPK signaling with calcium, auxin, and ethylene signaling in response to heavy metal stress. All these findings outline the regulatory function of MAPKs acting either upstream or downstream to other signaling molecules. In animals this regulatory network has been elaborately studied in metal stress than in plants. Hence, it will be highly advantageous to study the importance of this signaling crosstalk in heavy metal stress in plants. Additionally, this review also summarizes interplay between MAPK signaling and other regulators like miRNAs and transcription factors in conveying a response against metal stress. However, scarce reports on regulatory network of MAPKs with transcription factors suggest a need for more in depth experiments in response to heavy metal stress in plants. Though, there are ample numbers of reports on activation of different signaling components, studies on deciphering of a complete regulatory network in heavy metal stress in plants are still lacking. Furthermore, the studies on activation of MAPKs by metals and metal transporters and in turn their regulation by MAPKs in animals and yeast, suggests occurrence of this phenomenon even in plants. However, the fragmentary work performed keeps this area mysterious in plants. Exploring the regulators of these metal transporters will contribute significantly in unraveling the mechanisms of metal stress tolerance in plants.SJ, PB, DV, SN, ST, and DS: wrote the manuscript; SJ: conceptualize the overall structure, prepared the illustrations, and edited the manuscript; KS and PB: prepared figures; AS: conceptualized, edited and approved the final manuscript.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.


Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints  for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.The Malayan box turtle ( Farm-to-fork food safety and quality has long been a goal, but to ensure it, both regulatory and market monitoring measures must be transparent across the globe . Market Cuora genus, which encompasses a total of 12 turtle species, all of which are found in habitats in different geographical locations across the Asian peninsulas . Th. ThBfa1- samples . All of  samples . The expTarget stability is a key point to consider in the validation of any analytical results, especially for forensic samples where less stable analytes are frequently decomposed resulting in false negative identifications. Published reports reflect the significant effort that scientists have put into the development of short-length DNA targets, which are thermodynamically more stable than the longer targets and hence persist through extreme stresses that often break down longer DNA markers . In seveIn this study, the MBT-specific target was obtained from all of the thermally processed samples ; when MBSpecies-specific PCR assay is often conclusive , but it Bfa1-restriction endonuclease enzyme  because in silico analysis showed two restriction sites for the Bfa1 enzyme with unique fragment lengths: 72 bp, 43 bp and 5 bp. The digested products  obtained from the MBT-specific PCR products (120 bp) were separated and visualized by a micro-fluidic chip-based automated electrophoresis station   Figs , but becSA) Figs . The endSA) Figs . The MBT1 enzyme . We furt1 enzyme .Bfa1-restriction endonuclease enzyme and distinctive MBT fingerprints (72 and 43 bp) were obtained , which are claimed to provide multicure, has become a disastrous threats to the existence of several wild species, including MBT. Although most of them are claimed to be plant products, several highly endangered species, such as rhinos, crocodiles, turtles, tigers and elephants which are enlisted in the CITES Appendix I and II, have continued to be killed to supply the raw materials of these medicines which has got huge market in the Southeast Asia and Chinese communities around the world . In manyobtained   is one of 18 native freshwater turtle and tortoise species in Malaysia, and despite being protected, it has been overexploited for its various organs, such shells and bones, which are believed to have healing properties for use in analgesic, antipyretic and invigorating traditional medicines. The highest rate of exploitation has been reported in East Asian countries for use in sex stimulants and invigorating traditional Chinese medicines, so the population of this species has significantly declined, putting it at risk of extinction. Moreover, its slow reproductive cycle due to late maturity and the production of few eggs has placed the species on the verge of disappearance, causing enormous potential harm to biodiversity. Therefore, it is feared that continuous, high-volume exploitation combined with its vulnerable life history characteristics might lead to a serious population decline, making the MBT extinct, at least locally.The Malayan box turtle Bfa1-restriction profiles further authenticated the origin of the amplified products. Thus, it might be reasonable to claim that such a PCR-RFLP assay could be used by regulatory bodies, archaeologists and wildlife protection agencies for the detection of the Malayan box turtle under any matrices with great reliability and confidence.The Malaysian government banned the hunting of this species for sale in domestic or international markets, but it clandestine trade under the labels of permissible meat is greatly suspected. Thus, there is a need for a reliable tracing method for MBT identification before this vulnerable species disappears. This study has developed a PCR-RFLP assay for the detection of MBT under complex food matrices, and the features inherent to the short DNA target (120 bp) of multicopy mitochondrial cytochrome b have made the target extraordinarily stable under various environmental and food processing stresses. Furthermore, the use of an endogenous control (141 bp) effectively eliminated the chances of a false negative detection, and false positive identification was similarly avoided through the use of a negative control. A phylogeny against 64 species, a mismatch analysis against 28 species and an experimental validation against 21 different types have made the assay highly reliable and viable even under severe conditions. Additionally, the lower limit of detection (0.1% MBT meat and 0.0001 ng DNA) has made the assay suitable for the detection of marginal levels of adulteration in popular food products such as meatballs, burgers and frankfurters. The study emulated the real forms of adulteration practices by making binary and ternary admixtures with various percentages of the target meat in a wide range of food products. Commercially available meat products and traditional Chinese medicine samples were screened to optimize and establish the validity of the assay for the analysis of marketed food, and the distinctive S1 Fig1–44: turtles/tortoises; 48–57: snakes; 58–65: crocodiles; and 45–47 and 66–67: lizards. Phylogenetic tree was constructed using the Malayan box turtle-specific 120-bp site of the cytochrome b gene against 66 reptile species, of which 44 were turtle (1–44), 10 were snake (48–57), 8 were crocodile (58–65) and 5 were lizard species (45–47 and 66–67). Whereas a very narrow genetic gap was found among the 9 species of the Cuora genus, a wide genetic distance distinguished MBT from the other reptile species.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


One main function of central CB1 signaling is to maintain whole body energy homeostasis. Thus, cannabinoids functionally interact with classical neurotransmitters in neural networks that control energy metabolism and feeding behavior. The promotion of CB1 signaling can increase appetite and stimulate feeding, while blockade of CB1 suppresses hunger and induces hypophagia. However, in order to treat overeating, pharmacological blockade of CB1 by the inverse agonist rimonabant not only suppressed feeding but also resulted in psychiatric side effects. Therefore, research within the last decade focused on deciphering the underlying cellular and molecular mechanisms of central cannabinoid signaling that control feeding and other behaviors, with the overall aim still being the identification of specific targets to develop safe pharmacological interventions for the treatment of obesity. Today, many studies unraveled the subcellular localization of CB1 and the function of cannabinoids in neurons and glial cells within circumscribed brain regions that represent integral parts of neural circuitries controlling feeding behavior. Here, these novel experimental findings will be summarized and recent advances in understanding the mechanisms of CB1-dependent cannabinoid signaling being relevant for central regulation of feeding behavior will be highlighted. Finally, presumed alternative pathways of cannabinoids that are not driven by CB1 activation but also contributing to control of feeding behavior will be introduced.Cannabinoids are lipid messengers that modulate a variety of physiological processes and modify the generation of specific behaviors. In this regard, the cannabinoid receptor type 1 (CB Central regulation of feeding behavior is indispensable to life, since animals and men have to consume energy in terms of food to exert essential daily functions  in the brain  that further consists of endocannabinoids (eCBs) as intrinsic CB1 ligands, and of eCB synthesizing and hydrolyzing enzymes  in the hypothalamus -gamma and subsequent catalase activation .In addition to neurons, CB1 delivered unique mechanistic insights into central CB1 signaling, which provides an important prerequisite to uncover the physiological role of CB1 in distinct homeostatic and hedonic feeding centers of the CNS.Together, studies focusing on the cell type specific expression and subcellular distribution of CBHomeostatic feeding centers supervise the body's energy resources and are located in the hypothalamus and caudal brainstem  to nucleus accumbens (NAc) projections  neurons that acutely promote food intake, and POMC neurons that drive gradual onset of satiety  and palmitoylethanolamine (PEA), as close related lipids of AEA, bind to PPARs (Fu et al., 1, may deliver future targets for therapeutic interventions in control of feeding behavior. Indeed, targeted lipidomics of different brain regions derived from mice either deficient for CB1, the AEA degrading enzyme FAAH or the aforementioned 2-AG degrading MAGL revealed that AEA and 2-AG hydrolyzing enzymes, when compared to CB1, link the ECS to a broader lipid signaling network in contrasting ways, which again may open an avenue in altering neurotransmission and behaviors independently of CB1 signaling (Leishman et al., 1, such as OEA and PEA, that as mentioned before signal upon different metabolic relevant targets, such as PPARs (Leishman et al., Generally, eCBs do not resemble to classical neurotransmitters that are stored in synaptic vesicles (Piomelli, 1 signaling in control of feeding behavior. Despite the significant setback that occurred in the past on clinical use of CB1 inverse agonists in order to treat overeating, there still is strong confidence in the field that the recent discoveries on central CB1 signaling soon will leverage the therapeutic potential of CB1.Actually, there has been significant increase of knowledge about central CBMK designed this review, including Figure The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this review we bring together these novel aspects of the differential regulation of these Tet isoforms and the likely consequences on their activity.Discovery of the ten-eleven translocation 1 (TET) methylcytosine dioxygenase family of enzymes, nearly 10 years ago, heralded a major breakthrough in understanding the epigenetic modifications of DNA. Initially described as catalyzing the oxidation of methyl cytosine (5mC) to hydroxymethyl cytosine (5hmC), it is now clear that these enzymes can also catalyze additional reactions leading to active DNA demethylation. The association of TET enzymes, as well as the 5hmC, with active regulatory regions of the genome has been studied extensively in embryonic stem cells, although these enzymes are expressed widely also in differentiated tissues. However, TET1 and TET3 are found as various isoforms, as a result of utilizing alternative regulatory regions in distinct tissues. Some of these isoforms, like TET2, lack the CXXC domain which probably has major implications on their recruitment to specific  The family of ten-eleven translocation (TET) methylcytosine dioxygenases are considered to play a major role in maintaining the fidelity of DNA methylation patterns through mediating demethylation  in which its levels decrease during differentiation , but not at the TSS itself, where there is typically almost no modified cytosine  methylation . TET activity is determined not only by the levels and type of transcript produced, but also to some degree by post-translational modifications. Ubiquitination, acetylation, phosphorylation, GlcNAcylation, and PARylation have all been reported to modify these enzymes, and would be expected to affect variously the protein localization, interacting partners and catalytic activity, as well as altering protein stability (reviewed by Wu and Zhang, Lhb gene expression similarly, although activity of the full-length TET1 was lost in the hypomethylated state, indicating distinct mechanisms of recruitment (Yosefzon et al., loci (Zhang et al., It is still not clear how exactly the TET enzymes recognize their targets. The CXXC domains have been attributed a major role, as they are reported to bind unmethylated CpGIs (Xu et al., in vitro, to have similar binding properties. However, it was hypothesized that association with distinct CXXC domains in other proteins might moderate TET function, as some CXXC domain proteins shown to interact with TET3 are involved with known signaling pathways such as Wnt/β-catenin (Liu et al., The CXXC domain of the full-length TET3, which is the predominant form expressed in neurons (Colquitt et al., The single TET2 isoform completely lacks a CXXC domain, and it has been suggested that the neighboring gene, which encodes a CXXC4 protein, IDAX, was originally part of an ancestral TET2 gene before chromosomal rearrangement during evolution (Ko et al., Lhb gene promoter is not catalytically active, and in other somatic tissues in which this is the dominant isoform, and in contrast to ESCs, TET1 colocalization with 5hmC is more rarely seen (Neri et al., The distinct recruitment mechanisms of TET enzyme isoforms to modified or unmodified CpGIs, or CpGs in other contexts, would clearly play a role in determining their function. Notably only the global chromatin-binding, but not targeted binding of the short TET1 appears correlated with demethylation, and in mice expressing only the short isoform, PGC imprints were not erased and their progeny exhibited developmental defects (Zhang et al., via up-regulating the JMJD8 demethylase transcriptional repressor (Khoueiry et al., Lhb gene and its down-regulation is essential for this population of cells to complete differentiation. This effect is direct, and TET1 binds the upstream promoter at a region carrying H3K27me2/3 which was markedly reduced in cells in which TET1 was knocked down (Yosefzon et al., TET1 plays an important and very distinct role in regulating gene expression also, paradoxically, through repressing transcription, and its knockdown in mouse ESCs increased expression of many of its directly targeted genes (Williams et al., vice versa, and after knock down of either factor, many genes bound by these factors had increased expression, indicating that Sin3A is required for a subset of TET1-repressed target genes (Williams et al., TET1-mediated repression might also involve a role for the MBD3/NuRD repressor complex, which was shown in ESCs to co-localize with TET1, bind to 5hmC DNA with higher affinity than to 5mC, and its knockdown affected primarily expression of 5hmC-modified genes. Conversely, the knockdown of MBD3 also reduced levels of 5hmC, suggesting that it either plays a role in TET1 recruitment, or is recruited to 5hmC-modified DNA and plays another role in protecting them from complete demethylation (Yildirim et al., The localization of the full-length TET3 near the TSS of promoters with unmethylated CpGIs implies its role in protection of these regions from methylation. Because this isoform binds with higher affinity to 5caC, if aberrant 5mC methylation does occur, the TET3-mediated oxidation to 5caC would ensure that binding of TET3 to this location is further strengthened. In neurons, genes found to be regulated in this way include those involved in controlling key genes of the BER pathway, suggesting a TET3-dependent regulatory feedforward pathway for active DNA demethylation. This would likely be particularly important in post-mitotic neurons in which replication-dependent passive demethylation of aberrant 5mC would not occur (Jin et al., Since the identification of the TET enzymes and their modification of the DNA, a vast amount of research has contributed to our understanding of this family of enzymes and of the role of 5hmC in the genomic landscape, particularly in the context of ESCs and early development. Notwithstanding, their abundant presence in the brain together with high levels of 5hmC, as well as the discovery of TET2's role in AML, prompted examination of other differentiated tissues, revealing the wide spread expression of these enzymes which work to regulate transcription through diverse mechanisms in distinct contexts. As emphasized here, some of this diversity undoubtedly arises from the differential regulatory mechanisms which drive expression of distinct isoforms. However, it remains far from clear how the functions of this group of enzymes developed and diverged to such a degree, and the role of their aberrant expression in disease.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.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.