Introduction: In myopathies, the correlation of individual electromyographic and histopathologic findings remains poorly explored, as most previous studies have focused on the ability of muscle biopsy and electromyography to distinguish the neuropathic vs. myopathic nature of the underlying neuromuscular disease.Methods: We identified 100 patients who had a muscle biopsy and electromyography performed on identical muscles. We used a detailed grading system ranging from 0- normal to 4- severe; and graded 16 histopathologic findings in each biopsy. Electromyography findings were also graded from 0 to 4 according to the standard protocol in our EMG laboratory. We used Kendall's tau for non-parametric ordinal correlation analysis.Results: Fibrillation potentials correlated with atrophic, necrotic, and regenerating fibers, fibers harboring vacuoles, fiber splitting, fibers reacting for non-specific esterase, fibers with congophilic inclusions, inflammation , and increased endomysial connective tissue. Short-duration motor unit potentials correlated with atrophic, necrotic, and regenerating fibers, increased endomysial connective tissue, and perimysial inflammation. Long-duration motor unit potentials correlated with fiber-type grouping. Increased phases of motor unit potentials correlated with atrophic fibers, increased endomysial connective tissue, and fibers reacting for non-specific esterase; while increased turns correlated with atrophic and regenerating fibers, increased endomysial connective tissue and target formations. Rapid recruitment correlated with regenerating fibers, perimysial inflammation, and increased endomysial connective tissue.Discussion: By demonstrating a clear correlation of various electromyographic and histopathologic findings, this study improves interpreting electrodiagnostic testing in myopathies, and serves as the basis to further assess the correlation between clinical, electromyographic, and histopathologic findings. Electrodiagnostic testing is a cardinal component of the evaluation of patients with suspected myopathy . It can Previous studies have focused on the ability of muscle biopsy and EMG to distinguish the neuropathic vs. myopathic nature of an underlying neuromuscular disease, –9 with rWith Mayo Clinic Institutional Review Board approval, we reviewed our database for patients who underwent a muscle biopsy between October 2015 and October 2016 to identify 75 eligible patients. For a subject to be included, EMG and muscle biopsy had to be performed on the same muscle on opposite sides if the patient had symmetric weakness , or on the same side if EMG was performed 3 to 6 months prior to the biopsy after verifying clinical stability of the muscle strength in that timeframe . As certain rare disorders were expected to be under-represented compared to more common myopathies such as sporadic inclusion body myositis (sIBM), we also identified 25 patients with rare disorders  fulfilling the same inclusion criteria, but evaluated prior to October 2015. We included under the histopathologic diagnosis of “vacuolar myopathy” the muscle specimens where the predominant feature consisted of abundant vacuoles, whether rimmed (myopathy with rimmed vacuoles) or not. To avoid the controversial term of polymyositis, the term “inflammatory myopathy with autoaggressive features” was used to describe muscle specimens with inflammatory cells invading non-necrotic fibers and lacking other histopathologic features of inclusion body myositis.Per our Muscle laboratory protocol, muscle biopsy specimens were frozen in isopentane-cooled in liquid nitrogen with serial 10 μm sections stained with hematoxylin-eosin, trichrome, nicotinamide adenine dinucleotide dehydrogenase (NADH), succinate dehydrogenase (SDH), cytochrome c oxidase (CCO), ATPase , acid phosphatase, myophosphorylase, periodic acid-Schiff (PAS), oil-red O, non-specific esterase (NSE), and Congo red. Muscle biopsy slides were then reviewed by light microscopy and Congo red stained sections were viewed under rhodamine optics to better visualize amyloid deposits. Seventeen clinically relevant histopathologic findings were graded on each specimen. These included atrophic fibers defined as fibers having a diameter less than 25 μm; necrotic fibers; regenerating fibers; fiber splitting; fibers harboring vacuoles; ragged-red fibers; CCO negative fibers; fibers with target formations; fibers with increased glycogen content; fibers reacting for NSE; fibers with congophilic inclusions; amount of endomysial connective tissue; perimysial inflammation; endomysial inflammation; fiber type grouping, type 1 and type 2 fiber atrophy. With the lack of any published validated scale for muscle biopsy grading and to avoid utilizing a binary present/absent system, we, the authors, established a grading scale based on our clinical expertise and in keeping with the practice of our Muscle laboratory. The grading scale ranged from 0  to 4 (severe) as detailed in Table Per our electrodiagnostic laboratory routine, nerve conduction studies were obtained first, with at least one motor and one sensory nerve conduction study from one upper and one lower limb. EMG was then performed using a concentric needle and limiting the examination to one side allowing biopsy of contralateral side. A minimum of 20 motor unit potentials, recorded at low levels of activation were evaluated using a semi-quantitative approach and graded on a scale from 0  to 4 (severe) using the Mayo grading system  which is a non-parametric measure of the degree of concordance between two variables measured on an ordinal scale.RYR1 myopathy. Among patients with vacuolar myopathy, one had Pompe disease and five had myopathy with rimmed vacuoles (hereditary inclusion body myopathy): two of these patients had pathogenic mutations in VCP, one in GNE and one in TIA1. The six patients with necrotizing myopathy and one patient with myopathy NOS were diagnosed with necrotizing autoimmune myopathy based on clinical history, response to pharmacological treatment and 3-hydroxy-3-methylglutaryl–coenzyme A reductase or signal recognition particle antibody positivity.We identified 100 patients fulfilling the inclusion criteria. Ninety six patients had biopsy and EMG performed on the same muscle on the opposite side, and four patients on the same muscle and on the same side. EMG preceded the biopsy by a mean of 22 days . In eighteen patients, EMG and muscle biopsy were performed while the patient was on an immunomodulatory drug. The biopsy specimens had diverse histopathologic diagnoses as shown in Figure Fibrillation potentialsSixty eight patients had fibrillation potentials on EMG. The percentage of patients with fibrillation potentials in each histopathologic diagnosis were as follows: 100% in sIBM, dermatomyositis and myofibrillar myopathy; 77% in inflammatory myopathy NOS, 67% in denervation with or without reinnervation, 63% in inflammatory myopathy with auto-aggressive features, 57% in vacuolar myopathy, 42% in myopathy NOS, 25% in mitochondrial myopathy and 16% in type 2 fiber atrophy.There was a correlation between the presence of fibrillation potentials on EMG and the following histopathologic findings Figure : atrophiShort duration motor unit potentialsEighty five patients had evidence of short duration motor unit potentials on EMG. There was a correlation between short duration motor unit potentials and the following histopathologic findings Figure : atrophiLong duration motor unit potentialsp = 0.0243) but not with fiber type predominance (p = 0.4566) . Thirty three patients had fiber-type grouping and 19 additional patients had fiber-type predominance. There was a significant correlation between long duration motor unit potentials and fiber type grouping  Figure .Increased phases and turns of the motor unit potentialsThere was a correlation between increased phases and the presence of Figure : atrophiRapid recruitmentThere was a correlation between rapid recruitment and the presence of Figure : regenerMyotonic dischargesp = 0.0637), cytochrome C oxidase negative fibers (p = 0.0542) and glycogen accumulation (p = 0.0794).Myotonic discharges were recorded in only nine patients: necrotizing myopathy (4 patients), sIBM (1), myopathy with rimmed vacuoles (1), Pompe disease (1), MFM (1) and inflammatory myopathy NOS (1). They did not correlate with any of the studied histopathologic findings. A statistically non-significant correlation (trend) was noted between myotonic discharges and necrotic fibers  allowing to draw more detailed conclusions, that may help in diagnosis and treatment decisions. Adopting a detailed pathological grading system and subsequent ordinal correlation statistical analysis were crucial. As in practice, the detection of a specific histopathologic finding occurring rarely is often of unknown clinical significance and does not imply a specific pathological diagnosis . Furthermore, due to random sampling, the more frequent an abnormality is, the more likely it will be detected by electrodiagnostic testing and on muscle biopsy. Hence, a binary absent-or-present grading system would not be sufficient. In our practice, we select a muscle to biopsy based on clinical involvement, electrodiagnostic testing and sometimes muscle imaging. This resulted in a wide variety of muscles biopsied and the diverse clinical and histopathologic diagnoses enriched the studied sample.Fibrillation potentials correlated with a wide spectrum of histopathologic findings including: inflammation, active myopathy (necrosis and regeneration), fiber disruption , chronicity  and denervation (fibers reacting for NSE). While the presence of atrophic fibers highly correlated with fibrillation potentials on EMG, type 1, or type 2 fiber atrophy in isolation did not.Therefore, the presence of fibrillation potentials should be interpreted with caution and should not be considered as a surrogate for just inflammation or disease activity. The percentage of patients with fibrillation potentials per histopathologic diagnosis in this study may not necessarily reflect the true prevalence of fibrillation potentials in such disorder, in part due to the fact that a clinically-involved muscle with fibrillation potentials on EMG is usually a preferred target for biopsy.The duration of the motor unit potentials is a key component to determine the nature of an underlying process. However, it is not uncommon to have mixed short and long duration motor unit potentials especially in sIBM . In our Limitations of the present study include the retrospective design and the mostly semi-quantitative nature of the data. Furthermore, the pathological findings are not completely independent, as they tend to occur in patterns such as the association of necrotic and regenerating fibers. To better interpret histopathologic and EMG findings, the clinical context should be considered. For the purpose of this study, we limited the clinical information to the clinical diagnosis, the use of immunomodulatory drugs, time lag between biopsy and EMG, and ensuring strength stability between the two procedures.In this study, we demonstrated the correlation between various EMG and histopathologic findings. The results reflect the reasonable accuracy of the used methods especially the histopathologic grading system. Therefore, the findings and the methodology could serve as a platform for future studies to further assess the correlation with clinical or radiographic data as well as to better understand the muscle biopsy-EMG correlation in individual disorders.EN, MaM, MiM, and WL: study concept and design; EN: acquisition of data; EN, MaM, MiM, JM, and WL: data analysis and interpretation; EN and JM statistical plan and analysis. EN drafting the manuscript. EN, MaM, MiM, JM, and WL critical revision of the manuscript for important intellectual content.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.


CD40LG gene, well illustrates the dilemma between HSCT vs. long-term supportive treatment. This disease leads to higher risk of developing infections from bacterial and intracellular pathogens, especially Pneumocystis and Cryptosporidium spp. While supportive care allows improved survival during childhood, organ damages may develop with increasing age, mainly chronic lung disease and biliary tract disease (secondary to Cryptosporidium spp. infection) that may evolve later to sclerosing cholangitis, a severe complication associated with increased mortality. Early HSCT before organ damage development is associated with best survival and cure rate, while HSCT remains a risky therapeutic option for older patients, for those with organ damage, especially severe liver disease, and/or for those with limited or no donor availability. Prospective studies are needed to analyze risks of HSCT compared to those of life-long supportive therapy, including quality of life measures.Combined immunodeficiencies (CIDs) are a clinically and genetically heterogeneous group of primary immunodeficiencies (PIDs) that affect T-lymphocyte immunity with abnormal development or function. As compared to severe combined immune deficiencies (SCID), these patients are usually diagnosed later. They display a broad infectious susceptibility; immune dysregulation manifestations and chronic lymphoproliferation are also frequent. These complications and their specific treatments can lead to persistent damage to several organs. Prognosis of CIDs is worse as compared to other PIDs. The curative treatment is usually hematopoietic stem cell transplantation (HSCT), but difficult questions remain regarding the definitive indication of HSCT and its timing; the final decision depends on a conjunction of factors such as immunological parameters, severity of clinical manifestations, and natural history of the disease, when molecular diagnosis is known. CD40L deficiency, a CID caused by mutations in  Combined immunodeficiencies (CIDs) are a clinically and genetically heterogeneous group of primary immunodeficiencies (PIDs) that affect T-lymphocyte immunity with abnormal development or function. Humoral immunity may be intrinsically abnormal or defective as a consequence of an abnormal T-B lymphocyte cooperation .Clinically, CIDs are extremely variable in terms of manifestations, age of onset and severity. A broad susceptibility to opportunistic and non-opportunistic infections including viruses, bacteria, protozoan and parasites is a hallmark of these diseases. Defects of immune regulation are frequent and can lead to autoimmune and immune dysregulation manifestations that in some circumstances can be the predominant features. They occur in up to 40% of patients with CIDs, autoimmune cytopenia and inflammatory bowel diseases being the most frequent . ChronicInfectious, autoimmune, inflammatory and oncogenic complications and their specific treatments can lead to persistent damage to several organs including lung, liver, digestive tract, and/or kidney.There is a continuum between severe combined immune deficiencies (SCID) and CIDs without consensual demarcation between these two groups of diseases . CIDs diMolecular causes of CIDs are numerous and can affect either lymphopoiesis or T-lymphocyte function at several of the numerous developmental, activation or differentiation steps required for an efficient immune response  1). The. The1). Thanks to improved performance of next generation sequencing, there has been a rapid expansion in the identification of causative gene alterations among patients with CIDs in recent years. Genetic diagnostic is also shortened by these new approaches. However, a proportion of patients with CIDs remains without molecular diagnosis .Prognosis of CIDs is worse as compared to other PIDs such as humoral defects, innate, and granulocyte immune deficiencies . The curFor patients with early severe clinical presentations mimicking SCID, the recommendation to drive the patient quickly to HSCT will be straightforward, including with alternative donors. However, no threshold of T-cell immunity required to prevent severe complications has been established. When molecular diagnosis is known, it may guide the decision for diseases with well-known natural history, helping to weight the balance between risks of disease evolution with potential development of future organ damage vs. risks of transplantation. However, many genetic causes of CIDs have been recently described, involving a small number of individuals with limited experiences, as summarized in Retrospective studies performed in specific CIDs   advocateThe donor availability needs also to be considered for the transplant decision. Whatever the diagnosis, HSCT from a healthy matched sibling donor (MSD) remains the gold standard that offers the best results and allows to consider HSCT early in the course of the disease, even pre-emptively if poor outcome is predictable  [see revCautious evaluation by a multi-disciplinary team is important before transplantation. Previous recurrent or chronic infections may have led to severe and irreversible visceral damage, notably lung and liver. Renal impairment can also occur as a consequence of chronic or recurrent anti-infectious treatment or prolonged immunosuppression. Aggressive pre-emptive treatment and control of all treatable pre-existing infections needs to be proposed prior HSCT. Special attention to detect emergence of antimicrobial drug resistance need to be given, especially in heavily pre-treated patients. In case of EBV-related lymphoproliferation prior to HSCT, the degree of remission required before transplant is a question of debate . Optimizex vivo manipulation of the graft is proposed. Addition of serotherapy  is frequently recommended to prevent rejection and GVHD, but its omission can be discussed in HSCT from a MSD. In case of pre-HSCT autoimmunity, treatment with anti-CD20 monoclonal antibodies can be proposed.The residual T-cell immunity and the intact hematopoiesis of these patients increases the risk of allograft rejection, particularly in case of HLA disparity between donor and recipient. Some degree of myeloablation is usually required. Development of reduced toxicity conditioning regimens (CR) has contributed to reduce transplant-related mortality (TRM) in the last decade , 49. CurFull donor chimerism in all lineages is not always mandatory to correct the underlying disease with exceptions such as WAS, where mixed chimerism has been related to inferior long-term outcome (autoimmunity and thrombocytopenia) , 50.The risk of occurrence of graft-vs.-host-disease (GVHD) is dependent on the degree of compatibility between donor and recipient, but also on the clinical conditions of the recipient. Pre-existing viral infections or inflammation related to autoimmunity or immune dysregulation can predispose to onset of early and severe acute GVHD and therefore, effective GVHD prophylaxis is of primary importance. If GVHD occurs, it needs to be aggressively and rapidly treated.ex vivo graft manipulation, but using post-transplant cyclophosphamide (PTCY) as GVHD prophylaxis, have been increasingly used to treat adult patients with malignant diseases. Data on PTCY in children with PIDs and CIDs are still limited but encouraging  deficiency , 56 is aoprotein –61 mainloprotein , 63. GenCD40L deficiency is associated with high morbidity and mortality. Historically, long-term survival has been poor with supportive therapy only, with 20–50% of patients reaching their thirties –66 and oAt present, HSCT represents the only curative treatment for this disease. Historically, its outcome has not been optimal . In a fiInterestingly, de la Morena et al.  evidenceAll 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.


Exercise has a myriad of physiological benefits that derive in part from its ability to improve cardiometabolic health. The periodic metabolic stress imposed by regular exercise appears fundamental in driving cardiovascular tissue adaptation. However, different types, intensities, or durations of exercise elicit different levels of metabolic stress and may promote distinct types of tissue remodeling. In this review, we discuss how exercise affects cardiac structure and function and how exercise-induced changes in metabolism regulate cardiac adaptation. Current evidence suggests that exercise typically elicits an adaptive, beneficial form of cardiac remodeling that involves cardiomyocyte growth and proliferation; however, chronic levels of extreme exercise may increase the risk for pathological cardiac remodeling or sudden cardiac death. An emerging theme underpinning acute as well as chronic cardiac adaptations to exercise is metabolic periodicity, which appears important for regulating mitochondrial quality and function, for stimulating metabolism-mediated exercise gene programs and hypertrophic kinase activity, and for coordinating biosynthetic pathway activity. In addition, circulating metabolites liberated during exercise trigger physiological cardiac growth. Further understanding of how exercise-mediated changes in metabolism orchestrate cell signaling and gene expression could facilitate therapeutic strategies to maximize the benefits of exercise and improve cardiac health. Exercise promotes general metabolic wellness –3, improc 129–210 CE), who recognized that not all movement is exercise and that exercise is most beneficial when vigorous, with “the criterion for vigorousness [defined by a] change in respiration…those movements which do not alter respiration are not called exercise”  What kinds of exercise elicit changes in cardiac structure and function?(2) How does cardiac metabolism change during exercise?(3) How might exercise-induced changes in metabolism promote cardiac adaptation?Cardiac adaptations associated with exercise were first documented in 1899. Physical examination using auscultation and percussion revealed that Nordic skiers  and univIncreases in physical activity require changes in the distribution of oxygen and nutrients throughout the body. The increased work and ATP turnover of skeletal muscle  are faciWhereas the cardiac responses to endurance exercise are directly associated with the use of oxygen for ATP production in skeletal muscle, resistance exercises are more anaerobic in nature. In addition, resistance exercise generally increases blood pressure, which is due in part to mechanical restriction of blood flow during static contraction. These features of resistance exercise result in markedly different cardiac responses as compared with aerobic exercise. The modest increase in cardiac output initiated by resistance exercise is predominantly due to increases in heart rate, with virtually no change in stroke volume , 40. A hRepetitive bouts of strenuous exercise can promote mild cardiac hypertrophy and/or chamber enlargement , 44, 45,Although regular, intensive endurance exercise can decrease resting and submaximal heart rates e.g., , 54], th, th54)],Cardiac remodeling in response to exercise appears also to involve processes beyond cardiomyocyte hypertrophy. For example, exercise increases levels of circulating progenitor cells –75 and cAlthough too little exercise is currently a much more serious health problem than too much exercise , the popProlonged endurance exercise can promote “cardiac fatigue,” characterized by decreased cardiac output and ejection fraction , 105, alThe heart has a high energy demand, which requires continuous ATP generation to sustain contractile function, ion homeostasis, anabolic processes, and signaling –114. In Oxidation of fatty acids is the primary contributor to ATP production in the heart, with catabolism of lactate, glucose, ketones, and amino acids fulfilling the remaining energy demand –120. Thiin vivo.Substrates such as glucose, lactate, and pyruvate are generally more efficient energy sources for the heart. In the normal mammalian heart, glucose metabolism via glycolysis supplies approximately 2–8% and glucose oxidation contributes up to 30% to the ATP yield . InteresKetone bodies such as acetoacetate and β-hydroxybutyrate have received recent attention due to their potential importance in heart failure –154; howAmino acids have a relatively minor role in ATP production in the heart; however, they are essential for processes such as protein synthesis and cell signaling. In particular, branched chain amino acids  are major amino acids taken up by the heart, with uptake dependent primarily on circulating BCAA concentration . BecauseGlutamine, a “conditionally essential” amino acid, also appears to regulate the metabolism and health of the heart. In particular, it can activate mTOR in cardiomyocytes , and it An acute increase in workload during exercise has robust effects on the metabolism of striated muscle . In the An increase in myocardial workload is accompanied by increases in the catabolism of multiple substrates, in particular, fatty acids and lactate , 197–2002max) resulting in large increases in arterial lactate levels (2 max) does not increase circulating lactate levels remarkably  appears sufficient to partially phenocopy the exercise-adapted heart and regulate genes . The acMetabolite signaling is another mechanism that connects exercise-induced changes in metabolism to cardiac adaptation. In particular, glucose-derived metabolites regulate the activities of the prohypertrophic kinases mammalian target of rapamycin (mTOR) and AMP-activated kinase (AMPK) . The intCirculating metabolites are also important regulators of exercise-induced cardiac growth. Hormone-mediated adipose tissue lipolysis during exercise liberates palmitoleate (C16:1n7), which promotes cardiac growth potentially by activating G-protein-coupled receptors (GPCRs), Akt, or nuclear receptors . The carMetabolic changes caused by exercise are important for cardiac remodeling and adaptation. The integrative metabolic changes brought forth by exercise combine with changes in cardiac workload to regulate cardiac metabolism. In particular, exercise alters levels of competing substrates, and it changes the abundance of circulating hormones, which cue metabolic pathways that are critical for transcriptional changes and cardiac growth. In addition, changes in circulating and endogenous metabolites can trigger physiologic growth by activating prohypertrophic signaling pathways. Nevertheless, numerous questions remain, including questions of how to optimize the amount of exercise to produce beneficial, as opposed to deleterious, effects on cardiovascular health  as well 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.


Bacterial pathogens and commensals are surrounded by diverse surface polysaccharides which include capsules and lipopolysaccharides. These carbohydrates play a vital role in bacterial ecology and interactions with the environment. Here, we review recent rapid advancements in this field, which have improved our understanding of the roles, structures, and genetics of bacterial polysaccharide antigens. Genetic loci encoding the biosynthesis of these antigens may have evolved as bacterial diversity-generating machines, driven by selection from a variety of forces, including host immunity, bacteriophages, and cell–cell interactions. We argue that the high adaptive potential of polysaccharide antigens should be taken into account in the design of polysaccharide-targeting medical interventions like conjugate vaccines and phage-based therapies. Recent advances in glycobiology emphasise the importance of surface carbohydrates in the evolution of bacterial commensals and pathogens.Epidemiological and genomic analyses highlight the remarkable diversity and adaptability of bacterial surface polysaccharides, often driven by horizontal DNA transfer.Functional heterogeneity of bacterial polysaccharide antigens points to multiple factors which have shaped their diversity.Antigenic diversity is a challenge for polysaccharide conjugate vaccines which strive to eliminate some bacterial diseases, like the pneumococcal disease. Therefore, even though most bacteria produce extracellular polysaccharides, the biology of polysaccharide production has more exceptions than rules. Nevertheless, a cross-sectional look at different bacterial species reveals many similar characteristics of carbohydrate antigens, and these similarities have important implications for their evolution. Here we compare the main aspects of polysaccharide antigen biology between different antibiotic-resistant global priority pathogens as defined by the World Health Organization in 2017 , which is an important component of the outer cell membrane and often includes a highly variable O-antigen at the end. Gram-negatives can also produce an additional capsular polysaccharide (CPS), which forms a surface layer. Gram-positive bacteria can synthesise teichoic acids, and like Gram-negatives, can also produce CPS, but they do not produce LPS. In addition to these major antigens, both Gram-negative and Gram-positive bacteria can synthesise exopolysaccharides (EPS), which are released into the environment and are not attached to the cell.Neisseria meningitidis is not included as it does not appear on the WHO list; however, this is also an important encapsulated pathogen for which capsule-targeting vaccines are in use Streptococcus pneumoniae capsules facilitate escape from mucous entrapment, thus affecting bacterial ability to colonise and transmit Haemophilus, Neisseria, Campylobacter, or Enterobacteriaceae Escherichia coli or the alginate in Pseudomonas aeruginosa, forms the basis of biofilms – a self-produced polymeric matrix which enables bacterial adhesion to surfaces and protects colonies from environmental dangers, including desiccation, bacteriophages, antibiotics, or the host immune system All of these extracellular polysaccharides play a variety of important roles in bacterial lifestyle and pathogenesis. Polysaccharide capsules enable bacterial evasion of the host immune system by shielding bacteria from the complement system, antibodies, or engulfment by macrophages wzx and wzy genes). The typical design of a polysaccharide antigen locus in bacteria is shown in Box 1in silico serotyping has now largely overtaken the standard serotyping methods, which require specialised reagents and expertise. (in silico serotyping.) The flip side of this is that the gap between serologically-determined diversity and genetically-determined diversity is widening. Thus, biochemical characterisation of polysaccharide antigens remains important, and a good understanding of the complex genotype-phenotype map in polysaccharide antigens remains one of the great challenges of polysaccharide biology .Even though the sequence of genes located in the polysaccharide biosynthesis locus is highly predictive of the expressed polysaccharide, the genotype is not a perfect predictor of the phenotype for two main reasons. First, genetic mutations within those genes can alter specificity of enzymes encoded by them, thus altering a biochemical linkage and producing a new serotype. Second, additional genes located outside the synthesis locus can influence or direct the final sugar structure. This has important consequences for public health. With increasing adoption of high-throughput sequencing for strain characterisation by research and public health labs, Alt-text: Box 1In spite of its length, a polysaccharide chain consists of a relatively small number of sugar molecules. The polymer can be biosynthesised in different ways, but in the large majority of cases it is done via one of three different mechanisms: the wzy-dependent, ABC-dependent, or synthase-dependent pathway , or an exchange leading to a new combination of enzymes  – often with the help of transposable elements – as has been widely observed in well studied The evidence therefore points to polysaccharide antigen loci having universally evolved as unique bacterial adaptive weapons, able to diversify at different speeds l-rhamnose or l-fucose, which are not typically found in animal cells The genetic and phenotypic plasticity of polysaccharide biosynthesis loci can give rise to many serological types within a single species , but it K. pneumoniae expresses both K- and O-antigens, but exhibits far more capsular variation than LPS variation; whereas the closely related enterobacterial species Salmonella enterica (mostly unencapsulated) and E. coli (variably capsulated) display extensive LPS diversity. However, in general, our understanding of the relative importance of the three ecological forces for bacterial evolution and the resulting antigenic diversity is far from complete.The strength of diversifying selection will depend on the species ecology, which can be subdivided into three different classes of ecological factors: host immunity, bacteriophages, and cell–cell interactions . ImportaN. meningitidis and Haemophilus influenzae, or variable capsule expression in Bacteroides), which may promote colonisation and transmission by escaping the immune system N. meningitidis and S. pneumoniaeBoth capsules and O-antigens are known to interact with the immune system and can be immunogenic K. pneumoniae and E. coli. Furthermore, extensive capsule diversity is also found in commensal bacteria . Phages often encounter physical barriers preventing them from infecting bacterial cells: capsules, peptidoglycans, or extracellular polysaccharides in biofilms. Thus, phages have evolved strategies to overcome these barriers, and often carry receptors containing depolymerase – an enzyme which degrades polymers like extracellular polysaccharides present on bacterial surfaces. These receptors are typically encoded in tail fibres or base plates, and are highly specific to the antigen type. Consequently, diverse serotype-specific depolymerases have been identified in phages isolated from many different bacterial species Cells are surrounded by carbohydrate structures known as glycans, and interaction between those glycans and proteins of other cells is expected to be an important ecological factor shaping their diversity N. meningitidis serogroup B, chondroitin or heparosan in E. coli, hyaluronic acid in some species of Streptococcus  Helicobacter pylori or Campylobacter jejuniHost tissues, such as gut or respiratory epithelia , are covered by a dense layer of glycans called the glycocalyx, which impacts many colonising bacteria. Consequently, microbes have evolved various ways of interacting with these structures to facilitate colonisation, including attachment to host glycans S. enterica, these predators exhibit different feeding preferences towards different O-antigens Acanthamoeba castellanii, and E. coli has been attributed to the interaction between LPS and the predator’s surface mannose-binding protein In addition to host cells, bacteria colonising new hosts may encounter other prokaryotic and eukaryotic cells sharing the same niche. First, successful colonisation requires compatibility with host-associated commensal bacteria – for example, the mutualistic streptococcal communities found on oral mucosal surfaces. These communities are shaped by interactions between lectin-like adhesins and polysaccharides which act like receptors, thus connecting multiple cell–cell adhesions into large complex networks known as biofilms S. pneumoniae it is widely accepted that immune selection is an important driver of antigenic population structure. However, it is not entirely clear whether occasionally appearing capsule switches are evolutionary ‘mistakes’, or whether they are (at least partly) driven by interactions with bacteriophages. Pneumococcal capsule-specific depolymerase enzymes have been isolated before S. pneumoniae. In Klebsiella, some K-antigens are known to interact with the adaptive immune system Klebsiella, the ability to exchange K-antigens permits adaptation during environmental shifts. In S. enterica, host-specific distribution of O-antigens was previously argued to be a consequence of the the varying feeding preferences of intestinal predators present in different animal hosts Box 2Mathematical models serve as powerful tools for predicting the effects of medical interventions. For a model to be a successful prediction tool, one needs to choose the right balance between the model simplicity and its biological accuracy.H. influenzae and S. pneumoniae, mathematical models have been widely used to predict their effect on the control of bacterial disease Since the development and introduction of the conjugate vaccination programmes against Studies modelling the impact of conjugate vaccines have mostly focused on their short-term effects, namely their effect on bacterial transmission and disease. Some papers have examined the impact of conjugate vaccines on the bacterial population structure in vivo dynamics in vivo bacteria–phage coevolution. Such quantification will be facilitated by recent advances in microbiome research and genomics While phage therapy is fundamentally different from vaccination, previous studies have mostly relied on epidemiological models to predict its dynamics, and thus likely success Alt-text: Box 2A schematic summary of the factors discussed above, and their expected timescales, is given in S. pneumoniae, which emphasises the challenge of robust vaccine design against other diverse bacteria like Klebsiella or Acinetobacter , but in fact in all three species there has been a rise in the reported frequency of unencapsulated strains The simplest form of vaccine escape is by losing the capsule, which is an acceptable outcome since unencapsulated bacteria are unlikely to cause disease. This is most likely to happen when a vaccine spans the entire capsule repertoire . These approaches impose strong selective pressures on a fraction of the bacterial population by targeting only a small subset of the capsular repertoire. Given the discussed potential of polysaccharide antigens to rapidly generate novel diversity, a question arises about the long-term efficacy of such interventions (see Outstanding Questions). We have seen some evidence of vaccine-driven adaptation in the antigenically diverse tobacter . HoweverUltimately, long-term effectiveness of any polysaccharide-targeted medical intervention will depend on a good understanding of the evolutionary dynamics of the relevant bacterial system, as well as on clever formulation of mathematical models to predict the impact of such approaches see also . HoweverOutstanding QuestionsWhat is the impact of within-host evolutionary dynamics on bacterial population structure? We know relatively little about within-host forces driving antigenic diversification, and about trade-offs between within-host and between-host bacterial fitness and transmission.How important are polysaccharides in carriage and disease? Some capsule types are strong predictors of carriage and virulence, but their role in pathogenesis is not fully understood.Can we efficiently predict phenotypes from genotypes? Genetic typing permits rapid identification of well known serotypes but it tells us little about the genotype–phenotype map.How important are commensal and nonclinical strains in driving evolution of pathogens? Nonpathogenic bacteria are largely understudied, and we need to understand their role in pathogen evolution and diversification.How fit are different serotypes in different environments? Environment-dependent measures of serotype fitness would help us predict the emergence of new serotypes in the future.What impact do bacteriophages have on bacterial population dynamics? We need to understand how phages interact with bacteria and how they shape population structures of bacterial populations.How common are interactions between eukaryotic predators and bacteria? We need more studies highlighting the nature of interactions between protists, bacteria, and their ecology, and need to understand how they impact evolution of non-host-associated bacteria.What is the impact of host glycan diversity on pathogen evolution? Glycan diversity may affect the structuring of bacterial populations, both within-host and between-host.Polysaccharide antigens, such as capsules, O-antigens, or teichoic acids, are common in pathogenic and commensal bacteria. Our understanding of how these structures are synthesised and how they interact with hosts, viruses, and the environment have largely improved over the last two decades. Altogether, they paint a picture of genetic loci which are highly adapted to become modified and exchanged between different bacteria. Such diversity is likely a result of millions of years of coevolution with bacterial viruses, hosts, predators, and other bacteria, and is unlikely to change drastically on epidemiological timescales. However, the introduction of vaccines, antibiotics, or phage therapies may dramatically alter the structure of bacterial populations due to the resulting strong selective pressures. It is thus conceivable that these antigens could rapidly evolve, undermining the long-term efficacy of therapeutic interventions. Therefore, the ability to predict the long-term consequences of these interventions and inform public health will be conditional on our understanding of the biology, epidemiology, and ecology of the system in question, quantification of evolutionary and epidemiological parameters, and the resulting accurate design of mathematical models.


Several challenges present themselves when discussing current approaches to the prevention or treatment of pancreatic cancer. Up to 45% of the risk of pancreatic cancer is attributed to unknown causes, making effective prevention programs difficult to design. The most common type of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), is generally diagnosed at a late stage, leading to a poor prognosis and 5-year survival estimate. PDAC tumors are heterogeneous, leading to many identified cell subtypes within one patient’s primary tumor. This explains why there is a high frequency of tumors that are resistant to standard treatments, leading to high relapse rates. This review will discuss how epigenetic technologies and epigenome-wide association studies have been used to address some of these challenges and the future promises these approaches hold. Consistently across sources, the top identified risk factors for pancreatic cancer include: Smoking, obesity, age, recent onset diabetes mellitus, recent pancreatitis, hereditary pancreatitis and hereditary pancreatic cancer. These risk factors are only present in about 50–60% of pancreatic cancer cases, with known genetic alterations only accounting for about 5–10% ,3,4. InvGlobally in 2018 for pancreatic cancer, the estimated number of new cases was over 1,000,000, with 65,000 deaths [PDAC is characterized by well-defined genetic modifications; however, epigenetic alterations have recently been recognized as important contributors to PDAC development and progression , as wellEpigenetics refers to processes that take action on the DNA, rather than modifying the DNA sequence . Epigenomics only refers to the analysis of these epigenetic processes across multiple genes or the entire genome . Several schematics exist to demonstrate the interrelationship between genomic, epigenomic and signaling pathway alterations in PDAC ,15,16,17In this section, several types of epigenetic processes are discussed, along with current laboratory methods used to detect markers of each type of process. The processes that are discussed include nucleosome remodeling complexes and nuclear architecture, histones, transcription factors, methylation and non-coding RNA.Nuclear DNA exists in a very compact configuration because of its interaction with an array of proteins, including histones. During the process of transcription and replication, chromatin is relaxed, remodeled and recovered in a cascade of events concisely referred to as chromatin remodeling (CR). CR can be initiated by various types of histones or through an ATP-dependent mobilization of nucleosomes; the latter is termed as nucleosome remodeling (NR). NR complexes are categorized into four families: SWI/SNF, ISWI, CHD and INO80, each harboring a family-specific ATPase, BRM or BRG1 (SWI/SNF), SNF2H (ISWI), CHD3 (CHD) and INO80 (INO80). Increasing evidence now suggests that NR has fundamental roles in transcriptional regulation and DNA-damage repair . The subThe basic biological properties of nucleosomes and NR are investigated using atomic force microscopy (AFM) . AdditioHistone modifications, including acetylation and methylation, are another major epigenetic mechanism responsible for regulating gene expression. The acetylation of lysine residues within histone proteins prevents the positively charged lysine from interacting with the negatively charged DNA, resulting in a more open chromatin structure that promotes gene expression. Conversely, deacetylation has the opposite effect and results in a more closed chromatin structure that suppresses gene expression. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are the key enzymes associated with the removal and addition of acetyl groups to lysine residues in histone proteins ,27. IncrThe aberrant expression levels of mucinous glycoproteins, such as MUC17, has earlier been correlated with PDAC pathogenicity and progression . EpigeneTechniques to evaluate histone modifications frequently involve targeted antibodies . AlthougTranscriptional factors (TFs) have the functional role of directing and attaching transcriptional machinery to specific segments of the DNA within the cell . SeveralChIP-Seq can be used to survey interactions between proteins, DNA and RNA. ChIP-Seq using next generation sequencing (NGS) enables researchers to identify the binding sites of multiple protein targets, including transcription factors and histones, across the entire genome. Comprehensive tools such as Spatial Clustering for Identification of ChIP-Enriched Regions (SICER)  and ModeDNA methylation involves the addition of a methyl group to the 5-carbon position of cytosine within the DNA sequence and most frequently occurs in CpG sites where a cytosine (C) is positioned next to a guanidine (G) nucleotide. DNA methyltransferases (DNMTs) initiate and maintain the methylation state of the CpG sequences, which are commonly present in the promoter regions of genes. Promoter hypermethylation is primarily associated with gene silencing, as methylation interferes with the transcription factor binding to the promoter, resulting in the suppression of gene expression . ConversTechniques to evaluate DNA methylation have evolved since the 1970’s, when the first approaches were developed . These eRecently, several studies (last two in preprint stage) have observed increases in both widespread and targeted gene-specific 5-hydroxymethylation in PDAC. 5-hydroxymethylcytosine (5-hmC) is a residue that is generated from the well-studied cancer associated 5-methylcytosine (5-mC). The first study used a mass spectrometry method to compare 11 patient derived xenografts (PDX), 11 PDAC cell lines, and two control pancreas cell lines. Their results suggested an increase in 5-hmC in the enhancer region of key PDAC oncogenic genes  . The secProtein-coding genes constitute only 2% of the human genome, while a major proportion is transcribed into non-coding RNAs (ncRNAs). NcRNA refers to a broad category of RNA molecules that includes circular-RNA (circRNA), piwi-RNA (piRNA), micro-RNA (miRNA), small-interfering RNA (siRNA), enhancer-RNA (eRNA) and long non-coding RNA (lncRNA). Substantial breakthroughs have elucidated the functions of ncRNAs and have supplied plentiful evidence suggesting the roles of ncRNAs in all fundamental cellular processes ,73,74. FDatabases harboring methylome and epigenome data from resected tumors and biopsy samples have unlocked new avenues for genome-wide epigenetic research. The International Cancer Genome Consortium (ICGC)  and The Tissue biopsies are traditionally used to evaluate epigenetic changes in tumors. However, the inability of tissue biopsies to completely characterize tumors has shifted attention to new diagnostics and minimally invasive techniques, like liquid biopsies. Liquid biopsies involve the analysis of analytes from biological fluids, namely blood ,96. The PDAC tumors are known to be very heterogeneous and include a vast amount of stroma. Therefore, capturing gene expression data on a PDAC tumor will only provide an overview of the transcriptional activity of the tumor and not necessarily reflect what is occurring in the cancer cells. Approaches have been developed to elucidate the cell-to-cell variability in tumors, including PCR amplification of complimentary DNA from a single cells ,105. WitStudies investigating candidate epigenomic components and their associations with PDAC are increasing exponentially, with many review articles already published ,16,114. When genome-wide associations are investigated, numerous statistical tests are performed, therefore, multiple testing adjustment approaches such as the Bonferroni correction  or the fEWAS studies looking at methylation and PDAC are the most common because of the early implementation of technologies. As a consequence, researchers have a better understanding of the functional role that methylation plays in transcription and gene expression compared to other epigenetic factors. The key characteristics of the reviewed methylation EWAS studies are presented in p-value but also the beta value, and the methylation level was correlated to gene expression, thereby reducing the likelihood of false-positives and increasing the chances of identifying functionally relevant results. Any methylation markers appearing in multiple studies would be candidates for further study, since issues such platform technology, sample selection and statistical methods would be minimized.The eight reviewed studies have focused on comparing methylation marker patterns for cancer to either normal or cell line samples. The sample sources were generally from pancreas tissue, but also included blood, cell lines and mouse models. The earlier studies had small sample sizes, limited methylation markers and poor genome coverage. All studies experienced difficulty with the validation of markers, multiple testing and clinical factor adjustments. In the later studies, significance was based not only on the NcRNAs, originally considered “transcriptional noise”, are classified into two groups (short (sncRNA) and long (lncRNA)) based on the length of the RNA fragment . These RThe six reviewed studies have focused on comparing lncRNA markers for cancer to either normal or cell line samples. Among these studies, there was a much heavier focus on treatment response and survival as outcomes. The sample sources were generally from pancreas tissue, but also included cell lines and mouse models. All studies have been published since 2017 and have experienced difficulty with the validation of markers, multiple testing, clinical factor adjustments and determining the functional role of identified lncRNA markers. Any lncRNA markers appearing in multiple studies would be candidates for further study, since issues such platform technology, sample selection and statistical methods would be minimized.With the availability of public EWAS datasets with reasonable sample sizes for PDAC, researchers have started to take a more system-wide approach to understanding the complexity and interconnectedness of several types of epigenetic markers. These datasets are an important source for exploratory studies or the validation of potentially important marker sets. The key characteristics of the reviewed studies are presented in The four reviewed studies have focused on comparing at least two marker datasets  for cancer to either normal or cell line samples. The sample sources were generally from pancreas tissue or cell lines. These types of studies for PDAC started appearing in the literature around 2015 and have benefited immensely from the availability of data through the public databases. Multi-omic studies experience difficulty with the use of non-standard statistical/bioinformatic approaches, multiple tests, clinical factor adjustments and arbitrary filters to reduce dimensionality ,121,122.To date, EWAS studies have mostly focused on assessing the relationship between PDAC diagnosis or prognosis with methylation or ncRNA markers  across the genome. The hypothesized role of epigenetic markers is to regulate gene expression by controlling access to DNA , and markers are often correlated with gene expression. However, recent evidence suggests that this widely held assumption may not always hold true, as transcription factors have been shown to bind to both methylated and unmethylated DNA .The assessment of epigenetic markers associated with PDAC has been performed for both diagnostic ,128 and Multi-omic approaches are going to be key to furthering our biological/mechanistic understanding of the development and metastatic behavior of PDAC. Studies have started to identify cell subtypes based on epigenetic signatures in PDAC, and these subtypes have differential survival rates and responses to treatment (reviewed in Reference ). ThrougSample collection and selection are key factors that require transparent discussion of strengths, limitations and study hypotheses whenever researchers are presenting study results. For example, the methylation marker sets that we measured from the cell-free DNA that was collected from a PDAC patient will tell us different information than the methylation marker set which we would measure from the primary tumor or in circulating exosomes in the same patient ,140. In Candidate gene/pathway studies in the laboratory using multiple tissues/cells will continue to provide further biological and mechanistic evidence of statistical associations observed at the genome-wide level. These type of laboratory studies will also provide insight into biological pathways and can use knockout/knockdown models to clarify the functional relationships observed through statistical associations and computational modeling .Genome-wide based technologies and techniques are creating new datasets at a fast pace. As scientific researchers, we need to be conscious and always highlight the key limitations of the sample collection and lab-based methods, along with the results we are presenting. Each sample type and technology provides us with a snapshot view of the biological system and disease state. Therefore, attempting to further understand the biological processes and mechanisms of a complex and heterogeneous disease like PDAC will require us to take a more systematic approach through the integration of multi-omic datasets and laboratory results.A search was conducted using the database PubMed in order to identify all published studies with a combination of the following keywords in the abstract or title: “Pancreatic adenocarcinoma”, “pancreatic cancer”, “methylation”, “RNA”, “epigenetic”, “multi-omics”, “epigenomic”, “epigenomes-wide”, “genome-wide”. Abstracts were then screened to determine which articles were EWAS studies for full review and inclusion in


Inborn errors of metabolism (IEMs) are a group of inherited diseases with variable incidences. IEMs are caused by disrupting enzyme activities in specific metabolic pathways by genetic mutations, either directly or indirectly by cofactor deficiencies, causing altered levels of compounds associated with these pathways. While IEMs may present with multiple overlapping symptoms and metabolites, early and accurate diagnosis of IEMs is critical for the long-term health of affected subjects. The prevalence of IEMs differs between countries, likely because different IEM classifications and IEM screening methods are used. Currently, newborn screening programs exclusively use targeted metabolic assays that focus on limited panels of compounds for selected IEM diseases. Such targeted approaches face the problem of false negative and false positive diagnoses that could be overcome if metabolic screening adopted analyses of a broader range of analytes. Hence, we here review the prospects of using untargeted metabolomics for IEM screening. Untargeted metabolomics and lipidomics do not rely on predefined target lists and can detect as many metabolites as possible in a sample, allowing to screen for many metabolic pathways simultaneously. Examples are given for nontargeted analyses of IEMs, and prospects and limitations of different metabolomics methods are discussed. We conclude that dedicated studies are needed to compare accuracy and robustness of targeted and untargeted methods with respect to widening the scope of IEM diagnostics. The measurement of metabolites in the diagnosis of inborn errors of metabolism (IEMs) was established 55 years ago by Dr. Robert Guthrie for phenylketonuria (PKU) after its discovery by Dr. Asbjørn Følling three decades earlier . PKU is In parallel to measurements by mass spectrometry, genetic screening of IEMs started in the 1990s . Today, Nowadays, targeted metabolite assays represent the key technology in NBS worldwide . HoweverThe use of untargeted metabolomics could complement current targeted metabolite assays. Untargeted metabolomics has the prospect of studying a wider range of metabolic pathways and to provide a broader view of the true metabolic phenotype of diseases . UntargeIn this review, we will discuss IEM diseases with emphasis on screening and diagnosis using targeted versus untargeted mass spectrometry (MS) approaches. We will give an overview of studies, including application of matrices analyzed, instrumentation, and data processing methods. We will also discuss challenges and obstacles to adapt untargeted mass spectrometry in IEMs screening and diagnosis.IEM diseases are a group of inherited genetic diseases resulting from total or partial absence or deficient activity of an individual enzyme, structural protein, or transporter molecule . Recent A recent attempt for classification associated with the prevalence of each category of IEMs has been reported by Ferreira and his colleagues, . They us. These discrepancies of IEM categories appear in overall incidence but also for specific categories including organic acid disorders, lysosomal storage disorders, fatty acid metabolism disorders, mitochondrial disorders, urea cycle disorders, amino acid metabolism disorders, and carbohydrate metabolism disorders.Studies from different countries have found wide disparities in the reported incidence rates of IEM diseases . These dHowever, there is a lack of data regarding the causes of disparities in overall incidence and rates of individual disorders worldwide. Higher rates of consanguineous marriage in some countries or regions can increase the incidence rates up to 50-fold . RegionaMost IEMs are inherited in an autosomal recessive (AR) manner . In AR dIEM disorders are often characterized by alteration of multiple overlapping metabolic pathways. Different classifications have emerged to categorize IEM diseases to enable easier clinical and laboratory diagnosis and facilitate treatment  such as The Society for the Study of Inborn Errors of Metabolism (SSIEM) considers clinical aspects of IEMs in a specific classification. The SSIEM classification assorts large numbers of individual disorders according to their biochemical pathways and common pathophysiological mechanisms ,44. MetaIEMs may present at various ages in different ways. Clinical presentation of the disease can occur even before birth, at birth, or during the first days of life as deterioration after normal birth and delivery . Errors This phenomenon is caused by changes in catabolism which occurs normally in the first days of life, leading to an accumulation of metabolites that cause toxic manifestations. The rate of deterioration is variable according to disease type, the extent of the disease and the most affected organ. For example, inborn errors in metabolism may appear as neurological symptoms, disorders of acid-base balance, unexplained hypoglycemia, cardiomyopathy, hepatic deterioration, or sudden death. Other diseases have more subtle presentations, such as a characteristic odor which is not commonly detected ,57. In gClinical manifestations of IEMs are overlapping. Therefore, clinical approaches to diagnosing IEMs are very difficult, especially for rarer disease variants. Presenting symptoms in infants can have a wide variety of possible causes. Distinctive facial abnormalities are linked to a group of diseases such as lysosomal storage disorders, pyruvate dehydrogenase deficiency, glutaric aciduria type II, cholesterol biosynthesis defect, glycolysalation disorders, and disorders of peroxisomal biogenesis . InfantsDiagnosis of IEMs is based on biochemical tests that are divided into two approaches: (1) screening tests to detect possible abnormal levels of metabolic biomarkers in blood or urine before the disease manifests and (2) tests to detect specific pathognomonic biomarkers . All bioScreening is an important tool for primary disease prevention. In contrast to diagnostic investigations, the screening tests aim to detect diseases in the latent asymptomatic stage of the disease to facilitate intervention and improve outcomes . IEM scrIn the symptomatic stage of the disease, severity and onset of symptoms influence the investigations and diagnostic methods used. However, as many IEMs present with very similar symptoms, it is necessary to perform multiple diagnostic investigations concurrently . The invMetabolomics seeks to measure the complement of endogenous and exogenous small molecules in a given sample. Metabolomics analysis in cells, tissues, and body fluids reflects the biochemical status of the sample a direct readout of the current phenotype  of the living organism ,81. ThesMetabolomics analysis in clinical medicine is most commonly performed using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) . NMR speMass spectrometry is used in two different modes in IEMs screening: either by direct injection of the sample to the ionization source of the mass spectrometer or by separation mixtures of compounds by gas chromatography (GC) or liquid chromatography (LC) prior to MS detection. GC or LC methods reduce the complexity of chemical mixtures prior to mass spectrometry analysis  and is wThe introduction of ultrahigh-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS/MS) have improved sample throughput and analytical sensitivity of a wide range of metabolites ,94,95. RMetabolomics in biomedical studies generally uses biofluids, cells, and tissues as the primary matrix to generate metabolic signatures. In clinical practice, urine, serum, and plasma are easy to collect and to prepare. They present the most commonly used biofluids ,100,101.In addition, other biosamples could be used, for example cerebrospinal fluid (CSF)  and saliMetabolomics is divided into two approaches: studies looking only at specific compounds (targeted metabolomics) and studies that aim at measuring all compounds (untargeted metabolomics), Traditionally, the study and diagnosis of IEMs is performed using a panel of targeted analyses with dedicated analytical protocols. These protocols cover a selected panel of diseases by quantifying the metabolites in a disease pathway and metabolite levels are then compared to the range of healthy  metabolic concentrations ,119,120.Despite its unquestionable role, current target-based newborn screening programs yield only a snapshot of all metabolic alterations. Many IEMs cannot be identified by current routine targeted metabolite analyses . Even whFalse interpretation of disease manifestations by the parents or family doctors as well as incomplete description of patient symptoms by the parents may lead to inappropriate diagnostic panels. Hence, there is a high risk that improper tests are performed by metabolite target screening methods, increasing the rate of false negative results and delaying accurate diagnoses ,124. UsiThe economic evaluation of targeted newborn screening programs has been studied in different areas over the last decade. Thiboonboon et al. described extended metabolic screening as not cost-effective in Thailand . In the In addition to limits of testing policies, targeted metabolomic analysis in classic newborn screening programs limits the discovery of novel metabolic defects due to its focus on a single panel of known metabolic pathways. Providing a broader understanding of the pathophysiology and the metabolic interactions behind the phenotype of the disease is important to design better treatment regimens ,137. Cur3) and globotriaosylceramide (Gb3), has proven challenging to be correctly diagnosed. Auray-Blais et al. analyzed plasma and urine samples of Fabry disease patients using LC-time of flight mass spectrometry and found that more than 20 isoforms of Gb3 were responsible for the disease severity and prognosis. Such detailed results were previously missed by targeted approaches [With recent characterizations of novel IEMs, researchers are beginning to recognize the drawbacks of targeted metabolite methods in newborn screening program, especially for diseases that are difficult to diagnose. Lysosomal storage diseases have a high degree of phenotypic and genetic variability and are characterized by multisystem effects . Fabry dproaches ,145,146.proaches .In contrast to targeted metabolomics approaches, untargeted metabolomics aims to measure all detectable analytes in a sample, including unidentified metabolites . Untargem/z) and MS/MS fragmentation spectra. Chromatographic retention times represent the degree of interaction of chemicals with the column adsorbent material, while accurate mass m/z and MS/MS spectral data can be compared to authentic chemical standards to verify claims on compound identification in IEM reports [Sample preparation for untargeted metabolomics aims at broad scale metabolome coverage without discriminating against specific classes of small metabolites while still removing proteins and other macromolecules . Typical reports ,159.For untargeted assays, high resolution mass spectrometers provide the best sensitivity, specificity and coverage. Quadrupole time-of-flight MS (QTOF MS or TTOF MS) and quadrupole Orbitrap MS (Q-Exactive) instruments have full scan modes and ability to fragment ions to provide a panoramic view of metabolites in biological samples. Triple quadrupole MS (QQQ MS) are most useful for targeted metabolite newborn screening, especially for absolute quantifications . Yet, onUntargeted metabolomics produces large and chemically diverse datasets. Such datasets comprise signals of both of known and unknown chemical structures and require careful interpretations. Statistical analyses of large datasets must account for false discovery rates to adjust for multiple testing. In addition to univariate analysis, data can also be processed by multivariate data analysis, including enrichment statistics . StatistMany clinical and biomedical studies have reported using global untargeted metabolomics ,165, TabUntargeted metabolomics widens the range of metabolites associated with IEMs and discovers new compounds that could be potential biomarkers. Using untargeted metabolomics in phenylketonuria of patient plasma led to the identification of two new biomarkers, glutamyl-glutamyl-phenylalanine, and phenylalanine-hexose . These nMetabolites can be divided per their physicochemical properties such as water-soluble (hydrophilic) and lipid-soluble (lipophilic) molecules. Hydrophilic molecules are the domain of primary metabolism such as sugars, amino acids, organic acids, or nucleotides. Screening lipophilic compounds has been termed lipidomics . It routAcylcarnitines are important classical targets for diagnosis of IEMs such as organic aciduria, mitochondrial and fatty acid oxidation defects . Few stuWith current advances in analytic methods, thousands of peaks and metabolites with good sensitivity are revealed using different platforms. The need to identify these peaks  is a grand challenge for untargeted metabolomics researchers . The firA number of studies compared results between targeted and untargeted techniques ,203. A rO-sulfate, vanillylmandelate, and 3-methoxytyramine sulfate that are not screened in targeted assays [Conversely, classic NBS using targeted metabolomic approaches did not show much improvement over the past years with respect to the magnitude of false positive results. Two distinct studies reported one false positive case per every 50–300 true positive cases in the USA ,205. Thed assays .If targeted metabolomics yields inconclusive diagnosis results, additional tests are required such as analysis of further targets, DNA analysis, or more invasive techniques such as individual enzyme assay in cells. Such analyses require additional costs and time to achieve the accurate diagnosis . These pScreening of IEM diseases using untargeted metabolomics gives a broad overview on aberrant pathways. A large screen of multiple metabolic pathways provides more diagnostic confidence, . For exaTargeted metabolomics in screening and diagnosis of IEMs give a narrow view of the diseases assayed. We need to consider untargeted metabolomics as a new tool to improve the scope of IEM disease categories associated with pathophysiology, early symptoms, therapy options and follow-up strategies . Untarge


Microparticles (MPs) play important roles in intercellular communication, including adhesion, signal transduction, cell activation, and apoptosis. They possess a wide spectrum of biological effects in the immune responses. MPs could be immunotolerogenic or immunogenic depending on the contents and composition. Elevated levels of MPs have been reported in many forms for rheumatic diseases. This review focuses on the immunopathogenic and therapeutic role of MPs in rheumatic diseases. Platele MPs/ml) . NumerouMPs represent distinct subcellular structures and serve a prominent role in homeostasis and intercellular communication including immune activation. They can transfer bioactive molecules from parental to target cells, allowing for regulation and amplification of several biological mechanisms such as activation, apoptosis, coagulation, and proliferation. They can be released actively at early stages of apoptosis and emerge preferentially from regions of the membrane containing lipid rafts where accessory proteins are sorted with specific function . StudiesMPs may reposition nuclear constituent in a form that may be more accessible to the immune system. Free RNA is sensitive to RNase digestion and free DNA is abundant in the blood but inactive. The incorporation of DNA and RNA into the lipid complexes of MPs increases the stability of the nucleic acids by protecting them from degradation. The bone marrow is a site of extensive cell turnover. The central tolerance to self-nucleic components might partially owe to the enhanced capacity of MPs to present DNA/RNA in the bone marrow during B cell development. Their special structure and membrane components may make MPs more effective in inducing central B-cell tolerance through clonal deletion by presenting autoantigens to immature B cells, along with signalling through non-TLR sensors (reviewed in ). The spA number of MPs generated from apoptotic and activated cells/platelets are phosphatidylserine (PS) positive . PS is tin vitro by apoptotic cells [Under certain circumstances , MPs may present auto antigens to APCs in an immunogenic way. Studies on patients with a wide variety of rheumatic diseases have demonstrated significant elevations in circulating MPs compared to control populations. MPs display diverse pro-inflammatory and pro-thrombotic activities that can influence the course of rheumatic diseases and may serve as potentially important mediators of disease pathogenesis. SLE patients have elevated type I interferon (IFN-I) and IFN-inducible gene expression (IFN signature) . Activatic cells ,12. In Sic cells . Under sMPs may also contribute to the pathogenesis of rheumatic diseases by the formation of immune complexes. As components of immune complexes, DNA and RNA are rendered more stable and are protected from DNases and RNases, respectively. DNA and RNA could potentially stimulate immune responses, especially when in the form of immune complexes. Lupus plasma contains MPs with IgG binding properties and the number of IgG-positive particles was correlated with anti-DNA levels . NielsenDuring activation and apoptosis, the contents of cells undergo extensive modification, degradation, and translocation. As shown in other studies, particles from various sources may differ in functional properties. MPs in rheumatic diseases may contain similar amounts of DNA/RNA compared to normal controls, but the modification of nucleic acids may make them potential immunogens and provoke cytokine production . SeveralIncreased amount of MPs can also occur in synovial fluid, where MP levels can far exceed those of blood level. MPs can act locally to drive synovitis and systemically to promote vascular disturbances. In the study of 19 patients, platelet-derived MPs from plasma of clinically active as well as inactive patients with RA were higher than those of healthy controls and levels of PMPs (platelet-derived MPs) also correlated with disease activity . Joint fvs. PS-, endothelial derived, monocyte derived, lymphocyte-derived, platelet-derived et al. Second, studies have shown a paradoxical relationship of MPs to disease activity, which may be at least partly influenced by methodological differences. Third, MPs may bind to target cells or be sequestered within tissue compartments, especially under chronic inflammatory conditions found in autoimmune diseases. Fourth, increased phospholipase activity in autoimmune diseases may lead to increased lysis of MPs. Lastly, discrepancies between the MP studies in SLE and other rheumatic diseases could be partly explained by differences in patient selection and small numbers of patients in some studies. It may also reflect the lack of standardization of MP analyses and our current insufficient understanding of the complexity of the biology of MPs, including their tissue turnover and their exact role in homeostasis. The mechanisms of shedding MPs remain poorly understood, although cytoskeleton rearrangement and calcium signalling are involved in the process. Discriminating between MPs that arise through apoptosis versus activation and characterization of the MP antigen composition is an important goal of future studies.Although the correlation of increased MP numbers with rheumatic disease suggests a role in immunopathogenesis, elucidating the mechanism is difficult. First, MPs are heterogeneous populations, and different studies focus on diverse subpopulations of MPs, e.g. PS+ Most research of MPs in autoimmune diseases focused on SLE and RA. Few studies investigated MP levels in other forms of autoimmune disease. Sellam et al. investigated the plasma levels of total, platelet, and leukocyte derived MPs by prothrombinase capture assay and flow cytometry in 43 primary Sjögren's syndrome (pSS) patients and 44 healthy controls . PatientMPs have received increased attention as universal markers of activation in eukaryotic cells. They carry markers of their parent cells, including those induced by activation, apoptosis, cell lysis, or oxidative stress. These properties permit detection of specific subpopulations. Increased neutrophil-, endothelial-, and platelet-derived MPs have been described in rheumatic diseases. MPs can provide important information about ongoing pathogenic processes that might be valuable clinically for diagnosing, accessing disease activity, and evaluating the effects of treatment.EMPs are produced by endothelial cells in response to a variety of triggers and may act as biomarkers for endothelial activation and damage. Active SLE is associated with increased endothelial damage. Damaged endothelial cells often release EMPs into the blood. EMPs were significantly elevated in ANCA-(anti-neutrophil cytoplasmic antibody) associated small vessel vasculitis (AAV) . ReductiMPs may serve as biomarkers to define subsets of RA patients and provide information for disease activity and prognosis. Ostergaard et al. identified 531 unique circulating proteins from SLE patients and showed highly statistically significant differences of 248 proteins compared to healthy controls. SLE MPs showed an unique protein profile that could be distinguished from RA and SSc and from diseases controls . MPs froHowever, a study from Van Eijk et al. reported that circulating MPs associated with complement activation were not affected despite intensive anti-inflammatory therapy in early RA patients . CrookstCurrent treatment options for autoimmune diseases often involve nonspecific immunosuppression that may result in enhanced patient susceptibility to opportunistic infections. The number of MP therapies is growing, recent advances in understanding the function of MPs in homeostasis and inflammation have reinforced the potential of MP therapies in controlling inflammation while restoring the immune tolerance in autoimmune disease. Micro-mediated drug delivery system, including polymeric particles, liposomes, and hydrogels are well established as methods for sustained release of therapeutics . A numbeGetts et al. coupled myelin proteolipid protein (PLP) epitope to carboxylated 500 nm polystyrene beads (PSB) and successfully induced tolerance with the prevention of experimental autoimmune encephalomyelitis (EAE) ,44. ImpoMicro particle systems that selectively deliver drugs to inflamed synovium have the potential to improve drug efficacy while leaving extra-synovial tissues unaffected. Liposomal use has been widely studied as a potential carrier system for drug delivery for RA. An early study using liposome-encapsulated clondronate, an anti-inflammatory therapy that reduces bone resorption, resulted in a halt in disease progression and a reduction in inflammation . SimilarMPs consist of a communication network in transducing intercellular signals and maintaining the system bio physiological homeostasis. They display important biological properties that can mediate disease pathogenesis and provide important information of on-going pathogenic process. Though a consensus protocol to measure MP does not yet exist, most studies showed markedly altered concentrations and distributions of MPs subpopulations in patients with rheumatic diseases. In addition, the impact of circulating MPs is not well understood. However, MPs mediated drug delivery system could prolong drug retention time, increase patient compliance as well as therapeutic effect. Sustained therapeutic drug concentrations can also be achieved. The use of MPs as immune tolerogenic and as therapeutic agent has only recently initiated and is rapidly expanding. The design and safety of MPs will have to be addressed to achieve desirable outcome. The use of antigen-conjugated MPs appears to be therapeutically promising in experimental animal models . An impr


Comprehensive reports of malaria in Menoreh Hills, Central Java, Indonesia, a unique district cross-boundaries area under three districts and two provinces have been published previously. However, no study was performed to identify the hotspots of malaria in this cross-boundaries area, Kaligesing and Bagelen Subdistricts in Purworejo, Jawa Tengah Province and Kokap Subdistrict in Kulon Progo, Yogyakarta Province, using a longitudinal spatial data.Monthly reports of malaria cases at primary health centres during 2005–2015 were collected and processed with ArcGIS and SaTScan to identify the malaria distribution at the village level. Malaria distribution was analysed using global spatial autocorrelation (Moran index) in ArcGIS. Cluster analysis was conducted using SaTScan purely spatial clustering and purely temporal clustering. Cluster characteristics resulted from three different approach were compared and analysed.During the last 11 years, 3812 malaria cases were reported and the number of high case incidence (HCI) villages were increased continuously. Malaria spatial distribution in Menoreh Hills was clustered spatially. Using three different approaches of time period ranges, consistent conclusion was found i.e. most likely clusters always occurred in the Purworejo district while the secondary clusters appeared later in the cross-boundaries districts.Spatiotemporal analysis of an 11 years surveillance data showed that hotspots of malaria cases in Menoreh Hills were continuously located in Purworejo district. The success of malaria elimination in the cross boundaries area of Menoreh Hills might be depended on the success in malaria case management and surveillance in this hotspot area. Although annual parasite incidence (API) of malaria in Java has decreased, some pocket areas still exist and remain difficult to control. One of them is Menoreh Hills that belongs under three districts  of two provinces (Jawa Tengah and Yogyakarta). Malaria is persistently endemic in this cross-boundaries area. In the last decade, malaria in Menoreh Hills was first documented as endemic and in fact, was the largest contributor of malaria cases in Java . MalariaThe Menoreh Hills area consists of mainly Kulon Progo and Purworejo Districts that belong to Yogyakarta and Jawa Tengah Provinces, respectively. In Kulon Progo, malaria incidences were found in Kokap, Girimulyo, Samigaluh and Kalibawang sub-districts located in Menoreh Hills. The Kokap region is an area with dense vegetation, where temperature and humidity do not fluctuate sharply, providing a suitable environment for mosquito breeding areas , 9. ThusPurworejo District was the largest contributor to malaria cases in Jawa Tengah Province with API number 0.49 per 1000 population in 2010, which increased to 1.31 per 1000 population in 2011. Malaria cases in Purworejo occurred around the hills, border areas, rivers, and paddy fields  showed a clustering of malaria cases in cross-border areas of Menoreh Hills. Long-term analysis showed clustering patterns, while two periods of analysis showed random patterns, especially during 2008 and 2012. Spatiotemporal analysis has been commonly used to identify clustering or non-clustering patterns and the locations e.g., a study in Hubei Province, China, which showed non-random purely spatial cluster in 2004–2011 and identified 11 regencies as malaria high risk locations , a studyArcGis analysis only analysed the coordinates of clustering distribution while SaTScan analysed both coordinates and its proximity time, thus autocorrelation using ArcGIS was unable to determine cluster location while SaTScan analysis could provide information about cluster radius, allowing comparison between disease risk inside and outside the cluster . Using tThe three approaches analysis using SaTScan showed that the main target of the malaria problem in cross-border areas of Menoreh Hills (the most likely cluster) was the Purworejo District, while secondary clusters might involve both districts. The areas of most likely clusters and secondary clusters were villages with HCI and MCI categories. There were villages that were always involved in primary clustering, that is villages in Purworejo District whereas villages in Kokap sub-district, Kulon Progo District were only secondary cluster villages. Based on the data, the main problem of malaria was concluded to be in the cross-border areas of Menoreh Hills, specifically in Kaligesing and Bagelen Sub-districts in Purworejo, resulting in a cross-border transmission towards Kokap Sub-district, Kulon Progo. SaTScan result for long-term (11 years) analysis identified two significant spatial clusters during 2005–2015, while two periods analysis identified two significant spatial clusters in both 2005–2010 and 2011–2015 periods. The annual analysis identified 19 significant spatial clusters. Analysis of spatiotemporal studies is often used to conduct an intervention particularly to target villages and cities , within Due to the retrospective approach, visiting all cases and collecting geo-coordinate of malaria cases at home was not possible. Therefore, in this study, cases were aggregated by the village. Satscan allows for spatial analysis by area (thematic map), even though the accuracy was less accurate than the individual coordinate. However, previous papers in Malaria Journal also performed spatiotemporal analysis using thematic map , 19. SatPurely temporal cluster analysis was used to show the duration of malaria risk. From the three approaches of analysis, it is shown that long term analysis produced longer duration of malaria clustering compared to annual analysis. The annual analysis is used to determine which month in a year is involved in the malaria cluster. No consistent pattern was found from the result of purely temporal cluster analysis in Menoreh Hills for 11 years. Cluster duration was different every year. However, malaria clustering generally occurred more often in July–January. This clustering pattern was similar to the well-documented monthly malaria case incidence pattern, which increased from July–January then gradually decreased. This situation leads to a supported recommendation to do malaria intervention before July in the adjacent areas.Aside from the receptive status area , 31–33, A participatory approach to address within-country cross-border Malaria: the case of Menoreh Hills in Java, Indonesia” has been done sponsored by a donor agency. The outcome of the study was strategic actions developed by the involved parties.The finding of this study has been presented to the provincial stakeholders, malaria programmers at Districts and sub-district level related and national authority. All three districts and the two provinces committed to building a partnership to eliminate cross-border malaria. To facilitate the stakeholders in translating this finding into the implementable strategic plan, an operational study “The use of long-term, intermediate and annual spatiotemporal analyses using ArcGIS and StatsCan in Menoreh Hills lead to the conclusion that primary clusters of malaria in the cross-boundaries areas persistently occurred in Purworejo District. Neglecting this hotspot could potentially delay the malaria elimination in the entire area. The cross-boundary partnership needs to be properly outfitted and reinforced to support malaria elimination in all related districts.


Raised blood pressure is a major problem in older adults. Using a random-effects model, a recent meta-analysis reported statistically significant reductions in both resting systolic blood pressure (SBP) and diastolic blood pressure (DBP) as a result of aerobic, resistance, and combined aerobic and resistance exercise in adults ≥65 years. To provide more objective information regarding this nonpharmacologic approach, this study applied more robust methods to this data. Meta-analysis of 41 randomized controlled trials representing 96 groups . Any location where a randomized controlled trial could be conducted. Adults ≥65 years. Trials ≥2 weeks that included aerobic, resistance, and/or combined aerobic and resistance exercise as the intervention. The recently developed inverse heterogeneity model (IVhet) was used to pool findings and the Doi plot was used to examine for small-study effects. Absolute and relative differences between the IVhet and random-effects model were also calculated. Data were reported using the mean difference (exercise minus control) with nonoverlapping 95% confidence intervals considered statistically significant. Statistically significant reductions in resting blood pressure were found as a result of aerobic exercise , SBP but not DBP for resistance training , and both SBP and DBP for combined aerobic and resistance training . Exclusive of changes in DBP congruent with resistance training, exercise  reduces resting SBP and DBP in older adults. These findings have practical implications when considering exercise for the prevention and treatment of raised blood pressure in older adults. Raised blood pressure, defined as a resting systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg, is a major risk factor for cardiovascular morbidity and mortality . In adulData for this brief communication were derived from a recent systematic review with meta-analysis focused on the effects of exercise  on any level of resting SBP and DBP in older adults, details of which have been described elsewhere . BrieflyThe effect sizes for the current study were derived using the original metric (mmHg) from previously reported exercise minus control group changes in resting SBP and DBP along with their 95% confidence intervals . I2 statistics [Changes in resting SBP and DBP according to type of intervention  were pooled using the recently developed IVhet model . Brieflyatistics , respectatistics . Small-satistics , an appratistics . This platistics . Values atistics . SensitiA summary of changes in resting SBP and DBP using the IVhet model is shown in When compared to meta-analytic results from the original study using the random-effects model , findingUsing a more robust model, the findings of the current brief report suggest that, with the exception of changes in DBP as a result of resistance training, exercise  reduces resting SBP and DBP in older adults. Importantly, these findings are generally smaller than those previously reported and include wider confidence intervals , resultsWhile the reductions are generally smaller than those previously reported, they not only were statistically significant, but also appear to be practically important at the population level, especially with respect to the 4.7 to 7.0 mmHg reductions observed for resting SBP. For example, at the population level, a reduction of 5 mmHg in resting SBP has been associated with a 9%, 14%, and 7% reduction in coronary heart disease, stroke, and all-cause mortality, respectively. FinallyThe major strength of this study was the use of the IVhet model to provide more robust information regarding the effects of aerobic, resistance, and combined aerobic and resistance exercise on resting SBP and DBP in older adults . An addiThe overall findings of this brief report suggest that, with the exception of changes in DBP as a result of resistance training, exercise  reduces resting SBP and DBP in older adults. These findings provide important information when considering exercise for the prevention and treatment of raised SBP and DBP in older adults.


Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's disease, is a rare, X-linked, late onset neuromuscular disorder. The disease is caused by a CAG trinucleotide repeat expansion in the first exon of the androgen receptor gene. It is characterized by slowly progressive lower motor neurons degeneration, primary myopathy and widespread multisystem involvement. Respiratory involvement is rare, and the condition is associated with a normal life expectancy. Despite a plethora of therapeutic studies in mouse models, no effective disease-modifying therapy has been licensed for clinical use to date. The development of sensitive monitoring markers for the particularly slowly progressing pathology of SBMA is urgently required to aid future clinical trials. A small number of outcome measures have been proposed recently, including promising biochemical markers, which show correlation with clinical disability and disease-stage and progression. Nevertheless, a paucity of SBMA-specific biomarker studies persists, delaying the development of monitoring markers for pharmaceutical trials. Collaborative efforts through international consortia and multicenter registries are likely to contribute to the characterization of the natural history of the condition, the establishment of disease-specific biomarker panels and ultimately contribute to the development of disease-modifying drugs. Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's disease, is a rare, X-linked, adult onset, neuromuscular disorder  characteDespite several promising therapeutic studies , no diseLimb weakness is present in 97% of SBMA cases. It usually appears at the of age of 35–40 and starts typically proximally in the lower limbs , 17. HowBulbar impairment occurs in about 10–30% of patients at the onset of the disease , but it The presence of a distal sensory neuropathy is a hallmark feature of the disease  which haComplex multi-organ involvement is a hallmark feature of SBMA. The core non-neurological features of SBMA include gynecomastia, testicular atrophy, reduced fertility and erectile dysfunction. Dysfunction of the AR protein leads to partial androgen insensitivity , manifesA biomarker is a parameter that can be measured accurately and reproducibly and used as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention . An ideal biomarker should have a predictive value and capture subtle changes over relatively short periods of time. Additional requirements to biomarkers include cost-effectiveness, non-invasiveness, and reproducibility , 39. It In recent years, an unprecedented interest has developed in the standardized assessment of neuromuscular performance in SBMA, evaluation of novel therapeutic strategies  and in tThe 6-minute-walk-test (6MWT) was proposed as an accurate marker of disease progression . It measThe Adult myopathy assessment tool (AMAT) is a performance-based instrument composed of functional and endurance subscales . AMAT prThe SBMA functional rating scale (SBMAFRS) SBMAFRS is a recently validated scale , 49, whiThe 1234-scale is another questionnaire-based scale based on the ALSFRS-r, which focuses on SBMA-associated motor disability . It inclManual muscle testing (MMT) is commonly used to describe muscle weakness in neuromuscular conditions even though it is highly evaluator-dependent . A numbeVideofluoroscopy (VF) is routinely used to evaluate dysphagia in a range of neurological conditions. In SBMA, VF can reliably detect the impairment of the oral phase of deglutition confirming large amount of oral barium residue . VF has Fiber endoscopic evaluation of swallowing has also been assessed as a candidate biomarker of bulbar impairment, but the diagnostic and prognostic value of the technique is yet to be validated .Tongue pressure measurements using an electronic device has been proposed as a biomarker of dysphagia in SBMA, and has been shown to be a low-cost and reliable way of detecting tongue weakness early in the course of the disease . An impoStandard electrophysiology measures are routinely used in the diagnostic work-up of SBMA, but they exhibit limited sensitivity to longitudinal changes . This isWhile quantitative muscle MRI would be an obvious candidate marker of disease progression in SBMA, there is a surprising scarcity of such studies. Existing studies have shown that muscle imaging can effectively detect muscle pathology in distal leg muscles which is less obvious on clinical assessment .Spinal cord imaging has seen unprecedented advances in recent years and has been applied successfully to other motor neuron diseases such as ALS –75, and Quantitative brain imaging studies demonstrated white matter alterations in the corticospinal tracts (CST), limbic system , 79, braIncreased serum CK levels have been reported by almost every SBMA study and support the hypothesis of a primary myopathy in SBMA , 88. EleTransaminases levels have also consistently been shown to be raised in SBMA including the pre-symptomatic phase of the disease , but theSerum creatinine level has also been proposed as a potential biomarker  despite Hormones levels and ASI (Androgen Sensitivity Index) have also been repeatedly proposed as markers of SBMA. Free testosterone levels correlate with muscle strength in one study (Proxies of metabolic syndrome and insulin resistance are considered closely associated with primary molecular disease mechanisms. The homeostasis model assessment of insulin resistance (HOMA-IR) index correlated significantly with motor function parameters in one study , but thine study  but it dne study . DHEAS lne study .Skin biopsies have been performed in some clinical trials to evaluate changes in the frequency of anti-polyQ antibody-positive cells after treatment . This inA recent study proposed adipose tissue quantification using whole-body MRI and reported significant subcutaneous fat accumulation in SBMA patients. This correlated both with CAG repeat lengths, disease duration and progression rates . These dInterest in SBMA biomarkers has grown steadily in recent years, fuelled both by accruing knowledge about pathogenesis and novel therapeutic strategies , 42. SBMFurthermore, the comparison of the specificity profile of candidate biomarkers seems essential to define their roles in clinical applications. The establishment of national and international SBMA registers is a clear priority which will be an invaluable resource for future SBMA research . As in oThe paper was drafted by GQ and PB and has been reviewed for intellectual content by VM-P and P-FP.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.