The phylogenetic analysis showed that Kuwaiti isolates of group A rotavirus clustered with human rotaviruses. Taken together, it seems that rotavirus was present in most of the dairy farms in Kuwait. The high occurrence of the virus in calves in Kuwaiti dairy farms and the close phylogenetic affinity with human isolates warrants urgent action to minimize and control its spread between calves in farms.Rotavirus, one of the main pathogens causing morbidity and mortality in neonatal dairy calves worldwide, is responsible for 30–44% of cattle deaths. It is considered to be the most common etiologic agent of diarrhea in neonatal dairy calves and children, the dominant type being group A. Two hundred seventy animals from 27 farms from 2 regions of Kuwait were tested for the presence of Rotavirus serogroup A (RVA) using latex agglutination test (LAT) and reverse transcription–polymerase chain (RT-PCR) testing. RVA non-structural proteins NSP1-2, NSP4-5 and capsid protein genes VP1-7 were characterized by next generation sequencing. LAT was positive in 15.56% of the animals, and RT-PCR in 28.89%. Using RT-PCR as a reference method, LAT was 100% specific but only 83.33% sensitive. ANOVA analysis showed correlation only with the location of the farms but no significant correlation with the age and sex of the animals. Although there was a tendency of clustering of RVA positive animals, it did not reach statistical significance ( Neonatal and young calve's diarrhea (NCD) is a common syndrome and major health issue in the livestock industry, worldwide, resulting in both short- and long-term economic consequences that stem from increased morbidity and mortality. Decreased growth rate, increased caring needs, prolonged calving time, and incidental death are the main causes of the substantial increase in the overall costs of treatment and present a serious obstacle for a sustainable livestock industry. Given that in many countries this industry is a major economic resource, the constant monitoring of livestock health is of paramount importance. Moreover, as the industry is constantly growing due to the high demand for cattle meat, milk, and milk products, the requirement for verified, consistently healthy animals is increasing.Reoviridae, that acts and transmits through the gastrointestinal route. Full genomic classification has revealed a common origin for porcine, bovine and human rotavirus strains , a species of a double stranded RNA virus family, the  strains , 2. As w strains . Further strains .RVA infections are a constant health issue across the cattle farming industry, worldwide. A decade-long study, in Brazil, revealed that the RVA cattle infection rate remains constant through the years, despite the progress in our understanding of health epidemiology, the worldwide compliance clauses and the rigorous animal testing . In UrugIn Kuwait, the Public Authority of Agriculture and Fisheries (PAAF), reports approximately 9,004 dairy cattle, with a production rate of 19.78 liters/cow . RVA infThe present study aims to assess the current occurrence of rotavirus A infection in Kuwaiti cattle livestock, using the routine diagnostic tools that are used in real life situations (namely agglutination and RT-PCR), as well as novel next generation sequencing (NSG). We believe that this study will update the current situation of livestock in the country, help promote RVA surveillance and awareness campaigns and assist animal health authorities and policy makers draw up relevant guidelines and recommendations.Fecal samples (triplicates) from 10 random calves, each <1-year old, were collected from 27 randomly selected farms from two regions, namely 25 from Sulaibiya (north) and 2 from Kabed, during a period of 12 months (January-December 2016), resulting in a total of 810 samples. Sulaibiya is a major livestock region, where 47 dairy farms and the Kuwait Cattle Research Centre of the Public Authority for Agricultural Affairs, and Fish Resources are located.Samples of calve's feces were collected, randomly, regardless of the calf health condition or the presence/absence of any symptoms. The total number of calves in dairy farms was 852. Upon which the samples size was calculated to be around 265 and was rounded to 270 to test 10 calves from each farm so that we could achieve a statistical accuracy of 95% and a confidence interval of +/−3.45%, was calculated , 16.Approximately 10 g of calf feces was suspended in 5 ml of phosphate buffered saline solution, mixed by vortexing 30 sec, then 2 drops of fecal suspension were placed into a specific well, in order to perform latex agglutination (LAT) RVA detection, using the LTA kit Virotect-Rota, , according to supplier instructions.RNA was extracted using QIAamp Viral RNA Mini Kit . Viral RNA extraction was performed according to the manufacturer and has been previously reported . The totRT-PCR amplification was performed to detect RVA capsid VP7 RNA sequences, using the primers previously described. RT-PCR was first optimized to ensure the highest sensitivity . BrieflyFor NGS, the non-structural proteins NSP1-2, NSP4-5 and capsid protein genes VP1-7 were selected. RNA sample concentration was adjusted to 100 ng. An RNA library was then generated using NEBNext® Ultra RNA Library Prep Kit for Illumina  following manufacturer recommendations. After library quality assessment using a Bioanalyser , sequencing was carried out on a MiSeq sequencer . MiSeq reporter software  was then used to mine the generated data. Reads were aligned with the sequence of targeted rotavirus non-structural proteins NSP1-2, NSP4-5 and capsid protein genes VP1-7 and consensus sequences of the protein genes used as reference sequences in CLC Genomics Workbench . The assembled gene sequences were compared to reference rotavirus gene sequences using the ClustalW program within MEGA 5.05 package . Using Phttps://www.medcalc.org/calc/diagnostic_test.php.Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values were calculated as described at The data were analyzed using the free access statistical software Jamovi 1.8.2.n = 209) with only 61 males.The characteristics of the participating animals are summarized in p < 0.001), but the reverse does not apply. All triplicates showed the same result; thus, reproducibility of both LAT and PCR is 100%. Out of the 270 animals 42 were positive for LAT (15.56%), almost double were positive for RT-PCR (28.89%). All LAT positive samples were RT-PCR positive, accounting for a strong correlation  and specificity is 100.00% (97.97 to 100.00%). The PPV was 100.00%, while the NPV was 93.66%. Therefore, the accuracy of LAT was 95.19%.p = 0.035 for LAT; p = 0.264 for RT-PCR), although it seems that the farm location is significant (p < 0.001 for both LAT and RT-PCR), with Kabed being associated with higher viral presence . Age also does not correlate with RVA infection .Another observation from presence . The smaMH717372, MH717373, MH717374, MH717375, MH717366, MH717367, MH717368, MH717369, MH717370 and MH717371) (All generated sequences have been submitted to GenBank (H717371) . Blast aPhylogenetic analysis of Kuwaiti isolates of rotavirus group A gene sequences using either non-structural protein NSP4 or capsid protein genes VP4, 6 and 7 in single gene trees allowed confirmation that Kuwaiti isolates of rotavirus group A cluster with rotavirus group A isolates collected around the world. NSP4, VP4 and VP7 sequences showed close relationships with bovine isolates of the rotavirus, while NSP4, VP4, VP6 and VP7 additionally suggest close relationships with rotavirus isolates of simian, porcine, avian and human origins. Phylogenetic analysis using concatenated NSP4, VP4, VP6 and VP7 gene sequences unambiguously documented the close relationship of the Kuwaiti isolates with rotaviruses of human origin . The seqRotavirus in calves in Kuwait represents a major challenge to dairy producers, since it is one of the contributing reasons that leads to a high rate of economic losses, e.g., in Kuwait $252 per year per dead calf . The livSalmonella, E. coli, and Pasteurella). The calculated financial loss in Kuwait per dead calf was $252  makes our findings valuable, as trans-border infections are possible.The present study provides baseline data for occurrence of group A rotavirus in calves under 1 year of age in Kuwait. Further in-depth studies are required to determine any correlation of the calve's viral strain with the human viral strain.Rotavirus is highly present all the year in calves regardless of the animal's location in Kuwait, and the virus should be included in detection and control programs by farmers and Public Authority of Agriculture and Fisheries Resources (PAAFR).LAT could be used by farmers because of its simplicity, rapidity, and low cost when compared with molecular techniques.Since there are advantages of LAT, it is recommended to use it for initial screening of rotavirus in dairy farms, and if there is a positive sample(s) in a farm, extend the detection using RT-PCR in all calves in the same farm to determine the precise number of calves infected with the virus.Rotavirus screening is recommended to be in the list of pathogens by PAAFR for all dairy farms in Kuwait.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by Kuwait Agricultural and Fisheries Agency. Written informed consent for participation was not obtained from the owners because the authorization was taken from the Governmental Agency.MA: project leader. AH: samples collection. SA-A, HA-A, and EH: RNA extraction, quantification, PCR amplification, gel electrophoresis, and documentation. AC, LB, and FA: performed dissemination, genetic analysis, and interpretation for viral genome. Genetic sequencing was completed in University of Leicester, UK. All authors contributed to the article and approved the submitted version.This research was completely funded and supported by Kuwait Institute for Scientific Research (Kuwait).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.


Functional neurorehabilitation promotes neural reorganization by stimulating subjects without deep pain perception, leading to a faster recovery when compared to spontaneous recovery, and achieving fewer compensatory errors, or even deviations to neuropathic or adaptive pain pathways, such as spasticity. The present study demonstrates the importance of intensive and repetition-based functional neurorehabilitation, which is essential for subjects classified as grade 0 according to the modified Frankel scale. n = 5) recovered from ambulation, 44% of which (4/9) did so through functional spinal locomotion by reflexes, while one achieved this through the recovery of deep pain perception. These results suggest that intensive neurorehabilitation can play an important role in ambulation recovery, allowing for a better quality of life and well-being, which may lead to a reduction in the number of euthanasia procedures performed on paraplegic animals.This article aimed to evaluate the safety and efficacy of intensive neurorehabilitation in paraplegic cats, with no deep pain perception , with more than three months of injury. Nine cats, admitted to the Arrábida Veterinary Hospital/Arrábida Animal Rehabilitation Center (CRAA), were subjected to a 12-week intensive functional neurorehabilitation protocol, based on ground and underwater treadmill locomotor training, electrostimulation, and kinesiotherapy exercises, aiming to obtain a faster recovery to ambulation and a modulated locomotor pattern of flexion/extension. Of the nine cats that were admitted in this study, 56% ( Locomotion is based on the synchronization between the flexion and extension of the limbs and reflex circuits of the spinal cord. However, the spinal cord alone is not sufficient to enable walking, and the input of the brain centers is also necessary . The altLocomotor training after spinal cord injury is based on the principle that sensory stimuli reactivate and reorganize the spinal locomotor circuit . Over thIn this study, intensive neurorehabilitation is used to promote neural locomotion control. This control is based on spinal cord autonomy, the influence of sensory stimuli on the spinal cord autonomy, neuromodulation, the learning capacity of the spinal cord locomotor circuit, and, finally, the importance of the descending pathways in the control of locomotion . This paThis prospective clinical study was conducted in HVA/CRAA, from 15 September 2014 to 15 March 2021, according to the Faculty of Veterinary Medicine, ULHT ethics committee, after the owner’s consent.For the present study, only cats with a severe contusion of traumatic origin, hit by a car or injured from a fall, were included. The diagnosis was obtained by radiology and myelotomography. At 24/48 h after SCI, imaging exams showed edema (a presence of hypoattenuating fluid/tissue adjacent to the lesion site) and an absence of vertebral spine damage (fracture and/or luxation). The lesions were localized between T3 and L3 and had to be more than 3 months old. In addition, cats had to exhibit grade 0 in the modified Frankel scale (MFS), i.e., be paraplegic with an absence of deep pain perception (DPP) and a lack of superficial sensitivity (SS) at the level of the tail (base and tip) and perineum. The upper motor neuron (UMN) injury led to a neurogenic bladder.Cats with compressive myelopathies, intervertebral disc disease (IVDD), mainly spinal cord extrusion, neoplastic disease, and others were excluded from the study. Cats presenting severe contusions with neurolocalization beyond T3–L3 as well as cats with contusions at T3–L3, but with a degree higher than 0, according to MFS, were also excluded.Upon admission to the study, the neurorehabilitation consultation was performed, and the subjects were redirected to CRAA. The intensive neurorehabilitation protocol (INRP) was initiated 24 h after admission, allowing for accommodation to the new environment. The INRP was performed 6 days a week, from 10 a.m. to 6 p.m. In addition to the re-evaluations of the INRP in accordance to the neurological development, cats were evaluated over a period of 2 to 3 months, always by the same person, in the same environment, and at the same time, with a video recording of steps taken on alternate floor types, using a Canon EOS Rebel T6 1300D camera. The reevaluation was only recorded at specific time points, as described later. The cats that reached ambulation before 3 months (time limit for INRP) were discharged from the hospital and redirected to the neurorehabilitation follow-up appointment.Cats were classified as grade 0 in the MFS and as paraplegic. Peripheral spinal reflexes were evaluated through percussion with Taylor’s hammer. In the evaluation of peripheral spinal reflexes for the pelvic limb, the patellar reflex (PR), the tibial cranial reflex (TCR), the crossed extensor reflex (CER), and the flexor reflex (FR) were assessed. With cats in sternal recumbency, the cutaneous trunci reflex (CTR) was evaluated. Contracture or shortening of the femoral quadriceps muscle, associated with hypotonicity of the flexor muscle group, was observed in all subjects. The muscle tone of the rectus abdominis (RA) was also evaluated. Finally, sensitivity to superficial pain was assessed, i.e., the dermatomes of the pelvic limb, followed by an evaluation of deep pain perception in the medial and lateral digit of each limb. Twelve centimeter straight-ended Halstead mosquito forceps were used for this procedure.The INRP consisted mainly of locomotor training and electrostimulation. Regarding locomotor training, there were the following two modalities: bipedal locomotor training (BLT) and quadrupedal locomotor training (QLT) .In BLT, the subject was placed on the land treadmill with the thoracic limbs on a smooth surface platform, and gait movement was assisted only in the pelvic limbs. The therapist sat behind the cat and performed cycling movements at a constant pace. Initially, the BLT was executed 3–6 times a day for 2–5 min, increasing progressively to 3 times a day, for about 20 min, 6 days a week. Regarding the velocity of the treadmill, in the initial stage, we started at 0.8 km/h (0.22 m/s) and increased to 1.2 km/h (0.33 m/s), without a slope.In the first stage, QLT  was perfIn both modalities, the therapist stimulated the perineal region and base of the tail to induce stepping and progressively supported the weight by holding the tail. In the initial stage, the therapist performed cycling movements in alternation with the perineal and base of the tail stimulation.In the underwater treadmill locomotor training (UWT), cats were fitted on a harness in such a way that a guidance rope system was provided by the rehabilitation technician outside the underwater treadmill. This type of training was conducted once a day, 5 days a week, from 5 min up to 40 min, according to subject compliance, with a 10% slope. The temperature of the water was 24–26 °C, and after UWT, cats were dried with an absorbent towel in a calm environment.To complement the locomotor training, passive, passive-assisted, and active kinesiotherapy exercises were administered, such as postural standing stimulation , gradually introducing difficulties with postural standing on the trampoline or on balance boards  , and on The locomotor training was associated with electrical stimulation. Segmental electrical stimulation was prescribed according to the sciatic nerve path, with one electrode in the lumbosacral anatomical region (L7—S1/S2 )and the other in the ventral medial region of the thigh, close to the motor point of the hamstring muscles  . The parAll cats were evaluated at the time of inclusion in the study (M0). The neurological evolution up to the ambulatory state was evaluated after 15 days of hospitalization (M1) and then, reevaluated after 4 weeks (M2), 8 weeks (M3), and 12 weeks (M4) of hospitalization. Follow-up appointments were made 15 days (R1), 1 month (R2), 3 months (R3), 6 months (R4), 1 year (R5), 2 years (R6), and 3 years (R7) after medical discharge. The temporal space between neurorehabilitation appointments is schematically shown in At every appointment (from M0 to M4 and from R1 to R7), cats were evaluated according to the following: active postural standing capacity, the presence of a flexor reflex, a crossed extensor reflex, CTR, rectus abdominis muscle tone, superficial sensitivity in the perineum and tail (base and tip), evaluations of deep pain perception in the medial and lateral digits, and gait stimulation on different floors.Ambulation is related to motor capacity, whereby one can perform more than 10 steps independently of the floor and perform curves with a greater support base, demonstrating slight squat secondary to muscle weakening, without ever falling, to perform, for example, micturition, automatic or voluntary urination.® .The database and statistical analyses were performed with Excel 2013 n = 9), the cats had a mean age of 3.9 years, 67% (n = 6) were female and 33% (n = 3) were male, with a mean body weight of 4.4 kg.In this study (n = 8) of the cats presented a flexor reflex, and only one (11%) presented a clonic flexor reflex, as seen in Four cats (44%) presented the flexor reflex bilaterally, four (44%) presented a diminished flexor reflex, and one (11%) presented an exaggerated flexor reflex. After the INRP, 89% (n = 5) had a CTR at the lesion site , 33% (n = 3) exhibited an absence of a CTR at the lesion site, and one showed a CTR in the lumbosacral region. Following the INRP, 56% (n = 5) demonstrated a recovery, with the CTR in the lumbosacral region, 33% (n = 3) presented a CTR at the lesion site, and one showed no signs of neurological improvement, with the CTR remaining absent at the site of the lesion.Regarding the CTR, at admission, 56% (n = 9) were paraplegic, without DPP, at admission; whereas, after undergoing INRP, 44% (n = 4) of the cats presented functional spinal locomotion by reflexes, i.e., ambulation, 22% (n = 2) had non-functional spinal locomotion by reflexes, 22% remained paraplegic without DPP, and 11% (n = 1) had recovered DPP and stayed paraparesic ambulatory with proprioceptive ataxia.Regarding the neurological grade , all of In the study, non-functional spinal locomotion was considered when the cats exhibited a flexor-extensor pattern, moving on the floor, or with weight support by holding the tail, without being able to stand up.n = 4) of the cats, the remodeling regarding superficial sensitivity was evident, because three cats had superficial pain at the perineum, although two of them also presented this at the tip and base of the tail. Only one cat regained DPP, although when comparing the deep pain of the thoracic limbs, a slower reaction was found. The results of the present study are summarized in In 44%  ,8,23,24.−1, which was a slow speed when compared to our study (0.22 m/s increasing to 0.5 m/s). The maximum land treadmill speed at which spinal locomotion could be obtained was reported from 0.5 to 0.6 m/s, only after three weeks of training [The main difference between those studies, when compared to ours, is the velocity applied on the treadmill. In our study, locomotor training was performed, in some cases, bipedal training that progressed to quadrupedal training, which allowed the spinal cats to adapt to the treadmill and made it possible to reach higher treadmill speeds. Lovely et al., 1990  achievedtraining . Our stun = 3) reached a plateau performance in one month.Lovely et al., 1990  also perBarbeau and Rossignol, 1990  introducSmith et al., 1982  demonstrn = 5). Thus, only 10% of the white matter of the spinal cord is required to initiate and maintain the locomotor pattern, in a voluntary way, on the land treadmill [When compared to our study, on both modalities of the locomotor training (BLT and QLT), combined with FES, ambulation was obtained in 56% of cats  ,43 throuThe QLT presumably increases the synaptic activity by interneuron stimulation, which is responsible for locomotor patterns and intrinsic electrophysiological proprieties ,50 that The QLT promotes stimulation of the afferent pathways by receptors, located in the muscles , joints (nerve endings), and skin. This type of stimulation allows a dynamic interaction with the CPG circuit ,14,57, aThe vestibulospinal and reticulospinal tracts are responsible for posture, meaning that they have a greater positive effect on the extensor muscles , so the Locomotor training that allows neural plasticity should be performed for 30–60 min ,20, whicThe cat that had recovered DPP showed fast progression when performing these exercises, supporting the notion that DPP is considered a favorable prognostic factor ,66,67,68The limitations of our study are the small population sample and the fact that the cats had greater difficulties adapting to the INRP, requiring a longer period.The present prospective study demonstrates the importance of intensive and repetition-based functional neurorehabilitation in spinal cord injuries, since the INRP allowed 56% of chronic paraplegic cats with no DPP to recover ambulation with functional spinal locomotion reflexes.The protocols of intensive functional neurorehabilitation are safe and can allow a significant improvement in the neurological state of spinal cats.Thus, in cats with grade 0 in the MFS, it is suggested that early locomotor training for at least 30–60 min, attaining speeds of 0.5–0.6 m/s, associated with FES, is needed, allowing for the stimulation of the descending motor tracts and the afferent inputs, inducing neuromodulation, and promoting the learning ability of the spinal network. Given its limitations, this study should be continued in a clinical setting.


Chronic obstructive pulmonary disease (COPD) is a widespread, heterogeneous disease characterized by chronic inflammation of the airway and the gradual blockage of air flow due to bronchial obstruction. At present, a large number of traditional Chinese medicine injections (TCMIs) has been applied in the clinical treatment of COPD. However, there is insufficient evidence of evidence-based medicine of the interaction between them. Therefore, the purpose of this study is through the network meta-analysis to evaluate the efficacy and safety of the different TCMIs treatment of COPD, offering reference and evidence for clinical application.1), forced vital capacity (FVC), and FEV1/FVC will be the primary outcomes, FEV1 as a percentage of the estimated value (FEV1%pred), maximal voluntary ventilation (MVV), MVV as a percentage of the estimated value (MVV%pred), 6 minutes walking distance, The St. George's Respiratory Questionnaire score, and safety/adverse event will be evaluated as secondary outcomes. Mean differences or odds ratios will be used for statistical analysis. We will ensure the reliability of the results through node-split model and heterogeneity analysis. In addition, methodological quality will be evaluated based on the Cochrane Collaboration's tool, and the quality of evidence will be evaluated according to the Grading of Recommendations Assessment, Development and Evaluation system.We will search 7 databases for randomized controlled trials of TCMI for the COPD, including PubMed, the Cochrane Library, EMbase, China National Knowledge Infrastructure, China Biological Medicine, Chinese Scientific Journals Database, and Wan-fang databases, from the date of the establishment of each database to October 31, 2021. The network meta-analysis will be implemented through Aggregate Data Drug Information System 1.16.8 and Stata 13.0 software. Pulmonary function included forced expiratory volume in 1 second (FEVThis study will provide reliable evidence for the clinical selection of TCMI in the treatment of COPD.The results of this study will evaluate the efficacy and safety of TCMI in the treatment of COPD, and provide decision-making references for future clinical and scientific research. In 2005, COPD caused more than 3 million deaths and is considered one of the third leading causes of death, accounting for 6% of all deaths worldwide.,4 In addition, globally, as one of the causes of chronic morbidity and death, most patients with COPD for many years die from the disease itself or complications caused by it. Cough and sputum are the most common symptoms of COPD, occurring in about 30% of patients. At present, the treatment measures of COPD are mainly to relieve patients’ symptoms, reduce exacerbations, improve patients’ quality of life, and exercise tolerance.–8Chronic obstructive pulmonary disease (COPD) is a widespread, heterogeneous disease characterized by chronic inflammation of the airway and the gradual blockage of air flow due to bronchial obstruction.,10 Traditional Chinese medicine injections (TCMIs) treating COPD at present mainly include Tanreqing injection (TRQI), Danhong injection (DHI), Xuebiqing injection (XBQI), Shenmai injection (SMI), Huangqi injection (HQI), etc. Studies have confirmed that XBQI can regulate the levels of SP-D and CCL18 in patients with COPD and alleviate the clinical symptoms of patients. TRQI can inhibit the release of tumor necrosis factor-α (TNF-α), IL-1β, interleukin-6 (IL-6), and IL-8, and reduce airway inflammation.Existing evidence method of traditional Chinese medicine in the treatment of COPD has gained rich experience, and combining traditional Chinese and western medicine treatment can further improve the prognosis of patients.At present, a large number of TCMIs has been applied in the clinical treatment of COPD. However, there is insufficient evidence of evidence-based medicine of the interaction between them. Therefore, the purpose of this study is through the network meta-analysis (NMA) to evaluate the efficacy and safety of the different TCMIs treatment of COPD, offering reference and evidence for clinical application.22.1https://osf.io/dkgcq), registration number: DOI 10.17605/OSF.IO/DKGCQ. This protocol follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols guidelines.The NMA protocol has been registered on the Open Science Framework platform , intervention (I), comparator (C), outcome (O), and study design (S) are the 5 main factors determining the inclusion and exclusion criteria of this research.2.3.1This study is a systematic review with NMA of randomized controlled trials (RCTs) on TCMI for the COPD. All relevant RCTs using TCMI for the COPD will be included. Quasi-RCTs will be excluded such as those allocating by medical record number. The specific participants, interventions, comparators, outcomes criteria are as follows.2.3.2: Patients with COPD whose forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) after bronchodilator inhalation were less than 70% were defined as COPD, and the condition was stable/acute exacerbation. Patients with other serious complications were not included, and the course and severity of the disease were approximately the same regardless of sex, age, nationality, or educational background.All patients met the diagnostic criteria recommended by the Global Initiative for Chronic Obstructive Pulmonary Disease2.3.3Treatment group TCMI in combination with western medicine or taking TCMI alone, the control group did not intervene, placebo or western medicine. TCMI include TRQI, Reduning injection, XBQI, SMI, Shenfu injection, DHI, HQI, etc.2.3.42.3.4.1(1)1;FEV(2)FVC;(3)1/FVC;FEV2.3.4.2(1)1 as a percentage of the estimated value (FEV1%pred), maximal voluntary ventilation (MVV), MVV as a percentage of the estimated value (MVV%pred), 6 minutes walking distance;FEV(2);The St. George's Respiratory Questionnaire score(3)Serum inflammatory factors include superoxide dismutase, IL-6, and TNF-α;(4)Safety and adverse event monitoring.2.4Computer retrieval of published RCTs of TCMI for the COPD is conducted in PubMed, the Cochrane Library issue 10, 2021), EMbase, China National Knowledge Infrastructure, China Biological Medicine, Chinese Scientific Journals Database, and Wan-fang databases. The time limit of document retrieval is from the establishment of each database to October 31, 2021. The language is limited to English and Chinese. In addition, inclusive literature from the field and references from previous evaluations will be manually retrieved to find other potentially relevant articles. Search terms mainly include: “chronic obstructive pulmonary disease”, “chronic obstructive lung disease”, “COPD”, “traditional Chinese medicine”, “injection”, “Tanreqing”, “Danhong”, “Huangqi”, “Shenmai”, “Reduning”, “Xuebijing”, etc. Taking PubMed as an example, the initial retrieval strategy is shown in Table 0, 2021, 2.5https://endnote.com), the duplicate literature was eliminated; Conduct a preliminary screening by reading the headline summary to exclude literature that does not meet the inclusion criteria; Reading the full text and making final selections; Data extraction using a pre-designed data extraction table for the included literature and cross-checking the results; In case of disagreement, the third researcher Kun Yang will be called upon to assist in judgment. Microsoft Excel  will be used to extract the data and collecting relevant information. Data extraction mainly included basic information of the literature , basic information of study subjects . The outcomes included FEV1, FVC, FEV1/FVC, FEV1%pred, MVV, MVV%pred, 6 minutes walking distance, St. George's Respiratory Questionnaire score, superoxide dismutase, IL-6, TNF-α, AR, etc. At the same time, the key factors of bias risk assessment are extracted. We will contact the corresponding authors for additional information if necessary.As shown in Figure 2.6: random sequence generation, allocation concealment possibility; blinding of participants and personnel; blinding of outcome assessment; incomplete outcome data; selective reporting; and other bias. Based on the above 7 entries, the judgment is divided into 3 levels: low risk of bias, high risk of bias, and unclear risk of bias.Methodological quality will be assessed based on the bias tool (ROB) in Cochrane Handbook 5.1.0. Two trained researchers Zhongli Sun and Xingying Li will independently evaluate the risk of bias of the included studies. In case of dispute, submit to corresponding author Penglong Yu for arbitration. Cochrane bias risk assessment tool will be used to assess the risk of RCTs being included in NMA, 7 items are included2.72.7.1 Based on Bayesian framework, the software uses Markov Chain-Monte Carlo algorithm to prior evaluate and process the extracted data, which provides support for further research and decision making. Preset model parameters: 4 chains will be used for simulation analysis, with an initial value of 2.5, a step size of 10, 20,000 annealing times, and 50,000 simulation iterations. Mean differences or odds ratios will be used as the effect sizes for statistical analysis, both with 95% credible intervals. Firstly, the network evidence plot is generated according to different outcome indicators. The network evidence plot consists of boxes and lines. The box represents each intervention included in the analysis, while the line represents RCT evidence for a direct comparison between the 2 interventions, and the number represents the number of studies for the direct comparison. According to the results of the NMA, rank probability plot of various modeling methods is generated and sorted by dominance, with Rank top1 being the optimal sequence. However, if the lower score is better in the score standard of the outcome, the lowest ranked prediction sequence is the optimal sequence.This study will use Aggregate Data Drug Information System 1.16.8 for NMA.2.7.2P > .05) between direct comparison and indirect comparison, the consistency model is used, whereas the inconsistency model will be used for analysis. If the consistency model is adopted, then the stability of the results is verified by the inconsistency model: when the inconsistency factors including 0, and at the same time inconsistency standard deviation including 1 says the result of consistency model is more stable and reliable. At the same time, various analysis models are iterated with preset parameters, and the convergence of iteration effect will be judged by potential scale reduced factor (PSRF). When the PSRF value is close to or equal to 1 (1 ≤ PSRF ≤ 1.05), it indicates that the convergence is complete, the model has good stability, and the conclusion of analysis is reliable. If the PSRF value is not in this range, the iteration continues manually until the PSRF value reaches the range standard.Node-split model will be used to verify the consistency of the corresponding data. If there is no statistical difference (2.7.3https://www.stata.com). Evaluate the heterogeneity between studies through I2. When I2 > 50%, it indicates that the heterogeneity between studies is large, using a random effect model; when I2 < 50%, it indicates that the heterogeneity between studies is small or there is no difference qualitative, using a fixed effect model. When the heterogeneity is greater, the source of heterogeneity should be further sought.Before the combination of effect size, the heterogeneity of the included literature is tested using STATA 15.0 software (Stata Corp, College Station, TX, 2.7.4If necessary, sensitivity analysis will be used to assess the impact of the studies on the random effects model. After each study was excluded one by one, the data analysis was carried out again to determine the stability of the results. If there is no qualitative change in the combined effect showed in the results, the results are stable.2.7.5If there is clinical and methodological heterogeneity, we will conduct a subgroup analysis of the patient's age, the degree and stage of COPD, smoking history, duration of treatment, or study quality.2.7.6If 10 or more studies are included in the NMA, a comparison-adjusted funnel plot is developed using Stata to evaluate the presence of small sample effects or publication bias in the intervention network. If the plot is asymmetric and there is no inverted funnel shape, it indicates that there may be publication bias. The reasons may be related to the small sample size, allocation concealment, and insufficient implementation of blind method.2.7.7If the literature information is clearly incorrect or incomplete, we will contact the first author or the first author of the literature via email address. If no response is received, the document should be deleted.2.7.8 Based on bias, inconsistent, inaccurate, indirect, and the risk of publication bias 5 degradation factors, the quality classification for the 4 level of evidence: high, medium, low, and very low.To grade evidence quality and understand the current situation of evidence rating thereby analyzing possible problems, The Grading of Recommendations Assessment, Development and Evaluation instrument will be used to assess the quality of evidence in the NMA.2.7.9There was no patient or public involvement in the preparation of this protocol.3In recent years, more and more RCTs of TCMIs in the treatment of COPD, and studies have confirmed that TCMIs play an important role in the application of effective treatment of COPD. However, these results have not been fully confirmed. Therefore, this NMA was designed to evaluate the efficacy and safety of TCMI in the treatment of COPD. The purpose of this study is to provide a basis for clinical practice of traditional Chinese medicine, as well as to help doctors make clinical decisions for the treatment of COPD patients and strengthen the treatment strategy of COPD. Our study used a comprehensive analysis of all RCTs of TCMIs for COPD, including SMI, XBQI, DHI, HQI, etc, to synthesize the evidence and rank the available TIMIs for COPD. There are some potential limitations to this study, such as the fact that most studies published in Chinese journals do not have a strictly randomized design. In addition, due to the limited ability of the authors, only English and Chinese literature was retrieved, which may create a risk of bias.Conceptualization: Zhongli Sun, Wei Zhao, Penglong Yu.Data curation: Zhongli Sun, Wei Zhao, Kun Yang.Formal analysis: Zhongli Sun, Wei Zhao.Funding acquisition: Kun Yang.Methodology: Zhongli Sun, Wei Zhao, Xingying Li.Project administration: Zhongli Sun, Wei Zhao, Kun Yang.Writing – original draft: Zhongli Sun, Wei Zhao.Writing – review & editing: Zhongli Sun, Wei Zhao, Kun Yang, Xingying Li, Penglong Yu.


Herein, for the first time, the potential relationships between the cytoskeleton-associated proteins DAAM1 and PREP with different testicular disorders, such as classic seminoma (CS), Leydig cell tumor (LCT), and Sertoli cell-only syndrome (SOS), were evaluated. Six CS, two LCT, and two SOS tissue samples were obtained during inguinal exploration in patients with a suspect testis tumor based on clinical examination and ultrasonography. DAAM1 and PREP protein levels and immunofluorescent localization were analyzed. An increased DAAM1 protein level in CS and SOS as compared to non-pathological (NP) tissue was observed, while LCT showed no significant differences. Conversely, PREP protein level increased in LCT, while it decreased in CS and SOS compared to NP tissue. These results were strongly supported by the immunofluorescence staining, revealing an altered localization and signal intensity of DAAM1 and PREP in the analyzed samples, highlighting a perturbed cytoarchitecture. Interestingly, in LCT spermatogonia, a specific DAAM1 nuclear localization was found, probably due to an enhanced testosterone production, as confirmed by the increased protein levels of steroidogenic enzymes. Finally, although further studies are needed to verify the involvement of other formins and microtubule-associated proteins, this report raised the opportunity to indicate DAAM1 and PREP as new potential markers, supporting the cytoskeleton dynamics changes occurring during normal and/or pathological cell differentiation. In the adolescent and young adult male population (between 20 and 40 years), testicular cancers (TC) represent one of the most common solid tumors ,2,3,4. TTC is a heterogeneous pathology, which includes several types of cancer, such as germ cell tumors (GCT), sex cord–gonadal stromal tumors (SCGST), and secondary testicular tumors . TherefoSCGST ) are found in less than 5% of all TCs , while LGrowing evidence from the literature is pointing out that infertility is one of the consequences of TCs ; on the In this scenario, the complexity of the features of TCs and other testicular disorders is evident; thus, the knowledge of the biological mechanisms leading to a cancerous phenotype enables potential therapeutic and screening targets to be found. One of the major key events occurring during the neoplastic transformation, differentiation, and proliferation is the modification of cell shapes and their loss of polarity, determined by changes in the fundamental cytoskeletal proteome. Thus, the cytoskeleton, as well as the countless associated proteins, plays a pivotal role in this dynamic process .It is worth remembering that the cytoskeleton, which consists of three major components , and its associated/regulatory proteins, is an adaptive and dynamic network in the cytoplasm, which determines the shape and the structural support for cells. Moreover, cytoskeletal dynamics is one of the key events occurring during the spermatogenic cycle, assisting germ cells (GC) in their development from a round diploid cell into a haploid highly specialized mature spermatozoa . At the The purpose of this study was to evaluate, for the first time, DAAM1 and PREP expression and localization in CS, LCT, and SOS to elucidate potential relationships between these cytoskeleton-associated proteins and the above-mentioned testicular disorders.Although they account for nearly 1% of all cancers in men worldwide, TCs are the most common solid tumor among the 15–40 age group . FortunaWorth remembering is the prominent role played by the cytoskeleton and its dynamics in all the proliferative and differentiative cellular processes, especially during the spermatogenesis, where immature GC undergo both mitotic and meiotic divisions, followed by the spermiogenesis, allowing the production of good-quality spermatozoa. Therefore, to obtain more information on the relationship between the role of cytoskeleton dynamics changes and testicular disorders, we analyzed, in five non-pathological (NP), six CS, two LCT, and two SOS testicular tissue samples, the expression and localization of two cytoskeleton-associated proteins that, previously, we demonstrated to be involved in the regulation of the male germinal compartment cytoarchitecture: DAAM1 ,26 and PTissues samples were obtained at the “Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica—Università Vanvitelli”, and the diagnosis was confirmed by histological evaluations.The representative histological features of each sample are shown in The non-pathological (NP) testicular tissues were characterized by the presence of all the germ and somatic cells, exhibiting a normal spermatogenesis , NP. CS Western blot (WB) analysis showed that the protein levels either of DAAM1 and PREP changed in the different testicular pathologic conditions .p < 0.05 and p < 0.01, respectively), was evidenced. No significant differences in LCT samples as compared to NP group were observed , while in CS and SOS samples (p < 0.01), it decreased as compared to NP tissues  analysis , which rIn NP samples, a specific DAAM1 localization was observed in the cytoplasm of SC  and hormonal profile .The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of “Università degli Studi della Campania Luigi Vanvitelli” (protocol code 206 approved on 15 April 2019).2HPO4; 18.4 mM KH2PO4; 0.9 mM CaCl2; 0.5 mM MgCl2; pH 7.4)) in the presence of protease inhibitors . The homogenates were sonicated twice by three strokes (20 Hz for 20 s each); after centrifugation for 30 min at 10.000 g, the supernatants were stored at −80 °C [The frozen testicular tissue samples were lysed in a specific buffer , 100 mM sodium orthovanadate, 0.5% sodium deoxycholate in phosphate-buffered saline , and photographs were taken using the Leica DFC320 R2 digital Camera.For immunofluorescence staining, testicular tissue sections were permeabilized with PBS containing 0.1% Triton-X-100 for 30 min after deparaffinization and rehydration . Antigenp < 0.05. Analyses were performed using unpaired Students’ t-test or one-way ANOVA; Tukey’s post hoc t-test was applied when appropriate with Prism 5.0, GraphPad Software .Data were reported as mean ± standard error (SEM). Differences between the groups were considered statistically significant at In conclusion, this is the first report on the altered expression and localization of the cytoskeleton-associated proteins, DAAM1 and PREP, in the testicular pathologies CS, LCT, and SOS. These modifications may be the direct consequence of changes in the hormonal status, as well as by the altered seminiferous epithelium environment, evidenced by the loss of GC and, consequently, their interaction with SC, and the leakage of normal activity and connections between the two compartments. Although this study is preliminary, above all due to the reduced number of the used samples, it highlighted interesting new insight into the CS, LCT, and SOS biology, also supporting the important role of DAAM1 and PREP in the cytoskeleton dynamics changes occurring before, during, and after normal and/or pathological cell differentiation. Further studies are needed to confirm all the above results, not only to verify if other formins and microtubule-associated proteins may be involved in the testicular differentiative changes but also to raise the possibility of using DAAM1 and PREP as new potential markers to confirm the diagnosis of testicular diseases.


While emerging studies have suggested an association of cardiorespiratory fitness (CRF) with stroke risk and overall health outcomes, little is known regarding the optimum methods of CRF measurement in patients with mild acute ischemic stroke.The aim of this study was to explore the association between the 6-min walk distance (6MWD) and other measurements related to CRF in patients with mild ischemic stroke at the acute stage.A total of 30 patients with stroke and 71 healthy subjects matched for age and grip strength (GS) were prospectively recruited. All patients were within 14 days after stroke onset and presented mild motor impairment (with a full score of Fugl-Meyer Motor Assessment). Demographic data of both groups and clinical information of the stroke group were documented, and the CRF comparison between the two groups was conducted. Each participant underwent a one-time assessment of 6MWD and a series of measurements related to CRF, including GS, 10-m walk test (10mWT), five-times sit-to-stand time (FTSST), functional reaching test (FRT), Berg Balance Scale (BBS), and waistline. Pearson's product-moment correlation coefficient test and multiple linear regression were performed to explore the indicators of CRF.r <0.6) were found between 6MWD and GS of left hand (GS-left) , GS of right hand (GS-right) , FTSST , 10mWT , and FRT  in the patient group. While 6MWD displayed significant moderate correlations with waistline , 10mWT , FTSST , and FRT  in the healthy group. All these dependent variables were entered into a stepwise multiple linear regression analysis to evaluate their values in estimating CRF as measured by 6MWD in each group. Analyses suggested that GS-left (p = 0.002) and FTSST (p = 0.003) were the indicators of CRF in the patient group with stroke and explained 51.4% of the variance of 6MWD (R2 = 0.514); FTSST (p < 0.001), 10mWT (p < 0.001), and FRT (p = 0.021) were the indicators of CRF in the healthy group and explained 58.9% of variance of 6MWD (R2 = 0.589).Significant moderate correlations (0.3 < Our data confirmed that CRF is impaired in patients with mild ischemic stroke at the acute phase. Moreover, GS-left may be an optional indicator of CRF in patients with mild acute ischemic stroke, but not in healthy people.www.chictr.org.cn, identifier: ChiCTR2000031379. Low CRF level is an independent risk factor for all-cause mortality , is widely accepted as the gold indicator of CRF  , five-times sit-to-stand time (FTSST), functional reaching test (FRT), Berg Balance Scale (BBS), and grip strength (GS). Meta-analysis of cross-sectional studies reported a moderate correlation between the walking speed measured by 10mWT and VO, 95%CI) . A prospr stroke . FTSST ir stroke , which ir stroke . FRT andr stroke   experienced a clinically diagnosed mild ischemic stroke within 3–14 days from the onset; (2) with the NHISS  updated updated updated updated  of participants with stroke at the ratio of 1:3. Finally, a total of 90 healthy participants were screened and 71 were eventually recruited. The sample size was calculated based on the previously reported data and our pretest. The assumption was that the mean difference between the healthy group and the patient group with stroke would be 80 m in the 6WMD with a common SD of 120 m. The final target sample size was a total of 84–100 participants with 25–28 patients with stroke and 56–75 healthy subjects when setting a power of 80%, a significance level of 0.05, and a ratio of 1:2–1:3 between the patient group with stroke and the healthy group , 45.Ethical approval for this study was obtained from the Review Board of Ethics Committee of Huashan Hospital  and registered at the Chinese Clinical Trial Registry (Chi-CTR-2000031379). Written informed consent was obtained from all participants before recruitment.via a series of structured questionnaires. All recruited patients and healthy subjects underwent a single-time assessment of indicators related to CRF, including 6MWT, GS of left hand (GS-left), GS of right hand (GS-right), 10mWT, FTSST, FRT, mRS, waistline, and BBS. All assessments were performed by four well-trained physiotherapists, one in each research center , according to the standardized evaluator manual.Participants were initially screened by an interview, then the information on demographic features , degree of neurologic deficits, stroke history, and cognition was collected The 6MWT (m) is a well-validated, easy-to-administer submaximal exercise test . The 6MWGrip strength (kg) was measured using a hydraulic hand dynamometer  following a standardized measurement process according to a previous study . The parThe 10mWT was performed to obtain the maximal walking speed when the participants walked for the middle 10-m section of a 14-m straight corridor. The first 2 m and the last 2 m were used for acceleration and deceleration, respectively. According, timing started and stopped when the participant's first foot crossed the 2-m mark and the 12-m mark . EncouraThe FTSST is a quick and convenient test to evaluate mainly the functional lower extremity strength and balance of an individual . The parThe FRT(s) was used to assess the limit of stability by measuring the maximum distance which the participants can reach forward while standing in a fixed position. The participants were required to stand next to but not touch a wall with their feet shoulder-width apart, and to position the arm at 90° of shoulder flexion with a closed fist . After rThe BBS is a functional balance scale consisting of 14 functional balance tasks . It assePrevious studies have suggested the inverse relationship between waist circumference and CRF , 55. Thet-tests. Non-parametrical tests were conducted when the values did not follow a normal distribution. A correlation analysis between 6MWD and other indicators related to CRF was performed using the Pearson correlation analysis in both groups, respectively. We defined r > 0.6 as a strong correlation, 0.3 < r <0.6 as a moderate correlation, and r <0.3 as a weak correlation. All indicators, which demonstrated significant moderate or higher correlations  with 6WMD, were entered into stepwise multiple linear regression analysis to evaluate their potential values in predicting CRF as measured by 6MWD in each group. Multicollinearity (strong correlations among independent variables) was examined by collinearity diagnostic statistics. The value of p < 5% was considered significant in this study with symbols presenting as * for p < 0.05, ** for p < 0.01, and ***for p < 0.001.Statistical analyses were conducted using SPSS 25.0 . Continuous data are presented as the mean ± SD, and frequencies were calculated for categorical variables. The normality of data distribution and residuals from linear regressions were evaluated using the Shapiro–Wilk normality test. The differences between the groups were evaluated using the independent samples Data from 30 patients with mild acute ischemic stroke  and 71 healthy control subjects [38F/33M] were included for data analysis. All recruited participants successfully underwent the whole procedure of interview and assessment, and none of them withdrew from the study. The details of demographic and indicators related to CRF of patients with stroke and age- and GS-matched healthy subjects are displayed in t = 3.190, p = 0.002) and waistline  than the healthy group. In addition, there were significant differences in FTSST, FRT, BBS, and 6WMD between the two groups. The stroke group had a significantly higher FTSST score , while it had a significantly lower score of FRT , BBS , and 6WMD . However, there was no statistical difference between the two groups in 10mWT .As shown in r = 0.573, p = 0.001), GS-right , 10mWT , and FRT , showing that better GS, 10mWT, and FRT correlated with a higher CRF as measured by 6MWT in the stroke group. In addition, a significant negative correlation was found between 6MWD and FTSST , suggesting that better functional lower extremity strength and balance were associated with a higher CRF. There were no correlations between 6MWD and other variables.The results of Pearsons' Correlation Coefficient analysis between 6MWD and other variables related to CRF are shown as a correlation matrix in p < 0.01, r > 0.3) with 6WMD, were entered into stepwise multiple linear regression analysis. As illustrated in *GS-left-8.880*FTSST (r2 = 0.514) .Subsequently, these candidates' CRF-related indicators , which demonstrated significant moderate correlations , 10mWT , FRT , and BBS , showing that larger waistline as well as better 10mWT, FRT, and BBS implied a higher CRF in healthy subjects. Similar to the stroke group, a significant negative correlation was found between 6MWD and FTSST  in the healthy control group. There were no correlations between 6MWD and other variables.The correlations between 6MWD and other CRF-related variables are shown as a correlation matrix in p < 0.01, r > 0.3) with 6WMD, were entered into stepwise multiple linear regression analysis. As is shown in r2 as the optimum model. It included three indicators and manifested as 6MWT = 429.645-19.409*FTSST+96.972*10mWT+2.25*FRT (r2 = 0.589) , which showed significant moderate correlations (= 0.589) .In this study, we performed a series of measurements related to CRF in a group of patients with mild ischemic stroke at the acute phase and a group of age- and GS-matched healthy subjects. To our knowledge, this is the first study looking into the status of the CRF level in patients with mild ischemic stroke at the acute phase. Overall, we made three levels of observation. First, we compared the demographic and clinical characteristics related to CRF between the two groups. The comparison results of CRF-related variables suggested that patients with mild ischemic stroke at the acute phase did show a lower level in CRF even though we only recruited patients with no evidence of neurologic deficits. Second, the correlation analysis showed that quite a few variables were significantly associated with CRF value as measured by 6MWT in both groups. We also found that the patient group with stroke and the healthy group demonstrated the two distinct patterns of a correlation matrix, implying that the different dimensions of CRF were distributed differently in each group. Third, a multilinear regression analysis demonstrated that GS-left and FTSST were the indicators of CRF in the patient group with stroke, while FTSST, 10mWT, and FRT can partially represent the CRF level in the healthy group.2peck through CPET in further studies. Though the associations between 6MWD and VO2peak of patients with stroke range from 0.29 to 0.74, one meta-analyses including 13 studies involving 454 patients with stroke showed combined correlation coefficients for VO2peak and walking distance of 0.52  . For the GS, the mean value in the stroke group was about 29 kg and the mean value in the healthy group was about 30 kg, both of which were slightly lower than the normal value of 60 years old , 56. Mor2, 0.62) . More imanalysis , poor coanalysis . TherefoAdditional results of comparison between the groups are very intriguing as well. Previous studies showed that both waistline and BMI are the indicators of obesity, which is associated with an increased risk of ischemic stroke . In thisAs for the results of a correlation analysis, our results showed that several variables related to CRF were associated with 6MWD in two groups, but the two groups demonstrated two distinct patterns of correlation matrix. Specifically, significant positive correlations were found between 6MWD and GS-left, GS-right, FRT, and 10mWT in the stroke group, and between 6MWD and waistline, 10mWT, FRT, and BBS in the healthy group. Moreover, a significant negative correlation was found between 6MWD and FTSST in both groups. This result once again suggested that all CRF-related variables selected in this study were closely related to the CRF level as measured by 6MWT. Whereas, the difference in a correlation matrix between the group implied that the different dimensions of CRF  were distributed differently in each group, and the ability of each variable to predict CRF was not the same in different groups of people.2peak was used as the index for CRF, while we used 6MWT. In future studies, we should use VO2peak to further verify the relationship between CRF and GS.Stepwise multiple linear regression analysis furthers revealed that GS-left and FTSST were associated with 6MWD in patients with mild ischemic stroke at the acute phase, while FTSST, 10mWT, and FRT were associated with 6MWD in healthy subjects. GS is commonly used as a simple, an inexpensive, and a useful indicator of muscular fitness. But so far, few studies have investigated its value in predicting CRF . AlthougThe FTSST, which is a valid measure of balance performance in people with stroke , was lonSeveral limitations exist in our study. First, the sample size of the stroke group was relatively small. Future studies with larger sample sizes are necessary to confirm the findings of this study. Second, we focus only on patients with ischemic stroke at the acute phase with an intact function (mRS <2). In future studies, subjects with different levels of functional status should be recruited and studied. In addition, future studies are needed to verify our findings using CPET as the gold standard for CRF measurement.Returning to the question posed at the beginning of this study, it is now possible to state that the CRF is impaired in patients with mild ischemic stroke at the acute phase. Moreover, GS-left has a positive relationship with CRF measured by 6WMD and may be a complementary method for CPET to assess CRF in patients with stroke.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Review Board of the Ethics Committee of Huashan Hospital (KY2020-017). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.QQ and JZ contributed to the data collection, statistical analysis, result interpretation, and manuscript writing. HW contributed to the data collection and statistical analysis. QF, YZ, ZY, and QZ contributed to the data collection. YW, CZ, YH, and LY also participated in this study. JJ contributed to the conception and revision of the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Key Research & Development Program of the Ministry of Science and Technology of the People's Republic of China (Grant Nos. 2018YFC2002300 and 2018YFC2002301), the National Natural Science Foundation of China (Grant No. 91948302), the China National Nature Science Young Foundation (Grant No. 82102665), the Shanghai Sailing Program (Grant No. 21YF1404600), and the Innovative Research Group Project of National Natural Science Foundation of China (Grant No. 82021002).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.


DPP6) gene in chromosome 7, rs10224371, rs2316242, and rs10268004, respectively. The results of the mediation analysis showed that all of the selected SNPs have indirectly affected eGFR through a mediation of Al concentrations. Our analysis revealed the association between DPP6 SNPs, plasma Al concentrations, and eGFR. However, further longitudinal studies and research on mechanism are in need. Our analysis was still be the first study that explored the association between the DPP6, SNPs, and Al in plasma affecting eGFR.Aluminum (Al) toxicity is related to renal failure and the failure of other systems. Although there were some genome-wide association studies (GWAS) in Australia and England, there were no GWAS about Han Chinese to our knowledge. Thus, this research focused on using whole genomic genotypes from the Taiwan Biobank for exploring the association between Al concentrations in plasma and renal function. Participants, who underwent questionnaire interviews, biomarkers, and genotyping, were from the Taiwan Biobank database. Then, we measured their plasma Al concentrations with ICP-MS in the laboratory at Kaohsiung Medical University. We used this data to link genome-wide association (GWA) tests while looking for candidate genes and associated plasma Al concentration to renal function. Furthermore, we examined the path relationship between Single Nucleotide Polymorphisms (SNPs), Al concentrations, and estimated glomerular filtration rates (eGFR) through the mediation analysis with 3000 replication bootstraps. Following the principles of GWAS, we focused on three SNPs within the dipeptidyl peptidase-like protein 6 ( Aluminum (Al), which is the third most common element on earth, is widely used in many daily necessities and energy development . Even daPPP1R3B, but the biological information of this SNP was unknown [A genome-wide association study showed that the most significant SNP for Al in serum was at chr8:9095620, which is near  unknown . Moreove unknown . Some ep unknown ,14. In a unknown , which c unknown . On the  unknown , and dif unknown ,17. To oUsing the genome-wide data of Taiwan’s Han population, we could provide some evidence to look for potentially susceptible SNPs of a southern Chinese population to Al toxicity and understand the relationship between Al levels and genetic variants. Therefore, the goal of this study was to explore potential SNPs that involve plasma Al-affected renal function.The subjects of this cross-sectional study consisted of 500 Taiwan Han Chinese subjects from Taiwan Biobanks ,19, inclEach subject was administered questionnaires about demographic information, personal health behavior, present medication, drug use, diet, female-related questions, family history, lifestyle, economic status, and personalized information about the living environment.®, Waltham, MA USA). The resolution was set to 0.8 and 0.4 amu at 10% peak height, which complied with the typical Al analysis. Radio Corporation of America (RCA) cleaning standard was used on all equipment used in the laboratory. For sample preparation, 1% HNO3 was added to the plasma samples to make a 1:10 dilution, and then we waited for 10 min. For checking the high linearity, ICP-MS calibration standard solution  was diluted to 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, and 3000 ug/L to estimate the calibration curve, which showed a high correlation coefficient (r > 0.995). Before analyzing the unknown concentrations, we conducted quality assurance (QA) and quality control (QC) to ensure precision and accuracy. QA was used to analyze standard reference materials (SRMs). To ensure consistency of the laboratory test, we took random SRMs to conduct repeated analyses. Each result had to fit the reference between 90% and 110%. QC was to ensure the stability of the system by triple testing the SRM sample, of which the coefficient of variance (CV) should be less than 3%.Biomarkers of the Taiwan Biobank were all analyzed in the Linkou Chang Gung Memorial Hospital. However, the plasma Al concentrations were measured in the laboratory at Kaohsiung Medical University by an inductively coupled plasma mass spectrometry  to obtain 645,918 single-nucleotide polymorphisms (SNP). The TWB chip was based on Affymetrix databases, the HapMap Project , the 100p-value of the Hardy–Weinberg equilibrium (HWE), and SNPs were excluded if the p-value of HWE was < 10−6. Furthermore, we calculated identity by descent (IBD). To understand whether our sample was independent, we excluded individuals if IBD was > 0.1875. All quality controls were conducted using PLINK software (version 1.9) [To increase quality control in the genotype data, we looked for the subjects with high missing genotyping rates and extreme heterozygosity rates. The sample was excluded if the missing genotype rate was >5%, and SNP were excluded if the missing rate was >5%. We used (N-O)/N to calculate the mean heterozygosity rate, where N was the number of non-missing genotypes and O was the observed number of homozygous genotypes. Individuals with more than 3 standard deviations from the mean heterozygosity rate indicated that the DNA sample was contaminated or inbred; therefore, we excluded the subjects with extreme heterozygosity rates. We estimated the exact ion 1.9)  and R laion 1.9) .We used the mean ± SD and quartiles to represent continuous data and n (%) to represent categorical data. Blood measurements were log-transformed to generate normal distribution such as Al concentration. The MDRD equation was used to evaluate renal function :eGFR = 1x-axis and Al in plasma as the y-axis to make the scatter plot with a regression line to understand the distribution of eGFR and Al levels (log-transformed) in plasma. All descriptive analyses were conducted with SAS software (version 9.4).We used eGFR as the Association testing for log-transformed Al was conducted in the linear regressions through an additive model: SNP types were coded by assigning 0, 1, and 2 for homozygous genotypes with 0 minor alleles, heterozygous genotypes with 1 minor allele, and homozygous genotypes with 2 minor alleles, respectively.p-value of multiple testing. The corrected p-value = 0.05/numbers of SNPs, therefore the corrected p-value was (0.05/645918) = 8 × 10−8 and the suggested p-value was 1 ×∙10−6. In addition, a Manhattan plot was used to visualize the results of association testing. The vertical axis was −log10 , where a higher location meant a smaller p-value, and the horizontal axis represented the numbers of the chromosomes, and each dot represented each SNP. Meanwhile, we generated a QQ-plot to check the quality of the GWA results, where the Y-axis represented the observed −log10 , X-axis meant the expected −log10 , and each dot represented each SNP. We used g-profiler [We performed the crude model for log-transformed Al to avoid Type I errors because of multiple comparisons. The Bonferroni Correction  was usedprofiler  to finisAll association tests were performed with PLINK 1.9 , and R sWe performed a mediation analysis to examine if there was an indirect effect between SNPs and renal function .According to the assumption of Baron et al. , there wTo generate the normalized residual, we used log-transformed Al concentration and assigned 0, 1, and 2 for homozygous genotypes, heterozygous genotypes, and homozygous genotypes with 2 minor alleles to fit an additive model. The causal mediated effect was estimated using the product-of-coefficient method to generate the mediation coefficient ,32,33,34p-value <0.05 was considered statistically significant.We used the bootstrap method to estimate the indirect effect because bootstrap CIs did not require the assumption of normal distribution and did not require the sample size to be too large ,36. We uAfter collecting the questionnaires and blood samples, 500 subjects were involved in this study; however, there was a subject who rejected to participate in this study. In the beginning, there were 645,918 SNPs in the Taiwan Biobank chip (TWB chip) and 500 subjects . As illustrated in The demographic characteristics of the study population are shown in p-value < 8 × 10−8) and three variants between the significant level and suggestive level  in the genome-wide association test. The most significant SNP was rs9857275, which was an intron variant located in ZBTB38 (zinc finger and BTB domain containing 38), a protein encoded by this gene was associated with DNA methylation [BAZ2B (bromodomain adjacent to zinc finger domain 2B) gene [dipeptidyl peptidase-like protein 6 (DPP6) gene were three intron variants that were at the suggestive level (p < 10−6). However, the minor allele frequencies of SNPs  were too small (both MAF = 0.001), and the first 2 SNPs beyond the significant level were so rare that it was difficult to estimate the true effect of the rare variants based on the current sample size . Hence, we focused on the variants with bigger MAF, and we decided to focus on DPP6 variants  using a medication analysis.We did not use the SNPs of the Y chromosome. In hylation . The secDPP6, was significantly correlated with ln(Al) after controlling for potential confounders, and ln(Al) was correlated with eGFR . The evidence indicated that rs10223471 within DPP6 would lean to increased eGFR through a mediated effect of ln(Al) ; however, the direct effect was non-significant . This polymorphism only had an indirect effect. what it meant was that there was to for the exposure to Al which thus resulted in increased eGFR.As shown in As shown in DPP6, would cause a significant increase in ln(Al) by 0.311 units , and a unit of increase in ln(Al) would cause eGFR to increase by 4.64 units . A bootstrap with 3000 replications suggested that rs10268004 would result in increased eGFR by 1.443 units  through a mediated effect of ln(Al). However, no direct evidence indicated that rs10268004 could result in increased eGFR directly.As shown in Some studies have been based on subjects who had occupational exposure to Al ,3,4, whiDPP6. This gene, DPP6, has been proven as a candidate gene for amyotrophic lateral sclerosis (ALS) in the patients with ALS against the non-ALS population [DPP6 is associated with the regulation of the potassium ion transport channel and further causes neurodevelopmental impairment [DPP6 with suggestively significant association levels of Al concentration in plasma. Based on the biological evidence, Al may replace iron ions and calcium ions in the brain and interfere with other trace elements [DPP6 regulates the potassium ion transport channel and causes abnormal synapse activity [PPP1R3B, and three SNPs were found in chr7:89168136, chr7:73882011, and chr7:136945107, which report genes located at ZNF804B, GTF2IRD1, and PTN. The differences in our findings were probably due to race and type of experimental sample; Esther Ng et al. used the serum to measure the Al levels, and the study population was from Uppsala, Sweden [A GWAS technique was used to find out the potential SNPs of the interested phenotype; therefore, we used plasma Al levels as a phenotype to perform the association analysis. There were three SNPs found in the same linkage disequilibrium (LD) blocks, rs10224371, rs2316242, and rs10268004, within pulation ,42,43 anpulation  and postpulation . Therefopairment ,47 and mpairment ,45. In oelements , and theactivity ,47. EsthThe previous studies indicated that metal toxicity caused renal tubular damage ,49 and mDPP6 was observed. This represented that DPP6 would have no effect on eGFR if there was no Al in human plasma. This proved that DPP6 was associated with some neurological diseases, including cognitive diseases and memory impairment [DPP6, and eGFR. To our knowledge, there were few studies that explained the indirect effect of DPP6 on eGFR. Moreover, in our findings, three variants in DPP6 were within the same LD-blocks. Therefore, it may be interesting to further investigate the probable causal effect through deep sequencing and the gene expression of DPP6 on renal function through molecular biology experiments.In addition, the indirect effect of pairment ,45,47,51pairment ,53,54,55The first limitation of this study was its relatively small sample size. To detect the rare variant with enough statistical power, increasing the sample size is needed. Meanwhile, statistical power would be limited because of multiple-testing correction; therefore, only large-scale gene-versus-phenotype effects can be discovered. The second limitation was the study design. Since a cross-sectional study could not ensure a causal relationship, longer follow-up data is in need to evaluate the long-term impact of Al levels on renal function. Eventually, due to the low exposure to Al in the normal population, the data from occupationally exposed workers will provide more information for this kind of study.DPP6 variants played a mediator between Al in plasma and renal function. Though, longitudinal data or data of occupational exposure are in need. Nevertheless, these findings can provide additional information and medical care for the decision making of occupational medicine.To the best of our knowledge, this is the first GWA study to explore the association between candidate variants and Al concentration in plasma on the Han Chinese population. Furthermore, we found that


Spinal cord injury (SCI) is a common but severe disease caused by traffic accidents. Coronary atherosclerotic heart disease (CHD) caused by dyslipidemia is known as the leading cause of death in patients with SCI. However, the quantitative analysis showed that the cholesterol and lipoprotein concentrations in peripheral blood (PB) did not change significantly within 48 h after SCI. Due to the presence of the Blood spinal cord barrier (BSCB), there are only few studies concerning the plasma cholesterol metabolism in the acute phase of SCI. Exosomes have a smaller particle size, which enables them relatively less limitation of BSCB. This study uses exosomes derived from the plasma of 43 patients in the acute phase of SCI and 71 patients in the control group as samples. MS proteomics and bioinformatics analysis found 590 quantifiable proteins, in which 75 proteins were upregulated and 153 proteins were downregulated, and the top 10 differentially expressed proteins are those including downregulating proteins: HIST1H4A, HIST2H3A, HIST2H2BE, HCLS1, S100A9, HIST1H2BM, S100A8, CALM3, YWHAH, and SFN, and upregulating proteins: SERPIND1, C1QB, SPTLC3, IGHV4-28, C4A, IGHV4-38-2, IGHV4-30-2, SLC15A1, C4B, and ACTG2. Enrichment analysis showed that the largest part of proteins was related to cholesterol metabolism among the downregulated proteins. The main components of cholesterol  were changed in exosomes derived from plasma of patients. ELISA analysis showed that some components were disordered in the acute phase of SCI. These results suggested that the exosomes might be involved in cholesterol metabolism regulation in the acute phase of SCI. Spinal cord injury (SCI) is a common and severe disease, and the morbidity is continuously increasing, especially along with the increasing amount of traffic accidents, which usually cause physical, psychological, and social impairment Byra, . TraumatThe secondary injury of traumatic SCI is temporally divided into four phases, namely acute phase (within 48 h), subacute phase (2–14 days), intermediate phase (14 days−6 months), and chronic phase (more than 6 months)  and the BSCB, there has been very limited research on peripheral blood (PB) related to SCI. Previous serological analysis showed that total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), very low-density lipoprotein (VLDL-p), and triglyceride (TG) were increased in patients with SCI, while high-density lipoprotein cholesterol (HDL-c) was reduced  and was given official approval by the Ethics Committee of the Nantong First People's Hospital, and all the patients were informed and signed a paper informed consent form for scientific research. The experimental group in this study was patients with both vertebral fractures and SCI because patients with only SCI are relatively rare and much more common are patients with both vertebral fractures and SCI caused by violence in the clinic. To exclude the constitutive influence on the expression of exosomes, we choose patients with only vertebral fractures as the control group, not the healthy ones. The control group has no other comorbidities, including neurological dysfunction, numbness, and dyskinesia of lower limbs, difficulty urinating, or sexual dysfunction. According to the imaging data of the patient , clinical symptoms (Limb movement and skin sensation), and professional scoring standards , two spine surgeons and two radiologists will divide the patients into experimental or control groups. Experimental groups (Marked as Sbef48h):43 persons in total, 13 females and 30 males, average age 58 ± 13.0 years. Control groups (Marked as Cbef48h): 71 persons in total, 24 females and 47 males, average age 45 ± 10.5 years.All enrolled patients underwent surgery and had no history of long-term use of any drugs before the injury. All patients were not diagnosed with any diseases by professional doctors, including dyslipidemia, osteoporosis, nervous system diseases, tumors, and cardiovascular diseases. In both the experimental group and the control group, blood samples were taken immediately after hospitalization. The time limitation for collecting the blood samples was within 48 h after the injury, without any treatment or medication. One part of the blood samples (5 ml) was sent to the laboratory within 30 min to detect the plasma lipid composition, and the other part (20 ml) was centrifuged  for the supernatant (plasma), and then was stored in a deep freezer at −80°C.Exosomes were extracted from pooled plasma samples through the QIAGEN exoEasy Maxi kit (76064), strictly following the instructions of the manufacturer. The integrity of exosome vesicle morphology by using electron microscopy was observed. The PBS buffer that dissolves the exosomes were prefiltered with a 0.2 um aqueous filter to remove impurities such as crystals. About 20 μl of the PBS to resuspend the precipitated sample was taken and dropped on the copper mesh, and then it was allowed to stand for 30 min. The exosome solution with filter paper was aspirated. Staining with 2% phosphotungstic acid (PH5.0) for 60 s, and then the samples were examined by transmission electron microscopy (TEM). Nanoparticle tracking analysis technology was used to detect the size of exosomes . Western blot was used to detect the surface proteins of exosomes . Each experiment was repeated three times.About 1% protease inhibitor was added to the sample and ultrasonic lysis was used to extract protein. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. The peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum dried. The peptide was reconstituted in 0.5 M TEAB and processed according to the protocol of the manufacturer for the TMT kit/iTRAQ kit. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The m/z scan range was 350–1,800 for a full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. The resulting MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). Quantitative proteomics analysis was repeated three times.http://www.ebi.ac.uk/GOA/). The ontology covers three domains: cellular component, molecular function, and biological process. We use the KEGG pathway database to annotate protein pathways: first, use the KEGG online service tool KAAS to annotate the submitted proteins, and then use the KEGG mapper to match the annotated proteins into the corresponding pathways in the database. We use the software WoLF PSORT, which predicts subcellular localization, to annotate the subcellular localization of the submitted proteins. We first collected the functional classification information and corresponding P-values enriched by the protein groups were used, and then screened out the functional categories that were significantly enriched (P < 0.05) in at least one protein group. The P-value data matrix obtained by screening is first subjected to logarithmic transformation with –log10, and then the transformed data matrix is applied to each function classification by Z transformation. Finally, the data set obtained after the Z transformation is used for the hierarchical clustering  method for unilateral cluster analysis. The clustering relationship is visualized using the heat map drawn by the function heatmap.2 in the R language package (Gplots).We annotate proteins through Gene Ontology from the UniProt-GOA database , ApoB-100) and Elabscience (ApoB-48). The experimental samples were the plasma of patients (Sbef48h and Cbef48h were 40 samples in each group). The ELISA experiment was carried out following the instructions of the manufacturer, and the dilution concentration was as follows. The expression of Apo(a) is related only to LP(a), so we did not check its concentration in plasma. The detection wavelength was 450 nm. The data of OD values and cholesterol concentrations were analyzed using the Independent-Sample Test through SPSS20. The software used to make the chart is GraphPad Prism 6.01 and Adobe Photoshop CS6. In this study, we performed a quantitative analysis of the cholesterol and lipoprotein in PB of patients with SCI in the acute phase. Within 48 h after SCI, the cholesterol and lipoprotein concentrations did not change significantly, including the clinically common indicators TG, TC, HDL-c, LDL-c, ApoA, ApoB, and Lp(a) . This reExosomes have relatively less limitation of BSCB within 48 h after SCI, which makes them easier to be secreted to PB. Exosomes derived from the PB plasma of the patient were analyzed by TEM, nanoparticle tracking analysis, and western blotting. TEM observation showed the spherical vesicles and a typical cup shape in plasma exosomes . The speP < 0.05)  . These d < 0.05) . Gene On < 0.05) . The staThis study lists the top 10 differentially expressed proteins in The cholesterol metabolism pathway (hsa04979) is a signal pathway obtained by KEGG pathway enrichment analysis, which is closely related to downregulated proteins in exosomes. The expression of ApoB-48 and ApoB-100 increased, and the expression of ApoA-I, ApoA-II, ApoA-IV, ApoC, ApoE, and Apo(a) decreased in exosomes . ELISA aSpinal cord injury is a common and severe disease caused by accidents and it would bring catastrophic consequences and huge economic burden to the patients. The treatment of SCI mainly relies on surgical operations and postoperative rehabilitation. However, the recovery of nerve function and the occurrence of complications are still unpredictable , TG, TC, HDL-c, and LDL-c] did not change significantly, which is different from the previous research results in chronic phase. Is this because of the relatively short bedtime or other reasons? We extracted exosomes from peripheral plasma from patients with SCI within 48 h and performed proteomics and bioinformatics analysis. Surprisingly, we found that all the downregulated proteins were related to lipoprotein particles and parts of them were related to lipid transporter activity. The expression of ApoB-48 and ApoB-100 increased, and the expression of ApoA-I, ApoA-II, ApoA-IV, ApoC, ApoE, and Apo(a) decreased in exosomes. RT-PCR analysis confirmed the results that the expression of these main components of lipid and cholesterol was changed in exosomes derived from the plasma of patients with SCI in the acute phase. These results suggested that exosomes in peripheral plasma had been already participated in regulating lipid metabolism in the acute phase of SCI. However, these results did not cause changes in the PB lipid concentrations. ELISA analysis of individual components of lipid showed that some components were disordered in the acute phase of SCI. This might be because the lipid components change caused by exosomes had not yet reached the corresponding threshold. The different expression trend of lipid components between exosomes with PB could be interpreted as a stress response that might be made in advance by exosomes for resisting external stimuli.In addition, we also found the top 10 differentially expressed proteins in exosome derived from plasma of patients with SCI in acute phase through proteomics and bioinformatics analysis, including downregulating proteins: HIST1H4A, HIST2H3A, HIST2H2BE, HCLS1, S100A9, HIST1H2BM, S100A8, CALM3, YWHAH, and SFN, and upregulating proteins: SERPIND1, C1QB, SPTLC3, IGHV4-28, C4A, IGHV4-38-2, IGHV4-30-2, SLC15A1, C4B, and ACTG2. These proteins may also be involved in regulating lipid metabolism or other signaling pathways in the acute phase of SCI. Data results of this study suggested that the expression of cholesterol and lipoprotein components had changed, and exosomes might be involved in their metabolism regulation in the acute phase of SCI. These differentially expressed proteins might have been regulated at the gene level. Therefore, there must be a large number of differentially expressed genes in exosomes, which would be the next research content, including RNA-seq and biological informatics analysis.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the Nantong First People's Hospital. The patients/participants provided their written informed consent to participate in this study.All the authors confirmed they have contributed to the content of this paper and have completed the following three aspects: significant contributions to the experimental design and conception, acquisition, analysis, and interpretation of data, drafting or revising the article, and final approval of the published article.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.


This prospective cohort study aimed to evaluate whether COVID-19 lockdown caused biological, psychological, and/or physical performance variations in footballers. We compared the 2018/2019 and 2019/2020 seasons evaluating the plasma volume, hematological parameters, iron/ferritin, creatine kinase, vitamin D, cortisol, testosterone, and physiological state of players of the Italian football major league (Serie A). Measurements were performed before the preparatory period (T0), at the beginning (T1) and in the middle (T2) of the championship, and in March (T3) and at the end of season (T4). The results showed that in the 2019/2020 season affected by the lockdown, the weight, BMI, and fat mass percentage were higher than in the previous season. Hematocrit, hemoglobin, red blood cells, and ferritin decreased during both seasons, more significantly than in the regular season. During both seasons, creatine kinase increased from T2 whilst iron concentrations decreased in T3. Testosterone increased in both seasons from T0 to T3 and returned to initial levels at T4; cortisol increased in T2 and T3 during the 2018/2019 season but not during the COVID-19 season. Physical performance tests revealed differences associated with lockdown. Thus, although from a medical point of view, none of the evaluated changes between the two seasons were clinically relevant, training at home during lockdown did not allow the players to maintain the jumping power levels typical of a competitive period. On 11 March 2020, the World Health Organization (WHO) officially declared a pandemic status, caused by the new coronavirus (SARS-CoV-2). The official name given by the World Health Organization to the syndrome caused by the virus is COVID-19 (short for Coronavirus Disease—2019). The pandemic of the viral disease caused by the new coronavirus is still ongoing and returning to normal activities is still a challenge .For the total 25(OH)D measurement, an Abbott Architect 25-OH D reagent on an i2000 Architect analyzer  was used with a chemiluminescent competitive delayed phase immunoassay (Chemiflex) standardized according to the NIST SRM 2972 . As previously reported , serum tIntra- and inter-assay coefficients of variance for cortisol were 4.6% and 7.6%, respectively. The intra- and inter-assay coefficients of variance for testosterone were 3.7% and 5.6%, respectively. Serum testosterone and cortisol reference ranges were 10–75 ng/dL and 7–25 g/dL, respectively.−1, Hb > 14 g dL−1), iron depletion , iron deficiency , and iron deficiency anemia  were defined according to population references for iron status measures in males, 24.25.Normal iron storage  and Mognoni test.To minimize any effects of diurnal variation, the three testing sessions were conducted within 2 h of the same time of the day.Then, each player performed maximal CMJ on the contact platform from a standing position and with the hands on the hips. At the start, the subjects made a preparatory movement: from the extended leg position, they made a rapid bending of the knees until reaching the 90° angle, keeping the heels in contact with the ground and the trunk erect. After the jump, keeping the hands on the hips, the fall was performed with the knees extended, on the tip of the toes with subsequent cushioning to avoid trauma.The ground reaction force generated during these vertical jumps was estimated with an ergo jump . The height of the jump (cm) was the maximal height reached during the flight phase.The Mognoni test is a simple method to evaluate the speed at which the athlete reaches OBLA (Onset of Blood Lactate Accumulation).The test execution protocol provides that the subjects must travel 1350 m in 6 min, maintaining a constant speed of 13.5 km/h . In the t-test and Spearman correlation were used. p < 0.05 was accepted as a level of statistical significance. All data obtained from the study were expressed as mean ± standard deviation.Results obtained were stored in Microsoft Office Excel 2016 and statistically analyzed by GraphPad PRISM 5 software (GraphPad Software). All variables used in this study were checked for the normality of distribution before the analyses (Kolmogorov–Smirnov tests). Student’s paired The anthropometric characteristics of the players  measured during the two seasons analyzed are shown in p < 0.05) between the various points of the season. In addition, in the 2019/2020 season (COVID-19), there were higher weight values (p = 0.01) and BMIs (p = 0.03) and lower percentages of FFM (p = 0.01), due to the forced stop period.The reported values show some statistical differences . However, decrements in HB, RBC, and HT during both seasons ; in the 2019/2020 season (COVID-19), only vitamin D levels were also low in T2 (p > 0.05 by Spearman’s rank correlation). Ferritin levels decreased in T1 and T2 during the 2019/2020; however, a major decrement was observed in T2-T4 periods during the 2018/2019 season. Iron concentrations decreased in T3 in both seasons , it was higher in the 2018/2019 season  increased only in T1 of the 2018/2019 season; in all other periods, it did not show significant changes G,H.The clinical chemistries shown in No significant changes were observed for the CMJ Test . HoweverAnalysis of blood lactate concentrations during the competitive seasons showed a significant decline, but a significant increase was observed following COVID-19 lockdown. Consequently, between-period differences were significant when the COVID-19 lockdown period was compared with the 2018–2019 competitive period .Due to physiological and performance adaptations to training, professional football players are subjected to several alterations in health ,17 and pData from anthropometric, blood values, and hormonal parameters showed differences between the two seasons, proving that the forced stop period affected the physical and physiological state of professional football players. According to numerous studies, anthropometric and body composition indicators are important factors, which can predict the specific footballer’s performance, already in his adolescence . In the It is well established that training may alter homeostasis, hematological parameters included. Hb is a key determinate of oxygen transport and consumption , which iTo date, only a few studies have evaluated Hb and HT values during an entire competitive season in football players ,29. Part2max), and high sports performance in football [In the present study, we hypothesized that in the period of congested games (between T3 and T4), in which the championship resumed after the COVID-19 lockdown, the football matches would have negatively affected the plasma volume and hematological parameters. Instead, apart from a slightly decreased Hb during the COVID-19 season, we did not find significant changes in the hematological variables tested. In addition, it should be remarked that during the COVID-19 season , the hemodilution was absent. This phenomenon observed during the 2018/2019 season could be a favorable adaptation to training because decreased blood viscosity allows greater cardiac output. Instead, erythrocytes, Hb, HT, and plasma volume values decreased during the 2018/2019 season, without any interruption. Regarding erythrocytes, in agreement with our results in the 2018/2019 season, many studies have suggested that the number of erythrocytes decreased at the end of a competitive period . This isfootball . Severalfootball . The irofootball . In factfootball . In accofootball , in our football .Vitamin D, mainly synthesized by the skin when exposed to ultraviolet B radiation (UVB), is involved in several physiological processes as the maintenance of calcium, phosphate, and iron homeostasis . During −1, set as the highest reference value for the general population. High values of CK have been suggested as a symptom of overreaching or overtraining [Creatine kinase (CK), cortisol, testosterone, and the testosterone/cortisol ratio (T/C) have been used to assess athletes’ response to training load ,50. Prectraining  and unustraining . This matraining . In additraining .Plasma cortisol and testosterone are sensitive to training periods that differ in volume and intensity and to the frequency of matches during the competitive period . Many faThough we would have expected a significant increase in plasma cortisol after the lockdown, it should be noted that the absence of a significant change in its concentration could be attributed to the lack of stress due to the weekly official sports competitions, rather than the high level of fitness of the players and/or their usual high daily and weekly training volume over the course of the season. Several studies have documented the effects of COVID-19 on the psychological stress experienced in the general population , and it The change in anabolic hormonal concentrations could be due to interactive modifications of various endocrine parameters, and to the effects of modifications of the hypothalamic–pituitary axis on the testicles and adrenal glands. The training-induced increases in serum testosterone reflect the anabolic activity increment related to the volume of strength training and to physical performance improvements . In our In addition to training volume, training intensity is another key factor that may influence aerobic adaptations . SpecifiThe findings from this study are related to only one Italian football team, but different restrictions in other countries might have had different effects on football players’ physical performance and psychological implications.In addition, different home-based training strategies (including the different equipment) could result in different adaptations, in biological and physical performance, on football players.This study was conducted to provide reference data in professional football players, especially under stressful conditions, such as the pandemic period. Despite the spontaneous variability in most parameters, there were significant changes during both seasons in hematocrit, hemoglobin, iron, ferritin, vitamin D, and testosterone.However, from a medical perspective, none of the changes were considered clinically relevant to a player’s health or training status, because all were within normal values and most likely typical modulations of homeostatic concentrations in response to a stressful environment, training, and competition. In addition, this study showed that the training volume during home confinement in the COVID-19 period was probably insufficient to allow professional football players to maintain the jumping power of competitive periods. These changes might be not relevant enough to possibly interfere with clinical decisions in football players.