Herein, a sandwich ELISA for the specific detection of Aβ42 oligomers (oAβ42) and total Aβ42 (tAβ42) was developed based on sequence- and conformation-specific antibody pairs for the evaluation of plasma samples from a Chinese CSVD community cohort. After age and gender matching, 3-Tesla magnetic resonance imaging and multidimensional cognitive assessment were conducted in 134 CSVD patients and equal controls. The results showed that plasma tAβ42 and oAβ42 levels were significantly elevated in CSVD patients. By regression analysis, these elevations were correlated with the presence of CSVD and its imaging markers . Plasma Aβ42 tests further strengthened the predictive power of vascular risk factors for the presence of CSVD. Relative to tAβ42, oAβ42 showed a closer correlation with memory domains evaluated by neuropsychological tests. In conclusion, this sensitive ELISA protocol facilitated the detection of plasma Aβ42; Aβ42, especially its oligomeric form, can serve as a biosensor for the presence of CSVD and associated cognitive impairments represented by memory domains.Due to the heterogeneity of amyloid β-42 (Aβ Cerebral small vessel disease (CSVD) is common in the elderly, with an estimated prevalence of ~5% in 50-year-olds that increases sharply to almost 100% in 90-year-old individuals ,3. Recen38, Aβ40 and Aβ42 appeared, which were associated with imaging markers cross-sectionally and longitudinally ; the unpaired Student’s t-test was used for continuous variables with normal distribution, and results were expressed as Mean standard ± deviation (SD); the one-way analysis of variance (ANOVA) was used for multigroup comparisons, followed by Tukey’s post hoc test for between groups.The SPSS 20.0 software package was used for data management and analyses. A 42 and oAβ42. To measure the associations of risk factors with the presence of CSVD and its imaging markers, WMH was divided into limited (Fazekas score ≤ 2) and serious (Fazekas score 3–6) conditions. Other imaging markers were considered present when: PVS ≥ 11, CMB ≥ 1, and lacune ≥ 1. Aβ42 and neuropsychological scores alike were divided by quintiles. Binary or ordinal logistic regression was performed after adjustment for associated variables. Odds ratios (ORs) with their 95% confidence intervals (CIs) were obtained.For data from all participants, Spearman’s rank correlation was performed to show the correlation between tAβP-trends for continuous linear trends per stratum were analyzed using the Mantel–Haenszel chi-square test. Compound domain scores were calculated from the Z-scores of each neuropsychological test. Linear regression was performed after averaging the Z-scores within each domain. Tests to assess the memory domain included AVLT  and Digit Span Forward.To evaluate the possible predictive value for the presence of CSVD, receiver operating characteristic (ROC) curves were plotted and the area under the ROC curve (AUC) was calculated. 42, combinations of antibody pairs were screened. tAβ42 detection is based on the antibody pair 1F12/2C6 with different epitopes, of which 1F12 has single epitope, while 2C6 has four epitopes in mAβ42; this ensured that both mAβ42 and oAβ42 could be detected . Either tAβ42 or oAβ42 was much higher in the CSVD group compared with that in the control group (42 quintiles (p-trend < 0.05) . Furthermore, the elevation of either tAβ42 or oAβ42 quintiles correlated with CSVD presence when adjusted by age, gender, previous stroke, and hypertension  (A positive correlation was evident between tAβol group . A linea < 0.05) B. In parrtension , suggesto 0.699) .42 quintiles correlated with the presence of severe WMH, whereas the elevated oAβ42 quintiles also correlated with the CMB .Neuropsychological impairments were more prevalent in CSVD cases compared to controls . It was 42 quintiles correlated with impaired executive domain according to the digit span test and short-delayed recall domain of AVLT. Elevated oAβ42 quintiles correlated with delayed recall and recognition domains of AVLT , education (p = 0.000), and age (p = 0.015) were all associated with composite scores of memory domain .After adjusting for age, gender, education, and Hamilton depression/anxiety scores, elevated tAβ of AVLT . Predict42 have been reported previously, most are of partial selectivity for conformations such as oAβ42, pAβ42 or fAβ42, listed in 42, ranging from LMW to HMW, is neurotoxic [42 is of great significance for the diagnosis and deep understanding of the pathology. To date, accurate differentiation of all forms of oAβ42 from tAβ42 remains a challenge, because it suffers from the mis-detection of mAβ42 and lacks a pair of conformation-specific antibodies. Previously developed Aβ-based diagnosis tools were mainly based on a commercial antibody 6E10 reacting with almost all forms of Aβ at the N terminus but not oAβ42 and pAβ42 [40) [42) [42) [42 subspecies or tAβ42. Thus, we developed a sandwich ELISA for the precise and accurate analysis of different forms of Aβ42 including mAβ42, oAβ42 subspecies ranging from LMW to HMW, and tAβ42, which addressed the challenge of the current plasma Aβ42 detection and made it possible to research the correlations between Aβ42 and CSVD.Although several detection methods for the measurement of Aβurotoxic ,38. Thernd pAβ42 ,40,41,42β42 [40) , mAb 15840) [42) , and mOC42 and oAβ42 appeared in CSVD cases and was associated with the presence of CSVD imaging makers such as WMH and CMB. Notably, both tAβ42 and oAβ42 are candidates to be plasma biosensors for CSVD. The elevation of Aβ42, especially oAβ42, was further correlated with cognitive impairments, as represented by the memory domain, which usually appeared in CSVD.Based on the results of this sandwich ELISA, our findings showed that the elevated plasma tAβElevated Aβ deposition is commonly seen in AD and CAA. The pathology of AD and CAA intersects at the increased Aβ generation and impaired Aβ clearance . However42 [42 does not necessarily change in patients with subcortical CSVD despite reduced CSF levels of soluble APP-β [In contrast, although brain Aβ was associated with CSVD burden cross-sectionally, CSVD does not appear to directly influence Aβ accumulation longitudinally, as shown after 1-year follow-up in the elderly . A thoro42 . Howeverle APP-β .42 levels [40/Aβ42 were significantly associated with increased lacunar and microbleed counts, as well as increased WMH volume and poorer cognition [42 was associated with a higher risk of extensive WMH progression over the follow-up of dementia-free older persons in the three-City Dijon Study [42 levels in CSVD cases. In particular, these levels were significantly correlated with the presence of severe WMH. Hence, as one independent predictor for the presence of CSVD, testing plasma Aβ42 levels might enhance the predictive power of historic risk factors, albeit with compromised performance.With the development of high-sensitivity assays, Aβ can now be detected in plasma in addition to brain tissue and CSF. Several recent studies have shown that plasma Aβ may be a promising early biomarker for the ongoing amyloid pathology in AD ,52,53. Hognition . Howeveron Study . Here, t40/Aβ42 ratio predict the development of AD-related dementia, despite significant heterogeneity [40 level is associated with poorer memory and information processing speed, as well as cognitive decline in cognitively normal adults [38 and Aβ40, as well as the Aβ40/Aβ42 ratio, are associated with worse memory domains of AVLT in asymptomatic older adults [42 and cognitive domains. Digit span forward and backward tests, as well as the AVLT, showed that worse memory domains were correlated with increased plasma Aβ42, particularly oAβ42.It is well known that either Aβ or CSVD is correlated with cognitive impairment in the elderly. A moderately significant correlation between brain fibrillar amyloid load and episodic associative memory tasks has been reviewed elsewhere . The incogeneity . Some dal adults . For CSVr adults . Our stu42, the most toxic species, can effectively induce neuronal death [42 correlates with CSVD and the impairments of memory domains reflected by digit span and AVLT. As reported, the plasma concentrations of Aβ42 are unstable partly because of the transformation, and they vary in individuals depending on gene background [42 individually, oAβ42 was stably detected here. This supports that oAβ42 is more likely to be a plasma indicator for CSVD. As a subtype of CSVD, CAA has been known to closely correlate with Aβ deposition [42 in vascular dementia.Mounting evidence indicates that soluble oAβal death . Previoual death . Aβ oligal death ,61. Whilal death , their pckground . Relativposition . CAA wouThere are several limitations to the current study. First, because CSVD presents age-dependently, we selected matching controls from community populations, which could lead to potential biases. Second, other historic factors including involvement in sports, prior medication, and diet were not considered. Genetic tests to confirm the sporadic background were also not performed totally, and it remains uncertain whether AD was concomitant in the cohort only from MR imaging and clinical follow-up. Third, although positive results are described, further investigations with increased sample size are necessary to gain greater reliability. Deeper research using animal models or other multimode imaging would be beneficial. Future steps to assess causality, predictive models, and interactions between biosensors and CSVD would also be preferred.42 was associated with the presence of CSVD and cognitive impairments represented by memory domains. Testing plasma Aβ42, especially oAβ42, may be helpful for closely monitoring CSVD-related CNS damage. With continued investigation, oAβ42 may have the potential to be a plasma biomarker for the presence of CSVD and related cognitive impairments.In the present study, plasma Aβ


The results showed that the four typical “hot” property herbs could significantly improve physical signs, thyroid function, and the metabolic profile in hypothyroidism rats, the four typical “cold” property herbs did not show any benefit. Moreover, the four typical “hot” property herbs could improve lipid metabolism, energy metabolism, and thyroid hormone levels by the PI3K-Akt signaling pathway, Ca2+- AMPK signaling pathways, purine metabolism, and tryptophan metabolism. Additionally, the levels of UCP-1, Na+/K + -ATP enzyme, and ACC1 were ameliorated by the four typical “hot” property herbs in hypothyroidism rats. Therefore, a metabolomics strategy combined with network analysis was successfully performed and interpreted the mechanism of the four typical “hot” property herbs on hypothyroidism based on the theory of “cold and hot” properties of TCM well.Herbs with a “hot” properties are frequently used to treat cold symptoms in TCM. However, the underlying mechanisms of the herbs with “hot” properties on hypothyroidism have not been investigated. This study aimed to explore four typical “hot” and “cold” property herb on hypothyroidism. Firstly, the difference efficacy between the four typical “hot” property herbs and the four typical “cold” property herbs was assessed by physical signs, thyroid function, and the metabolic profile using multivariate statistical analysis. The influence of the four typical “hot” property herbs on hypothyroidism was validated pathologically. The impact mechanism of the four typical “hot” property herbs on hypothyroidism was investigated through a metabolomics method combined with network analysis. Na Hypothyroidism is on of the most common thyroid diseases affecting people worldwide, particularly during pregnancy and childhood . ObesityTCM has been applied in preventing and treating the diseased for thousands of years . ComplexAconitum carmichaeli Debeaux (FZ), Zingiber officinale Roscoe (GJ), Cinnamomum cassia (L.) J. Presl (RG), and Euodia ruticarpa (A. Juss.) Benth. (WZY), were selected. Meanwhile, the four typical “cold” property herbs , Coptis chinensis Franch. (HL), Gardenia jasminoides J. Ellis (ZZ), and Rheum palmatum L. (DH) were also selected as a contrasting experiment to explore the medication principles of the “cold/hot” property. Finally, the results of biochemistry, metabolomics, and the network analysis were integrated to clarify the effect of the four typical “hot” property herbs on hypothyroidism.Therefore, an integrated metabolomic strategy combined with network analysis methods was applied to reveal the effects of four typical “hot” property herbs and four typical “cold” property herbs on hypothyroidism. According to the records in ancient books of past dynasties, pharmacopoeia of the People’s Republic of China and modern research in the “cold/hot” property, the four typical “hot” property herbs , includity herbs , includiScutellaria baicalensis Georgi (HQ), Coptis chinensis Franch. (HL), Gardenia jasminoides J. Ellis (ZZ), and Rheum palmatum L. (DH), and the four typical “hot” property herbs: Aconitum carmichaeli Debeaux (FZ), Zingiber officinale Roscoe (GJ), Cinnamomum cassia (L.) J. Presl (RG), and Euodia ruticarpa (A. Juss.) Benth. (WZY), which were purchased from Beijing Tongrentang Co., Ltd . The herbs were authenticated by Professor Zhenyue Wang  and all voucher specimens were preserved at Heilongjiang University of Chinese Medicine.Acetonitrile  and formic acid  were purchased from Fisher Chemical. The four typical “cold” property herbs: 6-propyl-2-thiouracil (PTU) was purchased from Sigma-Aldrich , and L-thyroxine (L-T4) was purchased from Aladdin Biochemical Technology Co., Ltd., .The ELISA kits for rat triiodothyronine , thyroxine , and thyroid-stimulating hormone  were purchased from Wuhan Huamei Biotechnology Co., Ltd., . The ELISA kits for the Na+/K + -ATP enzyme (batch Number: 202109) and Acetyl CoA carboxylase 1  were purchased from Jiangsu Meimian Industry Co., Ltd. .The four typical “hot” property herbs  and the four typical “cold” property herbs  were extracted by decocting with water three times . Then, the combined decoctions were concentrated and dried in a vacuum to obtain a FZ/GJ/RG/WZY/HQ/HL/ZZ/DH crude extract.Sprague–Dawley rats  were obtained from Liaoning Changsheng Biotechnology Co., Ltd., . All the rats were housed under a 12 h/12 h light/dark cycle, at a temperature of 20 ± 3°C, with 60% ± 10% relative humidity. They had free access to standard food and sterile-filtered water. All the animal experiments were approved by the Experimental Animal Ethics Committee of the Heilongjiang University of Chinese Medicine and performed in accordance with relevant guidelines.n = 7), hypothyroidism model group , positive drug group , and treatment group by the four typical “hot” property herbs, including Hypo + FZ (n = 8), Hypo + GJ (n = 8), Hypo + RG (n = 8), and Hypo + WZY (n = 8), as well as the contrasting experimental group by the four typical “cold” property herbs, including Hypo + HQ (n = 8), Hypo + HL (n = 8), Hypo + ZZ (n = 8), and Hypo + DH (n = 8). With the exception of the rats in the control group, the hypothyroidism model was established in all rats by intraperitoneal injection PTU (10 mg/kg/d) for 28 days consecutively , the thyroid gland were obtained and fixed in 4% paraformaldehyde for histological examinations. Moreover, approximately 0.1 g of the liver tissue was homogenized at a 0.9% saline solution, which was used to detect the NaThe urine sample was mixed with four-fold cold acetonitrile, vortexed for 2 min, and centrifugation at 4°C at 13,000 rpm for 10 min. After high-speed centrifugation, the prepared supernatant was transferred into a fresh 2 ml LC-MS glass vial. In addition, the quality control (QC) sample was prepared by mixing an equal amount of every sample from an identical experiment group. Finally, 2 μL of the supernatant for each sample was injected into the UPLC-Q-TOF/MS system for metabolomic analysis.An ACQUITY UPLC system  in tandem with a Q-TOF synapt G2-SI mass spectrometer  was acquired for LC-MS/MS analyses with an ACQUITY UPLC HSS T3 column . The chromatography separation was performed at an ambient temperature of 40°C. The mobile phase included acetonitrile with 0.1 % formic acid (A), and deionized water with 0.1% formic acid (B), and the gradient of eluent was used: 0–15 min, 5%–68 % (A); 15–18 min, 68 %–95 % (A); 18–19 min, 95 %–5 % (A); and 19–20 min, 5 % (A). The flow rate was set at 0.3 ml/min, and the injection volume was 2 µL.The key parameters of Q-TOF/MS were optimized as follows: scan type: positive and negative, acquire mass over the range 100–1300 Da, scan time: 0.25s, collision energy: 25–35 V, cone voltage: 40 V. the electrospray capillary voltage was 3.2 kV in the positive and negative ionization modes, the ion source temperature was 320°C, and the auxiliary heater temperature was 350°C.http://www.hmdb.ca/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp/kegg/) in the Progenesis QI software  for peak detection, alignment, deconvolution, and normalization. The resultant data matrices were linked to EZ info  for the principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The list of ions that contributed to the grouping was obtained from loading the S-plot and variable importance plot (VIP). The biomarkers were identified by MS/MS fragment ion and accurate mass by searching reliable online biochemical databases such as the Human metabolome database (HMDB) . The sections were visualized using light microscopy , and digital images were captured and analyzed.+/K+-ATP enzymes in the liver were detected by a commercial ELISA kit  in accordance with the manufacturer’s protocols. The rectal temperature of the rats was measured by an electronic standard rectal thermometer and the bodyweight of the rats was weighed by electronic scales at days 1, 7, 14, and 28.A commercial ELISA kit  was used to analyze the levels of T3, T4, and TSH in the serum in accordance with the manufacturer’s protocol. ACC1 and NaThe extracted total RNA from BAT, the primers designed, and the cDNA synthesis were references reported in the literature, which was performed by Wuhan Servicebio Biotechnology Co., Ltd.  . PrimersAssay of the Western blot, as previously reported , the levhttps://tcmsp.com/) database were obtained. The database included a network of chemicals, targets, and drug targets, as well as the pharmacokinetic properties of natural compounds with respect to oral bioavailability, drug similarity, intestinal epithelial permeability, the blood–brain barrier, and water solubility (https://www.omim.org/), Drug bank, and Gene Card (https://www.genecards.org/). Moreover, the intersection of the four typical “hot” property herb targets and hypothyroidism-associated targets were analyzed by a Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/). And, the PPI network was performed on the STRING database (https://string-db.org/cgi/input.pl). The STRING database is used for searching known and predicted interactions between proteins. In addition to containing experimental data, it contains text mining results from PubMed abstracts, the integration of other database data, and it also uses bioinformatics methods to determine the predicted result. There are already many protein interaction databases, and STRING covers the most species among them. The specific settings in this system are as follows: select “Homo sapiens” in the organism column, select “evidence” in the meaning of network edges column, set the confidence to >0.9, remove the free protein, obtain the correlation data between the targets, and import the acquired data into Cytoscape 3.7.2 for the PPI network (https://david.ncifcrf.gov/home.jsp) for Gene Ontology (GO) and KEGG pathway enrichment analyses, which were mainly involved in the molecular function (MF), biological process (BP), and cellular components (CC). The cut-off criterion of the DAVID analysis was p < 0.05, which was considered statistically significant  and the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  was assessed in comparison with the respective control for each experiment using one-way analysis of variance.All the data were analyzed by using the SPSS 20.0 software . Statistical significance . Firstly, an unsupervised principal component analysis (PCA) was carried out to explore the influences of the four typical “hot and cold” property herbs on the hypothyroidism model metabolic profile. As the result shows in In the present study, combining the results of physical signs, thyroid function, and metabolic profile, we can conclude that the four typical “cold” property herbs showed no benefit in the hypothyroidism model. Therefore, the four typical “hot” property herbs that affected hypothyroidism were used for further mechanism analysis.The effect of the four typical “hot property” herbs on the histopathological changes in the thyroid tissue of hypothyroidism rats is shown in p <0.05). It was suggested that the metabolism state or metabolites have altered. Combined with the results of PCA, those changed metabolites could be the key potential biomarker of working mechanisms. In this study, a total of common 15 specific metabolic biomarkers , and the four typical “hot” property herb groups not only responded to the importance of metabolites but also validated the previous analysis results. As shown in In order to further analyze how the four typical “hot” property herbs work on the hypothyroidism model by those different potential biomarkers, the altered metabolites were annotated by the KEGG database, and the pathway was analyzed by the Metabo-Analyst. It was found that 7 perturbed metabolism pathways were involved , mainly 2+ signaling pathway, VEGF signaling pathway, NF-κB signaling pathway, sphingolipid signaling pathway, AMPK signaling pathway, insulin signaling pathway, cAMP signaling pathway, cytochrome P450, and PPAR signaling pathway  could improve hypothyroidism in rats with cold syndrome by adjusting the substances and energy metabolism . But the four typical “cold” herbs were shown to have no benefit, which was consistent with the TCM theory. In the metabolism profile analysis, the QC sample was closely gathered together in the PCA score scatter plots both in the positive and negative modes, which suggested the great stability of the instrument and the acquiring methods . Meanwhide novo lipogenesis,” is induced by the thyroid hormone, and then produces fatty acids, triacylglycerol, and VLDL  promoting thyroid hormone synthesis through purine metabolism; (ⅲ) acting on the tryptophan metabolism to improve the declined energy metabolism and thyroid hormone levels. These common potential biomarkers might reflect the characteristics of the body after the administration of the four typical “hot” property herbs, in other words, these biomarkers represent the “hot” property of Chinese medicines.In this study, we firstly reported that all of the four typical “hot” property herbs could ameliorate the hypothyroidism model to different degrees, whereas no significant amelioration effect was noted in the four typical “cold” property herbs. Moreover, a metabolomic strategy combined with network analysis was successfully performed and well interpreted the mechanism of the four typical “hot” property herbs on hypothyroidism based on the theory of “cold and hot” properties of the TCM. Specifically, the four typical “hot” property herbs might influence hypothyroidism by (ⅰ) acting on the PI3K-Akt signaling pathway, Ca


Differential DNA methylation is a feature of numerous physiological and pathological processes. However, the extent to which single-base cytosine methylation modifies cellular responses to various stimuli has not been well characterized. In this study, we carried out a systematic analysis of methylome data derived from human blood and immune cells and constructed the ImmuMethy database. ImmuMethy allows interrogation of DNA methylation plasticity (MPL) at the single cytosine level. MPL, which refers to the variability of DNA methylation, is quantitatively measured in multiple ways, such as quartiles and standard deviations. ImmuMethy comprises over 36 000 samples from the Human Methylation450 and MethylationEPIC BeadChips platforms and provides multiple applications, such as an overview of methylation status and plasticity, differential methylation analysis, identification of methylation markers and sample stratification. An analysis of all datasets revealed that DNA methylation is generally stable, with minimal changes in beta values. This further supports the characteristics of DNA methylation homeostasis. Based on the beta value distribution, we identified three types of methylation sites: methylation tendency sites, unmethylation tendency sites and dual tendency or nonbiased methylation sites. These sites represent different methylation tendentiousness of DNA methylation across samples. The occurrence of multiple methylation tendencies in a site means split methylation, which generally corresponds to high MPL. Inverted methylation tendencies from methylation tendency sites to unmethylation tendency sites, or vice versa, represent strong differential methylation in response to conditions. All these sites can be identified in ImmuMethy, making it a useful tool for omics-based data-driven knowledge discovery.Database URL: http://immudb.bjmu.edu.cn/immumethy/ DNA methylation is a major epigenetic modification involving the addition of a methyl group to the C5 position of cytosine to form 5-methylcytosine (5mC). In mammals, 5mC is predominantly seen at cytosine-phosphate-guanosine (CpG) dinucleotides, although methylation events can also occur in non-CpG contexts . In mostHigh-throughput technologies, such as microarrays and next-generation sequencing (NGS), have been widely used to identify differentially methylated sites in various contexts, including normal human development, aging, tumorigenesis, autoimmune disorders, neurological disorders, immunodeficiency, species evolution, environmental adaptation and genetic and epigenetic diseases. As a result, there is an abundance of epigenome-wide DNA methylome data available. Therefore, the systematic collection, integration and utilization of these data will undoubtedly contribute to the field, enabling an overview of gene methylation levels under various conditions, identification of the underlying regular methylation patterns and emerging new biological insights into methylation roles. In addition, re-evaluation of methylation biomarkers, such as cell type-specific and disease-associated methylation biomarkers, is also an important task for omic big data usage. Database construction is an excellent way to share large datasets and promote data usage in further experimental research. Currently, several methylation databases have been built, including MethDB , 4, PubMhttp://immudb.bjmu.edu.cn/immumethy/), which aims to collate available methylome data for human blood and immune cells. Integration of mouse methylation data will be considered in subsequent versions of ImmuMethy once sufficient data have been compiled. It currently comprises over 36 000 samples from both the Human Methylation450 (or 450k) and MethylationEPIC (or 850k) BeadChips platforms.Blood and immune cells are often used in methylation studies due to the ease of sample collection, resulting in abundant methylome data for these cells. Although there are some databases, such as epigenome BLUEPRINT , 10 and ImmuMethy provides data for DNA methylation at single cytosine bases. It can provide information on DNA methylation levels in human blood and immune cells derived from different disease states and tissue sources. ImmuMethy emphasizes a global view of methylation profiles due to the large sample size. Samples are categorized into different study datasets based on sample source, cell type and disease state. Therefore, the database can illustrate methylation profiles and plasticity and permits examination of the dynamic changes in DNA methylation that occur under various experimental conditions. In addition, further analyses, such as differential methylation profiles, methylation marker evaluation and tracing experimental conditions based on the methylation level, can also be carried out using the database.https://support.illumina.com/array/array_kits/infinium_humanmethylation450_beadchip_kit/downloads.html and https://support.illumina.com/array/array_kits/infinium-methylationepic-beadchip-kit/downloads.html. Chromosomal coordinates of methylation sites were converted from the human genome hg19 to hg38 assembly using the liftOver program (https://genome.ucsc.edu/cgi-bin/hgLiftOver). The 450k array interrogates over 450 000 methylation sites (predominantly CpG dinucleotides) across the human genome at a single-nucleotide resolution, covering 99% of reference sequence (RefSeq) genes and comprises the most abundant sample type across all current methylation platforms The 850k array covers the majority of the sites from the 450k array but adds 333 265 CpGs located in enhancer regions; therefore, it interrogates a total of over 850 000 CpG sites for methylation status assessment (All data related to the Infinium Human Methylation450 (450k) and MethylationEPIC (850k) BeadChips, as well as the platform annotation files, were downloaded from the GEO  and Arrasessment .http://www.r-project.org/) package “minfi”  algorithm . During quality control, samples comprising abnormal beta values less than zero, larger than 1 or without a beta value were removed.http://www.r-project.org/) software environment was used to calculate the minimum (Min.), first quartile , median, third quartile  and maximum (Max.) values. To systematically estimate the variability in DNA methylation or methylation plasticity (MPL), we calculated the standard deviation and quantile range to quantify the amount of dispersion or spread of beta values. In this study, we used two types of quantile ranges: “quantile range (Q75–Q25)” or the interquartile range (IQR), which is the difference between the 75th and 25th quantiles of the beta values, and “quantile range (Q95–Q5),” which is the difference between the 95th and 5th quantiles of the beta values. Quantile ranges are robust to outliers and allow enough samples to support methylation variability effectively and avoid potential negative biases due to experimental and probe design. The relationships among the three measures were compared by using the Pearson correlation method.For the normalized beta values in each study dataset, the “fivenum” function in the R  curve, which connects all sample ratios on the https://www.rdocumentation.org/packages/fpc/versions/2.2-9/topics/pamk) with default parameters was further used to determine the numbers of clusters. The function uses the most common k-medoids clustering method “pam” algorithm to perform partitioning around medoids clustering and returns a suggested number of clusters.Duda–Hart tests were performed using the “dudahart2” function in the flexible procedures for clustering (fpc) package  to deterP-value was less than or equal to 0.05 if more beta values were located in the range of 0.5–1 for MTS or in the range of 0–0.5 for UTS. In contrast, a site was considered a Dual Tendency methylation Site (DTS) or nonbiased methylation site (NTS) when the P-value was larger than 0.05. The methylation tendency of a queried site can be clearly illustrated via a methylation profile curve when the peak will be located mainly on the right (MTS), left (UTS) and middle or both of the left and right (DTS) sides of the curve with almost equal sample proportions.Methylation tendency refers to the overall tendency of a cytosine site to be methylated or unmethylated in the sample populations. Because a beta value of ∼0.5 indicates balanced signal levels between the methylated and unmethylated probes based on the formula of beta value calculation, beta value ranges of 0.5–1  and 0–0.5  reflect biased methylated (0.5–1) or unmethylated (0–0.5) probe signals, respectively. We used a chi-square goodness of fit test with the R function “chisq.test” to determine if there was a statistically significant difference in beta value distribution in the two data regions. We considered a site as a methylation tendency site (MTS) or an unmethylation tendency site (UTS) when the U-test  was automatically conducted in response to the user’s operation with the function “MannWhitneyUTest” (http://commons.apache.org/proper/commons-math/javadocs/api-3.0/org/apache/commons/math3/stat/inference/MannWhitneyUTest.html). For offline analysis, the R function “wilcox.test” was used for the Wilcoxon rank-sum test. The null hypothesis of the Wilcoxon test is that the two populations have the same distribution with the same median. A P-value less than or equal to 0.05 is generally statistically significant. However, considering the multiple comparison problem and hundreds of thousands of cytosine sites from both platforms, individual online tests with a P-value  < 1.00E-7 are suggested to be significant enough for differential methylation levels according to the widely used Bonferroni correction method. The formula for a Bonferroni correction is as follows:αoriginal indicates the original α level and n represents the total number of comparisons or tests being performed. In addition to a Bonferroni correction, a less stringent Benjamini and Hochberg’s false discovery rate controlling method, which takes both the total number of tests and the individual P-value’s rank into consideration, was also used for the differential methylation analysis. The R function “p.adjust” returns the adjusted P-value s with the “bonferroni” and “BH” (aka “fdr”) methods for multiple comparisons.For an online differential methylation analysis among methylation sites, the Mann–Whitney Some transcription factors (TFs) recognize methylated DNA and play an important role in gene regulation. Therefore, we integrated TF binding information into ImmuMethy. Data on the interactions between methylated DNA and TFs were downloaded from the MeDReaders database , which cImmuMethy was implemented based on the Client Browser/Web Server/Database Server three-tier architecture. It was built with the model–view–controller development framework. Servlet was used as a controller, with Java server page as a view component and Java Bean class as a model. The data were stored and managed by a MySQL relational database (version 8.0.17). The current version of ImmuMethy runs on an Apache Tomcat web server (version 6.0.45). It accesses the database using Java Database Connectivity.ImmuMethy uses beta values to quantify methylation levels. To include as many experimental conditions as possible for the downstream DNA methylation analysis, two types of beta values are reported: the uniformly processed values, which were recalculated in this study from the raw IDAT format files, and the preprocessed beta values, which were previously determined by the data submitter for which no IDAT files were provided. ImmuMethy integrates blood and immune cell data from 308 studies with different sample tissue sources, cell types and disease states , involving 36 272 samples from the two platforms, 450k and 850k . Each ce+ T cell class is separated into several subclasses, including naive CD4+ T cells, memory CD4+ T cells, central memory CD4+ T cells and effector memory CD4+ T cells. These subclasses are derived from different human peripheral blood samples representing different disease states, such as naive CD4+ T cells in peripheral blood derived from healthy individuals  or systemic lupus erythematosus (SLE) patients (PB_CD4Tnaive_SLE).For example, for the 450k platform, the CD4For the 450k platform, there were a total of 32 234 nonredundant samples, including 12 193 (37.83%) samples with uniformly processed beta values and 20 041 (62.17%) samples with preprocessed beta values. These samples were divided into 236 study datasets based on tissue source, cell type and disease state . For theImmuMethy provides a user-friendly interface to query DNA methylation at single cytosine sites. It supports gene symbols/aliases (e.g. CD4), methylation site IDs (e.g. cg00000029) and chromosome positions (e.g. 16 000 000–16 010 000 on chromosome 1) as search terms as output, both beta values and methylation profile queries are provided. Step 1. Select a platform (Human Methylation450 and MethylationEPIC BeadChips platforms). Look-up tables are provided for the two platforms provided below the click buttons .Step 2. Enter the query . As an eStep 3. Select study datasets of interest . SelectiOption 1. Click on the submit button at the bottom of the query web page to retrieve beta values. This result page is shown in Option 2. Click on the “Show” button to see the methylation profiles , which aP-values are directly from the Mann–Whitney U-test, which is automatically conducted in response to the user’s operation. The lower the P-value, the greater the statistical significance of the observed differential methylation levels. However, statistical comparisons are conducted only for uniformly processed data.Step 4. For Option 2, click on the “p values” button to retrieve statistical comparisons between the site in the indicated row and that site in the other rows . The P-vStep 4. Click on the “Download” button seen in Step 4. Click on the “see more” button if there are TFs predicted to bind the indicated cytosines with either high or low methylation levels. The TF–DNA interaction information, such as tissue source, predicted binding sequence, CpG site in the sequence and its methylation level, is shown. The information is directly derived from the MeDReaders database (see below for more details).Step 5 . New profiles were added to the current methylation profiles  sites from the 450k platform and 3 027 610 210 (16.67%) sites from the 850k platform. In addition, there were 93 455 099 methylation profiles, with 43 210 568 (46.24%) profiles from the 450k platform divided into 89 study datasets and 50 244 531 (53.76%) profiles divided into 58 study datasets from the 850k platform , all of The main aim of ImmuMethy is to facilitate a global study of DNA methylation across samples of immune cells to identify differential methylation patterns and the underlying mechanisms. In addition to the storage of a huge number of methylation datasets, ImmuMethy allows several applications as follows. In addition, we performed an integrative analysis to support big data-driven knowledge discoveries.+ T cells , methylation sites located in CGIs, first exons and TSS200 (0–200 nt upstream of the TSS) generally show a hypomethylated status, whereas sites in gene bodies , 3ʹ UTRs and shelves (2–4 kb from CGIs) tend to be hypermethylated , quantile range (Q95–Q5) and standard deviation as MPL scores, were used to quantitatively measure MPL for the uniformly processed data. Therefore, MPL levels of each site can be evaluated and compared according to these scores. Larger MPL scores indicate larger variability and higher plasticity. A cytosine site with extreme plasticity means it is highly or even fully methylated in some samples, whereas it shows very low or even no methylation in some other samples.+ T cells ” is observed in these datasets , suggestIn ImmuMethy, in addition to the above measures, the r-beta curve can directly illustrate MPL. High plasticity results in high quantile ranges and standard deviations of beta values and is generally indicated by a wide-shaped r-beta curve or a curve with multiple peaks. In contrast, low plasticity corresponds to low variability and is indicated by a narrow, single-peak r-beta curve.+ T cells, CD8+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, dendritic cells and neutrophils, as well as their subsets (if methylome data are available). Therefore, differential methylation among different cell types or diseases can be analyzed by comparing their methylation profiles. A shift in the peak of the methylation profile in combination with the statistical tests can help to effectively identify differentially methylated sites.ImmuMethy integrates different but functionally related studies into one study dataset; therefore, for differential methylation, it aims to illustrate large differences at a greater scale based on methylation distribution but does not focus on minor differences with very small beta value changes under specific conditions, as many of the source studies do. ImmuMethy comprises numerous blood cells subjected to various experimental conditions (including hundreds of disease states) and all main immune cell types, including B cells, CD4ImmuMethy contains data from large numbers of control samples from normal individuals, such as normal blood, leukocytes, lymphocytes and peripheral blood mononuclear cells PBMCs; . These aThe site cg02489202 , for example, was previously reported as being the most significant Parkinson’s disease (PD)-related CpG . This si+ and CD8+ T cells, whereas lowly methylated in B cells , which represents a specific marker gene in B cells, they are highly methylated in human CD4 B cells . More exImmuMethy pays much attention to the most reliable differential methylation using large datasets based on beta value distribution. There was no overlap between any two r-beta curves, and the farther the peak distance was, the greater the difference in methylation intensity was.However, because ∼62.29% (147/236) and ∼15.94% (11/69) of all study datasets for the 450k and 850k platforms, respectively, are not uniformly processed , only thIn the current study, we classified methylation sites into three categories based on the biased beta value distribution . This classification is based on the sample population but not individual levels. The MTS and UTS reflect that the majority of the sites tend to be methylated and unmethylated, respectively, although unmethylated status for MTSs and methylated status for unmethylated tendency sites might also be observed in certain circumstances across samples. We did not use “hypermethylation” or “hypomethylation” to describe the tendencies in that these terms only represent an increase or decrease during DNA methylation comparison and do not consider beta value distribution in a sample population. However, for NTSs, it means that the cytosine sites are partially methylated with methylation levels adjacent to 0.5, or that similar sample proportions are observed to be, respectively, methylated and unmethylated across samples.P-values were ∼0.37 and ∼0.08 for cg01836137 and cg00454305, respectively, during the tendency test in normal human peripheral blood cells . Therefore, both of the sites belong to nonbiased methylation.For example, as shown in These three types of methylation sites can be easily discerned in ImmuMethy. More examples of MTSs, UTSs and NTSs are listed in For the 450k platform based on our analysis, ∼54.67% (21 763 967/39 811 984), ∼42.92% (17 088 023/39 811 984) and only ∼2.41% (959 994/39 811 984) of all sites in the 82 study datasets with sample sizes larger than 3 were identified to be MTSs, UTSs and NTSs, respectively. This result means that ∼97.59% of the total cytosine sites present a significant tendency toward methylation or unmethylation. Furthermore, when we set a strict filter condition that all (100%) of the beta values of a site across samples were required to be larger than 0.5 for MTS or less than 0.5 for UTS, the result revealed that there were still ∼83.71% of all cytosine sites that showed strict tendentious distribution with ∼45.61% (18 159 128/39 811 984) of MTSs and ∼38.10% (15 170 118/39 811 984) of UTSs, respectively.Similar results were also observed for the 850k platform. There were ∼62.30% (25 904 134/41 576 916), ∼33.51% (13 930 735/41 576 916) and ∼4.19% (1 742 047/41 576 916) of all sites that were identified to be MTSs, UTSs and NTSs, respectively. Under the above strict conditions, ∼56.80% (28 540 379/50 244 531) and ∼31.59% (15 874 047/50 244 531) of all sites presented methylation tendencies and unmethylation tendencies, respectively.+ T cells . For example, among the MTSs  in the normal peripheral B cells of the 450k platform , there w T cells  and CD8+ T cells  from the T cells  from theAn interesting finding was that similar results were also observed when the preprocessed beta values were used to calculate the methylation tendency. For example, among the 23 study datasets that had both preprocessed and uniformly processed data values, ∼97.33% (10 606 101/10 897 038) of all shared sites were identified to have the same methylation tendency. This result suggested that although the preprocessed beta values could not be used for precise differential methylation among different datasets, they could still be used for a general quick determination of methylation tendency.Through ImmuMethy, methylation tendencies can be identified through a quick view of methylation profiles, which can be further checked based on the online downloaded beta values. A large sample size will make the methylation profile curves smoother and make it easier to discern distributional tendencies. The above results suggest highly biased tendencies of methylation distributions and further support a general limited variation in DNA methylation level in response to various experimental conditions. This means the absolute difference of methylation levels for differentially methylated sites is generally small; therefore, differential methylation should often occur in the sites with the same tendencies.+ T cells. Interestingly, methylation profiles from highly plastic sites generally have multiple peaks; however, relatively stable methylation status can still be observed for each peak of the same site ” as a measure of MPL (see section “Discussion”), that is, MPL score, ∼0.06035% of sites (293/485 512) have an MPL score ≥ 0.5 and ∼0.8401% of sites (4079/485 512) have an MPL score ≥ 0.3 in normal peripheral blood CD4ame site . For exaame site , whereasame site . This suTo systematically analyze the data distribution of beta values across samples for each site, we used the R package “fpc” with k-medoids algorithms to detect data homogeneity and further split beta values into the optimal clusters. As a result, ∼97% (450k) and  ∼ 99% (850k) of all sites in all datasets are homogeneous, which means that only one cluster for these sites was identified. This suggests that there are only a few cytosine sites that have obvious multiple peaks in their methylation profiles. For example, only 23 740 cytosine sites in human normal blood cells  have multiple clusters (or groups) based on the beta value distribution. For the sites in + T cells  and male (n = 2306) blood samples, respectively. Therefore, two typical aggregated distributions of beta values at the site represent the underlying differential methylation in female and male populations , cg10482512 (rs2021033/MAF = 0.466482) and cg00211215  in Split methylation represents high plasticity and highly differentiated methylation. For different split methylation sites, the underlying mechanisms need further investigation because they represent real biological effects, whether it comes from genetic polymorphisms, gender differences or something else.DNA methylation status can influence methylation-mediated protein–DNA interactions. In addition to the classical methyl-CpG binding domain (MBD) proteins, many TFs lacking an MBD, such as Kaiso and CCAAT/enhancer-binding protein-α (CEBPα), can interact with methylated DNA to mediate methylation-mediated biological processes . Therefohttps://www.ncbi.nlm.nih.gov/snp/?term=rs12728349). As expected, the methylation profiles of cg00439656 in many study datasets in ImmuMethy, such as whole blood cells and PBMCs, comprise three typical peaks centered on approximately 0.89, 0.45 and 0.04 on the x-axis, representing methylated (genotype CC), partially methylated (genotype CT) and unmethylated (genotype TT) status, respectively. The high methylation status of the site is predicted to bind CTCF (CCCTC-binding factor) and RAD21 (RAD21 cohesin complex component) in H1-hESCs, although low methylation may also bind CTCF in HCT116 cells  from the gene ARHGAP30 (Rho GTPase activating protein 30) is a site of split methylation owing to SNPs with high MAFs in different populations ” in this study. In contrast to the IQR, the standard deviation takes each value in a dataset into account and represents the average amount of variability. A high standard deviation indicates that values are generally far from the mean, whereas a low standard deviation represents that values are close to the mean. In this study, we found that beta values are frequently skewed with outliers, particularly for sites with split methylation or sites with high plasticity. In addition, we find that the distribution of “quantile range (Q75–Q25)” is more similar to standard deviation than that of “quantile range (Q95–Q5),” although both quantile ranges are closely correlated to standard deviation in all of the study datasets. Therefore, the “quantile range (Q75–Q25)” could represent a good choice as a quantitative measure of MPL, i.e. MPL score, particularly when enough samples are required to support MPL.However, neither the “quantile range (Q75–Q25)” nor the “quantile range (Q95–Q5)” represent absolute plasticity, as the extreme difference or the range  does. For example, for the 450k platform, there are ∼75%, ∼50%, ∼37%, ∼28%, ∼17%, ∼11% and ∼7% of all sites with extreme differences larger than or equal to 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5, respectively, whereas for the 850k platform, there are ∼70%, ∼46%, ∼30%, ∼20%, ∼9%, ∼4% and ∼2% of all sites with extreme differences larger than or equal to 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5, respectively. These ratios are much higher than those obtained by other measures, such as “quantile range (Q75–Q25)” and “quantile range (Q95–Q5).” However, the extreme difference method is not able to distinguish an outlier from true values that are too large or too small and offers insufficient sample support. For example, for highly plastic sites with the same MPL score, the use of “quantile range (Q75–Q25)” will obtain more sample (50% of the data outside the range) support variability than the use of “quantile range (Q95–Q5)” (only 10% of the data outside the range); however, there are none of the data outside the range to support the plasticity when the range is used. Therefore, there are multiple ways to measure methylation variability, depending on how large the expected variability and the amounts of data samples to support the variability. Therefore, the values from the extreme difference are not currently integrated into ImmuMethy; however, users can still perform the analysis based on the downloaded data.Methylation homeostasis is used to describe the finely tuned balance between de novo methylation, maintenance methylation, active demethylation and passive dilution of DNA methylation during cell replication . Cells n+ T cells , we performed a preliminary analysis, and the results also support the current conclusions about limited DNA MPL (data not shown).Arrays and NGS are dominant tools in the epigenome-wide association study field. WGBS has the greatest advantage in the quantitative determination of the methylation states of all CpG sites in the human genome, but it is very expensive. Reduced representation bisulfite sequencing is more cost effective, but datasets based on this protocol still await accumulation. All current data are array-based, as the sample sizes are much larger than those of HTS. However, studies have revealed that the methylation values determined by arrays and HTS platforms are highly correlated , with thHigh reproducibility at the 450k CpG sites in the 850k platform has been observed . HoweverTraditionally, an individual CpG site is classified as fully methylated (close to 1), unmethylated (close to 0) and partially methylated (close to 0.5) based on a variable cut-off of DNA methylation levels. However, different studies use different cut-offs to define methylation status. For example, a CpG site is classified as methylated and unmethylated based on DNA methylation levels higher than 80% and less than 20%, respectively, and others (20% to 80%) are classified as partially methylated , 47. In In this study, we divided methylation sites into three categories: MTSs, UTSs and NTSs. The classification aims to reflect the tendency of methylation level distribution at the population but not individual levels. Therefore, although the categories are closely associated with differentiated methylation status, the classification does not determine whether a site is fully methylated or fully unmethylated under a specific condition. This is because of the dynamic characteristics of DNA methylation and the various functional states of single cells contained in bulk samples. Therefore, even for MTSs, an unmethylated status may also be observed under certain conditions at some cytosine sites. In contrast, for unmethylated sites, methylation may occur under some other conditions. Bulk samples comprise multiple single cells with various methylation statuses, even for the same cell types; therefore, the methylation tendency reflects an overall probability of being methylated or unmethylated.In contrast to a previous study , which pMPL reflects the dynamic change in methylation levels in response to conditions. Low plasticity indicates a small change in methylation levels across samples, whereas high plasticity indicates a large change in methylation levels. Inverted methylation tendencies, such as from unmethylation to methylation, or vice versa, indicate higher plasticity than that of differentiated methylation within the same tendency sites. Plasticity is tightly associated with layering, and high plasticity provides the chance of sample division or stratification through component deconvolution. These divisions, as well as their further subdivisions, lead to sample heterogeneity, which implies dissimilarity and diversity. Therefore, plasticity explains the underlying mechanism of heterogeneity. Therefore, the plasticity analysis facilitates “marker” evaluation, which implies homogeneity and stability . For example, DNA methylation markers imply that they are lowly plastic.In our previous studies, we performed the quantitative measurement of gene plasticity, which was referred to as expressional gene plasticity , and potbaac020_SuppClick here for additional data file.


In particular, the isotherm diagrams for the AlxGa1−xAsySb1−y quaternary alloy at low temperatures were calculated . The AlxGa1−xAsySb1−y quaternary alloy was formed from four binary compounds such as GaAs, AlAs, AlSb, and GaSb, all with direct bandgaps. The regular solution approximation was used to find the quaternary isotherm diagrams, represented in four linearly independent equations, which were solved using Parametric Technology Corporation Mathcad 14.0 software for different arsenic and antimony atomic fractions. The results support the possible growth of layers via liquid-phase epitaxy in a range of temperatures from 500 °C to 350 °C, where the crystalline quality could be improved at low temperatures. These semiconductor layers could have applications for optoelectronic devices in photonic communications, thermophotovoltaic systems, and microwave devices with good crystalline quality.This work presents the theoretical calculation of isotherm diagrams for quaternary alloys of III–V semiconductor compounds with the form III The world is currently living a technological revolution in electronic communications, where silicon has been a fundamental aspect of technology for a long time. However, when the operation of a laser diode was demonstrated in 1960 ,3,4. GaAxGa1−xSb alloy were obtained via LPE, which were tuned with a wavelength of 1.55 µm (0.80 eV), where the silica fibers have their lowest losses [On the other hand, III–V semiconductor compounds have generally been obtained using methods such as MOCVD , MBE (molecular beam epitaxy), and LPE (liquid-phase epitaxy). MOCVD and MBE might have some disadvantages due to the use of highly toxic gases and high operating costs in their growth systems, respectively. LPE is an easy method to operate, with low cost, and it is widely used for the growth of heterostructure semiconductor devices ,6. Avalat losses . Figure t losses . The lowxGa1−xAsySb1−y quaternary alloy lattice-matched to GaSb substrates is an attractive material in optoelectronic devices such as photodetectors and thermophotovoltaic systems [0.6Ga0.4As0.05Sb0.9 layer lattice-matched to GaSb substrates was also grown via LPE at 635 °C to obtain a p–n junction. Toginho et al. reported a study of the photoluminescence properties in undoped and Te-doped AlGaAsSb alloys synthesized by MBE [xGa1−xAsySb1−y quaternary alloys [xGa1−xAsySb1−y quaternary alloy via LPE at temperatures above 700 °C [a−2.5 (a = lattice parameter) in semiconductors to calculate the mixing enthalpies with good results, principally in AxB1−xC ternary alloys with x = 0.5 [xGa1−xAsySb1−y quaternary alloy showed growth temperatures above 500 °C, which could produce structural defects during layer deposition at high temperatures. This work presents the isotherm diagrams obtained for AlxGa1−xAsySb1−y quaternary alloy, indicating the possible growth of layers via liquid-phase epitaxy lattice-matched to GaSb substrates at temperatures below 500 °C. The theoretical analysis of isotherm diagrams was conducted using the regular solution approximation, which has been extensively studied and proven in quaternary alloys such as AlGaPAs [In particular, Al systems . The imp systems . In anot systems , a type d by MBE . On the y alloys , while Pe 700 °C . However x = 0.5 . Then, d AlGaPAs ,15. The  AlGaPAs .One of the most important thermodynamic models to calculate isotherm diagrams for a quaternary system is the regular solution approximation proposed by Jordan and Ilegems . This thi , in the solid and liquid phase. Furthermore, ix is the atom fraction of component i in the solid [−1), R the universal gas constant (8.314472 J·K−1·mol−1), T is the temperature (K), and It is important to mention that the equations presented below were obtained from previous studies ,15, in wThen, the Gibbs free energy for a quaternary solid solution according to Equation (1) can be expressed as follows:ij can be obtained; therefore, Equation (4) can be represented in the following form:From the mass balance, the relationships of x, y, and xThen, the expression for the Gibbs free energy of mixing from four pure binary compounds,  Ilegems , can be tal data ,14,17. TWe can obtain the explicit relationships for xIII1−xVyV1−y were obtained, which were used in particular for the AlxGa1−xAsySb1−y quaternary alloy to evaluate its possible growth via LPE at low temperatures, according to the following equations:Therefore, the explicit equations of the solid–liquid equilibrium for the form IIIMoreover, Considering that the fusion enthalpy can be written in terms of the fusion entropy xGa1−xAsySb1−y quaternary alloy, the interaction parameters for the binaries, fusion entropies, fusion temperatures, and interaction parameters of the ternaries were searched in the literature, assigning the conditions Al = 1, Ga = 2, As = 3, and Sb = 4 [Lastly, for the Ald Sb = 4 ,15,17,18−8 for 350 °C to 10−5 for 500 °C. On the other hand, as the aluminum, arsenic, and antimony atomic fractions were small, the quaternary alloy was in the gallium-rich region as the solvent, while the other elements were solutes in the metallic solution. The aluminum fraction values in xGa1−xAsySb1−y quaternary alloy, the aluminum atomic fraction would continue to decrease; accordingly, the antimony and arsenic atomic fractions would continue to increase until reaching the GaAsSb ternary alloy. Furthermore, when the temperature decreased, the arsenic fraction also decreased until reaching the AlSbGa ternary alloy, whereas the GaAsSb ternary alloy could be obtained upon increasing the temperature.xGa1−xPyAs1−y quaternary alloy (see graph 1 in their study) [In r study) . This be−4 mol, along with an aluminum atomic fraction in the liquid of 0.002 mol, and an antimony atomic fraction in the liquid of approximately 0.03 mol . −5), with values for the atomic fractions of aluminum and antimony in the liquid of 0.0025 mol and 0.01 mol (450 °C), respectively. −6 mol for the arsenic atomic fraction in the liquid, with values for the atomic fractions of aluminum and antimony of 0.0035 mol and 0.004 mol, respectively, in the liquid at 400 °C. Lastly, −7 mol for the arsenic atomic fraction in the liquid, with values for the atomic fractions of aluminum and antimony of 0.0041 mol and approximately 0.001 mol , respectively, in the liquid at 350 °C.Therefore, it was observed again that, when the temperature decreased, the arsenic atomic fraction continued to decrease, approximating the AlGaAsSb quaternary alloy to the AlGaSb ternary alloy, as mentioned in xGa1−xAsySb1−y quaternary alloy were calculated using a regular solution approximation for possible growth of layers at temperatures below 500 °C, via liquid-phase epitaxy. A similar behavior in AlxGa1−xPyAs1−y quaternary alloy has been reported, agreeing with the results obtained in our study and suggesting their accuracy. Isotherm diagrams showed that, when the temperature increased from 350 °C to 500 °C, the aluminum atomic fraction decreased from 0.0041 mol to 0.002 mol, whereas the arsenic and antimony atomic fractions increased. On the other hand, graphs of the atomic fractions in solids x and y, as a function of the arsenic atomic fraction in the liquid, showed that, at low temperatures, the x fraction increased, while the y fraction decreased. Furthermore, the arsenic atomic fraction maintained a very small value until reaching the AlGaSb ternary alloy.Isotherm diagrams of the Al


Assessment of bimanual movements, which are frequently impaired in children with cerebral palsy, is highly challenging in clinical practice. Instrumented measures have been developed to evaluate and help to understand impaired upper limb movement during bimanual tasks in these children. The aim of this review was to report instrumented measurement tools  used for bimanual task movement analysis, and the metrological properties of the measures in children with cerebral palsy.A systematic review was conducted (Prospero CRD42022308517). PubMed, Web of Science, Cochrane and Scopus databases were searched with relevant keywords and inclusion/exclusion criteria. Article quality and biomechanical methods were evaluated with a customized scale and metrological properties with the COSMIN checklist.In total, 452 children, mostly with unilateral cerebral palsy, mean age 10.9 (SD 3.2) years, underwent quantitative bimanual assessments in the 31 included studies . The tools used were 3D motion analysis (n = 26), accelerometers (n = 2), and other instruments  (n = 3). Children performed 1–5 bimanual tasks in laboratory settings, mostly activities of daily living or game scenarios. Analyses focused mostly on spatiotemporal variables, 6 of which were specifically developed for bilateral measures . These instrumented measurements had moderate to good discriminant and convergent validity, but reliability and responsiveness assessments were lacking.A large number of quantitative bimanual assessments involving different tools, bimanual tasks and specific variables developed to evaluate bimanual function were found. Development of other relevant variables and validation of these tools are needed to further determine their usefulness, both as research outcomes and to guide therapies in clinical practice. Future research, involving younger children and real-life assessments, will improve our understanding of bimanual function in children with cerebral palsy.The online version contains supplementary material available at 10.1186/s12984-023-01150-7. Most daily activities require both hands . For children with cerebral palsy (CP), accomplishing these daily activities can be challenging because of the motor deficit, limited range of motion, lack of motor control and spasticity of their impaired upper limb (UL) –3. BimanInstrumented measurements of UL movements are being developed . 3D moveBimanual movement assessment with these quantitative tools is promising, increasingly used and required by both the research and clinical communities. However, a review of available tools, protocols, variables and metrological validation is lacking. Such a review would improve the understanding of impaired movements and would help to more accurately tailor interventions in children with CP. Moreover, few measures have been validated, although this is necessary to determine what is really measured.The purpose of this systematic review was therefore to report instrumented tools  and the metrological properties of the measures used to evaluate bimanual movements in children with CP. This systematic review aimed to (1) describe the types of instrumented tools used; (2) identify how data were collected and analyzed (protocols and variables); and (3) report the available evidence of validation of these measures  and (4) identify relevant outcomes for research and clinical application. Based on this review, we wished to draft a set of recommendations for the instrumented measurement of bimanual movements for future clinical development and research.This systematic review is reported according to the PRISMA guidelines. A PRISMA checklist was completed and the review protocol was published in Prospero (CRD42022308517).Searches were conducted in English in the following databases: PubMed (1996 to March 2022), Scopus (2004 to March 2022), Cochrane Library (1995 to March 2022) and Web of Science (2004 to March 2022). To ensure the search was exhaustive, the following combinations of keywords were used: (1) keywords relative to children and adolescents: “child”, “adolescent”, “teen”, “infant”, “baby”, “newborn”; (2) “upper limb”, “upper extremities”, “upper body”, “arm”, “hand”, “bimanual”, “interlimb”; (3) “movement”,”motion”, “motor”, “biomechanical”, “kinematic”, “instrumented measurement”, “inertial”, “sensors”, “accelerometer”, “technologies”, “spatiotemporal”, “temporo-spatial”, “smoothness”, “fluidity”, “trajectory”; 4) “Cerebral Palsy”, “hemiplegia”, “hemiparesis”, “quadriplegia”, “tetraplegia”, “stroke”, “cerebrovascular accident”  Design: full papers; 2) Participants: children and adolescents 0–18 years old with uni- or bilateral cerebral palsy; (3) Construct: exploration of bimanual movements  during symmetrical or asymmetrical tasks; (4) Type of instruments: quantitative instruments ; (5) Outcome measures: quantitative/instrumental measures of UL characteristics   score risk of bias checklist 2017 , 22 was Number of children and adolescents and demographics  were extracted.The following motion analysis data were extracted: type of instruments used, model; number, size and position of markers or sensors; sampling frequency (Hz); biomechanical model; specific algorithms and data analysis; protocols performed; starting position of the child; number of trials/sessions; velocity; duration of the protocol. The metrological properties  were collected for each study. Construct validity was evaluated, including convergent validity (correlation with a gold standard measure or comparison with other outcome measurement instruments), and discriminant validity (ability to discriminate children with CP from another population). Articles in which children with CP were compared to TDC were considered to assess discriminant validity. Within/between rater/session reliability  was evaluated with the measurement error and responsiveness (ability to detect a change before and after therapy). Studies that determined pre- and post-therapy effects and did not specifically assess the responsiveness of the measure were not considered to assess responsiveness.The following outcome measures were explored: spatiotemporal , kinematic , quality of movement  and actimetry (intensity and time). Variables measuring unilateral movement  and bilateral measures  were distinguished.A descriptive analysis of samples, type of instruments and outcome measures was performed. Quantitative results are expressed as mean (standard deviation [SD]) and categorical results as number (%). We did not conduct a meta-analysis because of the large number of protocols, heterogeneity of samples and varied outcome measures that prevented such an analysis.The initial search identified 2015 papers published since 1995 after removal of duplicates. Following screening of titles and abstracts, 285 articles were deemed appropriate for full text screening. Of the 285 articles reviewed, 254 were excluded. The remaining 31 were included in the review . Seven articles had a score above 80% –29, nine.7. SevenIn total, 452 children and young adults with CP, aged from 2 to 25 years (mean age 10.9 years [SD 3.2]) performed bimanual movements in the 31 studies and were included  [®) [®) [The 31 studies used 3 different types of measurement systems: (1) 3DMA, including optoelectronic systems (Optotracksearch®) , 49–53 ach®) [®) , 29), (2 [®) [®) , 26 and  [®) [®) , Kinect  [®) [®)  or digit [®) [®) ) (Table  [®) [®) . They us [®) [®) , 26.TablMost of the protocols assessed functional tasks Table . This inIn most studies, the starting position was almost the same: sitting on a chair at a table with the knees and elbows flexed at 90° , 42–53. (Table ). Movement time, mean and peak velocity were the most frequently measured. Trunk and upper limb joint (mostly elbow and wrist) kinematics were evaluated in 15 studies [Instrumented measurements were mainly used to calculate spatiotemporal variables during bimanual movements 26/32 studies) –44, 48 , , 42, 48)Among those that used unimanual clinical assessments, no correlation was found between MACS level and movement time or kinematic values . ModeratAmong those that used bimanual clinical assessments, the highest correlation was found between the AHA score and Arm Profile Score (APS) (a kinematic index)  . Low to The mean COSMIN score of the studies that assessed discriminant validity was 8/9 points.Twelve studies compared children and adolescents with uCP and TDC , 43, 48.Children and adolescents with uCP had altered spatiotemporal variable values, with longer movement durations , 42, 48 The mean COSMIN score of the studies that assessed reliability was 14/15 points.Reliability was evaluated in 2 studies, either within-session  or both The mean COSMIN score of the studies that assessed responsiveness was 8/12 points.The main objective of the studies was to evaluate the effect of an intervention but not specifically the responsiveness of the measures. Seven studies performed assessments pre- and post-intervention: Constraint-Induced Movement Therapy (CIMT) , 35, HanSpatiotemporal variables were evaluated both for research and clinical practice. Bilateral variables were used for research , 49–52 aThe outcome measures that underwent the most validity assessments were ROM, MAX, APS, SPARC and IOC  for research purposes , 25. AccThis systematic review of studies that used instrumented assessments of bimanual movements, most often 3DMA, in children and young adults with uCP aged from 2 to 25 years, found that the majority of studies were of good to very good quality (mean Q score 22/32). The protocols evaluated were varied, involving 1 to 5 bimanual tasks that mostly represented activities of daily living or were part of a game scenario. Spatiotemporal variables were most often evaluated, including 6 variables specifically developed for the assessment of bimanual movement: task completion time, goal synchronization, movement overlap time, interlimb coupling, continuous relative phase and asynchrony. The instrumented measures demonstrated moderate to good discriminant and convergent validity, but reliability and responsiveness assessments were lacking. All types of variables discriminated between children and adolescents with CP and TDC and were used to assess UL therapy efficacy with relevant results, therefore demonstrating their potential for clinical and research purposes.A wide variety of instruments has been used to quantitatively evaluate bimanual movements, depending on the objective or variables of interest (ex: accelerometers for actimetry). Most studies used 3DMA, probably because this was the first tool to be developed and because it has been shown to be consistent and accurate for gait analysis, for which it has become the gold standard . HoweverThe protocols lacked standardization, they involved different data collection procedures and, most importantly, different bimanual tasks. The choice of tasks for bimanual movement assessment is a real challenge because of the large variety of movements that can be performed with both ULs . Hung et al. proposed a unique task, ‘the drawer-opening task’ –35, 38, Spatiotemporal variables were the most often analysed. They were mainly used to describe the characteristics of bimanual movements and to evaluate the effect of therapies. Six bilateral variables were specifically developed to assess bilateral movements. Most bilateral variables were spatiotemporal and were designed for the ‘drawer-opening task’ –35, 38 wThe results of this review showed that most measures have not been fully validated. Reliability and responsiveness properties have not been sufficiently assessed, despite their importance. A representative example is the “drawer task”: it was used in 7 studies but only convergent and discriminant validity have been confirmed. Discriminant validity was the most evaluated: all the tools were able to differentiate between children with CP and TDC for all types of variables and bimanual tasks. Moreover, other types of discriminant validity were also evaluated, i.e. comparison between uni and bimanual movements , 49–52 aThis review has some limitations. The lack of standardization of protocols and systems made direct comparisons of results between studies challenging. The quality of the articles included ranged widely (from 13 to 30/32): full descriptions of data acquisition and adherence to biomechanical recommendations  were frequently missing. Furthermore, no studies reported a priori sample size calculations, therefore the statistical power is unknown. Among the articles included, 8 were from the same team –36, 38. This systematic review reported a large number of instrumented measurements of bimanual movements in children with CP, in good- to high-quality studies. These assessments involved different systems  and different bimanual tasks , and spatiotemporal parameters were mostly calculated in bimanual conditions. Some specific variables were developed for the evaluation of bimanual function . However, the metrological properties of these instruments were not fully evaluated, especially reliability and responsiveness. The complementary information provided by instrumented measures in relation to clinical assessments of bimanual function was highlighted. Development of other relevant variables and validation of these tools are required before they can be used as research outcomes or in clinical practice. Studies that involve younger children and real-life assessments will improve our understanding of bimanual function in these children.Additional file 1: Table S1. Search strategy used for systematic review.Additional file 2: Table S2. ‘Design, Participants, Construct, Type of instruments, Outcome measures’ following the COSMIN standard for systematic reviews of Patient‐Reported Outcome Measures [Measures .Additional file 3. Article quality was evaluated with a customized scale.Additional file 4: Table S3. Results of the Q-score  of each article.Additional file 5. Synthesis of recommendations for quantitative bimanual assessments.


Specifically, we used a genomic best linear unbiased prediction (G-BLUP) model to merge phenotypic characterization of 17 morpho-agronomic traits with genetic screening of 13 highly polymorphic SSR markers in a diverse panel of 104 avocado “criollo” “plus trees.” Estimated additive genetic parameters were validated at a 5-year-old common garden trial , in which 22 OP half-sib seedlings from 82 elite “plus trees” served as rootstocks for the cv. Hass clone. Heritability (h2) scores in the “criollo” “plus trees” ranged from 0.28 to 0.51. The highest h2 values were observed for ribbed petiole and adaxial veins with 0.47 (CI 95%0.2–0.8) and 0.51 (CI 0.2–0.8), respectively. The h2 scores for the agronomic traits ranged from 0.34 (CI 0.2–0.6) to 0.39 (CI 0.2–0.6) for seed weight, fruit weight, and total volume, respectively. When inspecting yield variation across 5-year-old grafted avocado cv. Hass trees with elite OP half-sib seedling rootstocks, the traits total number of fruits and fruits’ weight, respectively, exhibited h2 scores of 0.36 (± 0.23) and 0.11 (± 0.09). Our results indicate that elite “criollo” “plus trees” may serve as promissory donors of seedling rootstocks for avocado cv. Hass orchards due to the inheritance of their outstanding trait values. This reinforces the feasibility to leverage natural variation from “plus trees” via OP half-sib seedling rootstock families. By jointly estimating half-sib family effects and rootstock-mediated heritability, this study promises boosting seedling rootstock breeding programs, while better discerning the consequences of grafting in fruit tree crops.Grafting induces precocity and maintains clonal integrity in fruit tree crops. However, the complex rootstock × scion interaction often precludes understanding how the tree phenotype is shaped, limiting the potential to select optimum rootstocks. Therefore, it is necessary to assess (1) how seedling progenies inherit trait variation from elite ‘plus trees’, and (2) whether such family superiority may be transferred after grafting to the clonal scion. To bridge this gap, we quantified additive genetic parameters (i.e., narrow sense heritability— Grafting is an ancient technique used for vegetative propagation, especially in perennial fruit crops. This method, used on woody and herbaceous plants, can improve several agronomic characteristics, such as yield and vigor, as well as tolerance to biotic and abiotic stresses . In fruiFrom a physiological perspective, such confounding factors are pervasive, too. Broadly speaking, grafting affects three main processes at the tree level: uptake and transport of water and nutrients , productPersea americana Mill.) cv. Hass  half-sib interracial seedling rootstocks derived from selected “criollo” “plus trees” . TherefoPhytophthora cinnamomi Rans. Yield and adaptive traits are then left as second and third steps within avocado rootstock breeding. To guarantee that selected rootstocks exhibit a stable phenotypic effect, avocado nurseries in temperate latitudes often rely on clonal rootstock propagation  identify promissory avocado “criollo” “plus trees” as seed donors for rootstock production based on their genetic-estimated breeding values for heritable morphological and agronomic traits. We hypothesize that heritable variation for key traits would enable half-sib seedling rootstock selection from elite “criollo” “plus trees.” Specifically, we have relied on a total of 104 avocado “criollo” trees between 40 and 50 years old, identified as “plus trees” in the province of Antioquia (northwest Colombia). These avocado trees are long-term survivors across different remote agro-ecological zones, so we expect that they may serve as potential donors of seedling rootstocks due to their presumably outstanding adaptability and trait values . These “criollo” avocados had previously been characterized with microsatellite  markers to study their genetic diversity, as well as in terms of their morpho-agronomic traits  estimate additive genetic parameters  across promissory avocado “criollo” “plus trees,” (2) quantify the inheritance of agronomic traits of avocado “criollo” “plus trees” in cv. Hass c traits ,b. Therein situ characterized by taking morphological data, and carrying out their molecular fingerprinting (section below on “microsatellite markers characterization”). Collection of yield data was additionally carried out in these trees during four consecutive years (2014–2017).A total of 104 avocado “criollo” “plus trees” were selected in different remote agroecological zones up to 2,400 m asl of the province of Antioquia, northwest Andes of Colombia, during the years 2014 and 2015 , as well as in their OP half-sib seedling progenies used as rootstocks for the Hass scion (2017–2021).The seeds produced by selected elite avocado “criollo” trees were germinated at greenhouses, and obtained 3-month-old seedlings were grafted with cv. Hass scions. In the year 2015, the cv. Hass grafts were planted at a common garden  at AGROSAVIA’s research station La Selva, located in Rionegro, province of Antioquia, at 2,100 m asl. Morphological data, such as length and diameter of the rootstock, the canopy length and diameter of the scion, and the morphology of the graft scar were collected during the years 2017–2021. The production data of the cv. Hass grafts were collected in the same period from 2017 to 2021. Since half-sib seedling rootstocks grafted with the Hass clone have been in production during five consecutive years in the same environment , we were able to minimize environmental variance and equal phenotypic variance to the rootstocks’ additive genetic component, which is the main benefit of carrying out common garden or provenance tests. These trials intend to neutralize environmental effects over phenotypic expression, by planting diverse genotypes in a single locality . Our setcor.test and heatmap functions in R v.3.4.4 (R Core Team). Complete phenotypic measures were possible for a total of 82 “criollo” “plus trees” from the original sampling.A total of 17 morphological and agronomic traits were measured during the sampling trips of 2014 and 2015 in the avocado “criollo” selected “plus trees,” according to descriptors that are specific for avocado based on the International Plant Genetic Resources Institute (IPGRI). These descriptors included tree, leaf, fruit, and seed traits . Fruit pFurthermore, a total of 22 OP half-sib seedling progenies of 82 elite avocado “criollo” “plus trees” were used as rootstocks for the Hass scion. Grafted trees were characterized at AGROSAVIA’s La Selva research station in terms of their early growth traits , as well as in terms of their production  5 years after grafting (2017–2021). The analyses focused on yield traits because they allowed for a direct comparison with the recorded production traits in the “criollo” “plus trees,” besides the fact that yield traits are of paramount interest for commercial avocado cv. Hass orchards, especially those aiming for exportation markets.Total genomic DNA was obtained from avocado “criollo” leaves based on the extraction method standardized by ® ADN polymerase . Forward primers were labeled with WellRed fluorescent dyes on the 5′ end . The reactions were carried out in an I-cycler  thermo cycler using the following temperature profile for multiplex PCR 2 and 3: an initial step of 1 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 50°C, and 1 min at 72°C, and a final step of 5 min at 72°C. For multiplex PCR 1, the same temperature profiles were used, except for 55°C for annealing temperature that was used instead. The PCR products were analyzed by capillary electrophoresis in the equipment ABI PRISM® 3130 Genetic Analyzer . Allele sizes were estimated in base pairs with a Peak Scanner , allowing for a maximum of two alleles per sample.Three multiplex PCR amplifications were performed in 10 μl volume containing 16 mM (NH4)2SO4, 67 mM Tris-HCl pH 8.8, 0.01% Tween20, 2 mM MgCl2, 0.1 mM each dNTP,0.4 μM of each primer, 25 ng genomic DNA and GoTaqG-BLUP (genomic best linear unbiased predictor) model to quantify genetic parameters  for key agronomic traits across the avocado “criollo” “plus trees” selected as seed donors for the OP half-sib progenies used as rootstocks for the Hass clone. Genetic parameters are a baseline of any breeding program  a distance tree by means of the Neighbor Joining algorithm  enabled the use of the G-BLUP predictor. Hence, we relied on this G relationship matrix to compute the genetic-based G-BLUP model, following The admixed origin of the avocado “criollo” genepool, due to avocado’s protogynous dichogamy, is known to favor an adequately variable Genebank . The vary corresponds to the phenotypic trait vector, a is the vector of individual random additive genetic effects with a normal distribution [in other words, a ∼ N , where G is the among-tree pedigree-free SSR-based relationship matrix, and a2σ corresponds to the additive genetic variance , e is the vector of residual effects, and X and Z are the incidence matrices for fixed effects and additive genetic effects  implemented in the BGLR  software (Parameter estimation for the software  under anh2), which is the proportion of the overall phenotypic variance accounted for additive genetic variance, was then calculated by retrieving the additive (aσ2) and residual (eσ2) variances from the RKHS algorithm, following Narrow sense rootstock-mediated heritability  was computed per trait using Pearson’s correlation between observed and predicted (μ + GEBV) trait values .The vector of t values . Predicth2 scores for key agronomic traits in the OP half-sib seedling progenies of avocado “criollo” “plus trees” grafted with the Hass scion. This heritability analysis included 5 years of data in the OP half-sib seedling progenies of avocado “criollo” “plus trees” used as rootstocks for the Hass scion via the mother vs. half-sib regression analysis  were significant for the 11 measured morphological traits and for the 6 agronomic traits, according to the 95% CIs from the posterior distribution, all of which were above absolute zero  for the 11 morphological traits ranged from 0.28 to 0.51. The tree shape and branching traits presented the h2 lowest scores, 0.28 and 0.29, respectively. The highest h2 values were presented for ribbed petiole and adaxial veins with 0.47 and 0.51, respectively. The 6 agronomic traits evaluated presented very similar h2 values that ranged from 0.34 to 0.39. Seed weight was the trait with the lowest h2 score (0.34), while fruit weight and total volume traits exhibited the highest values, both with 0.39 (The estimated heritabilities (ith 0.39 .r = 0.64) pulp weight , adaxial veins , and ribbed petiole . Meanwhile, the trait that presented the narrowest confidence interval was the shape of the tree with 0.1–0.4 and r = 0.69  pointed toward “criollo” trees ANSS9 and ANNE55. Similarly, the trait vectors for total fruits’ volume, fruits’ length, and fruits’ weight, respectively pointed toward the avocado “criollo” “plus trees” encoded as ANDA62, ANCI75, and ANCH65. Meanwhile, the avocado “criollo” tree encoded as ANTU57 was closed to the vectors for the traits fruit diameter and pulp weight, which also makes ANTU57 a promising donor for seedling rootstock families . On the h2 scores for key agronomic traits, such as total number of fruits and fruits’ weight after 5 years of production. The estimation of overall h2 scores (spanning both the mother-offspring and the rootstock-mediated inheritance) for both agronomical traits followed a mothers vs. grafted half-sib offspring linear regression. In this sense, the dependent variable corresponded to the average half-sib family trait values for total number of fruits and fruits’ weight measured in avocado cv. Hass trees grafted on top of OP seedling rootstock families obtained from the avocado “criollo” “plus trees.” The trait values for the total number of fruits and fruits’ weight measured in the original mother “criollo” “plus trees” were depicted in the X-axis . The trait fruits’ weight exhibited a slope value of 0.056 with an r score of 0.18, and therefore an overall h2 estimate of 0.11 , and their OP half-sib seedling families. The SSR markers that were used for the molecular characterization of avocado “criollo” “plus trees” allowed obtaining G-BLUP heritability predictions of morphological and agronomic traits. After all, highly polymorphic SSR markers screened across variable genotypes , enable BLUP predictions, as demonstrated by via G-BLUP work either on the basis of shared relatedness  or on the basis of genetic hitchhiking due to linkage disequilibrium (LD) among marker loci. In the absence of demonstrated LD , shared relatedness as inferred by variable SSR markers across a diverse panel  and their OP half-sib seedling progenies used as rootstocks for the Hass scion (0.11), exhibiting a reduction of almost half in heritability. This indicates that nearly 50% of “plus tree” genetic gain for this trait may be inherited to cv. Hass through seed-mediated grafting.We estimated heritability values for 17 morpho-agronomic traits in avocado “criollo” “plus trees” selected as seed donors for rootstocks of the cv. Hass. These values were significant according to their 95% confidence intervals and heritabilities were found to be in a medium range according to h2T) of potential genetic gain for a key yield trait as is the total number of fruits. This overall effect totalizes the additive genetic contribution from OP half-sib seedling rootstock families to the Hass clonal scion . Ultimately, these estimates reinforce the feasibility of utilizing selected avocado “criollo” “plus trees” as seed donors for rootstocks.A long-standing debate in the breeding of fruit tree crops is whether seedling rootstocks are capable to enhance productivity in the absence of more uniform clonal rootstocks, which in turn are thought to lack sufficient genetic variability for long-term adaptation. As an intermediate solution, replacing seedlings genotypes by a diverse panel of elite clones is expected to convey major improvements while controlling adaptive genetic erosion . Nonethein situ superior “criollo” “plus trees” as donors of seedling rootstocks. Alternatively, buds from these selected “criollo” “plus trees” may be grafted to establish ex situ seed orchards. In any case, avocado cv. Hass nurseries may still face uncertainty while sorting the OP half-sib offspring from those “criollo” “plus trees” families in the absence of age-age correlations at the seedling stage. To assist early within-family selection of superior half-sib progenies, heritability estimates above 35%  also invite implementing last-generation high-throughput genomic selection (GS) schemes , a major fungal threat for avocado plantation worldwide  seedling screening at nurseries of avocado cv. Hass, as well as within the avocado rootstock breeding cycles per se. Nonetheless, a question persists, which is whether the studied SSR molecular markers are enough to capture sufficient additive variance as to make predictive breeding reliable. This potentially remains open judging by the r scores (> 0.54) of our pedigree-free SSR-inferred estimation of genetic parameters , as well as from these half-sib rootstock families to the avocado cv. Hass clonal scion . These future studies should also envision collecting further data across years . Altogether, these efforts will permit strengthening our modeling effort. Such baseline knowledge will further allow implementing predictive breeding platforms to guide before-grafting seedling selection at nurseries, while targeting diverse climates , such as number of fruits with exportation quality, as well as those discarded because of low weight or thrips” damage. The feasibility to harness “criollo” seedling diversity within a rootstock-breeding program reinforces the utility of native avocado trees to source cryptic genetic variation and adaptive potential (Our work has innovated the implementation of a pedigree-free marker-inferred heritability prediction model to assess the potential of avocado “criollo” “plus trees” as seed donors to produce OP half-sib rootstock families for the Hass clonal scion. The otential , ultimatotential  of clonaThe raw data supporting the conclusions of this article will be made available by the authors upon request, without undue reservation.GC-G, AN-A, and AC conceived the original sampling and research questions. GC-G and SS-O performed phenotypic data collection and sampling of leaves for genotyping. GC-G carried out DNA extractions, SSR genotyping, and alleles size estimation. GC-G, FL-H, and AC filtered input datasets and carried out data analyses. FL-H and AC plotted diagrams. GC-G, FL-H, AN-A, and AC interpreted heritability scores. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Increased fish consumption reduces the risk of dementia. However, it is unknown whether fish consumption reduced all-cause mortality in people with dementia. The purpose of the study is to investigate the association of fish consumption with all-cause mortality in older people with dementia versus those without dementia.n = 406). They were followed up for vital status until 2012.Using a standard method of the Geriatric Mental State, we interviewed 4165 participants aged ≥ 60 years who were randomly recruited from five provinces in China during 2007–2009 to collect the baseline data of socio-demography, disease risk factors, histories of disease, and details of dietary intakes, and diagnosed dementia  and 224 were those without dementia (22.3). In all participants, the risk of all-cause mortality was reduced with fish intake at “ ≥ twice a week”  and at “once a week or less”  compared to “never eat” over the past two years. In participants without baseline dementia, the corresponding HRs for all-cause mortality were 0.57 (0.33–0.98) and 0.85 (0.55–1.31), while in participants with dementia were 1.36 (0.28–6.60) and 1.05 (0.30–3.66), respectively.This study reveals that consumption of fish in older age reduced all-cause mortality in older people without dementia, but not in people with dementia. Fish intake should be increased in older people in general, prior to the development of dementia in the hope of preventing dementia and prolonging life. Consumption of fish reduces incidence of cardiovascular disease (CVD) , respiraThe study population was derived from the Anhui province cohort  and the four provinces' health survey study in China . Their mBriefly, in the Anhui cohort study, we recruited a random sample of 1810 older people aged ≥ 65 years who had lived for at least 5 years in Yiming subdistrict of Hefei city in 2001, and 1709 older people aged ≥ 60 years from all 16 villages in Tangdian District of Yingshang County in 2003. Our trained survey team from the Anhui Medical University used the standard methods of the Geriatric Mental State (GMS) questionnaire  to interThe methods employed in the four-province study have been fully described before , 24. In We took 6071 participants (4314 from the four-province study and 1757 from the Anhui wave 3 survey) as baseline cohort members, since their interview included the dietary intake questionnaire. In 2010–2012, we followed up the cohort to monitor their vital status and re-interviewed surviving participants using the same questionnaires as those at baseline , 19, 25.Descriptive statistics were used to examine the characteristics of the participants. Distributions of sociodemographic and risk factors between surviving and deceased were assessed by a t-test for continuous variables and a Chi-square test for category variables. Cox proportional hazards regression models were employed to assess all-cause mortality in relation to consumption of fish over the past two years at baseline. According to this cohort data, we divided the participants into three groups based on their consumption of fish at baseline; (1) “Never eat”, (2) “A little” (including those of “Once a week or less”), and (3) “A lot” . We computed the hazard ratios (HR) and 95% confidence intervals of all-cause mortality at each group level of fish consumption. In the models, we adjusted for different sets of confounding co-variables, including age, sex, province, urban–rural living, educational level, occupational class, income, smoking status, alcohol consumption, body mass index (BMI), marital status, frequency of visiting children or other relatives, activity of daily living (ADL), hypertension, heart disease, diabetes, depression,  dementia, and consumption of meat, vegetables, and fruits. Following the data analysis for all participants, we stratified data of participants with and without dementia at baseline for analysis and tested the differences in HRs between two groups of participants according to those we did in previous papers .All data analyses were conducted using SPSS version 26 software .Of 4165 participants, the mean age (SD) was 72.1 (7.3) years, 55.3% were women, 58.5% lived in rural areas, and 47.6% were illiterate. The details of the baseline characteristics of the study participants are shown in Table p = 0.011): 34.4 per 1000 person-years in participants who “never eat” fish over the past two years, 28.4 in participants with “a little” fish intake and 20.8 in participants with “a lot” fish intake. Compared to those with “never eat” fish over the past two years, the age-sex adjusted HR of all-cause mortality in participants with “a little” fish consumption was 0.70 (95% CI 0.53–0.93) and in “a lot” 0.56 (0.42–0.74). After further adjustment for socioeconomic status, social support, lifestyles, and BMI, these HRs were slightly increased , p = 0.308.However, the data from 405 participants with dementia at baseline showed no association of fish intake with all-cause mortality , quantity of fish, and the omega-3 supplements consumed, like some other studies , 41. ThuThis study has demonstrated an inverse association of fish intake at older age with all-cause mortality in older people, but not in people with dementia. The findings suggest that it would be better to increase the consumption of fish in older people in general, prior to the development of dementia in the hope of preventing dementia and prolonging life.