Metastasis is the last stage in the development of cancer and usually results in mortality. Cutaneous metastases (CMs) account for 2% of all skin malignancies. Nearly 70% of CMs in women originate from breast cancer (BC). Since CM is usually associated with poor prognosis, the development of management strategies for these patients remains an important clinical challenge. Identifying molecular markers in primary BC that predict CMs and determining the molecular differences between primary tumors and their metastases is of great interest for designing new therapeutic approaches.Cutaneous metastases (CMs) account for 2% of all skin malignancies, and nearly 70% of CMs in women originate from breast cancer (BC). CMs are usually associated with poor prognosis, are difficult to treat, and can pose diagnostic problems, such as in histopathological diagnosis when occurring long after development of the primary tumor. In addition, the molecular differences between the primary tumors and their CMs, and between CMs and metastases in other organs, are not well defined. Here, we review the main clinical, pathological, and molecular characteristics of breast cancer CMs. Identifying molecular markers in primary BC that predict CM and can be used to determine the molecular differences between primary tumors and their metastases is of great interest for the design of new therapeutic approaches. Breast cancer (BC) is the most prevalent neoplastic disease among women worldwide. In women, it represents around 30% of all new cancer cases and is the leading cause of cancer death ,2.Metastatic disease is the last stage in the development of cancer, and the outcome is usually fatal. It is a complex process in which the cells of a primary tumor propagate to distal organs by invading local tissues and blood and/or lymphatic vessels, followed by uncontrolled growth in these distal tissues ,4. BC ceCutaneous metastases (CMs) account for only 2% of all skin cancers ,14. In wThe frequency of CM varies according to the molecular subtype of BC. In their study, Lefebvre et al.  includedAlthough there are differences among series, a finding common to all of them is the relative overrepresentation of TNBC, since its frequency in the general population of BCs is around 15%, but it is between 23% and 39% in BCs with CM.The median age of women who have BC with CM is 74 years ,17,24,25The location of CMs in BC patients is not completely random but occurs more frequently in the vicinity of the primary tumor, perhaps by direct extension of the underlying tumor or by lymphatic spread . CMs froThe most common clinical presentation of skin metastases is in the form of nodules, with an incidence of 80% . They raThe telangiectatic pattern may appear as an erythematous patch with prominent telangiectasias or as a lymphangioma circumscriptum-like pseudovesicular lesion . It is cThe erysipeloides or erysipelatoides pattern is also known as inflammatory metastatic carcinoma ,36. ThisCarcinoma en cuirasse, also named scirrhous carcinoma, appears as scattered and firm erythematous and indurated plaques on the chest wall ,37,38. CNeoplastic alopecia presents as circular indurated areas of alopecia on the scalp due to the hematogenous spread of BC . The areThe main BC histological type that produces CMs are an invasive carcinoma of no special type (NST). In the series reported by Mayer et al. , 95.2% oThe pattern of infiltration of dermal tissue influences the clinical pattern of presentation. Histologically, nodules are composed of atypical neoplastic cells arranged in small nests and cords surrounded by fibrosis, usually in a single-file line within the collagen bundles of the dermis ,31. In tFor CMs, it is recommended that immunohistochemical analysis detection of ER, PR, and HER2  be repeaHistologically, the primary characteristics of the neoplasia can be observed in CM, but CMs are often very poorly differentiated variants of the original tumor, and a microscopic study may be insufficient to arrive at the etiological diagnosis . CertainThe difficulty in recognizing the tumor as being of mammary origin mainly depends on the profile of primitive BC. In luminal tumors, which present intense expression of HRs, and in HER2 tumors, the diagnosis can be made more easily, since these tumors present the characteristic immunophenotype of mammary origin with positivity for GCDFP (gross cystic disease factor protein), mammaglobin, GATA3, HRs, or HER2, if applicable.TN cases are the most difficult group to diagnose. This subgroup of tumors presents much lower positivity for typical breast markers, such as mammaglobin (17–24% positivity), GCDFP-15 (0–5%), and GATA3, which show a lower expression than that identified in luminal carcinomas or HER2 .GCDFP and mammaglobin are frequently used as immunohistochemical markers of mammary origin. Mammaglobin is more sensitive and GCDFP more specific, but both markers are expressed in most luminal and HER2 tumors. Staining is cytoplasmic and heterogeneous, so the entire stained slide should be carefully examined. However, these two markers are much less sensitive for TN tumors  and therefore have less diagnostic utility for them. Cutaneous adnexal tumors can also express these two markers, which must be taken into account for differential diagnosis .GATA3 is a transcription factor that plays a role in the differentiation of many tissues, including the mammary luminal epithelium. In surgical pathology, GATA3 is a sensitive marker of BCs since GATA3 is involved in ER signaling, but it is also expressed in urothelial, trophoblastic, salivary, and pancreatic tumors, among others . GATA3 iSOX10 is a transcription factor that plays a role in the differentiation and survival of neural crest cells. Under normal conditions, SOX10 is expressed in normal salivary gland tissue, in bronchial cells, and in myoepithelial cells of the breast. In surgical pathology, SOX10 has mainly been used as a marker for melanoma and nerve sheath tumors. In breast, the tumor expression of SOX10 has been related to the activation of progenitor cells and the epithelial–mesenchymal transition. SOX10 positivity has been described in 66–74% of TN and metaplastic BCs but only 5% of non-TN carcinomas. This marker can be useful even in the absence of GATA3 expression .It is important to note that both ER-positive and ER-negative tumors can show intense S100 expression, which implies a differential diagnosis of melanoma.Lastly, the androgen receptor plays a role in the normal development and proliferation of breast cells. It is expressed more frequently than ER and PR, but like GATA3, its expression is closely linked to ER expression, with a positivity between 25% and 35% in TN carcinomas .SOX10, androgen receptor, and GATA3 expression studies have recently been carried out in TNBC, both in primary tumors and in metastatic carcinomas  Figure . In the Another important differential diagnosis of CM is primary epithelial cutaneous tumors, mainly when metastasis develops a long time after the primary tumor. The distinction between primary adnexal carcinoma (PAC) and metastatic BC to the skin can be quite challenging, despite adequate clinical history ,51. The The poor prognosis conferred by the presence of CMs in women with BC is highlighted by the short median survival of these patients. There are few differences in prognosis regarding whether the lesions are single or multiple, with a mortality exceeding 70% in the first year after the diagnosis ,52.When BC spreads to the skin, it cannot be cured. The purpose of treatment is to relieve symptoms, improve quality of life, and slow the growth of the cancer. The CMs are systemically treated based on the molecular type . A HR-positive tumor can be treated with a variety of endocrine-based strategies. Chemotherapy is the preferred option for CMs that are HR-negative and/or rapidly progressing. HER2-directed therapy should be applied to HER2-positive tumors, depending on the extent of disease. For the local control of skin metastases, external beam radiation therapy is an option for palliation but would not usually be used in previously irradiated areas due to the issue of cumulative dose .Debridement may be advised when lesions are bleeding. Other options are possible and can be helpful, such as imiquimod, showing good results in localized lesions; trastuzumab, in tumors that are HER2-positive; systemic chemotherapy; radiation therapy; and immunotherapy ,54. SurgIn a number of cases, it has been highlighted that cryotherapy and topical immunomodulators may be useful for the management of CM of BC, possibly by eliciting improved responses to traditional systemic chemotherapy, as may emerging therapies including targeted agents and immune-checkpoint blockers. Trials evaluating combination therapy in metastatic melanoma (NCT03276832) are ongoing. Clinical and correlative studies to develop a therapeutic protocol for CM of BC based on these observations are underway .Another option for the treatment of CM in BC is electrochemotherapy (ECT). This is an effective treatment for cutaneous BC lesions that have proven refractory to standard therapies. As smaller lesions were found to be more responsive, Bourke et al.  suggesteElucidating the molecular landscape of CMs could help in selecting new targeted therapies for this ominous manifestation of BC.A potential diagnostic and treatment algorithm for CM is presented in the Metastases are associated with the acquisition of additional driver mutations compared with the primary tumor . DiffereESR1 mutations in metastasis . Additionally, they observed a metastatic enrichment of previously unreported, lower-prevalence ESR1 mutations in the ligand-binding domain, implying that these mutations may also be functional. The type of ESR1 mutation differed according to the histological type  and the metastatic site. Other alterations enriched across all the metastases include the loss of function of CDKN1B and mutations in the transcription factor CTCF, NF1, and KRAS. Mutations enriched at specific metastatic sites affected genes that are important in the development of primary tumors in that site, suggesting the local adaptation of BC metastasis. They included 118 cases of CM and demonstrated that only changes in NOTCH1 were specific for skin metastases. Other examples included PTEN and ASXL1 in brain metastases and KRAS, KEAP1, STK11, and EGFR mutations in lung metastases.Rinaldi et al. , who expESR1 mutations in metastases than in primary tumors, as well as more mutations in RB1. In this study, other genes that were more frequently observed to be mutated in metastasis from BC as compared with early BC were FSIP2, FRAS1, OSBPL3, EDC4, PALB2, IGFN1, and AGRN.Lefebvre et al. , in theiESR1, AKT1, ERBB2, and FGFR4 and that LM matched three prominent mutation signatures: APOBEC cytidine deaminase, ultraviolet exposure, and defective DNA mismatch repair. Huang et al. [TP53, ERBB2, RAD21, NF1, BRCA1, and ESR1. They also demonstrated significantly increased levels of immune-checkpoint-inhibitor biomarkers, microsatellite instability, and CD274 amplification in the metastasis cohort.Focusing on studies that analyzed specific metastases, Tian et al. , using tg et al.  studied TP53 and RB1, were observed in the progression of primary tumors to metastasis. Roy-Chowdhuri et al. [TP53, PIK3CA, KRAS, PTEN, BRAF, and AKT1. However, their group of patients focused primarily on the ER-positive subtype and included only 2% that were ER-negative/HER2-positive breast tumors and 25% that were TN breast tumors. Other studies have also shown that TP53 and PIK3CA are the genes most frequently mutated in metastases [MYLK, PEAK1, SLC2A4RG, EVC2, XIRP2, PALB2, and ESR1—five of which are not significantly mutated in any type of human primary cancer. Kjällquist et al. [AKAP genes in metastatic BCs, suggesting the involvement of AKAPs in the metastatic process. Furthermore, Van Geelen et al. [AKT1, BRCA2, CHEK2, ESR1, FGFR1, KMT2C, NCOR1, PIK3CA, and TSC2 that were significantly enriched in metastases when compared with paired primary tumors. On the other hand, they observed agreement in both types of samples in cases of TP53 and ERBB2 amplification. Paul et al. [MYLK, PEAK1, SLC2A4RG, EVC2, XIRP2, PALB2, and ESR1 (five of which are not significantly mutated in any type of human primary cancer). The most relevant CNVs were a loss of STK11 and CDKN2A/B and gain of PTK6 and the membrane-bound PR PAQR8. PAQR8 gain and ESR1 mutations were mutually exclusive, suggesting a role in treatment resistance.Some studies have compared mutations between paired cases of primary breast tumors and their metastasis. Schijver et al.  includedi et al.  analyzedtastases ,64. Paultastases  studied t et al. , who incn et al.  studied l et al.  includedAKT1, ARID1A, ESR1, ERBB2, or NF1 only in 22% of patients with metastasis; PTEN and NF1 deletion and CYP19A1 amplification were seen in 19% of patients [A recent specific retrospective study of 94 paired cases of lobular carcinoma demonstrated mutations in patients .Some of the studies that have analyzed mutations in metastatic lesions and mutations in their respective primary tumors specify additional mutations observed in skin metastases not seen in the primary tumors ,58,60. DADAM9, IKBKB, PRDM14, CCND1, MED1, ERBB2, CDC6, C11ORF30, CDH1, TRAF4, CPD, CDC6, MAPT, and CCNE1). These genes had been implicated in different pathways, including the development of therapy resistance. Focusing only on paired skin cases, they observed more gains of the ESR1, MTDH, MED1, and ERBB2 genes in metastasis samples than in the primary tumors and more CDH1 losses in the metastasis compared with the primary tumor. Curigliano et al. [Referring to copy number alterations, Moelans et al. , who inco et al.  identifiIn order to further explore the possible specific molecular alterations of CMs in comparison with other visceral metastases, we analyzed the mutational differences between CMs and LMs of BC. We chose LMs because they are the metastases most frequently found in the analyzed series, probably because they are easier to biopsy. We included 58 sequenced cases of skin metastasis from five series ,57,58,69First, we analyzed whether there were differences in the immunophenotypes of BC according to the metastatic site. The percentages of each phenotype of BC with skin metastasis with respect to those with LMs is presented in Although the differences in the immunophenotype were not statistically significant, which could be due to the relative low number of cases, we observed a higher proportion of the TN phenotype in tumors with CMs than in those with LMs.The mutations more frequently found in CMs and LMs are shown in RUNX1, ROBO1, MLL2, DYNC2H1, and CHD1 were mutated in 7% of CMs but no LMs , and TP53 was mutated in 50% of skin metastases and 31% of LMs   . This anAccording to the alterations observed in CM and LM, the biological mechanisms underlying their development can be hypothesized. To improve the knowledge of the biology of BCM, it would be necessary to carry out more sequencing studies with a greater number of cases from patients with CM of mammary origin. More paired cases of primaries with their corresponding CM should be studied with a broad panel of genes, including all those most frequently involved in BC, to find the additional mutations in CM that lead to their development, to identify not only metastasis mechanisms but also potential therapeutic targets. Another possible future projection in this area would be using whole exome sequencing to study the primary BC tumors of patients who have developed CM, attempting to find a specific mutational profile in such patients in comparison with tumors that developed metastases in other sites. Studies of this type could have clinical impact since knowing the pathways that lead to the development of skin metastases or knowing a specific genetic profile in the primary tumor of the patients who develop this type of metastasis would help to act against specific targets or help to predict and prevent tumor development to the skin.CM is a factor for poor prognosis of BC, and new therapeutic approaches are needed to improve patient survival. A better understanding of the pathological and molecular characteristics of the primary tumor and their CMs will help in achieving this objective.


Purpose: High heterogeneity of the response of cardiorespiratory fitness (CRF) to standardized exercise doses has been reported in different training programs, but the associated mechanisms are not widely known. This study investigated whether changes in the metabolic profile and pathways in blood serum and the skeletal muscle are associated with the inter-individual variability of CRF responses to 8-wk of continuous endurance training (ET) or high-intensity interval training (HIIT).Methods: Eighty men, young and sedentary, were randomized into three groups, of which 70 completed 8 wk of intervention (> 90% of sessions): ET, HIIT, or control. Blood and vastus lateralis muscle tissue samples, as well as the measurement of CRF [maximal power output (MPO)] were obtained before and after the intervention. Blood serum and skeletal muscle samples were analyzed by 600 MHz 1H-NMR spectroscopy (metabolomics). Associations between the pretraining to post-training changes in the metabolic profile and MPO gains were explored via three analytical approaches: (1) correlation between pretraining to post-training changes in metabolites' concentration levels and MPO gains; (2) significant differences between low and high MPO responders; and (3) metabolite contribution to significantly altered pathways related to MPO gains. After, metabolites within these three levels of evidence were analyzed by multiple stepwise linear regression. The significance level was set at 1%.Results: The metabolomics profile panel yielded 43 serum and 70 muscle metabolites. From the metabolites within the three levels of evidence , the variance in MPO gains was explained: 77.4% by the intervention effects, 6.9, 2.3, 3.2, and 2.2% by changes in skeletal muscle pyruvate and valine, serum glutamine and creatine phosphate, respectively, in ET; and 80.9% by the intervention effects; 7.2, 2.2, and 1.2% by changes in skeletal muscle glycolate, serum creatine and creatine phosphate, respectively, in HIIT. The most changed and impacted pathways by these metabolites were: arginine and proline metabolism, glycine, serine and threonine metabolism, and glyoxylate and dicarboxylate metabolism for both ET and HIIT programs; and additional alanine, aspartate and glutamate metabolism, arginine biosynthesis, glycolysis/gluconeogenesis, and pyruvate metabolism for ET.Conclusion: These results suggest that regulating the metabolism of amino acids and carbohydrates may be a potential mechanism for understanding the inter-individual variability of CRF in responses to ET and HIIT programs. As a consequence, international agencies recommend that adults accumulate ≥150 min·wkIn the past decade, with the technological advance of the comprehensive methods that make up the omic sciences  have been described in details previously , smoking, metabolic disorders, heart diseases, musculoskeletal problems interfering with exercise or significant chronic respiratory conditions : 24.8 ± 2.5 kg·mensaiosclinicos.gov.br; RBR-3rh38g).Written informed consent was obtained from each participant. The study was approved by the University's Research Ethics Committee  and included in the Brazilian Clinical Trials Registry (n = 30), HIIT (n = 30) and Control . This unequal randomization strategy was used to ensure adequate sample size for correlation analysis and subsequent analysis of high and low responders in the intervention groups. After the 4th wk of intervention, cardiorespiratory assessment was performed to adjust the training's intensity. In the end, after 48 h of the last training session, the assessments referred to in the pre-training moment were repeated , followed by cardiorespiratory assessment and retest 48 h later , the participants consumed a standardized and balanced meal , with an energy value corresponding to 30% of the total individual energy expenditure estimated in order to avoid effects of dietary variations on the metabolic profile of the blood and muscle tissue samples  , respectively. Body density was then assessed using a full-body plethysmograph calibrated according to the manufacturer's recommendations . Heart rate (HR) was continuously monitored by a cardio-frequency monitor . The subjective perception of effort was recorded at the final 15 s, using the 6–20 Borg scale  was defined as the mean value in the test's final 10 s. The reserve HR (HRR) was estimated by subtracting the values at rest from the respective maximum values achieved in the incremental test ], where W is the last load reached and t is the number of seconds in the test's final load , with 40 min per session, for eight wk, divided into Stage 1 (first four wk) and Stage 2 (last four wk). At the end of Stage 1, a cardiorespiratory assessment was performed to adjust the training intensity to be prescribed in Stage 2. For ET, the participants exercised for 40 min at 70% of HRR, three times a wk, in Stage 1; and for 40 min at 75% of HRR, four times a wk, in Stage 2. For HIIT, the participants exercised for 40 min, with 5 min at 50% of HRR, followed by five 4-min intervals at 90% of HRR (effort phase) interspersed with 3-min intervals at 50% of HRR (recovery phase), three times a week, in Stage 1; and 5 min at 60% of HRR, followed by five 4-min intervals at 90% of HRR interspersed with 3-min intervals at 60% of HRR, four times a week, in Stage 2. For the control, the participants were instructed not to perform physical exercises for eight wk. After four wk, the control participants were contacted again to remind them about the importance of remaining sedentary and to schedule the tests for the end of the eight-wk period.All training sessions were supervised by an experienced professional to ensure that the target HR and pedaling cadence (70–80 rpm) were maintained. All training sessions were carried out in a reserved environment, with temperature controlled between 21–23°C.2O, followed by centrifugation at 14,000 rpm and 4°C for 10 min. After the fifth wash, spin was performed  to eliminate any remnants of Milli-Q H2O. Subsequently, 500 μl of blood serum were added to the filter and centrifuged at 14,000 rpm and 4°C for 45 min. After this period, the filtered serum (250 μl) were diluted in an deuterium oxide solution  containing a phosphate buffer , 0.5 TMSP-d4 (3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionic acid from Sigma-Aldrich), and added to a 5 mm NMR tube  for immediate acquisition of the spectra on the spectrometer  were washed with 500 μl of Milli-Q Hg). The top phase of the mixture  was collected and completely dried in a vacuum concentrator . The remaining solid phase was rehydrated in 0.6 ml deuterium oxide containing phosphate buffer  and 0.5 mM of TMSP-d4. Finally, the samples were added to a 5 mm NMR tube  for immediate acquisition of the spectra on the spectrometer.The muscle tissue samples were processed following the Le Belle protocol  using the VnmrJ software (Varian NMR Systems) and an Inova Agilent NMR spectrometer , operating at a resonance frequency of 1H 600 MHz and a constant temperature of 298 K (25°C). A total of 256 free induction decays (FID) with 32-k data points over a spectral width of 8,000 Hz were used, with an acquisition time of 4 s and 1.5-s intervals between scans (relaxation delay). The spectral phase and baseline corrections, as well as the identification and quantification of the metabolites present in the samples, were performed using the Suite 7.6 Chenomx NMR software , with TMSP-d4 (concentration known) as a reference for quantifying the concentrations of other metabolites. All NMR spectra were processed with 0.5 Hz line broadening (lb) to smooth out the noise in the spectral signals. To inhibit any bias, the samples were randomly profiled by a blinded evaluator. Metabolites (methanol and ethanol) involved in the reagents used in the samples' collection and preparation were not considered for analysis. In addition, in relation to serum metabolites included in the study  were used to improve normality of distributions when appropriate . All transformed data were presented in their original scale for easier interpretation.For all variables, the data distributions were checked for major deviation from normality. Logarithmic transformations , and the significance level adopted was 1% for these hypothesis-generating analyses, assuming that a Bonferroni correction would be too conservative, leading to a high rate of false-negative results. In addition, this approach was supplemented by estimating the effect size and 95% confidence interval for each comparison between LRE and HRE. Thus, when the confidence intervals did not cross zero, the differences were considered significant  in the metabolites' concentration levels and participant's physical characteristics with MPO gains. Afterwards, the ET and HIIT groups were fragmented, separately, into new subgroups, based on the first tercile  and third tercile  of the distribution of MPO gains in response to training ; (2) significant difference between HRE and LRE; and (3) contributing to significant metabolic pathways associated with MPO gains. Afterwards, multiple linear regression models with stepwise selection were used to determine the variance in MPO gains explained by the changes observed in each serum and/or skeletal muscle metabolites retained by the three levels of evidence and to point out the key metabolites. To validate the models, the assumption of multicollinearity of measures between the independent variables was assessed by the variance inflation factor (VIF 1); the normality of residue distribution was determined by inspecting the frequency histograms; and the global influence of each case in the model was analyzed by inspecting the standardized residues and Cook's distance  correlation with MPO gains ; body fat ; and fat-free mass .There were no significant correlations between MPO gains and pretraining to post-training changes in: body mass (ET: r) ≥ |0.2| for the association between MPO gains and the pretraining to post-training changes in metabolites' concentration levels of ET and HIIT programs, respectively , asparagine, glycine, threonine, succinate, 3-hydroxybutyrate, xanthine, carnitine, and propylene glycol , while for HIIT, the most correlated were creatine (P < 0.01), guanidinoacetate and creatine phosphate (P < 0.05 for both). These correlations were moderate  and negative for all serum metabolites.Of the 43 metabolites quantified in blood serum, 24 and 19 showed correlation coefficients  ≥ |0.2| for the association between MPO gains and the pretraining to post-training changes in metabolites' concentration levels of ET and HIIT programs, respectively , tyrosine, threonine, 2-phosphoglycerate, anserine, glycolate, glycine, N-nitrosodimethylamine, alanine, trimethylamine N-oxide, lactate and carnitine , while for HIIT, the most correlated were glycolate, nicotinate (P < 0.01 for both), histamine and 2-phosphoglycerate (P < 0.05 for both). These correlations were moderate  and negative for all these skeletal muscle metabolites, except for glycolate in both training programs and nicotinate in HIIT, which were positive.Of the 70 metabolites quantified in the skeletal muscle, 42 and 18 showed correlation coefficients .As demonstrated in previous study  for these comparisons was classified as wide, ranging from 0.99 to 1.98  and skeletal muscle  in each exercise program. A total of 18 distinct and significantly changed pathways were identified and related to MPO gains, at a false discovery rate of 0.1. From these pathways, only 6 in serum  and 4 in skeletal muscle  were similar between ET and HIIT programs. The complete list of significant pathways and their related metabolites for each training program are summarized in detail in For pathway analysis, pretraining to post-training changed metabolites that were correlated at r ≥ |0.2|); (2) significant differences between LRE and HRE; and (3) contribution in significant altered pathways related to MPO gains.The altered metabolites associated with gains in MPO were identified based on three levels of evidence, previously described: (1) correlation with MPO gains  explained by each metabolite supported by the three levels of evidence . For ET,This study investigated whether changes in the metabolic profile and metabolic pathways of blood serum and the skeletal muscle are associated with the trainability of CRF, based on MPO, in response to ET and HIIT programs. The results were based on the commonality of three levels of evidence and the main findings were: (i) differences in the metabolic changes associated with the MPO gains between training programs, as well as between LRE and HRE; (ii) associations between changes in the metabolic profile and MPO gains, which were: negative for serum asparagine, glutamine, succinate, glycine, histidine, methionine, threonine, creatine phosphate, guanidinoacetate, ornithine, citrate, 3-hydroxybutyrate, betaine, choline, glycolate, and skeletal muscle pyruvate and histamine in the ET program; negative for serum succinate, valine, creatine, creatine phosphate, and guanidinoacetate in the HIIT program; and positive for skeletal muscle valine in ET and glycolate in both ET and HIIT programs; (iii) identification of key altered metabolites that were able to explain the interindividual response variance of the MPO gains, adjusted by random errors and intervention effects: 14.7%, based on changes of skeletal muscle pyruvate and valine, serum glutamine and creatine phosphate in the ET program; 10.5%, based on changes of skeletal muscle glycolate, serum creatine and creatine phosphate in the HIIT program ; (iv) thovertraining , while an increase in metabolites precursor of intermediates of the citric acid cycle via glyoxylate and dicarboxylate metabolism was found.Some important limitations to present study should be highlighted. Both training programs tested consisted of eight wk of training, which is generally not sufficient to achieve the maximum response to a given dose of exercise . The patients/participants provided their written informed consent to participate in this study.AC and MC-M conceptualized and designed the study. AC, RD, and SO-N conducted training program, experimental data collection, and metabolomics analysis. AA performed the muscle biopsies. MC-M and CC provided technical assistance and/or conceptual advice. AC performed statistical analysis and wrote the first draft of the manuscript. All authors have read, edited and approved the submitted version.MC-M is supported by grants from the São Paulo Research Foundation  and Support Fund for Teaching, Research and Extension . AC also was supported by the National Council for Scientific and Technological Development , Coordination for the Improvement of Higher Education Personnel , and São Paulo Research Foundation .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Oreochromis mossambicus ♀ × Oreochromis urolepis hornorum ♂), high-throughput Illumina deep sequencing technology was used to investigate the expression profiles of miRNAs under salinity stress , alkalinity stress  and salinity–alkalinity stress  challenges. The results showed that 31, 41, and 27 upregulated and 33, 42, and 40 downregulated miRNAs (P < 0.05) were identified in the salt stress, alkali stress, and saline–alkali stress group, respectively, which were compared with those in the control group (C). Fourteen significantly differently expressed miRNAs were selected randomly and then validated by a quantitative polymerase chain reaction. On the basis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, genes related to osmoregulation and biosynthesis were enriched in the three types of osmotic stress. In addition, three miRNAs and three predicted target genes were chosen to conduct a quantitative polymerase chain reaction in the hybrid tilapia and its parents during 96-h osmotic stress. Differential expression patterns of miRNAs provided the basis for research data to further investigate the miRNA-modulating networks in osmoregulation of teleost.Researchers have increasingly suggested that microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression and protein translation in organs and respond to abiotic and biotic stressors. To understand the function of miRNAs in osmotic stress regulation of the gills of hybrid tilapia ( Caenorhabditis elegans  are a class of 18–25 nucleotide length non-coding small RNAs that are partially or completely complementary to untranslated regions of their target genes. It played regulatory roles by repressing messenger RNA (mRNA) translation or by inducing mRNA degradation post-transcriptionally and involved various biological processes . Because elegans , numerou elegans . Accordielopment , abioticelopment , pathogeelopment . Also, FOryzias latipes  is the target gene of miR-429, which participates in osmoregulation in response to osmotic stress  for 2 weeks until they can well feed. The average weight of the experimental fishes was 51.27 ± 3.78 g. Three treatment groups with three replicates of each were set up. According to the previous research results of our research group , the tilO. mossambicus and O. hornorum were obtained from the Gaoyao Experimental Base of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China. The hybrid tilapia and its parents were put in salinity, alkalinity, and salt–alkalinity change water. After 6, 12, 24, 48, 72, and 96 h of three types of osmotic stresses, with three replicates of each time point, five tissues: gill, intestine, liver, kidney, and muscle of three type fishes were dissected and then used to analyze the relative expression levels of miRNAs and their possible target genes by quantitative polymerase chain reaction (qPCR).The ® Multiplex Small RNA Library Prep Set for Illumina®), next-generation sequencing libraries were constructed using 2 μg of total RNA with an RNA integrity number value above 7.5. Polyacrylamide gel electrophoresis was used to purify the total RNA of each sample; the RNA fractions ranging from 18 to 32 nt in size were collected. Then, the collected RNA fractions were ligated with adaptors and used to generate complementary DNA (cDNA) by reverse transcription, which was amplified subsequently by PCR. Lastly, according to the manufacturer’s instruction, the deep sequencing of the four small RNA libraries was performed based on Illumina HiSeq 2000 at the GENEWIZ .The total RNA used in miRNA library construction was extracted using TRIzol Reagent (Invitrogen). Then, Agilent 2100 Bioanalyzer , NanoDrop (Thermo Fisher Scientific Inc.), and 1% agarose gel were used to detect the concentration and quality of total RNA of each sample. In strict accordance with the manufacturer’s protocol (NEBNext2 using Bowtie 2 (V.2.1.0). miRDeep2  was extracted using a miRcute miRNA Isolation Kit  according to the manufacturer’s protocol. The first-strand cDNA was synthesized using the miRcute miRNA First-Strand cDNA Synthesis Kit (TianGen) according to the operating manual. RNase-free water was used to dilute the concentration of the cDNA before further PCR analysis.–ΔΔCt method through IBM SPSS Statistics 22. The results are presented as the mean ± standard error. The relative expression levels of miRNAs were analyzed using the one-way analysis of variance method. The workflow of miRNA-Seq and bioinformatics analysis is shown in According to the operating instructions of the miRcute miRNA qPCR Detection Kit , qPCR was performed in an ABI Step One Plus PCR System and in the following conditions: 95°C for 15 min, 40 cycles of two steps . All primer sequences are listed in During the saline and alkali stress, some fishes showed different levels of abnormal behaviors and morphological characteristics. First, some fishes were motionless underwater, and then the tilapia would roll over and be unable to maintain their balance. Also, the body color darkened gradually and secreted a lot of mucus on the body surface. Moreover, the effect of the alkalinity stress and saline–alkali stress on tilapia was more serious than that of salinity stress. Under the alkalinity stress and saline–alkali stress, the head was black, and the gills were bleeding. Only four fishes died in the saline–alkali stress group with swollen abdomen during the experiment. Dissecting the dead fishes observed different degrees of hydrops in the intestines.To identify miRNAs in hybrid tilapia in response to osmotic stress challenges, miRNA libraries derived from four groups were constructed and sequenced. After data quality analysis of the obtained sequence, the total raw reads were obtained from four groups with 12 different libraries, respectively . After tAmong the 12 libraries, the size distribution and frequency percentage of the clean reads were similar, and 21–23-nt lengths were accounted for approximately 84.2% . The 22 P-value < 0.05, we identified 64 significantly differentially expressed miRNAs in groups C vs. S, including 31 upregulated and 33 downregulated miRNAs , 421 (37.2%), and 397 (35.1%) genes were assigned to the cellular components, biological processes, and molecular functioning categories, respectively (P < 0.05) identified 33, 34, and 34 GO terms in the S group, A group, and SA group, respectively. In the salinity group, 64 (20.4%) and 61 (19.4%) genes were enriched in membrane and membrane parts, respectively, within the cellular component ontology, whereas 6 (1.4%) were enriched in response to a stimulus within the biological process ontology. Many genes that may have osmotic regulation functions were enriched in GO terms. Moreover, 32 (8.1%), 192 (48.4%), 3 (0.8%), and 4 (1.0%) were enriched in transporter activity, binding, electron carrier activity, and molecular transducer activity, respectively, within the molecular function ontology.The Gene Ontology (GO) database was used to annotate the predicted target genes . Among tectively . The resvs. S  and the mTOR signaling pathway (26 predicted target genes) in group C vs. S , indicating the credibility of the miRNA sequencing results.To validate the reliability and validity of the miRNA sequencing, 14 miRNAs were randomly selected from the significantly differentially expressed miRNAs for qPCR validation. Melting-curve analysis revealed that primers of all tested miRNAs were specific. The results of qPCR were compared with the miRNA-Seq analysis results . The rescant1 , novel miRNA-766–LOC100709869 , and novel miRNA-1352–LOC100694683  were detected by qPCR. The novel miR-766 and novel miR-1352 expression levels first decreased and then increased during 96 h of alkali stress   , cAMP si sapidus , tight jer baeri  have beeO. mossambicus, O. hornorum, and the hybrid tilapia were detected through qPCR. The integrated analysis of the expression levels of miRNAs and their predicted targets revealed that the expression trends of miRNAs had negative correlations with their target genes, and the results were coincident with the observations that miRNAs repressing regulates target gene expression through transcript cleavage or translation repression  in cell membrane phospholipids to maintain intracellular osmotic pressure balance . In recemo salar , Siganusiculatus , and Pagus major ; the LC-ty acids , arachidty acids . Numerouhe liver . Chuppa  studies . In the  studies , and in  studies , leadingPiT-1) in the five tissues of the hybrid tilapia and its parents within 96 h of alkaline stress both showed a trend of increasing first and then falling and was opposite to miRNA-1352 expression. This indicates that there may be a targeting relationship between miRNA-1352 and PiT-1, which needs further validation. Studies have shown that PiT-1 is a protein encoded by SLC20A1 and has functions in phosphate homeostasis, contributes to cellular uptake, cell homeostasis, and mineralization  database under SRA accession number The studies involving fish were reviewed and approved by the Animal Care and Use Committee of the Centre for Applied Aquatic Genomics at the Chinese Academy of Fishery Sciences.DM and HS designed the experiment and drafted the manuscript. HS and HZ performed the experiment and analyzed the data. JF participated in experiment and coordination and contributed to the manuscript preparation. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Furthermore, a higher degree of browning was observed in dark-colored honey, such as buckwheat (3.1) and heather (1.35 mAU), than in light ones. Moreover, L* and b* parameters had a greater value in the honey of multiflorous, linden, and rapeseed, and a* was higher in buckwheat and heather. The variables of browning and TPC, ACW, and FRAP were positively correlated with each other. It can be concluded that the browning index strongly contributed to parameters of honey appearance, bioactive compound content, and antioxidant activity.Honey is a source of sugars, amino acids/proteins, and polyphenols, which are the main substrates and reactants in the Maillard reaction. Several bioactive molecules are formed and sequestered to the brown polymeric melanoidins, resulting in a gain and loss of antioxidant function in honey. Therefore, the relationships between the browning index and total phenolic contents, color, and antioxidant activity of Polish-originated honeys, namely acacia, buckwheat, heather, linden, multiflorous, and rapeseed, obtained from three local beekeepers, were addressed in this study. The Total Phenolic Content data showed the following order: buckwheat > heather > acacia > multiflorous > linden > rapeseed. The buckwheat honey also had the highest ability to scavenge free radicals in the range of 207.1–289.3 and 40.9–52.3 µmol Trolox g Honey is a sweet and aromatic natural food product that is widely consumed. According to the consumer study of Kopała, Kuźnicka, and Balcerak , in PolaThe antioxidant activity has already been measured using different methods, such as 1,1 diphenyl-2-picrylhydrazyl (DPPH), ABTS , ferric reducing antioxidant power (FRAP), photochemiluminescence assay for antioxidant compounds, water-soluble (ACW), and lipid-soluble (ACL) modes ,10,11. RTherefore, the objective of this study was to describe the relationships between browning index and total phenolic contents, color, and antioxidant activity of Polish-originated honeys, namely acacia, buckwheat, heather, linden, multiflorous, and rapeseed. The formation of brown pigments e.g., melanoidins, was measured arbitrarily at 450 nm. Then, the Folin–Ciocalteau assay was used for the determination of the total phenolic content (TPC). The color of the honey was obtained considering three parameters, L*, a*, and b*, of the CIELAB  system. The color was also characterized by professional panelists during a sensory session. The antioxidant activity was measured by photochemiluminescence assay in the mode of water-soluble antioxidant compounds (ACW) and by ferric reducing antioxidant power (FRAP) assay. Moreover, differences/similarities between the honey types and three honey producers were also studied using principal component analysis (PCA), and were widely discussed.3·6H2O); sodium acetate; and acetic and hydrochloric acids were purchased from Sigma . A PCL kit for hydrophilic antioxidants (ACW) was purchased from Analytik Jena . Ethanol was purchased from POCH S.A. . Water was purified by a Milli-Q system .6-Hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid (Trolox); 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ); iron (III) chloride hexahydrate (FeClAcacia Mill.), buckwheat (Fagopyrum esculentum Moench.), heather , linden (Tilia cordata Mill.), rapeseed (Brassica napus L.), and multiflorous. Honey jars were kept in a dark place at room temperature (20–22 °C) before analysis.Honey samples were collected during the 2017 season in the Warmia and Mazury region (northeast part of Poland) and supplied from three local and professional beekeepers, who are representatives of Culinary Heritage of Warmia, Mazury, and Powiśle. Honey variety was declared by the producers according to the location of the beehive and available floral source: acacia . While L* is an index giving information about lightness, a* is positive in reddish colors and negative in greenish colors, and b* is positive in yellowish colors and negative in bluish colors . Before The evaluation of color was also carried out by a seven-person team selected, trained, and monitored following ISO 8586:2012 , with apHoney samples were extracted according to the methodology described by Wilczyńska  with 96%2−). Therefore, the ACW measurement was performed using PHOTOCHEM apparatus  according to protocols elaborated by Zieliński, Zielińska, and Kostyra [−1).The ACW method is used to measure the ability of antioxidants from extracts to scavenge superoxide anion radicals (O Kostyra . The lag−1). The antioxidant tests were performed in triplicate for each sample.The FRAP method is based on the reduction of ferric ions by antioxidant compounds. The FRAP assay was conducted according to the experiment of Horszwald and Andlauer . The samv/v) were added, and the mixture was incubated for 10 min in the dark at room temperature. Then, 25 μL of 20% sodium carbonate were added to each well. The absorbance of the mixture in the measurement of TPC was evaluated with the Folin–Ciocalteu reagent at 755 nm after 5 min of reaction in microplates. The results were calculated according to prepared standard curves of gallic acid in the range of 0.03–1.0 mg L−1 and presented as milligrams of gallic acid equivalent (GAE) per gram of sample (mg GAE g−1). Three replicates were analyzed for each honey type.The measurements of TPCs were performed in microplates  using the procedure of Horszwald and Andlauer . The TPCp < 0.05) using STATISTICA 13.1 . The principal component analysis (PCA) was performed using Addinsoft .The data are presented as mean values and standard deviations of triplicate measurements. The differences between the samples were analyzed by a one-way ANOVA with Tukey’s multiple comparison test (p < 0.05). The only browning index in multiflorous honey was not dependent on the producer localization. The average degree of browning that could be related to melanoidin content in Polish-originated acacia honey from the three different producers was the same as in India-originated acacia ones, while multiflorous honey was twice as rich [450) for buckwheat honey, but this could be related to the measuring of 50% diluted honey samples.The degree of browning of honey from three producers is shown in  as rich . Beretta as rich  presenteOur results confirmed that the Maillard reaction and melanoidin formation occurs in unheated Polish-originated honeys. It was reported that concentrations of catalytic sugars and a pool of free amino acids promote and facilitate the final stage of the reaction, resulting in the formation of brown melanoidins of a high molecular weight that exhibit antioxidant activity ,27. MoreColor is the first attractive attribute of honey, and, as such, it is very important for commercialization. It is an important parameter in the quality, acceptance, and preference of consumers. Color can be measured using the CIELAB system. It expresses color as three values: L* for lightness and a* and b* for the red, green, blue, and yellow colors. The lightness of honey plays an important role due to consumer preferences. According to our consumer survey (data not published), respondents preferred light honeys (58.2%) over dark ones (14.9%). However, color is mainly determined by its botanical origin. The Codex Alimentarius Committee on Sugars  stipulatThe studied Polish honeys according to color measurements can be divided into light, medium, or dark, differing in the number of melanoidins produced. Light-colored honeys, like acacia and rapeseed, produced the lowest amounts of melanoidins, medium amounts were noted in multiflorous and linden, and the highest content was shown in buckwheat and heather . Moreovep < 0.05).The highest lightness values were ascribed to rapeseed and multiflorous honeys originated from the three producers, whereas the lowest ones were noted for buckwheat and heather honeys. In our study, the lightness values for acacia honeys from producer 1 and 2 were significantly lower compared to that of producer 3 and the available literature, whereas comparable results were observed for multiflorous honey . MoreovePita-Calvo, Guerra-Rodríguez, and Vázquez  classifiThe total phenolic content determined by the Folin–Ciocalteu method varied greatly among the honey types, as shown in −1, respectively) compared to the other tested varieties (p < 0.05). The TPC of multiflorous, rapeseed, and acacia honeys was determined to be in the range of 37.7–45.0, 13.5–28.9, 42.3–65.4 mg GAE 100 g−1, respectively. It was noted that, amongst the three producers of Polish-originated honeys, the TPC varied significantly at least from one producer (p < 0.05). The TPC values for multiflorous honey were similar to those obtained in a previous study by Sawicki, Bączek, and Starowicz [−1, but significantly higher for acacia honey than reported by Attanzio et al. [Heather and buckwheat honeys were characterized by a significantly higher content of phenolic compounds  to ferrous iron (Fe2+).In the present study, the photochemiluminescence technique (ACW) and FRAP method for reducing antioxidant power were applied for the determination of the antioxidant capacity of the honey samples. The ACW assay is based on the photo-induced autoxidation inhibition of luminol by antioxidants, mediated from the radical anion superoxide, and is suitable to measure the radical scavenging properties of single antioxidants, as well as more complex food systems . The FRA−1. Jasicka-Misiak et al. [−1) and multiflorous (26.2–27.7 µmol Trolox g−1) honey. These findings were in agreement with earlier research conducted by Wilczyńska [−1 (rapeseed honey) to 47.13 mmol Trolox g−1 (buckwheat honey). These results are in agreement with Socha et al. [The superoxide anion radical scavenging capacity of honey varied from 26.94 to 255.40  in the ACW system . The higk et al.  indicatelczyńska  and Wesolczyńska , where aa et al.  obtainedThe results of ACW obtained for honeys from producers 1, 2, and 3 were highly correlated with the respective FRAP data . However, what is more important is that the antioxidant capacity of honeys obtained from producers 1, 2, and 3 was highly positively correlated with the TPC  and melanoidin content . Similar highly positive correlations were also noted between the FRAP and TPC and melanoidin content, thus confirming the contribution of melanoidins to the formation of the antioxidant capacity formed by water-soluble antioxidants. Chua et al.  found thThe PCA was prepared to find similarities and differences between melanoidin content, antioxidant activity (ACW and FRAP assays), total phenolic content (TPC), and color obtained in the CIELAB system and during sensory evaluation (brown and yellow color) obtained by the panel. The combination of PCA accounted for 63.22% for F1 and 18.91% for F2 of the variance in the data. The first two principal components (F1 and F2) explained 82.13% of the total data variance. Karabagias and Karabournioti  achievedUsing PCA, Kaygusuz et al.  successfEvidence is growing that honey may have antioxidant potential through scavenging the reactive oxygen species and reducing the prooxidative metal cations. We have demonstrated that the Maillard reaction and melanoidin formation occurred in unheated Polish-originated honey, and brown pigment formation was strongly correlated with antioxidant potential. Generally, dark honeys showed better antioxidant capacity and higher melanoidin content compared to light honeys. Moreover, our study indicates a link between the TPC and melanoidin formation in honeys. Among the Polish-originated honey, buckwheat, heather, and linden showed the highest ability to scavenge superoxide anion radicals. A highly positive correlation noted between the browning index and antioxidant capacity provided by the ACW assay confirmed the contribution of brown pigments to the formation of antioxidant capacity formed by water-soluble antioxidants. A better understanding of these findings will help reveal the mechanisms of honey functions and predict honey’s effect in different biological systems. As was demonstrated by Vela, de Lorenzo, and Pérez , honey,


Plant growth rhythm in structural traits is important for better understanding plant response to the ever-changing environment. Terrestrial laser scanning (TLS) is a well-suited tool to study structural rhythm under field conditions. Recent studies have used TLS to describe the structural rhythm of trees, but no consistent patterns have been drawn. Meanwhile, whether TLS can capture structural rhythm in crops is unclear. Here, we aim to explore the seasonal and circadian rhythms in maize structural traits at both the plant and leaf levels from time-series TLS. The seasonal rhythm was studied using TLS data collected at four key growth periods, including jointing, bell-mouthed, heading, and maturity periods. Circadian rhythms were explored by using TLS data acquired around every 2 hours in a whole day under standard and cold stress conditions. Results showed that TLS can quantify the seasonal and circadian rhythm in structural traits at both plant and leaf levels. (1) Leaf inclination angle decreased significantly between the jointing stage and bell-mouthed stage. Leaf azimuth was stable after the jointing stage. (2) Some individual-level structural rhythms  were consistent with leaf-level structural rhythms. (3) The circadian rhythms of some traits  were not consistent under standard and cold stress conditions. (4) Environmental factors showed better correlations with leaf traits under cold stress than standard conditions. Temperature was the most important factor that significantly correlated with all leaf traits except leaf azimuth. This study highlights the potential of time-series TLS in studying outdoor agricultural chronobiology. Plant growth rhythm depicts biological and physiological behaviors of plants, such as “tree sleeping” at night –3, whichPlant growth rhythm has been extensively and accurately studied in plant physiology –9, but hLight detection and ranging (LiDAR), an emerging active sensing technology less influenced by the natural illumination conditions, measures the three-dimensional 3D) structure of a plant with high precision and accuracy. LiDAR has been recognized as a cutting-edge tool in quantifying plant structure across different environments , 14. DurD structuRecently, a few studies have tried to use hypertemporal LiDAR data to study short-term  structural rhythm in trees. For example, Puttonen et al.  used terThese investigations using multitemporal and hypertemporal LiDAR applied to trees , 28, 29 In this study, we aim to quantify the seasonal and circadian rhythms in maize structural traits at the plant and leaf levels under standard  and cold stress conditions in field environments from TLS time series. Specific questions are targeted. (1) Can we quantify the seasonal rhythms of structural traits? (2) Can we quantify the circadian rhythms under standard and cold stress conditions? (3) What are the possible relationships between the environmental factors and the circadian rhythms of structural traits under standard and cold stress conditions?″N, 116°10′46″E), Beijing, China. The planting area was around 600 m2 (Zea mays L.) on Mar. 25, 2019 , and Aug. 25, 2019 , respectively. A total of 55 plots were planted in the two experiments. The plot size was 3 m × 3 m, and the row and column spacing between adjacent plots was both 1 m. In each plot, plants were planted at an interval of 0.6 m in both row and column directions. Exp. 1 grew well with the standard cultivation and management, which was used to collect data for studying seasonal and circadian rhythms under standard conditions. Exp. 2 did not have a complete growth period due to cold stress, and it was only used for studying the circadian rhythm.The study area was located at the Xiangshan breeding base of the Institute of Botany, Chinese Academy of Sciences  days belong to key growth periods and (2) weather condition was stable with no rain, no water condensation, and no wind/occasional gusts (human observation) during data acquisition.In this study, TLS data were collected using a high-throughput phenotyping platform, Crop3D, developed by the authors' team  31, 32], 3231, 3https://www.timeanddate.com/. In this study, the moments of sunset (D89), sunrise (D90), noon (D90), and sunset (D90) were 19:46, 04:46, 12:16, and 19:46, respectively. The scanning moments were trying to cover the moments of sunset, sunrise, and noon. However, to avoid interference from environmental factors , the interval of scanning moments was not exactly but around 2 hours. Finally, data were collected at 16 moments from D89 to D90, including 19:49, 21:48, 23:48, 01:48, 3:48, 4:48, 6:48, 8:48, 10:48, 12:00, 12:48, 14:48, 16:48, 18:48, 19:48, and 21:48 . Data for studying circadian rhythm were scanned at the late bell-mouthed stage around every 2 hours from sunset on June 22, 2019, to sunset on June 23, 2019 (D89-D90). The moments of sunset, sunrise, and noon  were refnd 21:48 .Exp. 2 was only used to acquire data at the bell-mouthed stage for studying circadian rhythms under early cold stress. During this period, the plant did not die but showed certain symptoms of chilling injury, such as frostbite on the edge of the leaf. Data for studying circadian rhythm were scanned around every 2 hours from sunset on Nov. 9, 2019, to sunset on Nov. 10, 2019 (D76-D77). The moments of sunset, sunrise, noon, and data scanning were determined using the same method in Exp. 1 . In thisμmol·m−2·s−1), temperature , and relative humidity , every half hour through the whole growing season. PAR was recorded using SPN1 Sunshine Pyranometer . T and RH were recorded using an in situ weather station .In addition to LiDAR data, this study recorded environmental data, including the incoming photosynthetically active radiation . The clipped data were preprocessed with a statistical outlier removal method integrated in LiPlant software . The noise-removed data was filtered and normalized using a digital-terrain-model-based method described in Jin et al. [The single station data was first manually clipped to obtain data in an area of 3 m × 3 m where the point density is high  period, resulting in a decrease in the number of leaves. The average value of the several traits investigated was computed for the four periods and was used for studying seasonal rhythm . In addiPrevious studies mainly extracted height quantiles as structural traits to study the tree dynamics , 3. In aHmax, m), mean height , 99% height quantile , crown size, plant azimuth angle, projected leaf area , volume, projected area index , and three-dimensional profile index (3DPI). The plant azimuth angle was defined as the angle between the maximum eigenvector of the plant and the north direction on the vertical projection plane , PLA, leaf inclination, and leaf azimuth. Of which, the definitions and formulas of leaf length, leaf width, leaf area, and leaf inclination can be found at Jin et al. . Leaf hep value were used to analyze the relationship between these environmental factors and the circadian rhythms in plant and leaf structural traits under standard and cold stress conditions. The circadian rhythm observation time and the climate data recording time were not always fully synchronized. The raw half-hour climate data were therefore interpolated using a cubic spline function to predict the climate data corresponding to the circadian rhythm observation.To show whether TLS can characterize the seasonal rhythm in structural traits, Tukey's multicomparison method was used to test the difference among the measurement dates at the 0.05 confidence level. Besides, because the circadian rhythm is driven by local environmental signals  , 36, theHmax, Hmean, and H99), PLA, volume, and 3DPI increased significantly in the first three stages and became stable at the mature stage. Crown size showed a significant increase in the first three stages but decreased significantly at the mature stage. PAI was stable at the first two and the last two stages, but it showed significant growth from the second (bell-mouthed) to the third (heading) stage. Interestingly, the azimuth angle of individual plants was stable throughout all stages.At the individual plant level, results show that TLS can describe the seasonal rhythms of several structural traits of maize . Height-At the leaf level , resultsHmax between two adjacent sunsets  and leaf traits  showed a similar pattern that increased at night and decreased during the daytime. PLA increased at night and decreased during the daytime at both plant and leaf levels, which was contrary to the circadian rhythm of PLA under standard conditions. Additionally, most rhythms under cold stress  were clearer (less fluctuation) than circadian rhythms under standard conditions.Overall, TLS can quantify the circadian rhythm in structural traits at both individual plant and leaf levels under cold stress. Many individual traits  grow during the vegetative growth periods and become stable at the mature stage. The pattern of volume and 3DPI has rarely been reported because it is difficult to measure these two traits using traditional methods, such as measuring with a ruler or optical image. Further, the seasonal rhythms revealed some new findings. For example, the crown size decreased significantly at the mature stage, which is almost consistent with the trends of PLA  and is sAt the leaf level, the seasonal dynamics have rarely been studied because they have higher requirements on data quality and processing methods. Our results showed that the leaf-level seasonal rhythms were more diverse. This is possibly explained by the variability in the rhythm of the leaves of a different order , 51, whiStudying circadian rhythm is much more difficult because plants may have a slight movement during a short interval . MeanwhiHmean decreased at night, which is consistent with previous findings about “tree sleeping” at night [Hmax and H99 increased at night, which indicates that crop height reduction  may mainly occur in the lower layer that is consistent with the dynamics of height percentile in Zlinszky et al. [Under standard conditions, most findings are easy to understand. Some findings are interesting and deserve further exploration. For example, at night , 3. Howey et al. . Previouy et al. , 28, 56.Besides, we can see that the rhythm of the day is not as obvious as the rhythm of the night because environmental conditions are more complex during the daytime as mentioned in previous studies . We founHmax, H99, crown size, azimuth, PLA, and volume) than under standard conditions. This may be because plants harden their tissues to prevent freeze injury, making circadian rhythm less disturbed [Hmax, H99, leaf length, leaf width, and leaf area) showed a similar pattern that increased at night and decreased during the daytime under cold stress  circadian rhythms  than the rhythm during the daytime. Leaf-level circadian rhythms are more clear than those at the plant level. Circadian rhythms under cold stress are more clear than those rhythms under standard conditions.This study demonstrated that TLS can be used to analyze the seasonal and circadian rhythms of structural traits of maize at the plant and leaf levels under natural and cold stress conditions. To our knowledge, this is the first effort that uses TLS to study the structural rhythms of maize. Besides, this study analyzed the structural rhythm not only at night but also during the daytime. Moreover, structural rhythm under cold stress was studied and compared with standard conditions, which contributed to better understanding of the structural response of crops to different environments.However, some challenges still need to be improved in future works. First, we did not set a rigid marker as a reference in the time-series analysis to quantify the observational error during LiDAR data collection. Puttonen et al.  used theHmax and H99) under cold stress and standard conditions are opposite. Additionally, we found environmental factors have more significant correlations with leaf trait rhythms under cold stress than standard conditions, especially air temperature under cold stress conditions. This study highlights the potential of time-series TLS in studying crop chronobiology in the outdoor environment. By combining structural rhythms with plant biology theories, it will allow improving our understanding of the mechanism driving plant rhythms and the survival strategy of plants in the context of environmental changes.The research of plant rhythms has a long history, but the use of LiDAR for studying structural rhythms is indeed a novel and interesting topic in recent years. This study explored the potential of TLS in the study of seasonal and circadian rhythms at the plant and leaf levels under standard and cold stress conditions. The seasonal rhythms in structural traits are clear and consistent with our expectations. Some structural traits such as azimuth and PLA show good consistency between plant- and leaf-level rhythms. However, the overall seasonal rhythms at the leaf level are more diverse, such as the decreasing of leaf inclination angle between the jointing stage and the bell-mouthed stage. By contrast, circadian rhythms are more complicated. Circadian rhythms of some traits (e.g.,


Objectives: This study aimed to identify variables and develop a prediction model that could estimate extubation failure (EF) in preterm infants.Study Design: We enrolled 128 neonates as a training cohort and 58 neonates as a validation cohort. They were born between 2015 and 2020, had a gestational age between 250/7 and 296/7 weeks, and had been treated with mechanical ventilation through endotracheal intubation (MVEI) because of acute respiratory distress syndrome. In the training cohort, we performed univariate logistic regression analysis along with stepwise discriminant analysis to identify EF predictors. A monogram based on five predictors was built. The concordance index and calibration plot were used to assess the efficiency of the nomogram in the training and validation cohorts.Results: The results of this study identified a 5-min Apgar score, early-onset sepsis, hemoglobin before extubation, pH before extubation, and caffeine administration as independent risk factors that could be combined for accurate prediction of EF. The EF nomogram was created using these five predictors. The area under the receiver operator characteristic curve was 0.824 . The concordance index in the training and validation cohorts was 0.824 and 0.797, respectively. The calibration plots showed high coherence between the predicted probability of EF and actual observation.Conclusions: This EF nomogram was a useful model for the precise prediction of EF risk in preterm infants who were between 250/7 and 296/7 weeks' gestational age and treated with MVEI because of acute respiratory distress syndrome. With the development of perinatology, there has been a rapid increase in the survival rate of premature neonates annually, followed by the increasing incidence of neonatal respiratory distress syndrome (RDS). Although early use of pulmonary surfactant (PS) and non-invasive ventilation  have been proven effective in the prevention and treatment of RDS, some premature neonates with acute RDS still need mechanical ventilation through endotracheal intubation (MVEI). In addition, MVEI is a vital way to save premature neonates after PS and non-invasive ventilation failed. Fifty percent of neonates born at <28 weeks' gestation who received CPAP failed, and 37% of those who received PS failed . More thMechanical ventilation is a double-edged sword, i.e., although it can save the life of neonates, it also might result in adverse outcomes, including ventilator-associated pneumonia (VAP), pneumothorax, pulmonary hemorrhage, and so on . If MVEI2), lower pre-extubation partial pressure of carbon dioxide (PCO2), lower peak FiO2 within the first 24 h of age, non-small for gestational age status, and passing spontaneous breathing trials were associated with a higher likelihood of extubation success (ES). Wang et al. /success (ES) may avoid reintubation and improve clinical outcomes. Studies , 11–13 sg et al. . FurtherThis was a retrospective cohort study approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University, Hefei, China.0/7 to 296/7 weeks' gestational age; (2) born in the obstetrics department at the same hospital with NICU, transferred to NICU immediately after birth and treated with MVEI within the first 24 h of postnatal age (to reduce the heterogeneity of the study population). The exclusion criteria were as follows: (1) died before extubation; (2) accidental extubation; (3) received surgical treatment within 5 days after extubation; (4) congenital malformation of vital organs or genetic/metabolic diseases; (5) the lack of important clinical data.Neonates who were born between June 2015 and December 2020 and met the inclusion criteria were recruited from three medical centers, including the First hospital of Anhui Medical University, Second Xiangya Hospital of Central South University, and First Hospital of Jilin University. The neonates recruited from the First Affiliated Hospital of Anhui Medical University born between June 2015 and December 2019 served as the training cohort, and the neonates recruited from all three medical centers born between January 2020 and December 2020 served as the validation cohort. The inclusion criteria were as follows: (1) 252)] within 0.5 h before intubation, non-invasive ventilation before intubation, age of starting enteral feeding, and early-onset sepsis (EOS). Peri-extubation characteristics were collected, including ventilator mode at extubation , hematology examination  within 48 h before extubation, arterial blood gases within 0.5 h before extubation , highest fraction of inspired oxygen (FiO2) after intubation, FiO2 at extubation, first MVEI duration, weight at extubation, postmenstrual age (PMA) at extubation, and caffeine treatment. Clinical outcomes were collected, including death, BPD, neonatal necrotizing enterocolitis (NEC), late-onset sepsis (LOS), VAP, pulmonary hemorrhage, pneumothorax, retinopathy of prematurity (ROP), age at total enteral feeding, length of time on MVEI, length of time on hospital stay, length of time on oxygen, antibiotic utilization rate, daily weight gain, and daily hospitalization cost.We collected clinical characteristics  and clinical outcomes of the infants from the medical records. Demographics were collected, including gender, gestational age (GA), birth weight (BW), small for gestational age (SGA), assisted reproduction, cesarean delivery, multiple births, and maternal age at pregnancy. Perinatal characteristics were collected, including hypertensive disorder complicating pregnancy of the mother, gestational diabetes mellitus of mother, antenatal steroids, prolonged rupture of the membrane [>18 h], 1-min Apgar score, 5-min Apgar score, intubated in the delivery room, epinephrine in the delivery room, PS administration, hypoglycemia, arterial blood gases  was performed to select the useful combination of factors that could precisely predict EF. A nomogram for EF was created based on the multivariate logistic regression model using the selected factors. Receiver operator characteristic (ROC) curve was performed to evaluate the predictive value of the model. Next, we carried out Harrell's concordance index (C-index) and calibration plot in both training and validation cohorts to assess the performance of the model. A concordance index is a numerical measure of discriminative ability and calibration plots are graphic evaluations of predictive ability that compare observed probabilities with nomogram-predicted probabilities  and the R software .The preterm infants in the training cohort were divided into EF groups and ES groups according to whether to reintubate within 5 days after extubation. For the infants who failed extubation, Kaplan-Meier survival curves were plotted to explore the timing of EF. Univariate logistic regression analysis was performed to identify the risk factors for EF. All bilities . FinallyWe eventually enrolled 128 infants as training cohort. Of these 128 infants , they han = 35) and the ES group (n = 93). In the EF group, 28/35 (80%) infants were reintubated within 3 days (2 retention or/and hypoxemia including severe infection 11/35 (31.43%), atelectasis 7/35 (20.00%), and pulmonary hemorrhage 5/35 (14.29%).In the training cohort, 55/128 (43.0%) of the infants who received MVEI treatment were reintubated during hospitalization, and 35/55 (63.6%) were reintubated within 5 days after extubation , the medn 3 days , and theP = 0.614, Fisher's exact test). The rate of PS administration in this study was 97.66%. Further analysis of the data showed that 35 infants in the EF group used PS and 90 infants in the ES group, there was no statistical significance between the two groups .In clinical practice, ventilator mode at extubation and the administration of PS might affect the occurrence of EF. In this study, 96.09% of infants were weaned with CMV, 3.91% were weaned with HFV. Among those who were weaned with HFV, 2 were in the EF group and 3 were in the ES group, there was no statistical significance between the two groups (2 before extubation (95%CI: 1.01–1.07), and caffeine treatment (95% CI: 0.11–0.55) were identified as the risk factors of EF. We selected 5-min Apgar score, EOS, pH before extubation, hemoglobin concentration before extubation, and caffeine treatment as the best combination of factors to predict EF through stepwise discriminant method. A nomogram for EF was constructed based on the multivariate logistic regression model using these selected factors ( factors .P < 0.05).Compared with the outcomes of the two groups, infants in the EF group had a higher rate of mortality (14.3 vs. 3.2%), higher rate of VAP (51.4 vs. 20.4%), higher rate of pulmonary hemorrhage (37.1 vs. 18.3%), longer time on MVEI among survivors , higher antibiotic administration rate , slower weight gain , and higher hospitalization costs  . The aboThe nomogram which could predict the probability of EF was created using the factors of 5-min Apgar score, EOS, pH before extubation, hemoglobin concentration before extubation, and caffeine treatment . The areThe usage of the predictive model was drawn with an assumptive preterm infant with a 5-min Apgar score of 6, EOS, HB before extubation of 160 g/L, pH before extubation of 7.35, and no caffeine treatment. The points for 5-min Apgar score, EOS, HB before extubation, pH before extubation, and caffeine treatment were 50, 53, 25, 0, and 60, respectively. The total point added up to 188 for this infant, which represented ~0.56 of the probability of EF.The performance of this nomogram was assessed by C-index and calibration plots. Next, we collected 58 preterm infants who met the inclusion and exclusion criteria as a validation cohort to validate our model. The C-index in the training and validation cohorts was 0.824 and 0.797, indicating a high discrimination of the nomogram. The calibration plots also showed high coherence between the predicted probability of EF and actual observation , which iInfants with EF are known to be more likely to have a higher rate of mortality and morbidity, including VAP, pneumothorax, BPD, neurological damage, etc. Currently, the decision to extubate is based on clinicians' discretion and is influenced by clinical evaluation, blood gas variables, oxygen requirement, and level of ventilator support. There is a lack of strong evidence to support the use of any predictor of extubation readiness in preterm infants over clinical judgment alone. Although current studies on spontaneous breathing trials are highly sensitive to ES, they provide little benefit in the identification of EF , 14. Notn = 1,071, <28 weeks GA) showed that the rate of EF within 5 days in preterm infants was 37–50%. Chen et al.  suggested that the rate EF within 7 days was 15.5–33.9%. Wissam et al.  showed that the cumulative rate of EF was high within 3–7 days after extubation. In our study, we found that 55/128 (43.0%) neonates with MVEI were reintubated during hospitalization, and 35/128 (27.3%) were reintubated within 5 days after extubation. More than half (35/55) neonates were reintubated within 5 days of extubation, which was consistent with the results of Wissam et al. A study of Wang et al. , invasive procedures . Thus, the higher incidence of EOS seems reasonable. This study provides data on the incidence of EOS, including culture-positive and culture-negative septicemia, on the condition that epidemiological data are limited.EOS in our study included culture-positive and culture-negative septicemia. Many neonates are diagnosed with a “probable or possible” sepsis or a “presumed symptomatic infection but no bacterial cause identified”; conditions often referred to as “culture-negative sepsis” . Robust Our study showed that the lower HB within 48 h before extubation was associated with EF, which was not mentioned in the published literature that we can retrieve. The result suggests that if infants with invasive ventilation have anemia, indications for red blood cells transfusion might need to be appropriately broadened. As we know, hemoglobin is responsible for transporting oxygen and carbon dioxide as the important component of red blood cells. Previous studies have reported a significant association between anemia and respiratory disease. Watanabe et al.  showed tOur results were not consistent with the study by Manley et al.  on extreThe primary limitation of this study is that the included predictors were not comprehensive. Researches by Ding et al.  and CapaThe high C-index and the good coherence between the predicted probability and actual observation of the model indicate that the modeling has better performance. The model with highest Youden index defined EF risk as 0.207 and predicted extubation failure with a sensitivity of 0.83 and a specificity of 0.67. The study of Shalish et al.  shows thOur study noted that neonates who failed extubation had higher rates of mortality, VAP, pulmonary hemorrhage, and other negative outcomes among survivors, including longer time on MVEI, higher antibiotic administration rate, and higher hospitalization costs. These findings are consistent with published researches that suggest a relatively poor prognosis for EF.0/7~296/7 weeks: (1) In the delivery room, the best resuscitation teams and facilities should be ensured to improve the 5-min Apgar score; (2) After transferring to the NICU, it is important to take measures to prevent sepsis, especially for neonates with a low 5-min Apgar score; (3) Reduce unnecessary venous blood collection. When a neonate passes the spontaneous breathing trial, clinicians should monitor the neonate's HB level before weaning. If the 5-min Apgar score is found to be low or sepsis has been diagnosed, and the hemoglobin is at a relatively low level, indications for red blood cells transfusion could be appropriately broadened; (4) Extubation at pH <7.25 is not recommended; (5) It is recommended to administer caffeine before extubation and maintain it for over 2 days after extubation.Our study suggested that a lower 5-min Apgar score, EOS, lower pH before extubation, lower HB before extubation, and non-use of caffeine treatment before and after extubation were the key factors that determined EF for infants treated with MVEI. Based on our research, we conclude that the following measures might reduce the rate of EF for a preterm infant with a gestational age of 25Our study provides a reference basis for clinicians to choose the timing of extubation and improves the situation that extubation relies on the subjective judgment of clinicians in some way. Our cohort was not representative of all preterm infants with invasive mechanical ventilation; thus, qualified medical centers could try to organize a multi-center prospective study to verify the conclusion and explore better models.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Anhui Medical University. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.ZC, ZD, and QZ: conception and study design, collection, analysis, and interpretation of data, and writing the first draft of the manuscript. JZ, SH, and JG: collection, analysis, and interpretation of data and drafting the manuscript for important intellectual content. YW: conception and study design, drafting the manuscript for important intellectual content, review and editing of the manuscript, and the decision to submit the paper for publication. All authors contributed to the article and approved the submitted version.This work was supported by the Public Welfare Technology Application Research Linkage Project of Anhui Provincial Science and Technology Department (grant number 1704f0804018).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SOD1G93A mutant. In this model, landmark stages of the disease can be recapitulated at specific time points, making possible to understand changes in gene expression across time. While there are several works reporting TE activity in ALS models, they have not explored their activity through the disease progression. Moreover, they have done it at the expense of losing their locus of expression. Depending on their genomic location, TEs can regulate genes in cis and in trans, making locus-specific analysis of TEs of importance in order to understand their role in modulating gene expression. Particularly, the locus-specific role of TEs in ALS has not been fully elucidated. In this work, we analyzed publicly available RNA-Seq datasets of the SOD1G93A mouse model, to understand the locus-specific role of TEs. We show that TEs become up-regulated at the early stages of the disease, and via statistical associations, we speculate that they can regulate several genes, which in turn might be contributing to the genetic dysfunction observed in ALS.Transposable Elements (TEs) are ubiquitous genetic elements with the ability to move within a genome. TEs contribute to a large fraction of the repetitive elements of a genome, and because of their nature, they are not routinely analyzed in RNA-Seq gene expression studies. Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease, and a well-accepted model for its study is the mouse harboring the human  Transposable Elements (TEs) are genetic elements capable of mobilization within a genome. They can be classified into retrotransposons (that involve the reverse transcription of their mRNA) and DNA transposons . Because of their activity, they are found repetitively and ubiquitously in almost every genome, representing almost 50% of the human, and 40% of the mouse genomes. Due to the potentially deleterious activity of transposition in gene expression, most TEs have accumulated mutations that render them inactive and/or are repressed via epigenetic mechanisms . DespiteSOD1 gene was the first to be associated in a causally manner to ALS. In turn, the first stablished ALS model is the SOD1G93A transgenic mouse, in which the spinal cord MNs become the most affected. [G93A mutant stablished in [G93A mice model is the most extensively used to study ALS, and has been used in the development of drugs to treat the pathology. Thus, for the conditions relevant for this study, the SOD1G93A mouse model, the pre-symptomatic stage is defined during the first 4–8 weeks of age, where no motor deficit signs are visible. The onset stage of the disease begins at 12 weeks of age, with distinguishable phenotypic and histopathological hallmarks that impact directly on the ability to move. Afterwards, the disease worsens (symptomatic) reaching the end stage at 17 weeks of age with severe loss of MNs, resulting in hind limb paralysis [Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive loss of upper (motor cortex) and lower  motor neurons (MNs) . As a coffected. , 15. As ished in . Despitearalysis , 17.SOD1G93A mice, and the corresponding Wild-Type (WT) control mice [SOD1G93A mouse disease stages. Multiple genes and signaling pathways, such as TGF-Beta, were found to be affected, all potentially contributing to neurodegeneration. However, the work did not examine whether TEs become transcriptionally active through the disease progression, and whether they can potentially alter genes linked to the disease, as they focused in generating a catalogue (“fingerprint”) of gene expression and pathway changes in ALS. Earlier works have shown over-expression of the Reverse Transcriptase (RT) protein of Human Endogenous Retroviruses (HERVs) in the brain of ALS patients compared to non-ALS patients [To understand the changes in gene expression that takes place in ALS spinal cord tissue, Phatnani et al., performed bulk RNA-Seq assays at Weeks 4, 8, 12 and 17, in patients  and thatpatients . Recent patients , howeverpatients –24. Morepatients  and TEcapatients  to analySOD1G93A mice and the corresponding Wild-Type (WT) control mice at Weeks 4, 8, 12 and 17. The reads were paired-end, with each pair having a read length of 36 bp  database was utilized (accession GSE43879) . This daof 36 bp .Mus musculus mm10 genome version was used. Differential expression analysis were done using DESeq2 [SOD1G93A samples with their respective Wild-Type/Control samples.RNA-Seq quality control was done with FastQC . RNA-Seqg DESeq2 , compariin silico experiment. First, to estimate the TE Divergence from consensus sequences, we used RepeatMasker, and further processed the results using in-house scripts. This was needed to check if there was a bias in the Differentially Expressed TEs towards younger instances (<10% divergence from consensus sequence). Statistical analysis of this bias was performed with a Test of Given Proportions, using R, setting the significance level at 5%. [To assess the ability of SQuIRE to correctly estimate expression of young TEs at the locus level, we performed an l at 5%. .Then, rel at 5%. , with 20Classification of TEs according to their genomic location and overlap with genes was assessed with BEDtools . BrieflyProtein-Protein interaction networks for selected genes were obtained from the STRING database .All plots were generated using ggplot2 .Mus musculus genome, in order to conduct differential expression analysis of TEs using SQuIRE and DESeq2  in one time point, might not be differentially expressed in another, a time course analysis of all the DE-TEs was performed . This reG93A model. This finding was limited to the L1orl elements, with others such as the L1spa not showing significant changes in expression [SOD1G93A mice  in the murine SOD1G93A model [Considering that transposition-competent TEs might generate DNA damage that might in turn be linked to neurodegeneration, we checked if there is any TEs amongst the DE-TEs that could be transpositionally active. Most of the TEs have predicted protein sequences with Stop codons, with only 36 TEs not having any Stop codon . Of thes3A model . It is pMus musculus genome versus the same distribution of the DE-TEs , which may improve the mappability somehow, major doubts about the certainty of the locus-specific expression of TEs can be raised. This is, particularly, an issue for younger TEs (TEs having less than 10% divergence from consensus sequences) and SQuIRE, the tool used to estimate TE expression is less accurate for these TEs . To assee DE-TEs . We founde novo transcriptome assembly, to generate synthetic long reads which may improve the mappability. Thus, TEcandidates is designed to predict which TE locus might be the origin of expression, and in turn, generates a lower amount of predictions. Also, TEcandidates does not estimate TE expression levels. We hypothesized that a combination of both tools might allow us to find out a more reliable subset of expressed TEs. To this end, we performed a simple experiment: we simulated reads from the DE-TEs, then we used SQuIRE to estimate TE expression with different score thresholds, and we evaluated the Precision  and Recall  using SQuIRE alone and SQuIRE+TEcandidates. In this experiment we also varied the read length to see how our results would improve if we had access to a set with longer read length  and in trans (in genes up to thousands of nucleotides away). Thus, at least for Intergenic TEs, the approach we used might not reveal all of their regulatory potential. Nonetheless, without further data for this model this cannot be addressed. Amongst the Significant Associations, all of the Exonic TEs have a positive correlation with their corresponding Gene. This result was expected, as Exonic TEs are already genetically fixed. Thus, if its host gene is transcribed, then the TE will also appear as transcribed. For Intronic TEs, there are both positive and negative correlations. In this case, positive correlations might be indicative of co-transcription and/or intron retention, without apparently negative consequences on the host genes. On the other hand, negative correlations might be indicative of transcriptional interference. That is, the disruption of appropriate transcription of the Gene by the TE. Finally, for Intergenic TEs, all correlations were positive. Except for 1 Intergenic TE, all the others where in the vicinity of 10 kbp or less of their closest gene  doesn’t seem to have a clear pathway associated , Lrrc47 (leucine rich repeat containing 47), Slc25a33 (solute carrier family 25 member 33) and both Sox5 and Sox13, which correspond to the SRY-Box Transcription Factor family. Cep85 colocalizes with isoform A of Nek2 (Nek2A) at centrosomes and forms a granule meshwork enveloping the proximal ends of centrioles. Nek2, in turn, has been implicated in centrosome disjunction at the onset of mitosis to promote bipolar spindle formation [Cep85 would mean a destabilization of the cell and its death. It is worth noting that ALS MNs are post-mitotic, suggesting that there might be either an alternative role for Cep85 in the disease, or perhaps its deregulation occurs in glial cells. Lrrc47, has been linked to ALS, and it has been speculated that it may play a role in protein-protein interactions in the disease [Lrrc47 plays a role in apoptosis, which is a well-known “pathogenic pathway” in ALS. [Cep85 might have influence on apoptosis, and in turn, contributing to the disease. Slc25a33, is part of the family of solute carriers, and adequate expression of this family is required for correct neuronal activity. This is mainly due to these solute carriers being responsible for neurotransmitter transport. While Slc25a33 has not been directly linked to ALS, dysfunction in other members of the solute carrier family have [Slc25a33 might also be contributing to the disease progression. Finally, Sox5 has been associated previously with ALS [Sox5 or Sox13 at the gene level, it is possible that if Cep85 is impacting them, it is doing so at the protein level.Of the interaction partners of ormation . As the  disease . It is w in ALS. . Thus, tily have . It coulwith ALS , 44. WhiCep85, while not directly linked to the disease before, is part of an important protein-protein interaction network. Thus, the potential misregulation of this gene by a TE might indicate that TEs could play a role in the disease. As ALS MNs are post-mitotic, and Cep85 is involved in mitotic progression, the separation of neuron populations from glia population is required to test if there is an alternative role in neurons or if Cep85 is preferentially expressed in glial cells. Thus, future works using single cell resolution are required to better understand the potential role of Cep85, and to identify if there is specificity of TE expression across cell types.The locus-specific role of TEs in ALS has not been explored so far, despite several works suggesting that TEs might contribute to disease. Here, by utilizing state of the art approaches for assessing the locus-specific expression of TEs, we found several putative TE-Gene regulation events. Interestingly, after all our statistical analysis, we only found 1 gene. This gene, The main limitation of this work is that due to the high stringency used in all of our analyses, we discarded results that could represent other gene-TE regulation events, in order to eliminate all False Positives. Based on our results, we suggest that with the advent of newer sequencing techniques that generate long reads, the locus-specific role of TEs during ALS could be more clearly elucidated in future works. Until then, our work highlights the importance of these type of analysis in expanding our knowledge in how TEs might alter gene regulation during disease.S1 Fig(PDF)Click here for additional data file.S2 FigA. DE TEs locus distribution. B. DE TEs class distribution.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.