Alismatis rhizoma (AR) has been used as an herbal medicine in China for over a thousand years. Crude AR, salt-processed AR (SAR), and bran-processed AR (BAR) are recorded in the Pharmacopoeia of the People′s Republic of China. However, the differences of chemical composition between crude AR and its processing products remain limited. In this study, triterpenes were identified from crude AR, SAR, and BAR by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight-mass spectrometer (UHPLC-QTOF-MS/MS). Subsequently, the differences of triterpenes between the crude AR and processed ARs were compared via a targeted metabolomics approach. Finally, a total of 114 triterpenes were identified, of which 83, 100, and 103 triterpenes were found in crude AR, SAR, and BAR, respectively. After salt-processing, there were 17 triterpenes newly generated, 7 triterpenes with trends of increasing, and 37 triterpenes decreased. Meanwhile, 56 triterpenes including 21 newly generated and 35 with significant increases were observed in BAR. This study could be benefit to investigate the processing mechanism of AR, as well as support their clinical applications. In Traditional Chinese Medicine (TCM), processing (“Paozhi” in Chinese) refers to certain types of pharmaceutical technology that promote the use of Chinese herbal medicines to fulfil specific requirements of therapy . Under tAlismatis rhizoma , recorded in Chinese Pharmacopoeia (2020 edition), is the dried rhizome of Alisma orientale Juzepzuk, belonging to family Alismataceae + ion at m/z 515.3700 in the full mass spectra scans. The product ions at m/z 497.35, m/z 455.35, and m/z 419.33 were formed by successive loss of H2O and AcOH. The m/z 383.29 ion was observed in the product ion related to the dissociation of the C23–C24 bond, and the product ion at m/z 365.28 was acquired by a further loss of H2O from the ion of m/z 383.29. In addition, the characteristic fragment at m/z 339.26 was obtained by losing C2H2 from m/z 365.28. Therefore, the product ion at m/z 339.26 can be used as characteristic diagnostic ions for screening the same type of triterpenes. Similarly, the other types of triterpenes could be identified based on the fragmentation rules and corresponding characteristic product ions.According to the different structural types, the collision-induced dissociation fragmentation patterns for representative triterpenes of each type skeletons were shown in 1, 11, 20, 29, 32, 37, 41, 65, 73, 80, 90, 92, 93, 104, 105, 107, and 109. As for BAR, 21 new triterpenes were found in BAR  were identified in AR. Among these triterpenes, 10 were type I-1 triterpenes, 8 were type I-2 triterpenes, 6 were type I-3 triterpenes, 43 were type II-1 triterpenes, 15 were type II-2 triterpenes, 6 were type II-3 triterpenes, 22 were type III-1 triterpenes, and 4 were type III-2 triterpenes. In addition, the results indicated that 17 new triterpenes were discovered in SAR , includid in BAR , includi2X = 0.73, Q2 = 0.666), which indicated a difference in the levels of triterpenes between crude AR and processed ARs. Thus, the results indicated that different changes in triterpenes had occurred after processing compared with crude AR. The supervised partial least squares-discrimination analyses (PLS-DA) was used to further explore the differences between crude and processed ARs.A targeted metabolomic analysis was used to compare the triterpenes between AR and processed ARs. In the unsupervised principal component analysis (PCA) model a, the se2Y and Q2 were 0.99 and 0.976 for A group and S group, and 0.993 and 0.953 for A group and B group, respectively, which indicates that the models had good abilities for both prediction and reliability. Moreover, the Q2 of the 999-time permutation tests of the aforementioned models were both negative, indicating the two PLS-DA models were not overfitting , 44 triterpenes occurred significant differences in SAR, including 37 decreased and 7 increased triterpenes. Meanwhile, 35 triterpenes were found to increase significantly in BAR and there were no significant differences in other triterpenes. Compared to crude AR, there were 17 triterpenes newly generated in SAR. The majority of them were type II-1 and type III-1 triterpenes, accounting for 35.29 and 29.41%, respectively. As for BAR, 21 triterpenes newly generated and more than half of them were type II-1 triterpenes (accounting for 61.90%). As depicted in Further, the triterpenes with significant differences between AR and its processed products were illustrated as the error line diagram of fold changes based on eight types see . The folTo explore the differences between crude and processed ARs, the UHPLC-QTOF-MS/MS combined with multivariate statistical analysis was developed to identify and distinguish the difference in triterpenes in crude AR and processed ARs. In this study, a total of 83, 100, and 103 triterpenes were identified in crude AR, SAR, and BAR, respectively. There were 17 and 21 newly generated triterpenes were detected in SAR and BAR. Meanwhile, compared with the content of triterpenes in crude AR, 44 triterpenes had significantly changed after salt-processing, including 37 decreased and 7 increased triterpenes. While after bran-processing, 35 triterpenes were significant differences and all of them showed an increase in contents.14, 17, 18, 19, 22, 23, 25, 27, 33, 35, 43, 46, 49, 53, 57, 64, 71, and 75, which all possessed a C24, C25-oxide three-membered ring of the side chain were massively reduced in type II triterpenes compared to type I triterpenes. It was speculated that the type II triterpenes with a C24, C25-oxide three-membered ring of the side chain were more unstable than others. A previous study indicated that owing to the presence of a carbonyl group at C16, cracking and ring opening of triterpenes at position 24 and 25 was more easily than others [48, 50, 54, 56, 62, 70, and 81) and type III  triterpenes with hydroxyl or acetyl groups at C23, C24, and C25 were significantly reduced after salt-processing. It was reported that the compounds with substituting groups, such as hydroxyl and acetyl groups, would lose H2O and AcOH under high temperature [Many methods of processing, such as stir-frying, steaming, and boiling, need to be carried out at the heating conditions, which probably leads to complex chemical changes . In thisperature . However, the reduction in certain triterpenes after heating was not observed in BAR, on the contrary, triterpenes with significant differences showed an increasing trend after bran-processing. Our findings indicated that triterpenes in crude AR had different change trends after two processing of SAR and BAR. In this study, AR was processed at two methods with different excipients , temperature ranges (80 to 120 °C and 150 to 220 °C) and heating durations (around 30–40 min and around 8 min); thus, these parameters might be responsible for the different change trends of triterpenes between SAR and BAR. In addition to the heating temperature, heating duration was also an important factor in AR processing. A report about triterpenes suggested that the contents of alisol A 24-acetate and alisol B 23-acetate initially increased as the duration of heating time, but after 40 min of heating, this increasing tendency ceased and the concentrations rapidly decreased . Conside79), 11-deoxy-alisol B (compound 97), 11, 25-anhydro-alisol F (compound 101), and 11-deoxy-alisol B 23 acetate (compound 108) were found with significance increases after processed with salt. In addition, the therapeutic efficacy of SAR might benefit from synergistic actions of multiple ingredients. During the processing, complicated changes in herbal components may occur, and the pharmacological activity of a certain herbal medicine may be changed accordingly [For the effectiveness of TCM, herbal may profit from synergistic functions of multiple chemical ingredients ,25. Althordingly ,26. Thisordingly ,28.79) is one of the main active triterpenes in AR and possesses many biological activities including antibacterial, antiviral, lipid regulating, and diuretic effects [16 of the parent nucleus and a C24, C25-oxide three-membered ring of the side chain had relation with the lipid-lowering activity [24, C25-oxide three-membered ring of the side chain no matter in SAR or BAR. Moreover, processing Chinese medicine can enhance the efficacy and change the trend of action [Generally, the main purposes of processing are reducing toxicity and enhancing the effects of crude drugs. Previous studies indicated that the hydroxyl and acetyl groups on the side chain of triterpene were the essential groups to exert lipid-lowering effect ,30. For  effects ,31,32,33f action . The diff action . In addif action . In concAcetonitrile  and formic acid  were purchased from Fisher Scientific  and Sinopharm Chemical Reagent Co., Ltd. , respectively. Purified water was obtained from Millipore using a Milli-Q system . The dried rhizomes of cultivated AR were collected from Sichuan province of China in April 2019 and identified by Prof. Keli Chen of Hubei University of Chinese Medicine. The dried rhizomes of AR were cut into 2–4 mm slices before used. The reference standards  were previously isolated and purified from the dried rhizome of AR. The purities of these standards were tested to be above 98% using HPLC coupled with UV detector. w/v) until fully absorbed in a sealed container. Then the moistened AR were stir-fried in a metallic pan over a low flame (80–120 °C) around 30–40 min until the water evaporated. Similarly, the bran (100 g) was placed in a preheated stir-frying pan. When the bran was fuming, added 1 kg of AR and stirred rapidly around 8 min until the color of AR turned yellow, and removed the bran. After cooling to room temperature, SAR and BAR were obtained and used for further analysis.SAR and BAR were prepared according to the procedure recorded in the general principles of Chinese pharmacopoeia (2020 edition) . Briefly−1) as internal standard was injected and analyzed by the UHPLC-QTOF-MS/MS. SAR and BAR were extracted with the same procedure as above. Thus, the samples were separated into 3 groups: raw AR group (A group), salt-processed AR group (S group), and bran-processed AR group (B group). Meanwhile, the quality control samples (QC) were prepared by pooling each solution from all samples. To assess of the stability of analytical systems, the QC samples were performed at the beginning, the end, and every 5 injections throughout the whole UHPLC-MS/MS analysis.Seven batches of AR were smashed and sieved (24 mesh) to obtain homogeneous powders separately. Sample powder (0.5 g) was ultrasonically extracted with 5 mL acetonitrile at room temperature for 30 min. Then, the supernatant of these samples was centrifuged at 14,000 rpm for 10 min followed by filtration, after which 5 μL of supernatant of each sample with fexofenadine (100 ng·mL−1 and the injection volume was 5 μL with a column temperature at 40 °C. The mobile phase of the eluent was 0.1% formic acid in water (A) and acetonitrile (B), and the gradient conditions were as follows: 0–18 min, 20–77% B; 18–25 min, 77–95% B; 25–27 min, 95% B; 27–28 min, 95–20% B; 28–30 min, 20% B.UHPLC-MS/MS analysis was performed on a Waters ACQUITY Ultra Performance LC system. A C18 column  was used for separation. The flow rate was 0.3 mL·min−1; desolvation gas flow, 600 L·h−1; desolvation temperature, 500 °C; source temperature, 100 °C; cone voltage, 60 V; capillary voltage, 3 kV. The collision energy was set at 30 eV and the collision energy spread was 40 eV for MS/MS experiments. The mass ranges were set at m/z 100–1200 for full scan with a scan duration of 1 sec.Mass spectrometric analyses were performed on a Waters Xevo G2-XS QTof system with an electrospray ionization source and the positive ion mode was used for data acquisition. The following MS conditions were set: cone gas flow, 50 L·hThe raw spectra data were acquired and managed using MassLynx software . Quantitative results were obtained by dividing the peak area of each triterpene by the peak area of the internal standard fexofenadine. After this procedure, the resulting dataset was on multivariate statistical analysis and one-way ANOVA using the SIMCA-P software  and GraphPad Prism .2Y and Q2 were calculated to evaluate the goodness-of-fit and predictive ability of PLS-DA model. Variable importance in the projection (VIP) was a parameter that showed the importance of a variable in a model. Usually, a compound with VIP > 1.0 was considered to play an important role in distinguishing groups [p value < 0.05) calculated by one-way ANOVA.To evaluate the differentiation of chemical compounds caused by different processing methods, crude AR, SAR, and BAR were set as groups A, S, and B, respectively. QC samples were set as group QC and used to evaluate the repeatability and stability of the whole sample set . To deteg groups ,38. TritIn this study, UHPLC-QTOF-MS/MS combined with a targeted metabolomics analysis was applied to investigate the changes in triterpenes between crude AR and processed ARs. As a result, a total of 83, 100, and 103 triterpenes were identified in crude AR, SAR, and BAR, respectively. Except for the newly generated triterpenes, there were 44 and 35 triterpenes were changed significantly after salt and bran processing. Our results suggested that triterpenes in crude AR had different change trends after two processing of SAR and BAR. In SAR group, significantly changed triterpenes were mainly decreased in content, while all 35 triterpenes with significant differences had an increased in content after bran-processing. The different change trends between SAR and BAR could be attributed to different heating durations, temperatures, and excipients. The results of this study could provide scientific evidences for the variations of triterpenes in AR under different processing methods, and be valuable to elucidate the processing mechanisms of AR. Further studies will focus on the relationship between the differences of triterpene and pharmacological activity after AR processing.


A customized transcription factor array identified that signal transducer and activator of transcription 3 (STAT3) functioned as the main downstream effector. Mechanistically, dual-luciferase report analysis and RNA antisense purification (RAP) assay indicated that lnc-C2orf63-4-1 directly decreased the expression of STAT3, which was depend on the reduced stabilization of STAT3 mRNA. Importantly, up-regulation of STAT3 efficiently reversed the protective role of lnc-C2orf63-4-1 against Ang II-mediated vascular remodeling. Therefore, lnc-C2orf63-4-1 negatively regulated the expression of STAT3 and prevented the development of aortic dissection. Our study revealed that lnc-C2orf63-4-1 played a critical role in vascular homeostasis, and its dysfunction exacerbated Ang II-induced pathological vascular remodeling.Emerging evidence indicates that long non-coding RNAs (lncRNAs) serve as a critical molecular regulator in various cardiovascular diseases. Here, we aimed to identify and functionally characterize lncRNAs as potential mediators in the development of thoracic aortic dissection (TAD). We identified that a novel lncRNA, lnc-C2orf63-4-1, was lowly expressed in aortic samples of TAD patients and angiotensin II (Ang II)-challenged vascular smooth muscle cells (VSMCs), which was correlated with clinically aortic expansion. Besides, overexpression of lnc-C2orf63-4-1 significantly attenuated Ang II-induced apoptosis, phenotypic switching of VSMCs and degradation of extracellular matrix both  Thoracic aortic dissection (TAD) is the most serious form of acute aortic syndrome with an incidence of about 15 cases per 100,000/year . The chaThe advancement in large-scale whole-genome sequencing technologies suggests that less than 2% of the human genome encodes for proteins, whereas much of the remaining genome is transcribed into non-coding RNAs . Long noIn the present study, we utilized next-generation high-throughput sequencing (HTS) for TAD patients and control subjects  and found that the expression of lnc-C2orf63-4-1 was reduced during the development of aortic dissection. Moreover, we showed that overexpression of lnc-C2orf63-4-1 significantly prevented the incidence and severity of aortic dissection in angiotensin II (Ang II)-infused mice, potentially via decreasing VSMC apoptosis, matrix metalloproteinase (MMP) activity, and accumulation of collagen fibers. Specifically, we identified that lnc-C2orf63-4-1 affected aortic dissection through mediating the function of signal transducer and activator of transcription 3 (STAT3), which exerted pro-apoptotic effects in VSMCs, leading to subsequent vascular remodeling.Study protocols were approved by the ethical committees of the First Affiliated Hospital of Harbin Medical University (No. HMUIRB20170034) and complied with the declaration of Helsinki. Written informed consents were obtained from all participants.n = 24), who were identified without Marfan syndrome, Loeys-Dietz Syndrome, familial aortic dissection, and bicuspid aortic valve. A reference control group (n = 13) was set up from patients who had coronary artery bypass grafting  and heart transplant donors (n = 3) without hereditary cardiovascular disease, aortic dissection, previous history of inflammatory aortic disease, or known connective tissue disorder.The tissue samples of ascending aorta were collected from Stanford type A aortic dissection patients (n = 3) and heart transplant donors (n = 3). The other aortic wall samples from control and TAD groups were prepared for quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting analysis, and histological analysis. The specimens were immediately sliced into small pieces, soaked in RNAlater solution , and preserved at −80°C until further assays. The gene expression analysis was performed using lncRNA in the aortic wall from Stanford type A aortic dissection patients  following the manufacturer’s instructions, the RNA integrity was examined using an RNA integrity number (RIN) by an Agilent Bioanalyzer 2100 , and samples with an initial RIN >6.5 were used for the gene expression profiling. Qualified total RNA was further purified by RNAClean XP Kit  and RNase-Free DNase Set .p values of <0.05.Aortic wall tissues were processed to synthesize double-stranded complementary DNA (cDNA), and the cDNA was labeled and hybridized to lncRNA by using an Illumina HiSeq 2500  at Shanghai Biotechnology Co., Ltd. . The expression levels of whole samples were presented as Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) values, which is the recommended and most commonly used method to estimate the level of gene expression . The difhttp://www.noncode.org/) showed that nine lncRNAs were conservative with mice, and we further randomly selected seven among these differentially expressed lncRNA transcripts for validation. Total RNA was extracted from the control (n = 13) and TAD (n = 24) aortic tissues using TRIzol reagent  and then reversely transcribed to cDNA. qRT-PCR was conducted using a standard SYBR Green PCR kit  on an ABI 7500 real-time PCR System . β-actin was selected as the housekeeping gene, and all experiments were performed in triplicate. The relative expressions of the target genes were calculated by the 2−ΔΔCt method. The operations were carried out as previously described  (8 TU/ml) of overexpression lentivirus (OE), inhibition of expression lentivirus carrying shRNA (sh), or empty lentivirus (NC) after 24 h of plating. The blank group was transfected with the same volume of culture medium. At 72 h after the lentivirus were transfected into VSMCs, the Ang II  at a concentration of 1,000 nM  and maintained in DMEM  supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin  at 37°C in a humidified atmosphere containing 5% CO, China) . Cells w1,000 nM  was addein situ hybridization (RNA-FISH) Cy3-labeled lnc-C2orf63-4-1 was purchased from RiboBio . Frozen tissues were sectioned by Cryotome E . The final signal was developed with diaminobenzidine , and the nuclei were counterstained in hematoxylin for 3 min. The sequence of lnc-C2orf63-4-1 probe used was as follows: 5′-TCGTCTAAACAAACATTTCATTTCAGAAAATCTGCATCAATCTACACGGAC-3′.RNA fluorescence 2, 0.125% N-lauroylsarcosine, 0.025% sodium deoxycholate, and 2.5 mM TCEP). Eluted RNA complexes were used in qRT-PCR to quantify the RNA yield and enrichment.RNA antisense purification (RAP) was performed based on a previously published method . The lysSTAT3-WT and STAT3-Mut were cloned into the luciferase vector V80-PmirGLO . For luciferase reporter assays, the lnc-C2orf63-4-1 was co-transfected into mouse aortic VSMCs with the luciferase constructs described above using Lipofectamine 2000 . The luciferase activity at 24 h post-treatment was measured by the Dual-Luciferase Reporter Assay System  as previously described .5 cells/well), digested with trypsin , washed with phosphate-buffered saline (PBS) three times, suspended in 500 μl of binding buffer, and then incubated with 5 μl of fluorescein isothiocyanate (FITC)-conjugated Annexin V and 3 μl of propidium iodide (PI) for 15 min at room temperature in the dark. After incubation, the samples were tested using a flow cytometer . Early apoptotic chondrocytes were defined as annexin V-positive and PI-negative cells, and late apoptotic chondrocytes were defined as annexin V-positive and PI-positive cells. The proportion of apoptotic cells was calculated using the FASC Calibur MT flow cytometer .Apoptosis was determined by flow cytometry analysis using the Annexin V-FITC/PI Apoptosis Kit . Cells were seeded in 6-well plates  chromogenic substrate at room temperature for 2 min, and the band density was quantified using Image Lab software.Histological staining was performed by using a standardized protocol as previously described. Aortas were isolated and fixed in 4% formalin overnight at 4°C. Paraffin cross-sections (4 μm) from organs were stained with hematoxylin and eosin, elastic van Gieson (EVG) staining, and Masson staining. The interstitial fibrotic areas were calculated using Image-Pro Plus software . Collagen volume fraction was calculated as collagen area/total area ×100%.Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Mice were anesthetized using 2% isoflurane (Vet One) and laid supine on a heated plastic pillow (37°C), the osmotic minipumps were implanted after used microscopic scissors to cut the back skin. The osmotic minipumps  containing either Ang II  to construct aortic aneurysm and dissection (AAD) model or saline were implanted in 22∼26 week-old ApoE−/− male mice (control group). Only male mice were used because of the male predominance of human AAD and the potential influence of female sex hormones on AAD models (12 genome copies of AAV9 encoding lnc-C2orf63-4-1 sequence in murine (http://www.noncode.org/) via the tail vein for lnc-C2orf63-4-1 overexpression (AAD + OElnc-V group) (n = 10) or control vectors (AAD + NC-V group) (n = 10) in 0.1 ml of PBS, or the same volume of PBS (AAD group) (n = 10). The pAAV9-CMV-MCS-lnc-C2orf63-4-1 and pAAV9-CMV-MCS-3FLAG-WPRE vectors were generated and packaged by OBiO Technology  Co., Ltd. Following final transthoracic echocardiography, mice were sacrificed by intravenous injection of a lethal dose of pentobarbital sodium (100 mg/kg), and aortic sections were removed for further experiments. The aortic dissection in AngII treated mice were defined by histopathological staining. When rupture and thrombosis of the media aorta were observed under light microscope during the autopsy, which confirmed that the mice were occurred aortic dissection.All animal experiments were reviewed and approved by the Animal Care and Use Review Committee of Harbin Medical University . The study conformed to the D models . Adeno-aD models . Among vD models . At 3 daAt 28 days after Ang II induction, the aortic arch diameter was detected by B-mode US imaging. Mice were anesthetized using 2% isoflurane (Vet One) and laid supine on a heated plastic pillow (37°C). The two-dimensional B-mode US imaging was performed after setting up a real-time micro-visualization scan head (RMV 704) with a central frequency of 40 MHz, a frame rate of 30 Hz, a focal length of 6 mm, and a 20 × 20 mm field of view . All images were acquired for multiple cardiac cycles and digitally stored in the hard drive for offline analysis. All aortic diameters were measured in anterior-posterior direction during the diastolic phase. US image analysis was performed using the accompanying Vevo770 software . Measurements were repeated on two separate occasions using a random selection of each dataset and operator blinding to prevent recall bias. For these parameters, results were validated by a comparative analysis of one independent observer blinded to the treatment groups.p < 0.05 was considered statistically significant.Data were presented as means ± SEM, unless stated differently. Groups were compared using Student’s t-test. Normality was tested to ensure that parametric testing was appropriate. When comparing multiple groups, data were analyzed using analysis-of-variance (ANOVA) with Bonferroni’s post-test. The association of the two variables was evaluated using a two-tailed Pearson’s correlation analysis. A value of p < 0.05). We comprehensively analyzed the expression profile of lncRNAs in TAD aortic wall tissues by using the HTS analysis and compared it with that in normal aortic wall tissues from healthy donors. A total of 53 lncRNAs (24 up-regulated and 29 down-regulated) were significantly aberrantly expressed in TAD tissues compared with the normal tissues  construct NC-V (the NC RNA engineered into the AAV9 vector), and they were infused with Ang II for 4 weeks to develop AAD. The efficiency of AAV-lnc-C2orf63-4-1 to rise endogenous lnc-C2orf63-4-1 in AAD mice was also confirmed by qRT-PCR, showing that the transcript level of lnc-C2orf63-4-1 in the aorta was significantly repressed in the aortic dissection group, while it was increased by AAV9-lnc-C2orf63-4-1 treatment (AAD + OElnc-V) , suggesting that overexpression of lnc-C2orf63-4-1 in VSMCs partly attenuated Ang II-induced AAD.Chronic infusion of Ang II recapitulates many aspects of aortic dissection and is widely used to study AAD . Here, AOElnc-V) . In the OElnc-V) . More imOElnc-V) . In addiOElnc-V) , sugges−/− mice receiving chronic treatment of Ang II, while AAV9-lnc-C2orf63-4-1 remarkedly reduced the thickness of tunica media  database was used to predict the RNA targets of lncRNAs. The analysis concluded that KIAA0895, homeobox genes B9 (HOXB9), CCR4-NOT transcription complex subunit 7 (CNOT7), histone cluster 1 H4A family member C (HIST1H4C), MMP-7, MutS Homolog 2 (MSH2), B9 protein domain 1 (B9D1), and STAT3 were possibly involved in the molecular function of lnc-C2orf63-4-1 , indicating that lnc-C2orf63-4-1could directly bind to STAT3. In addition, using an RAP assay-based approach, we further confirmed that the bioinformatic analysis predicted lnc-C2orf63-4-1 binding mRNA, the STAT3, was indeed bound by lnc-C2orf63-4-1 in VSMCs , thus reinforcing the idea that lnc-C2orf63-4-1 maintained the VSMC homeostasis by negatively regulating STAT3.To further confirm the effect of lnc-C2orf63-4-1 on STAT3 in VSMCs, we performed a dual-luciferase reporter assay using a vector carrying wild-type (WT) or mutant (Mut) STAT3 3′-UTR and co-transfected them with lnc-C2orf63-4-1 mimics or NC. Luciferase reporter assays showed that lnc-C2orf63-4-1 significantly suppressed the luciferase activities in VSMCs transfected with the STAT3 3ʹ-UTR WT reporter, whereas they did not have any significant influence on the luciferase activities of the mutant reporter , indicain VSMCs , thus rThen, we explored whether deletion of STAT3 via siRNA could reverse lnc-C2orf63-4-1-induced apoptosis in VSMCs. As expected, inhibition of STAT3 reversed apoptosis-related markers and regulators of structural ECM degradation induced by silencing of lnc-C2orf63-4-1 in VSMCs . Howeverde novo protein synthesis. As shown in Therefore, we applied actinomycin D (ActD) to test whether STAT3was post-transcriptionally regulated by lnc-C2orf63-4-1 in VSMCs. Intriguingly, the half-life of STAT3 mRNA was markedly reduced in response to overexpression of lnc-C2orf63-4-1 , indicat−/− mice exhibited more severe apoptosis, greater MMP-2/9 activity, less contractile phenotype of SMC, and increased collagen deposition compared with the control mice. Mechanistic studies revealed that Ang II infusion resulted in a marked increase in the expressions of apoptosis-related gene caspase-3/9 and Bax, elevated expressions of interstitial fibrosis-related genes collagen type I/III, and enhanced STAT3 transcription activity, which was partially restored by pre-treatment of lnc-C2orf63-4-1. Moreover, STAT3 was identified as a downstream target of lnc-C2orf63-4-1 through bioinformatics analysis, and lnc-C2orf63-4-1 also attenuated the increased expression of STAT3 mediated apoptosis and synthetic phenotypic transformation in VSMCs induced by Ang II stimulation. Collectively, our findings demonstrated that lnc-C2orf63-4-1 played an important role in regulating vascular remodeling and pathogenesis of aortic dissection. Recently, new research has been proposed that Ang II-mediated up-regulation of lncRNA lnc-OIP5-AS1 results in increased expressions of apoptotic genes as a competing endogenous RNA of microRNAs in human aortic VSMCs and exacerbates aortic injury (Acute AAD is a severe and life-threatening cardiovascular disease without effective pharmacotherapy at present. Hence, it is necessary to investigate the biological basis of aortic dissection and identify novel targets for prevention and therapy. In the present study, we identified an uncharacterized lncRNA, lnc-C2orf63-4-1, which was down-regulated in the aorta of TAD patients and functionally required for the vascular remodeling. We generated mice non-specifically overexpressing lnc-C2orf63-4-1 using the AAV-9 vector and showed that up-regulation of lnc-C2orf63-4-1 greatly attenuated Ang II-induced AAD. Following Ang II infusion, the ApoEc injury , and a nc injury . From thin vivo or in vitro study. These studies indicate that the differentially expressed lncRNAs may play an important role in the development of AAD, and it is urgently necessary to understand the potential mechanisms and pathways of target lncRNAs to affect the function of VSMCs. In the present study, we identified 53 lncRNAs that were aberrantly expressed between healthy donors and TAD patients by using HTS analysis. The accuracy of transcriptome sequencing was confirmed by a relative large number of samples. Moreover, we found that the lnc-C2orf63-4-1 was a lowly expressed novel lncRNA in the aorta of TAD patients, which was significantly correlated with increased aortic arch width, suggesting that lnc-C2orf63-4-1 played a critical role in vascular remodeling during AAD.LncRNAs are defined as transcripts longer than 200 nt without evident protein-coding function, and they are located in both the nucleus and cytoplasm and participate in the regulation of gene expression at both the transcriptional and post-transcriptional levels to affect numerous physiological and pathological processes . For exa−/−mice by Ang II stimulation compared with the control group. Moreover, the AAD mice showed enhanced vascular interstitial fibrosis, which was mainly attributed to the activation of collagen type I and III proteins in VSMCs. In addition, changes in MMPs are another important mechanism for the development of AAD. When VSMCs switch from a contractile phenotype to a synthetic phenotype under pathological conditions, they promote a proinflammatory response and increase the production of MMPs. Both aorta samples from TAD patients and AAD animals exhibited an enhanced production and secretion of MMPs, especially MMP-2 and MMP-9, leading to augmented elastin degradation and aortic wall weakening, and ultimately rendering the aorta prone to rupture and AAD progression (AAD is characterized by extensive molecular changes in vascular remodeling and loss of the contractile phenotype of VSMCs. Multiple mechanisms have been proposed to promote the pathogenesis of AAD, including VSMC apoptosis, ECM degradation, and vascular interstitial fibrosis. As the predominant cells in the AAD of the aorta, VSMCs are essential for the maintenance of the aortic structure and function. Apoptosis of VSMCs would reduce the tensile strength and elasticity of the aorta, resulting in tunica media susceptible to rupture and contributing to the development and progression of AAD . Herein,gression . Herein,de novo synthesis of mRNA or protein in VSMCs, respectively. Intriguingly, the half-life of STAT3 mRNA was markedly reduced in response to lnc-C2orf63-4-1, while the level of STAT3 protein was not affected by lnc-C2orf63-4-1, indicating that lnc-C2orf63-4-1 regulated the STAT3 expression via destabilizing mRNA.LncRNAs have been reported to be located in both the nucleus and the cytoplasm, and subcellular localization patterns of lncRNAs reveal fundamental insights into their biology and provide hypotheses for potential molecular roles . In the −/− mice depends at least in part on the Ang II-mediated activation of STAT3 (It has been reported that Ang II can elicit phosphorylation and activation of STAT3, and then STAT3 forms homodimers or heterodimers translocated to the nucleus and activates the transcription of target genes . Tissuesof STAT3 . Moreoveof STAT3 . Upon exof STAT3 . Indeed,Our study also has some limitations. First, we only detected the expression of lnc-C2orf63-4-1 from aorta samples but not in the blood samples of TAD patients. Multiple confirmations would enhance the evidence of lnc-C2orf63-4-1 as a clinical biomarker in AAD. Second, we only identified that lnc-C2orf63-4-1 could negatively regulate the expression of STAT3 at the transcriptional level through stabilizing STAT3 mRNA in VSMCs. Whether lnc-C2orf63-4-1 could function as a sponge lncRNA to affect the expressions of other key regulators in AAD remains largely unexplored. Third, we did not provide evidence of whether the lnc-C2orf63-4-1-transgenic mice could prevent Ang II-induced AAD. Nonetheless, the present findings in human aortic dissection samples and AAD mouse model generated novel hypotheses about the role of lnc-C2orf63-4-1 in the progression of AAD.


Background: Behçet’s disease (BD) is a chronic autoimmune disease. The early diagnosis of BD is very important to avoid serious and/or fatal complications such as eye damage, severe neurological involvement, and large vessel occlusion. New, sensitive biomarkers would aid in rapid diagnosis, the monitoring of disease activity, and the response to treatment.Methods: This study’s aim is to identify two immune system-related BD biomarkers. We measured long non-coding RNAs (lncRNAs) NEAT1 (nuclear-enriched abundant transcript 1), and lnc-DC (lncRNA in dendritic cells) in serum by real-time polymerase chain reaction (RT-PCR) in 52 BD patients and 52 controls. We analyzed the association between NEAT1 and lnc-DC and the clinical parameters of BD. Receiver operating characteristic (ROC) curve analysis was performed to explore the diagnostic performance of the studied genes.Results: Compared to controls, the significant upregulation of NEAT1 {median [interquartile range (IQR)] = 1.68 (0.38–7.7), p < 0.0001} and downregulation of lnc-DC  were detected in the sera collected from BD patients. Higher serum expression levels of NEAT1 and lnc-DC were significantly associated with the following clinical presentations: cutaneous lesions, vascular manifestations, articular manifestations, neurological manifestations, and higher disease activity score. Also, high NEAT1 levels were significantly associated with a negative pathergy test, while higher lnc-DC was significantly associated with a positive family history. ROC curves showed that NEAT1 and lnc-DC levels in serum could be used as predictors of BD with high specificity and fair sensitivity. NEAT1 had an area under the curve (AUC) of 0.692 , and lnc-DC had an AUC of 0.615 .Conclusion: Serum lncRNAs NEAT1 and lnc-DC are biomarkers for BD. Behçet’s disease (BD) is a chronic multi-systemic inflammatory disease with a wide spectrum of clinical presentations varying from mucocutaneous ulcerations and ocular inflammation to more severe and potentially fatal manifestations such as the vascular system, nervous system, and gastrointestinal tract lesions. Different theories posit that BD is a result of immune system dysfunction due to environmental or infectious triggering factors in genetically susceptible individuals. To date, there is no specific laboratory test for BD diagnosis, and so the identification of essential genes underlying the susceptibility, activity status, or manifestations of the disease is of great importance and may provide new regimens for controlling or treating the disease .NEAT1 (nuclear-enriched abundant transcript 1) (lnc-DC (lncRNA in dendritic cells) (Long non-coding RNAs (lncRNAs) are a group of RNAs that are longer than 200 nucleotides in length and do not encode proteins . Althougcript 1) , and lncc cells) ].NEAT1 is an lncRNA that localizes to and plays a critical role in maintaining the formation of paraspeckles, an interchromatin nuclear body with unknown function   . In addi pathway , suggestLnc-DC is expressed in dendritic cells (DCs) and mediates DC maturation by phosphorylating signal transducer and activator of transcription 3 (STAT3)  . As suchnd IL-12 .NEAT1 and lnc-DC, with BD susceptibility, activity, and clinical manifestations. We also determined the diagnostic performance of NEAT1 and lnc-DC in differentiating BD patients from healthy controls.Based on this background, we investigated the relationship between the expression levels of two immunity-related lncRNAs, This study was conducted per the Declaration of Helsinki and was approved by the Ethical Committee of the Faculty of Medicine, Fayoum University (protocol code M400). Written consent was obtained from all participants after the declaration of all involved procedures and steps. Participants were categorized into two groups. For Group I, we selected 40 male and 12 female patients fulfilling the criteria of the International Study Group (ISG) for the diagnosis of BD . ParticiMedical history and clinical examination were performed for all patients to detect disease features and complications. Patients underwent a complete medical history , general examination, specific examination to detect the symptoms and signs of BD disease, involving oral or genital ulcers, the presence of cutaneous lesions, Duplex and color-coded Doppler examination on arterial and venous systems, and fundus examination by slit lamp. Central nervous system (CNS) and joint examination by computed tomography and magnetic resonance imaging were performed when indicated to detect any vascular, ocular, CNS, or articular lesions. The pathergy test was done for all patients, and a positive pathergy test was indicated by the formation of a small papule or pustule (3–10 mm) within 1 or 2 days at the site of sterile needle prick . All patVenous blood samples were obtained from all patients and healthy individuals under sterile conditions. The collected venous blood was kept undisturbed at room temperature to allow clotting. The samples were subsequently subjected to centrifugation for serum separation. The clear supernatant present in the upper part of the tube (serum) was transferred to a clean tube and stored in the freezer at −80°C until RNA extraction.NEAT1 and lnc-DC, was extracted from the serum using the miRNeasy extraction kit  by following the manufacturer’s protocol.Total RNA, including g for 15 min at 4°C. Next, we transported the upper aqueous phase to a new collection tube, followed by the addition of 1.5 times its volume of 100% ethanol. After that, we pipetted 750 μl of the mix to the 2 ml RNeasy Mini spin column and centrifuged it at 8,000 ×g at room temperature for 15 s. When the mixture had totally passed the column, we discarded the flow through water and reused the tube to repeat this step with the remaining part of the mix. Then, we used the washing buffers in the kit (RWT and RPF) as follows: firstly, we added 700 μl of the RWT buffer to each spin column. Then, we centrifuged it at 8,000 ×g at room temperature for 15 s, discarded the flow through, and reused the column for the next step. Secondly, we pipetted a 500 μl buffer RPE to the spin column and again centrifuged it at 8,000 ×g at room temperature for 15 s, discarded the flow through water, and repeated the previous step. To finish, we transported the spin column to the new collecting tube and centrifuged the tube for 2 min at full speed for dehydration, and for elusion, the column was transferred to a clean Eppendorf tube and 50 ul Rnase-free water was pipetted directly onto the column and centrifuged for 1 min at 8,000 ×g. A NanoDrop (ND)-1000 spectrophotometer  was used to quantify extracted RNA purity and concentration; about 1–2 μl of eluted RNA samples were sufficient, and the ratio of absorbance at 260 and 280 nm is used to assess the purity of RNA; a ratio of ∼2.0 is generally accepted as “pure” for RNA.The steps of RNA extraction include the following: first, we cleaned the working place with 70% ethanol. Then, we added 1 ml QIAzol lysis reagent to a 200 μl sample and incubated it for 5 min at room temperature. Next, we added 200 μl chloroform, vortexed the tube for 15 s, and incubated the mixture at room temperature for 2 min. Afterwards, we used cool centrifugation to avoid the destruction of RNA at 120,00× Reverse transcription to obtain cDNA was performed using the RT2 First Strand Kit  according to the manufacturer’s protocol in a final volume of 20 μl (10 μl reverse-transcription mix was added to each tube containing a genomic DNA elimination mix that has the extracted RNA and DNA elimination buffer).NEAT1 and lnc-DC in serum was measured using the RT2 SYBR Green PCR kit  using predesigned primers obtained for NEAT1 , lnc-DC , and GAPDH as an internal housekeeping gene  under the following conditions: 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 60 s. The relative fold changes of the target genes in patients affected by BD compared to the controls were calculated using the 2equation . For theuals one .NEAT1 and lnc-DC was 52 in each group.The sample size was calculated using G power version 3.0.10. The minimum sample size of patients needed to get a power level of 0.80, an alpha level of 0.05, and a medium effect size of 0.50 for t-test was used to test significance. NEAT1 and lnc-DC data were presented as median and interquartile range (IQR). The Mann–Whitney U and Kruskal–Wallis tests were used to compare between any two or three groups, respectively. Spearman correlation was performed to identify any relationships between NEAT1 and lnc-DC with study parameters among patient cases. ROC curve analysis was used to determine the cut-off point differentiating between case and control with the highest sensitivity and specificity for NEAT1 and lnc-DC. Categorical data were presented as frequencies and percentages, and chi-square (χ2) was used as a test of significance. For the interpretation of results, the significance was determined to be p ≤ 0.05.The collected data were organized, tabulated, and statistically analyzed using SPSS software package version 22 . Age and other basic data were presented as mean and standard deviation (SD). Independent p = 0.524) and sex .The demographic data of the studied groups are presented in Family history, clinical data, activity index, and treatment options are presented in NEAT1  and significantly less lnc-DC  were detected in the sera collected from BD patients relative to the controls.Relative target gene expression is shown in NEAT1, lnc-DC, and patient demographics and clinical data were analyzed. We determined that higher serum expression levels of NEAT1 and lnc-DC were significantly associated with the following clinical presentations: cutaneous lesions, vascular manifestations, articular manifestations, neurological manifestations, and higher activity score. NEAT1 was also significantly associated with a negative pathergy test, and higher lnc-DC was significantly associated with a positive family history . NEAT1 and lnc-DC expressions also showed significant positive correlation with disease duration , pulse rate , and with BDCAI activity score . However, the patient’s age was only significantly positively correlated with NEAT1  (There was a strong positive correlation between the two studied genes (= 0.003) .NEAT1 and lnc-DC in serum had diagnostic value. RNA levels of these two genes could be used to distinguish BD patients from healthy controls. For NEAT1, AUC = 0.692, 95% confidence interval (CI) = 0.591–0.794, cut-off point = 1.09, p = 0.001, with sensitivity = 69.2%, and specificity = 100%. For lnc-DC, AUC = 0.615, 95% CI = 0.508–0.723, cut-off point = 0.66, p = 0.043, with sensitivity = 61.5%, and specificity = 100%  and pro-inflammatory cytokines , resulting in an immune response in inflammatory and immune diseases . Moreover, it is interesting that NEAT1 positively correlated with disease activity score (BDCAI), demonstrating that increased inflammation may lead to higher expression of NEAT1.Our results showed that the expression of NEAT1 expression and disease activity in SLE. In addition, NEAT1 results in the improvement of skin lesions associated with herpes simplex infection. NEAT1 is also hypothesized to contribute to neurodegenerative diseases  and contributes to the disease via different mechanisms that result in the increased production of inflammatory cytokines and the increased apoptosis of chondrocytes . The serum level of lnc-DC was also positively correlated with the duration of disease and pulse rate.In the present study, our results showed that the lnc-DC is exclusively expressed in human conventional DCs, promoting their differentiation and increasing the capacity of DCs to stimulate T cell activation. lnc-DC mediates these effects by activating the transcription factor STAT3  since they observed a high level of lnc-DC in the serum of patients with MS. The discrepancy between upregulation vs. downregulation of lnc-DC in different autoimmune diseases is hard to definitively clarify as few studies examine the role(s) of lnc-DC in the pathogenesis of autoimmune diseases. The differences in lncRNA expression may be due to tissue specificity in the expression of some lncRNAs  and fair sensitivity . The AUC for NEAT1 and lnc-DC was 0.692 and 0.615, respectively.A positive correlation between NEAT1 and lnc-DC in the prediction of BD. Future studies are required to verify our results and elucidate the involvement of NEAT1 and lnc-DC in the pathogenesis of BD. Such future studies are needed to overcome the limitations of the current study, which include 1) small sample size due to the rare pattern of the disease and 2) the lack of scientific literature illustrating the exact role or underlying molecular contributions of lncRNA generally and of NEAT1 and lnc-DC specifically in the pathogenesis, propagation, and prognosis of BD. Larger-scaled studies that explore the exact functions of these genes in BD pathogenesis are required.The results presented here offer a potential new significance for lncRNA NEAT1 and lnc-DC can be used as novel biomarkers for BD. In addition, their high transcript levels were associated with disease activity score and could be targets of therapy in patients with BD.We showed that


Due to demographic change with an ageing workforce, the proportion of employees with poor health and a need for medical rehabilitation is increasing. The aim was to investigate if older employees with migrant background have a different need for and utilization of medical rehabilitation than employees without migrant background. To investigate this, self-reported data from older German employees born in 1959 or 1965 of the first and second study wave of the lidA cohort study were exploratory analyzed (n = 3897). Subgroups of employees with migrant background were separated as first-generation, which had either German or foreign nationality, and second-generation vs. the rest as non-migrants. All subgroups were examined for their need for and utilization of medical rehabilitation with descriptive and bivariate statistics . Furthermore, multiple logistic regressions and average marginal effects were calculated for each migrant group separately to assess the effect of need for utilization of rehabilitation. According to our operationalizations, the foreign and German first-generation migrants had the highest need for medical rehabilitation while the German first- and second-generation migrants had the highest utilization in the bivariate analysis. However, the multiple logistic model showed significant positive associations between their needs and utilization of rehabilitation for all subgroups. Further in-depth analysis of the need showed that something like under- and oversupply co-exist in migrant groups, while the foreign first-generation migrants with lower need were the only ones without rehabilitation usage. However, undersupply exists in all groups independent of migrant status. Concluding, all subgroups showed suitable use of rehabilitation according to their needs at first sight. Nevertheless, the utilization does not appear to have met all needs, and therefore, the need-oriented utilization of rehabilitation should be increased among all employees, e.g. by providing more information, removing barriers or identifying official need with uniform standards. Due to the demographic change and prolonged working lives, the proportion of older employees is increasing in Germany , 2 and oIn Germany, in order to be eligible for medical rehabilitation, the objective need must be assessed first. The need for rehabilitation is not automatically officially acknowledged by the psychological or physical impairment, but mainly from the continuing or expected impairment of participation in social and working life –8.The concerned person must submit an application himself, so that individual need can be proven. The validation is jointly done by the rehabilitation providers , who coordinate their responsibilities among themselves. Within an objective socio-medical evaluation, information provided by the applicant, doctors, psychological psychotherapists and other therapeutic professions in social work and care are taken into account. However, no uniform procedure to assess the objective need for rehabilitation  exists aIn particular, groups of employees who have worked as factory workers are burdened by monotonous, repetitive work and physically demanding tasks , 19, 21.Compared to those without a migrant background (non-EMB), employees with a migrant background (EMB), especially foreign nationals, are more frequently exposed to such health-endangering working conditions which our own data has also shown –25. CompThe group of EMB comprises employees born outside of Germany  and employees born in Germany, but with one or both parents born abroad  . A post-hoc test revealed that the need for rehabilitation was statistically higher for German G1 EMB  and foreign G1 EMB  compared to non-EMB (3.66 ± 2.25). There was no statistically significant difference between G2 EMB and non-EMB (p = 1.0).In contrast, foreign G1 EMB showed the highest need for rehabilitation when comparing means of the need score . The meaTo answer the second research question, logistic regressions were conducted separately for each migrant group to investigate further behavioral or migrant-group-specific differences see . In bivaIn the model adjusted for sex, year of birth and education, foreign G1 EMB had the highest odds  and the highest probability (4.2% for each unit change) to utilize medical rehabilitation when the need increased. The other groups showed ORs from 1.25 to 1.30 each. When testing narrowed models, in detail only one sociodemographic control variable at a time to follow the “one in ten rule”, nearly no change in the coefficients was detected.Our bivariate analyses revealed that foreign G1 EMB have a higher need but lower utilization Tables , which wIn the present study, we analyzed the need for and respective utilization of rehabilitation for employee groups with and without migrant background. For the primary research question, we identified that foreign and German G1 EMB had the highest need for rehabilitation when measuring with our need score. The highest utilization of rehabilitation was reported by German G1 EMB and G2 EMB with 17%, while foreign G1 EMB showed the lowest with 11%. Secondarily, when considering the respective need in multiple logistic regressions, significant positive associations with the utilization were found for each subgroup. Foreign G1 EMB showed the highest association between need and utilization.However, the results of foreign G1 EMB have to be carefully interpreted as the case number of this group was quite low , as wellThis analysis is the first in Germany to identify the need for rehabilitation in different migrant groups compared to non-migrants. The findings of a higher need for rehabilitation in G1 EMB and especially in foreign G1 EMB match our assumptions before analysis as this group often experience unfavorable working conditions, as mentioned in the introduction. Our results showed, when assessing need with the help of our need score: the higher the need, the higher the utilization of rehabilitation for all groups. However, further barriers for instant utilization of healthcare and rehabilitation e.g. language problems, illiteracy or cultural aspects might exist for foreign G1 EMB due to own migration experiences and potential different health beliefs within their cultural background. Hence, when assessing need for medical rehabilitation with our need score, this group showed zero utilization of rehabilitation when having low need, compared to the other groups in In more detail, our additional analysis for interpretational purposes detected that over 70% of the people with higher need did not utilize rehabilitation services but instead 7–10% of those with lower need used rehabilitation in the past. Only those with lower need did not use rehabilitation at all in foreign G1 EMB. This raises the question as to whether health services like medical rehabilitation are truly authorized according to the need in Germany. Especially in older working age, health services like medical rehabilitation should be provided depending on the existing need. Only in this way, equal opportunities to stay healthy and actively in work and prevent early exit can be assured and unnecessary costs avoided. While there have been some projects in Germany in the past, aiming at improving the information about and access to medical rehabilitation where needed e.g. , accordiHowever, the main dependent factor is, of course, the instrument to assess the need for rehabilitation, as the term “need” is not distinct and results are highly dependent of the chosen instrument. As described before, there are several operationalizations within rehabilitation research in Germany suggested to support the identification of need. In the presented study, we decided to orientate the operationalization towards the checklist of Deck et al. , as it cIn the future, the operationalization should definitely be standardized and so we are calling for a harmonization of the assessment procedure for the need of rehabilitation as other rehabilitation researchers , 8. As rAnother important aspect, especially for the further outlook, constitutes the timing of needs assessment, as people with need should be identified and allocated early enough to medical rehabilitation. Schlöffel and colleagues  tested aOur study has several strengths. First, the sample is representative for the German population of socially insured employees of the considered two age cohorts , 38. SecDespite these strengths, the study also has its limitations. First, there is no gold standard for the assessment of the need for medical rehabilitation in Germany. So, we could not test the validity of our assessment instrument derived from the check list suggested by Deck et al. 2009) for general practitioners more comprehensively. Furthermore, certain aspects which influence the official need were discussed in previous rehabilitation research, but could not be considered, as there were no suitable items within the first and second lidA study wave assessed. These include the overall rehabilitation prognosis, the participant’s motivation and therapy options, such as the local infrastructure for rehabilitation. Yet, these factors might be more relevant in estimating the long-term success of medical rehabilitation and to a lesser degree in assessing the actual need for rehabilitation, which was the focus of our investigation. Overall, the items of the need score were based on subjective information of the study participants and not on an objective assessment of the need for rehabilitation. Nonetheless, the items were collected independently and without this purpose. Another limitation is the restriction to two age cohorts within socially insured employees due to sampling, where the lidA study does not include sworn civil servants and self-employed persons. While the majority of employees in Germany are socially insured 009 for g, employeAccording to our results and operationalizations, all subgroups showed suitable use of rehabilitation according to their needs at first sight, foreign G1 showed the highest association. However, when looking more in detail, something like under- and oversupply co-exist in all subgroups, while foreign G1 employees with lower need were the only ones without rehabilitation usage. Yet, undersupply exists in all groups independent of migrant status. Therefore, the need-oriented utilization of rehabilitation should be increased among all employees, e.g. by providing more information, removing barriers or identifying official need with the same standards. The findings highlight the necessity for distinct operationalization and investigation of migrant groups in research and resulting policy.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Virtual reality (VR) gaming offers an immersive experience that can enrich physical therapy for the burn patient by introducing variability, enjoyment, and reward during functional motions of the upper extremities. In this study, we aim to support a proof-of-concept for VR gaming in upper extremity burn rehabilitation by characterizing shoulder and elbow kinematics during VR gaming in a healthy volunteer.A healthy volunteer without burn injuries played two games, a virtual rhythmic baton and virtual boxing game, on a commercially available VR gaming platform. Kinematics during play were assessed using two external cameras placed orthogonally, to the player’s front and left, so that 3-dimensional motion of the player’s left arm could be captured. Video of each gaming session was processed using an open-source perceptual computing software that dynamically tracks the user’s upper extremity during play. Kinematics at the left shoulder and elbow were characterized with respect to range of motion (ROM) and time spent in composite positions.During the rhythmic baton game, the player achieved 157 degrees of elbow flexion ROM and 90 degrees shoulder elevation ROM. During the boxing game, the player achieved 156 degrees of elbow flexion ROM and 123 degrees shoulder elevation ROM. The baton game was associated with more time spent in the “rest” position  while boxing was associated with more time in the “Guard” position  . Both games demonstrated simultaneous movement at both the shoulder and elbow during play.The two VR games investigated in this kinematic assessment challenged players to achieve a wide range of motion at the upper extremity with functional, multi-joint movements. These findings support a potential role for commercial VR gaming in burn rehabilitation and future research in burn patients is required to demonstrate its therapeutic value.


The non-thyroidal illness syndrome (NTIS) was first reported in the 1970s as a remarkable ensemble of changes in serum TH (TH) concentrations occurring in probably any severe illness. Ever since, NTIS has remained an intriguing phenomenon not only because of the robustness of the decrease in serum triiodothyronine (T3), but also by its clear correlation with morbidity and mortality. In recent years, it has become clear that  feeding in patients with critical illness should be taken into account as a major determinant not only of NTIS but also of clinical outcome. Moreover, both experimental animal and clinical studies have shown that tissue TH concentrations during NTIS do not necessarily reflect serum low TH concentrations and may decrease, remain unaltered, or even increase according to the organ and type of illness studied. These differential changes now have a solid basis in molecular studies on organ-specific TH transporters, receptors and deiodinases. Finally, the role of inflammatory pathways in these non-systemic changes has begun to be clarified. A fascinating role for TH metabolism in innate immune cells, including neutrophils and monocytes/macrophages, was reported in recent years, but there is no evidence at this early stage that this may be a determinant of susceptibility to infections. Although endocrinologists have been tempted to correct NTIS by TH supplementation, there is at present insufficient evidence that this is beneficial. Thus, there is a clear need for adequately powered randomized clinical trials (RCT) with clinically relevant endpoints to fill this knowledge gap. Soon after the development of radioimmunoassays for thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) in the 1970s, a drop in serum thyroid hormone (TH) levels was reported both during starvation and illness. In mild illness, only a decrease in serum T3 levels was seen but with increasing severity and duration of the illness there appeared to be a drop in serum T4 as well. This decrease of serum TH levels was reported in a variety of species and pathologies including sepsis, myocardial infarction, and many more diseases. In contrast to the situation during primary hypothyroidism, however, decreased serum TH concentrations did not coincide with increased serum TSH. This typical ensemble of changes in serum TH concentrations was called the low T3 syndrome, the euthyroid sick syndrome (ESS) or the sick euthyroid syndrome (SES). A more appropriate designation, without presuming the metabolic status of the patient, is non-thyroidal illness syndrome, or NTIS. The focus in publications on NTIS has traditionally been on the change in serum TH concentrations in relation to particular pathologies. Early studies pointed to increased serum reverse T3 (rT3) resulting from decreased degradation of rT3 during NTIS, when several labs with access to immunoassays for both T3 and rT3 observed this discrepancy between these two metabolites of T4. However, it is now generally accepted that serum rT3 may be normal or even reduced in some NTIS cases. Once elevated, rT3 does not increase much more in patients with more severe illness.This review will focus on more recent studies mainly centered around differential changes in TH metabolism depending on the organ and tissue studied. TH availability in these tissues during NTIS appears to be independent of decreased serum concentrations to a large extent. To illustrate our increasing understanding of the pathogenesis of these complex changes we will zoom in on effects of NTIS on organ-specific TH transporters, receptors and deiodinases. The role of inflammatory pathways in these non-systemic changes will be addressed, including recent studies on a novel role for TH metabolism in innate immune cells. Finally, clinical implications of NTIS, including the role of nutrition, will be briefly touched upon.Slc16A2 (MCT8) and Slc16A10 (MCT10) mRNA expression, whereas chronic inflammation results in a combination of decreased Slc16A2 and increased Slc16A10 mRNA expression. Unexpectedly, lethal bacterial sepsis affects hepatic TH transport only marginally [In the past two decades it has become clear that TH needs to be taken up in cells by specific transporters including monocarboxylate transporter 8 (MCT8), monocarboxylate transporter 10 (MCT10) and organic anion transporting polypeptide family 1C1 (OATP1C1) to exert its action at the cellular level . In the rginally . In theoIntracellular TH is metabolized via iodothyronine deiodinases, a group of selenium cysteine containing enzymes capable of removing an iodine atom from the inner or outer ring of TH. Iodothyronine deiodinases comprise type 1 deiodinase (DIO1) catalyzing inner- and outer-ring deiodination, type 2 deiodinase (DIO2) catalyzing only outer-ring deiodination (converting its preferred substrate T4 into T3), and type 3 deiodinase (DIO3) which catalyzes only inner-ring monodeiodination  . ChangesA variety of changes in TH transporters, deiodinase and TR action have been observed in different organs and tissues both during acute and chronic illness, while serum TH levels are uniformly downregulated , 11. ChaHypothalamic MCT10 and OATP1C1 expression increases during critical illness in experimental animals while hypothalamic MCT8 mRNA expression does not change. Of note, these alterations are unrelated to local tissue T3 concentrations and their functional consequences are unknown at present .TH transport in the human liver is differentially affected during illness: MCT8 expression increases in prolonged critical illness compared to acute illness, while MCT10 expression does not differ between these groups . LikewisLike the liver, muscle is an important T3 target tissue and muscle TH metabolism is known to be affected during NTIS. In mice, muscle TH transporter expression is low during acute inflammation and bacterial sepsis but remains unchanged during chronic inflammation. Similar changes were observed in patients, as MCT8 expression was remarkably low in muscle tissue of patients during acute surgical stress compared to patients with prolonged illness . ProlongThe balance of deiodinase activities is an important determinant of TH availability in the cell. Deiodinase expression and activity are markedly affected during illness, depending on the severity and duration.In the hypothalamus, DIO2 is expressed in glial cells and tanycytes and DIO3 is expressed in neurons of the paraventricular nucleus (PVN), supraoptic nucleus (SON) and IFN . IllnessA healthy liver predominantly expresses DIO1, with very low DIO3 expression. Illness results in marked downregulation of DIO1 both in humans and experimental animals , 20, 21.Dio2 mRNA expression, while bacterial sepsis decreases Dio2 mRNA expression both in mice [In muscle, local conversion of T4 toT3 by DIO2 is important for muscle function both in rodents and in humans , 27. Mus in mice  and huma in mice . By cont in mice . Acute i in mice  while ch in mice . The samStreptococcus pneumonia [Recently, it became clear that the cellular function of neutrophils and macrophages, both important players of the innate immune system, highly depends on the availability of intracellular TH, regulated via DIO3 and DIO2 respectively. DIO3 protein is highly expressed in infiltrating murine neutrophils during both bacterial infection and sterile inflammation , 33. DIOneumonia  and cleaneumonia . These fneumonia . Activatneumonia . In addineumonia , 37. FinTH action is determined by intracellular T3 concentration and TR status. Both components have been studied during NTIS in both humans and experimental animals.Hypothalamic TR mRNA expression does not change dramatically during NTIS , but theLiver TRα and TRβ mRNA expression are differentially affected during illness, depending of its severity and duration. Liver TRα and TRβ expression in rodents decrease during acute illness , 39 but The effects of illness on T3 signaling in muscle also depends on the type of illness. UCP3, which is a T3 target gene, increases in skeletal muscle of mice during acute sepsis  and inflThe differential regulation of TH transporter, deiodinase and TR expression in acute inflammation, chronic inflammation and sepsis supports the concept that the net result of altered cellular TH metabolism during NTIS determines organ function during specific stages of illness.Multiple mechanisms are known to be simultaneously involved in the pathogenesis of NTIS. One key element is the role of inflammation. Inflammatory mediators like cytokines and inflammatory signal transduction have been extensively studied in humans, experimental NTIS animal models and in vitro models . An earlRecent studies indicate a role for the inflammatory signal transduction pathway nuclear factor κB (NF-κB) in the pathogenesis of NTIS. The NF-κB protein family consists of several subunits of which the 65kD subunit of NF-κB (RelA) activates the Dio2 promoter , 62. InhOne of the key features of NTIS is the absence of increased serum TSH in the face of markedly decreased serum T3 and T4, pointing to altered set-point regulation of the hypothalamus-pituitary-thyroid (HPT) axis. One of the mechanisms involved is central downregulation of the HPT axis, resulting in persistently low serum TH concentrations. Indeed, reduced hypothalamic TRH expression, portal serum TRH and pituitary TSH content have been observed both in NTIS patients  and NTISBesides inflammatory mediators, glucocorticoids and energy status, alternative mechanisms involving the peripheral metabolism of TH have been reported. Liver DIO1 expression and activity are downregulated during NTIS. The Dio1 gene is positively regulated via a TR/RXR heterodimer which suggests that decreased TR or/and reduced RXR expression may play a role in the downregulation of hepatic DIO1 during NTIS . Indeed,The clinical endocrinologist is frequently asked to provide endocrine consultation in case of a severely ill patient in the ICU without a (family) history of thyroid or pituitary disease, in whom a very low (F)T3 is found in combination with low or normal (F)T4 and low or normal TSH. It is clear that the typical clinical features of severe hypothyroidism with or without the presence of a goiter are absent in patients with NTIS, even in the presence of a very low serum T3. Thus, the combination of these features should lead to a high suspicion of NTIS. If both T3 and T4 are very low a potentially fatal outcome may be assumed. Potential caveats include a clearly elevated TSH suggesting the presence of prior hypothyroidism. There is no doubt that overt primary hypothyroidism in a patient with NTIS in the framework of severe illness should be treated with levothyroxine (LT4). Finding positive anti thyroid peroxidase (TPO) antibody titers supports the diagnosis of primary hypothyroidism but does not prove it. In addition to a high serum TSH concentration, a relatively high ratio of serum T3–T4, a low thyroid hormone-binding ratio and a low serum concentration of rT3 favor the presence of hypothyroidism over NTIS and vice versa. The determination of rT3 is, however, not routinely available in many hospitals and does not reliably distinguish between hypothyroidism and NTIS. As a potential complicating factor, serum TSH may be relatively low in critically ill patients with concomitant hypothyroidism as a result of a variety of drugs, for example dopamine which inhibits TSH release . MoreovHyperthyroidism is the typical cause of suppression of TSH below 0.01 mU/L, but it is rarely difficult to exclude this diagnosis in the setting of severely depressed T4 and T3.The question whether interventions aimed at normalizing TH concentrations in patients with NTIS in the framework of protracted critical illness are beneficial has not been answered to date. Only a small number of RTCs involving heterogeneous NTIS patient groups, ranging from protracted critical illness to elective heart surgery in otherwise healthy participants, have assessed the effects of treatment with THs. Both T3 and levothyroxine have been used. The results of TH treatment in critical illness have been largely negative in terms of clinical benefit  . This maAn unresolved issue is whether there is a place for TH treatment in patients with heart failure. Although the use of TH treatment in patients with heart failure has not been adequately studied, some trials have reported encouraging findings. A small RCT  in patieante mortem plasma TSH and T3 concentrations [An important question raised by dr Van den Berghe and colleagues is whether hypothalamic neuropeptides , growth hormone releasing peptide 2 (GHRP2), gonadotropin releasing hormone (GRH), and TRH) can be used in patients with long-term critical illness in an attempt to stimulate the anterior pituitary gland, thereby restoring endocrine function in terms of plasma concentrations and hormone pulsatility . A numbetrations . HoweverIn sum, no definitive conclusion about the efficacy of TH treatment in patients in the ICU with NTIS can be made at present. The reported studies thus far were not adequately powered to detect clinically meaningful differences. Furthermore, most studies used rather high doses of either T4 or T3, probably inducing further suppression of pituitary TSH release. In this respect, the use of neuropeptides, including TRH, to stimulate the HPT axis may be more promising. Large RCTs in well-defined patient groups will be needed to assess possible positive effects of this approach in terms of clinical outcome. High priority should be given to RCTs comparing the effect of hypothalamic neuropeptides including TRH with placebo, because this approach has already been shown to partly normalize concentrations of serum THs and at the same time improve metabolic markers . AnotherCritical illness is associated with loss of appetite and poor nutritional intake. As fasting in healthy individuals induces complex changes in the HPT axis including low serum T3 without increased TSH, decreased caloric intake during severe illness is likely to contribute to the development of NTIS .A large clinical trial reported comparisons between two nutritional regimens in ICU patients, i.e., an early parenteral nutrition group receiving parenteral nutrition within 48 h of admission to the ICU to supplement insufficient enteral nutrition, and a parenteral nutrition group who did not start this feeding regimen before day 8 in the ICU. Interestingly, the toleration of a nutritional deficit during the first week of critical illness resulted in fewer complications and accelerated recovery . AbilityTogether, these findings suggest that thyroid economy is affected by decreased nutritional intake during acute critical illness and that the inactivation of T4–rT3 and T3–T2, as part of the fasting response, might be a beneficial adaptation during acute illness. Teleologically, the reduced amount of circulating active T3 could be interpreted as an attempt by the body to decrease the metabolic rate, reduce energy expenditure, and prevent protein breakdown, thereby promoting survival. By contrast, the central lowering of T4 could be harmful  and peripheral (thyroid gland and target organs) components of the HPT axis show robust changes during NTIS. Studies in mice on the time course of the changes in thyroid hormone metabolism in the HPT axis during acute illness induced by bacterial endotoxin  showed a rapid induction of interleukin-1beta mRNA expression in the hypothalamus, pituitary, thyroid and liver. This was followed by almost simultaneous molecular changes in the pituitary, the thyroid gland and the liver. In the hypothalamus, D2 mRNA was strongly increased whereas preproTRH mRNA expression did not change after LPS. Serum T3 and T4 fell only after 24 h. Together, this suggested almost simultaneous involvement of the whole HPT axis in the downregulation of thyroid hormone metabolism during acute illness .In the clinical setting, less information is available on the timing and the relative role of central versus peripheral mechanisms in the pathogenesis of NTIS. Peripheral mechanisms related to macronutrient restriction occur already in the acute phase in critical illness and appear beneficial. In the prolonged phase of critical illness, hypothalamic thyrotropin releasing hormone (TRH) expression is suppressed as shown both in animal and human studies, contributing to reduced TSH secretion and reduced thyroidal hormone release. The clinical implications of NTIS in the prolonged phase of critical illness when patients receive nutrition but continue to depend on intensive medical care, is uncertain.Many experimental and clinical studies pointed to a biphasic neuroendocrine pattern in critical illness as conceptualized by dr Van den Berghe and colleagues . InDuring prolonged critical illness, several tissue responses reported from both animal and human studies could be interpreted as compensatory to low thyroid hormone availability. These responses include increased tissue expression of monocarboxylate transporters, upregulation of type-2 deiodinase activity, and increased sensitivity at the receptor level . InfusinNTIS has traditionally been seen as a syndrome defined by lower plasma TH concentrations of unknown significance. However, more recent studies have clearly shown that NTIS rather represents a complex set of marked and differential changes in TH physiology, both at the level of the HPT axis and at the organ and tissue level Fig. .Fig. 1ScWhether the observed changes in critically ill patients defined as NTIS are beneficial or deleterious is currently unknown. This may depend on disease type and severity, the need of prolonged vital support, and environmental factors including parenteral nutrition. At present, there is no clinical evidence to support TH treatment of NTIS in critically ill patients. There is a clear need to perform adequately powered RCTs to define whether pharmacological management of NTIS, e.g., using neuropeptides including TRH, may yield benefit in terms of clinical outcome.