Here, we investigated how β2‐AR signaling regulates the protein abundance of FoxO and its transcriptional activity in skeletal muscle. We observed that stimulation of β2‐AR with its selective agonist, clenbuterol, rapidly decreased FoxO1 mRNA expression, and this was accompanied by a decrease in either FoxO1 protein level or FoxO transcriptional activity. We subsequently observed that miR‐374b‐5p and miR‐7a‐1‐3p were rapidly upregulated in response to β2‐AR stimulation. Transfection with mimics of either miRNA successfully decreased FoxO1 mRNA. Moreover, because β2‐AR stimulation increased cAMP concentration, pretreatment with an adenylyl cyclase inhibitor canceled out these effects of β2‐AR stimulation. These results suggest that β2‐AR stimulation results in rapid upregulation of miR‐374b‐5p and miR‐7a‐1‐3p in myotubes, which in turn results in a decrease in FoxO1 mRNA expression via the β2‐AR–cAMP signaling pathway.β 2‐adrenoceptor (β2‐AR) signaling regulates the protein abundance of forkhead box O (FoxO) and its transcriptional activity in skeletal muscle. β2‐AR stimulation resulted in rapid upregulation of miR‐374b‐5p and miR‐7a‐1‐3p in myotubes and consequently decreased FoxO1 mRNA expression via the β2‐AR–cAMP signaling pathway.We investigated how β ChangesAtrogin‐1/MAFbx by AKT‐mediated phosphorylation [The forkhead box O (FoxO) transcription factors have a critical role in the transcriptional regulation of Atrogin‐1/MAFbx, one of the muscle‐specific Ub ligases, and thus, they play an important role in controlling the degradation of muscle protein . The trarylation , 18, 19.Atrogin‐1/MAFbx mRNA [1‐AR, β2‐AR, and β3‐AR, and the injection of β2‐AR agonists  also reduces the gene expression of Atrogin‐1/MAFbx, consequently exerting an anabolic effect [2‐AR selective agonist, clenbuterol, suppressed the transcriptional activity of FoxO by AKT‐related phosphorylation [Atrogin‐1/MAFbx mRNA in murine skeletal muscles [2‐AR agonists or norepinephrine regulates FoxO1 transcriptional activity by controlling not only phosphorylation level but also protein abundance in skeletal muscle. However, the mechanisms behind the adrenergic signaling‐induced downregulation of FoxO protein abundance in skeletal muscle remain unclear.In skeletal muscle, either epinephrine or norepinephrine decreases the rate of Ub–proteasome system‐dependent protein degradation by reducing the expression of Fbx mRNA , 21. Thrc effect , 23. Ourrylation . Recentl muscles  also rep muscles , 27. The2‐AR‐induced suppression of muscle protein degradation, this study investigated how β2‐AR signaling regulates the protein abundance of FoxO and its transcriptional activity in skeletal muscle.On the basis of these findings, to elucidate the role of FoxO in controlling β® CRL‐1772™) were purchased from ATCC  and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S) for 3 days. Cells were grown in 10 cm dish (for measurement of Nτ‐methylhistidine release and protein content) or 6‐well plate  at 37 °C in 5% (v/v) CO2 in a humidified environment. To induce myotube formation, the medium was replaced with DMEM supplemented with 2% horse serum and 1% P/S for 5 days. All drugs were made up in phosphate‐buffered saline (PBS) and subsequently added to the appropriate tissue culture wells (1 : 1000). The vehicle‐control wells received just PBS in the same volume as the treatment groups. To study the effects of β2‐AR agonist treatment, C2C12 myotubes were treated with clenbuterol (1 μm), because this concentration of clenbuterol has been reported to successfully decrease Atrogin‐1/MAFbx mRNA expression in C2C12 myotubes [m) was used to block cAMP synthesis.Murine myoblasts  and all primer pairs had equivalent PCR efficiency (from 98% to 105%). The melting curves revealed a single peak for all primer pairs. The coefficient of variation was 7–11%. Amplification, dissociation curves, and gene expression analysis were performed using the Dissociation Curves software . The level of 18S ribosomal RNA was used as an internal standard. The primers used in this study are listed in Table C2C12 myotubes were homogenized in ISOGEN II , in accordance with the manufacturer’s instructions. Real‐time PCR was performed as described previously . Each saAnti‐FoxO1 (#9454) and anti‐phospho‐FoxO1  were purchased from Cell Signaling Technology . Anti‐glyceraldehyde 3‐phosphate dehydrogenase , anti‐rabbit IgG (sc‐2030), and anti‐goat IgG (sc‐2020) were purchased from Santa Cruz Biotechnology .m Tris, 150 mm NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mm ethylenediaminetetraacetic acid, and 1% protease inhibitor cocktail , pH 8.0. The lysate was centrifuged at 20 000 g for 10 min at 4 °C, and the supernatant was collected.C2C12 myotubes were homogenized in 500 μL of a lysis buffer consisting of 50 mimagej software .Western blot analysis was performed as described previously . In brie® Luciferase Assay System  and normalized to the internal control.C2C12 myotubes were transfected with the FoxO reporter and negative control reporter  using the TransIT‐X2 Dynamic Delivery System. Fold induction of normalized reporter activity by FoxO  in C2C12 myotube culture medium after 3 h of clenbuterol stimulation was calculated. The myotubes were also transfected with miRNA mimic . At 24 h post‐transfection, luciferase activity was measured with the Dual‐Glom) for 24 h in DMEM. The medium was then collected, and Nτ‐methylhistidine concentration was determined as described previously [g for 5 min. The supernatant was recovered and evaporated under reduced pressure. The residue was dissolved in 0.2 m pyridine and applied to a cation‐exchange column . After most of the acidic and neutral amino acid had been washed out with 0.2 m pyridine, Nτ‐methylhistidine was eluted with 1 m pyridine and collected. The solvent was evaporated, and the residue was dissolved in mobile phase (15 mm sodium 1‐octanesulfonate in 20 mm KH2PO4). An aliquot was injected into an HPLC system  equipped with an Inertsil ODS‐80A column . The column was attached to an oven at 50 °C. A fluorometric detector set at an excitation wavelength of 365 nm and an emission wavelength of 460 nm was used to monitor the fluorescence derived from the reaction with ortho‐phthalaldehyde. The protein content was determined by the Bradford method with bovine serum albumin as a standard [C2C12 myotubes were incubated with clenbuterol (1 μeviously . Culturestandard .m) for 24 h in DMEM. Total protein and DNA were simultaneously isolated from myotubes using ISOGEN™ reagent (Nippon Gene), in accordance with the manufacturer’s instructions. The protein content was determined by the Bradford method with bovine serum albumin as a standard [C2C12 myotubes were incubated with clenbuterol . Microarray analysis of miRNAs was performed using the Affymetrix GeneChip® miRNA 4.0 Array (Thermo Fisher Scientific). These arrays were scanned with the GeneChip® Scanner 3000 7G (Thermo Fisher Scientific). Differentially expressed miRNAs were analyzed using the Affymetrix® Expression Console™ Software (Thermo Fisher Scientific).Using the mirVana™ miRNA Isolation Kit , miRNAs were isolated in accordance with the manufacturer’s instructions. All samples were diluted to a final concentration of 3 ng·μL® miRNA reverse transcription kit (Applied Biosystems), and real‐time PCR of miRNAs was performed using the TaqMan Universal Master Mix II (Applied Biosystems), in accordance with the manufacturer’s instructions. Quantitative analysis of the expression was carried out using the TaqMan Assay Kit (Applied Biosystems). The level of snoRNA142 was used as an internal standard. The Taqman assay IDs used in this study are as follows: snoRNA142 (Taqman assay ID: 001231), miR‐7a‐1‐3p (Taqman assay ID: 001338), and miR‐374b‐5p (Taqman assay ID: 001319). Each miRNA expression result is expressed as the ratio of the experimental treatment value to the corresponding untreated control value.The miRNAs were reverse transcribed using the Taqmanm mirVana miRNA mimic  or AccuTarget™ miRNA Mimic Negative Control #1  was separately used in each transfection. The assay IDs of mirVana miRNA mimics used in this study are as follows: miR‐374b‐5p (MC11339), miR‐7038‐3p (MC26508), miR‐7016‐3p (MC30144), and miR‐7a‐1‐3p (MC10440). After transfection, cells were added to prewarmed medium and immediately placed in an incubator at 37 °C and 5% CO2. Cells were harvested at 24 h after transfection.Transient transfection of C2C12 cells was performed using the TransIT‐X2 Dynamic Delivery System , in accordance with the manufacturer’s instructions. Briefly, 30 μcAMP levels were determined using an Enzyme Immuno Assay Kit , in accordance with the manufacturer’s instructions.t‐test, Dunnett’s test, or Tukey’s multiple comparison test. P values under 0.05 or 0.01 were considered to indicate statistical significance. These analyses were performed using r software .The data are expressed as mean ± standard error (SE). Statistical comparisons were performed using Student’s 2‐AR activation by 1 μm clenbuterol resulted in decreases of FoxO1 mRNA expression after 1 and 3 h of stimulation . We found that 3142 miRNAs were detectable in these myotubes. The differentially expressed miRNAs among them are shown as heat maps in Fig. We performed a microarray analysis to identify miRNAs that were differentially expressed between C2C12 myotubes stimulated with 1 μFoxO1. As shown in Fig. FoxO1 might be targeted by four of the nine miRNAs (each having one to three putative binding sites). We therefore focused further on these four miRNAs: miR‐374b‐5p, miR‐7038‐3p, miR‐7016‐3p, and miR‐7a‐1‐3p. To investigate their functional roles, we examined FoxO1 expression in C2C12 myotubes transfected with their mimics. Among them, transfection with the miR‐374b‐5p and miR‐7a‐1‐3p mimics successfully decreased FoxO1 mRNA expression in the C2C12 myotubes  before clenbuterol stimulation Fig. . In addiion Fig. .FoxO1 mRNA, while this was not observed in C2C12 myotubes pretreated with SQ22536  predicted to target the 3′UTR of FoxO1.It has been reported that the half‐life of myocytes ; howevern output . During n output . In thiser cells , 35, 36.FoxO1 mRNA expression in myotubes transfected with their mimics. Among the four mimics, either miR‐374b‐5p or miR‐7a‐1‐3p successfully decreased FoxO1 mRNA expression in the C2C12 myotubes. In addition, we confirmed that the transcriptional activity of FoxO was decreased in the C2C12 myotubes transfected with these two mimics. Indeed, decreased Atrogin‐1/MAFbx mRNA expression was confirmed in the C2C12 myotubes transfected with either miR‐374b‐5p or miR‐7a‐1‐3p mimics. These results suggest that, in addition to the previously reported miRNAs , either miR‐374b‐5p or miR‐7a‐1‐3p is rapidly upregulated by β2‐AR stimulation and destabilizes FoxO1 mRNA, consequently affecting its transcriptional activity against its target gene  in myotubes. However, TargetScan analysis also predicted that the 3′UTR of Atrogin‐1/MAFbx would be targeted by both miRNAs, which raises the possibility that these miRNAs directly destabilize Atrogin‐1/MAFbx mRNA, even though β2‐AR stimulation did not affect Atrogin‐1/MAFbx mRNA expression in myotubes within 1 h after stimulation (data not shown).To investigate the functional activity of these miRNAs, we examined 2‐AR couples with the G protein, which activates adenylyl cyclase, catalyzing the formation of cAMP [2‐AR stimulation by clenbuterol increased the cAMP concentration in rat skeletal muscle [2‐AR stimulation rapidly increased cAMP concentration. Then, to inhibit the β2‐AR agonist‐induced increase in cAMP, we employed an adenylyl cyclase inhibitor, SQ22536. Pretreatment with SQ22536 successfully canceled out the β2‐AR agonist‐induced increases in miR‐374b‐5p and miR‐7a‐1‐3p expression, in concert with the increase of cAMP concentration in C2C12 myotubes. In addition, pretreatment with SQ22536 also canceled out the β2‐AR stimulation‐induced decreases in FoxO1 mRNA expression in C2C12 myotubes. Silveira et al. [in vivo muscle‐specific activation of PKA, a cAMP‐dependent protein kinase, inhibited FoxO transcriptional activity. These results suggest that β2‐AR stimulation induced either miR‐374b‐5p or miR‐7a‐1‐3p via β2‐AR–cAMP signaling and that these increased miRNAs in turn contributed to suppressing FoxO1 transcriptional activity in C2C12 myotubes.As mentioned above, norepinephrine stimulation decreases FoxO1 protein abundance via the cAMP–PKA pathway in murine skeletal muscle . β2‐AR c of cAMP . It has l muscle . In this2‐AR–cAMP signaling increases either miR‐374b‐5p or miR‐7a‐1‐3p expression in C2C12 myotubes remains unclear. One possible explanation for this might involve the endoribonucleases  required for miRNA processing [FoxO1 mRNA [2‐AR stimulation and Ftx transcript expression remains unclear, it has been reported that β2‐AR stimulation increased Hnrnpk protein levels in the skeletal muscle of human with high‐intensity training [2‐AR stimulation‐induced increase in either miR‐374b‐5p or miR‐7a‐1‐3p. Further investigations are needed to gain insight into the mechanisms by which β2‐AR stimulation recruits these miRNAs in myotubes.It has been reported that cAMP elevation changed the expression of a large number of miRNAs. For example, in human placental cells, forskolin, an inducer of intracellular cAMP, significantly changed the expression of 116 miRNAs , 40, 41.ocessing . In isolxO1 mRNA . Furthertraining . It was 2‐AR stimulation by clenbuterol rapidly upregulated miR‐374b‐5p and miR‐7a‐1‐3p in myotubes and then decreased the mRNA expression of FoxO1 via the β2‐AR–cAMP signaling pathway. β2‐AR–cAMP signaling suppresses FoxO1 transcriptional activity that would otherwise promote the expression of genes encoding Atrogin‐1/MAFbx mRNA, and consequently suppresses protein degradation via the dual mechanisms of altering FoxO1 protein abundance and phosphorylation in skeletal muscles.In conclusion, βThe authors declare no conflict of interest.SS, KN, AO, and DI conceived and designed the experiments. SS, KN, NN, and RK performed the experiments and contributed reagents/materials/analysis tools. SS, KN, and DI wrote the paper. AO and SF reviewed and revised the paper.


Background: Although anterior cruciate ligament reconstruction (ACLR) is a well-established procedure and is standardly performed by orthopedic surgeons all over the world, there does not seem to be a standard protocol for early rehabilitation. The purpose of this study was to give answers to the following questions: (i) Does (a) the use of a specific tendon graft, and (b) potentially additional therapy of concomitant pathologies influence surgeons’ choice of a distinct postoperative rehabilitation protocol after ACLR? (ii) To what extent do these rehabilitation recommendations differ? Methods: Retrospective analysis of currently used early rehabilitation protocols after ACLR in German-speaking countries  was conducted. Rehabilitation criteria included weight bearing, range of motion (ROM), the utilization of braces, continuous passive/active motion therapy (CPM/CAM), rehabilitation training and sport-specific training. Tendon grafts were differentiated as hamstring (HAM) and bone–patellar tendon–bone grafts (BTB). Concomitant pathologies included meniscus injuries (+M) and unhappy triad injuries (+UTI). Results: Most of the surveyed protocols were differentiated according to the used tendon graft or additional therapy of concomitant injuries . When comparing ACLR-HAM and ACLR-BTB, significant differences were found regarding weight bearing (p = 0.01), ROM (p = 0.05) and the utilization of braces (p = 0.03). Regarding ACLR+M, an overall significant decelerated rehabilitation could be detected. After ACLR+UTI-therapy, a significant delayed start to full weight bearing (p = 0.002) and ROM (p < 0.001) was found. Conclusions: Most orthopedic surgeons from German-speaking countries differentiate early rehabilitation after ACLR according to the tendon graft used and therapy of concomitant pathologies. No consensus about early rehabilitation after ACLR is available. However, tendencies for an accelerated rehabilitation after ACLR-BTB and a more restrained rehabilitation of multiple injured knees were detected. Injuries of the anterior cruciate ligament (ACL) are among the most common ligamentous injuries of the knee joint ,2,3,4,5.Looking at orthopedics worldwide, there are numerous national and international associations and research groups that provide post-treatment recommendations after ACLR, which are updated regularly, e.g., by MOON , KNGF , the DOA  or the DGOU . However, they differ not only in terms of content concerning the recommendations, but also in terms of quality and applicability to clinical practice. What is striking here is that most of them do not address different graft types or concomitant injuries ,20,21,22Therefore, the research questions for this study were:(1)Does (a) the use of a specific tendon graft, and (b) potentially additional therapy of concomitant pathologies influence surgeons’ choice of a distinct postoperative rehabilitation protocol after ACLR?(2)To what extent do these rehabilitation recommendations differ?Design—The medical device company OPED  developed a tabular template for creating a post-treatment protocol after ACLR, which OPED made freely available to orthopedic departments and outpatient orthopedic centers in German-speaking countries, so that the associated orthopedic surgeons could use the template to create their individual post-treatment rehabilitation standard using the categories described in OPED itself was not involved in the content of the rehabilitation protocols. All protocols were collected and blinded for study purposes. The investigated protocols contain general early rehabilitation recommendations of orthopedic surgeons given to their patients and physiotherapists. The investigation of these protocols focused on the described use of different tendon grafts, such as HAM grafts or BTB grafts, as well as the presence and additional therapy of concomitant injuries such as meniscus lesions or UTIs and the categories of early rehabilitation as described in Participants—Rehabilitation protocols for early rehabilitation after ACLR in written form (n = 205), which are currently used in daily routines in German-speaking countries , were surveyed for this qualitative study. These originated from 120 different orthopedic institutions, of which 63 were outpatient centers and 57 were clinical centers, 4 of which in turn were university medical centers.Measures—The included early rehabilitation protocols were analyzed regarding the surgery performed, particularly the type of tendon graft used, the therapy of concomitant pathologies and the following rehabilitation categories . Data are presented as mean ± SD or absolute and relative frequencies. Continuous data between two or more groups were compared by an analysis of variance (ANOVA). Categorical data were compared by using the chi-squared test of independence. In case of significance, further post hoc tests (according to Fisher’s least significant difference) were performed. A probability (p) value of ≤0.05 was considered to be significant for each test. Graphical illustrations were generated with GraphPad Prism®  and Microsoft PowerPoint 2013® .In total, 205 early rehabilitation protocols after ACLR were analyzed. Of these, 57 protocols provided information about early rehabilitation after ACLR using HAM grafts (ACLR-HAM), 21 for BTB grafts (ACLR-BTB), 52 for ACLR with concomitant meniscus repair (ACLR+M) and 17 for UTI (ACLR+UTI). Another 58 protocols were available for ACLR without clear differentiation of the used graft type (ACLR-w/o-gd). The respective evaluation and comparison of the early rehabilitation categories is described below.p = 0.01). The one-week earlier return to full weight bearing (ACLR-HAM after four weeks and ACLR-BTB after three weeks) was found in all post-surgery recommendations . When comparing ACLR+UTI to ACLR-w/o-gd, the delay of increase in weight bearing was one week . A two-week delayed return to full weight bearing was found in all post-surgery recommendations (full consensus)  see .p = 0.050) . The mean time of recommended full ROM after ACLR-BTB was found to be one week earlier compared to ACLR-HAM, which was statistically significant  see .p < 0.001; ACLR+UTI: 4.6 ±1.5 weeks, p < 0.001)  see .n = 5; 23.8%). In contrast, almost half of the evaluated concepts after ACLR-HAM recommended the use of braces . Even if the recommended wearing time of the brace differed by almost two weeks, no significance was detected when comparing ACLR-BTB (4.8 weeks ± 1.6 weeks) to ACLR-HAM   see a,b.n = 41; 79% of the evaluated ACRL+M protocols). In contrast, just half of the rehabilitation concepts utilized braces after ACLR-w/o-gd  and ACLR+UTI . Analyzing the brace-wearing time, the mean recommended period ranged from six to seven weeks   see a,b.p = 0.71). After ACLR, in combination with therapy of meniscus injuries or UTIs, just a few protocols recommended the use of CPM for the first three postoperative days. In contrast, CAM therapy was strongly recommended. Recommendation for CAM was limited to six postoperative weeks  (see p < 0.001). In contrast, rehabilitation training after ACLR-w/o-gd and ACLR+UTI was recommended to start after 4.5 weeks  (see p = 0.17). The recommended start of specific training after ACLR+M was delayed by almost two weeks (14.2 ± 4.5 weeks) in comparison to ACLR-w/o-gd  and ACRL+UTI   see a. In the.65) see a. The st.85) see b.The main finding of this study is that almost three-quarters of the evaluated orthopedic recommendations differ in the early rehabilitation of ACLR in regard to different graft types and therapy of concomitant pathologies. This is an important fact and shows an awareness of orthopedic surgeons of the requirement of individualized rehabilitation concepts to achieve optimal clinical outcomes and return to sports and work. This finding is of particular interest, since current national and international guidelines for rehabilitation after ACLR only provide a basic rehabilitation description, without connecting it to different graft choices or adaptations due to the additional therapy of concomitant injuries ,22,26. IIn the analyzed early rehabilitation protocols, no general consistent recommendations were found in regard to weight bearing, ROM or the utilization of braces after ACLR and possible additional treatment of concomitant pathologies. Similarly, wide disparities have already been found in a survey of members of the “American Orthopedic Society of Sports Medicine” and the “Arthroscopy Association of North America” . HoweverIn the case of ACLR+M and ACLR+UTI, a significantly decelerated start and increase in weight bearing, as well as ROM, was recommended compared to isolated ACLR. Although the principles of ACL healing are the same, orthopedic surgeons acknowledge the additional injuries of the meniscus and collateral ligament as a potential multiple trauma and therefore assume longer healing times. As a consequence, prolonged periods of partial weight bearing and prolonged ROM limitations are implemented. However, there is no evident data in the current literature concerning advantages for accelerated or conservative rehabilitation strategies after meniscus repair ,37. In oCertainly, the present study has some limitations. Due to the retrospective design, the results lack factors such as individual modification of the rehabilitation protocol by orthopedics, clinical outcome, the patients’ compliance or sociodemographic data such as gender or age. Furthermore, technical details such as meniscus lesion patterns and the kind of meniscus repair, as well as therapeutic information concerning the therapy of medial collateral ligament injuries in context to UTI, are not available. Rehabilitation data about concomitant cartilage injuries are also missing, as they were not available in the protocols. The evaluated rehabilitation protocols cover the early period of rehabilitation up to 12 weeks post-surgery, while regeneration and return to work/sport require a longer period of rehabilitation and are therefore not covered by this data sample. However, this study reveals the clinical routine and daily practices of today’s orthopedic health care providers and therefore shows the current state of play in the postoperative care of ACL-related knee injuries.In summary, this study presents detailed information about the status quo of currently used early rehabilitation concepts after ACLR for the first time. Almost three-quarters of orthopedic surgeons in German-speaking countries implement into their postoperative care protocols the choice of a tendon graft and the treatment of concomitant injuries. Even if no consistent rehabilitation algorithms are available, there are trends respecting the individual pathology and consecutive therapy. However, further prospective research is required to investigate the evidence of the currently evaluated rehabilitation concepts and to create a guideline outlining all different aspects of ACLR and the treatment of concomitant injuries.


A insuficiência valvar aórtica (IA) é um distúrbio valvar comum2 que também pode ser um dos mais desafiadores para quantificar-se com precisão. O ecocardiograma ajuda a avaliar a gravidade da IA utilizando várias técnicas bidimensionais, tridimensionais e de Doppler colorido, mas o mais importante é que oferece uma oportunidade única para a avaliação hemodinâmica, que é extremamente importante na classificação da gravidade da IA.O ecocardiograma continua sendo o padrão-ouro para o diagnóstico e classificação das cardiopatias valvares,3 os autores testaram, como prova de conceito, a correlação entre a integral velocidade-tempo (IVT) da IA e a gravidade da insuficiência aórtica em uma análise multivariada e mostraram a relação inversa entre a IVT da IA e a gravidade da IA, independente do diâmetro e volume do ventrículo esquerdo, frequência cardíaca, pressão arterial diastólica ou fração de ejeção do ventrículo esquerdo. Eles também mostraram que a IVT da IA é um método que pode ser facilmente obtido e reproduzido para avaliar a gravidade da IA, em comparação com outros métodos comumente utilizados, como a Área de Superfície da Isovelocidade Proximal (PISA). Este estudo apresenta um conceito interessante e promissor que aumentará o nível de confiança ao avaliar a gravidade da IA. Também faz sentido fisiologicamente, pois os pacientes com IA grave têm um gradiente diastólico menor entre a aorta e o ventrículo esquerdo  que teoricamente resultarão em um valor menor de IVT da IA devido à rápida equalização pressão entre a aorta e o ventrículo esquerdo.No artigo intitulado: “Integral velocidade-tempo da regurgitação aórtica: um novo marcador ecocardiográfico na avaliação da gravidade da insuficiência aórtica”,Vale ressaltar, porém, que existem algumas limitações para este estudo. Primeiro, há uma falta de padrão ouro para a avaliação da gravidade da IA, além da “opinião de especialistas”. Segundo, o grupo com IA grave é provavelmente composto de dois grupos separados: o grupo IA grave aguda e o grupo IA grave crônica. É importante distinguir esses dois grupos, pois a IA grave, bem compensada e crônica é provavelmente hemodinamicamente semelhante à IA moderada, com um valor de IVT da IA maior do que aqueles com IA grave aguda. A falta de associação entre a gravidade da IA e o meio-tempo de pressão neste estudo confirma que o grupo com IA grave é provavelmente uma mistura de pacientes com cronicidade variável. Terceiro, alguns jatos de IA serão muito desafiadores para obter uma amostra por Doppler de onda contínua, dada sua excentricidade. Isso é principalmente significante em pacientes com válvulas aórticas bicúspides ou unicúspides que tendem a ter jatos regurgitantes muito excêntricos. Por fim, será necessário um acompanhamento clínico para avaliar a sobrevida, a necessidade de cirurgia valvar aórtica ou outros eventos adversos com base no valor da IVT da IA para verificar a utilidade do conceito.A introdução de novos conceitos ou técnicas que ajudam na classificação da gravidade da IA é um recurso valioso. A IVT da IA é um conceito promissor que é fisiologicamente correto e parece ser reproduzível. Serão necessários ensaios clínicos maiores para avaliar melhor seu papel e, mais importante, seu valor prognóstico e correlação com os resultados clínicos.1É indispensável, no entanto, ter em mente que é muito improvável que encontremos um marcador ecocardiográfico único que seja um padrão ouro na avaliação da gravidade da IA. Os ecocardiografistas devem manter a mente aberta e integrar todos os dados disponíveis para chegar a uma conclusão final. Isso inclui o seguinte:Dados clínicos Avaliação bidimensional e tridimensional da válvula aórtica  e câmaras cardíacas 4 largura do jato comparado à largura da VSVE, avaliação PISA5 quando possível e quantificação 3D com Doppler colorido6 )Doppler colorido .Entretanto, como em outras lesões valvares, ecocardiografistas e estagiários devem evitar diagnosticar a gravidade da IA com base apenas no Doppler colorido, mesmo que seja tentador fazê-lo inicialmente. A avaliação das consequências hemodinâmicas da IA deve ser um componente-chave da avaliação. Por exemplo, é improvável que o diagnóstico de IA grave com tamanho diastólico final normal do ventrículo esquerdo, sem reversão do fluxo diastólico na aorta torácica descendente ou abdominal ou com uma pressão de pulso normal seja preciso e deve ser reavaliado.7 A tomografia computadorizada (TC) cardíaca também pode ser útil para identificar vazamentos periprotéticos e orientar procedimentos cirúrgicos e percutâneos.8 Finalmente, um aortograma bem executado, realizado no laboratório de cateterização, pode ser muito valioso quando outras modalidades de teste são inconclusivas.Além disso, a utilização de outras modalidades para avaliar a gravidade da IA pode ser necessária quando os dados ecocardiográficos e clínicos forem inconclusivos ou contraditórios. A ressonância magnética cardíaca (RMC) tem um papel importante e promissor na avaliação da gravidade da IA, especialmente com jatos excêntricos ou vazamentos valvares periprotéticos. Ajuda a avaliar a fração regurgitante, utilizando imagens de contraste de fase e o tamanho e a função dos ventrículos esquerdo e direito com boa precisão. 1 despite the development of other imaging modalities. Aortic valve regurgitation (AR) is a common valvular disorder,2 which can also be one of the most challenging to accurately quantify. Echocardiography helps in assessing the severity of AR utilizing multiple two-dimensional, three-dimensional and color Doppler techniques, but most importantly, offers a unique opportunity for hemodynamic evaluation, which is extremely important when grading the severity of AR.Echocardiography remains the gold standard for the diagnosis and grading of valvular heart diseases,In the article entitled: “Velocity-time integral of aortic regurgitation: a novel echocardiographic marker in the evaluation of aortic regurgitation severity”, the authors tested, as a proof of concept, the correlation between AR velocity-time integral (VTI) and the severity of aortic valve regurgitation in a multivariate analysis and showed the inverse correlation between AR VTI and AR severity, regardless of left ventricular diameter, volume, heart rate, diastolic blood pressure or left ventricular ejection fraction. They also showed that AR VTI is an easily obtainable and reproducible method to assess AR severity when compared to other commonly used methods, such as Proximal Isovelocity Surface Area (PISA). This study introduces an interesting and promising concept that will add to confidence level when assessing AR severity. It also makes physiological sense, as patients with severe AR will have a smaller diastolic gradient between the aorta and the left ventricle (higher left ventricular end diastolic pressure and lower diastolic blood pressure), which will theoretically result in a smaller AR VTI value due to rapid equalization of pressure between the aorta and the left ventricle.It is worth noting, however, that there are a few limitations to this study. First, there is a lack of a gold standard for the assessment of AR severity other than an “expert opinion”. Second, the severe AR group is probably consisted of two separate groups: the acute severe AR group and the chronic severe AR group. It is important to distinguish between these two groups as chronic, well compensated, severe AR is likely to be hemodynamically similar to moderate AR with a larger AR VTI value, when compared to those with acute severe AR. The lack of association between AR severity and pressure half time in this study supports the fact that the severe AR group is probably a mix of patients with variable chronicity. Third, some AR jets will be very challenging to sample by continuous wave Doppler, given their eccentricity. This is mainly notable in patients with bicuspid or unicuspid aortic valves, which tend to have very eccentric regurgitant jets. Finally, a clinical follow-up to assess survival, need for aortic valve surgery or other adverse events based of AR VTI value will be needed to verify the utility of the concept.Introducing new concepts or techniques that can help grading AR severity is a valuable resource. AR VTI is a promising concept that is physiologically sound and appears reproducible. Larger clinical trials will be needed to further assess its role and, more importantly, its prognostic value and correlation with clinical outcomes.1It is imperative, however, to keep in mind that it is very unlikely that we will find a single echocardiographic marker that will be a gold standard when assessing AR severity. The echocardiographist has to keep an open mind and integrate all available data to come to a final conclusion. This includes the following:Clinical data Two-dimensional and three-dimensional evaluation of the aortic valve  and cardiac chambers 4 jet width compared to LVOT width, PISA5 evaluation when feasible and 3D color Doppler quantification6 )Color Doppler .However, as with other valve lesions, echocardiographists and trainees should refrain from diagnosing AR severity based on color Doppler alone, even if it is tempting to do so initially. Assessing the hemodynamic consequences of AR should be a key component of the evaluation. For example, diagnosing severe AR in the setting of a normal left ventricular end diastolic size, without diastolic flow reversal in the descending thoracic or abdominal aorta, or with a normal pulse pressure is unlikely to be accurate and should be reassessed.7 Cardiac Computed Tomography (CT) can also be helpful to identify peri-prosthetic leaks and guide surgical and percutaneous procedures.8 Finally, a well-performed aortogram carried out in the catheterization laboratory can be very valuable, when other testing modalities are inconclusive.Furthermore, utilizing other modalities to assess AR severity might be needed when echocardiographic and clinical data are inconclusive or contradictory. Cardiac Magnetic Resonance Imaging (MRI) has an important and promising role in assessing AR severity, especially with eccentric jets or peri-prosthetic valvular leaks. It helps assess the regurgitant fraction utilizing phase-contrast imaging and left and right ventricular size and function with good accuracy.


B decays endure. We present results of an updated global analysis that takes into account the latest experimental input – in particular the recent results on The anomalies in rare  B mesons based on the neutral current Since several years there exist persistent discrepancies between the Standard Model (SM) predictions and the experimental results for rare decays of B decay data find consistently very strong preference for new physics in the form of the operator Intriguingly, the simplest new physics scenarios that address the theoretically clean hints for LFU violation simultaneously explain also the other discrepancies. Parameterizing the new physics in terms of four fermion contact interactions, global fits of rare mentclass2pt{minimwork see ).Very recently, the LHCb collaboration presented updated results for two theoretically clean observables that have previously shown tensions with the SM predictions: the LFU ratio nt}RK is 2\documenmentclasspt{minimaThe branching ratio of the nd to be , 143\doc37)×10-9 –18. Comp.3)×10-9 , the newThe main goal of this paper is to interpret the impact of the new experimental results in a model independent way, using the well established effective Hamiltonian approach. We parameterize new physics contributions by Wilson coefficients of dimension 6 interactions evaluated at the renormalization scale he SMEFT . We alsohe SMEFT , 21.A critical aspect of global fits is the treatment of theory uncertainties. In our previous studies , 22–24 wThe paper is organized as follows: in Sect. Our global fits are based on a  follows  and is sWe have developed a computationally efficient method to determine the new physics dependence of B decay amplitudes are linear functions of the Wilson coefficients it is possible to express the branching ratios as second order polynomials in the Wilson coefficients. The coefficients of the polynomials are independent of new physics and their correlated uncertainties can be described by a covariance matrix that needs to be determined only once. The covariance matrix of the branching ratios can then be expressed in a straight forward way in terms of the covariance matrix of the polynomial coefficients and the Wilson coefficients.As the rare The CP averaged angular observables The new error treatment is particularly relevant for quantities that are predicted with very high precision in the SM but that have non-negligible uncertainties in the presence of new physics. In that case, the corresponding entries in the theoretical covariance matrix evaluated in the SM and the ones in the presence of new physics may differ significantly. The most important examples can be grouped into three categories: (i) lepton flavor universality tests, (ii) CP asymmetries, (iii) observables that vanish in the absence of right-handed currents. In most cases, the current experimental uncertainties of these observables are considerably larger than the theory uncertainties both in the SM as well as in viable new physics scenarios and the impact of the theory uncertainties in the global fit is moderate. However, with the expected improvement in experimental sensitivity, the theoretical uncertainties will become more and more important and their new physics dependence needs to be taken into account.Among the lepton universality tests, ratios of branching ratios, like ument}1% , 26. In ment}∼4% , c.f. Eqnt}2.8%)  assuming(B→K∗ee)  , hant}Qi in  it becomIn Figs. relation . For \dotainties , 32, as Similarly, the plot in Fig. ll large , we showmentclass2pt{minimWith regards to CP violation, we note that results on CP asymmetries in n 1 data . The mos physics  (Interesussed in ). In the∼O(10-3) . The avaument}5%  both at In Fig. tainties , as wellThe update of the rom LHCb . The \do results . IncludeThe new analysis . While tThe latest results on from CMS  and the from CMS , 14. We from CMS , as descfrom CMS , our comThe recent update of ument}RK . The newmentclasspt{minimaThe latest results from LHCb and CMS on the effective ×10-12 s , 14 and ×10-12 s   , 40.The recent update of the ng ratio  that conng ratio  with theOur numerical code is based on the Python package flavio [In comparison to our previous fit in , we impre flavio , which pi v2.3.1 , which bWe start by considering simple one parameter new physics scenarios, switching on one real new physics Wilson coefficient at a time. We consider several fits, including certain subsets of observables. In Table In the column “mentclass2pt{minimtainties , it cannIn the column “LFU, mentclass2pt{minimB decays” in Table Finally, in the the column “all rare entclass1pt{minimaTo illustrate the impact of our improved treatment of theory uncertainties, we compare in Table Next, we discuss scenarios where two Wilson coefficients are turned on simultaneously. In Fig. e update  is shownIn Fig. nomalies  or from As clearly seen in the plots of Figs. see e.g. ). In Figectively . A similectively . We do nWe also consider more generic scenarios with more then two Wilson coefficients. In particular, we consider a four parameter scenario including the muon-specific semi-leptonic Wilson coefficients In the four parameter scenario we perform two fits: (1) a fit including only the  iminuit , 48 PythWe find sizable correlations among the Wilson coefficients. One of the main contributors to the correlations is the new precise measurement of The central values of our global four parameter fit agree within uncertainties with the central values of a similar fit performed in . Compareation in . We chec done in ) improve done in  to some We find similar results in the six parameter scenario. The parameters of the multivariate Gaussian that approximates the likelihood function in the vicinity of the best fit point of the global fit is reported in Table In the presence of new physics, the contributions to the flavor changing Wilson coefficients can generically be CP violating. While the observables that show tensions with SM predictions are CP conserving, it is interesting to investigate the impact that imaginary parts of Wilson coefficients have on the fit, and to which extent imaginary parts are constrained by existing data  can significantly improve the agreement between data and theory predictions. The various best fit points show comparable pulls, and it is therefore interesting to identify predictions that allow us to distinguish the new physics scenarios.We consider six different two parameter new physics scenarios: (i) The first set of rows shows the predictions for the LFU ratios The second set of rows shows predictions for LFU differences of The last set of rows shows predictions for the In Figs. With the recent updates of Finally, we give new physics predictions for a large set of observables including LFU ratios, LFU differences of CP averaged Note Added Another model independent interpretation of the new results can be found in . First i


Importance: The need for cancer rehabilitation is expected to continue to dramatically increase with the aging population and increasing number of cancer survivors. These survivors experience a wide range of physical limitations and symptoms that negatively affect their health and quality of life. Research is needed to determine the rate of adherence, reasons for non-adherence, and interventions to improve adherence to acute inpatient rehabilitation among patients with cancer.Objective: To evaluate the rate of adherence and reasons for non-adherence to acute inpatient rehabilitation in patients with cancer.Design, Setting, and Participants: This was a secondary analysis of a retrospective study that assessed medical complications in 165 patients with cancer who had a median length of stay of 11 days (interquartile range of 8-14) in acute inpatient rehabilitation. We reviewed the records of all consecutive patients who underwent acute inpatient rehabilitation from September 1, 2017 through February 28, 2018 at a large academic, quaternary National Cancer Institute-designated Cancer Center.Main Outcomes and Measures: We calculated the rehabilitation session adherence rate and descriptively summarized the reasons for non-adherence.Results: There were 78/165 (47%) patients that had 1 or more incomplete rehabilitation sessions due to medical complications. These patients had a median of 2 (interquartile range of 1-4) incomplete rehabilitation sessions. We noted other reasons for incomplete rehabilitation sessions in 146/165 (89%) patients, who had a median of 3 (interquartile range of 2-4) incomplete rehabilitation sessions. The median total number of days with incomplete rehabilitation sessions in the entire cohort was 2 (interquartile range 1-3).Conclusion and Relevance: Among patients with cancer undergoing acute inpatient rehabilitation, the adherence rate to 1-hour long intensive rehabilitation sessions were low due to medical complications and other reasons. This in turn affected compliance with the 3 hours of rehabilitation a day requirement for acute inpatient rehabilitation. Patients with cancer undergoing acute inpatient rehabilitation are medically complex and further research at multiple institutions with larger cohorts may be beneficial in further assessing adherence rates and reasons for non-adherence to improve participation in acute inpatient rehabilitation. For admission to acute inpatient rehabilitation, the Centers for Medicare & Medicaid Services (CMS) require that the patient 1) undergo an intensive rehabilitation program generally consisting of 3 hours of therapy per day at least 5 days a week with multiple therapy disciplines; 2) is medically stable and expected to benefit significantly, with measurable functional improvement from active participation within a prescribed period of time; 3) is supervised by a rehabilitation physician with face-to-face visits at least 3 days a week; and 4) undergoes the program with a coordinated, interdisciplinary team approach In a systematic review, the adherence rate for supervised physical therapy in outpatient settings for patients with cancer was greater than 64% This is a secondary analysis of a retrospective study that included 165 patients, whose demographic and clinical characteristics along with data collection procedures were described and published previously see  4. ApproProgress notes from rehabilitation clinicians  were reviewed to measure adherence and identify reasons for incomplete therapy sessions, which were defined as fewer than 45 minutes of participation out of 60 minutes scheduled per rehabilitation session. At our institution, the rehabilitation sessions are provided as 1-hour sessions 3 times a day on weekdays and an additional 1-hour session on a weekend day. Sessions on weekends, holidays, admission days, and discharge days were excluded from this study because patients did not consistently undergo 3 hours of rehabilitation sessions on those days. The duration of each rehabilitation session is documented in each progress note by physical and occupational therapists. Speech and language pathology sessions were assumed to be complete sessions (unless explicitly stated as a missed session) as their durations were not recorded.th) day beyond the requirement of 3 hours of rehabilitation sessions per day at least 5 days out of the week. In identifying reasons for incomplete rehabilitation sessions, problems identified as moderate or high risk of complications in the medical decision-making section of CMS guidelines We assessed adherence by measuring the frequency and proportion of patients who were noted to have 1 or more incomplete rehabilitation sessions on days when they should have had 3 hours of rehabilitation sessions. We did not assess adherence based on the total number of rehabilitation sessions since patients had an additional rehabilitation session on a weekday  in acute inpatient rehabilitation. Among these patients, there were 61 (37%) patients who had metastatic solid tumors and 69 (42%) who had solid tumors without metastasis. Baseline demographic and clinical characteristics including neoplasm types were previously published see  4. ThereSeventy-eight out of 165 (47%) patients had 1 or more incomplete rehabilitation sessions due to medical complications Table  with a mTo our knowledge, this is the first report of rates of adherence and reasons for non-adherence to acute inpatient rehabilitation among patients with cancer. We noted a high frequency (47%) of patients having incomplete rehabilitation sessions due to medical complications and a very high frequency (89%) of patients having incomplete rehabilitation sessions due to other reasons. This resulted in a median of 2 days with less than 3 hours of rehabilitation sessions. Incomplete rehabilitation sessions can prolong rehabilitation length of stay and decrease the effectiveness of acute inpatient rehabilitation.In order to maximize patient adherence to rehabilitation sessions, several interventions can be considered. Some interventions such as optimization of medical comorbidities, pharmacological management for improved symptom control, and coordination of care with acute care oncology services to ensure medical stability, can be applied before transfer to (and during) acute inpatient rehabilitation. Ideally, these interventions should be performed before starting an intensive acute inpatient rehabilitation program to minimize interruptions during rehabilitation participation. Other steps, such as improvement of documentation of incomplete rehabilitation sessions, communication to allow for flexibility with schedules, and coordination of rehabilitation sessions and planned medical care , can help improve adherence to rehabilitation sessions.Although an acute inpatient rehabilitation program generally consists of at least 3 hours of rehabilitation per day at least 5 days a week, in well-documented cases, it can be provided as at least 15 hours of intensive rehabilitation therapy over 7 consecutive days When patients are transferred from acute medical care to acute inpatient rehabilitation, their medical condition should be stable This study is limited by its small sample size and assessment at a single institution. Because this was a retrospective study, it depended on the accuracy of the rehabilitation clinicians' progress notes. Factors such as the phase of oncological treatment, patients' motivation or level of education, and living conditions were not documented consistently in the majority of the studied population before transfer to acute inpatient rehabilitation. Unlike physical and occupational therapy progress notes, speech and language pathology progress notes did not routinely document session duration. Therefore, we could have underestimated the true rate of adherence and overall assessment of various reasons for non-adherence. Nevertheless, this pilot study provides a framework for understanding adherence to acute inpatient rehabilitation.Among patients with cancer undergoing acute inpatient rehabilitation, the number of patients unable to adhere to 1-hour long rehabilitation sessions to complete 3 hours of rehabilitation sessions a day was high. Further research is needed to assess adherence and reasons for non-adherence and improve participation to maximize functional gains from acute inpatient rehabilitation.Supplementary table S1.Click here for additional data file.


In both in vitro and in murine models of DDP-induced nephrotoxicity, lnc-MEG3 exacerbated DDPIN by negatively regulating miRNA-126 subsequently causing a decreased AKT/TSC/mTOR-mediated autophagy. By silencing lnc-MEG3 or incorporating miRNA-126 mimetics, the proliferation and migration of DDP-treated cells were restored. In vivo, we identified Paeonol to alleviate DDPIN by the inhibition of lnc-MEG3. Taken together, lnc-MEG3 represents a novel therapeutic target for DDPIN and Paeonol may serve as a promising treatment by inhibiting lnc-MEG3 and its related signaling pathways.Cis-Diamminedichloroplatinum (II) (DDP)-induced nephrotoxicity (DDPIN) may cause irreversible renal injury associated with high morbidity and mortality. Current standard therapies have not achieved satisfactory clinical outcomes due to unclear molecular and cellular mechanisms. Therefore, exploring potential therapies on DDPIN represents an urgent medical need. Present study characterized the role of lncRNA maternally expressed gene 3 ( Cisplatin (cis-diamminedichloroplatinum (II), DDP) is a widely used chemotherapy for the treatment of many malignancies lnc-MEG3), transcripts 1.6kb, is an imprinted gene locus located at chromosome 14q32.3 in humans and chromosome 12 in mice. Previous studies have demonstrated that lnc-MEG3 was reduced in renal fibrosis and renal cell carcinoma lnc-MEG3 has been shown to significantly alleviate lipopolysaccharide-induced renal tubular epithelial cell apoptosis and hypoxia-elicited kidney injury in acute renal allografts lnc-MEG3 in DDPIN has not been established yet.Long non-coding RNAs (lncRNAs), molecules longer than 200 nucleotides, directly or indirectly interact with target genes at the transcriptional level and play an important role in biological processes lncRNAs usually function as a competing endogenous RNA (ceRNA) sponge to regulate microRNA expression and downregulate specific proteins such as AKT  Moutan CortexPaeonol  is an active ingredient derived from the root bark of the Chinese herbal medicinelnc-MEG3 in DDPIN was noted both in vitro and in vivo and subsequently suppressed the activation of miRNA-126. Inhibition of miRNA-126 activated the AKT/TSC pathway, leading to suppressed mTOR-mediated autophagy, inflammatory responses, apoptosis and renal tubular epithelial cell damage. We further uncover a novel mechanism in which Pae exhibits protective effects on DDPIN by inhibiting the ability of lnc-MEG3 to regulate miRNA-126.In this study elevated level of lnc-MEG3 was drastically in renal injury based on publicly available NCBI Gene Expression Omnibus (GEO) database (GSE124622)  and creatinine (SCr)  A; severene (SCr) B; increane (SCr) C; accumurophages D and altrophages E were evabase GSE24622 (Fiabase GSE24622 (Fiin situ hybridization (FISH) assays were used to examine the location and expression of lnc-MEG3 mRNA and our results demonstrated that lnc-MEG3 localized primarily in the cytoplasm fractionation of proximal tubule epithelial HK-2 cells -induced renal fibrosis lnc-MEG3 and kidney injury, studies of lnc-MEG3 in the context of DDPIN are lacking. It was reported that lnc-MEG3 knockdown alleviates apoptosis or inflammation in human bronchial epithelial cells and macrophages lnc-MEG3 was increased in DDPIN, which was associated with increased apoptosis and inflammation. These can be reversible following silencing lnc-MEG3. Expression of autophagy-related proteins and autophagy-associated AKT/TSC/mTOR signaling was also significantly increased upon lnc-MEG3 knock down and lnc-MEG3 silencing. Collectively, our results suggest that lnc-MEG3 functions as a critical mediator of DDPIN by regulating AKT/TSC/mTOR-mediated autophagy.We then explored how cisplatin regulates autophagy levels and subsequent renal injury. We found that lnc-MEG3 regulates autophagy-related signaling pathways based on a bioinformatic prediction that lnc-MEG3 may function as a competing endogenous RNA sponge to regulate the miRNA-126 expression. In fact, miRNA-126 was negatively correlated with lnc-MEG3 both in vitro and in vivo and the downregulation of miRNA-126 expression was observed in DDPIN, whereas miRNA-126 overexpression reversed characteristic features of DDPIN. To clarify the downstream signaling pathways mediated by miRNA-126 we analyzed the activation of intracellular autophagy signaling components. As expected, AKT was highly phosphorylated in DDPIN mice and was accompanied by increased lnc-MEG3 and decreased miRNA-126 expression. AKT is known to phosphorylate TSC2, which destabilizes the TSC2 protein and results in TSC complex  dissociation from the lysosome lnc-MEG3 negatively regulates miRNA-126 expression which leads to the activation of AKT/TSC/mTOR signaling pathway that is required for autophagy suppression.We then studied how Mudan Cortex, has protective effects on chemotherapy drugs-induced oxidative stress, nitrative stress and autophagy in the heart, liver and kidney lnc-MEG3 and reduced miRNA-126 were reversed in DDPIN mice following Pae treatment. Pae also altered multiple aspects of the AKT/TSC/mTOR signaling pathway, thereby partially ameliorating pathogenic levels of autophagy. Nonetheless, the underlying mechanism in which Pae downregulates lnc-MEG3 remained elusive. Future studies should also look into autopsy specimens of patients who suffered from cisplatin-induced nephropathy. In doing so, the clinical relevance of lnc-MEG3 expression could be further highlighted, inspiring novel therapeutic interventions to alleviate the disease.We have previously found that Pae, an active component isolated from lnc-MEG3 contributed to DDPIN pathogenesis both in vitro and in vivo. Mechanistically, lnc-MEG3 functions as an endogenous miRNA-126 sponge which inhibits AKT/TSC/mTOR-mediated autophagy. Impaired autophagy in HK-2 cells and kidney tissues of DDPIN mice then leads to increased apoptosis and inflammation. We also demonstrated that Pae could inhibit the expression of lnc-MEG3 to attenuate renal injury in DDPIN mice. Our study defines a novel role of lnc-MEG3-mediated DDPIN progression and identifies Pae as a promising therapeutic agent in cisplatin-induced nephrotoxicity.To conclude, present study found that This study was approved by the Medical Ethics Committee of the Shandong University Medical School  and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals A-0028).In situ hybridization was performed using the BOSTER kit  according to the manufacturer's instructions.Acute kidney injury (AKI) was induced in male C57BL/6 mice aged 6-8 weeks via a single intraperitoneal injection of cisplatin at a dose of 30 mg/kg  The protocol was performed as described previously The Agilent 1100 HPLC system  was used to measure blood urea nitrogen (BUN), and serum creatinine (SCR).Kidney sections were fixed using formalin, embedded in paraffin and sliced into 5 μm pieces.The samples were stained with hematoxylin and eosin (H&E). The percentage of tubules in the corticomedullary junction that displayed cellular necrosis and loss of brush border were counted and scored 0-5 in a blinded manner as follows: none, 0-10%, 11-25%, 26-45%, 46-75% and >75%. More than 6 high power fields (200 X) per section for each sample were examined Renal tissues from AKI mice were fixed with formalin, embedded in paraffin and sliced into 5 μm pieces. The slides were stained with different antibodies. . ResultsApoptosis in the kidney tissues after DDP (or and Pae) treatment was detected by TUNEL assay, which was performed according to the manufacturer's protocol . Apoptotic cells were identified by green nuclear staining counted with one hundred cells from 6 random fields. Results were analyzed via diaminobenzidine staining  under a microscope .The human tubular epithelial cell line HK-2 was purchased from the Cell Bank of the Chinese Academy of Sciences . HK-2 cells were cultured and treated as described previously Total RNA was isolated from tissues or HK-2 cells using a TRIzol reagent  according to the Manufacturers' instructions as described previously Pre-treated HK-2 cells in different conditions were collected and stained with 7AAD-Annexin V for 30 min. After washed with PBS twice, the samples were detected by a Novocyte flow cytometer (ACEA biosciences). All the data were analyzed by a Flowjo software (BD).Total protein lysates of kidney tissues or HK-2 cells were prepared for Western blot analysis as described previously The membranes were incubated with primary antibodies overnight at 4 °C after blocking. Following incubated with secondary antibodies, the protein bands were visualized by an ECL kit (Millipore) and calculated using the Image Lab software . Specific antibodies used are listed in Immunofluorescence staining was performed as described previously Small interference RNAs (siRNAs) and mimics were synthesized at GeneParma  . Transfe3 HK-2 cells were seeded in a 96-well plate after different treatment conditions. The absorbance of each well was detected at 560 nm.HK-2 proliferation was determined by the MTT assay. Briefly, 5×10CCK8 assay was performed to measure the cell proliferation. After treating different chemicals, cell viability was measured at 0 and 24 h, respectively. Specifically, 10 μL of CCK8 reagent  was added and cells were incubated at 37 °C for 1 h. The absorbance at 450 nm was measures by a microplate reader.HK-2 cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for an additional 30 min following different treatments. Visible colonies were counted in a blinded manner and captured using a microscope .5 cells were plated in the upper chamber and the lower chambers were filled with RPMI 1640 medium containing 10% FBS. Following different treatments, the migrated or invaded cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for an additional 20 min.Transwell chambers  were used for cell migration and invasion assays. Specifically, 1×10The MEG3-WT plasmid was cloned the sequence of MEG3 into the dual luciferase reporter vector pMIR-Glo, MEG3-MUT plasmid was cloned into the pMIR-Glo after mutation of the binding site of MEG3 and miR-126. The miR-126 mimic, MEG3-WT or MEG3-MUT and miR-NC were co-transfected in HK-2 cells using lipofectamine 2000 transfection reagent (Invitrogen) for 48h. Luciferase activities were measured with a Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Data are normalized for transfection efficiency by dividing firefly luciferase activity with that of Renilla luciferase.P< 0.05; **P < 0.01; ***P < 0.001; #P< 0.05; ##P < 0.01; ###P < 0.001; NS, not significant.Data of mean determinants were presented as ± SEM. No statistical methods were used to predetermine sample sizes, and the exact values of n (sample size) are provided in each figure legends. Statistical analysis of data was performed using a standard Unpaired Student's t-test by GraphPad Prism 8.0. * Supplementary figures and tables.Click here for additional data file.


The rehabilitation to the walk remains the basic treatment of the arteriopathy of the lower limbs. Different walking protocols can be proposed. For similar effectiveness, vascular rehabilitation consists of an outpatient or specialized institution management program based on a comprehensive approach involving all or many of the following techniques: relaxation, active analytical exercises, gait rehabilitation, exercise re-entry, physical activities adapted to the patient's daily life and illness, respiratory physiotherapy, therapeutic education of the patient, smoking cessation aid.Vascular rehabilitation is an essential and effective treatment of peripheral arterial obstructive disease (PAOD). It is recommended in the first line by the European and American scientific societies.Vascular rehabilitation of arteritis requires a variety of skills but still faces a clear lack of suitable structures; it remains poorly prescribed and poorly known by usual prescribers  •Vascular rehabilitation is the first-line treatment of stress ischemia in patients with peripheral arterial obstructive disease (PAOD).•It allows to significantly increase the maximum walking distance.•Its prescription must be integrated into the management networks of cardiovascular diseases. The patient with PAOD has a poly arterial disease that can affect other areas including the heart and brain. In the international register REACH, half of the patients with PAOD had at least one other arterial territory affected against 1/3 of cerebrovascular patients and 1/4 of patients with coronary artery disease ,6., its frequency was of the order of 5% by the age of 50, then increases in both men and women to reach a frequency of the order of 20% after 80 years in high-income countries [Indeed, a claudication questionnaire was used in European and American studies which objected that the prevalence of intermittent claudication was 1% before the age of 50 and reached only 6% after the age of 65. The prevalence of PAOD also increased by almost 25% between 2000 and 2010ountries ., the first double or triple the risk of PAOD, and the second double or quadruple with an increase in the risk related to the seniority of diabetes. High blood pressure, high cholesterol in LDL are also associated with PAOD but less so. [The main vascular risk factors that promote the development of PAOD are: smoking and diabetesless so. .1.2•Mechanisms for improving walking distance include:With aging, all sclerosis including the arteries. This inevitable evolution is pathogenic. With age, the hardening of the artery wall extends. This arteriosclerosis transforms the arterial tree into a rigid “pipe ”, less and less able to change its caliber and regulate blood flow. Little by little, the periphery is less well fed , this pr1.3PAOD causes mitochondrial dysfunction by accumulation of intermediate metabolites (acylcarnitin).Regular gait training reduces the concentration of acylcarnitin and improves mitochondrial function .1.4Physical exercise induces an increase in VEGF and HIF-1 (factors induced by hypoxia), which promote angiogenesis .1.5It has been shown that the trained patient will decrease the muscle oxygen consumption by betterSupport of the movement and the walking pattern .1.6The blood viscosity and plasticity of red blood cells are improved by exercise .1.7Classically raised in the PAOD and markers of a pejorative cardiovascular evolution, they decrease after 3 months of rehabilitation .1.8Several studies have shown an improvement in endothelial function after re-training.The importance of this function has been highlighted for its role in improving walking distance in patients working only the upper limbs .1.9Found in a study by Gardner, it allows a better adhesion of the patient .1.10Exercise improves blood pressure profile, lowers LDL cholesterol, regulates diabetes and helps stabilize body weight .1.11With increased activities of daily living, decreased reactive depressive syndrome, improved cognitive function .1.12Vascular rehabilitation is currently recommended in first line (level A) in patients with intermittent claudication - ischemia of effort-by many scientific societies for many reasons, first, the impact on quality of life through the improvement of self-esteem inherent in the improvement of functional abilities, and then the action on morbidity-mortality through participation in a therapeutic education programme ,14. ThisThe main mechanisms involved in improving walking distance include cardiorespiratory, circulatory, biochemical adaptations (action of growth factors such as VEGF and FGF-2) and musculoskeletal as well synthesized by Brenner et al., in a recent study .The rehabilitation can be done in a rehabilitation centre specialized in vascular pathologies in the context of a complete hospitalization or ambulatory; it can also be done at home, Some authors have also shown a substantially identical effectiveness over walking distance, in patients re-educated in centres versus in homes under supervision ,19 Regul1.13-Effort ischemia, especially for femoral lesions, patients with iliac or distal lesions also but with less satisfactory results .-Resting ischemia in a patient with surgical contraindication. In this case, it is preferable to carry it out in a specialized centre .-Following surgery or endovascular techniques or even amputation. This therapeutic orientation should be considered in conjunction with the vascular surgeon .The contraindications of vascular rehabilitation are the presence of an unstable cardiac, pulmonary or cerebral pathology. That is, any contra-indication to effort . It is nFor many years, scientific societies have advocated the “Gardner protocol”  of walkiThe proposed protocol must of course adapt to each patient characteristics and any associated diseases. Walking rehabilitation with the onset of pain at each walking episode leads to better results, but may discourage fragile patients who do not want to suffer. Gardner A.W. has shown that, at equivalent work volume, a lower intensity of effort (40% versus 80%) allows getting identical results .The training interval method consists of a split-type workout alternating a period of high intensity work and an active recovery period with sub-maximum intensity effort. This method is used in cardiology with good results. In 2011, Villemur B. et al., in a preliminary study, tested this protocol in stress ischemia and showed a doubling of walking distance in 2 weeks. This result requires confirmation .The required duration of the support is indeterminable as the factors are numerous and variable and so the anomalies persist and grow. A consensus nevertheless recommends an initial program of supervised exercises  consisti-Measurement of walking distance on flat ground until the onset of cramp: poorly standardized test and low reproducibility . This st-6-min walking test: standardized and validated in the assessment of chronic disabilities. Explore enduring capabilities with good reproducibility .-Treadmill tests to monitor patient progress under standardised and therefore reproducible speed and slope conditions. Most often these are tests with a gradual increase in work . Treadmi-Ergometric bicycle test: May be useful in case of obesity or walking disorders related to orthopedic or neurological impairments. This test is interesting in respiratory and cardiology, but does not test the handicap related to arterial disease-Upper limb ergometer stress test: Is a good alternative when lower limbs are not not useable with a sensitivity close to that of a treadmill test .A vascular rehabilitation programme consists, at a minimum, of three weekly 1-h sessions for a period of three to six months in a specialized centre, under supervision at home or in a physiotherapy practice . In all The second phase of retraining is personalized, adapted to each case and takes into account the results obtained by the patient during the evaluation phase. The protocol put in place may evolve according to the evolution of arterial disease. It is important to set appropriate and realistic goals so as not to discourage patients .There are two main types of techniques that are often associated:The overall training and analytical reconditioning.The overall training consists of performing various walking exercises either on a treadmill or on a cycloergometer in order to solicit the lower limbs, the upper limbs and thus recruit a maximum of muscle volume. Ergometric reconditioning involving the upper limbs and trunk may be useful when the lower limbs are not useable with a positive systemic effect, improving tissue oxygenation at lower limb level .The analytical  reconditioning of the lower limbs is the set of techniques that target muscle building by muscle contraction against progressive resistance as well as increased blood flow in the arterial network. It is less often advocated because systemic effects are less important compared to overall training . DynamicOther rehabilitation techniques can be applied according to situations such as respiratory physiotherapy in case of associated COPD, lymphatic drainage in case of edema, mobilization in case of retractions and stiffness, postural rehabilitation in case of balance disorders, such as limb swaying and proprioception exercises, so-called reflex massages to stimulate microcirculation, interesting in case of trophic disorder or very close claudication, or walking in an aquatic environment when the walking perimeter is very limited. Finally, credo therapy, which is part of the physical therapeutic arsenal, can be proposed ,45.1.14The work of relaxation or stretching is an essential prerequisite for muscular and endurance work. It also improves flexibility ,45.1.15Segmental muscle building improves the strength of the muscles and functional capacity, the strength of the contractions is fixed at 20–50% of the maximum voluntary force previously evaluated so as to stay on aerobic work ,45.1.16Walking exercises are daily and based on the initial assessment of the perimeter of embarrassment. The walking distance worked is fixed at 80% of the perimeter of discomfort, as well as the recovery time which is fixed at 80% of the time of disappearance of the pain ,45.1.17Sessions last from 30 to 45 min. The intensity of re-training is always based on the training heart rate and the dyspnea threshold. The sessions include a 30-min endurance phase at the target heart rate, preceded by a warm-up phase and followed by an active recovery phase. The endurance phase can be maintained in a constant load plateau or by alternating periods of lower intensity and peaks of activity ,45.The effort re-training can be done on different ergometers  but can also be an outdoor walk, outdoor bike, swimming … ,45.1.18The work of ventilatory control is interesting, throughout the rehabilitation, in patients often « heavy smokers».It allows the awareness of the abdominodiaphragmatic breathing and thus improve the intake of O2 ,45.1.19Therapeutic education is an integral part of the care program. It should allow the sustainability of the results achieved during the program.The main topics covered are treatment, dyslipidemia, excess weight, blood pressure control, diabetes, tobacco, physical activity, foot care ,45.1.20Gardner's meta-analysis shows a 180% increase in the distance of onset of pain with a result from the first 2 months. Watson et al. .In a 2008 Cochrane review, which included 22 randomized studies, walking distance improved by more than medical treatment and by 200% with no cardiovascular complications and maintained over 2 years .In rehabilitation centres, with a higher volume of work, the walking distance can be increased from 4 to 5 times. Femoral lesions perform best, but iliac and distal lesions can also improve significantly. The lameness of buttocks is more moderate. Fokkenrood  shows inThe recent ERASE study compared the increase in maximum walking distance in patients with claudication related to iliac or femoral artery injury treated either by endovascular treatment alone or by the combination of endovascular treatment and gait rehabilitation Gardner protocol. The combination of endovascular treatment and walking rehabilitation resulted in a better result at one year with a maximum walking distance multiplied by 5.7. In the walk-alone rehabilitation group, maximum walking distance was also significantly improved (WMD 4.35)  The 20132Vascular rehabilitation is the first-line treatment of stress ischemia in patients with PAOD. It allows to significantly increase the maximum walking distance especially in case of femoral injuries, it also allows to improve the balance of risk factors, cognitive functions and activities of daily life. It is just as effective as endovascular treatment for long-term femoral lesions. It should be carried out preferentially in a supervised manner but can be prescribed as an outpatient with minimal supervision, it must be adapted and multidisciplinary. It corresponds to an obvious need but not satisfied by the manifest lack of suitable structures. It remains poorly prescribed and poorly known to usual prescribers . Its prescription must be integrated into the management networks of cardiovascular diseases.Not commissioned, externally peer reviewed.We have the agreement of the ethics committee, university Mohammed first faculty of medicine and pharmacy OUJDA MOROCCO.We have any sponsors.AHMED AMINE EL OUMRI: concept. MOHAMMED NOUMAIRI, AMINE BOUALLALA, SIHAM EL MIR: collection. ALLAM ABDALLAH: analysis. MOHAMMED NOUMAIRI: writing the paperWi did just review not study so we have not any consent of patients.Name of the registry:Unique Identifying number or registration ID:Hyperlink to your specific registration (must be publicly accessible and will be checked):MOHAMMED NOUMAIRI et al.-We have any conflicts of interest-All authors agree to publication.