The Y-chromosomal short tandem repeat (Y-STR) is an effective forensic tool in familial searches and patrilineal relationship evaluation. However, currently available Y-STR panels often lack sufficient discriminatory power to resolve genetic relationships between distant relatives or within patrilocal populations. This study aims to establish a novel Y-STR amplification system for forensic casework analysis and database construction, which contains 44 slowly and moderately mutating and one rapidly mutating Y-STR. The validation of the assay was conducted following the recommendations of SWGDAM developmental validation guidelines. Different types of casework samples were tested and reliable profiles were obtained. Furthermore, we genotyped and analyzed 141 unrelated Han Chinese male samples. The results showed that this Y45 kit could improve the performance of identifying male individuals, higher haplotype diversity, and discrimination capacity when compared to the previous widely used Yfiler Plus kit. In general, the validation study demonstrated that the newly developed Y45 kit possesses high sensitivity, inhibitor tolerance, male specificity in a mixture, species specificity, and precision and is capable of forensic casework analysis and database construction. Various × 10−2) .Currently, the common commercial Y-STR kits mainly include MM and RM Y-STRs. A previous study indicated that added RM Y-STRs would improve the efficiency of the system  but mistIn this study, we constructed a new 45-plex Y-STR typing system, including 21 Y-STRs from a Yfiler Plus system  and 24 Y-STR loci with low to moderate mutation rates. Various tests were performed to evaluate the efficiency of the system, including PCR condition study, sensitivity, inhibitor study, mixture, species specificity, and stutter calculation. The developmental validation was conducted following the guidelines issued by SWGDAM .® QuantStudio™ 7 Flex Real-Time PCR System following guidelines as recommended by the manufacturer and diluted to approximately 2 ng/μL with TE buffer.This study was approved by the Ethics Committee of Southern Medical University, and all the procedures were carried out following the recommendations of the Declaration of Helsinki . ControlThe Y-STR loci with slow and moderate mutation rates were selected based on the studies of ® PCR System 9700 thermal cycler (Thermo Fisher Scientific). Each amplification reaction contained 4 μL premix, 2 μL primer, 0.1–2 ng template DNA or 1.2 mm punch of the FTA card sample, and sdH2O to obtain a final reaction volume of 10 μL. Standard thermal cycling was performed under the following conditions: initial denaturation at 95°C for 10 min, 30 cycles of 94°C for 30 s, 60°C for 1 min, and 66°C for 1 min, followed by a final extension at 60°C for 20 min. Electrophoresis was performed on the Applied Biosystems 3500xl Genetic Analyzer (Thermo Fisher Scientific) and analyzed with GeneMapper® ID-X Software v1.6.DNA samples were amplified on a GeneAmp2O to fill the total volume to 10 μL.To validate the recommended PCR parameters of the user’s manual and optimize the PCR amplification condition, the cycling number, annealing temperature, and final extension time were tested. For each study, 0.5 ng of control DNA 9948 was prepared in triplicate, with 4 μL reaction mix, 2 μL Y45 primers, 0.4 μL C-Taq, and sdHFor the cycle number test, the system was amplified in series of 28, 29, 30, 31, and 32 cycles; the recommended thermal cycling number is 30.For the annealing temperature test, the temperature was designed in increments of 59, 61, 63, 65, and 67°C; 63°C is the recommended temperature in the protocol.For the final extension test, the final time was respectively held in the gradient of 10, 15, 20, 25, and 30 min; 15 min is the recommended time.To evaluate the sensitivity of the Y45 kit, the male control DNA 9948 was serially diluted with TE buffer to 1.0, 0.5, 0.25, 0.125, 0.0625, and 0.03125 ng each 1 μL.Four common inhibitors, namely, hemoglobin, indigo, humic acid, and EDTA were employed to assess the stability of the Y45 kit. The mixture template contained constant 0.5 ng male control DNA 9948 with inhibitors at following concentrations: 100, 200, 400, 600, 800, and 1,000 μmol/L of hemoglobin; 2, 8, 16, 20, 24, and 30 mmol/L of indigo; 10, 20, 40, 60, 80, and 100 ng/μL of humic acid; and 0.2, 0.4, 0.8, 1.0, 1.2, and 1.5 mmol/L of EDTA. The tests of all concentrations were performed three times.Male/male DNA mixtures were prepared with control DNA 9948 and 2800M, and the mixture ratios varied at 1:1, 1:3, 1:9, and 1:19. Female/male DNA mixtures were tested with control DNA 9947A and 9948 at 1,000:1, 100:1, 10:1, and 1:1 ratios. Each of the samples above was tested with a total DNA template of 0.5 ng and amplified in triplicate.Several non-human genomic DNA samples, including dog, cat, pig, rabbit, chick, duck, rat, and chimpanzee, were tested for cross-reactivity. The quantity of the DNA samples mentioned before was permanently held at 0.5 ng.A total of 50 samples accumulated from daily work were prepared for the reproducibility study. Three laboratories (Guangzhou Forensic Institute and two of its affiliated institutes) were employed to genotype the same DNA samples, and the generated results were compared to estimate the reproducibility.® ID-X Software v1.6 were used to conduct the standard deviation calculation on the observed allelic sizes. To determine the stutter ratios, 50 samples from the population study were randomly selected to calculate the stutter percentage by dividing the height of the stutter peak by the main allele peak height.A 24-injection run of the AGCU Y45 allelic ladder with internal ladder standard (ILS) was performed on a 3500xl Genetic Analyzer. The AGCU Marker SIZE-600 size standard and GeneMapperA total of 30 common casework samples collected from the daily work were tested aiming to access the efficiency of the kit. These samples included muscle tissue (two), old bone (three), hair (five), nail (four), bloodstain (six), semen (two), teeth (three), saliva on cigarette (three), swabs of bottles (one), and swabs of ropes (one).To conduct the population study, 141 unrelated healthy Han Chinese males were genotyped using the Y45 kit with informed consent. Additionally, all 141 male samples had been genotyped by Yfiler Plus  beforehand, and the concordance was evaluated by comparing the alleles obtained by these two kits. Relevant forensic parameters such as the haplotype diversity (HD), match probability (MP), and discrimination capacity (DC) were calculated.2O to fill the total volume to 10 μL. The amount of DNA template was 0.5 ng, and all experiments were repeated three times.In this study, each reaction mixture consisted of 4 μL reaction mix, 2 μL Y45 primers, 0.4 μL C-Taq, and sdHnd cycle, so the recommended optimal cycle number was set to 30 cycles.The full profiles for all replicates were obtained at different cycle numbers (28–32 cycles). The corresponding average peak heights were 1792, 4,068, 5,826, 9776, and 17147, respectively. An expected increase of peak height has occurred under the additional cycles. The cycle numbers had no significant influence on the color balance. Off-scale peaks were detected at the 32Amplification specificity was determined by adjusting annealing temperature. Higher annealing temperature within the range of melting temperature could significantly reduce the non-specific binding between the primer and template. No locus dropout was detected in the annealing temperature between 56°C and 62°C . Allele The final extension step aimed to ensure all double-stranded PCR products to be completely extended. The results demonstrated that the effect of varying final extension time by ±20 min from the optimal extension time of 20 min on the genotypes of the Y45 kit could be negligible. Generally, 20 min is the most recommended extension time .Complete and accurate profiles were observed with DNA inputs ranging from 1 ng down to 61.5 pg by the threshold of 50 RFU. As shown in Inhibitors that usually exist in the crime scene may affect the amplification of DNA samples and even cause failure. To evaluate the inhibitor tolerance of the Y45 kit, four common inhibitors including hemoglobin, indigo, humic acid, and EDTA were tested at verified concentrations. Complete profiles were obtained with 200 μM hemoglobin, 20 ng/μL humic acid, 0.2 mM EDTA, and all tested indigo concentrations (from 2 mM to 30 mM). When the concentration of humic acid was raised to 40 ng/μL, dropouts occurred in the loci with long fragment–sized alleles. With the concentration of hemoglobin exceeding 400 μM, allele dropouts were observed .As expected, the primate DNA sample, chimpanzee, yielded a reproducible detectable product , which sMixtures are commonly encountered in forensic casework. It is of high necessity to distinguish the major and minor components and figure out the contributor proportions. Female/male mixtures were prepared to access the amplification specificity and sensitivity of male samples in this system, while male/male DNA mixtures were used to evaluate the allele calling ratio of Y-STR typing of DNA samples at low concentrations in mixed samples. For the female/male mixtures, full profiles were obtained at ratios of 1:1, 10:1, and 100:1, and 95.65% alleles were called at 1,000:1 . For theA total of 30 samples consisting of ten case types were tested using the Y45 kit. The results showed that most of the case samples could generate complete DNA profiles, except for epitheliums on some samples. Full and accurate profiles were observed on muscle tissues, old bones, hair, nail, blood stains, semen, tooth, and saliva on cigarettes, while dropouts of some large size fragments were detected at DYS510 and DYS443 from DNA on the swabs of bottles and ropes .Three laboratories were involved in the reproducibility study by genotyping the same samples. The results showed that the same samples got consistent genotypes among three participating laboratories, which demonstrated that profiles generated from the Y45 kit are reliable and suitable for comparison between laboratories .Stutter is a by-product commonly generated in the amplification step owing to the slippage, which is usually one repeat unit shorter than the true allele. It may not seriously impact single sample typing but brings difficulties to mixed sample identification. The stutter ratios and relative parameters were calculated for 50 individuals from the population study. As presented in A total of 141 unrelated Han Chinese males were tested using the Y45 typing system, which was genotyped by the Yfiler Plus kit beforehand, and relevant allelic frequencies and forensic parameters were obtained. A total of 139 different haplotypes were generated, of which 137 haplotypes were unique, and 2 haplotypes were observed twice. The number of alleles at single-copy Y-STRs ranged from 2 for DYS645 to 11 for DYS557 . For mulOur study designed and developed a novel Y-STR system with increased loci number and high polymorphism in Han Chinese to satisfy the need for Y-STR database construction and forensic pedigree searches. A series of validation experiments were performed, and the results demonstrated that the Y45 kit is sensitive and robust, which demonstrated that the kit is capable of producing male-specific, reliable, and accurate profiles.


Diagnostic cutoff points for sarcopenia in chest computed tomography (CT) have not been established although CT is widely used for investigating skeletal muscles. This study aimed to determine reference values for sarcopenia of thoracic skeletal muscles acquired from chest CT scans and to analyse variables related to sarcopenia using the cutoff values determined in a general Asian population.th vertebral region (T4CSA); T4CSA divided by height2 (T4MI); pectoralis muscle area (PMCSA); and PMCSA divided by height2 (PMI) at the 4th vertebral region. Sarcopenia cutoff was defined as sex‐specific values of less than −2 SD below the mean from the reference group.We retrospectively reviewed chest CT scans of 4470 participants  performed at a check‐up centre in South Korea (January 2016–August 2017). To determine cutoffs, 335 participants aged 19–39 years  were selected as the healthy and younger reference group, and 4135 participants aged ≥40 years  were selected as the study group. We measured the following: cross‐sectional area (CSA) of the pectoralis, intercostalis, paraspinal, serratus, and latissimus muscles at the 4CSA, T4MI, PMCSA, and PMI cutoffs for sarcopenia were 100.06cm2, 33.69cm2/m2, 29.00cm2, and 10.17cm2/m2 in male, and 66.93cm2, 26.01cm2/m2, 18.29cm2, and 7.31cm2/m2 in female, respectively. The prevalence of sarcopenia in the study group measured with T4CSA, T4MI, PMCSA and PMI cutoffs were 11.4%, 8.7%, 8.5%, and 10.1%, respectively. Correlations were observed between appendicular skeletal mass divided by height2 measured by bioelectrical impedance analysis (BIA) and T4CSA /T4MI , and ASM/height2 measured by BIA and PMCSA /PMI . In the multivariate logistic regression models, sarcopenia defined by T4CSA/T4MI were related to age  and diabetes . Sarcopenia defined by PMCSA/PMI were related to age , male sex , diabetes , history of cancer , and sufficient physical activity .In the reference group, T4The reference cutoff values of a general population reported here will enable sex‐specific standardization of thoracic muscle mass quantification and sarcopenia assessment. The decrease in skeletal muscle function or strength and muscle mass by aging is defined as sarcopenia.Conventionally, when analysing muscle quantity using CT, skeletal muscle measurements at the level of the third lumbar vertebra are used.CSA) correlated with quadriceps size and limb muscle strengthCSA to predict the prognosis in idiopathic pulmonary fibrosis patients.CSA) have shown a relationship with prognosis in a smoking population,Various methods for analysing thoracic skeletal muscles from chest CTs have shown clinical importance. Among the methods, the CSA of pectoralis, intercostalis, paraspinal, serratus, and latissimus muscles  determine reference values for sarcopenia from thoracic skeletal muscles acquired from chest CT scans, (ii) compare the determined values with those acquired by BIA, and (iii) analyse variables related to sarcopenia, in a general Asian population.n = 122), (ii) incomplete smoking status records (n = 84), (iii) having any current pathological disorders including cancer, liver cirrhosis, chronic renal insufficiency, uncontrolled asthma, cardiovascular disease, or cerebrovascular accident (n = 242), (iv) did not undergo BIA (n = 204), and (v) did not undergo pulmonary function test (n = 24). As a result, 4470 participants were enrolled in our study, of which, 335 participants aged 19–39 years  were selected as the reference group for determining T4CSA, T4MI, PMCSA and PMI cutoffs in accordance with previous studiesFigureWe performed a retrospective, cross‐sectional study based on participants aged 19 years or older who voluntarily visited the Health Promotion Center of the Severance Hospital, Seoul, South Korea and underwent a health checkup programme that included chest CT scans between January 2016 and July 2017. The exclusion criteria were as follows: (i) incomplete past medical history records  were included in the questionnaire. Smoking history was categorized as ‘never/ex‐smoker/current smoker’ and alcohol history was calculated as cc/day. Aerobic exercise was defined as ‘moderate activity that makes you out of breath a little more than usual’, and the examples provided for aerobic exercises included brisk walking, tennis (doubles), cycling at slower than 10 miles per hour, and dancing. Intensive exercise was defined as ‘strenuous activity that makes you out of breath much more than usual’, and the examples provided for intensive exercises included running, aerobic dancing, cycling at faster than 10 miles per hour, tennis (singles), and hiking uphill. Sufficient physical activity was considered as performing intensive exercise ≥75 min/week and/or aerobic exercise ≥150 min/week according to the recommendations of WHO and AHA.CSA) and CSA of pectoralis muscle area (PMCSA) at the fourth vertebral region. All chest CT scans were performed in the standardized position with the arms positioned at the sides of the trunk as per the protocol of the Health Promotion Center of the Severance Hospital. Measurements of the skeletal muscles were performed as in our previous study.FigureCSA) was quantified followed by CSA of only the pectoralis muscles (PMCSA). Second, T4 muscle index (T4MI) was calculated as T4CSA divided by height2 and pectoralis muscle index (PMI) was calculated as PMCSA divided by height2. The measurement of CSAs was performed by 3 radiology technicians with 4, 6, and 10 years of experience. Afterwards, 500 samples were randomly selected from the data and another technician performed measurement of CSAs to validate the reliability of the data. The intraclass correlation coeffiencts for the initial values and the re‐measured values were 0.993 (P < 0.001) in the T4CSA and 0.999 (P < 0.001) in the PMCSA. Radiology technicians measured CSAs without access to patient information. Both contrast‐enhanced and non‐contrast CT scans were used as there was no difference in muscle CSA measurements between these in the previous study.We measured CSA of the pectoralis, intercostalis, paraspinal, serratus, and latissimus muscles at the 4th vertebral region . Receiver operator characteristic (ROC) curves and AUC (area under the ROC curve) were generated using the MedCalc software .Descriptive statistics were reported as numbers with proportions or as means with SDs. Chi‐square tests were conducted to compare categorical variables; CSA, T4MI, PMCSA, and PMI were 100.06 cm2, 33.69 cm2/m2, 29.00 cm2, and 10.17 cm2/m2 in men, respectively and 66.93 cm2, 26.01 cm2/m2, 18.29 cm2, and 7.31 cm2/m2 in female, respectively. A total of 4135 participants aged ≥40 years  were selected as the study group. Baseline characteristics of the participants in the study group stratified by presence of sarcopenia defined by T4CSA and T4MI, and baseline characteristics of the study participants stratified by the presence of sarcopenia defined by PMCSA and PMI are provided in TableCSA, T4MI, PMCSA, and PMI cutoffs was 11.9% (319/2690), 9.4% (252/2690), 6.2% (168/2690), and 8.4% (226/2690) in male, respectively; and 10.7% (154/1445), 7.5% (108/1445), 12.8% (185/1445), and 13.4% (193/1445) in female, respectively. The sarcopenia group had higher BMI than that in the normal group . There was no significant difference in body weight between the sarcopenia group defined by T4MI and the normal group ; additionally, no significant difference in height was found between the sarcopenia group defined by PMI and the normal group . The proportion of chronic underlying diseases such as hypertension and diabetes was significantly higher in the sarcopenia group compared with that in the normal group . Laboratory and pulmonary function test results of the study patients stratified by presence of sarcopenia defined by T4CSA, T4MI, PMCSA, and PMI are provided in TableP < 0.050). The distribution of the T4CSA, T4MI, PMCSA, and PMI values are shown in FigureCSA, T4MI, PMCSA, and PMI decreased as age increased. The prevalence of sarcopenia increased with older age in both sexes. High correlations were observed between T4CSA and PMCSA , and T4MI and PMI . Similarly, correlations were observed between ASM/height2 measured by BIA and T4CSA /T4MI , and ASM/height2 measured by BIA and PMCSA /PMI   were selected as the reference group. When values less than –2SD below the mean were invested in the reference group as the cutoff for defining sarcopenia, the sex‐specific cutoff points of T41) Table.TableCSA, T4MI, PMCSA, and PMI. Sarcopenia defined by T4CSA was related to older age  and diabetes , and was inversely related to body mass index (BMI)  and sufficient physical activity ; sarcopenia defined by T4MI was related to older age , diabetes , history of liver disease . Sarcopenia defined by PMCSA was related to older age , history of hypertension , diabetes , pulmonary disease , and cancer  and was inversely related to male sex , BMI , alcohol consumption , and sufficient physical activity ; sarcopenia defined by PMI was related to older age , history of diabetes , cancer , alcohol consumption , and was inversely related to male sex , history of cerebrovascular accident , and sufficient physical activity .CSA, T4MI, PMCSA, and PMI cutoffs by identifying correlations with BIA results and showing relationships with known variables. The muscle mass estimates by BIA are known to be highly correlated with those measured by DXAThis study attempted to establish reference cutoff values for thoracic skeletal muscles at the level of T4 that can be universally used in clinical settings and other sarcopenia studies. This study validated sarcopenia defined by T4CSA, T4MI, PMCSA, and PMI measured on chest CT scans. At the level of L3, the International Consensus of Cancer Cachexia proposed sarcopenia cutoff—defined as L3 muscle CSA divided by height2—to be less than 55 cm2/m2 for men and less than 39 cm2/m2 for women.et al. reported L3 muscle CSA and L3 muscle CSA divided by height2 cutoff as 144.3 cm2 and 45.4 cm2/m2 in men and 92.2 cm2, 34.4 cm2/m2 in women.To our knowledge, this is the first study that reports the reference cutoff values of T4CSA, T4MI, PMCSA, and PMI was 11.4%, 8.7%, 8.5%, and 10.1%, respectively. It should also be considered that the sarcopenia study group was set as ≥40 years compared to other studies that set sarcopenia study population at age of 50–70 years.The prevalence of sarcopenia in a healthy general population assessed through the CSA in CT has not yet been reported. Estimates of sarcopenia prevalence vary from 1.7 to 40.4%; in a meta‐analysis of 35 studies, the overall estimate of prevalence was 10%.2 and CSAs divided by height2 to define sarcopenia. Determining the ideal adjustment method including height has been a long debate in the field of sarcopenia.2, by weight, or by BMI.2. This study used both raw CSA and CSA divided by height2 to define sarcopenia on chest CT, as both these values have shown a relationship with clinical outcomes and/or sarcopenic measures.2. The purpose of height adjustment is to remove the correlation between muscle CSA and height. At the level of L3, Derstine et al. proposed skeletal muscle area divided by height for optimal height adjustment and proposed z‐score for optimal height and BMI adjustment.TableCSA, T4MI, PMCSA, and PMI. This may mean that although the T4CSA, T4MI, PMCSA, and PMIs have shown clinical importance, more detailed studies including BMI adjustments may help determine optimal body size‐adjusted muscle indexes.This study used raw CSAs not divided by heightCSA, T4MI, PMCSA, and PMI were similar, but there were also some differences. Male sex, history of cancer, and alcohol consumption were associated with sarcopenia defined by PMCSA but were not associated with sarcopenia defined by T4CSA in the multivariate analysis. Male sex, history of cerebrovascular accident, cancer, and sufficient exercise were associated with sarcopenia defined by PMI but were not associated with sarcopenia defined by T4MI in the multivariate analysis. Compared to PMCSA and PMI, T4CSA and T4MI includes the CSA of intercostalis muscles, which are involved in breathing; paraspinal muscles, which support the back; and serratus and latissimus muscles, which are involved in the movement of the scapula and arm; and the differences in the muscles included may be the cause of differences between T4CSA/T4MI and PMCSA/PMI. More detailed studies are therefore needed.Variables associated with sarcopenia defined by T4CSA, T4MI, PMCSA, and PMI. Fifth, we could not evaluate the impact of low muscle quantity on long‐term clinical outcomes.This study has some limitations. First, the study population comprised participants that voluntarily visited one health check‐up centre for regular medical check‐ups, which can limit generalizability and lead to selection bias. Second, the study population comprised only Asian participants. It is well‐established that body composition differs between major races.CSA, T4MI, PMCSA, and PMI measured on CT scans and to suggest cutoff points for diagnosis of sarcopenia in a large population of the general Asian participants. The sex‐specific cutoff points of T4CSA, T4MI, PMCSA, and PMI were 100.06 cm2, 33.69 cm2/m2, 29.00 cm2, and 10.17 cm2/m2 in men, respectively, and were 66.93 cm2, 26.01 cm2/m2, 18.29 cm2, and 7.31 cm2/m2 in women, respectively. Correlation between the BIA results and the values of T4CSA, T4MI, PMCSA, and PMI were observed. The relationship between the variables and sarcopenia defined by T4CSA, T4MI, PMCSA, PMI were similar to known sarcopenia‐related factors. Reference cutoff values for thoracic skeletal muscle measured from chest CT scan in a general population reported here will enable sex‐specific standardization of thoracic muscle mass quantification and sarcopenia assessment that can be universally used in clinical settings, and this will promote further sarcopenia research.In conclusion, this is the first study to report the reference values of T4All authors declare that they have no competing interests.This research was supported by funds (2019‐2) provided by the Department of Internal Medicine, Yonsei University College of Medicine. The funding body had no role in the design of the study, collection, analysis, interpretation of data or writing of the manuscript.https://ocr.yuhs.ac, IRB No. 4‐2018‐1220) and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Because this study was a retrospective analysis of existing administrative and clinical data, informed consent was not required.The study procedures were approved by the Severance Hospital Institutional Review Board. (Table S1. Correlation between T4CSA, T4MIa, PMCSA, and PMIb, and ASM divided by height2 measured by bioelectrical impedance analysisTable S2. Sex‐specific Pearson's correlation, p‐value shown for T4CSA/PMCSA versus BMI, height, and weightFigure S1. Flow diagram of participants in this study.Click here for additional data file.


Diabetes mellitus (DM) is associated with left ventricular remodeling and incident heart failure, but the association between glycated hemoglobin A1c (HbA1c) and subclinical cardiac disease is not established. We aimed to determine the associations between HbA1c and (1) echocardiographic measures of left ventricular structure and function, and (2) cardiovascular biomarkers: cardiac troponin T, NT‐proBNP , and CRP (C‐reactive protein).2, and 49% were women. Higher HbA1c was associated with worse left ventricular systolic  and diastolic (E/e'‐ratio) function, myocardial injury (cardiac troponin T), inflammation (CRP), and impaired neurohormonal homeostasis (NT‐proBNP) . The associations between HbA1c and cardiovascular biomarkers were independent of the echocardiographic variables, and vice versa. Associations were nonlinear (P<0.05 for nonlinearity) and appeared stronger in the pre‐DM range of HbA1c than the no‐DM and DM range.Participants (n=3688) born in 1950 from the population‐based ACE (Akershus Cardiac Examination) 1950 Study were classified as DM (HbA1c≥6.5% or self‐reported DM), pre‐DM (HbA1c 5.7%–6.5%), and no‐DM (HbA1c<5.7%). DM, pre‐DM, and no‐DM were classified in 380 (10%), 1630 (44%), and 1678 (46%) participants, respectively. Mean age was 63.9±0.7 years, mean body mass index was 27.2±4.4 kg/mHbA1c was associated with indexes of subclinical cardiovascular disease, and this was more pronounced in pre‐DM. Our results suggest that cardiovascular preventive measures should be considered also in subjects with hyperglycemia and HbA1c below the established DM cutoff.clinicaltrials.gov. Identifier: NCT01555411. However, the study group welcomes initiatives for cooperation, and data access may be granted on application. More information is available on the study website (The ACE (Akershus Cardiac Examination) 1950 Study is an ongoing epidemiologic cohort study that recruited 3706 men and women born in 1950 from Akershus County, Norway . The baseline visit, which makes the basis for the current analysis, was conducted in 2012 to 2015 at 2 field centers: Akershus University Hospital and Bærum Hospital, Vestre Viken Hospital Trust. Details of the study design and methods have previously been published.or self‐reported DM or use of antidiabetic medication), pre‐DM (HbA1c 5.7%–6.4% and no self‐reported DM or use of antidiabetic medication), and no‐DM (HbA1c<5.7% and no self‐reported DM or use of antidiabetic medication).For the present analysis, we excluded 18 participants with missing measurements of HbA1c, cardiac troponin T (cTnT), CRP (C‐reactive protein), or NT‐proBNP , leaving a study cohort of 3688 participants. We classified participants according to the recommendations from the American Diabetes Association 2019 GuidelinesHbA1c, glucose, CRP, creatinine, cTnT, and NT‐proBNP were measured from blood samples drawn from participants at the baseline visit. HbA1c was measured immediately in EDTA whole blood, and glucose, CRP, and creatinine in serum, at the central routine laboratory at each hospital. Estimated glomerular filtration rate was calculated by the Chronic Kidney Disease Epidemiology Collaboration formula.Comprehensive 2‐dimensional Doppler, tissue Doppler imaging, and speckle‐tracking echocardiography were performed at the baseline visit using GE Vivid E9 with the M5S probe, and stored in EchoPAC 201  for subsequent off‐line analysis, as previously described.P value <0.05 was considered significant.Participants were categorized on the basis of DM status, and clinical characteristics, echocardiographic variables, and cardiac biomarker concentrations were compared for trend, using linear or logistic regression, across the 3 groups . For echocardiographic parameters and cardiac biomarkers, further adjustments were made for clinical variables selected a priori: age, sex, body mass index (BMI), smoking status, prevalent hypertension, atrial fibrillation, coronary artery disease, and estimated glomerular filtration rate. To assess independent predictors of HbA1c, we performed multivariable linear regression models with the echocardiographic parameters and cardiovascular biomarkers as dependent variables. The regression analyses for echocardiographic variables were adjusted for cTnT, NT‐proBNP, and CRP, in addition to the clinical variables described above. The biomarker regression analysis was adjusted for key echocardiographic measures of LV structure, systolic function, and diastolic function: LV mass index, LVEF, and E/e', in addition to the clinical variables. There was substantial missing data for GLS and TRVmax, and high degree of collinearity between GLS and TRVmax and the 3 echocardiographic variables already included. Hence, GLS and TRVmax were adjusted for in additional sensitivity analyses. We did not adjust for multiple testing. We performed interaction analysis for sex and DM category to adjust for effect modification. Univariable and multivariable logistic regression analyses were also conducted with outcome variables dichotomized at the median . We used restricted cubic splines to assess the nonlinear association between HbA1c and each biomarker concentration. We used the lowest Akaike Information Criterion to determine the number of knots with the best model fit. We did not force the position of the knots or adjust for possible confounders to assess the natural association between each biomarker concentration and HbA1c. We performed sensitivity analysis in participants free of self‐reported HF and by replacing BMI with waist‐hip ratio. Statistical analysis was performed using STATA software v15.1 . A 2‐sided 2. DM was present in 380 (10%), pre‐DM in 1630 (44%), and no‐DM in 1678 (46%) participants. Of the participants with DM, 265 (70%) self‐reported established DM, of which 20 (8% of the DM population) were type 1 DM, 238 (90%) were type 2 DM, and 7 (2%) were not classified. Two hundred of the participants with DM used antidiabetic medication (53% of the DM population), with 160 participants using noninsulin medications, 23 participants using insulin, and 17 participants using a combination of insulin and other antidiabetic medication. Mean HbA1c in total population was 5.8±0.7% , and mean fasting glucose was 5.5±1.2 mmol/L .Among the 3688 participants included, 1795 (49%) were women, mean age was 63.9±0.7 years, and mean BMI was 27.2±4.4 kg/mParticipants in higher glycemic categories were more likely to be men, to be obese, and to have preexisting hypertension, hypercholesterolemia, coronary artery disease, and HF. These participants were more frequently using cardioprotective and antidiabetic therapy and had lower cholesterol concentrations Table .P=0.002] and GLS [P<0.001]), worse LV diastolic function (higher E/e'‐ratio [P<0.001]), and higher concentrations of cTnT (P<0.001), NT‐proBNP (P<0.001), and CRP (P<0.001) (Table P=0.23) or TRVmax (P=0.09) in adjusted models.After adjusting for clinical characteristics, higher categories of DM  were associated with worse LV systolic function (lower LVEF [1) Table . There wP=0.009), worse GLS (P<0.001), and higher E/e'‐ratio (P=0.001) after adjusting for clinical characteristics and cTnT, NT‐proBNP, and CRP (Table P<0.001), higher CRP (P<0.001), and lower NT‐proBNP (P<0.001) concentrations, after adjusting for clinical characteristics and LV mass index, LVEF, and E/e' (Table P<0.001) and NT‐proBNP .To assess independent associations of HbA1c with echocardiographic variables and cardiac biomarkers, we included these in the same model . Higher HbA1c remained associated with lower LVEF , with stronger associations for cTnT and E/e' with HbA1c in men and stronger associations for NT‐proBNP with HbA1c in women . The best fit for all 6 associations using restricted cubic spline yielded an S‐shaped association for these associations , except for LVEF (P=0.71)  Table . In gene1) Table . These rP<0.01 for all). The associations did not have the same S‐shaped association as demonstrated for HbA1c , LV diastolic dysfunction (E/e'), inflammation (CRP), myocardial injury (cTnT), and impaired neurohormonal homeostasis (NT‐proBNP), and that these associations are beyond obesity and measures of cardiac structure, function, injury, and inflammation. NT‐proBNP concentrations above the median (>66 ng/L) were 50% less likely in participants with DM compared with non‐DM.The association between HbA1c and measures of cardiac function and structure has previously been investigated in elderly subjects from the ARIC (Atherosclerosis Risk in Communities) study, where they found GLS, E/e'‐ratio, and LV mass to be the most important echocardiographic correlates of chronic hyperglycemia status.The recently updated American Diabetes Association definition of pre‐DM and DMDysglycemia is an independent risk factor for HF, irrespective of DM status.The number of subjects with DM or undiagnosed DM (HbA1c ≥6.5%) was relatively low, and associations within this group must be interpreted with caution. Missing data on GLS and TRVmax, primarily because of poor image quality,HbA1c is nonlinearly, and independently, associated with markers of myocardial injury, inflammation, neurohormonal dysregulation, and LV systolic and diastolic dysfunction, in middle‐aged subjects from the general population. Particularly strong relationships were seen in participants with pre‐DM, which could indicate a need for lifestyle and possibly pharmacological interventions also in subjects with HbA1c levels below the current diagnostic cutoff for DM.Dr Myhre and Dr Lyngbakken are supported by grants from the South‐Eastern Norway Regional Health Authority. The ACE (Akershus Cardiac Examination) 1950 Study is funded by the 2 health trusts: Akershus University Hospital Trust and Vestre Viken Hospital Trust.Dr Myhre has served on advisory boards for Novartis and Novo Nordisk, and has received consulting honoraria from Novartis, AmGen, and Novo Nordisk. Dr Omland has served on advisory boards for Abbott Diagnostics, Roche Diagnostics, and Bayer, and has received research support from Abbott Diagnostics, Novartis, Roche Diagnostics, Singulex, and SomaLogic via Akershus University Hospital, and speaker's or consulting honoraria from Roche Diagnostics, Siemens Healthineers, and CardiNor. Dr Røsjø has received personal fees from Novartis and Thermo Fischer BRAMS, CardiNor, and SpinChip. The remaining authors have no disclosures to report.Tables S1–S3Figures S1–S2Click here for additional data file.


The maintained high power factor is due to the markable enhancement in the Seebeck coefficient, which could be attributed to the significant suppression of hole concentration and the valence band convergence upon Sb doping, while the low thermal conductivity stems from the suppression of electronic thermal conductivity due to the increase in electrical resistivity and the lowering of lattice thermal conductivity through strengthening the phonon scattering by lattice distortion, dislocations, and twin boundaries. The excellent thermoelectric performance of Ge0.90Sb0.10Te shows good reproducibility and thermal stability. This work confirms that Ge0.90Sb0.10Te is a superior thermoelectric material for practical application.Lead-free and eco-friendly GeTe shows promising mid-temperature thermoelectric applications. However, a low Seebeck coefficient due to its intrinsically high hole concentration induced by Ge vacancies, and a relatively high thermal conductivity result in inferior thermoelectric performance in pristine GeTe. Extrinsic dopants such as Sb, Bi, and Y could play a crucial role in regulating the hole concentration of GeTe because of their different valence states as cations and high solubility in GeTe. Here we investigate the thermoelectric performance of GeTe upon Sb doping, and demonstrate a high maximum zT value up to 1.88 in Ge T = S2Tσ/κ, where S is the Seebeck coefficient, σ is the electrical conductivity, T is the absolute temperature, and κ is the thermal conductivity, which usually stems from the crystal lattice vibrations (lκ) and the charge carriers drift (eκ). Hence, a good thermoelectric material should have a large σ and S along with a low κ [Thermoelectric (TE) materials can convert heat directly into available electricity based on the Seebeck effect, providing an alternative way to utilize fossil energy more efficiently ,3,4,5,6. a low κ .zT exceeds 2.5 and it has been attained in PbTe and SnSe TE materials [lκ [zT, e.g., the maximum zT over 2 is attainable in , , , and  codoping systems [In recent decades, IV–VI semiconductors with a narrow band gap have made a great progress in terms of theory and TE performance, showing a bright prospect in TE application ,9,10,11.aterials ,15,16,17aterials . Alternaaterials ,19,20. Gnd 700 K . It is b00 K [κl ,23. Addi systems ,26,27,28S and high κ, i.e., the large number of Ge vacancies causes a high hole concentration (~1021 cm−3), a low S (~34 μV K−1), and a high κ (~8 W m−1 K−1) at room temperature [20 cm−3 could be obtained by Sb/Bi/Y doping, and the available maximum zT is up to 1.7–1.85 [S. It has been demonstrated that In/Ga/Ti doping could introduce the resonant level around the Fermi level [However, the pristine GeTe commonly exhibits an inferior TE performance due to its low perature . To supp1.7–1.85 ,31,32,33a low S ~ μV K−1, mi level ,36,37, ami level ,23,25. Ia low S ~ μV K−1, mi level ,38,39.lκ by the reinforcement of phonon scattering [lκ of pristine GeTe (2.7 W m−1 K−1) can be decreased within the range of 1.0–1.5 W m−1 K−1 by doping 10 at% Sb/Bi/Pb [−1 K−1 in codoping systems, for instance, Ge0.88y−Sb0.08PbyTe and Ge0.85Bi0.05Sb0.10Te alloys [lκ was mainly attributed to the atomic mass and lattice strain fluctuations induced by point defects. Yet, with the substitution of extrinsic atoms on Ge sites, the evolution of microstructures is complicated, often involving precipitates [In addition to the optimization of electrical transport properties, doping or alloying can introduce significant lattice disorder, resulting in atomic mass and lattice strain fluctuations that endow a low attering . For exaSb/Bi/Pb ,42. Thise alloys . This pripitates ,31,44,45ipitates ,47, inveipitates ,49,50, aipitates ,51. Thesx1−SbxTe , which were prepared by the solid-state reaction and spark plasma sintering (SPS) route. We proved that a maximum zT of 1.88 at 773 K could be attained in Ge0.90Sb0.10Te with good reproducibility and thermal stability. With Sb substitution on Ge sites, the Seebeck is greatly improved due to the reduction in hole concentration and valence band convergence. Moreover, a significantly reduced κ is achieved, which originates from both the decrease in eκ and lκ. Transmission electron microscopy (TEM) observations verify the existence of nanoscale twins and dislocations in the matrix, which together with point defects  result in a low lκ, i.e., ~0.89 W m−1 K−1 in Ge0.90Sb0.10Te at 773K. This work confirms Ge0.90Sb0.10Te is a reliable candidate for TE application.Here, we systematically investigated the TE performance of Gex1−SbxTe  were prepared through a solid-state reaction followed by spark plasma sintering (SPS) procedure. High-purity (>4 N) elements of Ge, Sb, and Te were weighted in the corresponding mole ratio and flame-sealed in quartz tubes under a vacuum lower than 5 × 10−4 Pa. The tubes were heated to 1173 K and soaked for 6 h, both of heating and cooling rate at 1 K min−1. These as-synthesized ingots were hand-ground into fine powders and then consolidated into cylindrical bulks by SPS at 823 K for 3 min under a uniaxial pressure of ~50 MPa in a vacuum (˂10 Pa). The relative density for these sintered samples is no less than 95%. Rectangular specimens (~2 mm × 2 mm × 10 mm) and square-shaped pellets (~6 mm × 6 mm × 1.5 mm) were cut off for electrical and thermal transport properties measurements, respectively. Polycrystalline samples Geα, λ = 0.154 nm) operated at 45 kV and 40 mA with a step size of 0.01313°. The high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and the energy-dispersive X-ray (EDS) mapping were performed on a probe-corrected transmission electron microscopy  equipped with a super EDS system at 300 kV. The backscattered electron image (BSE) and EDS mapping scanning were employed with electron microscopy  equipped with a super EDS system.The crystal structure of sintered samples was analyzed using a PANalytical X-ray diffractometer (Cu Kp) and mobility (μH) were examined using a direct-current (dc) Hall effect measurement system  at the excitation current up to 100 mA and magnetic field of 1.5 T. Electrical resistivity (ρ) and Seebeck coefficient (S) were measured by a static dc method  under a helium (99.999%) atmosphere. The thermal conductivity (κ) is calculated by κ = pD d C, where D is the thermal diffusivity, pC is the specific heat, and d is the volume density. The thermal diffusivity (D) was measured by a laser flash method ; the specific heat (pC) was estimated based on Dulong–Petit law, which is approximate to the measurement result; and the volume density (d) was determined by the Archimedes method.Room temperature carrier concentration . Particularly, the presence of double peaks in the 2θ range of 23–27° and 41–45° further confirms the rhombohedral phase of pristine GeTe. The peaks located at ~27.3° and ~45.3° correspond to cubic Ge (JCPDS 065−0333) secondary phase. The obviously enlarged Ge peaks with increasing the Sb content indicates more Ge precipitates in the matrix, which is probably due to that Sb atoms occupy on Ge sublattice sites. To examine the impact of Sb doping on the crystal structure of GeTe, we calculated lattice parameters of the sintered Gex1−SbxTe samples and the results are shown in a and decrease in c with increasing the Sb content, suggesting a smaller c/a ratio. This phenomenon indicates that Sb doping could lower the structural transition temperature of GeTe and promote the formation of cubic phase, which is consistent with the convergence of (024) and (220) diffraction peaks  of the samples Gex1−SbxTe. The positive S suggests that all the samples have a p-type conductive behavior and the inability of single Sb doping in inversing such a conductive behavior. As expected, the S is greatly improved upon Sb doping, increasing from ~31 μV K−1 for the pristine GeTe to ~119 μV K−1 for Ge0.90Sb0.10Te, and further to ~173 μV K−1 for Ge0.80Sb0.20Te at 323 K. A maximum S up to 249 μV K−1 at 574 K is attained in Ge0.80Sb0.20Te.S of GeTe upon Sb doping, the Pisarenko curve based on a two-valence-band model is plotted in S in the Sb-doped GeTe samples could be attributed to the valence band convergence and the hole concentration suppression, as the S is inversely coupled with the carrier concentration.To find out the underlying reason for the enhanced PF) of the samples Gex1−SbxTe. The maximum PF as high as ~4.74 × 10−3 W m−1 K−2 at 773 K is attained in the pristine GeTe. The high PF is maintained when the Sb content does not exceed 10 at% due to the greatly enhanced S, despite the evident increase in ρ. The maximum PF up to ~3.23 × 10−3 W m−1 K−2 achieved in Ge0.90Sb0.10Te is comparable to V-, Y-, Bi-, and Sb-doped counterparts [terparts ,32,33,47κ) of the samples Ge1x−SbxTe. The κ is greatly reduced over the whole temperature range (323–773 K) upon Sb doping, which decreases from ~7.40 W m−1 K−1 for the pristine GeTe to 1.61 W m−1 K−1 for the sample Ge0.90Sb0.10Te at 323 K. To understand the details regarding the great reduction of κ, the κ is generally divided into two parts of electronic thermal conductivity (eκ) and lattice thermal conductivity (lκ). The eκ is calculated according to the Wiedemann–Franz law, eκ = L ρ−1 T, where L is the Lorentz number estimated based on a two-valence-band model , dislocations, and twin boundaries.To illustrate the low  phonons . OverallzT up to 1.88 is achieved in the sample Ge0.90Sb0.10Te (The TE performance of GeTe is greatly enhanced upon Sb doping as a result of holes concentration optimization, band convergence, and the significant decrease in thermal conductivity. A highest Sb0.10Te a, which 0.90Sb0.10Te is reproducible, which is verified by the repeatable zT of the different baches samples, as shown in 0.90Sb0.10Te, the electrical properties remain unchanged during the 2 times heating and cooling cycles (0.90Sb0.10Te. These results indicate Ge0.90Sb0.10Te has promising application in commercial TE devices.More importantly, the high TE performance of Geg cycles , suggestx1−SbxTe samples were fabricated by the solid-state reaction followed by spark plasma sintering procedure. Sb doping not only leads to an optimized hole concentration approaching 1020 cm−3 but also results in valence band convergence for the GeTe system. Moreover, the thermal conductivity is greatly reduced due to the suppression of both electronic and lattice thermal conductivity. While the reduction in electronic thermal conductivity is due to the increase of electrical resistivity, the decline in lattice thermal conductivity is caused by the modification in microstructures, that is, the phonon scattering is remarkably strengthened by substitutional point defects, dislocations, and twin boundaries. As a result, a maximum zT up to 1.88 is achieved in Ge0.9Sb0.1Te. This work demonstrates that Ge0.90Sb0.10Te is a promising candidate for TE commercial application.The Ge


Clematis species are attractive ornamental plants with a variety of flower colors and patterns. Heat stress is one of the main factors restricting the growth, development, and ornamental value of Clematis. Clematis lanuginosa and Clematis crassifolia are large-flowered and evergreen Clematis species, respectively, that show different tolerance to heat stress. We compared and analyzed the transcriptome of C. lanuginose and C. crassifolia under heat stress to determine the regulatory mechanism(s) of resistance.C. lanuginose and C. crassifolia, respectively. The photosynthesis and oxidation–reduction processes of C. crassifolia were more sensitive than C. lanuginose under heat stress. Glycine/serine/threonine metabolism, glyoxylic metabolism, and thiamine metabolism were important pathways in response to heat stress in C. lanuginose, and flavonoid biosynthesis, phenylalanine metabolism, and arginine/proline metabolism were the key pathways in C. crassifolia. Six sHSPs , POD1 (c200317_g1), POD3 (c210145_g2), DREB2 (c182557_g1), and HSFA2 (c206233_g2) may be key genes in the response to heat stress in C. lanuginose and C. crassifolia.A total of 1720 and 6178 differentially expressed genes were identified from C. lanuginose and C. crassifolia. The results increase our understanding of the response mechanism and candidate genes of Clematis under heat stress. These data may contribute to the development of new Clematis varieties with greater heat tolerance.We compared important metabolic pathways and differentially expressed genes in response to heat stress between The online version contains supplementary material available at 10.1186/s12870-022-03497-w. Clematis L. (Ranunculaceae) are mainly perennial woody vines. There are many species, varieties, and flower patterns. The optimum growth conditions for Clematis generally involve cool to moderate temperatures. There are approximately 355 species of Clematis worldwide and more than 147 species in China [Clematis varieties are mostly cultivated in Poland, Britain, and other European countries.in China . The horClematis species also have medicinal value. Their chemical components include triterpenoid saponins, alkaloids, flavonoids, lignans, and steroids. Clematis spp. are plant sources of many pharmaceutical active ingredients [Clematis aethusifolia showed moderate toxicity to five human solid tumor cell lines, including A-375 and SK-OV-3 [Clematis aethusifolia inhibited the growth and development of Plutella xylostella in crops, and may have potential for insecticide development [Clematis is also an important genus of garden greening vines that increases the diversity of vertical greening plants in gardens. They can be used as ground cover plants, potted plants, and garden greening plants, and are excellent ornamentals.redients –5. Flavo SK-OV-3 . The triClematis show symptoms such as leaf wilting and plant wilt after suffering from heat stress. Heat stress significantly restricts the ornamental uses and growth environment of Clematis. Therefore, understanding the heat resistance mechanism of Clematis and the breeding of heat-resistant varieties are important topics.With global climate change and increased temperatures, extreme heat stress has become a problem that threatens plant growth and development. Heat stress can cause physiological, molecular, and biochemical changes in plants and interfere with the growth and development process of cells and the entire plant. Heat stress can limit the growth, metabolism, and yield of plants –10. HeatClematis lasiandra provide effective anti-tobacco mosaic virus (TMV) activity through the TMV-CP target [Agrostis stolonifera [Plants can adapt to, or resist, heat stress by morphological changes, photosynthesis, protective enzyme activity, and osmotic substance regulation , 14. PlaP target . Heat stlonifera . Activatlonifera .AtHsfAs were the key transcription factors for Arabidopsis thaliana to increase its heat tolerance [AtHsfA1a and AtHsfA1b could upregulate HPSs and other related genes, and protect plants from cytotoxicity through the expression of related genes involved in osmotic regulation [AtHsfA2 expression in Arabidopsis thaliana resulted the downregulation of the HSPs expression, and AtHsfA2 could protect plants against heat stress induced oxidative damage, organelle dysfunction and subsequent cell death [Many transcription factors in plants are significantly upregulated or downregulated, and their expressions change the physiological and biochemical responses in plants to heat stress. The plant heat stress transcription factor family (HSFs) includes the most important transcription factors in plant responses to heat stress. They can induce the synthesis of heat shock proteins (HSPs) in plants to improve their heat tolerance –27. AtHsolerance . In the gulation . Silencell death .MADS, WRKY, MYB, bHLH, and other genes responding to heat stress signals have been studied [OsMADS87 is a heat-sensitive gene regulating the seed size of rice, and it has the potential to improve rice heat tolerance [AtWRKY39 could promote plant responses to heat stress through SA and JA mediated signaling pathways [Arabidopsis pif4 mutant was early flowering but did not show rapid extension of plant axes and leaf hyponasty under heat stress. This showed that PIF4 was an important component in heat stress response [The regulation mechanisms of transcription factors such as  studied –33. OsMAolerance . AtWRKY3pathways . The Araresponse .OsMYB55 transgenic maize, a significant number of genes involved in responses to abiotic stresses, such as high temperature, dehydration, and oxidative stress, were upregulated [HSP, cytochrome P450, and bHLH transcription factor were specifically induced [OsACT in different germplasm may be involved in the heat resistance of SDWG005 [Transcriptome sequencing provides important information on gene expression patterns, functional genes, and regulatory mechanisms involved in plant abiotic stress , 38. In egulated . Several induced . In the  SDWG005 .Clematis lanuginose was introduced to Europe in the nineteenth century and is the parent of many early varieties of Clematis. Clematis crassifolia, which is an evergreen Clematis with many flowers in winter, is sensitive to high temperatures in summer. In this study we used transcriptome analysis to compare different heat stress periods on C. lanuginose and C. crassifolia. We compared and analyzed the DEGs under heat stress and observed response differences between C. lanuginose and C. crassifolia. while also screening potential heat stress response genes to cultivate Clematis varieties with strong heat resistance.C. lanuginose had leaf tip wilting and withered new leaves 4 d after heat stress. C. crassifolia leaves turned yellow and wilted 2 d after heat stress  database, 35,990 genes were annotated to the Gene Ontology (GO) database, 5,098 genes were annotated to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, 81,459 genes were annotated to the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database, and 75,064 genes were annotated to the Swiss-Prot database. Of these, 3,795 genes were annotated in all databases  genes had significant hits for Vitis vinifera, followed by Hordeum vulgare subsp. vulgare , Cajanus cajan , Malus domestica , Klebsormidium flaccidum  and Beta vulgaris subsp. vulgaris . A total of 60, 943 genes were homologous to other species  genes were homologous to ccidum 166, 1.82% C. lanuginosa and C. crassifolia under heat stress. After 1, 2, and 4 d of heat stress treatment, 327, 536 and 209 genes were upregulated and 454, 255 and 500 genes were downregulated, respectively, in C. lanuginosa . Among all the differentially expressed genes, 421 (5.6%) genes were significantly differentially expressed in both C. lanuginosa and C. crassifolia, 1299 (17.4%) genes were specifically expressed in C. lanuginose and 5757 (77.0%) genes were uniquely expressed in C. crassifolia  in C. lanuginosa and C. crassifolia. In the comparison groups of HS d 1 vs. HS d 0, 314 (40.20%) and 809 (25.31%) DEGs were annotated into the GO terms in C. lanuginose and C. crassifolia, respectively. For HS d 2 vs. HS d 0 comparison groups, 173 (21.87%) and 628 (26.21%) DEGs were annotated in C. lanuginose and C. crassifolia, respectively. In the groups of HS d 4 vs. HS d 0, 320 (45.13%) and 625 (24.53%) DEGs in C. lanuginose and C. crassifolia were annotated, respectively , PODP7 (c204229_g1) and PsbY (c200811_g3) showed different expression trends in C. lanuginosa and C. crassifolia , bHLH112 (c203571_g1), JAZ1 (c194555_g1), MYBR1 (c204139_g1), MYC2 (c208293_g1), TCP15 (c195225_g2), WRKY40 (c189120_g1), WRKY41 (c200654_g1), and WRKY51 (c187717_g1). Four transcription factors including DREB2 (c182557_g1), PIF3 (c209598_g2), WRKY7 (c206794_g2), and WRKY72 (c203219_g1) showed opposite expression trends in the two Clematis species , HSP26.5 (c200771_g1), HSP70 (c204924_g1), HSP18.1 (c199407_g2) and HSP20 (c201522_g2) were significantly upregulated from d 1 to the end of heat stress, and HSP17 (c192936_g1) and HSFA2 (c206233_g2) were significantly upregulated on d 1. DREB2 (c182557_g1), POD1 (c200317_g1) and POD3 (c210145_g2) were significantly upregulated at 4 d of heat stress. JAZ1 (c194555_g1) was significantly downregulated from d 1 to d 3, and upregulated at d 4 , HSP17.8 (c176964_g1), HSP26.5 (c200771_g1), HSFA2 (c206233_g2), DREB2 (c182557_g1) all increased significantly after 2 d of heat stress, and HSP70 (c204924_g1), HSP18.1 (c199407_g2), HSP20 (c201522_g2) were significantly upregulated on the first day. POD1 (c200317_g1) and POD3 (c210145_g2) were significantly upregulated under heat stress  were specifically and significantly expressed in the leaf. There were 6178 DEGs, among which 5757 (93.19%) genes were significantly expressed only in C. crassifolia. These results indicate that the gene expression in C. crassifolia was more sensitive to heat stress  system oxygen releasing complex , 43. Anaess Fig. b. Duringis Figs.  and 6. Pose Fig. .1O2, O2−, H2O2, and OH− under heat stress, which will impair chloroplast and mitochondrial functions, subject the plant cells to oxidative damage, including lipid peroxidation, protein oxidation, and DNA damage. H2O2 is one of ROSs which is closely related to oxidative stress. It is derived from superoxide anion disproportionation, and the product of H2O2 has strong oxidation ability [SlMAPK3 was a negative regulator of thermotolerance in Solanum lycopersicum, slmapk3 mutants have higher activities and transcript levels of POD, SOD, CAT, and APX than wild type plants [2O2 in C. lanuginosa, which might be related to the maintenance of antioxidant enzyme activity. In C. crassifolia, the increase of SOD and POD activities on d 1 and d 2 maintained ROS balance at the early stage of heat stress  was significantly upregulated in the early and late stages of heat stress in C. crassifolia  was also observed in C. lanuginosa and C. crassifolia  [JAZ1 (c194555_g1) was significantly downregulated during heat stress in C. lanuginosa and C. crassifolia  and WRKY72 (c203219_g1) were upregulated in C. lanuginosa and C. crassifolia, respectively  . JAZ1 . C. lanuginosa and C. crassifolia plants were grown in the Zhejiang Institute of Subtropical Crops, China. Healthy, two-year-old plants were grown in a grown chamber under 25/20 °C (16:8 h (L:D) photoperiod); 65% humidity) conditions for two weeks. After two weeks of pretreatment, C. lanuginosa and C. crassifolia plants were transferred to a growth chamber for cultivation at 45/40 °C temperature and 16:8 h (L:D) photoperiod. The heat stress treatment duration was 4 d. During the treatment period, 500 ml of water was given to each plant every 2 d, to ensure sufficient soil moisture. The leaves sampled before the heat stress were labelled as “HS 0d,” and those after heat stress were labelled as HS 1d, HS 2d, and HS 4d. Experimental treatments were repeated three times. All methods, including plant experimental research, were in compliance with the relevant guidelines, regulations and legislation.The −2 s−1, the CO2 concentration was 400 ppm, and the relative humidity was 65%.Healthy and fully developed leaves were randomly chosen for photosynthetic parameter measurements, using LI-6400 XT portable photosynthesis system , and equipped with a 6400–18 RGB LED light source. The measurements were carried out from 9:00 to 11:00 am, the photosynthetic photon flux density was 1200 μmol mFor peroxidase enzyme activity analysis, fresh leaves (0.1 g) were ground in liquid nitrogen and suspended in 8.0 ml solution containing 50 mM phosphate buffer (pH 7.4). The homogenate was centrifuged 15 min  at 4 °C, and then the supernatant was collected to obtain crude enzymes.2− produced. SOD activity was determined at 550 nm in absorbance after 40 min of reaction at 37 °C. One-unit of SOD activity (U) was defined as the amount of enzyme that resulted in 50% inhibition of reduction of nitrite in 1 ml of reaction solution.SOD activity was analyzed by measuring the inhibition rate of the enzyme to O2O2 with CAT, and the yellow MA-H2O2 complex was generated by adding ammonium molybdate to quickly stop the reaction. CAT activity was calculated at 405 nm. One-unit was defined as the amount of enzyme that resulted in the decomposition of 1 µmol H2O2 per second in 1.0 g fresh tissue.CAT activity was determined by the hydrolysis reaction of H2O2 based on the change of absorbance. One-unit was defined as the amount of enzyme that resulted in the change of 0.01 at 470 nm per minute by 1.0 g fresh tissues in the reaction system. Leaf tissue amounting to 0.2 g was finely ground with 25 mL acetone and homogenized at 0 °C; the content was calculated using H2O2 as the standard [POD activity was measured at 470 nm by catalyzing Hstandard .2O2 content was measured according to the method described by Patterson [2O2 content was calculated using H2O2 as the standard.The Hatterson . A 0.2 gC. lanuginosa and C. crassifolia was extracted using TRIZOL reagent . The concentration and purity of the total RNA was tested with an Agilent 2100 Bioanalyzer. The mRNA from total RNA was purified by the polyA structure unique to mRNA and mRNA with the polyA structure was enriched by Oligo(dT) magnetic beads. The first strand of cDNA was synthesized using 6-base random primers and reverse transcriptase using mRNA as a template. The second strand of cDNA was synthesized using the first strand of cDNA as template. The chain-specific library was established, and the quality of the library was detected by Agilent 2100 Bioanalyzer. Next Generation Sequencing (NGS) was used to perform paired-end (PE) sequencing .Total RNA from different leaf samples of Clematis, so Trinity software (r20140717) was used to splice clean reads to obtain the transcript for subsequent analysis. Trinity is a De Novo assembly software for transcriptome splicing, splicing high-quality sequences based on the DBG (De Bruijn Graph) splicing principle [There was no reference genome in the transcriptome sequencing of rinciple . The lonwww.kegg.jp/kegg/kegg1.html) were used to annotate all the unigenes  [NR, Swiss-Prot, eggNOG and KEGG databases (1.0 e−5) –83. GO aGene expression were analyzed using the FPKM (Fragments Per Kilobase of exon model per Million mapped reads) method. The criteria for screening DEG were p-value ≤ 0.05, false discovery rate (FDR) < 0.001, Fold Change ≥ 2, or Fold Change ≤ 0.5. Subsequently, GO and KEGG databases were used to analyze the main functions and metabolic pathways of the DEGs.GAPDH gene. Each primer pair was validated the specificity by melt curve analysis, and the gene expression levels were calculated by the 2−△△Ct method.Total RNA was extracted from leaves and the cDNA was synthesized using the Revert Aid RT Kit , Primers designed with Primer Premier 5.0 are shown in Table SP ≤ 0.05.Data were analyzed by one-way or two-way ANOVA using the SPSS 10 program . Different letters on the histograms between different treatments indicate a significant difference at Additional file 1: Fig. S1. Gene Ontology classification and KEGG analysis of the transcriptome. Fig. S2. Enriched GO terms of DEGs. Fig. S3. Enriched GO terms (cellular component) (P < 0.05) of DEGs. Fig. S4. Clustering analysis of gene expression. Additional file 2: Table S1. Statistics of splicing results. Table S2. Annotation of unique DEGs related to heat stress in C. lanuginose. Table S3. Annotation of unique DEGs related to heat stress in C. crassifolia. Table S4. The GenBank number of the unigenes. Table S5. Primer sequences for gene expression analysis.


The experimental group was given 5 days per week physical activity, for 3 months, and a diet supplement of finger millet-based sweet balls (ragi laddu), 3 days per week for 3 months. The above parameters were measured as the posttest. Physical activity was assessed by the General Practice Physical Activity Questionnaire. A 24 h recall assessment was carried out for the diet supplement, and self-reported activity checklist was maintained for physical activity. Among 720 women, 163 (22.6%) showed BMD, t-score < −1.0, and calcium <9.0 mg/dL (p < 0.001). The serum phosphorus and alkaline phosphatase were also low (p < 0.001). After the supplementation to the experimental group, all the biochemical parameters, BMD, and physical activity score showed significant improvement in the posttest (p < 0.001). This study showed significantly low BMD and calcium among premenopausal women. Physical activity and finger millet supplement improved the calcium level and BMD.The effectiveness of physical activity and finger millet-based food supplement on biochemical parameters and bone mineral density (BMD) among premenopausal women were studied. Serum calcium, phosphorus, alkaline phosphatase, and BMD of 720 women (30–40 years) were analyzed. From them, 150 women with low BMD ( Osteoporosis is a global public health problem leading to increased bone fracture . A studyEleusine coracana, ragi in Hindi) is rich in calcium, protein, and essential amino acids [To prevent osteopenia and osteoporosis, calcium and vitamin D supplements are required. Food containing high calcium, potassium, magnesium with vitamins and other minerals, viz., phosphorus, iron, and zinc are preferable for the normal bone metabolism to control osteopenia and osteoporosis . Nutritino acids . Finger no acids , 10. Finno acids . Finger no acids . Finger no acids .Physical activity is essential for bone gain, and activities such as weight-bearing activities and muscle strengthening are recommended for a healthy bone , 15. PhyHealthcare professionals should be aware of bone loss in various disease conditions and should prevent it with proper intervention supported by clinical biomarkers . Osteopot-score of −1 to −2.5, and serum calcium <9.0 mg/dL were included in this study. They were divided into control and experimental groups, equally by a random number table. Women with mental health problem and other systemic disease were excluded from the study. This study was approved by Institutional Ethics Committee of Saveetha Medical College and Hospital, Chennai (SMCH-IEC/006/04/2019), and carried out between May 2019 to April 2020. An information sheet was provided in English and local language (Tamil) to each participant, and signed consent to participate in the study was obtained.720 women of premenopausal age (30–40 years), members of a private women's organization , were initially screened for BMD and biochemical parameters. Among them, 150 women with BMD, t-score of −1 to −2.5 and serum calcium <9.0 mg/dL) were excluded, and 13 women who did not volunteer to the osteoporosis prevention program due to personal reasons were also excluded. The 150 women who gave the signed consent for the intervention study were allocated randomly to the control and experimental groups. Pretest information on demographic profile by the questionnaire was recorded. The osteoporosis prevention program for the experimental group consisted of general awareness, physical activity, and diet supplementation. Structured education was provided once, on osteoporosis prevention (flash cards for 20 min). The physical activity consisted of spine (resistance), hip (strengthening), shoulder (resistance), and knee (strengthening). Pamphlets were given to the experimental group women after demonstration and was instructed to continue for 5 days a week for 3 months. Ragi laddu  supplement was given to the experimental group, three days per week for 3 months. The laddu was prepared using ragi flour (800 g), powdered jaggery (400 g), and ghee (300 g). 100 g of ghee was heated in a frying pan and ragi was roasted for 5 min in low flame. Powdered jaggery was added slowly and remaining ghee was poured, and mixed thoroughly. After cooling, the mixture was divided into 10 equal parts and made into laddu (150 g). All hygienic precautions were adhered in the preparation of the laddu by one of the investigators. The laddu was prepared every week fresh and 3 pieces were distributed to each experimental group women during the weekly meeting. A 24 h recall assessment was carried out for the diet supplement, and self-reported activity check list was maintained physical activity. The prepared laddu was certified by an approved food testing laboratory . At the end of the experimentation, analysis of serum calcium, phosphorus, and alkaline phosphatase and BMD were carried out for both control and experimental groups as the posttest.Serum calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) were analyzed by an autoanalyzer as per manufacture's procedure , and BMD was measured by an ultrasound bone densitometer  for all the 720 women. The 557 premenopausal women who did not meet the criteria . For the assessment of physical activity, the General Practice of Physical Activity Questionnaire (GPPAQ) can be used which consists of seven self-explanatory questions and can be answered within one minute. The GPPAQ is a self-assessment physical activity questionnaire and is commonly used in primary care. The GPPAQ includes type of physical activity, duration, and walking speed and is designed for adults. A physical activity index can be computed and categorized as active, moderately active, moderately inactive, and inactive. , 21. Theχ2 test, Mann–Whitney rank sum test, Wilcoxon signed rank test, one-way ANOVA, and two-way repeated measures ANOVA with Bonferroni t-test for comparison of the variables. Linear regression was used for correlating the variables. Since the physical activity was assessed with a questionnaire, nonparametric statistics was used. For the biochemical and BMD measurements, parametric statistics was used assuming normal distribution. SigmaPlot 13  was used for the statistical analysis and for plotting the graphs.The data were analyzed by the t-score with serum calcium less than 9.0 mg/dL . The serum phosphorus and ALP levels were also significantly low (p < 0.001), though there was no significant difference in the age (p=0.554).Among the 720 women evaluated, 163 (22.6%) showed BMD less than −1.0 .0 mg/dL . χ2 analAnalysis of the laddu revealed 4.66 mg, 370 mg, and 133 mg of iron, calcium, and phosphorus, respectively, per 100 g. The protein content and fiber content were 4.0 g and 3.8 g per 100 g of laddu. The calculated energy was 372 kcal/100 g of laddu.p < 0.001), pretest and posttest (p < 0.001), and the interaction (p < 0.001). There was no significant difference in the pretest (p=0.074), but the posttest showed a significant difference (p < 0.001) between control and experimental groups. The control pretest and posttest did not show a significant difference (p=0.446), while the experimental pretest and posttest showed a significant increase in the serum calcium level (p < 0.001). The mean ± SEM values of serum phosphorus of the control pretest, control posttest, experimental pretest, and experimental posttest are 1.86 ± 0.02, 1.84 ± 0.02, 1.84 ± 0.02, and 2.59 ± 0.02 mg/dL, respectively. Similar to serum calcium, serum phosphorus also showed a significant increase in experimental posttest only (p < 0.001). The ALP level of the control pretest, control posttest, experimental pretest, and experimental posttest was 29.9 ± 0.24, 29.7 ± 0.23, 29.2 ± 0.26, and 39.7 ± 0.30 U/L, respectively. Though the experimental posttest showed an elevated ALP level, it was within the clinical range.The control and experimental groups serum calcium, phosphorus, and ALP levels and BMD are shown in 2, respectively. Similar to serum calcium, there was significant improvement in the experimental posttest in BMD. There was no significant difference in the pretest (p=0.153), but the posttest (p < 0.001) showed a significant difference between control and experimental groups. The control pretest and posttest did not show a significant difference (p=0.928), while the experimental pretest and posttest showed a significant increase in BMD (p < 0.001).The mean ± SEM values of BMD of the control pretest, control posttest, experimental pretest, and experimental posttest are −1.80 ± 0.03, −1.80 ± 0.03, −1.73 ± 0.04, and −0.92 ± 0.04 g/cmp < 0.001) in the physical activity (from median of 0 to 3.2) compared to the control group (from median of 0 to 0) based on the GPPAQ. Osteoporosis is not generally considered as a serious disease when compared to other communicable and noncommunicable diseases . NeverthPhysical exercise has been shown to improve flexibility, strength, posture, and balance in women and also improve bone health . PlannedEleusine coracana) is a rich source of calcium and phosphorus and easily digestible [t-score of BMD, and a positive correlation was obtained with serum calcium and BMD (r = 0.761). Dairy products fortified with calcium and vitamin D to postmenopausal women has been shown to significantly influence BMD [Several dairy products, vegetables, cereals, millets, and pulses are rich sources of calcium. Diet-based calcium supplement would be more acceptable and easier to practice . Finger gestible . It has gestible . Finger gestible . Jaggerygestible . In the gestible . In the ence BMD . The daience BMD . 150 g oThe present study shows significant low BMD and calcium among premenopausal women. Physical activity and finger millet supplement improved the calcium level and BMD. Women in the premenopausal stage should be aware of low BMD and its risk. They should improve their nutrition, particularly calcium from naturally available food products.


Similarly, the Enterobacteriaceae counts of candies from formal processors (nondetectable) were considerably lower (p = 0.015) than those from informal processors (1.8 ± 0.56 log 10 CFU/g). The ergosterol content in these baobab product samples ranged between 0.46 and 1.92 mg/100 g while the aflatoxin content ranged between 3.93 and 11.09 × 103 μg/kg, respectively. Fungal and aflatoxin contamination was detected in 25% and 5% of pulp from formal and informal processors, respectively, and in 5% of candies from informal processors. Microbial contamination in processed baobab products shows an unhygienic processing environment while the fungal and aflatoxin contamination may indicate poor postharvest handling, transport, and storage conditions of baobab fruits along the baobab value chain.Baobab fruit demand has been on the rise in the recent past, and in an attempt to match the demand, farmers and middlemen are forced to harvest immature fruits which are not fully dried. To ensure an acceptable moisture content, baobab fruits are subjected to solar drying, which is a slow process and often carried out in open and unhygienic conditions raising safety concerns. This study was conducted to investigate the microbial and aflatoxin contamination levels in ready-to-eat baobab products from selected formal and informal processors in specific counties of Kenya. Selected processed baobab products were sampled randomly from formal and informal processors and analyzed for the total aerobic count, Enterobacteriaceae, yeast and molds, ergosterol, aflatoxins, moisture, and water activity. The moisture and water activity of baobab pulp and candies from formal processors ranged between 7.73% and 15.06% and 0.532 and 0.740 compared to those from informal processors which ranged from 10.50% to 23.47% and 0.532 to 0.751, respectively. In this study, baobab pulp from formal processors had significantly ( Adansonia digitata) candies and pulp are ready-to-eat snacks processed from baobab fruit mostly consumed in a number of sub-Saharan Africa countries. The baobab fruit pulp is naturally dried and is rich in vitamin C, calcium, and antioxidants [Baobab . HoweveSome of the handling practices of baobab pulp may also expose the pulp to extrinsic environmental factors such as high humidity. The pulp is hygroscopic and is thus susceptible to moisture absorption leading to mold growth and subsequent spoilage and/or contamination by mycotoxins rendering the commodity unsafe for consumption . DetermiAspergillus parasiticus and Aspergillus. flavus L. strains were recovered from baobab fruit obtained from the market in Zambia, though the average aflatoxin concentrations in the baobab were below maximum allowable levels in food [ in food . HoweverAdansonia digitata L.) products in eastern and coastal Kenya” [p′ = proportion ratio, x = the size of the proportion, n = the size of the total population.This study involved the use of two classification groups: the formal and the informal processors. The term formal was used in this study to indicate processors registered with the Kenya Bureau of Standards (KEBS), whereas informal represents processors not registered with KEBS. Total population sampling was applied for formal processors (control) due to the manageable size of the population and systematic sampling for the informal processors. Details of the formal baobab product processors were obtained from KEBS where a sampling list of 15 processors was obtained with only 10 of the processors selected for this study being fully operational. For formal processors, eight processors were located in Nairobi, one in Kilifi, and one in Mombasa counties. The details of the informal processors were gathered from a baobab processor survey carried out under the project “A value chain analysis of baobab  and moisture content measurements. The water activity was measured as described in, using HygroPalm HP23-AW-A Portable Water Activity Analyzer . The water activity meter was set at awQuick mode, and sealed samples were kept at relatively constant temperature (22 ± 3°C) for 5 minutes to allow for the temperature conditions of the sample and the probe to stabilize before the displayed aw reading was recorded.All samples were subjected to water activity  were made and analyzed for [The baobab samples were each analyzed in duplicates for the total aerobic count, Enterobacteriaceae, and yeast and molds according to the AOAC microbiological method number ISO 4833, ISO 21528-2, and ISO 7954, respectively . All medyzed for : total ayzed for , 18. Ther2) of 0.999 was used to demonstrate linearity. Based on the signal-to-noise ratios (S/N) of 3 : 1 and 10 : 1, respectively, the limit of detection (LOD) and limit of quantification (LOQ) were determined [μg/ml in acetonitrile solution. Working standards were prepared by serial diluting with acetonitrile, and a 6-point calibration curve was generated covering the range of 1.25–12.5 ppm for each aflatoxin group.Ergosterol standard (E6510-5G) was obtained from Sigma-Aldrich . The ergosterol stock solution was prepared at a concentration of 1 mg/ml, and further working standards were prepared by serial dilutions with ethanol. A standard calibration curve was then generated, and a correlation coefficient of . The HPLC system consisted of an auto-sampler model SIL-20A HT, quaternary pumps (Shimadzu model LC-20 AD), a reverse-phase SUPELCO C-18 (5 μm 260 mm × 4.6 mm) column, CTO-10AVP column oven, and a photodiode detector (SPD-M20A). The separation was achieved through isocratic elution at a flow rate of 2 ml/min, with a linear gradient solvent .Extraction and quantification of ergosterol were carried out on each of the 60 baobab samples. Two grams of the sample was placed in the saponification solution composed of 15 ml of methanol and 5 ml of potassium hydroxide solution (40 g/l KOH in ethanol) and agitated for 30 minutes. This was then filtered into evaporation tubes using a 0.2 nitrogen . The ergv/v) and centrifuged at 10,000 rpm for 5 minutes. Five millilitres of supernatant was transferred to a 50 ml centrifuge tube and diluted with 40 ml of 2% PBST  solution [μm millipore filters into vials. Aflatoxin quantification was done by injecting 20 μl of the samples into a SHIMADZU HPLC system  equipped with; RF20A fluorescence detector operated at an excitation wavelength of 350 nm and an emission wavelength of 450 nm; auto-sampler SIL 20AHT, a reverse-phase SUPELCO C-18 (5 μm 260 mm × 4.6 mm) column, CTO 10ASVP column oven set at 40°C, and LC20AD quaternary pump. A mobile phase of acetonitrile : methanol : water (10 : 30 : 60 v/v) was used at a flow rate of 1 ml/min.Two grams of the baobab samples was added to an extraction solution comprising of 1 g sodium chloride in 25 ml  than those from formal processors   . This cop ≤ 0.001, p ≤ 0.001) Enterobacteriaceae and yeast and molds counts (3.1 ± 0.70 log10 CFU/g and 5.3 ± 0.11 log10 CFU/g) than those from formal processors (0.7 ± 0.29 log10 CFU/g and 3.1 ± 0.38 log10 CFU/g), respectively. Production practices, extrinsic, intrinsic, and processing factors determine the microbial load of food products at the time of consumption [The presence of microbes in food does not imply unfitness for consumption, but rather an indication of the hygienic status during preparation and processing . Nonethesumption .The total aerobic count loads in this study (4.3 ± 0.22) in pulp were higher than those of studies done on microbial analysis of freeze-dried fruits  and the The baobab pulp contamination can be traced back to handling practices along the value chain and at the farm level . To meetp = 0.015) was observed in terms of Enterobacteriaceae counts between candies from formal (0.0 ± 0.00 log10 CFU/g) and informal processors (1.8 ± 0.56 log10 CFU/g). The Enterobacteriaceae counts in baobab products in this study were higher than those reported by Ntuli et al. [10 CFU-g)   in some 0 CFU-g) . During 10 CFU/g) in the candies from the informal processors  in the total aerobic count, yeast and molds, and Enterobacteriaceae loads, respectively, among products from the regions. The highest TAC contamination in candies was detected in samples collected from Mombasa County (5.4 ± 0.28 log10 CFU/g), whereas the highest Enterobacteriaceae contamination was detected in samples collected from Kilifi county (3.3 ± 0.11 log10 CFU/g)  . This co et al., ).10 CFU/g and 5.1 ± 0.01 log10 CFU/g, respectively. Similarly, candies from informal processors in Mombasa and Kilifi recorded the highest levels of yeast and molds at 4.3 ± 0.98 log10 CFU/g and 3.6 ± 0.05 log10 CFU/g, respectively, as shown in (10 CFU/g) and those from Mombasa and Kilifi (5.4 ± 0.30 log10 CFU/g and 5.1 ± 0.01 log10 CFU/g), respectively. A similar trend can be seen in a study done on the effect of temperature on microbial growth in food during storage, where high environmental temperatures increase the proliferation of microorganisms [The baobab pulp from informal processors in Mombasa and Kilifi recorded the highest yeast and mold contamination in baobab pulp at 5.4 ± 0.30 logshown in . A signirganisms . Kilifi rganisms ).μg/kg for AFB1 and 10 μg/kg for total aflatoxins. The EU standard for baobab pulp prescribes a maximum limit of 2 μg/kg for AFB1 and 4 μg/kg total aflatoxins. Temperature, water activity, and humidity play a vital role in fungal growth and mycotoxin production [r = 0.5019, p = 0.04) , , and , respectively. The results obtained are comparable to the prediction of mycotoxin deoxynivalenol (DON) contamination in Fusarium-infected wheat grains based on the determination of the ergosterol content [oduction . A posit = 0.04) . There wSamples 1, 7, 12, and 14 had a water activity of above 0.7 , which cThe percentage of the fungal and aflatoxin-contaminated baobab samples is shown in μg/kg in the analyzed samples indicating a potential health risk to baobab consumers [The results from the study show that there is a minimal inspection of the on-shelf baobab products by the regulatory agencies. Aflatoxin concentrations were ranging between 3.93 and 11.09 × 103 onsumers . The cononsumers . Anotheronsumers .In conclusion, informal baobab processor products recorded the highest microbial contamination in terms of Enterobacteriaceae and TAC, which could be associated with unhygienic handling during processing. The results of this study incriminate the unhygienic postharvest practices along the baobab value chain as the source of contamination of the baobab pulp and candies. Therefore to ensure the safety of dried baobab fruits and baobab products along the baobab value chain, remedial steps such as training on good hygiene and good manufacturing practices as well as Hazard Analysis Critical Control Points (HACCP) should be taken .To maintain the postharvest quality of baobab fruits, right postharvest management steps should be taken. For instance, baobab drying should be carried out using certain methods, such as convective air drying and vacuum drying, in an enclosed environment. However, if solar drying is to be used, the use of indirect solar drying or enclosed solar dryers is recommended as the product is covered with a transparent cover in a cabinet that protects it from dust, insects, rains, and rodents that could otherwise lead to contamination. Appropriate primary and secondary packaging technologies should also be incorporated especially hermetic bag packages.


Zea mays L.) is nutrient specific and associated with key vegetative and reproductive growth stages. The objective of this study was to determine the fate of foliar-applied B, Fe, Mn, Zn, and Fe/Zn together, evaluate the effect of foliar micronutrients applied at multiple rates and growth stages on maize grain yield, and determine their apparent nutrient recovery efficiency (ANR). Five Randomized Complete Block Design (RCBD) experiments were conducted in 2014 and 2015 at five locations across Nebraska. Total dry matter was collected at 5–6 stages, and separated into leaves, stalk, and reproductive tissue as appropriate to determine micronutrient uptake, partitioning, and translocation. Foliar B, Mn, Zn, and Fe/Zn had no effect on grain yield for most application time by rate levels, though, at the foliar Mn site, there was a 19% yield increase due to a V18 application of 0.73 kg Mn ha−1 which corresponded with reduced Mn uptake in maize grown in control plots. At the foliar Zn site, there was 4.5% decrease in yield due to a split foliar application of 0.84 kg Zn ha−1 total, applied at V11 and V15 stage, which increased leaf Zn concentrations greater than the established toxic level. Only the Fe site had consistent grain yield response and was the only experiment that had visual signs of micronutrient deficiency. Regardless of application time from V6 to R2, there was a 13.5–14.6% increase in grain yield due to 0.22 kg Fe ha−1 foliar application. Most micronutrients had limited or no translocation, however, early season applications of B, prior to V10, had significant mobilization to reproductive tissues at or after VT. Foliar Mn, Zn, and B application had ANR LSmeans of 9.5, 16.9, and 2.5%, respectively, whereas the Fe/Zn mix had negative ANR LSmeans of −9.1% Fe and −1.3% Zn which indicate suppression. These data highlight the importance of confirming a micronutrient deficiency prior to foliar application, guide specific growth stages to target with specific micronutrients, track the fate of foliar-applied micronutrients, and describe the variable effect of foliar-applied micronutrients on grain yield.Timing of micronutrient demand and acquisition by maize ( Zea Mays L.) is associated with key vegetative or reproductive growth stages  × 100.ANR (%) = [, and ANR were analyzed using PROC GLIMMIX SAS 9.3 software  . Block w−1). Only the 2015 foliar Fe application location had visual signs of deficiency  though neither soil or plant analysis Fe was below critical levels of 4.5 mg Fe kg−1 soil as reported by Ward [−1 leaf tissue for maize as reported by Mills et al. [−1 and 181 mg Fe kg−1, respectively). Soil pH was alkaline  which likely contributed to reduced micronutrient availability and the subsequent visual signs of deficiency. This location was fully irrigated and high yielding   which was in excess . Plant t by Ward  and 10 ms et al.   .−1 in leaf tissue for maize prior to tassel) as reported by Mills et al. [−1    .−1 leaf tissue critical value at V5 , however, plant tissue Mn was relatively low compared to other sampled locations in Nebraska  were above the soil critical levels of 0.75 and 4.5 as reported by Ward [−1 in control plots)  .−1, respectively, due to mid-day sampling as compared to morning sampling, but time of day has less effect on B concentration [Though none of the targeted micronutrients were below critical levels during early season leaf tissue samples (V5–V6), it has been shown that these nutrients fluctuate throughout the day within the growing season due to environmental stresses and in some samples, micronutrient concentrations fell below their respective critical levels  during the growing season . Leaf tintration  and thusp = 0.006) due to a V18 application of 0.73 kg Mn ha−1 and a 4.5% yield decrease (p = 0.02) due to a split application of foliar 0.84 kg Zn ha−1 applied at V11 and V15 compared to the control . There was a 14.6% increase (p = 0.04) due to the 2× application at V6 , a 14.2% increase (p = 0.04) due to a split application of the 2× rate at V6 and V15 , and a 13.5% increase (p = 0.05) due the 2× application at R2 . The 2× rate consistently outperformed the 1× rate across all treatments and time of application did not have a significant effect on yield (p < 0.05). Further, the single 1× application of foliar Fe  had consistently greater yield than the control and less than the 2× rate but not significantly. As there were only three Fe rates no response function could be calculated. Therefore, it is unknown if a greater rate of Fe application would have greater response. These data highlight the importance of confirming a micronutrient deficiency prior to applying a foliar micronutrient treatment and is consistent with the deficiency correction theory.The foliar-applied B, Mn, and Zn experiments showed these nutrients had limited effect on grain yield for most application time by rate level combinations though there was a 19% yield increase ( control . The fol−1 in leaf tissue for maize prior to tassel) as reported by Mills et al. [−1 in leaf tissue for maize after tassel)  in leaf tissue but this was not associated with a significant increase in grain yield (p < 0.05). The Mn location (Oakland) had significant grain yield increase due to the T2R1 treatment . This yield increase may be due to heavy rainfall  where there was standing water on the experiment for approximately 2 weeks  as reported by Mills et al. [p < 0.05.At the B location (Meadow Grove), there were no significant yield effects. The B location for the control plots reported V6 leaf tissue below critical B concentrations  a. Both t 2 weeks  causing s et al. . Though −1 grain yield when compared to the control (p = 0.006) at the foliar Mn location. This trend may hold true for the other micronutrients but was not confirmed in this study. Overall, the 1X rate of 0.73 kg Mn ha−1 had greater effect on grain yield than did the 2× rate of 1.46 kg Mn ha−1. Planned selected contrasts confirmed that the 1× rate had significantly greater effect (p = 0.0008) on grain yield than the 2× rate and two separate applications of the 1× rate  and had significantly greater effect (p = 0.005) on yield than one application at the 2× rate . Inversely, when there is excess soil supply of the foliar-applied micronutrient, such as in the case of the foliar Zn only location (Winside), there may be yield reduction. This is supported by the significant 4.5%, 0.62 Mg ha−1 yield decrease as compared to the control (p = 0.03). Mills et al. [−1. At the Zn only location, the split application of 0.84 kg Zn ha−1 at V11 and V15 significantly increased the leaf Zn concentration from the control from 24.7 at V14 and 27.3 mg kg−1 at V17 to beyond the upper limit of the sufficiency range to 62.0 mg kg−1 (p < 0.0001) at V14 and 91.3 mg kg−1 (p < 0.0001) at V17  at V17 g, which −1 detection limit. Partitioned plant sampling following foliar B treatments at rates of both 0.14 kg B ha−1 and 0.28 kg B ha−1 and at all application times showed significant increase (p < 0.05) in B uptake and B concentration in leaf, stalk, and reproductive tissues; however, for all treatments by R6 leaf, stalk, and total B uptake had declined and were not different than the control at p < 0.05 (−1 applied at V10) increased the R6 B uptake to the reproductive tissues as compared to the control by 10.3 g B ha−1 (p = 0.001) which was due to a 3.7 mg B kg−1 concentration increase (p = 0.0005)  a. These  0.0005) a. Though 0.0005) a. V10 or−1 and 0.84 kg Zn ha−1) foliar applications of Mn and Zn significantly increased (p < 0.05) the R6 total Mn and Zn uptake as compared to the control (p < 0.05) of Zn and Mn uptake at R6 as compared to the control (−1 (p < 0.0001) and reproductive tissues Zn concentration by as much as 18.8 mg Zn kg−1 (p < 0.0001) as compared to the control  than the control biomass)  .p < 0.05. There was a consistent trend for both Zn and Mn ANR at the 2X rate to always have a greater ANR than the 1X rate for treatments applied at the same growth stage . This study design could not exclusively define that all foliar applied micronutrients were taken up through areal plant tissues as opposed to reaching the soil and uptake through the roots. This fate of foliar applied nutrients should be further studied. These data could be used to calculate application rates with specific goals of increasing micronutrient concentrations in plant tissue from below critical values to above critical values. These results can also be used to guide the use of foliar micronutrients for agronomic biofortification of maize grain. In conclusion, this study showed that foliar applications of B, Mn, Zn, and Fe had limited effect on grain yield in regions with soils and conditions like those of this study unless there is a confirmed micronutrient deficiency. A summary of our recommendations is provided in


Primary pulmonary EWS/PNET is extremely rare. This report describes a 20 year‐old man with primary pulmonary EWS/PNET with TP53 germline and SKT11 somatic mutation. After four neoadjuvant chemotherapy cycles  combined with anlotinib, a left pneumonectomy was performed. Maintenance anlotinib monotherapy was then continued with no sign of recurrence to date. It is suggested that before the treatment and prognosis of children or young adults with primary EWS/PNET of the lung that consideration should be given to genetic testing. Primary pulmonary EWS/PNET is extremely rare. This report describes a 20 year‐old man with Primary pulmonary EWS/PNET with TP53 germline and SKT11 somatic mutation.After 4 neoadjuvant therapy, R0 resection was obtained. Then maintenance anlotinib monotherapy was continued, no sign of recurrence to date as an outcome. Ewing's sarcoma is a rare and highly invasive mesenchymal tumor which belongs to the neuroectodermal tumor family, and includes Ewing's osteosarcoma, extra‐skeletal Ewing's sarcoma, primitive neuroectodermal tumor (PNET), Askin tumor and atypical Ewing's sarcoma.2, intravenous glucose tolerance test [ivgtt], D1, D8 and D15; actinomycin D 1.5 mg/m2, ivgtt, D1; cyclophosphamide, ivgtt, 1.2 g/m2, D1) and IE  repeated every three weeks. The first three cycles were combined with anlotinib . After four cycles of neoadjuvant chemotherapy, the left mass had significantly reduced to 3.4x 2.5 cm on thoracic CT re‐examination  of PPES often presents with a high SUV value, smooth margins, and high fluorodeoxyglucose uptake. In addition, PET‐CT can help to rule out the possibility of pulmonary metastasis by extra‐pulmonary Ewing's sarcoma.On light microscopy,There is currently no standard treatment for PPES. Treatment includes surgery, chemotherapy and radiation. Standard first‐line treatment consists of alternating chemotherapy with vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide. Chemotherapy has previously been reported to improve patient survival rate following radical surgery.The authors report no conflict of interest.


Nature Communications 10.1038/s41467-021-25586-4, published online 06 September 2021Correction to: The original version of this Article contained errors in the subsection “Real-space density profiles of metastable clusters and critical nuclei” of the “Results” section. The hyperlinks to Fig. 3—Fig. 3, Fig. 3a, Fig. 3b, Fig. 3c—are mistakenly hyperlinked to Fig. 2.This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained errors in the subsection “Dynamics of metastable clusters, critical nuclei and supercritical nuclei in the transition” of the “Results” section. The hyperlinks to Fig. 4—Fig. 4a, Fig. 4b, Fig. 4c—are mistakenly hyperlinked to Fig. 3.This has been corrected in both the PDF and HTML versions of the Article.