Furthermore, the average weekly volume of training was 23.5 ± 1.4 h, with the highest training volume occurring on Monday and Tuesday and high variability within gymnasts. Our study also revealed that the most frequently injured joints that had the most negative impact on training volume, sport performance and pain status were the ankles and low back, followed by the knees and shoulders. This kind of yearly screening method is warranted to allow more definitive conclusions to be made on adjusted training and preventive strategies.Body composition (BC), training volume/pattern, and injury status are a few important factors affecting training quality and sport performance in female artistic gymnastics. We aimed to examine BC status, training volume/pattern, and injury status during the first competition period. Our cross-sectional study included 17 female gymnasts  who were high performance at the international and national level. The BC  parameters included body height and mass, body fat percentage (BF %), lean body mass (LBM), body mass index (BMI), total bone mineral density , and total bone mineral content . Training volume and pattern were assessed via an author-developed questionnaire, while injury status  was assessed via a modified questionnaire on overuse injuries used in sports injury epidemiology studies with elite-level athletes. Body composition parameters were as follows: the BMI was 21.5 ± 1.4 kg/m Body composition (BC) is used as one tool among several to ensure optimal preparation for competition. Body composition can be used as a guideline for weight management recommendations and concerns in competitive sports. The two main modifiable factors that directly influence body composition are (i) overall physical activity  and (ii) dietary intake  ,2,3. Onehigh performance level.Female gymnasts participating in this sport experience many difficulties regarding their BC and visual body image ,7,8. TheAdditionally, gymnasts commonly initiate systematic training at the age of six years, and concerns about body weight and BC are brought to their attention practically as soon as they are introduced to the sport . On mostAthletes, especially gymnasts, constantly seek a balance between adequate nutrition, an appropriate BC, a favourable visual appearance, effective recovery and a good immune system ,12,17,18There is a lack of recent data on various sport-related factors in high-performance-level artistic gymnasts, especially BC status during the competition period . Thihttps://clinicaltrials.gov . All participants and/or the legal guardians signed an informed consent form for inclusion in the study. The participants were not compensated financially to participate in the study. The study was conducted on 4 April 2018 (during the competition period) within three hours. The BC and other measurements were performed at a certified biomedical centre  by their medical staff  and a certified physician (BC measurements). All the assessments performed in the study were funded by the Slovenian Research Agency.This study was approved on 28 March 2018 by the Slovenian Medical Ethics Committee  and registered at We included 17 high-performance-level female artistic gymnasts from Slovenia, with a mean age of 17.4 ± 4.1 years. All the participants resided in Slovenia at a latitude of 46° N, and several of them were performing at the highest international competitive level . The female gymnasts were recruited through personal contacts with the National Team Coach and other gymnastic coaches working in the clubs around Slovenia. The criteria for inclusion in the study were as follows: competing at the international and national level, currently fully actively involved in a training programme, and not using any prescribed medication that affects bone metabolism. All the gymnasts who were recruited participated in the study, and none of them were excluded from the study analysis. The variables in this study included the basic characteristics of the athletes, anthropometric and BC parameters, training parameters , and the injury pattern . Of note, dietary intake, serum micronutrients and cardiovascular health status are summarized only to understand the obtained results and are presented in more detail in separate manuscript .The characteristics of the study participants  and the training status  were evaluated by a questionnaire developed by the authors.The basic anthropometric parameters included body height, body mass, and body mass index (BMI), all of which were measured by experienced technicians using standardized medical approved professional personal floor scale with stand  and the corresponding protocols .The body composition (BC) measures included body fat percentage (BF %), lean body mass (LBM), total bone mineral density , bone mineral density of the spine (BMD spine), bone mineral density in the left femoral neck (BMDLF neck), and total bone mineral content . For the evaluation of the BC parameters, DEXA  with EnCore software, version 13.31, was used.The injury location, symmetry and the rate of injury were evaluated via a modified questionnaire based on a method of assessing overuse injuries used in sports injury epidemiology studies  and an a12, 25-hydroxyvitamin D (25(OH)D), calcium, magnesium, phosphorus, potassium, and iron) as well as several cardiovascular health markers  using biochemical assays and blood pressure measurement. Nutritional and cardiovascular health information are provided in more detail in a separate article [We evaluated dietary and supplements intake using a standardized Food Frequency Questionnaire (FFQ), serum micronutrients . The participants started with regular gymnastics training at a mean age of 5.3 ± 2.7 years, and they had all been involved in systematic training for at least 7 years, with an average of 10.5 ± 3.4 years of systematic training. More details on the characteristics of the athletes are presented in 2, and 21.9 ± 4.7%, respectively. Importantly, the mean BMI and BF % of the two gymnasts who competed at the highest level before the study (World Cup) were lower than the average BMI and BF % . More details on the BC status of the gymnasts are presented in The average body height, body mass, BMI, and BF % were 159.8 ± 6.2 cm, 54.8 ± 5.3 kg, 21.5 ± 1.4 kg/mThe gymnasts reported training for an average of 23.5 h per week. The days on which the highest total training volume occurred for the gymnasts were Monday (4.0 h/day), Tuesday (3.9 h/day), and Friday (4.0 h/day); for the remaining days, the training volume was modestly lower on Wednesday (3.6 h/day) and was similar on Thursday (3.7 h/day) and Saturday (3.6 h/day). Sunday was mostly a rest day (0.7 h/day). However, 29% of gymnasts practised on Sunday; the duration of a single workout was two hours long, and the session primarily consisted of recovery or prevention training. Interestingly, at the time of the study, two of the most successful gymnasts reported practising 20  and 32 h per week . In addition, for the most successful of these two gymnasts, the practice volume on Monday, Tuesday, Thursday, and Friday was six hours daily, and on Wednesday and Saturday was four hours daily, and Sunday was a rest day.The areas of the body that were most commonly injured were the ankles and low back. Ankle and low back injuries limited the volume of gymnast training the most, while knee injuries decreased the training volume and negatively affected sport performance the most. Furthermore, the gymnasts felt moderate or severe pain in the ankles and lower back, followed by the knees and elbows. Due to ankle injuries, 35% of gymnasts had to limit training, and the second most-limiting injuries were low back injuries (23%). Importantly, 41% of the gymnasts decreased their training volume due to low back or shoulder injuries. A total of 41% of the gymnasts reported that pain in their low back  or knees  was the most common reason for a decrease in performance within the last 6 months. Moderate and severe pain were the most frequently reported severities of pain in the ankles (25% more cases on the right ankle) and low back (11% more cases on the right side). Injury assessment is presented in 12 (47%). Finally, all cardiovascular and safety variables  were within recommended ranges; however, 41.2% of them had low-density lipoprotein cholesterol above 3 mmol/L and 35% had triglycerides ≤0.6 mmol/L [The mean (± SD) energy intake was 1514 ± 258 kcal/day . Dietary supplements or sport drinks were consumed by 59% of gymnasts ). Dietary intakes from foods and supplements are presented in 6 mmol/L .In this study of the BC, training parameters and injury status of high-performance-level female artistic gymnasts, there were three important findings. First, the gymnasts’ BC differed from those found in previous studies and recent reviews. Second, training volume varied, even within the most successful gymnasts, but the average total training volume in the competition period was consistent with those reported in other studies. Third, the areas of the body that were the most susceptible to injuries and when injured, had the largest negative impact on training, training volume, sport performance, and pain (moderate or severe) were the ankles, low back, and knees.Mean BC in our sample was higher than other studies that reported on elite- or high-performance-level female athletes in artistic gymnastics ,26. The 2), a higher relative LM (81.7 vs. 75.2%), and a lower BF % (14.1 vs. 21.9%) [In a previous cross-sectional study on six artistic gymnasts, members of the Danish National team (aged 17.9 vs. 17.4 years in our study) had a higher mean body height than that found in our study (165 vs. 159.8 cm), a similar body mass (53.7 vs. 54.8 kg), a lower BMI (19.7 vs. 21.5 kg/m. 21.9%) . The res. 21.9%) . Intereselite level or high performance level used, and the associated level of competition, the high proportion of ED that may lead to lower BF %, differences in motor skills requirements in case the gymnast competes in a single discipline  or among all-round specialists. Lastly, female artistic gymnastics is not a sport of maximum effort. Body control is a more important component and consequently there is a possibility to compensate certain physical potentials with other abilities.The time in the season and the level of performance are likely very important considerations when comparing results of our study to previous results. For example, higher level of performance has been associated with a lower BF % ,26,36,372 for Slovenian and US gymnasts) [2 vs. 1.24 g/cm2) [2), we still found lower BMDLF values in our study than in the Danish study. The reasons for this inconsistency might be a suboptimal 25(OH)D level and dietary status among the gymnasts in our study [Furthermore, the BMD of the gymnasts in our study was somewhat higher than that of the 48 members of the National US team measured in the 2005 study mentioned above with respect to BF % (1.22 and 1.06 g/cmymnasts) . Importa4 g/cm2) . When weur study .The expected training volume/pattern was observed; the training volume was higher on Monday, Tuesday, and Friday, and on the other days (Wednesday and Saturday), training volume decreased slightly; Sunday was mostly a rest day. However, some gymnasts (29%) had a short training session on Sunday, but the session consisted of mostly recovery- or prevention-type training. Importantly, at the time of the study, the most successful gymnast  performed 32 h of training per week, while the next most successful gymnasts  performed significantly lower training volumes . These data indicate a relatively large and important inter-individual variability in training volume as well as individual conditioning training tapering during the competition period for training periodization.It is well established that a typical amount of training for elite-level gymnasts ranges from 20 to 30–40 h of training per week ,15. The In our study, we found that the most injured areas of the body were the ankles and back, followed by the knees. These three areas also had the most negative impact on training, training volume, and sport performance and led to moderate or severe pain. Overall, when we take into account all the measured variables, our results showed that the low back was as injury sensitive as the ankles.The high and repetitive intensity of practice and competitions, the large number of events, and the technical difficulties make gymnastics one of the most injury-prone sports . Our resThere is various evidence on how to effectively reduce the rate and severity of gymnast injuries while maximizing gymnastic performance, including implementing appropriate training modalities that include mental training to enhance self-esteem, maintaining the appropriate BC, maintaining adequate nutrition, using protective equipment, and spotting ,24,51,52The strength of our study was that we assessed all high-performance-level gymnasts from Slovenia, where gymnastics is commonly practiced, with many Olympic and world-class gymnasts being from Slovenia over the last 100 years . For BC However, the study has known limitations inherent to the nature of cross-sectional studies and the sample size of the high-performance-level gymnasts. Therefore, due to these limitations, the results should be interpreted with caution, replicate studies with larger sample sizes should be performed. Importantly, the questionnaires that characterized the participants  and their training status  were self-reported; and reliability and validity were not estimated for these questionnaires; therefore, further replication is needed. Only the volume and weekly training pattern were reported, while the intensity and content of each training session were not assessed. Similarly, only anatomical location and the rate of injury were assessed, and the injury type  and cause  were not reported, which may change how the injury data is interpreted. Our study also did not analyse motor skills  or neuromuscular fatigue that can affect performance. Future research using longitudinal research should also address the question about the relationship between various interrelated parameters such as training volume, dietary intake, BC, injuries and sport performance/success.The results demonstrated that the average BF % status was higher than found in most other studies on elite or high-performance-level female artistic gymnasts. Additionally, we found that the volume of training varied within gymnasts, even among the most successful ones, and should be viewed as an individual characteristic; training differs in response to individuals’ conditions, the competition period and their recovery as well as dietary support. However, the average total weekly training volume was found to be in line with the results reported in previous studies for the competition period. Similarly, in line with the results of most other studies, the anatomical location and the injury rate evaluation showed that the ankles, low back and knees are the most susceptible areas to injury, have the most negative impact on training volume and sport performance, and led to moderate or severe pain. Female gymnasts, coaches, researchers, and the gymnastic system urgently need to effectively solve the long-standing challenge of achieving and maintaining the desired BC, a healthy body image, and effective post-training recovery and improving long-term injury prevention, thereby improving gymnasts’ sport performance and health. Taken together, our findings call for periodic screening and potentially for adjusted strategies  to improve BC status and to decrease injury rates.


Research has the potential to influence US social policy; however, existing research in this area lacks a coherent message. The Model for Dissemination of Research provides a framework through which to synthesize lessons learned from research to date on the process of translating research to US policymakers.The peer-reviewed and grey literature was systematically reviewed to understand common strategies for disseminating social policy research to policymakers in the United States. We searched Academic Search Premier, PolicyFile, SocINDEX, Social Work Abstracts, and Web of Science from January 1980 through December 2019. Articles were independently reviewed and thematically analyzed by two investigators and organized using the Model for Dissemination of Research.The search resulted in 5225 titles and abstracts for inclusion consideration. 303 full-text articles were reviewed with 27 meeting inclusion criteria. Common sources of research dissemination included government, academic researchers, the peer reviewed literature, and independent organizations. The most frequently disseminated research topics were health-related, and legislators and executive branch administrators were the most common target audience. Print materials and personal communication were the most common channels for disseminating research to policymakers. There was variation in dissemination channels by level of government . Findings from this work suggest that dissemination is most effective when it starts early, galvanizes support, uses champions and brokers, considers contextual factors, is timely, relevant, and accessible, and knows the players and process.Effective dissemination of research to US policymakers exists; yet, rigorous quantitative evaluation is rare. A number of cross-cutting strategies appear to enhance the translation of research evidence into policy.Not registered. Contributions to the literatureThis is one of the first systematic reviews to synthesize how social policy research evidence is disseminated to US policymakers.Print materials and personal communications were the most commonly used channels to disseminate social policy research to policymakers.Several cross-cutting strategies  were identified that are likely to lead to more effective translate of research evidence into the policy making process in the United States.globally  AND [policy OR policies OR law OR laws OR legislation] AND [use OR utilization OR utilisation] OR [disseminate OR dissemination OR disseminating] OR [implementation OR implementing OR implement] OR [translate OR translation OR translating]. Our search was limited to studies in the United States between 1980 and 2019. We selected this timeframe based on historical context: the 1950s through the 1970s saw the development of the modern welfare state, which was (relatively) complete by 1980. However, shifting political agendas in the 1980s saw the demand for evidence increase to provide support for social programs ; we hopeAll titles and abstracts were screened by the principal investigator (LEA) with 20% reviewed at random by a co-investigator (DAQ) with total agreement post-training. Studies remaining after abstract screening moved to full text review. The full text of each study was considered for inclusion (LEA and DAQ) with conflicts resolved by consensus. The data abstraction form was developed by the principal investigator (LEA) based on previous research , 26 and Abstracted data was qualitatively analyzed using thematic analysis (LEA and DAQ) and guided by the Model for Dissemination of Research. The goal of the preliminary conceptual model was to synthesize components of dissemination for studies that evaluate the dissemination of social policy to policymakers.The search of the literature resulted in 5675 articles and 137 articles recommended by content experts for review with 5225 titles and abstracts screened after duplicates removed. Of those articles, 4922 were excluded due to not meeting inclusion criteria. Further, 303 full text articles were reviewed with 276 excluded as they did not meet inclusion criteria. Twenty-seven articles met inclusion criteria , while others used case studies (n = 6) or focus groups (n = 3). Most studies examined state-level policy (n = 18) and nine studies examined federal-level policy, with some studies looking at multiple levels of government. Included studies focused on the executive and legislative branches with no studies examining the judicial branch.Included studies are listed in Table We examined dissemination based on geographic regions and/or political boundaries . Sixteen of the 27 studies (about 59%) used national samples or multiple states and did not provide geographic-specific results . Two stuWe also analyzed whether dissemination at different levels of government  used unique channels. Six of included studies (about 22%) examined multiple levels of government and did not separate results based on specific levels of government –31, 53. Five studies (about 18.5%) examined dissemination at the federal level –36. At tn = 13). Four studies used the Weiss Typology [Roughly half of studies were atheoretical . We integrated themes from the studies into the Model see Fig. .Fig. 3An = 7) [n = 5) [n = 5) [n = 11) [n = 7) [The sources of knowledge varied across studies with some studies including multiple sources of social policy information. The most common sources of knowledge included research, as in peer-reviewed literature (n = 7) , 49, 54, [n = 5) , 34, 56, [n = 5) , 48, 55,[n = 11) , and org [n = 7) –54, 56.n = 12) [n = 6) [n = 4) [n = 3) [The majority of studies focused on health topics (n = 12) , 56, 58  [n = 6) , 52, 57. [n = 4) , 53, 54, [n = 4) , and gen [n = 3) , 32, 48. [n = 3) . One stu [n = 3) . This fi [n = 3) , 52, par [n = 3) , 34, 41. [n = 3) , 57.n = 9) [n = 12) [n = 3) [The audience included executive branch policymakers , adminisn = 9) , 55, 57,n = 9) . Studies[n = 12) , 53, 58  [n = 3) , 34, 36. [n = 3) , 54, 56 n = 10) [n = 3) [n = 9) [n = 7) [n = 6) [n = 2) [n = 4) [n = 2) [n = 3) [n =2) [Included studies examined a variety of channels with many including multiple channels. Print materials was the most commonly used channel, including reports (n = 10) , 57, 58  [n = 3) , 34, 38. [n = 9) , 56, 57. [n = 7) , 52, 54. [n = 6) , 52–54 a [n = 2) , 53 were [n = 4) , 57, ele [n = 2) , 57, onl [n = 3) , 58, and) [n =2) , 52.The majority of studies employed qualitative research methods  to evaluate the impact of scientific research on domestic social policy. Our review of the literature also identified nine quantitative and mixed-methods studies , 49, 58.Four studies highlighted the importance for early and ongoing engagement with policymakers throughout the research process in order to maximize interest and applicability. Researchers are encouraged to take the initiative to contact policymakers as early as possible in the research process. Many policymakers may be interested in accessing and using research but uncertain who or how to make connections in the academic or research community . InvolviIn addition to early policymaker engagement, three studies highlighted the need for researchers to garner outside support for their work, ideally involving a broad pool of experts and cultivating a broader coalition of supporters than typical academic endeavors . Often, champions or brokers; these are intermediary individuals or organizations who connect research suppliers  to research demand  [A common strategy for garnering support (as recommended above) is the use of evidence ymakers) . These cymakers) , 52, 53.ymakers) . One stuymakers) .Two studies specifically addressed the role of intermediary organizations or brokers in the translation of research evidence to policy. Hopkins et al.  exploredMultiple studies stressed the importance of research evidence being contextually relevant to the specific policy audience , 55, 57.Ideology of external advisors and brokers (as discussed above) and policymakers’ own personal beliefs and experiences  and the As with all research endeavors, timeliness and relevance are paramount. However, the typical timeline for academic research (years) is often too long for policymakers whose window for championing a policy action is much shorter (weeks or months) , 52. A fMultiple studies engaged with policymakers in an effort to determine how they receive research evidence and what strategies or formats are most desirable or effective . After pA study on state policymakers’ perceptions of comparative effectiveness research (CER) found that the most useful research is that which is consistent and specific to the needs of the policymakers . The samFinally, two studies described pilot projects focused on the delivery of research evidence directly to policymakers. The first cultivated researchers’ capacity to accelerate the translation of research evidence into useable knowledge for policymakers through a rapid response researcher network . This moPolicymakers are as much experts in their arena as researchers are in their academic fields. In order to build lasting working relationships with a target policymaking audience and maximize the relevance of research products for policy work, researchers must first understand the policy process , 30, 34.Public policy, whether legislative, executive, or judicial, affects all areas of daily life in both obvious and subtle ways. The policy process  does not exist in a vacuum; it is influenced by many factors, including public opinion , 60, speMany channels exist to disseminate evidence to policymakers, with the most common being print materials . This finding is surprising in our current digital age, as print materials are necessarily time-bound and rapidly evolving technology has created more channels  which may be preferred by policymakers. This shift creates an opportunity to optimize the content of print materials to disseminate in new mediums; it also offers a chance for authors to improve the accessibility of their work for broader audiences  , 69–71.Our review found strategies to increase effectiveness of research dissemination to policymakers includes starting early, drumming-up support, using champions and brokers, understanding the context, ensuring timeliness, relevance, and accessibility of research products, and knowing the players and the process. These themes align with existing knowledge about policymaker preferences including face-to-face engagement , 73, conMany of the barriers and facilitators to research dissemination that we identified in this review mirror those cited by policymakers as barriers and facilitators to evidence use; this overlap reasonably suggests that efforts to expand research dissemination may improve the other. Particularly relevant lessons from the evidence use literature that also emerged from our review include emphasis on the benefit of building personal relationships between researchers and policymakers , 75, 76,Our findings also highlight several unique elements of the US policy landscape, wherein significant power is reserved from the federal-level and afforded to state-level government. In some states, this power is further distributed to county and local governments. This system creates major variation across the country in both policy decisions and in resource availability for social policy implementation. Despite our relatively unique government structure, however, many of the effective strategies for dissemination we identified mirror strategies found in other countries , 80.Studies that focused on a specific level of government had some unique characteristics such as formality and reliance on print materials. For example, federal dissemination relied more heavily on formal legislative testimony while state level material relied on written policy materials . However, these results are limited by small sample sizes and limited evidence about effectiveness.A wide range of contextual variables may influence policy dissemination in the US at different levels of government. In the federal legislative context alone, multiple committees and subcommittees of both the U.S. House of Representatives and the U.S. Senate may exercise some control over programs and policies related to a single social policy issue  . At the Infrastructure differences may also represent important differences between the US social policy context and that of other developed nations. Each country has a distinct and perhaps unique policy context given available resources, political rules and regulations, and priorities. While models for infrastructure and dissemination interventions may be shared across policy contexts, it may be difficult to directly compare dissemination strategies in one country with dissemination strategies in another country.www.wisfamilyimpact.org) are an example of long-standing initiatives that provide the opportunity for researchers and policymakers to come together to discuss unbiased policy-relevant evidence [Several examples across western countries contribute to a stronger nexus between research evidence and the policy-making process. In the United States, the Wisconsin Family Impact Seminars  presents an opportunity for future work in this field to integrate quantitative and qualitative methodologies.The current study has several limitations—these illustrate opportunities for future research. First, we attempted to cast a wide net when searching for studies which examined the influence of research on social policy by including a broad search of the peer-reviewed literature, think tanks, and content experts. However, it is possible we missed some studies which examine how research influences policy. Second, we provide a rationale for focusing on US studies and that our findings may not be generalizable to other countries. Third, we were unable to assess the risk of bias for individual studies as current standards note difficulties in assessing quality and bias in qualitative research . Fourth,One significant challenge to increasing the rigor in dissemination research studies is the difficulty in choosing and then measuring an outcome. Many of the studies included in this review are either case studies or descriptive, making it difficult to determine what, if any, impact the given research had on policy. Bogenschneider and Corbett discuss this at length as one of the primary challenges to furthering this research , imploriDissemination and implementation are often described together; not surprisingly, is overlap in effective strategies for each. The current review identified six dissemination strategies and described their reported effectiveness, while the Expert Recommendations for Implementing Change (ERIC) Project identified 73 implementation strategies . One sucFinally, the dissemination of research to policymakers may raise certain ethical issues. It is imperative for researchers to critically assess when and how to disseminate research findings to policymakers, keeping in mind that promoting a specific policy agenda may result in a perceived or real loss of objectivity . SynthesWe summarize strategies and illuminate potential barriers to the research-to-policy dissemination process. Key findings are drawn from multiple disciplines and suggest that lessons learned may cut across both research topics and levels of government. The most frequently referenced channel for dissemination to policymakers was print materials, with personal communication  a close second. Corresponding strategies for effective dissemination to policymakers included starting early, drumming-up support, using champions and brokers, understanding the context, ensuring timeliness, relevance, and accessibility of research products, and knowing the players and the process. A shared feature of these strategies is the distillation of complex research findings into accessible pieces of relevant information that can then be delivered via multiple avenues.Interdisciplinary collaboration is a common practice in scientific research . Our finAdditional File 1. PRISMA Checklist.Additional File 2. Search Strategy.Additional File 3. Data Abstraction Form.


Drugs  can be designed to suppress premature translation termination by inducing readthrough (or nonsense suppression) at the premature termination codon. These findings have introduced new hopes for the development of a pharmacologic approach to cure this genetic disease. In the present review, we first summarize the principle and current status of the chemical relief for the expression of functional proteins from genes otherwise unfruitful for the presence of nonsense mutations. Second, we compare data available on readthrough molecules for β0-thalassemia. The examples reported in the review strongly suggest that ribosomal readthrough should be considered as a therapeutic approach for the treatment of β0-thalassemia caused by nonsense mutations. Concluding, the discovery of molecules, exhibiting the property of inducing β-globin, such as readthrough compounds, is of great interest and represents a hope for several patients, whose survival will depend on the possible use of drugs rendering blood transfusion and chelation therapy unnecessary.Several types of thalassemia (including β Screening small molecules able to induce nonsense suppression in β-globin, as well as NMD inhibitors is of great interest and represents a hope for several patients, whose survival will depend on the possible use of drugs rendering blood transfusion and chelation therapy unnecessary.Gene expression requires quality control mechanisms to ensure the synthesis of fully functional gene products. Many errors produced during the various steps of gene expression may cause genetic diseases. A significant subset of such errors consists of nonsense mutations, frameshift mutations, and mutations that result in alternative splicing events that interrupt the open reading frames by introducing premature termination codons (PTCs). Recent advances of genomic technologies, allowing complete inspection of the genome sequence and genome editing, constitute an unprecedented opportunity to diagnose genetic disorders and pave the way to gene therapy. It has been estimated that approximately 12% of inherited genetic diseases and several forms of cancer are caused by nonsense mutations, resulting in a single nucleotide change that converts a sense codon within protein coding genes into a premature termination codon , spinal utations ,29,30. IThe synthesis of a protein results from mRNA translation. This process initiates when the start codon AUG enters the A-site of the ribosome, with a complimentary base pairing of aminoacyl-tRNA anticodon, loaded with methionine. For each of 61 codons specifying an amino acid, a corresponding aminoacyl-tRNA anticodon is available in the cell s, Upf1, Upf2, and Upf3. interacting in the sequence Upf1-Upf2-Upf3 in the activation of NMD ,31,32,33Unravelling the mechanisms governing NMD is a crucial step in identifying targets and factors aimed at attenuating NMD, thus increasing the abundance of PTC mRNAs available for the nonsense suppression therapy.Most of the genetic disorders caused by nonsense mutations are characterized by a severe phenotype resulting from the combined effect of the premature arrest of translation and the accelerated degradation of the relevant mRNA. In principle, conditions leading to a decrease in the efficiency of translation termination at PTC could favor, to some extent, the synthesis of a full length protein and the stabilization of mRNA encoding it. Considering that even a small amount of the gene product  could be sufficient to alleviate the phenotype of a genetic disease , a favorSaccharomyces cerevisiae [The properties of aminoglycoside antibiotics to mediate phenotypic suppression of nonsense mutations were first evidenced in the yeast revisiae ,45. Thesrevisiae . Since trevisiae ,47. In crevisiae ,49,50,51revisiae . Among trevisiae  and in crevisiae ,55,56. Arevisiae ,58. A nerevisiae . Most rerevisiae . NB124 rNew pseudotrisaccharide derivatives of aminoglycosides, in addition, have been designed that exhibit enhanced readthrough properties, compared with gentamicin, on mutations underlying the genetic diseases CF, Usher syndrome, and Hurler syndrome . OverallA novel promising approach was recently described that was based in a high throughput screening for molecules able to potentiate the nonsense suppression mediated by aminoglycosides. Five compounds, named CDX3, CDX4, CDX5, CDX10, and CDX11 , were idmdx mouse cultured cells or mdx mice harboring a UAA PTC in the dystrophin gene showed the production of full length dystrophin protein and enhanced muscle function respectively. In cystic fibrosis (CF), another severe inherited disease caused by loss of function of the gene encoding the cystic fibrosis trans-membrane conductance regulator CFTR, approximately 10% of cases are caused by nonsense mutations. In the CF mouse model expressing a human CFTR with a nonsense mutation, treatment with ataluren restored the synthesis of a functional CFTR [Saccharomyces cerevisiae, as well as in human cells expressing multiple nonsense mutations including a CFTR nonsense allele [In the search for small molecules that could have readthrough properties, be safe, and orally administered, high throughput screens of 800,000 compounds were performed by using luciferase reporters stably expressed in HEK293 cells or rabbit reticulocytes . Subsequnal CFTR . The atanal CFTR ,68,69,70nal CFTR ,72, atalnal CFTR . Howevernal CFTR ,75. These allele ,43. Reade allele . In a vee allele , indicate allele . Howevere allele ,78. By ue allele . Surprismdx models [Novel non-aminoglycosides with readthrough properties have been identified, by using an ELISA based assay, in a screening of a library of 34,000 small compounds for searching for those capable of suppressing nonsense mutations in the ATM gene that causes ataxia telangiectasia (AT) . Two comx models ,84. In ax models . In addix models . The appx models . In addix models . In a rex models . Incorpox models . OverallIdua locus, resulted in increased enzyme function compared to aminoglycoside used as a single readthrough agent. In addition to NMDI-1, other NMD inhibitors, have been described as effective in genetic diseases and cancer caused by nonsense mutations [Introduction of a PTC within an open reading frame can generate the synthesis of a truncated protein that could be un-functional, partially functional, or aberrant for a gained negative-dominant function. In the latter case, NMD has a surveillance function preventing the synthesis of harmful protein by accelerating the turnover of the mRNA harboring the PTC. In doing this, even when the truncated protein still retains partial function, NMD precludes its expression, worsening the disease phenotype. In all cases, NMD decreases the expression of mRNA containing PTCs, limiting the potential of the nonsense suppression therapy ,22. The utations . NMD wasutations . Among tutations . This aputations . The eme−) or absent (β0) synthesis of the β-globin chains of adult hemoglobin (HbA).In the last few years, nonsense suppression therapy has been approached for a variety of genetic diseases based on nonsense mutations, including lysosomal storage diseases (LSDs) and genetic eye disorders. The first two diseases have been recently reviewed respectively ,23. HereThe β-globin gene is located in the distal portion of the short arm of chromosome 11, within the β-globin gene cluster. It extends for 1.6 Kb and contains three exons, two introns, and two sequences in the flanking 5′ and 3′ defined as untranslated regions (UTRs) . Its exp039-thalassemia, the CAG (glutamine) codon of the β-globin mRNA is mutated to the UAG stop codon [Nonsense mutations are among the most important alterations of the β-globin gene in β-thalassemia patients and are based on a change of an amino acid codon to a premature termination codon (PTC). For instance, in βop codon ,99, leadop codon ,102,103.op codon , β-globiop codon ,105,106.op codon  elicits op codon ,108.The disease is inherited in a recessive mode, and heterozygote subjects are asymptomatic, when the PTC is located at a position activating NMD. In this case, the NMD degradation of the defective β-globin transcript produces a reduced synthesis of the truncated protein . If the 0-39-thalassemia.In β-thalassemia, the absence or reduced synthesis of β-globin chains causes severe chronic anemia and treatment related complications. Patients are usually treated with chronic blood transfusions and iron chelation therapy. Alternatively, bone marrow transplantation has been proposed. Gene therapy is still not currently available. In addition, novel strategies such as the ribosomal readthrough could introduce new hopes for the development of a pharmacologic approach to cure β-thalassemia caused by PTC, such as βRecently, Salvatori et al.  reportedIn another article, Salvatori et al.  demonstr+) or when this approach is carried out in combination with other treatments, such as hydroxyurea, as inducers of HbF production [Moreover, an efficient production of β-globin by β°39-globin mRNA was reported in erythroid precursor cells isolated from six homozygous β°39-thalassemia patients and treated with erythropoietin (EPO) with or without G418. Using G418, the conversion of a high proportion of these cells from being negative for β-globin chain synthesis to β-globin producing cells was observed by both FACS and HPLC analyses . Obviousoduction  or NMD ioduction ,114,115.oduction . In addioduction .Recently, Kar et al.  investigFinally, the current clinical trials under investigation for β-thalassemia are relatives of gene therapies, HbF inducers, iron chelators, but not for readthrough molecules, which should be considered as potential and alternative approaches to be deeply investigated for this hemoglobinopathy. However, the possible application of readthrough molecules to β-thalassemia is clearly mentioned in some granted patents  in which the possible application of the invented compounds for personalized therapy of β-thalassemia is clearly proposed ,119,120.Moreover, β-thalassemia is characterized by ineffective erythropoiesis (IE) and anemia, so novel approaches should restore the tissue oxygenation producing adequate and functional red blood cells. From this point of view, the iron reduction can decrease erythroid cells’ production with abnormal Hb and improve erythropoiesis. In particular, novel molecules targeting iron  can be useful to improve IE ,120,121 Since the original evidence that nonsense suppression approach could provide a means to restore the synthesis and the function of CFTR, inactivated by a nonsense mutation, many studies have explored the feasibility of the nonsense suppression approach through numerous genetic diseases caused by PTC ,24,47. TS. cerevisiae, recently developed, could be useful for the identification of novel small readthrough compounds and/or NMD modulators [Thalassemia is one the most important health problems in developing countries mainly because of the absence of genetic counseling and prenatal diagnosis ,128,129.dulators ,131.In conclusion, while significant progress has been made in the development of a therapeutic strategy for the treatment of disparate genetic diseases and various forms of cancer, combining nonsense mutations, clinical issues still remain below expectations. In this context, we are aware that the use of readthrough mediating compounds is often associated with many risks and difficulties related to not only drug toxicity to the cell and to the organism, but also to the lack of certainty about the identity of amino acids decoded under the readthrough treatment. Nevertheless, considering the extreme severity of many PTC caused genetic diseases and considering that even a small response to the therapy could make the difference, any effort to improve the readthrough therapeutic approach should be produced. As an example, the small and safe molecule ataluren has been recently used in CF cells in combination with caffeine, an attenuator of NMD, and found to increase the recovery of a functional full-length CFTR protein ,131,132.A recent study, combining genomic, proteomic, and biochemical data, demonstrated that many common nonsense variants do not necessarily result in loss of protein production and provided experimental evidence for the existence of gene rescue mechanisms ,132,133.


Amongst other approaches, adipose-derived stromal cells (ASCs) have recently been tested with respect to their regenerative capacity for treatment of neuromuscular disorders. While beneficial effects of ASCs on muscle recovery were observed previously, their impact on regeneration of neuromuscular junctions (NMJs) is unclear. Here, we used a murine glycerol damage model to study disruption and regeneration of NMJs and to evaluate the effects of systemic application of ASCs on muscle and NMJ recovery. In mice that were not treated with ASCs, a differential response of NMJ pre- and post-synapses to glycerol-induced damage was observed. While post-synapses were still present in regions that were necrotic and lacking actin and dystrophin, pre-synapses disappeared soon in those affected areas. Partial regeneration of NMJs occurred within 11 days after damage. ASC treatment slightly enhanced NMJ recovery and reduced the loss of presynaptic sites, but also led to a late phase of muscle necrosis and fibrosis. In summary, the results suggest a differential sensitivity of NMJ pre- and post-synapses to glycerol-induced muscle damage and that the use of ASC for the treatment of neuromuscular disorders needs further careful evaluation. In order to induce muscle damage, 20 μl of a 50% glycerol solution diluted in physiological saline were injected into the tibialis anterior (TA) muscle of anesthetized 2 months old C57BL6 female mice. The decision to use female arose from two observations: first, females are experimentally easier to handle, since animals should preferentially be kept in groups for social interactions, but males under these circumstances often bite each other and this can lead to damage of muscles that is not due to the experimental protocol. Second, females seem to possess a slightly better regenerative capability . The sam2. After 1 day, the non-adherent and red blood cells were removed. Expanded cells were characterized regarding their proliferation capacity, immune phenotype, and adipo- and osteogenic differentiation potential, as described previously , as described before . Brieflyeviously . Before 2O, the slides were mounted in Mowiol and analyzed using the same microscope and objective used for the muscle cryosections. Antibodies, dyes, and dilution rates are listed in Freshly after dissection, TA muscles were covered with a thin layer of Frozen Section Compound (Leica) and frozen on liquid nitrogen. Transverse cryosections of 15–20 μm thickness were cut using a Leica CM1950 cryostat, carefully positioned on glass microscope slides (Thermo Fisher Scientific) and then immunostained for epitope detection. Cryosections were permeabilized with 0.1% TritonX-100/PBS for 10 min at room temperature and, after three washing steps in PBS (5 min each), incubated with 2% BSA/PBS for 2 h to avoid unspecific binding of primary antibodies. The sections were then incubated overnight at +4°C with primary antibodies diluted in 2% BSA/PBS. After three washing steps in 2% BSA/PBS, sections were incubated with the secondary antibodies/dyes/toxins in 2% BSA/PBS for 3 h at room temperature and in the darkness. Antibodies, dyes, and dilution rates are listed in Confocal images obtained from muscles cryosections were processed and analyzed using ImageJ (FIJI version). From maximum projections of the images, areas which showed the presence of mIgG, collagen I, or actin were manually thresholded, areas selected and measured. The entire area of the cryosection was contoured by hand using the background signal. The areas where dystrophin-outlined cells were present were obtained from maximum projected confocal images to which a median filter was applied and then thresholded. Subsequently the “analyze particles” function of ImageJ was used . The image obtained was then measured. Muscle fibers, CNFs, and eMHC+ fibers were counted manually. For each sample, a minimum of three cryosections was processed and analyzed. NMJs, identified by AChR and VAChT expression together with their shape and/or position with respect to muscle fibers, were analyzed and quantified manually. Where indicated, the areas positive for AChR and/or VAChT (per endplate) were thresholded, segmented and measured. Numbers of analyzed synapses are given in t-test or Welch test. Data compilation was done in Microsoft Excel for Mac, plots were made using either Microsoft Excel for Mac or Graph Pad Prism. Figures were prepared in Adobe Photoshop and then compiled in Adobe Illustrator.For statistical analysis, data were first screened for normality and homoscedasticity using Kolmogorov–Smirnov test and F-test. Mean and SD/SEM were calculated. Then, significance was assessed with Student’s To understand the effects of glycerol injection on muscle integrity and regeneration, a panel of markers for muscle necrosis, function, and regeneration was analyzed. Therefore, TA muscles were injected with 20 μl of either saline (control) or glycerol and then muscles were harvested after 18 h and 3, 5, 8, and 11 days. Sliced muscles were co-stained with DAPI for nuclei and antibodies against mouse IgG, collagen I, dystrophin, or eMHC to assess the status of necrosis, fibrosis, muscle fiber integrity, and regeneration. Co-staining with phalloidin served to visualize the presence of f-actin. Confocal images of these sections revealed a massive muscle damage already after 18 h of glycerol injection . As showTo visualize the effect of glycerol injection on NMJs, TA muscles injected with either glycerol or saline (control) were taken at days 3, 5, 8, and 11 after injection. Slices were stained against postsynaptic nicotinic AChR and presynaptic VAChT with fluorescent α-bungarotoxin (αBGT) and anti-VAChT antibody, respectively. Confocal microscopy analysis of these samples revealed that while AChR+ sites were present at all timepoints, many of these were negative for VAChT at 3, 5, and 8 days post glycerol injection . Quantitn = 3 muscles) at day 11 post injection, while it remained low in corresponding saline controls . In summary, these results suggested that ASC application induced a late-phase inflammatory response and basal regenerative processes.The second goal of this study was to address the effect of ASCs on muscle and NMJ regeneration. Thus, directly after intra-muscular application of either saline or glycerol, ASCs were administered systemically via the tail vein. Then, microscopic analysis was performed to evaluate the consequences of ASC treatment on muscle de- and regeneration. Therefore, muscles were harvested at days 3, 5, and 11 post injection and then sliced. Sections were stained for nuclei and mouse IgG, collagen I, dystrophin, eMHC, or f-actin. While fiber numbers, dystrophin and f-actin results were similar to the samples not treated with ASCs, the data for mIgG, collagen I, eMHC, and CNFs showed interesting differences . Indeed,n = 3 muscles), they reached 65.1% ± 10.3% and 83.4 ± 1.3%  upon ASC injection. However, in ASC-treated mice, the numbers dropped at day 11 to a value of 57.3 ± 3.7%  (n = 3 muscles). Thus, although ASC treatment showed some beneficial effect on NMJ recovery at early timepoints, this effect did not persist.Finally, the effects of ASC treatment on NMJs were quantified. Therefore, muscle slices from animals treated with glycerol plus ASCs or saline plus ASCs were labeled for AChR and VAChT and then analyzed with confocal microscopy . Countinmuscles) , insteadStem cells have become a widely studied option to treat neuromuscular disorders. However, while their effects on the sarcomeric part of muscles have been investigated regularly, little is known on their regenerative capacity with respect to NMJs. In this study, a glycerol-based muscle damage model was employed to address three biological questions: (i) If a low dose of glycerol could trigger a degeneration – regeneration process, (ii) how this affected NMJs, and (iii) if and how a systemic treatment with ASCs could modulate muscle and synapse recovery.Most studies based on glycerol damage applied quantities of 50–100 μl of glycerol per mouse TA muscle . This prAn analysis of post- and pre-synapses in the presence and absence of glycerol showed that the two synaptic compartments responded differently to the treatment: while a small but insignificant loss of post-synapses was detected over the entire experimental period, the pre-synapse marker VAChT was missing in around 60% of postsynaptic regions at 3 days post glycerol injection and then steadily recovered at the later timepoints . A more Adipose-derived stromal cells were previously found to be interesting for clinical application due to their differentiation capacity, their beneficial effects on other cell types involved in tissue regeneration, and their immunomodulatory properties. In the present work, systemic ASC injection plus local glycerol application led to two major differences in the muscle degeneration – regeneration profiles as compared to glycerol injection alone: First, the reduced amounts of mIgG and collagen I as well as the early detection of eMHC and presynapse regeneration jointly indicated a slightly less severe damage at day 3 post glycerol injection. Second, however, the increasing amounts of mIgG, fibrosis, and CNFs at day 11 post glycerol suggested a secondary damage or differing regeneration phase. The latter effect of ASC application might be due to a mobilization of immune and stem cells into the muscle, causing a stronger regeneration phase. Initially, ASC may dampen the initial degeneration and inflammation by reducing the degree of necrosis and consequently the intensity of neutrophil-induced inflammation. In the following, they may impact the repair and regeneration phase for instance by an IL-6 dependent mechanism, as direct injection of mesenchymal stem/stromal cells was found to increase muscle regeneration via promoting the switch from M1 to M2 macrophages, hence, stimulating muscle stem cell activity . FinallyIn conclusion, although our data suggest a partially positive effect of ASCs on the recovery of NMJs in the early regenerative phase, a secondary degenerative or aberrant regenerative period reverted these benefits at a later stage. Due to the manifold effects of ASCs on various phases of de- and regeneration, which were likely involving muscle and neuronal cells, endothelial cells, but also all kind of immune cells, future studies need to further explore the mechanisms involved and work around late degenerative processes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the Regierungspräsidium Karlsruhe.NG and RR contributed to conception and design of the study. MR, SH, and TS performed the experiments and statistical analysis. MR and RR wrote the first draft of the manuscript. KB and MH contributed to resources and materials. All authors contributed to manuscript revision and, read and approved the submitted version.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.


Objective: Segmental demyelination is the pathological hallmark of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), but other elementary lesions are frequently observed, configuring a series of different pathological pictures. In this article, we review the pathological findings of a large series of sural nerve biopsies from our cohort of CIDP patients. Patients and Methods: Patients with CIDP who underwent nerve biopsy were retrospectively selected from those referred to the Institute of Neurology of the “Università Cattolica del Sacro Cuore” in Rome, Italy, from 1982 to February 2020. Sural nerve biopsy was performed according to standard protocols. Results: Sural nerve biopsy was performed in 43/130 CIDP patients. Demyelinating abnormalities and axonal loss were found in 67.4% and 83.7% of biopsies, respectively. Conversely, onion bulbs and inflammatory infiltrates were rare . In three cases, we observed normal pathological findings. Conclusions: A pathognomonic pathological finding of CIDP cannot be established, but we confirm the utility of nerve biopsy in this setting to confirm the diagnosis  and to elucidate pathogenic mechanisms. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinically heterogeneous, roughly symmetric, sensory and motor neuropathy of likely immune origin ,2,3. ItsIn its classic presentation, CIDP appears as a mainly motor neuropathy, affecting both distal and proximal muscles of the four limbs, along with sensory involvement and generalized areflexia, evolving as a monophasic, relapsing, or progressive disorder in more than two months. However, there is a remarkable heterogeneity in clinical presentation, and several variants of CIDP have thus far been described, all characterized by electrophysiological and/or histopathological features of segmental demyelination . Demyelinating lesions are distributed in a multifocal pattern in the peripheral nervous system. The examination of sensory nerve biopsy represents a privileged instrument both for diagnostic purposes and for understanding possible pathogenic mechanisms ,15,16,17Segmental demyelination is typically considered as the pathological hallmark of CIDP, but other elementary lesions are frequently observed, including axonal degeneration, proliferation of Schwann cells leading to the formation of “onion bulbs”, and inflammatory infiltrates. In a minority of cases, endoneural and intramyelinic edema, and axonal shrinking have been described too ,16,17,18For many years now, macrophage-mediated demyelination has been described in CIDP. The first description by Prineas demonstrated, using electron microscopic examination of nerve samples from patients with recurrent idiopathic polyneuropathy, that myelin breakdown is initiated by macrophages penetrating Schwann cells, and in the following years, this mechanism was confirmed in other studies ,16,17,18Recently, autoantibodies against nodes of Ranvier and paranodes have been identified, and their association with distinct subgroups of CIDP patients has been described .All the described alterations can combine in many ways, configuring a series of different pathological pictures.In this article, we review the pathological findings of a large series of sural nerve biopsies from our cohort of CIDP patients in order to underline the most frequent pathological alterations and to make a correlation with clinical findings.Patients were retrospectively selected from those referred to the Institute of Neurology of the “Università Cattolica del Sacro Cuore” in Rome, Italy, from 1982 to February 2020.Diagnosis was defined according to the European Federation of Neurological Societies and the Peripheral Nerve Society (EFNS/PNS) diagnostic criteria for CIDP , includiAs regards the phenotype, based on clinical and electrophysiological features, patients were further divided into two classes: “typical” and “atypical” CIDP .Patients with an approximately symmetrical sensory-motor neuropathy, with motor involvement grossly more prominent than the sensory one, were labeled as having a “typical CIDP”. In contrast, in the second group, we included all the patients with any of the atypical variants of CIDP so far described: distal acquired demyelinating symmetric neuropathy (DADS neuropathy); multifocal acquired demyelinating sensory and motor neuropathy (MADSAM neuropathy or Lewis–Sumner syndrome); pure motor CIDP; sensory CIDP; and focal CIDP .Furthermore, according to the disease course after the initial phase, three different types of disease course were recognized: monophasic course, relapsing-remitting course, chronic progressive course .Sural nerve biopsy was performed, after obtaining informed consent, as previously described . Light aStatistical analysis of data was performed by SPSS  version 24.0 to assess any differences between the group of patients who underwent nerve biopsy and the group of patients for which nerve biopsy was not performed (biopsy performed vs. biopsy not performed). Moreover, considering only patients who underwent nerve biopsy, statistical analysis was carried out to assess any differences based on the clinical phenotype . Mann–Whitney U test and Fisher’s two-tailed exact test were used to compare numerical and nominal dichotomous variables, respectively. In case of categorical polytomous variables, a Chi-squared test was performed. Significance was set at 0.05.The study was approved by the “Fondazione Policlinico Universitario A. Gemelli IRCCS—Università Cattolica del Sacro Cuore” Ethics Committee, Rome (Prot. 23255/16 ID 1229). The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki , as reflected in a priori approval by the institution‘s Human Research Committee.A total of 130 patients  were diagnosed with CIDP in the period examined. Male-to-female ratio was 1.8. Mean age at onset was 48.7 years . Mean follow-up was 97.0 months . A typical presentation was observed in 84/130 (64.6%) patients; conversely, an atypical phenotype was observed in 46/130 (35.4%) patients .Demographic and clinical characteristics of the entire CIDP cohort are summarized in A total of 43 out of 130 (33.1%) patients  underwent a nerve biopsy. Male-to-female ratio was 1.9. Mean age at onset was 46.7 years . Mean follow-up was 139.5 months . Mean age at biopsy was 51.8 years . Mean time from disease onset to nerve biopsy was 68.8 months .According to diagnostic criteria, 38/43 had a definite CIDP, 2/43 had a probable CIDP, 0/43 had a possible CIDP, and 3/43 did not fulfil EFNS/PNS diagnostic criteria .A typical presentation was observed in 30/43 (69.8%) patients . Mean agAn atypical phenotype was observed in 13/43 (30.2%) patients . Mean agDemographic and clinical characteristics of CIDP patients who underwent nerve biopsy and their comparison with the remaining cohort are summarized Obvious demyelinating abnormalities in both semithin sections and teased fiber analysis were found in 16 biopsies (37.2%). Conversely, demyelination was detected only in semithin sections (fibers with very thin or absent myelin sheath) in seven cases (16.3%) a, and onElectron microscopy confirmed the alterations seen on light microscopy and in some cases showed macrophages loaded with myelin debris invading the basal lamina of myelinated fibers a–d. In three patients, evidence of demyelination and/or remyelination was not evaluable because of the severe axonal loss. In the remaining 11 patients (25.6%), there was not any feature suggestive of either demyelination or remyelination.Onion bulbs and inflammatory infiltrates, considered as other typical features of the disease, were present only in eight (18.6%) and two (4.7%) patients, respectively a–d.Subperineural edema was detected in four patients (9.3%), and endoneural edema in another patient (2.3%). Marked myelin swelling due to intramyelinic edema was observed in three other cases (7.0%) a–e.In the great majority of nerve specimens , we detected a variable degree of axonal loss , sometimes with focal distribution a. WallerDetailed pathological findings are summarized in In our cohort, a similar percentage of biopsies was performed in patients with typical and atypical phenotypes, and no specific pathological alteration was predominant in one group than in the other. Comparisons between pathological findings among the patients who underwent nerve biopsy based on the clinical phenotype are reported in Our series confirms that segmental demyelination is the pathological hallmark of CIDP, together with other elementary lesions, as widely reported in the literature on nerve biopsies and in rare autopsy studies  22]. Im. Im22]. Several factors contribute to such variability. Firstly, the observed picture on nerve biopsy represents only a snapshot of the cascade of events occurring over time. Therefore, the type of lesions observed is strictly dependent on the disease phase in which the sampling is performed. Secondly, CIDP is a multifocal disorder where the spatial distribution of the lesions follows the laws of variability and unpredictability typical of stochastic phenomena, including inflammatory processes . The nerSegmental demyelination is characterized by the destruction of the myelin sheath which generally involves short (less than one internode long) segments, while the axonal structures remain intact. In our series, demyelination was found in 67% of patients  4,6,7,8,15,16,17Demyelinating phenomena can be observed in light microscopy and/or electron microscopy, but teased fiber examination allows a more accurate identification and quantification of these phenomena a,b 4]. . 4]. The American Academy of Neurology (AAN) commission suggested quantitative criteria to establish “unequivocal evidence of demyelination and/or remyelination” in nerve biopsies of patients with CIDP: at least 12% of 50 dissociated fibers, minimum of four internodes each, must have these alterations, or by electron microscopy, a minimum of five fibers must show demyelination .Although demyelination represents the prevalent and probably primary event of CIDP, pathological examination shows that, in most patients, this lesion is associated with a significant loss of fibers a. In ourAxonal involvement can represent an “innocent by-stander” effect, a non-specific consequence of an inflammatory process that primarily affects myelin. However, it should be borne in mind that the distinction between axonal and demyelinating pathologies, while retaining an undoubted value in the diagnosis and classification of neuropathies, is an artificial simplification since neuritis and Schwann cells form a morpho-functional unit in which the two components are strictly interdependent .A “pure” demyelinating disease, in which the nerve biopsy showed only myelin destruction without the involvement of the axons, was observed in only 5% in our cohort (two patients) ,15,16,17It is important to underline that, in about 25% of our cohort, the nerve biopsy does not show clear demyelinating phenomena but only a loss of fibers, associated or not with axonal degeneration in the active phase. This finding may be a consequence of the multifocality of the disease. Fiber loss will be the only alteration when active inflammation, responsible for demyelination and destruction of the axons, occurs in the segments proximal to the site of the biopsy sample. Confirming this data, in all patients with pathological evidence of “axonal” neuropathy from our cohort, neurophysiological investigations  show signs of demyelination. However, rare cases have been reported in which both the pathological examination of the nerve and nerve conduction studies showed an exclusively axonal involvement . One posThe hypothesis of an inflammatory nature for CIDP is related to the observation that inflammatory cells, namely macrophages and T cells, actively participate in the pathogenesis of the disease . HoweverIn one patient in our cohort, we detected antibodies against Contactin-1 : the patIn a variable percentage , the morIn our cohort, 7% of biopsies were completely normal, a percentage similar to that reported in the literature, ranging from 10% to 20% of cases. This can be explained once again by the multifocal nature of CIDP, in which the nerve sample may not show any alterations because it is spared from the inflammatory process by chance. Furthermore, normal aspects of nerve biopsy, when associated with the absence of clinical and electrophysiological sensory involvement, occur in pure motor CIDP, which has been suggested to represent a distinct nosological entity ,34,35. FIn some patients , the nerIn conclusion, we confirm that nerve biopsy may be helpful in the diagnosis of CIDP and may probably contribute to improve our understanding of pathogenetic mechanisms and to identify proper treatments.


Snakebite envenoming (SBE) is a public health issue in developing countries. The estimated annual global incidence of snakebites is about 5.4 million snakebites per year, resulting from 1.8 to 2.7 million cases of SBE and from 81,000 to 138,000 deaths with 400,000 survivors suffering permanent physical and psychological disabilities. There are more than 3000 species of snakes around the world: 600 are venomous and over 200 are considered to be medically important because of their clinical effects. The severity of SBE depends on several factors among which bite localization, snake’s size, condition of glands and teeth, bite angle and bite duration, the microflora of the snake’s mouth and victim’s skin, age of the victim, weight, health status, and victim’s activity after a bite. Snake venoms are mixtures of protein families, and each of these families contains many different toxins or toxin isoforms. Based on their effects, snake venoms can be classified as hemotoxic, neurotoxic, or cytotoxic and they can all act together involving multiple tissues and organs. When the bite is fatal, the mechanism of death is primarily related to the paralysis of respiratory muscles, which causes asphyxia and hypoxic-ischemic encephalopathy, but also anaphylactic shock, hemorrhagic shock, cardiomyopathy, acute tubular necrosis (ATN). The purpose of this literature review is to evaluate epidemiological and post-mortem examination findings in fatal SBEs in order to better understand the pathophysiological mechanisms, thus helping pathologists in defining the correct diagnosis. SBE is a potentially life-threatening disease resulting from the injection of a mixture of different toxins (venom) that particularly afflicts the poorest people living in developing countries with the worst quality of life . In 2017There are more than 3000 species of snakes around the world: 600 are venomous and over 200 are considered to be medically important because of their clinical effects . New speThe search of PubMed, Scopus, Web of Science, and Google Scholar databases provided a total of 263 articles. After adjusting for duplicates and reviewing titles and abstracts, 228 studies were discarded. Finally, only 35 papers reporting, in total, 56 cases of fatal SBE satisfied the inclusion criteria. A flowchart depicting the selection of studies according to PRISMA standards  is reporViperidae (n = 28–50.000%) followed soon after by Elapidae (n = 23–41.071%) and Lamprophiidae (n = 1–1.786%). In four cases (7.143%) no additional information about the snake was available. Most of the fatal cases of SBE reported can be classified into category 1 (n = 28) according to the WHO Guidelines on production, control, and regulation of snake antivenom immunoglobulins [The most common families of snakes involved in fatal SBE were dae n = 2–50.000% lobulins ,33, the n = 35 cases; 62.500%) with a male/female ratio (M/F) of 1.67. Male victims were older than females. The median age in years for males was 40.77 and for females 28.71. The overlapping mean age was 36.25 years.Most of the SBE victims were males (The body part most frequently bitten by snakes was the lower limb in 31 cases (55.357%), the upper limbs in 15 cases (26.786%), in one case the face (1.786%), and in one other case the genitalia (1.786%). Unfortunately, in eight cases (14.286%) the site of the snakebite was not reported. The vast majority of the SBE was accidental, but in one case study involving three victims, the manner of death was assessed as homicide [n = 12) is reported from Australia, followed by Myanmar and India (n = 7 each), Brazil and Sri-Lanka (n = 6 each). However, all continents in the world are represented: 41.071% of victims were from Asia (n = 23), 21.429% from Oceania (n = 12), 19.643% from America , 14.286% from Africa (n = 8), and 3.571% from Europe (n = 2). It is worth mentioning that 16 out of 56 cases were related to work activities. Most of the cases occurred in a rural context .The largest number of victims  and before 6h in 19 cases (33.929%). Most of the victims, 40 out of 56 cases (71.429%), were hospitalized and the specific antivenom was administered to 26 individuals. In a few cases, the victim was at first treated by a local healer or with traditional remedies ,20,28,436% and beMost of the bite marks (19 out of 56 cases) are described as two puncture wounds from the animal’s fangs. Just a single puncture wound has been found in three cases, sometimes scratched or lacerated by the movement of the victim and/or his clothing deviating the fangs . AdditioIn this regard, it is worth mentioning that for the identification of the offending snake the number of puncture wounds must be related to the number of teeth, which is unfortunately not the same in all kinds of snakes. Different species of snakes can have different anatomical conformation of the teeth and fangs. Fangs are sharp, long, hollow, or grooved teeth that are connected to a small sack full of venom in the snake’s head behind its eyes but not all snakes have fangs. Most snakes have six rows of teeth in total: one row on each lower jaw and two rows on each maxillary and palatine or pterygoid bones of the upper jaw . It is an = 8) [With regard to the morphology of the fangs, sometimes the bite injuries are also described as linear or curvilinear stripes  fresh lin = 8) ,37,44,45n = 8) , a rash n = 8) , and a pn = 8) . The main = 8) ,27,39,44n = 8) ,28,34,36n = 8)  Myocardin = 8) ,27,35,39n = 8) . Signs on = 8) ,24,25 ann = 8) ,19,20,39n = 8) ,37,39,42n = 8)  and widen = 8) . Microthn = 8) ,42. PituBungarus spp., cobras—Naja spp., taipans—Oxyuranus spp., coral snakes—Micrurus spp., death adders—Acanthophis spp., and tiger snakes—Notechis spp.) and to the Viperidae family . According to the geographical latitudes, the species of snakes causing the largest numbers of bites and fatalities are Echis sp.  in Northern Africa, Bothrops asper and B. atrox (lance-headed pit vipers) in Central and South America, and Naja sp. (cobras) and Bungarus sp. (kraits) in Asia [SBE is an acute medical emergency and a major public health problem that kills thousands of people each year, particularly in rural territories . Most of in Asia . SBE is  in Asia . Snake v in Asia . The sev in Asia ,54,55.However, the main deadly factor is represented by the components of the venom. Snake venoms are mixtures of protein families, and each of these families contains many different toxins or toxin isoforms . A very Based on their effects, snake venoms can be classified as hemotoxic, neurotoxic, or cytotoxic and they can act all together involving multiple tissues and organs . NeurotoUsually, snakes from the Viperidae family are considered hemotoxic . ViperidIn the cases included in our review, the gross findings were non-specific. Autopsy findings reported are mostly non-specific pathologic changes consistent with a rapid death such as multi-visceral congestion, edema, and petechial hemorrhages. Signs of ischemic and hemorrhagic encephalopathy can be found mostly represented by intracranial or subarachnoid hemorrhages as well as myocardial hemorrhages. Widespread microthrombosis has been also described as the result of the coagulation disorders caused by the hemotoxic effects of SBE . A relatCrotalus durissus terrificus venom (CdtV) on the pulmonary mechanic events have been studied in male swiss mice [Crotalus durissus cascavella venom have been also investigated in mice by Azavedo et al. (2020) [Other than pulmonary edema and congestion related to acute cardiac failure, pulmonary injuries by SBE are mostly represented by DAD and widespread microthrombosis in the DIC scenario. In particular, the effects of iss mice ,79. The . (2020) . They re. (2020) .Acute nephrotoxicity is a relevant complication of SBE mostly represented by AKI that is a major cause of morbidity and mortality. AKI following SBE is mostly related to ATN or ACN and has multifactorial pathogenesis including, hypotension, coagulopathy, intravascular hemolysis, thrombotic microangiopathy (TMA), and DIC, capillary leak syndrome, rhabdomyolysis, complement activation, and sepsis . Once deSBE is a public health issue in tropical countries. The identification of snake species can be crucial for appropriate clinical management and can improve the prognosis. Although snake venom can be easily detected by toxicological analysis, diagnosis of SBE related death is challenging for the forensic pathologist. The cause of death from SBE is primarily related to the neurotoxic, cytotoxic, and hemotoxic effects of the venom components including the paralysis of respiratory muscles, which causes asphyxia and hypoxic-ischemic encephalopathy, hemorrhagic shock, cardiomyopathy, ATN, and death from anaphylactic shock . Key eleThe review of the literature was conducted independently by two examiners and carried out according to the PRISMA statement .This retrospective study was performed using the following electronic databases: PubMed, Scopus, and Web of Science. The following keywords were searched in all fields: “snake bite” AND “autopsy” OR “snakebite” AND “autopsy” to recognize relevant research available until 18 April 2020. No time limit was set. The systematic search was further extended by snowball search and hand searching by using Google Scholar.The following inclusion criterion was adopted: (1) papers reporting autopsy cases of fatal SBE with a description of the autopsy and/or histological findings. Exclusion criteria comprised the following: (1) animal studies; (2) scientific article not published in English, and (3) full text not available. For duplicate studies, only the article with more detailed information was included.Two examiners independently provided the initial selection of the articles to determine whether they might potentially fit the inclusion criteria. The title, abstract, and full text of each potentially pertinent study were reviewed. Disagreements on the eligibility of the studies were solved between the two examiners; if no agreement could be reached, it was resolved using a third co-author as a reviewer. A data extraction sheet was developed and for each selected study the following items were recorded: snake species; details of the victims ; location ; clinical history ; autopsy features .


The contribution of dysregulation of lipid signaling and metabolism to neurodegenerative diseases including Alzheimer’s and Parkinson’s is the focus of this special issue. Here, the matter of three reviews and one research article is summarized. Lipidomics is a newly emerged discipline that studies cellular lipids of biological systems on a large scale of pathways and networks by exploiting advanced technological tools ,2, whereIn the first review , Mesa-Hesubstantia nigra in the midbrain region and aggregation of the α-synuclein protein, which disturbs the function of the CNS. Despite occurring at different brain locations and reporting different clinical features, AD and PD share pathological intersections so that patients with AD prove in some cases a higher chance of developing PD. While the mutual pathological overlap may be linked to genes, mitochondrial dysfunction, neuroinflammation, tau protein, and α-synuclein protein [http://www.lirmed.com/tam2/) [In the second review , Rana et protein , common  protein . In the  protein , Rana etm/tam2/)  with theThe third review  focuses These changes of SL levels observed in biofluids may reflect alterations in nervous degenerating tissues, so a correlation has been inspected by comparing studies carried out on white matter of temporal cortex and cerebellum, prefrontal cortex, frontal gyrus, and anterior cingulate cortex. Again, in brain tissues, an increased content of Cer d18:1/16:0 is described in agreement with most of the case–control and longitudinal studies on biofluids. Indeed, the recurrent altered level of Cer C16:0 has been associated with its capacity to mix well with cholesterol and consequently influence the functionality of lipid rafts that, becoming enriched in ceramide, activate both intrinsic and extrinsic apoptotic pathways . Lipid msubstantia nigra. Skin fibroblasts are now broadly recognized as a suitable model of primary human cells, able to reflect the chronological and biological aging of patients since they share the genetic information of neuron sprouts [p < 0.05); among them, 8 lipids, namely, PI 38:3, PC 34:1, PE 38:2, and SM 36:2;2, and 4 plasmalogens seem to follow an ascending or descending trend as the severity of the disease increases. Statistical analyses show abnormality of GP metabolism in the PD group, suggesting that the downregulation of ethanolamine plasmalogen (pPE) supply in the circulation system, especially those containing PUFA, might be likely associated with neurodegeneration. However, for a definitive application of these potential biomarkers in clinical diagnosis, validation in a larger population should be further achieved.The fourth research paper of this SI  aimed to sprouts . A lipid sprouts ). By uni


Traditional Chinese medicine plays an irreplaceable role in the treatment and prevention of epidemic diseases in China. Acupuncture is an important part of Chinese medicine. During the coronavirus disease 2019 (COVID-19) epidemic, acupuncture and related interventions are used to treat COVID-19 patients in China. The systematic review aims to evaluate the efficacy and safety of acupuncture and relevant interventions for anxiety in COVID-19.We will search for randomized control and observational studies of acupuncture and related interventions for anxiety in COVID-19 in the 6 databases from inception to 31 October 2020. There is no language restriction. Two independent reviewers will screen and collect all trials, data extraction and evaluate the risk of bias of the researches. We will perform a meta-analysis if appropriate.Our findings will evaluate the feasibility of acupuncture and related interventions as adjunctive therapy for anxiety in COVID-19 patients, which will be disseminated in a relevant conference and published in a peer-reviewed publication.Our research will appraise the overall quality and evidence of whether acupuncture and related interventions are effective therapies for anxiety in COVID-19. Unfortunately, there is currently no specific antiviral therapy for this pandemic. The clinical manifestations of COVID-19 appear to be broad, including mild upper respiratory tract disease, severe pneumonia, and even death.,2Recently, an outbreak of the highly infectious novel coronavirus disease 2019 (COVID-19) has swept globally. Acupuncture is 1 of the TCM treatments. During the severe acute respiratory syndrome epidemic, the intervention of acupuncture has played a useful role. Clinical observation study found that acupuncture can relieve chest distress, fatigue, and other symptoms in patients with severe acute respiratory syndrome in convalescence. In the current Chinese medical system, TCM and western medicine are equally important, and TCM's participation in the treatment of COVID-19 patients is encouraged by the government. Chinese doctors and scientists have conducted many clinical studies on the treatment of COVID-19 with the combination of TCM and western medicine.,6,7 It is worth noting that most COVID-19 patients in China have received integrated Chinese and western medicine. The comprehensive application of acupuncture, moxibustion, and Chinese herbal medicine has provided new ideas for the treatment and prevention of COVID-19. A retrospective observational study has shown that acupuncture (in conjunction with routine treatment) significantly improved chest pain, fatigue, insomnia, anxiety, nervousness, nausea, vomiting, and other symptoms in patients with COVID-19.In China, historical records indicate that traditional Chinese medicine (TCM) plays an indispensable role in the prevention and treatment of epidemics. It is worth noting that the Chinese association of acupuncture and moxibustion has proposed and issued the guideline for acupuncture and moxibustion interventions on COVID-19 (Second edition). The main contents are shown in Table  A randomized controlled study on intervention for the patients under quarantine after close contact with COVID-19 is undergoing. The study uses the internet and mobile phone software to remotely guide patients to moxibustion themselves, reducing the contact between doctors and patients.Some of the intervention measures in clinical practice are a mixture of acupuncture and other relevant therapies, while acupuncture is delicately defined as stimulation of acupoints, with penetration into skin by thin metal needles. In other words, acupuncture and other relevant therapies also include moxibustion and transcutaneous electrical nerve stimulation. During the COVID-19 epidemic, the general public (including COVID-19 patients) and healthcare workers are all likely to experience anxiety and insomnia.,15 Acupuncture is an excellent symptom management therapy. Acupuncture and related interventions are often used to relieve common symptoms including vomiting, nausea, insomnia, dyspnea, fatigue, abdominal pain as well as anxiety disorders. –26 Possible related symptoms of COVID-19 treated with acupuncture is shown as Figure  Another systematic review revealed that there is good evidence to encourage the treatment of anxiety disorders with acupuncture, because it produces effective results and has fewer side effects than traditional therapies. Studies on data mining also summarizes the commonly used acupoints for anxiety treatment, as shown in Figure  Therefore, we intend to systematically review the clinical trials of acupuncture and related interventions for anxiety in COVID-19 patients. We aim to provide a more reliable evidence base for clinical practice in treating anxiety in COVID-19.The clinical presentation of COVID-19 has involved fever, cough, headache, abdominal pain, fatigue, and so on.22.1Our research was registered in PROSPERO (CRD42020190153) on 5 June 2020. Also, this protocol report follows Preferred Reporting Items for Systematic Review and Meta-analysis Protocols guidelines.2.22.2.1Randomized controlled trials (RCTs) and observational trials, including case control studies and cohort studies, that have tested acupuncture and related therapies interventions with or without western medicine for anxiety in COVID-19, will be included. Publications in any language will be included.2.2.2,32Participants who were diagnosed as COVID-19 with anxiety will be included, without limits on age, gender, and race. The diagnosis of COVID-19 includes Chinese or international diagnostic criteria.2.2.3Participants undergone acupuncture and related interventions treatment such as, electroacupuncture, acupuncture, fire needle, transcutaneous electrical nerve stimulation, auricular point therapy, and moxibustion will be included in the experimental group. There is no restriction on the duration.Patients who have treated with interventions such as sham acupuncture, routine care, or conventional therapy will be included in the control group.2.2.4The primary outcomes will be the therapeutic effects of treatment on anxiety. Both qualitative and quantitative outcomes, change in anxiety score from baseline to the last available follow-up, such as the state-trait anxiety inventory and shortened 10-item version of the state-trait anxiety inventory assessing the severity of anxiety will be allowed.–36 Furthermore, for COVID-19, which is an infectious disease, the risk of acupuncture application also needs to be considered. Therefore, the safety of physicians is also an outcome measure.The secondary outcomes will be evaluated based on adverse effects and adverse events.2.32.3.1We will search 6 electronic databases, including the VIP, China National Knowledge Infrastructure, Wan Fang Data, EMBASE, Medline as well as Cochrane Central Register of Controlled Trials (CENTRAL) from their inception till 31 October 2020. There will be no limitation on the language of the included studies. The combined use of medical subject headings and keywords is of considerable significance to the retrieval of related trials. Moreover, our searching strategy for Medline is shown in Table 2.42.4.1Two independent researchers will generate and apply a standardized selection form to select appropriate researches. In the event of a disagreement, the third investigator will make the final decision. We will describe and summarize the selection process for each study in the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols flowchart, with reasons for exclusion. The process of screening operation is shown in Figure 2.4.2All data extraction will be independently undertaken by 2 investigators using predesigned forms, including study design, general information, characteristics of patients, comparison interventions, and outcomes. For studies with uncertain data, we will contact the authors by telephone or email to obtain the complete data. And studies without sufficient information will be excluded. If there is any dispute during data extraction, it will be referred to a third reviewer.2.4.3 And disagreements will be discussed with another reviewer. Each RCTs will be assigned as unclear risk, low, or high of bias for 7 domains , and a revised version of the Newcastle–Ottawa Scale for observational studies.,39Two independent investigators will assess the risk of bias of included RCTs studies using the Cochrane Collaboration's risk of bias tool.2.4.4For continuous outcomes, data will be analyzed by using a weighted mean difference (WMD) or mean difference with 95% confidence intervals, while dichotomous data will be expressed as relative risk, with confidence intervals of 95%.2.4.5I2 statistics will be used for the evaluation of heterogeneity, with an I2 of >50% or P < 0.1 considered indicative of high heterogeneity.Both the Chi-squared and 2.4.6If a sufficient number of trials are included, we will assess small study biases (such as publication bias) with funnel plots. For continuous variables, Egger tests will be recommended. For the dichotomous data, we will select the Harbord and Peters test.2.4.7I2 and Chi-squared test statistics. If the included studies have no obvious heterogeneity (I2 < 50% or P > 0.1), we will use a fixed-effect model for calculation. Or else, a random-effects model will be used.,40–43Stata, version 14.0 (StataCorp LLC), will be used to analyze the data. Studies that examine the same interventions and outcomes in similar populations will be combined using the meta-analysis to estimate the combined intervention effect. Heterogeneity among trials will be identified by the 2.4.8To test the robustness of the review findings, we will perform a sensitivity analysis for the primary outcome according to the statistical model.2.4.9If sufficient studies can be identified according to age, duration of treatment and different interventions, we will conduct a subgroup analysis.2.4.10We will use the Grading method of Recommendations Assessment, Development and Evaluation to rate the quality of evidence for each outcome as low, very low, moderate and high level.2.5Ethical approval is not required because no individual patient or animal information is collected.3–47 This research will provide evidence-based medicine to determine whether acupuncture and related interventions are effective interventions for patients with anxiety in COVID-19.Considering that COVID-19 is an important public health issue, the efficacy of acupuncture combined with routine treatment of COVID-19 remains inconclusive, and the findings of this research add some evidence to the knowledge in this field. The main challenge with this study is the generally poor quality of clinical studies on acupuncture.Conceptualization: Feng Qi and Haowen Jia.Data curation: Kai Zhang and Zhenzhen Han.Formal analysis: Kai Zhang and Haowen Jia.Investigation: Haowen Jia, Hongwen Huang and Kai Zhang.Methodology: Kai Zhang and Hongwen Huang.Software: Qilin Tang and Kaihang Sun.Supervision: Qilin Tang.Writing – original draft: Haowen Jia, Kai Zhang and Haowen Jia.Writing – review & editing: Qilin Tang.