COVID-19 has negatively influenced industrial development, family consumption, and residents’ mental health. Unfortunately, it has not yet been studied whether this adverse situation can be alleviated after the relaxation of the COVID-19 control policy (RCC). Therefore, this study aimed to analyze the effect of the RCC on the resident’s mental health and the mediating effect of family tourism consumption. By using the PSM and mediating effetc model to research the panel data of two periods (April 2021 and April 2023) for Shaanxi province, China.The RCC negatively inhibited the mental health severity of residents, and the mental health severity decreased by 0.602. In particular, the RCC showed the most substantial negative effect on residents’ stress, followed by anxiety and depression. Meanwhile, it is found that the impact of the RCC on the mental health of residents is highly heterogeneous. The RCC indicates a linear significant effect on the mental health of residents under 60 years of age, while the results were found insignificant for residents above 60 years of age. Meanwhile, the RCC’s improvement effect on urban residents’ mental health is greater than that of rural residents. In addition, mechanism analysis showed that tourism consumption plays a mediating role in the influence of the RCC on the mental health of residents, and the mediating effect accounted for 24.58% of the total effect.Based on the findings, the study proposes that government and policymakers should strengthen mental health intervention, improve access to mental health counseling, stimulate economic development, expand the employment of residents, and track the mutation of the novel coronavirus. PreviouMoreover, much of the literature explored why COVID-19 affects mental health. Firstly, COVID-19 control policies such as wearing masks, restricting travel, keeping social distance, and quarantine policies have changed the way residents live, which may directly impact the mental health of residents through difficulties in breathing fresh air, increase in lonely time, and decrease of human interaction, as well as fear of isolation policy . SecondlIt is further believed that the COVID-19 pandemic has dampened the tourism industry and family tourism consumption due to travel restrictions, quarantine policies for suspected infections, and social distancing. The crash in international tourism due to the coronavirus pandemic also caused a considerable loss of more than $4 trillion to the global GDP for 2020 and 2021, according to a UNCTAD report published on 30 June , using tAlthough the tourism industry is a sunrise industry driven by the spiritual pursuit of residents, some scholars hold that long-distance travel requires a variety of individuals skills, such as physical fitness, concentration, understanding, decision-making, and confidence, and that mental health can affect the acquisition of these skills, which can affect tourists’ sense of experience, satisfaction, and happiness . HoweverIn summary, previous studies have not empirically tested the causal relationship between RRC and residents’ mental health. Moreover, the mediating mechanism of family tourism consumption has not been studied previously. To make up for the research gaps, the study’s main objects is to innovatively explore the effect of the RCC on residents’ mental health and the mediating effect of family tourism consumption. Furtherly, the main contribution include the following: First, we employed the differences-in-differences (DID) method to explore the impact of the RCC on residents’ mental health using micro-panel data from Shaanxi province, China. Second, considering age and urban–rural differences, this paper explored the heterogeneity of the effects of the RCC. Third, the mediating effect model was used to test the role of family tourism consumption concerning the impact of the RCC on residents’ mental health. Finally, the study provides valuable experience for other countries or governments to improve the mental health of residents in the post-epidemic era.2.2.1.Psychologists generally hold that individuals would continuously pursue happiness and success. Individuals are more likely to succeed if they can establish and maintain the personal characteristics and social status that can lead them to higher incomes and protect them from losses . Further2.2.According to COR theory, individuals experience emotional feedback when they perceive the threat of losing resources or experience the actual loss of resources , 58. WelH1: The RCC could significantly improve the mental health of residentsH1a: Compared to non-older adults, the RCC had a weak effect on improving mental health in the older adults.H1b: Compared with rural residents, the RCC had a stronger effect on improving the mental health of urban residents.2.3.COR theory holds that individuals are not passive in coping with resource loss and psychological stress but can adjust their strategies according to the resource situation . Family H2: Family tourism consumption had an intermediary effect concerning the RCC's influence on residents' tourism consumption.3.3.1.The study included the panel data for the two periods, April 1 to 7, 2021, and April 1 to 7, 2023, respectively, through a large-scale online survey of residents from Shaanxi Province, China. The main reasons for the selection of sample areas are as follows: Shaanxi Province is in the west of China, the income gap between urban and rural residents is large, and the differentiation of urban and rural sample areas is obvious. Moreover, the research group won the cooperation with China Mobile Shaanxi Co., LTD. to carry out online questionnaire survey. Specially, firstly, the Mobile Shaanxi Co., LTD. randomly informed residents of the purpose, main content, and rewards for phone calls through mobile phone messages. Secondly, if the respondent agreed, the interviewer contacted them by phone to complete the questionnaire. Finally, the research group protected the transferee’s mobile phone number information and collected panel data. The questionnaire’s main contents included the respondents’ characteristics, the characteristics of their families, their cognitive status, their employment and income, and mental health. Excluding 42 respondents who were unwilling to answer during the second survey, the questionnaire survey obtained data from 735 residents, among which 421 belonged to urban areas while 314 were from rural areas. Besides, since the questionnaire adopted the principle of random sampling and the mobile phone coverage rate of urban and rural residents in Shaanxi exceeded 90%, sample selection bias was minimized.3.2.3.2.1.In this study, the dependent variable is the residents’ mental health. Previous literature has characterized the residents’ mental health mainly from stress, anxiety, and depression. Referring to relevant studies such as Zhou et al.  and Huan3.2.2.The independent variable is the relaxation of COVID-19 control policy, categorized by the dummy variable. In December 2022, China relaxed its prevention and control measures and lowered the epidemic from A to B. Meanwhile, the novel coronavirus pneumonia was renamed novel coronavirus infection. Therefore, we assigned a value 1 to samples collected before December 31, 2022, and 0 to samples collected after that date.3.2.3.per capita, time to pay attention to COVID-19, chronic diseases, relationship network, urban or rural areas. Additionally, considering that other policy factors might directly influence the residents’ mental health, the study added the influence of government mental health counseling on residents’ mental health. The study used independent sample T to test the difference between samples A and B, and the results are reported in Referring to the studies of Xiong et al.  and Albidiff. = 0.326**), and they could not only realize the harm of COVID-19 but also treat the self-limiting disease objectively and rationally. Residents’ job satisfaction is also higher (diff. = 1.761***), and per capita disposable income has increased significantly (diff. = 0.041**). Moreover, residents have a more robust network of relationships (diff. = 0.614**). Further, they have substantially less time to pay attention to COVID-19 (diff. = −1.743*) and are less likely to receive mental health counseling from the government (diff. = −0.199*).According to 3.3.3.3.1.Since the outbreak of COVID-19 in December 2019, the Chinese government has adopted the class A management strategy for class B infectious diseases and implemented various control measures such as maintaining social distancing, wearing masks, and shutting down the infected areas. Until December 2022, the 3-year epidemic has seriously affected the mental health of residents. As the Omicron virus became less virulent, the Chinese government has adjusted and relaxed COVID-19 control. An issue worth studying is whether the mental health of residents is restored after the relaxation of the COVID-19 control policy. What are the possible mediating mechanisms? To this end, first, using the DID model, two periods of panel data from residents of Shaanxi, China, were used to empirically analyze the influence of the RCC on residents’ health levels. The model is constructed as follows:Where 3.3.2.Based on model verification (1), tourism consumption is further added to empirically test that the RCC could significantly improve residents’ mental health by improving family tourism consumption. The mediating variable “tourism consumption” is selected mainly given the following considerations: after the deregulation of COVID-19 control, the tourism industry showed retaliatory recovery and development. Meanwhile, travel is also a core mean to improve residents’ mental health. Consequently, the study employed the mediating effect model to analyze the mediating effect of tourism consumption. The hierarchical regression is constructed as follows:Where 4.4.1.According to per capita disposable income, relationship networks, and mental health counseling also negatively influenced residents’ mental health. If per capita disposable income is increased by 1 unit, their mental health severity would decrease by 0.206; if the relationship network is increased by 1 unit, their mental health severity would decrease by 0.208. Furthermore, if residents received mental health counseling from the government, the severity of their mental health would be reduced by 0.031. Additionally, some other factors could exacerbate residents’ mental health seriousness. If the proportion of the older adults and children and the time to pay attention to COVID-19 increases by 1 unit, the severity of the mental health of the residents would increase by 0.291 and 0.038, respectively.Additionally, it is believed that the mental health of the residents is also affected by certain other factors. For instance, the gender of the residents, the awareness of COVID-19, and job satisfaction are also likely to significantly and negatively influence the mental health of the residents. If the residents surveyed were men, their mental health severity would decrease by 0.206; If residents realized the harmfulness of COVID-19 and the self-limiting nature of the virus, their mental health severity would decrease by 0.406; If the residents’ job satisfaction increased by 1 unit, the severity of the residents’ mental health would decrease by 0.505. Meanwhile, 4.2.4.2.1.The DID method required that there was no significant difference in residents’ mental health before December 31, 2022, between the treatment group (samples after the RCC) and the control group (samples before the RCC). According to 4.2.2.p-value distribution in Further, exploring whether the estimation results of the DID method are biased or not due to missing variables, this paper conducted a placebo test through a randomized selection of treatment groups. Since the newly generated treatment group was random and would not affect the explained variable, its estimated coefficient should be around 0. This paper repeated the random generation process 1,000 times and reported the estimated coefficient of the random treatment group and its 4.3.Furthermore, 4.4.This study also used the mediating effect model to verify the mediating mechanism of tourism consumption related to the RCC that influences the mental health of residents. The study selected “amount of family tourism consumption in the first quarter ” to measure mediating variable “tourism consumption.” Using the hierarchical regression model, the results showed that the RCC positively affects tourism consumption. Meanwhile, the RCC and tourism consumption significantly influenced residents’ mental health. The intermediary effect of tourism consumption is 0.1492 (−0.785*0.190), and its proportion in the total effect is 0.2458 (0.1492/0.607). Therefore, 24.58% of the inhibitory effect of the RCC on residents’ mental health severity is found to be contributed by family tourism consumption. Thus, hypothesis H2 is also endorsed.5.In infectious disease prevention and control, humanity has experienced the longest, largest, and most damaging COVID-19 outbreak , 89. AgaUnlike the study of Wilding et al. , Razali In addition, considering the differences in the ages and regions of the residents, we further analyzed the heterogeneity of the impact of the RCC on the mental health of residents. Although many previous studies have confirmed a causal relationship between the COVID-19 pandemic and mental health in the older adults people , our stuWe also explored the impact of other factors on the mental health of residents. Consistent with the studies of Xiong et al.  and WallFinally, consistent with the studies of Gilbert and Abdullah , Chen etOf course, our research still has some shortcomings. For example, the RCC data was only available for 3 months, and the study only explored the short-term effects of the RCC on the mental health of the residents. Therefore, the long-term effect of the RCC on residents’ mental health needs further investigation. Moreover, some unobserved factors may also affect both the RCC and the mental health of residents, thus generating endogenous issues, which need to be further solved by obtaining survey variables and data. Besides, due to using the panel data of two periods (April 2021 and April 2023) for Shaanxi province, China, we do not have enough data points to test whether there was a pre-trend before the RCC lift.6.In the post-epidemic era, in addition to tracking the mutation of the novel coronavirus, designing targeted vaccines, and developing effective drugs, the residents’ mental health also deserves the international community’s attention. The RCC will inevitably lead to economic and social development recovery, but the residents’ mental health resilience is very weak. Whether mental health can recover effectively from RCC also needs theoretical and empirical exploration. In this paper, the panel data of Shaanxi, China, were used to analyze the effects of the RCC on residents’ mental health and the intermediary mechanism of tourism consumption, respectively, by employing the DID method and the mediating effect model. The following conclusions are drawn.per capita disposable income, relationship network, and mental health counseling also significantly and negatively influence the seriousness of mental health. However, the proportion of older adults and children and the time to pay attention to COVID-19 can exacerbate the severity of mental health. Thirdly, the RCC’s effects on residents’ mental health are heterogeneous. The RCC had a linear significant effect on the mental health of residents under 60 years, while it does not significantly influence the mental health of residents over 60 years. Meanwhile, the RCC’s improvement effect on the mental health severity of urban residents is greater than that of rural residents. Finally, tourism consumption plays a mediating role in the RCC’s influence on residents’ mental health. The intermediary effect of tourism consumption is 0.1492 (−0.785*0.190), and its proportion in the total effect is 0.2458 (0.1492/0.607).Firstly, the RCC significantly inhibited the mental health severity of residents, and the mental health severity decreased by 0.602. In particular, the RCC has a negative and significant influence on residents’ stress, anxiety, and depression, and the severity of stress, anxiety, and depression decreases by 0.805,0.406, and 0.315, respectively. Hence, the RCC has the strongest negative effect on residents’ stress, followed by anxiety and depression. Secondly, the gender of the residents, awareness of COVID-19, job satisfaction, In the post-epidemic era, it is necessary to strengthen mental health interventions. Firstly, the government should strengthen residents’ mental health identification and treatment. Community and public hospitals should increase the number of mental health clinics, expand the scope of identifying residents’ mental health issues, and initiate early intervention strategies. Meanwhile, the government should also improve access to mental health counseling through online consultation platforms, provide mental health relief and self-help information to residents, and help them improve their stress, anxiety, and depression. Secondly, the government should guide enterprises to increase tourism investment, develop tourism products, upgrade tourism formats through financial incentives, and encourage residents to expand tourism consumption to alleviate residents’ mental health issues caused by COVID-19. Thirdly, the government should stimulate economic development, expand the employment of residents, increase household income, and relieve the pressure and anxiety caused by family economic pressure. Finally, the government should continue to track the mutation of the novel coronavirus, forecast timely & provide early warning of new outbreaks, and strengthen the research and development of vaccines and drugs to reduce residents’ fear and concern about COVID-19. Besides, the government should treat residents’ mental health problems from the public health perspective, improve residents’ awareness of mental health, and eliminate social panic & discrimination against mental health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YH: conceptualization & methodology and writing original draft. HS and YD: data sources & review, revise and editing. SZ: funding. All authors contributed to the article and approved the submitted version.This work was supported by the National Social Science Foundation of China (Grant No. 21BKS171).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Triglyceride-glucose (TyG) index has been reported to be associated with various cardiovascular diseases in recent years. However, the conclusive association between the TyG index and hypertension was not established in the last meta-analysis. Furthermore, it remains unclear whether a linear relationship exists between these two variables.We conducted a comprehensive search of the CNKI, VIP, WanFang Data, CBM, PubMed, EMbase, Web of Science, and The Cochrane Library databases up until May 10, 2023, to identify relevant studies conducted in China. We used Stata version 17.0 and Rstudio version 4.2.1 to analyze the data and assess the association between the TyG index and the risk of hypertension, as well as the dose-response relationship between these two variables. We will select either a random-effects model or a fixed-effects model based on the results of the heterogeneity tests and report 95% confidence intervals accordingly.I2 = 69.0% P < 0.001]. Through meta-regression analysis of multiple covariates, we found that study type, study region, sample size, database source, and study quality score, the above five variables were able to explain 63.0% of the total heterogeneity. The results of the dose-response Meta-analysis showed: The TyG index has a linear relationship with the risk of developing hypertension, as indicated by non-significant coefficients of higher-order terms in the nonlinear model (P > 0.05). The linear trend analysis showed that for every one-unit increase in the TyG index, the risk of developing hypertension increased by 1.5 times [1.532 95%CI  P < 0.001]. However, this result is influenced by the number of studies included in the dose-response analysis and requires further corroboration.In the end, our analysis encompassed 22 studies involving a total of 668,486 participants, comprising 12 cross-sectional studies and 10 cohort studies. Meta-analysis results showed: Analysis of data from China revealed that an elevated TyG index was associated with a higher risk of developing hypertension, as indicated by an OR/HR of 1.36 [95%CI (1.28–1.45) In the Chinese region, there was an independent association between TyG index and the risk of developing hypertension, with a linear trend. However, the results of the linear trend need to be corrected by the more number of related studies.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023425836. The incidence of hypertension varies significantly across the globe, showing substantial differences. In the past 30 years, the prevalence and absolute burden of hypertension have continuously increased in low- and middle-income countries, including China and India—the two countries with the largest populations in the world. Blood pressure and hypertension incidence have been rising or have plateaued at most –4. In ChOur meta-analysis for this observational study was done under the PRISMA  statement  and MOOSWe searched Chinese and English databases for studies involving the Chinese region separately, and we also referred to the studies covered in the previous meta-analysis , which wArticles meeting the following criteria will be included in the meta-analysis: Inclusion criteria (1) Study design: Observational studies . (2) Methods and outcomes: Any study reporting associations between the TyG index and hypertension. Exclusion criteria: (1) Studies conducted outside of China; (2) Studies lacking relevant OR, RR, or HR values for necessary outcome reporting; (3) Studies without direct comparison of hypertension and the TyG index; (4) Studies without reporting of any excluded confounding factors; (5) Non-core journal papers published in Chinese databases; (6) The journal where the study is published has been removed from the database; (7) Repeatedly published articles, conference proceedings, and reviews; (8) Special groups, such as pregnant women, children, etc. Two researchers independently screened the studies and discussed and resolved the controversial ones.Two researchers independently extracted the study data using the same Excel spreadsheet. Any elements that generated controversy were referred to the third researcher(Q Fu) for discussion and resolution. The content of our subgroup analysis had been predetermined in advance, and thus, our main extracts included: (1) the First author's name and publication year of the study; (2) the specific region in China where the study was conducted; (3) the study design; (4) participant characteristics; (5) mean age ; (6) male to female ratio, along with corresponding effect sizes and intervals (only studies with relevant reports were extracted); (7) sample size. (8) Report format, cutoff value, and effect size with 95% confidence interval for TyG index {for studies that did not provide continuous variables, the effect sizes were obtained by reasonable selection or combination}. (9) Mean BMI {no studies providing mean BMI, obtained by calculation}. (10) Content of adjusted confounders. (11) Relevant data for dose-response analysis.Two investigators  independently evaluated the results of the included studies and the final meta-analysis, in which studies that were too controversial were referred to a third investigator (Q Fu) for discussion and resolution. Only two study types, cross-sectional studies and cohort studies, were ultimately included in this paper. Due to different types of studies requiring different assessment scales, therefore, the cross-sectional study was evaluated with The Agency for Healthcare Research and Quality (AHRQ) scale , which cI2 statistic, where P < 0.0001 was considered heterogeneous , P < 0.0001, I2 = 69.0%, indicating moderate heterogeneity in the meta-analysis (P < 0.0001). This indicates the need for subgroup analysis. Based on the results of individual covariate regression analysis, we found that study type, study region, sample size, database source, and study quality score may be sources of heterogeneity. Therefore, after including these variables in the multiple covariate regression analysis, we found that the results were statistically significant (P = 0.007). The heterogeneity among studies decreased from tau2 = 0.0122 to tau2 = 0.002693, indicating that the introduction of these five variables explains 63.0% of the total heterogeneity. Additionally, I2 also decreased to 10.63%, indicating that the remaining heterogeneity among different studies, relative to the unexplained variation, is 10.63% after the inclusion of these variables. This suggests that the heterogeneity is associated with these five variables. Finally, no type I error was found in the test using the Monte Carlo permutation method. Please refer to In the heterogeneity test results, we found that H = 1.8 > 1.5, analysis . Additioanalysis . In ordeStudy type may be a source of heterogeneity, with significantly lower heterogeneity results in cohort studies and higher heterogeneity results in cross-sectional studies . Sample P = 0.002. We speculate that the publication bias may stem from the regional nature of the study, as this study was based on the Chinese region. To assess the potential impact of publication bias on the results, we applied the trim-and-fill method to deal with publication bias. Finally, under the fixed-effect model, we found that the estimate before filling was 0.239 , and after filling, the estimate was 1.245  . Using the same method, we found that publication bias still existed , we later found through regression analysis that the partial heterogeneity was due to the study type, study region, sample size, database source, and study quality score, and these five variables can explain 63.0% of the total heterogeneity.Based on the results of our observational study's meta-analysis and dose-response analysis, we found a significant and linear association between TyG index and the risk of hypertension or the risk of elevated blood pressure in China [1.532 95%CI  P < 0.001]. In the subgroup analysis of gender, we found that the association between TyG index and hypertension is more pronounced in the female group. This may be because the average age of the participants in this study was around 50 years old, and there is study has shown that the prevalence of hypertension in women before menopause is significantly lower than in men, but after menopause, the prevalence doubles, and is significantly higher than in premenopausal women and age-matched men (According to the results of the regression analysis, the study type, study region, mean age, gender, sample size, mean BMI, database source, and study quality score have an impact on the final results, and subgroup analysis is required. In subgroup analysis, the north-south difference in China has a relatively small impact on the results, but its impact on the northern region is slightly higher than that on the southern region, which may be attributed to the fact that blood pressure is generally higher in the population of northern China than in southern China . In the ched men  while ma① (② (+/H+ transporter expressed in renal tubular cells while also promoting the deposition of intrarenal hyaluronic acid and lipids, which further increases intrarenal pressure. This leads to impaired urine outflow and reduced tubular flow, which in turn increases body fluid volume and drives hypertension. ③ (④ (In the present study, the ability of the TyG index to predict or monitor hypertension is mainly attributed to its effectiveness in reflecting the degree of insulin resistance. The relationship between insulin resistance and elevated blood pressure can be discussed from the following perspectives: ① , 52, 53 ① (② , 13, 53 nsion. ③ , 12 In ton. ③ (④ .The TyG index is often associated with insulin resistance or diabetes in predicting hypertension. However, more research is needed to explore other mechanisms that may explain the relationship between the TyG index and hypertension. One advantage of the TyG index over the HOMA-IR index is that it can reflect other factors and their relationship with cardiovascular disease to some extent . In addiHowever, there are still some limitations in this study. Firstly, the selection or processing of continuous variables included in the meta-analysis may have introduced some bias. Secondly, the results of the regional meta-analysis may not be representative of the association in all regions. Meanwhile, this study still has residual heterogeneity that has not been explained, which means that further in-depth exploration is needed. Finally, the small number of included studies in the dose-response analysis may have an impact on the final linear results.Due to the varying and significant differences in the incidence of hypertension worldwide, the hypertension status of populations in developed regions has greatly improved, while the blood pressure situation of populations in middle and low-income areas is not optimistic. Therefore, we conducted this regional discussion. In summary, there is substantial evidence to support the independent and linear association between the TyG index and the risk of hypertension within the middle and low-income populations in China. However, further large-scale clinical studies are needed to precisely determine the numerical value of this linear relationship. Additionally, it is crucial to expand research on the mechanisms underlying the association between the TyG index and hypertension beyond insulin resistance alone.


Epilepsy is the most common neurological disorder in dogs. It is increasingly recognized that ketogenic diets enriched with medium chain triglycerides (MCT) have a positive impact on dogs with idiopathic epilepsy by reducing the frequency and severity of seizure activity. Significant evidence points towards a relationship between gut microbiota and epilepsy, and that the MCT diet may alter this microbiota. The current study examined the effects of a MCT diet administered for one month on the fecal microbiota in dogs with IE and non-epileptic beagles. The diet reduced Actinobacteria in all dogs while reducing seizure frequency in epileptic ones. Different baseline microbiota patterns were found in dogs with drug-sensitive epilepsy (DSE) and dogs with drug-refractory epilepsy (DRE). The baseline microbiota pattern of dogs with DSE was similar to that of non-epileptic dogs. In them, the MCT diet decreased the relative abundance of bacteria from the Firmicutes phylum and increased that of the Bacteroidetes and Fusobacteria phylum. The opposite effect was found in dogs with DRE. These results suggest that the MCT diet could help reduce gut microbiota differences between dogs with DRE or DSE.n = 11)  and non-epileptic beagle dogs (n = 12). A significant reduction after diet in the relative abundance of bacteria from the Actinobacteria phylum was observed in all dogs. Epileptic dogs showed a higher relative abundance of Lactobacillus compared with non-epileptic dogs at baseline but these differences disappeared after diet. Epileptic dogs also showed a significantly higher abundance of Negativicutes and Selenomonadales after dietary intervention. Baseline microbiota patterns were similar in non-epileptic beagles and dogs with DSE but significantly different from dogs with DRE. In non-epileptic and DSE groups, the MCT diet decreased the relative abundance of Firmicutes and increased that of Bacteroidetes and Fusobacteria, but the opposite effect was observed in dogs with DRE. These results suggest that the MCT diet effect would depend on individual baseline microbiota patterns and that ketogenic diets could help reduce gut microbiota differences between dogs with DRE and DSE.Ketogenic diets have been successfully used in people and dogs with idiopathic epilepsy. This study examined the effect of a ketogenic medium chain triglycerides (MCT)- enriched diet administered for one month on the fecal microbiota of epileptic ( Epilepsy is the most common neurological disorder in humans and dogs with an estimated prevalence of 0.52–0.88% in humans ,2 and 0.People and animals with epilepsy show perturbed metabolic processing of glucose in epileptogenic brain areas, and the ketogenic diet (KD) provides ketone bodies that can be used as auxiliary brain fuel in addition to glucose . Adding Dietary supplementation with MCT oil in commercially produced diets or added to food is increasingly recognized as having a positive impact on epileptic dogs by reducing the frequency and severity of seizure activity ,14,15,16Enterococcus, Anaerostipes, Bifidobacterium, Bacteroides, and Blautia at the genus level in children with drug-refractory epilepsy (DRE) compared to matched healthy controls. However, the abundance of these genera was reversed after 6 months of KD treatment, thus concluding that intestinal dysbiosis could be involved in the pathogenesis of DRE in children. Furthermore, Olson et al. [Bacteroidaceae compared to a baseline diet (home owner-managed diet).Although evidence of a connection between gut microbiota and epilepsy has been recorded in human and veterinary medicine, further investigations are needed ,20. The n et al.  demonstrn et al.  observedSignificant evidence, therefore, points towards a relationship between gut microbiota and epilepsy, and that the KD can alter this microbiota and reduce seizure frequency. However, different taxonomic shifts have been observed, highlighting that the KD-induced microbiota likely depends on host genetics and baseline microbiota profiles. There is also variability in the results in terms of which specific taxa and relative abundance of bacteria protect against seizures.This study aimed to investigate the effect of a ketogenic MCT-enriched diet on the microbiota in canine IE. Changes in the phylogenetic composition and structure of the fecal microbiota from epileptic dogs were evaluated both before and after administering the diet for 30 days. Differences in the microbiota profiles between good responders to a single AED (DSE: drug-sensitive epileptic group) and those showing refractory epilepsy (DRE: drug-refractory epileptic group) were also analyzed. In parallel, the effect of the same KD on the fecal microbiota of a homogeneous population of non-epileptic dogs was studied and compared results with those of epileptic patients.This pilot study was a non-blinded, no placebo, prospective clinical trial conducted in two groups, sick (epileptic) and healthy (non-epileptic) dogs. The epileptic dogs were recruited from the neurology service of our veterinary teaching hospital . To be included in the study, dogs had to have been diagnosed with IE according to the International Veterinary Epilepsy Task Force (IVETF) Level I confidence level criteria  at leastThe non-epileptic dogs were a group of beagles owned by the University of Zaragoza (Veterinary Faculty) for research purposes. To be included in the study they had to be healthy , they had also to be correctly vaccinated and dewormed, and their feces had to have a normal appearance. EI was defined according to IVETF  and charDuring the study period, there were no changes in handling other than diet. Changes in treatment or any other problem during the development of the study constituted exclusion criteria.Before enrollment, owners were informed about the study and procedures. The selected diet for this study is commercially available for canine IE and cognitive dysfunction and its administration was always indicated by clinical criteria as part of the treatment. Owners were allowed the opportunity to ask any questions and to confirm or decline participation. All procedures were carried out under Project License PI27/18 approved on 17 July 2018 by the Ethics Committee for Animal Experiments from the University of Zaragoza. The care and use of control dogs were performed following the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes.®PetCare, ProPlan® Veterinary Diets, Barcelona, Spain). This diet was progressively introduced throughout a week. After this week of adaptation, all the dogs were fed exclusively with the new diet for one month. This new diet consisted of a high MCT oil (6.5%)—diet. Analytical constituents included 30% crude protein, 15% crude fat (MCT included), 7.5% crude ash, 1.5% crude fiber, 0.4% EPA + DHA, and 210 mg/kg vitamin B group.For the study, all dogs were fed a commercially produced KD enriched in MCT  were collected from both groups before (D0) and after 1 month on the MCT diet (D30) directly from the rectal ampoule with sterile gloves and immediately frozen at −80 °C to fix bacterial growth and preserve DNA content.® Kit for Illumina® . The library was then sequenced on an Illumina MiSeq platform and 250 bp paired-end reads were generated.Bacterial DNA extraction from fecal samples and sequencing of bacterial 16S rRNA gene procedures were the same as we previously published . BacteriBioinformatic analyses were the same as previously published . Paired-p) was set at 0.05.A one-tailed Wilcoxon matched-paired signed ranks test was used to compare alpha-diversity, the relative abundance of bacteria at different taxonomic levels, and clinical outcomes between two moments in time (D0 and D30) both in the epileptic and non-epileptic groups, as well as in the DSE and DRE-groups. A one-tailed Mann-Whitney U test was used to compare these variables between epileptic and non-epileptic dogs at D0 to rule out any potential biases, and at D30, when they were all with the same diet. In addition, a principal component analysis (PCA) was performed to reduce the larger set of bacteria (variables) into a smaller set which accounted for most of the variance. An unrotated factor solution was used and variables with eigenvalues >0.7 were extracted. The proportion of total variance accounting for the three first components was 55.7%. Finally, a multifactorial multivariate analysis of variance with repeated measures was carried out to assess the disease (IE) and diet (MCT-diet) effects and their interaction with the selected bacteria at different taxonomic levels. Statistical analyses were carried out with IBM SPSS 19.0 for Windows, and the type I error  and the remaining with imepitoin (dose range 10–20 mg/kg/12 h). The other five dogs were classified as having DRE, and were under different combinations of phenobarbital, imepitoin, KBr, and levetiracetam.p = 0.015). When the response to AED was considered, this signification remained only as a trend in both the DSE (p = 0.083) and the DRE (p = 0.059) groups. Of the six dogs with DSE, four achieved seizure freedom at the end of the study, and two animals remained at the same seizure frequency as at baseline. Of the five dogs with DRE, four showed a reduction in monthly seizure frequency during the period of study (50% reduction in three of them), one dog exhibited <50% reduction, and one dog maintained initial seizure frequency.A significantly lower seizure frequency was observed during the month of the study with the MCT-diet compared to the frequency in the previous month  weight reduction after one month on the new diet, along with a significant body condition reduction . Half of these dogs usually (30–50% of the study days) did not finish the food ration corresponding to their weight.The non-epileptic group consisted of 12 healthy beagles , aged between 2–9 years old, and weighing between 11.2–17.0 kg. All of them started from a fairly homogeneous weight and they showed a significant . Similarly, non-epileptic dogs were fed by their caregivers a commercial maintenance diet , given in amounts to cover maintenance energy requirements.p = 0.009) and after (p = 0.013) the MCT diet. Similarly, the Chao1 non-parametric estimator of species richness was significantly higher in epileptic than in non-epileptic dogs, both before (p = 0.011) and after (p = 0.006) the MCT diet. There were no significant differences between groups in the Shannon index.Alpha diversity indices were higher in epileptic than in non-epileptic dogs . The numr = 0.234; p = 0.004)  plots showed a different cluster for epileptic and non-epileptic dogs . Analysi= 0.004) .Bacilli (p = 0.006) class, Lactobacillales (p = 0.041) order, and Lactobacillus (p = 0.004) genus compared to non-epileptic dogs. After 1 month of dietary intervention (D30), epileptic dogs showed a significantly higher abundance of Negativicutes (p = 0.000) and Bacilli (p = 0.019) at the class level, Selenomonadales (p = 0.000) at the order level, Veillonellaceae (p = 0.000) at the family level and Megamonas (p = 0.000) at the genus level, and lower abundance of Peptostreptococaceae (p = 0.044) at the family level and Peptoclostridum (p = 0.009) at the genus level than non-epileptic dogs.Concerning the relative abundance of fecal bacteria in basal conditions (D0), dogs with IE showed a higher abundance of p = 0.004) phylum, Coriobacteria (p = 0.008) class, and Coriobacteriales (p = 0.008) order.The introduction of the MTC diet in this group of dogs did not modify the alpha  or beta p = 0.006) phylum, Negativicutes (p = 0.002) and Coriobacteria (p = 0.004) classes and Ruminococcus gnavus_group (p = 0.002) and Megamonas (p = 0.002) genera. The introduction of the MTC diet in this group of dogs did not modify the alpha  or beta Bacteroidia (p = 0.012) and Fusobacteria (p = 0.042) classes, as well as a lower relative abundance of Firmicutes (p = 0.019) phylum and Clostridia (p = 0.019) class, highlighting the reduction in the Blautia (p = 0.002) genus. After the MCT diet, the microbiota of the DRE and DSE groups tended to equalize and no significant differences were found in the relative abundance of bacteria.No significant differences were found in alpha diversity or beta diversity between the DRE and DSE groups, but there were some differences in the relative abundance of several bacteria at baseline (D0). Thus, dogs with DRE, compared to dogs with DSE, showed a higher relative abundance of Clostridia, Negativicutes, Gammaproteobacteria, Bacteroidiia, Fusobacteriia and Betaproteobacteria at class level, Clostridiales, Selenomonadales, Enterobacteriales, Bacteroidales, Fusobacteriales, and Bukholderiales at the order level, Lachnospiraceae, Enterobacteriaceae, Villonallaceae, Fusobacteriaceae and Bacteriaceae at the family level and Blautia, Megamonas, Bacteroides, and Fusobacterium at the genus level. When the multivariate analysis was applied, there were found significant differences between non-epileptic, DRE, and DSE groups at the phylum level with a significant diet effect (p = 0.048). Multifactorial multivariate analysis of variance with repeated measures did not find interactions between disease and diet, but at the univariate level, the phylum Firmicutes, Fusobacteria, Proteobacteria, and Bacteroidetes showed a significant interaction (p < 0.05), mainly due to the different microbiota patterns in the DRE group  compared to non-epileptic dogs. In particular, an increase within this class of Selenomonadales order, Veillonellaceae family, and Megamonas genus was observed in dogs with IE. Recently, a low abundance of Negativicutes class and Selenomonadales order has been linked to Parkinson’s disease in rats, with an increase of these taxa being observed when rats were treated with Tianqi Pingchan Granule, a clinically effective formula of traditional Chinese medicine that attenuates the production of peripheral inflammatory cytokines and inhibits the activation of microglia and astrocytes in substantia nigra [Furthermore, after one month on the MCT diet, epileptic dogs showed a significantly higher abundance of ia nigra .Clostridia class, as well as in the Blautia genus, compared with dogs with DSE before the introduction of diet. Despite the different disease-related microbiota patterns, these differences could be related to the higher phenobarbital doses usually used in DRE compared with DSE. In this line, Watanangura et al. [Clostridiales after three months of phenobarbital treatment in epileptic dogs.When considering the response to AED, dogs with DRE showed a lower relative abundance of bacteria included in the Firmicutes phylum and a et al.  found a The MCT diet’s effect differed depending on the epileptic subgroup and their basal microbiota composition. Thus, dogs with DSE showed very similar basal characteristics to the group of healthy beagles and in both cases, the diet decreased the relative abundance of Firmicutes and increased that of Bacteroidetes and Fusobacteria. However, the diet had the opposite effect in dogs with DRE, significantly increasing Firmicutes and decreasing Bacteroidetes, Fusobacteria, and even Proteobacteria. These differences in microbiota profiles between dogs with DRE or DSE could help explain the lack of response of dogs with DRE to AED. Moreover, these results suggest that the MCT diet could help to reduce microbiota differences between dogs with DRE and DSE.Some limitations of this study should be highlighted. First, the small number of subjects, especially when comparing the results of dogs diagnosed with DRE or DSE. Second, the fact of not having placebo group regarding the diet, since it was not possible to have an equivalent diet without MCT. Thus, this did not allow us to determine whether the observed changes in the gut microbiota were due to the MCTs or to the KD diet as a whole. In any case, the changes observed in the fecal microbiota after one month on the MCT-diet were consistent, especially in the group of healthy dogs, which started from a homogeneous diet and could contribute to the understanding of the effects of MCT-enriched diets. Finally, as already stated, the comparison between the groups of epileptic and non-epileptic dogs must be interpreted with caution, given the different characteristics of both groups. Thus, the homogeneity of the beagles group would make it possible to study the effect of the diet without the intervention of other factors under controlled environmental conditions, while the group of epileptic dogs could represent the real variability at the clinical level. Despite these limitations, this pilot study provides relevant information that can help guide further studies that are undoubtedly necessary to continue deepening our knowledge of the role of gut microbiota in the development of IE.Lactobacillus at baseline compared with non-epileptic dogs, but this difference disappeared after the dietary intervention for one month. Epileptic dogs also showed a significantly higher abundance of Negativicutes class and Selenomonadales order compared to non-epileptic dogs after diet, two taxa recently involved in Parkinson´s disease. Considering these findings, these bacterial taxa  should be further investigated in canine IE as possible biomarkers associated with the response to the MCT diet.Similar to studies in epileptic children, a significant reduction in the relative abundance of bacteria from Actinobacteria phylum was observed in both the epileptic and non-epileptic groups after consumption of the MCT diet for 30 days, which could contribute to reducing seizures in the latter. Moreover, epileptic dogs showed a higher relative abundance of When considering the response to AED, the MCT-diet effect varied depending on the baseline microbiota patterns. Thus, DSE dogs showed very similar basal microbiota characteristics to the group of non-epileptic dogs, and in both cases, the diet decreased the relative abundance of Firmicutes and increased that of Bacteroidetes and Fusobacteria. However, the diet had the opposite effect on DRE dogs. These preliminary results suggest that ketogenic MCT diets could help reduce differences in gut microbiota between dogs with DRE or DSE, but future studies with larger numbers of dogs will be required to confirm these findings.Clinically, epileptic dogs showed good acceptance and tolerance of the MCT-enriched diet, which could be beneficial for seizure control.


Ex group at 12 weeks. The knee flexor and extensor strength and the KOOS scores improved considerably in the Ex and the ColEx group (p < 0.05) at 12 weeks. Exercise therapy improved pain, strength and function in subjects with EOA, whereas the association of collagen seems to have accentuated the effects of exercise in bringing about clinical improvements.Athletic injuries are commonly implicated in the development of early osteoarthritic (EOA) changes in the knee. These changes have a significant impact on athletic performance, and therefore the early detection of EOA is paramount. The objective of the study is to assess the impact of different interventions on individuals with EOA, particularly focusing on recreational athletes. The study aims to evaluate the effectiveness of three treatment groups in improving various aspects related to knee EOA, including pain, range of motion, strength, and function. A study was undertaken with 48 recreational athletes with EOA who were assigned to one of three groups by the referring orthopedic surgeon: collagen (Col), exercise (Ex), or collagen and exercise (ColEx) groups. All the participants received their respective group-based intervention for 12 weeks. Visual analog scale (VAS), knee flexion range of motion (ROM) knee flexors and extensors strength, and KOOS were assessed at baseline, and after 4 weeks, 8 weeks, and 12 weeks of intervention. VAS for activity improved in all treatment groups, with no difference between groups. The between-group analysis for knee ROM revealed a significant difference ( Sports are physical activities that require skill. Sporting activities and their capacity to cause musculoskeletal problems have traditionally been the subject of much discussion . AthleteThe knee joint is a functional unit that is responsible for transmitting load during both extensive and daily activities. The magnitude of the load can range from exceedingly low to very large. Sporting activities place a large level of repetitive stress on the tibiofemoral joint. Hence, it becomes imperative to detect these arthritic changes within the knee joint early and introduce primary interventions and therapeutic approaches that could prevent further progression of the disease .Osteoarthritis (OA) is a global health problem that affects over 300 million people and is a major cause of disability. In India, the prevalence of knee OA is found to be 28.7% and approximately 80% of Indians who claim to have knee pain show signs of OA, out of which 20% report an incapability to carry out daily activities . Knee OA is important not only due to its high prevalence rate compared to other types of OA, but because it is increasingly being reported in the young and in athletic populations . The preThe identification of risk factors in EOA is crucial to initiate adequate and prompt conservative management and to prevent progression of the condition to the point where surgical intervention becomes necessary .Etiological considerations contributing to EOA can be broadly classified into endogenous and exogenous factors. Genetic factors, especially those coding for the structural proteins in type II collagen, gender, where studies show that females are more prone to developing osteoarthritis because of the anatomical difference that exists between the genders, various ethnic groups, and the age of the individual are the endogenous factors contributing to the disease ,10.External factors like meniscal tears, which may accelerate the process of arthritic changes, obesity, i.e., a BMI > 30, injuries involving the ligaments of the knee, which may lead to decreased joint stability and could cause joint degeneration, and acquired knee axis deviations and various nutritional deficits, are a few factors that could account for the early onset of the disease ,11.The first line of management for knee pain associated with arthritic changes is often pharmacological, followed by therapeutic intervention . AcetamiA 12-week program of collagen supplementation is known to significantly reduce self-reported pain in young athletes . ResultsManagement of knee OA can be broadly categorized as pharmacological and non-pharmacological. Therapeutic interventions are targeted to reduce pain, increase strength, and improve knee joint function. Exercise programs implemented for a period of 16 weeks are known to improve muscle strength, reduce pain, and improve the functionality of the knee joint . CombineAn agent with a reported positive effect on self-reported knee pain in athletes is hydrolyzed type I and native type II collagen. A 12-week program of collagen supplementation is known to significantly reduce self-reported pain in young athletes . ResultsIn light of the aforementioned considerations, the present study aims to investigate the effectiveness, superiority, and relative benefits of three distinct treatment paradigms for athletes diagnosed with early arthritic changes in the knee. Specifically, the study will evaluate the impact of collagen supplementation, exercise, and exercise along with collagen supplementation, as modes of intervention, on alleviating knee pain, improving joint function, and potentially slowing the progression of knee OA. Hence, against the above background, the present study is undertaken to understand the effectiveness and superiority, and to review the relative benefits, of three different treatment paradigms in athletes diagnosed with early arthritic changes in the knee.A double-blind, quasi-randomized trial was conducted in the hospital settings of Kasturba Medical college, Mangalore, between March 2021 and March 2022. The primary outcomes were measured using the pain scale and assessed using the visual analogue scale (VAS). The functional outcomes were measured using the knee injury and osteoarthritis outcome score (KOOS) and the secondary outcomes were knee muscle strength and knee muscle range of motion (ROM). A total sample size of 48 was included and a standard deviation of 0.74 was acquired from a previous study at a confidence level of 95% and precision d = 0.86 .Participants were randomly allocated by the referring surgeon using a random number table, and subjects were randomly allocated using the non-purposive sampling technique in one of the routinely followed and prescribed treatment groups, i.e., group (1) was the exercise therapy group, group (2) the collagen supplementation group, and group (3) underwent a combined intervention of exercise therapy and collagen supplementation .The inclusion criteria were as follows: (a) athletes above the age of 18 and below 55; (b) knee pain and stiffness that interferes with sports performance; (c) grades I and II on the Kellgren Lawrence scale, as diagnosed and confirmed by an orthopedician. The participants were screened based on the study’s eligibility requirements. The participants were excluded if they had any recent trauma or injury to the knee or any surgery in the past 3 months. A handheld dynamometer was used to assess the muscle strength, ROM was assessed using the universal goniometer, and the KOOS scale was used to evaluate the patient-reported outcomes. Group 1—exercise therapy group;Group 2—collagen supplementation;Group 3—combined intervention of exercise therapy and collagen supplementation.(1)hip muscle strengthening exercises, including hip flexors, abductors, adductors, and extensors;(2)knee muscle strengthening exercises, including dynamic quadriceps exercise, hamstring curls, gastrocnemius stretching, and squatting;(3)cycling.The exercises prescribed to both groups were:The study received ethical approval from the Institutional Ethics Committee, KMC Mangalore, Manipal Academy of Higher Education (IECKMC MLR 02-2021/54). The study was enrolled into India’s clinical trial registry under the identifier CTRI/2021/04/033091. Recreational athletes of either gender, aged from 18 to 52 years, with no current history of musculoskeletal injury, were recruited after providing their written informed consent. Demographic data and baseline data were noted on the day the patient was referred for physiotherapy. The data included a 10 cm visual analogue scale (VAS), the knee injury and osteoarthritis outcome score (KOOS), knee flexion range of motion (ROM), which was measured using a goniometer, and knee flexor and extensor muscle strength, which was quantified using a handheld dynamometer. All of the aforementioned outcome measures were assessed at 4 weeks, 8 weeks, and 12 weeks, respectively. A qualified physiotherapist delivered the exercise programs, following which the clinical endpoints were measured by the primary investigator, who was blinded to the study.t-test was used. The between-group comparison was made using repeated-measures ANOVA. Bonferroni post hoc analysis was used to correct the differences between groups. A p-value of <0.05 was considered statistically significant. The collected data were entered into the Statistical Package for Social Sciences (SPSS) version 21.0. A descriptive analysis of the baseline characteristics of the study subjects was carried out. Baseline data were compared between groups, and as the data were normally distributed, an unpaired Patient demographics: The study included 48 participants with a mean age of approximately 29–30 years, divided into three groups: collagen + exercise (ColEx), collagen (Col), and exercise (Ex).p < 0.001, Col: p < 0.001, Ex: p < 0.001. During activity, ColEx: p < 0.001, Col: p < 0.001, Ex: p < 0.001. No significant differences were found between the groups.Pain score: Pain levels significantly reduced in all three groups over 12 weeks , whereas the ROM in the Ex-group at baseline was 122.50 ± 9.129, which increased to 129.69 ± 5.313 by 12 weeks (p = 0.000). The baseline values for knee ROM in the Col group were 126.47 ± 5.491, but did not show a statistically significant improvement by 12 weeks 127.47 ± 5.197 (p = 0.111) (The knee flexion ROM  in the C= 0.111) .p < 0.001). No significant differences were observed in knee extensor strength between ColEx and Ex (p = 1.000), but there was a significant difference between ColEx and Col (p < 0.001) and Col vs. Ex (p < 0.001).The knee flexor strength at baseline was statistically significant in all three groups from baseline to 12 weeks . The betp = 1.000). However, significant differences were observed between the ColEx vs. Col group and the Col vs. Ex group in several KOOS components, indicating variations in treatment effects.KOOS Score:  For varip-value of <0.001 when compared to baseline. Pain during activity also significantly improved in all groups, with a p-value of <0.001, and there were no statistically significant differences between the groups. Knee flexion range of motion (ROM): knee flexion ROM significantly improved in the ColEx and Ex groups, with p-values of <0.001. In contrast, the Col group did not show statistically significant improvements in knee flexion ROM, with a p-value of 0.111. Between-group analysis revealed a statistically significant difference in knee ROM improvement between the Col group and Ex group, with a p-value of 0.022. Knee strength: Knee flexor strength showed statistically significant improvements in all groups, with p-values of <0.001. Knee extensor strength improved significantly in the ColEx, Col, and Ex groups, with p-values of <0.001. Knee osteoarthritis outcome scores (KOOS): For various components of the KOOS , there were no statistically significant differences between the ColEx and Ex groups, with p-values of 1.000. However, statistically significant differences were observed between the ColEx vs. Col group and the Col vs. Ex group in several KOOS components, indicating variations in treatment effects.All three groups exhibited significant reductions in pain levels over the 12-week study period. The ColEx group experienced a substantial reduction in pain at rest, with a The objective of this study was to determine the efficacy, superiority, and relative benefits of three different paradigms of treatment, i.e., exercise therapy, collagen supplementation, and a combination of collage supplementation with exercise therapy, in alleviating pain, and improving muscle strength, range of motion, and functional scores in athletes diagnosed with EOA.VAS on activity b was sigIn our study, athletes diagnosed with EOA were managed conservatively using either exercise therapy (routine exercises + proprioceptive exercises), oral collagen supplements, or both. The implications for exercise and pain reduction in an arthritic joint include Interleukin 1 beta , which sThe findings of our study are in line with another 12-week study of participants with knee OA, which concluded that strengthening and coordination exercises conducted three times a week reduced pain in 63% of sessions .In the present study, it was also observed that the collagen group did not demonstrate a statistically significant difference in pain relief when compared to the other groups . PreviouDifferences in joint mobility were measured using the ROM method and isometric muscle strength was quantified with a hand-held dynamometer. We observed a moderate to strong improvement in the ROM and knee muscle strength in individuals with EOA in the Ex-group. The ROM of the arthritic knee joint is thought to be a crucial determinant in the development of muscle weakness during isokinetic activity . PatientIn the present study, active knee flexion ROM increased significantly in the Ex-group as compared to the other groups , whereasIn our present study, the KOOS scores showed a statistically significant difference for the ColEx and the Ex-group at the end of 12 weeks . A plausEOA is a constellation of anatomical abnormalities that cause functional disability and pain. There are various risk factors associated with disease progression; however, it appears that early joint damage or trauma during sporting events is a more common cause in the athletic population. The assessment and effective management of EOA in an athlete is frequently difficult due to his or her increased exercise demands and requirement for an accelerated return to sport. Athletes who participate in sports that require quick acceleration and instant deceleration, who engage in continuous training with an impact on joints, or who compete at an elite level for extended periods are at a higher risk of developing EOA .EOA management should always include a variety of therapeutic options targeted at alleviating pain and improving function. The recommended management framework should prioritize nonpharmacological techniques, followed by nutritional supplements and analgesics. In the present study, the ColEx group displayed significant changes in all the investigated parameters; however, exercise therapy is a standalone treatment.Considerably significant changes were seen in the results when compared to individuals treated with collagen supplementation alone. Therefore, the authors highly recommend using oral collagen supplementation along with exercise as the treatment of choice in EOA.We accept that our study had several limitations. As subject randomization was achieved through allocation and not concealment, a possible selection bias could have occurred. Second, because this study was conducted on recreational athletes, the capacity to extend these findings to a larger population is limited, and lastly, the sample size within the included groups is relatively small.The influence of sports practice on the development of EOA is a cause for concern. The ability to treat OA in its early stages may be largely linked to alterations in specific extrinsic or intrinsic factors associated with the patient to spare the joint from further disease progression; these factors include sports practice, equipment, and load. Nonpharmacological management such as muscle strengthening, and training plays an important role in the care of athletes with EOA. The present study concluded that exercise therapy as a standalone treatment exhibited considerably significant changes; however, exercise therapy combined with oral collagen supplementation was effective and superior to the other interventions in improving the VAS scores, strength, and KOOS scores in people with EOA. Further studies need to be conducted, wherein the effect of exercise and collagen supplementation on cartilage health should be correlated with the radiological findings and clinical biomarkers for EOA.


Torpedo torpedo, Torpedo marmorata and Tetronarce nobiliana); and (2) the possible occurrence of other hidden species in the area. Our results suggest that (1) the Sardinian seas (Western Mediterranean) host populations characterized by high levels of genetic diversity, significantly different from other areas located in the Eastern Mediterranean basin, deserving special attention; (2) only three species occur in the investigated area; (3) inaccuracies exist in the current taxonomy of the three investigated species, such as the possible occurrence of cryptic species outside the Mediterranean Sea, as well as in other genera/families of the order Torpediniformes. Future studies are needed to address these issues and inform effective conservation measures.Electric rays are currently poorly investigated; therefore, their biological status, species identification and distribution are often hard to assess. The present work, using mitochondrial sequence data, aimed to investigate (1) the genetic diversity of the three species of electric rays reported in the Mediterranean Sea  to identify specimens caught during fishing operations, (2) to measure the diversity among and within these species, and (3) to shed light on the possible occurrence of additional hidden species in the investigated area. New and already published sequences of COI and NADH2 mitochondrial genes were analyzed, both at a small scale along the Sardinian coasts (Western Mediterranean) and at a large scale in the whole Mediterranean Sea. High levels of genetic diversity were found in Sardinian populations, being significantly different from other areas of the Eastern Mediterranean Sea due to the biotic and abiotic factors here discussed. Sardinian torpedoes can hence be indicated as priority populations/areas to be protected within the Mediterranean Sea. Moreover, sequence data confirmed that only the three species occur in the investigated area. The application of several ‘species-delimitation’ methods found evidence of cryptic species in the three species outside the Mediterranean Sea, as well as in other genera/families, suggesting the urgent need for future studies and a comprehensive revision of the order Torpediniformes for its effective conservation.The present study focused on the three species of electric rays known to occur in the Mediterranean Sea:  Elasmobranchs are characterized by an intrinsic and very low variation of morphological characters, able to hinder correct species identification ,2. It isElectric rays (of the order Torpediniformes) are still poorly investigated, and many aspects of their life history traits remain incomplete ,13. BaseMany Torpediniformes species are classified as Data Deficient (DD), Not Evaluated (NE) or threatened  according to the International Union for Conservation of Nature (IUCN) , and theTetronarce Gill, 1862 and Torpedo Dumèril, 1805. These comprise 13 and 12 species, respectively [Torpedo torpedo , the marbled torpedo Torpedo marmorata  and the great torpedo ray Tetronarce nobiliana  [Torpedo sinuspersici , has been reported in the Eastern Mediterranean Sea [Tetronarce tokionis  [Torpedo alexandrinsis  and Torpedo fuscomaculata Peters, 1855, have been reported [T. sinuspersici has been provisionally listed as a possible Lessepsian immigrant in Syria [Only the family Torpedinidae occurs in the Mediterranean Sea ,20 with ectively , even ifectively ,22. Three, 1835) ,20. At lnean Sea . In detaa, 1908) , as wellreported . Similarin Syria . These fin Syria . Apart fin Syria ; Turkey in Syria ; or in tin Syria ,33,34,35Tetronarce, while spiracles with slender, tentacle- or knob-like papillae on the posterior and lateral margins are present in the genus Torpedo. At the same time, the dorsal surface pattern may assist taxonomic identification, resulting in uniform or various light and dark markings in Tetronarce and Torpedo, respectively. Despite this apparently clear differentiation, Cariani et al. [Despite the very scarce commercial interest, electric rays are frequently caught as bycatch in the Mediterranean waters ,27,32,34i et al.  used molConsidering that the correct identification of specimens is the first essential step to properly assessing the impact of fisheries on populations and species, the present study aimed to improve the knowledge of the Mediterranean electric rays using genetic data (1) to molecularly identify specimens caught during fishing operations and assess the rate of eventual morphological misidentifications on the field; (2) to measure the diversity among and within these species and characterize the health status of populations; and (3) to shed light on the possible occurrence of additional hidden species in the investigated area or in general in the studied species and other related taxa.Firstly, sequences for the cytochrome oxidase subunit 1 (COI) and the nicotinamide adenine dehydrogenase subunit 2 (NADH2) mitochondrial genes were newly produced and used to measure the genetic diversity and connectivity for the three native species along the Sardinian coasts (Western Mediterranean Sea). Secondly, new and published sequences were integrated to characterize the genetic diversity of these species at the level of the whole Mediterranean Sea and in nearby Atlantic waters. Finally, given the finding of cryptic species in the three species, the final analyses were performed by retrieving and integrating all sequences available in public databases for Torpediniformes. Several ‘species-delimitation’ methods were also used to check on the possible occurrence of hidden species and taxonomic issues in other species/genera/families within this order.T. nobiliana, 39 to T. torpedo and 98 to T. marmorata  and thearmorata , followiA fragment of the mitochondrial gene COI was obtained from each specimen by PCR using the FishF2 and FishR2 primers . AmplifiThe new COI and NADH2 sequences obtained were imported in MEGA X  and all DNASP v.6  was usedSequences of COI and NADH2 for the three electric ray species, available on the GenBank and BOLD databases ,46,47, wTo estimate the occurrence of population structuring for the three electric ray species around the Sardinian coasts the Analysis of Molecular Variance  was perThe available sequences of COI and NADH2 for the three electric ray species from the GenBank and BOLD databases ,46,47 weThe software Bayesian Analysis of Population Structure (BAPS) v.6.0  was emplrenv [Homologous COI and NADH2 sequences were retrieved from public repositories for all species of Torpediniformes. The script-based search of available sequences was performed using a modified version of the Meta-Fish-Lib R scripts proposed by Collins et al. . Apart frenv . DuplicaA molecular taxonomic approach, implementing species delimitation methods, was applied to assess taxonomic uncertainties and accuracy of public data, and unravel the origin of detected errors.Firstly, to test the accuracy of specimen identification, all sequences were analyzed using a tree-based approach and four methods: (1) the Bayesian tree reconstruction based on the Yule speciation process , perforThe YSP analyses were carried out using the GTR+G substitution model (identified as the best model in MEGA), strict clock type, running 50,000,000 MCMC generations sampled every 1000 generations with a 20% burn-in. Log files were analyzed with Tracer v.1.7.2  to evaluhttp://www.atgc-montpellier.fr/phyml/ (accessed on 26 May 2023) [PTP, bPTP and mPTP were carried out using Maximum Likelihood trees (ML), which were obtained in the PhyML v.3.0 web server (ay 2023)  by applyay 2023) ) alreadyhttp://mptp.h-its.org (accessed on 31 May 2023) using default settings . Similarly, the bPTP analysis was conducted on a web server (http://species.h-its.org/ptp/ (accessed on 31 May 2023) set up for 200,000 generations, with a thinning of 100 and burn-in of 0.25. Bayesian trees used as input files of the bPTP analysis were computed by MrBayes v.3.2.7  was employed as outgroups for all analyses . Pteropanalyses .Secondly, the accuracy of species delineation was also tested using a distance-based approach by performing (a) the Best Close Match Analysis , (b) ththreshOpt function to identify the optimal threshold value for our dataset following the author’s tutorial indications [The BCMA analysis was carried out in the R package Spider v.1.5 , using tications . To perfhttps://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html (accessed on 31 May 2023). Default values of p between 0.001 and 0.1 were used. The number of steps was set to 10. Several values of X from 0.5 to 1.5 were evaluated. Simple distances are reported as these seemed to perform better than the Jukes–Cantor or Kimura 2-parameter method, as also previously suggested in Srivathsan and Meier [The ABGD method  was compnd Meier .https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html (accessed on 31 May 2023), choosing the simple distance (p-distance) to calculate the pairwise distance.The ASAP analysis was carried out using the ASAP webserver ; availabFor the COI sequences, MOTUs were also delimited using the Barcode Index Numbers as defined in BOLD .As the different methods can lead to conflicting results, we considered as consensus the results shared by the majority of all methods applied.T. marmorata to T. torpedo before the start of the subsequent analyses.DNA was successfully extracted, amplified and sequenced for 130 and 129 individuals for COI and NADH2 mtDNA markers, respectively . A singlT. nobiliana, T. torpedo and T. marmorata, respectively  showed a total of 109 polymorphic and 104 parsimony sites, counting two, four and seven haplotypes for ectively . The newectively . All theNADH2 = 0.898) and higher nucleotide diversity  than COI sequences   .T. torpedo and T. marmorata, we clustered the sequences in a single group, two groups (separating the north/western from the south/eastern locations) and three groups  to test for possible differentiation on a geographic basis. Signals of significant genetic differentiation were detected in T. marmorata  and T. torpedo , but not in the other scenarios investigated , obtaining no significant values of Φst in all two datasets analyzed  shared among the Sardinian and other Mediterranean samples, as well as the presence of private haplotypes in several Mediterranean areas  separating the Western and Eastern Mediterranean basins  were significantly different from the Western Mediterranean locations  and the Eastern Mediterranean  from the Mediterranean locations both in T. marmorata and T. torpedo (The NADH2 sequences confirmed the differentiation within the Mediterranean (Western vs. Eastern locations) in  torpedo .T. nobiliana, the AMOVA on a very few sequences allowed us to demonstrate only an overall differentiation  and distant species (within the order Torpediniformes). The sequence mining led to final datasets containing 468 and 184 sequences for COI and NADH2, respectively, associated with four families out of the five currently described for the order Torpediniformes: Narcinidae, Narkidae, Platyrhinidae and Torpedinidae. When collapsed into haplotypes, the two final datasets contain a total of 153 and 81 haplotypes for COI (34 species) and NADH2 (21 species), respectively.Considering the results from both delimitation approaches (tree-based and distance-based), a consensus number of 41 and 26 MOTUs were obtained for COI and NADH2, respectively, more than the initially associated species in the public repositories .T. torpedo, T. marmorata and T. nobiliana), they were recognized as separate MOTUs by most of the methods, and provisionally identified as MOTU 10, Torpedo bauchotae Cadenat, Capapé and Desoutter, 1978 and Tetronarce occidentalis , respectively  or between T. nobiliana and Tetronarce fairchildi   .Narke japonica , MOTU 5, Narcine brunnea , MOTU 6, Narcine timlei  and MOTU 7, Narcine maculate  and MOTUs 8, 9). On the contrary, contrasting results were obtained for Narcine brasiliensis  and Narcine bancroftii : they were identified as separated MOTUs only by the NADH2 data  appear distant from the other congeneric species  clustered with sequences of Narkidae (genus Typhonarke). Moreover, in the family Narcinidae, large genetic distances were computed within the genus Narcine, with species assigned to two well-distinct clusters, encompassing species from Indo-Pacific and South American origin in separate clades , new genetic information was acquired to complement the biological and fishery-related data recently compiled for the same area ,34.T. marmorata, and for the first time a total of 12 NADH2 haplotypes were obtained from T. nobiliana and T. torpedo specimens of Mediterranean origin . From a conservation perspective, this high diversity seems to indicate that the Sardinian electric rays are variable and hence apparently in ‘good health condition’. This potentially enables them to adapt to changes in their environment and/or to stressful conditions such as excessive fishing pressure more efficiently [Secondly, we measured moderate-to-high genetic diversities in the Sardinian electric rays . High NAiciently . On the iciently ,80 and aT. nobiliana and T. torpedo. These low numbers could reflect the rarity of the species at sea and/or the inherent difficulties in sampling them at the markets due to the low economic value and fishery interest.Finally, lack of significant genetic structuring was detected for the three species. It is well known that the level of genetic differentiation within marine species results from a complex equilibrium between structuring factors  and homogenizing factors  . In our Examining the three species of electric rays at a wider geographic scale, the network analysis revealed the occurrence of shared haplotypes among sequences from Mediterranean and extra-Mediterranean specimens, as well the occurrence of private and divergent haplotypes.The BAPS and AMOVA analyses detected the presence of significant genetic differentiation, even within the Mediterranean Sea between the Eastern and Western locations. This observed genetic structuring could depend both on historical processes , contemporary restrictions to dispersal  and biological characteristics of the species   with respect to the ‘true’ Mediterranean ocellate torpedoes, potentially representing a ‘still undescribed’ new species. These results confirm the need to further investigate the Senegalese waters, where additional undescribed elasmobranch species are possibly reported to occur [T. nobiliana , were identified as a different MOTU; they should be attributed to T. occidentalis as suggested in [Firstly, the NADH2 sequence from specimens of the Central Eastern Atlantic , originally deposited under the name Q518928; ), was clribed in . Similarribed in ,91, fromto occur . FinallyT. cf. nobiliana) and sequences from New Zealand/Australia (deposited under the name T. fairchildi/T. macneilli) are recognized as being the same MOTUs as the Mediterranean great torpedoes; they are all synonyms of T. nobiliana [On the contrary, the sequences from South Africa  are a common occurrence not only in torpedoes but in chondrichthyan species, making morphology-based identification often unreliable, and highlighting the importance of genetic tools for accurate identification, quantification of catches, delineation of units and definition of the best strategies for fisheries management and conservation .Tetronarce and Torpedo several ‘unidentified’ MOTUs were found .  authors ,96.N. brunnea, N. timlei and N. maculata, but are clearly different from them. Unfortunately, these sequences are often ‘unpublished’ (not included in any peer-reviewed publication), or they are not associated with a detailed description of the voucher specimen. This makes the proper attribution of these sequences to a species hard and ‘questionable’.Similarly, in the Indian Ocean, cryptic undescribed species  were identified in the families Narkidae and Narcinidae. Most cases are related to sequences deposited in the public repositories under the names of On the other hand, new species have been described in the Indian Ocean or reported for the first time in this same area ,99,100. Narcine family Narcinidae and (2) the position of T. marmorata within the genus Torpedo family Torpedinidae. In all our analyses, sequences of Narcine spp. were never grouped together but spread in three–five distinct clusters. Similarly, T. marmorata clustered within Torpedo but quite apart from other species of this genus. In both cases, the molecular data suggest the possible need for a taxonomic revision, eventually providing for separate subgenera or even distinct genera (to be defined).Finally, a few additional thoughts for future taxonomic studies can be derived from our results related to (1) the genus N. brasiliensis and N. bancroftii). Therefore, it is highly advisable to encourage the use of both markers, in particular to enlarge the number of NADH2 sequences, including underrepresented or lacking species. Moreover, a denser taxon sampling across the species of different genera and families is required to reach robust conclusions.As concerns the marker to be used, overlapping results were obtained with the two genes in this study. COI sequences were available for many species, producing a more complete picture of the taxon under study. Conversely, NADH2 sequences were more variable than COI and, in a few cases, outperformed them, such as by being able to distinguish even very close species  the urgency of quickly improving our knowledge on the distribution and connectivity of the three Mediterranean species and (b) the need for an interdisciplinary action for a reliable taxonomic revision at both the family (Torpedinidae) and order (Torpediniformes) levels for this iconic group.In brief, our data allowed us to identify Sardinian electric rays as genetically rich, and hence good candidates as priority populations/areas to be monitored through time and eventually protected within the Mediterranean Sea, if changes are recorded . Nevertheless, more data are needed to define the degree of differentiation within the species and among the Mediterranean basins. The use of very informative tools  could provide new insights into the mechanisms behind the population differentiation, the phylogeographic patterns, the evolutionary history, and the demographic status of the Mediterranean electric rays. Given the difficulties in having a large number of samples to analyze, the use of a large number of genome-wide SNPs could be of help, allowing us to reduce the number of samples required per area ,102, witIn recent years, a number of conservation strategies have been implemented or proposed for elasmobranchs, from gear/catch limitations to sanctuaries (discussed in detail in ). In parIn addition, our results highlighted several other areas/species deserving deeper study. Multiple markers  or even entire mitogenomes should be used to shed light on the known taxonomic uncertainties, impossible to disentangle with partial, incomplete datasets ,106,107.


No previous studies investigated the association between decrement of low-frequency repetitive nerve stimulation (LF-RNS) and amyotrophic lateral sclerosis (ALS) survival. We aim to study the relationship between decrement and survival in ALS.Sporadic ALS patients diagnosed at the Department of Neurology, the First Medical Center, Chinese PLA General Hospital from January 2018 to December 2019 were enrolled in this study. Experienced neurologists followed up the participants regularly every 6 months until January 2022. A decremental response of 10% or greater at least in one muscle was considered positive. According to the decrement, the participants were divided into LF-RNS (+) and LF-RNS (−) groups.p = 0.159, Kaplan–Meier method). In multivariate Cox regression analysis, age of onset, diagnostic delay, and ALS Functional Rating Scale-Revised (ALSFRS-R) score were associated with ALS survival, but the decrement was not correlated with ALS survival (p = 0.238).One hundred and eighty-one sporadic ALS patients were recruited in our study, including 100 males and 81 females. Among them, 10 cases (5.5%) were lost to follow-up, 99 cases (54.7%) died, and 72 patients (39.8%) were still alive at the last follow-up. The median survival time of all ALS patients in this study was 42.0 months. There was no significant difference of median survival in LF-RNS(+) group and LF-RNS(−) group (The decrement in accessory and ulnar nerves was not associated with the survival of ALS. The decrement of LF-RNS could not be an electrophysiological marker to predict ALS survival. ALS is caused by degeneration of upper and lower motor neurons, but the etiology is not yet clear . There iRepetitive nerve stimulation (RNS) is a technique widely used to assess the neuromuscular junction function. In 1959, Mulder et al.  first deIn addition, the relationship between decrement and disease progression in ALS has been studied. Wang et al.  found thDue to the high positive rate of LF-RNS in ALS patients, and the availability and repeatability of the procedure, it is worth exploring whether it can be used to predict the survival of ALS. Therefore, the purpose of this study was to explore the association between decrement and ALS survival.2.According to El Escorial revised criteria , consecuMuscle strength was assessed using modified Medical Research Council (MRC) scale. The electrophysiological studies were performed in a keypoint workstation machine , including RNS, electromyography (EMG) and nerve conduction studies. The measured limb was in a relaxed state. The skin temperature of the examined muscle was maintained at 32°C or above throughout the examination. On the side with more severe symptom involvement, LF-RNS was performed. For asymptomatic patients on both upper limbs, RNS examination was performed on the right side. LF-RNS was performed in trapezius for the accessory nerve and abductor digiti minimi (ADM) for the ulnar nerve in all ALS patients. The accessory nerve was stimulated on the posterior border of the sternocleidomastoid and the ulnar nerve was stimulated on the wrist. The surface recording electrodes were used to record over the belly of the trapezius and the ADM. The reference electrodes were placed on the tendon of the tested muscles. Compound muscle action potential (CMAP) was recorded in the trapezius and ADM of 10 stimuli at a low frequency of 3 Hz in the accessory nerve and ulnar nerve, separately. A decrement of the peak-to-peak amplitudes of the CMAP of the 4th–1st responses was measured. A decremental response of 10% or greater at least in one muscle was considered positive, as recommended by suggestions of the AAEM Quality Assurance Committee . None ofBaseline clinical data of ALS patients, including age at disease onset, diagnostic delay, site of onset , body mass index (BMI), ALSFRS-R score and disease progression rates, were obtained. Diagnostic delay was defined as the interval (in months) between symptom onset and diagnosis. Disease severity was assessed using the ALSFRS-R scale. Disease progression rates were calculated by the following formula: ΔFS = (48-ALSFRS-R score at time of diagnosis)/Duration from symptom onset to diagnosis (months).For patients who died, the survival time was from disease onset to death, tracheostomy, or censoring date . For patients who lost to follow-up, survival time was calculated from onset of disease to the last contact. All patients were followed up by neurologists every 6 months. If the neurologists failed to contact patients by phone twice, patients were considered to be lost at follow-up. This study was approved by the Ethics Committee of Chinese PLA General Hospital and written informed consent was obtained from participants.t-tests or Mann–Whitney U-tests. Enumeration data between groups were compared using Chi-squared test. Survival comparison between groups were performed with Kaplan–Meier curves and log-rank tests. Continuous variables were classified into appropriate forms to fit proportional hazards. Univariate and multivariate Cox regression analyses were performed to determine the variables associated with ALS survival. Data for continuous variables were expressed as mean ± standard deviation (SD) or median with quartile. The significance level was set at p < 0.05.All statistical analyses were performed using SPSS 22.0. Based on the decrement, participants were classified into LF-RNS(+) and LF-RNS(−) groups. Comparison of continuous variables between groups were calculated with Student’s 3.r = −0.301, p < 0.001) in ulnar nerve. According to the decrement, ALS patients were divided into two groups. No significant differences were shown in gender, site of onset, age of onset, diagnostic delay, BMI, ALSFRS-R score and disease progression rates between LF-RNS(+) and LF-RNS(−) groups  were lost to follow-up, 99 cases (54.7%) died, and 72 patients (39.8%) were still alive at the last follow-up. No patient underwent tracheostomy in our study. The epidemiological characteristics of all ALS patients were shown in ) groups .p = 0.159) , as shown in Kaplan–Meier analysis . In addi= 0.159) .In univariate Cox regression analysis , several4.To our knowledge, our study is the first large-scale study to explore the association between decrement and ALS survival. An association between decrement in accessory and ulnar nerves and ALS survival was not observed. We found that decrement could not predict the survival of ALS.Previous studies have shown that the disease progression rate in the LF-RNS(+) group was faster than that in the LF-RNS(−) group. Therefore, they considered positive decrements in RNS were an indicator of disease progression and active period . HoweverIn general, faster disease progression is associated with shorter survival. Previous studies and this study have found no significant difference in the rate of disease progression between the RNS (+) and RNS (−) groups, which also supported our findings to some extent. However, disease progression in ALS patients is not entirely linear and it may progress faster or slower at some stage. Therefore, the rate of disease progression does not fully predict the survival time of ALS. While previous studies have focused on decrement and the rate of disease progression in ALS, our study directly assesses the association between decrement and ALS survival time. Compared with previous studies, this study can more accurately show whether decrement can assess ALS prognosis.Clinical parameters such as age of onset, diagnostic delay, and ALSFRS-R score can be used to predict ALS survival . Our stuThere are some limitations in our study. First, although we followed up the outcomes of ALS patients and used a Cox model to analyze the relationship between decrement and survival, we did not measure the change of decrement during follow up. Second, RNS decrement abnormalities may occur in bulbar district of ALS patients. However, decrement of bulbar district was not studied. Third, RNS decrement of median nerve was not examined in this study.In conclusion, our study did not find any association between decrement of LF-RNS in accessory and ulnar nerves and ALS survival. Although LF-RNS is a well-tolerated and reproducible test, it can not be used as an electrophysiological marker for predicting survival in ALS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Ethics Committee of Chinese PLA General Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.YZ, JB, ML, HW, JW, and XH contributed to the study conception and design. Material preparation, data collection, and analysis were performed by YZ, JB, and ML. The first draft of the manuscript was written by YZ and JB. YZ, JB, ML, HW, JW, and XH commented on previous versions of the manuscript. All authors contributed to the article 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.