Many health benefits are claimed by individuals and facilities promoting sauna bathing; however the medical evidence to support these claims is not well established. This paper aims to systematically review recent research on the effects of repeated dry sauna interventions on human health. A systematic search was made of medical databases for studies reporting on the health effects of regular dry sauna bathing on humans from 2000 onwards. Risk of bias was assessed according to the Cochrane Collaboration guidelines.n < 40). Reported outcome measures were heterogeneous with most studies reporting beneficial health effects. Only one small study (n = 10) reported an adverse health outcome of disrupted male spermatogenesis, demonstrated to be reversible when ceasing sauna activity. Forty clinical studies involving a total of 3855 participants met the inclusion criteria. Only 13 studies were randomized controlled trials and most studies were small ( Regular dry sauna bathing has potential health benefits. More data of higher quality is needed on the frequency and extent of adverse side effects. Further study is also needed to determine the optimal frequency and duration of distinct types of sauna bathing for targeted health effects and the specific clinical populations who are most likely to benefit. Sauna bathing is a form of whole-body thermotherapy that has been used in various forms  for thousands of years in many parts of the world for hygiene, health, social, and spiritual purposes. Modern day sauna use includes traditional Finnish-style sauna, along with Turkish-style Hammam, Russian Banya, and other cultural variations, which can be distinguished by the style of construction, source of heating, and level of humidity. Traditional Finnish saunas are the most studied to date and generally involve short exposures 5−20 minutes) at temperatures of 80°C–100°C with dry air (relative humidity of 10% to 20%) interspersed with periods of increased humidity created by the throwing of water over heated rocks  of Alzheimer's disease, a 63% risk reduction  of sudden cardiac death, and a 40% risk reduction  of all-cause mortality  versus a control group [−0.01 L/s (−0.075–0.04 L/s)], p = 0.019, that received usual medical care. No other changes in spirometry parameters or 6-minute walk test distances were detected between the two groups  but this finding was not sustained through days 3, 5, and 7 of study. Fewer doses of cold and flu medications were taken by the treatment group on day 1 of assessment , compared to the control group ; upregulation of various heat-stress genes ; and upregulation of HSPs (heat shock proteins) and HSFs (heat shock factors) . However, all specified changes reverted back to normal 6 months after ceasing sauna activity and no significant changes in plasma sex hormones from baseline were detected directly after sauna or after 3 or 6 months [One longitudinal time-course study examined the effects of Finnish sauna activity on male sperm and fertility measures in 10 healthy men. After 3 months of repeated sauna (15-min saunas twice weekly), the investigators reported reduced sperm counts (93 ± 27.0 × 10n = 149) included symptomatic low blood pressure, hypovolemia, polyurination, weight loss, and, questionably, acute bleeding after a tooth extraction [n = 21) reported transient leg pain in one participant during a first infrared sauna session with the pain improving after completing a few sauna sessions and disappearing altogether by the end of the 6-week study [Of the 40 included studies, only eight reported any adverse symptoms from sauna bathing. Six studies recorded adverse effects graded as mild, meaning symptoms of complaint were noted which did not alter the study protocol or incur dropouts to the study. Mild heat discomfort was the major complaint , 61, 69.traction . Anotherek study . Pach etek study .n = 46) reported 2 patients dropping out of the treatment arm due to acute bronchitis and claustrophobia experienced in the sauna room [Two studies recorded moderate adverse effects, defined as symptom complaints that led to dropout of study participants or led to changes in study protocols. One study, involving fifteen women diagnosed with chronic fatigue syndrome/myalgic encephalomyelitis, reported enough heat intolerance in “most” of the participants such that the investigators reduced the temperature of the infrared sauna intervention from 60°C to 45°C to finish conducting the study . Anotheruna room . None ofThe findings of this review suggest frequent dry sauna bathing improves a variety of subjective and objective health parameters and that frequent Finnish sauna bathing is associated with improved outcomes such as reduced overall mortality and reduced incidence of cardiovascular events and dementia, at least in men , 39. Then = 149) reported improvements in all outcome measures except B-type natriuretic peptide (BNP) levels  classification) in the infrared sauna-treated congestive heart failure group compared to control over only 2 weeks of intervention [Cardiovascular disease has clearly been a focus for sauna researchers since 2000 despite Finnish-style sauna being considered by some in the past as a contraindication for patients with CHF and other cardiovascular diseases, most likely because of perceived intolerance to the high temperatures . Nearly rvention .While sauna bathing appears to show promise as a lifestyle intervention for cardiovascular disease, a majority of the cardiovascular disease-related sauna studies (16 of 19) were conducted by the same core Japanese research group and affiliates who employed “Waon therapy” , which iDespite differences in sauna types, temperature, frequency, and duration of interventions, the far-infrared sauna studies involving cardiovascular disease and congestive heart failure patients suggest favourable outcomes that reinforce earlier findings of interventional Finnish sauna studies and cardiovascular disease –79. Thisα2) [Several mechanisms of action have been proposed for the health effects of frequent sauna bathing. Exposure to heat increases cardiac output and reduces peripheral vascular resistance and induces other physiological changes in cardiovascular parameters such as decreased systolic and/or diastolic blood pressure , 60, 65,2α)  have bee2α) , 28, 81.The complexity of how sauna bathing may influence cardiovascular risk factors is suggested by the report of beneficial effects on total cholesterol and LDL (low density lipoprotein) cholesterol and conflicting results on HDL (high density lipoprotein) levels in healthy young men and women , 66, 67.α-tocopherol (antioxidant) levels and decreases in RBC catalase activity after an initial sauna were not maintained after 5 months of regular sauna [While there are likely to be many mechanisms of action influencing the physiological effects of sauna bathing, it has been suggested that sauna bathing may induce a general stress-adaptation response that leads to “hormetic adaptation” and the establishment of “sauna fitness,” possibly analogous to the hormetic adaptation responses of exercise. This is supported by newer, single-cell analysis methods that suggest sauna bathing increases generation of free radicals and reactive oxygenated species along with enhanced antioxidant activities via proposed nitric oxide- (NO-) dependent processes in blood  and uprear sauna , suggestImproved adaptation to stress with regular sauna bathing may be further enhanced by excretion of toxicants through heavy sweating. Many industrial toxicants including heavy metals, pesticides, and various petrochemicals may be excreted in sweat leading to an enhancement of metabolic pathways and processes that these toxic agents inhibit . Sweat-iIn addition to having profound physiological effects, sauna bathing is reported to have beneficial psychological effects that are reflected in the many reports of improved well-being, pain tolerance, and other self-assessed symptom-related scoring , 68, 69.In the medical literature at large, there are reports of severe adverse effects from saunas that include dry sauna-induced burns  and myocIn this review, adverse signs and symptoms of both Finnish-style and infrared sauna bathing were reported as mild to moderate heat discomfort and intolerance in 4 of the studies , 61, 69,To the best of our knowledge, this is the first systematic review to include both Finnish-style and infrared sauna studies. However, we did not include studies of steam sauna interventions and therefore may have overlooked some evidence of the effects of heat on health. Another limitation of this study is the inclusion of only English language, especially since sauna activity is frequent in non-English speaking countries. Furthermore, the quality of the reviewed studies was variable with many studies having small sample sizes, poorly described methodology, variable use of controls, differing types of sauna and sauna protocols, variable duration and frequency of sauna interventions, and inconsistent mention of cooling therapies or rehydration protocols along with heterogeneous outcome measures. The great heterogeneity of studies makes meaningful comparisons across studies difficult and provides insufficient evidence to recommend specific temperature, frequency, or duration of sauna bathing for any specific health outcome.In the months since this systematic review was conducted, a number of new research findings have been published, analyzing various subsets of the same Finnish prospective cohort of over 2000 men who regularly sauna-bathed, initially aged 42–60 years, followed over 20 years as part of the KIHD (Kuopio Ischemic Heart Disease) study, as detailed in two of the studies included in With the rise of single-cell analysis and “omics” platforms of analysis such as metabolomics and transcriptomics, especially applied to sweat, blood, urine, saliva, and other human biofluids, the ability to unravel the metabolic pathways at work during sauna or whole-body thermotherapy will certainly improve. Further study of these metabolic pathways might also help to elucidate the stress-related pathways of immune and inflammatory activity that may be involved in conditions such as chronic fatigue syndrome, chronic pain, rheumatoid arthritis, and ankylosing spondylitis.Studies examining heart rate variability (HRV) as an outcome assessment are increasing and further results may better inform the physiological models of what is thought to be happening with repeated sauna of either Finnish or infrared types. The concepts of hormetic stress and interrelating “sauna fitness” or habituation to the physiological effects of repeated sauna activity might have implications for preventive or therapeutic targets in the future. Conducting more studies of repeated sauna in healthy but nonathletic participants may further help to elucidate the similarities and differences in metabolic pathways between repeated sauna activity and regular exercise. Further studies are also needed to distinguish between the health effects of Finnish saunas, which often involve brief periods of increased humidity and dramatic cooling interventions, compared to the lower temperature infrared saunas that typically do not have such variations.Regular infrared and/or Finnish sauna bathing has the potential to provide many beneficial health effects, especially for those with cardiovascular-related and rheumatological disease, as well as athletes seeking improved exercise performance. The mechanisms for these effects may include increased bioavailability of NO (nitric oxide) to vascular endothelium, heat shock protein-mediated metabolic activation, immune and hormonal pathway alterations, enhanced excretions of toxicants through increased sweating, and other hormetic stress responses.Currently there is insufficient evidence to recommend specific types of sauna bathing for specific clinical conditions. While regular sauna bathing appears to be well-tolerated in the clinical setting with only minor and infrequent adverse effects reported, further data on the frequency and extent of adverse effects is required. Further studies are also required to explore the mechanisms by which sauna bathing exerts physiological, psychological, and metabolic effects, as well as to better define the benefits and risks of distinct types of saunas and the optimal frequency and duration of sauna bathing for beneficial health effects.


DMD gene that encodes for the protein dystrophin. Dystrophin has critical roles in myofiber stability and integrity by connecting the actin cytoskeleton to the extracellular matrix. Absence of dystrophin leads to myofiber fragility and contributes to skeletal muscle degeneration in DMD patients, however, accumulating evidence also indicate that muscle stem cells  are defective in dystrophic muscles, which leads to impaired muscle regeneration. Our recent work demonstrated that dystrophin is expressed in activated satellite cells, where it regulates the establishment of satellite cell polarity and asymmetric cell division. These findings indicate that dystrophin-deficient satellite cells have intrinsic dysfunctions that contribute to muscle wasting and progression of the disease. This discovery suggests that satellite cells could be targeted to treat DMD. Here we discuss how these new findings affect regenerative therapies for muscular dystrophies. Therapies targeting satellite cells hold great potential and could have long-term efficiency owing to the high self-renewal ability of these cells.Duchenne muscular dystrophy (DMD) is a genetic disease characterised by skeletal muscle degeneration and progressive muscle wasting, which is caused by loss-of-function mutations in the  In absence of dystrophin, myofibers are unstable and fragile, which causes progressive skeletal muscle degeneration.Duchenne muscular dystrophy (DMD) is an X-linked recessive disease that affects ~1 in 3,600 boys that is characterised by progressive debilitating muscle weakness resulting in gradual ambulatory disability, respiratory dysfunction and ultimately premature death in the second to third decade of life.4 In healthy condition, satellite cells possess a tremendous capacity to regenerate muscles with their ability to proliferate extensively, differentiate and self-renew.5 However, in dystrophic muscles, the proliferating capacity of satellite cell is reduced and the overall muscle regeneration is impaired.6–12 Satellite cell exhaustion have been suggested to contribute to the reduced regenerative ability.13 However, although a decrease in the number of satellite cells is observed during aging of mdx or DMD muscles, that number remains equal or higher in dystrophic muscles compared to aged-matched healthy muscles.15Skeletal muscle contains muscle stem cells, named satellite cells, that are the engine of muscle regeneration.Dag1, the transmembrane laminin-binding protein of the DGC, specifically in the myofibers induces a relatively mild degeneration phenotype compared with complete deletion of Dag1, i.e., in both the myofibers and the satellite cells.16 The authors suggested that Dag1 expression in satellite cells is essential to maintain their regenerative potential and, consequently, inadequate muscle repair caused by Dag1-deficiency in satellite cells is an important mechanism affecting the severity of muscular dystrophy.16 Similar results were obtained by analysing muscle regeneration of mice deficient in fukutin, a protein regulating Dag glycosylation and function.17 Conditional deletion of fukutin specifically in myofibers using muscle creatine kinase promoter also leads to a mild muscle phenotype compared with the severe muscle wasting observed when fukutin is specifically deleted in muscle precursor cells using the Myf5 promoter.17Pioneer work showed that deletion of 18 Dystrophin and Dag1 are expressed in a subset of activated satellite cells and are asymmetrically polarised prior to the first cell division. In activated satellite cells, dystrophin and Dag1 act as scaffolding proteins to which binds the cell polarity effector Mark2 . Dystrophin/Dag1-Mark2 interaction promotes the phosphorylation of the cell polarity regulator Pard3 leading to its asymmetric segregation at the opposite pole of the cell  is expressed in activated satellite cells where it regulates satellite cell fate and myogenesis.the cell . Asymmet20 These findings indicate that there is a cell-autonomous defect in DMD-mutant satellite cells, which contributes to the pathogenesis of DMD. Understanding the molecular mechanisms driving satellite cell dysfunctions in dystrophic muscles opens new therapeutic avenues for the treatment of DMD. Here we discuss the consequences of these findings on the therapeutic strategies targeting satellite cells as well as potential implications for other diseases.In absence of dystrophin, the cellular polarity of satellite cells is perturbed leading to the loss of asymmetric cell division and to an increase in the number of abnormal cell division. Lack of asymmetric cell division leads to a reduction in the number of myogenic progenitor cells and to impaired muscle regeneration .18,20 Th21 For instance, calmodulin was shown to bind to dystrophin and syntrophin to regulate the Akt pathway, while the growth factor receptor-bound protein 2 (Grb2) binds to β-Dag and activates the mitogen-activated protein kinases (MAPK) pathway.22 Microarray data comparing prospectively isolated satellite cells to differentiating myotubes showed that multiple components of the DGC such as dystrophin, syntrophin, Dag1 and sarcoglycan are expressed in satellite cells as well as in myofibers.18 Therefore, in addition to its role in the regulation of Mark2 activity, dystrophin and other members of the DGC could potentially activate various signalling cascades regulating satellite cell behaviour , was shown to bind to syntrophin and regulate nitric oxide (NO) production in myofibers.25 Notably, NO has been demonstrated to have a direct effect on satellite cell proliferation and self-renewal through a Vangl2-dependent noncanonical Wnt pathway.26 NO delivery was also shown to partially rescue satellite cell dysfunction in α-sarcoglycan-null dystrophic mice and to promote muscle regeneration.26 Altogether, these findings suggest that DGC components expressed by the satellite cells interact with multiple signalling effectors and could be involved in the regulation of satellite cell behaviour through additional mechanisms other than Mark2-dependent cell polarity establishment  delivery of CRISPR-Cas9 endonucleases coupled with paired guided RNAs can be used to remove the mutated exon 23 in mdx mice and restore Dmd expression in postnatal muscles.29–31 Depending on the delivery method , the level of dystrophin restoration is mild to moderate, but, interestingly, this technique was also shown to restore dystrophin expression into satellite cells, albeit to a lower level.29 AAV gene delivery is inefficient into quiescent satellite cells but its efficiency is increased into proliferating myoblasts.32 Further studies are needed to improve AAV transduction efficiency into satellite cells and, in this regard, the recent identification of the AAV receptor is a step forward in the development of new tools to improve AAV efficacy.33 Moreover, even with limited efficiency, it was showed that lentiviral-mediated gene delivery of micro-dystrophin into a small proportion of satellite cells can sustain dystrophin expression in myofibers for years.34 Therefore, satellite cells are a primary target to improve long-term efficacy of gene therapy due to their high self-renewal capacity.Many therapies aiming to restore dystrophin expression such as exon skipping or gene delivery are currently in the preclinical or early clinical phase.fukutin-deficient dystrophic mice, administration of an AAV vector containing the fukutin complementary DNA under the CMV promoter (leading to ubiquitous gene expression) leads to better muscle regeneration compared with the muscle creatine kinase promoter (myofiber specific), indicating that the restoration of fukutin expression in satellite cells improve their regenerative potential.17 However, restoring dystrophin expression in DMD is challenging considering the large size of the DMD gene. To circumvent this technical challenge, micro-dystrophin complementary DNA was created to fit into AAV vectors.35 Micro-dystrophin lacks most of the DMD rod domain and generates a truncated dystrophin protein that does not contain the Mark2-binding site (spectrin-like repeats R8–R9 corresponding to exon 26–30 of the DMD gene). These micro-dystrophin proteins are able to functionally restore myofibers stability, however, they cannot rescue the polarity defect in dystrophin-deficient satellite cells. Consistent with the important role of the DMD domain containing the Mark2-binding site (exon 26–30), analysis of the severity of Becker muscular dystrophy phenotype shows that mutations in the proximal rod domain of DMD (exon 10–32) causes a more severe muscle phenotype compared to mutations in the central rod domain (exon 33–45).37 Therefore, it may be beneficial for the development of micro-dystrophin vectors to include the Mark2 binding site in order to rescue satellite cell intrinsic defects and improving long-term efficacy.In addition to improve long-term engraftment, gene therapies designed to rescue the intrinsic defects of satellite cells could be an alternative approach. For instance, in 38 Recent case studies also showed that some DMD patients have the remarkable capacity to maintain their muscle function and walking ability despite the complete absence of dystrophin, however it remains unknown whether this effect is caused by overexpression in signalling pathways, such as Notch, that enhance satellite cell function.40 The Notch pathway is also involved in asymmetric cell division as demonstrated by the asymmetric inheritance of Notch ligand Delta-1 and the Notch interacting protein Numb in the committed daughter cell following asymmetric satellite cell division, but it remains to be determined whether Notch overexpression rescues the asymmetric polarity deficit observed in dystrophin-deficient satellite cells.41 Consistent with the positive role of Notch/Numb on satellite cell function, it was shown that inactivation of Numb in mdx mice promotes senescence of myogenic cells and exacerbates the dystrophic muscle phenotype.42 Therefore, the Notch pathway is an interesting target to improve satellite cell function, however, constitutive activation of the Notch pathway specifically in mdx satellite cells through Notch intracellular domain overexpression was shown to restore satellite cell self-renewal, but it did not translate into better regenerative potential.6 This discrepancy can be explained by the fact that overexpression of Notch pathway promotes self-renewal, but represses satellite cell myogenic progression, a process that is already insufficient in mdx satellite cells.18 Therefore, Notch is a potential therapeutic target to rescue satellite cell proliferation and self-renewal, however, it needs to be transiently activated and/or used in combination with other compounds promoting myogenic progression.The finding that dystrophin-deficient satellite cells are intrinsically defective suggest that therapies enhancing satellite cell myogenic capacity have the potential to improve muscle regeneration and ameliorate the quality of life of DMD patients . Consist44 Notably, the use of Stat3 inhibitors partially rescues satellite cell proliferation and muscle regeneration in dystrophic muscles.44 Another signalling pathway, the p38MAPK pathway was shown to be involved in the regulation of satellite cell asymmetric division.45 In aged muscles, p38MAPK signalling pathway is chronically upregulated in satellite cells and p38MAPK inhibitor was shown to partially restore the establishment of asymmetric cell polarity and improve self-renewal of satellite cells.46 p38MAPK is also aberrantly upregulated in regenerating mdx muscle and pharmacological inhibition of p38MAPK in dystrophic mice was shown to improve muscle phenotype.48 However, the exact effect of p38MAPK inhibitors on dystrophin-deficient satellite cells remains to be determined.Various other signalling pathways involved in cell polarity establishment were also shown to be dysregulated in pathological conditions and could be targeted to rescue satellite cell functions . For ins50 Noticeably, Wnt7a injection into the TA muscle of mdx mice was shown to increase the number of activated satellite cells (Pax7+/MyoD+), myofiber size and muscle force.51 Notably, a truncated variant containing only the C-terminal portion of Wnt7a was shown to retain its full biological activity and has enhanced dispersion compared with full-length Wnt7a, which is highly relevant for the development of therapeutic drugs.52 Another factor, the granulocyte-colony stimulating factor (G-CSF) receptor is asymmetrically segregated during asymmetric satellite cell division and treatment with G-CSF was shown to improve muscle regeneration in dystrophic mice.53 Altogether, these results suggest that alternative mechanisms exist to restore satellite cell function with the lack of dystrophin. However, whether the positive effects of Wnt7a or G-CSF on dystrophic muscle regeneration are mediated by the reestablishment of satellite cell polarity remained to be determined.The potential of biological molecules acting on satellite cell polarity should be evaluated as therapeutic approaches to restore dystrophin-deficient satellite cell function . For insVarious pharmaceutical components have the potential to improve the myogenic potential of dystrophin-deficient satellite cells by rescuing asymmetric division or by promoting alternate cell polarity establishment that rescues aberrant cell division. Many of these drugs are already approved for the treatment of different diseases, however, systemic delivery of these compounds is likely to have off-target effects and a cost-benefits analysis should be assessed. Moreover, some signalling pathways affected by these drugs are also important for myogenic progression and should be only transiently targeted. Nonetheless, these new findings indicate that satellite cells are an important therapeutic target that could improve muscle regeneration and preserve muscle function over time in DMD patients .54 Similar to what was observed in dystrophin-deficient satellite cells, these mutations could lead to intrinsic defects in satellite cell behavior. For instance, deletion of Dag1 in satellite cells was previously suggested to impair their regenerative potential.16 Conditional deletion of fukutin, which is important for Dag1 glycosylation and function, under the control of the Myf5 promoter was demonstrated to impair satellite cell proliferation, differentiation and skeletal muscle regeneration.55 Likewise, deletion of Large, which encodes for another glycosyltransferase that glycosylates Dag1, was shown to impair satellite cell proliferation in situ, however, when the satellite cells are removed from their niche and cultured in vitro the proliferation capacity is restored.56 A similar inconsistency between in situ and in vitro results was also observed in mdx satellite cells, where the decrease in satellite cell proliferation observed on isolated myofiber culture is not detectable when primary myoblasts are cultured out of their niche in a dish.18 These results suggests that the interaction of the DGC with its microenvironment is important in situ for cell polarity establishment and cell division, however, this interaction is not needed for cell division in vitro where there is a lack of polarity cues from the microenvironment. This hypothesis is supported by the fact that the expression of the DGC components is loss when primary myoblasts are cultured in vitro18 together with their ability to perform asymmetric cell division.57Muscular dystrophies (MDs) are a group of heterogeneous diseases (>30 different types of MDs) that are characterised by muscle weakness and dysfunction, but that are caused by different mutations in genes that encode for various proteins. Many mutations affect genes that encode for proteins interacting, directly or indirectly, with the DGC, such as Dag1, apha7-integrin (itga7), fukutin, Large, laminin and so on.18 Muscle deficient in itga7 shows classical symptoms muscular dystrophy.58 Double knockout of itga7 and dystrophin/Dag1 strongly increases the regeneration deficit suggesting that these two laminin-binding proteins have complementary roles.59 Similar to dystrophin-deficient satellite cells, absence of itga7 leads to a strong reduction in satellite cell proliferation and differentiation.60 Whether integrins regulates cell polarity establishment in satellite cell as it does in other stem cell types remained to be determined.61The DGC complex was also shown by proximity ligation assay to physically interact in myofibers and in satellite cells with itga7, another laminin-binding protein.62 Utrophin is widely expressed in various tissues, but its expression in healthy adult skeletal muscle is restricted at the neuromuscular and myotendinous junctions. In dystrophin-deficient muscles, utrophin expression is also detectable in the myofibers; and mice deficient for both dystrophin and utrophin display worst muscle phenotype, suggesting that these two proteins have complementary functions.64 Furthermore, overexpression of utrophin in mdx mice reduces the dystrophic phenotype.66 It is unknown whether utrophin is expressed in satellite cells, however, it would be interesting to determine since utrophin also possesses the binding site for Mark2.67 Notably, it was shown that satellite cells from mice double knockout for dystrophin and utrophin exhibit proliferation and differentiation defects stronger than in mdx mice, however, it is unclear whether this defect is cell autonomous.68Utrophin is a gene homologous to the dystrophin gene  with highly conserved sequence at the N-terminal and C-terminal ends, but with less similarities at the rod domain.Altogether, these findings indicate that interaction of satellite cells with their microenvironment provides critical feedbacks for the establishment of cell polarity and mutations in genes that encode for proteins of the DGC do not only lead to myofiber fragility, but also to intrinsic defects in satellite cells in a variety of MDs.DMD gene encodes for different isoforms of dystrophin generated by different promoters or by alternative splicing. These different dystrophin protein (Dp) isoforms are classified based on their size in kDa, i.e., Dp427, Dp260, Dp140, Dp116, Dp71 and Dp40. Some isoforms of dystrophin are ubiquitous such as Dp71, whereas others are tissue-specific such as Dp427 , Dp260 , Dp140  and Dp116 (Schwann cells). The point mutation in mdx mice is located at exon 23 leading to the lack of full-length dystrophin (Dp427) but it does not affect the other isoforms.69–71The 72 Impaired stem cell function could be partially responsible for other symptoms observed in DMD patients. For example, DMD patients and mdx mice have higher prevalence of cognitive impairment with disorganised central nervous system architecture and reduced number of neurons, a phenotype that is worsen by the absence of the Dp140 isoform.74 Brain-specific deletion of Dag1 using various Cre drivers leads to brain abnormality partially caused by reduced laminin-binding ability and neuronal migration errors.75 Furthermore, asymmetric division of neuroblasts is critical to generate the ganglion mother cell that will give rise to neurons and it could be hypothesised that dystrophin/DGC has a role to play in the regulation of neuroblast asymmetric cell division that is similar to what is observed in satellite cells. Therefore, intrinsic defects in polarity establishment and asymmetric cell division should be evaluated in stem cells from tissues other than skeletal muscles such as brain and retina.Expression of dystrophin in various tissues suggests that it could regulate polarity establishment and asymmetric division in stem cells from other tissues. For instance, expression of Dag1 was shown to be important to drive cell polarity establishment in epithelial cells.DMD gene have also been associated with higher incidence of various mesenchymal tumours, such as gastrointestinal stromal tumour, rhabdomyosarcoma and leiomyosarcoma.77 Dystrophin re-expression with mini-dystrophin vector reduced the invasiveness and the migration of myogenic sarcoma cells, which demonstrates that dystrophin has a tumour suppressive effect.76 Notably, mutations associated with myogenic cancers lead to deletion of the Dp427 dystrophin protein but preserved the Dp71 isoform, suggesting that Dp71 supports tumour formation and that expression of full-length dystrophin is required to preserve the tumour-suppressive effect.76 Consistent with these results, our findings indicate that absence of full-length dystrophin in satellite cells impairs asymmetric cell division,18 which has been related to formation of tumours in different tissues.78 Dystrophin-deficient satellite cells also display abnormal division patterns, which is consistent with previous studies showing that myoblasts isolated from mdx mice or dystrophic patients and cultured in vitro have higher proportion of cancer-like aberrations such as somatic aneuploidy and DNA damage response.77 Altogether, these results suggest that regulation of cell polarity by dystrophin is a common mechanism that is dysregulated both in DMD and in myogenic tumour.Mutations in the 79 A combined therapeutic approach that improves satellite cell function in DMD patients could transform this severely debilitating and lethal disease into a manageable chronic condition. Moreover, owing to their high self-renewal capacity, rescuing satellite cell dysfunction could also be beneficial for gene therapies and increase their long-term efficacy.The findings that dystrophin-deficient satellite cells are intrinsically defective in DMD patient indicate that they could be targeted by therapeutic compounds. Drugs aiming to restore polarity and proper mitotic division could be used as part of a global therapy for DMD, which could include other treatments such as anti-inflammatory and anti-fibrosis drugs.


Pinus kesiya forest is increasingly threatened by human disturbance in Yunnan Province. We observed that species richness had a positive impact on aboveground biomass across all forest vegetation layers, and this relationship was strongest in the herb layer. The asymptotic relationship between cumulative species number and aboveground biomass suggested that individual of Pinus kesiya trees with relatively large diameters contributed the majority of the aboveground biomass in the tall tree strata due to their strong competitive advantage over other tree species. Although aboveground biomass increased with stand age in the tall tree strata, climate factors and the soil nutrient regime affected the magnitude of the diversity-productivity relationship. Stand age had no significant effect on species richness and aboveground biomass in the forest understory. The effect of the positive diversity-productivity relationship of the tall trees on the shrub layer was negligible; the diversity-productivity relationship in the forest understory was significantly affected by the tall tree aboveground biomass. The tall trees have increased the strength of the positive diversity-productivity relationship in the forest understory.Both biodiversity and biomass are important variables in forest ecosystems, and the relationship between them is critical for ecosystem functioning and management. The primary  A clear forest biodiversity-biomass relationship has been shown in previous studies. Positive species diversity and biomass relationships are ubiquitous in most forest ecosystems around the world, and species loss in these ecosystems negatively impacts ecosystem functioning4. Most previous research has focused on the relationship between species diversity and productivity, not biomass, in forest ecosystems6. However, biomass is strongly correlated with productivity when the effects of stand age are taken into account8. An increasing number of studies have focused on the relationship between biodiversity and aboveground biomass, as well as the mechanisms resulting in variations in both species functional traits and environmental conditions in tropical, temperate and boreal forest ecosystems9.The relationship between biological diversity and ecosystem functioning has stimulated research on the relationship between terrestrial plant species richness and temporal variation in biomass production during the past two decades11. Accordingly, studies on the relationships between species richness and biomass across forest strata have been carried out in boreal and subtropical forests14. Reich et al. confirmed that a positive species richness and aboveground biomass relationship across forest strata was found, and the understory species richness is mediated by the indirect effects of the dominant producers on resource availability and heterogeneity in a boreal forest13. Further evidence from Canada’s National Forest Inventory studies shows that positive relationships between species richness and aboveground biomass in understory vegetation may not affect overstory tree species12. Simultaneously, similar findings have also indicated that diversity-productivity relationships in the forest understory were not associated or were negatively associated with the aboveground biomass of the overstory layer7. In contrast, the species diversity of the overstory layer significantly increased the species diversity of the understory layer in a subtropical forest, while the effect of the aboveground biomass of the overstory on the diversity and aboveground biomass of the understory was negligible14. The occurrence of shade species from the canopy trees in the understory layer is a common phenomenon, and the overstory-understory species richness relationship suggests that different functional groups have specific growth strategies11. There is no doubt that the size of the overstory, the interaction between species richness and aboveground biomass in this layer, and its indirect effects on resource availability and heterogeneity mediate the relationship between species richness and aboveground biomass in the understory layers13.Previous studies have shown that tree species in the overstory play important roles in mediating the effects of environmental conditions and disturbance on understory species richness15. A previous study demonstrated that the majority of productivity of the forest ecosystem occurred in the overstory, while understory plants have relatively low contributions to the total biomass stock16. The relationship between species richness and biomass is more complex in natural ecosystems because of species dominance and composition17. The selection effect suggests that species with the most productive traits will have greater opportunities to dominate the biomass of species-rich polycultures through interspecific competition, while the complementarity effect suggests that species can capture resources to maintain diversity as a result of niche partitioning or interspecific interactions when they occur together in plant communities18.The driving mechanisms of comprehensive causal relationships could be explained by both selection and complementarity effects7, biotic and abiotic factors affect the magnitude and quality of such relationships, and these effects should be considered, especially by increasing the range of variables included in multivariate studies, such as competition intensity, resource heterogeneity and population dynamics13. Stand age is closely associated with the species richness-biomass relationship9. Simultaneously, the consideration of environmental conditions may be crucial for understanding the relationship between species richness and biomass along with two drivers4: First, higher resource availability supports more biomass accumulation under more favourable climate and soil nutrient conditions15. Second, the diversity-productivity relationship shows a unimodal association from harsh to favourable climate and soil nutrient conditions4. The extents of the influences of climate and local site conditions on plant species diversity differ significantly among vegetation strata19. Climate, soil nutrient conditions and stand age are recognized as directly or indirectly affecting the species richness-aboveground biomass relationship, but were rarely explicitly considered because of the instability in environmental conditions12.Although positive species diversity and aboveground biomass relationships are mostly found across forest strata that exhibit higher species richness and many trophic levelsPinus kesiya forests have their largest distribution throughout northern India, the Philippines, Myanmar, Vietnam, Laos, Thailand and Yunnan Province, China21. In Yunnan Province, China, primary Pinus kesiya forests play an important role in local forest ecosystem functioning, including carbon sequestration and biological conservation, in addition to timber production and resin tapping. Deforestation has decreased the area of forest over recent decades. Degradation of the remaining stands and climate change have affected the regeneration, growth and distribution of primary Pinus kesiya forests, increasing species loss and threatening stable ecosystem functioning22. Pinus kesiya usually invades monsoon evergreen broadleaf forest areas after the broadleaf species have been cleared23, causing the vertical structure of the community to experience obvious species stratification. There is a distinct difference between a primary Pinus kesiya forest and a boreal or subtropical evergreen broadleaf forest, as that almost all Pinus kesiya individuals are distributed in the overstory layer in the primary Pinus kesiya forest, while most of the broadleaf species occupy the understory layer. There is little information regarding the relationships between the species richness and aboveground biomass across forest vegetation strata in primary Pinus kesiya forests.Primary Pinus kesiya forest. We also analysed the influences of the tall tree layer, stand age, soil nutrient regime and climate factors as mechanisms driving the relationship between species richness and aboveground biomass across forest vegetation strata based on existing theoretical frameworks7. Based on the existing study of Zhang et al.12 and our field data, we summarized theoretical frameworks to test the following paths: (1) the relationships between species richness and aboveground biomass across forest vegetation strata, including the tall tree, short tree, shrub, liana and herb layers; (2) the influences of the soil nutrient regime, stand age and climate factors on the species richness across forest vegetation strata and the effect of stand age on aboveground biomass; (3) the influences of species richness and aboveground biomass in the tall tree strata on the paths of (1) and (2) in the short tree, shrub, liana and herb layers; and (4) the influence of species richness in the short tree strata on aboveground biomass across all layers. The aim of this study was to disentangle the potential mechanisms that mediate the relationship between species richness and aboveground biomass across forest strata and to provide a reference for the management of natural subtropical coniferous forests under global climate change.In this study, we used a multivariate model to evaluate path hypotheses to assess the relationship between the species richness and aboveground biomass of the tall tree, short tree, shrub, herb and liana layers in a primary Pinus kesiya forest. The cumulative proportion of aboveground biomass showed that there was a strong asymptotic relationship with the cumulative species number   .The structural equation model linking the aboveground biomass and species richness of the tall tree layer showed a good fit , accompanied by variations in the tall tree species richness and stand age of up to 57.3%. Our results suggested that the tall tree species richness and stand age directly affected the aboveground biomass of the tall tree strata  and a dry season (from November to April). The annual mean temperature ranges from 17 °C to 18.5 °C. The annual precipitation ranges from 1100 mm to 1550 mm, with favourable hydrothermal conditions, and more than 80% of the annual precipitation falls during the rainy season23.The study was conducted in primary Pinus kesiya and Schima wallichii are the dominant canopy tree species in all the sampled stands. The subcanopy tree species in the forest are Castanopsis hystrix, Castanopsis echidnocarpa, Castanopsis calathiformis, Castanopsis fleuryi, Lithocarpus truncatus, Lithocarpus fenestratus, Machilus rufipes, Betula alnoides, Rhus chinensis, and Alnus cremastogyne. The shrub layer is dominated by Symplocos paniculata, Breynia fruticosa, Ficus hirta, Melastoma affine, Glochidion eriocarpum, and Inula cappa. The herb layer is dominated by Eupatorium adenophorum, Zingiber densissimum, Hedychium flavum, Imperata cylindrica var. major, Woodwardia japonica, Dicranopteris pedata, Scleria levis, and Dianella ensifolia. Mucuna macrocarpa, Craspedolobium schochii, Embelia ribes, Smilax spp., Gnetum montanum, and Celastrus monospermus are the dominant liana species in the primary Pinus keisya forest.2) based on the requirement in Yunnan vegetation23. All plots were measured between 2012 and 2014  was determined as the mean age of the three largest Pinus kesiya individuals. We fitted an age-DBH equation for Pinus kesiya using the formula y = 3.326DBH0.733 .The data were from 170 field sampling plots located in nine counties, including Simao, Jinghong, Menghai, Jinggu, Zhenyuan, Jingdong, Yunxian, Changning and Lianghe in southern and southwestern Yunnan Province, China. The plots ranged from 22°11′ to 24°38′ N and from 22°11′ to 24°38′ E. The plots were 20 m × 20 m (400 m−1) across forest vegetation strata was estimated as the sum of the stems, branches, and leaves using allometric equations including trees with DBH ≥ 5 cm, shrubs and small trees with DBH < 5 cm, and lianas (DBH ≥ 2 cm)  Schima wallichii , (3) Castanopsis hystrix and Fagaceae species with similar functional traits28, (4) Betula alnoides29, (5) Rhus chinensis30, (6) Alnus cremastogyne31, (7) other mixed tree species for less frequently occurring species32, (8) shrubs and small trees33, and (9) lianas34. We used a destructive harvesting method to measure the aboveground biomass of the herb layer, including herbs, shrubs and tree seedlings (DBH ≤ 1 cm)20.Aboveground biomass , growing degree days  and climate moisture index  were used to account for the effect of environmental conditions and stand growth on aboveground biomass. We used the N:P ratio as a measure of the soil nutrient regime to represent local site conditions. Climate data were obtained from the Climate AP software and have been revised for use in the Asia-Pacific region17. We used a structural equation model (SEM) in our analyses to determine the effects of response variables, which were related to the tall tree and environmental conditions on the relationships between the species richness and aboveground biomass of different short tree layers.We regressed the species richness on aboveground biomass across forest vegetation strata to assess the relationships between these two variables using ordinary least squares (OLS) regressions with a cubic smoothing spline. The cumulative value of aboveground biomass was plotted against the cumulative species number  according to the magnitude of the aboveground biomass of each species in all plots, which could indicate the relative contribution of the species to the aboveground biomass36. We explicitly defined two main paths: (1) the relationships between species richness and aboveground biomass within each layer, and (2) the effects of covariates, including GDD, SNR, CMI, SA and the tall tree layer, on the short tree layer. As recommended by Zhang et al.7, the GDD, SNR, SA, CMI and aboveground biomass across forest vegetation strata were transformed using the natural log scale. The standardized root mean square residual (SRMR) and goodness-of-fit index (GFI) were used to evaluate the goodness of fit of the SEMs with GFI values >0.95 and SRMR values <0.08. We report the standardized coefficient for each path from each component model. The total effects, as well as the direct and indirect standardized effects, were also calculated to strengthen the interpretation of our SEM results. The SEM analysis was implemented using the “lavaan” package in R 3.3.237 (R development Core Team 2016).In addition, we further fitted an SEM to interpret the casual effects and relative importance of stand age, soil nutrient regime, growing degree days, climate moisture index, species richness, and aboveground biomass of the tall tree layer on the species richness and aboveground biomass of the short tree, shrub, herb and liana layers Table . The SEMSupplementary Information


Ubiquitination is a highly dynamic process and can be reverted by deubiquitinases (DUBs), which cleave the isopeptide bond that links ubiquitin to the target proteins . DUBs caOncotarget, Fraile and colleagues (2016)[et al. identify USP54 as overexpressed in intestinal cells with stem cell expression profiles (high levels of Lgr5 and EphB2 receptors). Accordingly, silencing of USP54 in colorectal cancer (CRC) cells led to a significant decrease in cell proliferation, colony formation capacity, invasiveness, and tumorigenicity in an in vivo xenograft model, indicating that USP54 could promote CRC, potentially by controlling stem cell properties in intestinal cells. To further explore the role of USP54 in CRC, the authors generated USP54-hypomorphic mice, which were then subjected to a CRC-induction protocol. Mice with negligible levels of USP54 showed a decrease in adenocarcinoma formation and in overall colon inflammation when compared to the normal counterparts. Furthermore, the proportion of infiltrating adenocarcinomas and the number of infiltrating tumors per mouse was significantly reduced in USP54-deficient mice, confirming the role of USP54 in CRC initiation and progression. Interestingly, high expression levels of USP54 were significantly associated with poor survival in a cohort of 269 patients with intestinal cancer, further demonstrating the protumorigenic role of USP54.In the current issue of es (2016) unveil tUSP54 is deleted in malignant peripheral nerve sheath tumors, hinting at a putative tumor suppressor function in this tumor type. In contrast, in neuroendocrine prostate cancer USP54 undergoes genomic amplification, suggesting a potential oncogenic role. In addition, mutations in this gene are found in colorectal, pancreatic, and endometrial carcinomas as well as in melanoma. These findings prompted the authors to probe the function of USP54 in an in vivo melanoma model. Injection of murine melanoma B16F10 cells, with control or USP54-directed short-hairpin RNAs, into recipient mice led to a significant decrease in the number of metastases to the lung, corroborating the pro-metastatic role of USP54 in melanoma progression.To investigate the clinical relevance of USP54 beyond CRC, the authors explored publicly available cancer genome databases (cBioPortal, [While this study clearly shows the critical role of USP54 in tumorigenesis, several intriguing questions remain unanswered. What is the mechanism behind the oncogenic activity of USP54? What determines the pro-tumorigenic or putative anti-tumorigenic function of USP54 in different malignancies? One possibility is that the role of USP54 in cancer could be linked to the capacity of DUBs to interact with diverse substrates in different cell types . The ideFinally, this study has important therapeutic implications, as USP54 could potentially be a good target for CRC or melanoma. However, it might be difficult to develop effective and specific USP54 inhibitors. One of the challenges is that DUBs from the USP family present a high degree of structural conservation in the active sites of their catalytic domains, which could hinder the specificity of the inhibitors . The use


Streptomyces have been explored extensively over the past decades as these filamentous bacteria are highly efficient in producing bioactive compounds with human health benefits. Being ubiquitous in nature, streptomycetes can be found in both terrestrial and marine environments. Previously, two Streptomyces strains (MUSC 137T and MUM 256) isolated from mangrove sediments in Peninsular Malaysia demonstrated potent antioxidant and cytotoxic activities against several human cancer cell lines on bioactivity screening. These results illustrate the importance of streptomycetes from underexplored regions aside from the terrestrial ecosystem. Here we provide the insights and significance of Streptomyces species in the search of anticancer and/or chemopreventive agents and highlight the impact of next generation sequencing on drug discovery from the Streptomyces arsenal.Human life expectancy is rapidly increasing with an associated increasing burden of chronic diseases, such as neurodegenerative diseases and cancer. However, there is limited progress in finding effective treatment for these conditions. For this reason, members of the genus  Among primates, humans have the longest life expectancy. With the advancement in medical technologies and improved hygiene conditions, the global life expectancy of a human at birth has increased from 66.4 years in 2000 to 71.4 years in 2015 . In reality, S. antibioticus was first discovered from soil in 1940 by Waksman and Woodruff  was discovered from a soil-derived Streptomyces peucetius in 1957 , and Ectothiorhodospiraceae (a family within Gammaproteobacteria). Previous studies have also claimed that the high abundance of primary producers in mangrove sediments results in the sediment being enriched with high nutrient levels, supporting growth and encouraging species richness. On top of that, numerous studies have also suggested that the stress on the organisms due to the rapid fluctuations in environmental factors in the mangrove environment, such as temperature, salinity and oxygen availability could also promote emergence of novel species by encouraging metabolic pathway adaptations and production of valuable metabolites  mycelium which grow radially with frequent branching and subsequently form a young colony , but also by the addition of phosphate in fermentation medium. Phosphate concentration has profound effects on clavulanic acid production with alterations showing a biphasic response, in which low or high concentration of phosphate in media produced low clavulanic acid yield  to complicated instrumentations with separation columns like in high performance liquid chromatography (HPLC) -, and two cyclic dipeptides: pyrrolopyrazine-1,4-dione, hexahydro- and pyrrolopyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-. Phenolic compounds have been widely recognized as potent chemopreventive agents, acting as antioxidants and modulators of intracellular signaling processes involved in initiation/promotion of cancer  bottom-up strategy that focuses on seeking compounds/agents that affect molecules that might be critical to disease; and (b) top-down strategy which emphasizes searching for compounds/agents that affect cellular processes that might be critical to disease  at DNA synthesis stage by inhibiting DNA and RNA synthesis by intercalating between base pairs of DNA/RNA strand and/or preventing relaxing of supercoiled DNA ; (ii) at protein level via generation of reactive free radicals that damage cellular components; or (iii) at both levels through induction of histone eviction from chromatin  which may eventually cause DNA double strand breaks , some cancers are associated with environment and lifestyle behaviors, such as smoking, obesity and lack of exercise. The current therapy of cancer usually involves systematic therapy whereby drugs are administered intravenously or orally and/or “local” therapy that may include surgery and radiation therapy. Many currently available anticancer drugs exhibit their anticancer activity by inducing apoptosis of cancer cells Hickman, . ProgramT and MUM 256, there were several interesting cyclic dipeptides that have been detected in other Streptomyces species. Previously, our group has detected another novel Streptomyces species, MUSC 136T extract containing cyclic peptides that may induce p53 associated apoptotic cell death pathway through the downregulation of intracellular glutathione (GSH) content in colon cancer cells - have been featured numerous times in the literature, notably for its growth inhibitory effects on MCF-7, HT-29 and HeLa cancer cell lines  has opened a new window for researchers to tap into the genomic potential of bacteria including the drug prolific streptomycetes.The importance of isolating, and subsequently mass producing any compound of interest can be illustrated using penicillin as the classic example. Alexander Fleming was the first to observe the “killing” power of the mold, KEY CONCEPT 3Streptomyces sp., particularly in the identification and expression of “silent” or cryptic biosynthetic gene clusters.Along with media optimization, advances in next generation sequencing greatly benefit the drug discovery process in Streptomyces coelicolor A3(2) was available in 2002 —consisting of 8,667,507 bp—is more than two times larger than the genome size of Bacillus subtilis  and thus it was published as the largest completely sequenced bacterial genome  gene cluster in Streptomyces lividans. As previous work has failed to induce cluster expression in heterologous system, the team decided to employ a knock-in method on the native S. roseosporus host. Through this technique, the team successfully activated the expression PTM biosynthetic genes and stimulated production of two PTMs. The continual growth in genome sequencing and mining tools, such as the availability of genome editing technique like CRISPR-Cas9 has truly opened new windows in microbial drug discovery, especially for the identification of novel compounds from Streptomyces sp.The first complete genome of model actinomycete, g et al.  has rece4-105 unique species (which is frequently observed in soil samples) , polyketide synthase (PKS), isoprene, sugar, shikimic acid, alkaloid, ribosomal peptide), in which most of these classes show common, highly similar conserved biosynthetic domains  that have demonstrated potential to kill cancer cells. Further investigations to elucidate the action mechanisms of these bioactive compounds may hold promise as chemopreventive or chemotherapeutic agents; possibly with the application of advanced genomics and bioinformatics technologies which have revolutionized microbial natural products discovery. With the fast development of sequencing technologies and genome editing techniques, these streptomycetes hold tremendous promise for the development of new anticancer and/or chemopreventive drugs.In summary, The literature review, data analysis and manuscript writing were performed by HS, LT, and JL, while KC, AD, SS, PP, NA, TK, BG, and LL provided vital guidance, technical support and insight. LL and BG founded the research project.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.


Three-dimensional gait analysis (3DGA) is commonly used to assess the effect of orthopedic single-event multilevel surgery (SEMLS) in children with spastic cerebral palsy (CP).The purpose of this systematic review is to provide an overview of different orthopedic SEMLS interventions and their effects on 3DGA parameters in children with spastic CP.A comprehensive literature search within six databases revealed 648 records, from which 89 articles were selected for the full-text review and 24 articles (50 studies) included for systematic review. The Oxford Centre for Evidence-Based Medicine Scale and the Methodological Index for Non-Randomized Studies (MINORS) were used to appraise and determine the quality of the studies.Except for one level II study, all studies were graded as level III according to the Oxford Centre for Evidence-Based Medicine Scale. The MINORS score for comparative studies (n = 6) was on average 15.7/24, while non-comparative studies (n = 18) scored on average 9.8/16. Nineteen kinematic and temporal-distance gait parameters were selected, and a majority of studies reported improvements after SEMLS interventions. The largest improvements were seen in knee range of motion, knee flexion at initial contact and minimal knee flexion in stance phase, ankle dorsiflexion at initial contact, maximum dorsiflexion in stance and in swing phase, hip rotation and foot progression angles. However, changes in 3DGA parameters varied based on the focus of the SEMLS intervention.The current article provides a novel overview of a variety of SEMLS interventions within different SEMLS focus areas and the post-operative changes in 3DGA parameters. This overview will assist clinicians and researchers as a potential theoretical framework to further improve SEMLS techniques within different SEMLS focus groups. In addition, it can also be used as a tool to enhance communication with parents, although the results of the studies can’t be generalised and a holistic approach is needed when considering SEMLS in a child with spastic CP. Gait abnormalities are common in children with cerebral palsy (CP) and are generally caused by an abnormal muscle tone, loss of motor control and balance problems due to a non-progressive lesion of the developing brain . FollowiThe assessment and treatment of gait abnormalities in children with CP are challenging. Several complementary interventions are often used to develop the most optimal and energy efficient gait pattern in these children. These interventions range from physical and occupational therapy, neurosurgical and pharmacological interventions to reduce hypertonia and orthopedic interventions aiming to restore anatomical structures and musculoskeletal conditions .As a multi-level approach has proven to be the most effective treatment option, it is not surprising that within orthopedics, single-event multilevel orthopedic surgery (SEMLS) is the preferred method to treat musculoskeletal deformities in children with CP ,4. SEMLSRecently, McGinley et al.  conducteProviding an overview of which changes in gait parameters can be expected after SEMLS, is of great value for clinicians and researchers. Unfortunately, and due to the variety of SEMLS interventions with different focus areas and specific CP populations, it is impossible to perform a meta-analyses of the literature. However, a systematic review of SEMLS studies and their effect on 3DGA parameters in children with CP will result in an overview of gait changes that have been reported after different types of SEMLS techniques. A systematic review of these SEMLS outcome studies can provide valuable insight for clinicians, can assist in preoperative discussion with parents and form a platform to potentially further improve SEMLS techniques Therefore the aim of this study is to provide a systematic overview of which soft tissue and bony interventions have been performed as part of SEMLS intervention in children with CP, with a special focus on the post-operative changes in 3DGA kinematic and temporal-distance parameters.A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . A comprArticles were evaluated for eligibility based on the title and abstract. After the initial identification and screening, full-text articles were reviewed and independently assessed against the inclusion/exclusion criteria by two reviewers (NL and MB). Articles were selected when fulfilling the following criteria: 1) Study the effects of SEMLS on gait assessed by 3DGA; 2) Cohort consisted of ambulant children and adolescents diagnosed with spastic CP; 3) Only SEMLS interventions performed (e.g. not combined with botulinum toxin injections 6 months prior to surgery); 4) Detailed description of orthopedic procedures; 5) Reported number of operated sides; 6) 3DGA conducted before and after SEMLS interventions; 7) Mean follow-up time of at least 12 months; and 8) Include at least three temporal-distance and/or kinematic parameters.Two reviewers (NL and MB) graded the level of evidence of the selected articles by using the Oxford Centre for Evidence-Based Medicine Scale  and compTwo reviewers (NL and RL) extracted the demographic and SEMLS background information, as well as the pre- and post-operative temporal-distance and kinematic parameter data. Temporal-distance and kinematic gait parameters were included if they were used in at least 7 different studies. In addition, reference norm values from typical developing children were extracted where possible. Significant changes were defined as ‘improved’, a significant change getting closer to the reference norm values, or as ‘deteriorated’, a significant change moving away from the reference norm values. An experienced pediatric orthopedic surgeon (JDT) reviewed the selected articles with regards to data on surgical interventions performed, while a fourth reviewer (MB) verified all extracted information.The electronic databases search produced 648 initial references of which 24 articles met the inclusion criteria  8–31]. . 31]. ThAll the articles were graded as OXFORD  level IIThe sample size of the different study cohorts ranged from 8 to 45 children (14–82 operated sides) with a mean age between 8 and 13 years. Except for one article , all autIn total 19 gait parameters were identified within the systematic review, namely three temporal-distance parameters , thirteeSeventeen articles (32 studies) reported cadence, stride length and walking velocity before and after SEMLS intervention . In addiPelvic range of motion (ROM) and mean pelvic tilt were the most commonly used pelvic parameters as reported in 12 articles (24 studies) . Four arHip ROM, minimum hip flexion in stance and, maximum hip flexion in swing phase were reported in 11 articles 19 studies) . Four st studies Knee ROM, knee flexion at initial contact (IC), minimum knee flexion in stance and maximum knee flexion were reported in 19 articles (40 studies) . Six artAnkle dorsiflexion at IC, maximum dorsiflexion in stance and maximum dorsiflexion in the swing phase were reported in 15 articles (29 studies) . Five arKinematics in the transverse plane, which included mean pelvic rotation, mean hip rotation and foot progression angles, were reported in 8 articles 14 studies) . Four ar studies This is the first systematic review that provides an overview of the different SEMLS interventions within different SEMLS focus areas in children with spastic CP and, their effects on 3DGA gait parameters. For this 510 articles were screened of which 24 articles met the strict inclusion criteria for this systematic review , gait posture and muscle control, general SEMLS interventions included frequently psoas recessions –12, mediFollow-up studies showed good results with these techniques resulting in improved pelvic ,12, hip The main aims of lever-arm dysfunction SEMLS interventions are to improve gait patterns through a more neutral pelvic and hip alignment and external foot progression. Since lever-arm dysfunction is related to torsional deformities , the maiIn addition to the transverse plane, three of the five lever-arm dysfunction studies –16, alsoThree research groups –20 studiBernthal et al.  and SteiFive studies –25 perfoThe aim of Rodda et al.’s  study waStiff-knee gait, which is characterized by reduced knee ROM in the sagittal plane were specifically targeted with a SEMLS intervention by Cruz et al.  and PresDreher et al. ,24 studiAdolfsen et al.  studied Seven studies –31 focusThe studies by Svehlík et al. , Dreher Metaxiotis et al.  and DrehThompson et al.  studied Although this systematic review provides a good overview of which gait changes can be expected after a certain type of SEMLS intervention in children with spastic CP, the data need to be interpreted within the available literature and its detail. This review is based on 24 articles with limited demographic information and varying heterogeneity within the study cohorts. This limitation did not allow stratifying for age or functional level of the subjects, what could have provided interesting information. In addition, the wide variety of surgical techniques and range in patient populations made it impossible to conduct a meta-analysis with drawing clear overall conclusions. Although this is admirable in the future, the differences in surgical preference and approach by different surgeons around the world might prevent this. It also needs to be mentioned that only one study could be classified as an OXFORD level II , while mTherefore, this systematic review should be seen as an overview paper providing a framework for clinical discussions and research, and a summary of results that can be used by clinicians to enhance the communication with parents when considering SEMLS in their child. However, we want to emphasize that the outcomes of the studies can’t be generalised. Each child with CP and his/her situation is different and the influence of a variety of confounding factors has to be kept in mind when interpreting research studies. For example, the psychological and social well-being of the child and their families, rehabilitation procedures offered and financial situations  will influence the external validity of each study. Another consideration to take into account is that this systematic review is based on the change in gait parameters, but it is important to also look at other outcome measures and approach this holistically With regards to the gait analyses itself, 3DGA is seen as the gold standard, however, the gait data should be interpreted within the reliability of the gait measurement itself, and the subjective interpretation of the data might slightly vary between the different experts ,34. TherThis is the first systematic review article which provides an overview of the effectiveness of SEMLS interventions based on different 3DGA parameters in children with spastic CP. SEMLS interventions generally resulted in good improvement in most gait parameters, with the biggest improvements seen for knee ROM, knee flexion at IC and minimal knee flexion in stance phase, ankle dorsiflexion at IC, maximum dorsiflexion in stance and in swing phase, hip rotation and foot progression angles. However, based on the main focus of the SEMLS intervention  and the patient’s characteristics  changes in gait parameters might slightly vary. The current overview provides a framework for clinicians, researchers and parents, although individual factors and/or adaptations of SEMLS techniques need to be taken into account when interpreting the findings of this systematic review. In addition, future research should aim to have consensus on reporting 3DGA results, include outcome measures with a holistic approach and provide more specific information about the participants , rehabilitation programs and costs involved.


Congenital cytomegalovirus (CMV) remains a leading cause of disability in children. Understanding the pathogenesis of infection from the mother via the placenta to the neonate is crucial if we are to produce new interventions and provide supportive mechanisms to improve the outcome of congenitally infected children. In recent years, some major goals have been achieved, including the diagnosis of primary maternal CMV infection in pregnant women by using the anti-CMV IgG avidity test and the diagnosis and prognosis of foetal CMV infection by using polymerase chain reaction real-time tests to detect and quantify the virus in amniotic fluid. This review summarises recent advances in our understanding and highlights where challenges remain, especially in vaccine development and anti-viral therapy of the pregnant woman and the neonate. Currently, no therapeutic options during pregnancy are available except those undergoing clinical trials, whereas valganciclovir treatment is recommended for congenitally infected neonates with moderately to severely symptomatic disease. Our understanding of the pathogenesis of CMV during pregnancy continues to improve through the application of new technologies and interventional studies. However, there remain a number of outstanding questions relating to CMV infection in pregnancy, especially in the context of women who are already seropositive for CMV where multiple strains may be present in the face of a strong T- and B-cell immune response, the comparative pathogenesis in developed and developing countries, and the optimal therapeutic strategies to be deployed. Recently, there has been significant interest in establishing associations between genetic variants and strain pathogenicity of CMV. In 2013, Renzetteet al. created the most detailed map of human CMVin vivo evolution to date and demonstrated that viral populations can be stable or rapidly differentiate, depending on the host environment2. Furthermore, in 2015, Sijmonset al. provided an important compendium of data concerning human CMV strain diversity3. These studies support the hypothesis that human CMV strains may vary in virulence, tropism, and pathogenic potential, which in turn is probably related to the genetic variability exhibited in key genes important for pathogenesis among wild-type CMV strains. Identification of specific, more highly pathogenic, CMV variants could provide clinically useful information3.Cytomegalovirus (CMV) remains a major cause of congenital infection and disease during pregnancy around the worldThe present article will survey the progress that has been made in these areas, particularly over the last 4 years, and provide a succinct update on how our understanding of CMV in pregnancy has matured in recent years and the potentially beneficial effects of anti-viral therapy in managing congenital CMV infection and disease.7  and transmits the virus to the foetus. In this situation, transmission to the foetus occurs in 30–35% of cases and congenital disease in around 10–15% of those born with congenital infection6. In contrast, in women who are already seropositive, reactivation or reinfection gives rise to a foetal infection rate of about 1.2%, which whilst much lower than primary infection acts to be the main contributor to the total number of congenital infections (and disease) worldwide11. Lastly, increasing observations demonstrate the risk for symptomatic infection at birth, and sequelae, especially hearing loss, are similar upon primary and non-primary maternal CMV infection15.CMV seroprevalence across the globe varies substantially both between and within countries7 . As a ruin utero or shortly after birth and this is usually because of significant neurological damage and multi-organ failure16. Of the remainder, about 60% will have cognitive defects; sensorineural hearing loss (SNHL) and neurological impairment are two common manifestations16. Indeed, SNHL, which is prevalent at about 35%, is a progressive disease, and, even in neonates born with asymptomatic infection, there is now increasing evidence that a significant subset of these will develop SNHL19. Thus, early identification of children at risk of progressive SNHL is a priority, as interventions may provide a substantial benefit (an aspect that will be discussed later).In terms of pathogenesis, in children with symptomatic disease, approximately 4% will die20. Additional studies by targeted sequencing demonstrated that other genes of CMV have highly variable regions including proteins gN, gO, gH, and UL1443. However, the role that mixed genotype infection plays in transmission and the immune response against these different strains has not been fully investigated.In CMV seropositive women, the risk factors for congenital infection are less well defined, although in a recent study of pregnant Polish women, around 22% of seropositive women who transmitted to their neonate were infected with multiple CMV strains as judged by glycoprotein B genotyping21. In many studies of transplant recipients, a high frequency of functional  CMV-specific CD8 together with CD4 helper cells has been shown to be protective against high-level CMV replication and also disease25. Thus, the assumption might be that in pregnant women a similar T-cell phenotype may be protective against CMV disease. Recent data question this hypothesis; with IFN-γ ELISpot assay and CMV IgG avidity testing, it was shown that women who transmitted to their offspring were more likely to have a higher T-cell response and that the combination of low CMV IgG avidity and high ELISpot values gave an area under the curve of 0.8726.In healthy humans, the T-cell immune response against CMV is both multi-specific and high frequency in both CD4 and CD8 compartmentset al. proposed a novel normalisation method testing the individual IFN-γ response to CMV  detected by the QuantiFERON assay and found that the group of women with a low CMV IFN-γ RR did not transmit the virus to the foetus27. However, Forneret al. showed that the results of CMV cell-mediated immunity obtained with the ELISpot assay were more significantly associated with the risk of CMV transmission compared with those obtained with the QuantiFERON test28. Further studies in a larger number of CMV-infected pregnant women should be performed in order to verify the prognostic efficacy of determining the maternal CMV-specific T-cell immune response. Finally, data using a Rhesus animal model of CMV have clearly demonstrated that, in the absence of CD4 T cells, more severe CMV disease was observed in the offspring29.Eldar-Yedidiade facto screening31. One of the stated reasons why such an approach is not warranted is the incidence of congenital infection and disease amongst children born to women who are already CMV IgG positive. However, if there is clinical suspicion of primary CMV infection, appropriate testing of antenatal samples for seroconversion and investigating IgG avidity levels are standard practice32. To assess the risk of transmission to the foetus, prenatal testing of amniotic fluid at 20–21 weeks of gestation by real-time polymerase chain reaction (PCR) has also been investigated40. The foetal CMV diagnosis is reliable: a negative result in the amniotic fluid can rule out foetal infection with a high degree of certainty. Positive results in amniotic fluid identify CMV-infected foetuses but do not discriminate those infants who will have symptoms at birth. Studies have shown that low viral loads in amniotic fluid are associated with a lower risk of congenital disease, but it is clear that the positive and negative predictive values of qualitative and quantitative PCR on amniotic fluid are not sufficiently robust for routine deployment and should be considered with caution42.Screening of mothers for CMV IgG on a routine basis has not been universally adopted, although at present eight European countries have adopted a43, impairs cytotrophoblast-induced lymphangiogenesis and vascular remodelling in the placenta, and arrests the correct development of human trophoblast progenitor cells, thus interfering with the earliest stages in the growth of new villi. This results in increased hypoxia, which ultimately contributes to restriction in foetal growth44. In fact, immunohistochemical and virological studies of placental tissues suggested that severe placental infection was associated with diffuse villitis and necrosis, consistent with functional impairment and possible consequent hypoxic cerebral damage45.In recent years, our understanding of the complex interaction between the placenta and CMV and how it relates to congenital infection has improved significantly. Of particular note here is the work of the Pereira group. The recent observations show that CMV particularly infects amniotic membranessurvivin and Bclx-l through both STAT-3-dependent and -independent mechanisms.46. In addition, CMV has been shown to inhibit Wnt5a-stimulated migration of trophoblasts through increasing the expression of the WNT receptor ROR247.CMV infection also appears to persist in the amniotic epithelial cells and has been associated with increased expression of the anti-apoptotic proteinset al. highlighted how CMV infection modulates the placental immune environment, suggesting CMV-induced upregulation of monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor-alpha (TNF-α) expression as a potential initiator or exacerbator (or both) of placental and foetal injury48. In CMV-infected decidual cultures, there is a predominant induction of INF-γ and inducible protein 1 (IP-1) expression, reflecting the immune activation generated upon CMV infection49.CMV infection induces an innate immune response in the placenta, significantly altering the decidual cytokine and chemokine environment. In particular, Hamiltonet al.50 assessed each of the sequelae of congenital CMV and the evidence for different interventions making a positive impact. This study concluded that there was good evidence that non-pharmaceutical interventions for children with delayed hearing loss by 9 months of age would have an impact and that a moderate effect of pharmaceutical interventions would be observed on hearing loss between 9 and 24 months and on CMV-related cognitive deficits. However, the evidence for interventions affecting children with hearing loss of neurological impairment occurring after 24 months was weak. On this basis, the authors proposed that a combination of newborn screening and early detection and interventions would benefit thousands of children with congenital CMV in the USA alone.A comprehensive analysis by Cannon53. Further improvements in sensitivity can be achieved by standardised procedures of viral DNA extraction and nested PCR approaches, with a recent article showing a sensitivity of 81%54. As an alternative, saliva has been suggested as the sample of choice, but this would require a different sample to be taken at birth, and for routine screening this may prove to be too complex and costly to adopt55.At present, no European country routinely screens for congenital CMV infection. In four states in the USA, the efforts of non-profit organisations have determined the introduction and passage of legislation for infant CMV screening. Attention over the last decade has turned to the use of dried blood spots, since these are routinely taken to diagnose biochemical and genetic disorders in the newborn. However, the sensitivity of this approach using PCR-based detection methods is highly variable (28–100%), even when large sample sizes have been used57. Rates of congenital CMV infection in HIV-exposed but uninfected infants in Nairobi have been shown to be 6.3% and this increased to 29% if the infant was HIV infected58. To date, no large systematic studies have investigated the transmission frequency in HIV-infected women of CMV to the neonate in the context of both maternal CMV infection during pregnancy versus post-partum infection, which is known to be prominent through CMV in breast milk. However, one study has used data from a valacyclovir interventional study (for herpes simplex virus [HSV] infection) to investigate its effects on infant CMV acquisition59. Valacyclovir at the doses used for HSV-1 control has a relatively weak anti-viral effect on CMV replication. The upshot of this study was that maternal valacyclovir use had no effect on the timing or acquisition of infant CMV or on breast milk viral loads but that it did reduce cervical CMV shedding. Further studies using high-dose valacyclovir or safer anti-CMV drugs would be warranted in this patient group60. It has also been shown that maternal highly active anti-retroviral therapy (HAART) can reduce vertical transmission of CMV but does not reduce breast milk levels and so would be unlikely to impact on post-natal infection61.A major co-infection which impacts directly on CMV in pregnancy is HIV infection. Recent data highlight that HIV-1/CMV co-infected infants have a high risk of mortality, neurological defects, and HIV disease progression62. The study enrolled 96 neonates who were randomly assigned to either 6 months or 6 weeks of valganciclovir therapy. The primary endpoint  was similar between the two arms, although total ear hearing was more likely to have improved and be stable at 12 months in the 6-month valganciclovir arm. Interestingly, the 6-month treatment arm was also associated with significant improvements (P <0.004) in neurodevelopmental scores . This study builds on a previous anti-viral study of intravenous ganciclovir63 and, though encouraging, shows that until we have access to anti-CMV drugs with greater potency and improved side-effect profiles, it is unlikely that this area will move forward rapidly. Currently, valganciclovir treatment for 6 months is recommended for congenitally infected neonates with moderate to severe CMV disease and should be started within the first month of life.The most extensive study to date on the use of anti-viral therapy for symptomatic CMV disease was published in 201564. One non-randomised, single-blind clinical trial is currently investigating whether early treatment with valganciclovir of infants up to 12 weeks of age with both congenital CMV infection and SNHL can prevent progression of hearing loss . Two other clinical trials are being undertaken in order to provide evidence for treatment options in congenitally CMV-infected newborns .At present, there is no evidence for the potential benefit of treatment of asymptomatic infants or asymptomatically infected children with isolated sensorineural hearing loss (≥20 dB in one or both ears)in utero treatment of congenital CMV with high-dose valacyclovir (8 g/day) has recently been published. The interim analysis indicates that longer-term exposure to valacyclovir (median of 89 days) decreases foetal viral loads significantly and combined with historical controls decreases the proportion of symptomatic neonates from 57% to 18%60. However, the trial design using historical controls is not optimal and so further, more substantial analysis of this ongoing study is required and there is a need for others to replicate the study design in a controlled way.A preliminary report on an open-label phase II study of65. Modelling has shown that if vaccination was started in all toddlers and children aged 12, after 4 years a decline in infected babies occurs (owing to girls immunised at age 12 starting to enter child-bearing age) but that a more rapid decline is observed for babies with congenital infection born to seronegative women and also seropositive women who become re-infected during pregnancy66. To date, only a recombinant CMV glycoprotein B (gB) vaccine administered with the adjuvant MF59 has been evaluated in seronegative women; the vaccine showed an approximately 50% reduction in maternal infection in women vaccinated67. This phase 2 study also observed one congenital infection in the vaccine group compared with three in the placebo arm, although the sample size prevented statistically valid analysis of this observation.The basic reproductive number (Ro) for CMV for infections occurring in the developed world is around 2.4, meaning that for herd immunity a vaccine update rate (assuming 100% efficacy) would need to be 59–62% to achieve eradication68.Very recently, a study performed in a guinea pig model demonstrated that immunisation with a novel bivalent vaccine based on a non-replicating lymphocytic choriomeningitis virus (LCMV) vector expressing gB and pp65 did not show interference. Moreover, the bivalent vaccine elicited potent humoral and cellular responses and conferred protection, reducing the magnitude of maternal viremia and improving pup outcomes. These results support further testing of LCMV-vector-based human CMV vaccines in clinical trials69. In this study, 124 pregnant women with primary CMV infection at 5–26 weeks of gestation were randomly assigned to hyperimmune globulin or placebo every 4 weeks until 36 weeks of gestation or until CMV was detected in amniotic fluid. The rate of congenital infection was comparable between the two groups . Hyperimmune globulin had no effect on maternal CMV DNA levels in the blood or in time to clearance of DNA from the blood and did not impact on CMV DNA levels in the placenta. However, the results of this randomised placebo-controlled trial showed no agreement with those from a non-randomised study published in 2005 by Nigroet al., who showed that the administration of CMV-specific hyperimmune globulin to pregnant women with primary infection significantly decreased the rate of mother-to-foetus transmission, from 40% to 16% (P = 0.04)70. Further studies are needed given the biological plausibility of this approach70.An alternative approach using hyperimmune globulin to prevent congenital CMV has also been subjected to a randomised controlled trialCurrently, two randomised, phase 3 studies of the prevention of congenital infection are underway. One, sponsored by Biotest, is being conducted in Europe, and the second, sponsored by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, is ongoing in the USA .The hope is that these studies will further aid our understanding of the efficacy and safety of hyperimmune globulin administration as a means of preventing congenital CMV infection.et al. highlighted how, despite a number of case series and case control and observational studies, there is a significant lack of robust clinical trial data examining prophylactic interventions for congenital CMV infection71.A systematic review published by HamiltonThe last few years have seen real progress in understanding the basic biology of CMV in the placenta and the role that ongoing viral replication plays in the pathogenesis of CMV disease in the neonate. However, for a variety of reasons, screening of pregnant women for CMV, whilst supported, has not been implemented globally, and routine surveillance of neonates for evidence of CMV infection requires new methodologies or improvement of the current ones, especially with respect to simpler protocols and lower costs. The therapy of the newborn infant with CMV shows promise, but we need safer and preferably more potent drugs to make a large impact; new drugs such as letermovir are on the horizon, as are active vaccine development programmes, making the future of CMV in pregnancy a very active area of basic and translational research.CMV, cytomegalovirus; gB, glycoprotein B; HSV, herpes simplex virus; IFN-γ, interferon-gamma; LCMV, lymphocytic choriomeningitis virus; PCR, polymerase chain reaction; Ro, basic reproductive number; ROR2, receptor tyrosine kinase-like orphan receptor; RR, relative response; SNHL, sensory neural hearing loss; STAT, signal transducer and activator of transcription.