Early life stress (ELS) is known to modify trajectories of brain dopaminergic development, but the mechanisms underlying have not been determined. ELS perturbs immune system and microglia reactivity, and inflammation and microglia influence dopaminergic transmission and development. Whether microglia mediate the effects of ELS on dopamine (DA) system development is still unknown. We explored the effects of repeated early social stress on development of the dopaminergic system in male and female mice through histological, electrophysiological, and transcriptomic analyses. Furthermore, we tested whether these effects could be mediated by ELS-induced altered microglia/immune activity through a pharmacological approach. We found that social stress in early life altered DA neurons morphology, reduced dopamine transporter (DAT) and tyrosine hydroxylase expression, and lowered DAT-mediated currents in the ventral tegmental area but not substantia nigra of male mice only. Notably, stress-induced DA alterations were prevented by minocycline, an inhibitor of microglia activation. Transcriptome analysis in the developing male ventral tegmental area revealed that ELS caused downregulation of dopaminergic transmission and alteration in hormonal and peptide signaling pathways. Results from this study offer new insight into the mechanisms of stress response and altered brain dopaminergic maturation after ELS, providing evidence of neuroimmune interaction, sex differences, and regional specificity.The online version contains supplementary material available at 10.1007/s12035-022-02830-6. Adverse childhood experiences can modify trajectories of brain development, leading to aberrant maturation that increases risk for psychopathologies and neurological disorders , 2. AmonOne of the actors involved in guiding DA system development is the brain’s resident immune compartment, namely microglia. Studies have established that during brain development, microglia contribute directly to the correct maturation of the DA system and DA-dependent behaviors, by regulating outgrowth of dopaminergic axons in the mouse embryonic forebrain  and prunRecently, we reported that exposure to a threatening social environment  in the juvenile period in mice brings about microglia activation and aberrant DA-induced self-inhibitory responses of DA neurons in the developing VTA, and sensitivity towards the effects of cocaine later in life , 22. TreFollowing these results, here we used the SS model –25 to evprimipara) was used for producing only one litter to rule out the effects of changes in maternal behavior across pregnancies. Mice were kept at constant temperature (21 ± 1 °C) and humidity (55 ± 5 percent). Food and water were provided ad libitum, and mice were housed on a 12:12 light:dark cycle with lights on at 07.00 a.m. All experiments were carried out in accordance with Italian national law (DL 26/2014) on the use of animals for research based on the European Communities Council Directive (2010/63/UE) and comply with the ARRIVE guidelines. Experiments were approved by the ethics committee of the Italian Ministry of Health  and by local Ethical Committee of the Santa Lucia Foundation. All efforts were made to minimize the number of animals used and their suffering.Seven-week-old CD-1 (CD1) male and DBA2/J @Ico (DBA) male and female mice were purchased from Charles River Laboratories . DBA male and female mice were mated at 12 weeks of age, with max 3 females and 1 male per cage. Pregnant mothers were single housed around gestation day 16. Each male was used for producing max 2 litters, and each mother  injection of minocycline hydrochloride  , 26 dissAt PD22, 4–6 mice per sex per group were anesthetized with Rompun  and Zoletil  and perfused transcardially with 20-ml saline followed by 20 ml of 4% paraformaldehyde (PFA) in phosphate buffer (PB) . Brains were post fixed in 4% PFA overnight at 4 °C and then immersed in a 30% sucrose-PB solution until sinking. Afterwards, brains were cut into 30-µm-thick coronal sections using a freezing microtome.2O2 to block endogenous peroxidase and then incubated for 48 h at 4 °C with rabbit anti-tyrosine hydroxylase (TH) antibody  for detection of dopaminergic neurons. TH is the rate-limiting enzyme in the synthesis of dopamine and marks dopamine neurons in the VTA and SN. Then, sections were incubated for 2 h with a biotinylated donkey anti-rabbit antibody , followed by 2 h in an ExtrAvidin solution  at room temperature. As chromogen, 3,3’diaminobenzidine 0.05%  was used. Primary and secondary antibody solutions as well as ExtrAvidin solutions were prepared in PB and 0.3% Triton X-100. Each incubation step was followed by three 5 min rinses in PB. The specificity of immunohistochemical labeling was evaluated based on established guidelines on TH + staining in the midbrain [For stereological and morphological analyses of dopaminergic neurons, every second slice approximately between – 3.08 and – 3.80 mm from Bregma (based on ) containmidbrain . SectionFor immunofluorescence staining, a total of 4–6 regularly spaced sections of VTA and SN  were seSections processed for TH immunohistochemistry were used for obtaining unbiased estimates of total number and morphology of TH + neurons, and total surface area of the VTA and SN. The boundaries of these areas were defined by TH staining, according to published guidelines . In the https://www.mbfbioscience.com/help/si11/Content/Contours/contourmeasure.htm) were collected to evaluate somatic shape based on literature [Neuronal morphology analysis on sections processed for TH immunohistochemistry was performed using an optical microscope  equipped with a motorized stage and a camera connected to the Neurolucida 7.5 software (MicroBrightField) , 32. Thrterature , 33: celSections were examined through a confocal laser scanning microscope (Zeiss CLSM800). Acquisitions of one left or right hemisphere per section (hemisections) were performed by using a 20 × /0.50 objective  so that all samples within each experiment were captured using consistent settings for laser power and detection gain. Images were acquired at 1024 × 1024-pixel resolution (image size 638.9 μm per side) and 8-bit depth and exported in carl zeiss image format (.czi). PBP and PN subareas of the VTA, and distinction between VTA and SN were identified with the aid of an atlas . AnalyseN = 3 mice/group) was performed as described previously [2/5% CO2 gas mixture at a constant flow rate (2.5 mL/min). Slices were visualized with a microscope’s in-build CCD camera (Photometrics Evolve). Referring to the mouse practical map of DA neuron location in distinct VTA subregions previously reported by our group [Preparation of midbrain slices  co-transported with 2 Naradients –42, and radients –45. DA  and brains were dissected, deprived of cerebellum and pons-medulla, and stored at − 80 °C. Punches of VTA were obtained from coronal brain slices no thicker than 300 μm according to Ventura et al. [Male pups were decapitated at PD22 . Universal Plus mRNA-Seq kit  has been used for library preparation following the manufacturer’s instructions. Final libraries were checked with both Qubit 2.0 Fluorometer  and Agilent Bioanalyzer DNA assay or Caliper . Libraries were then sequenced on single-end 75 bp mode on NextSeq 500 . On average 30 million reads were produced per sample.ftp://ftp.ensembl.org/pub/release-102/gtf/mus_musculus/). Read counts were then input into DESeq2 in R, and differential expression analysis was conducted with default options [p-value was less than 0.05. Subsequent to scaling of raw read counts and execution of differential expression analysis, read counts were transformed by DESeq2’s variance stabilizing transformation (VST), in order to stabilize read count variance as a function of total reads number. Transformed gene expression for genes with adjusted p-value less than 0.05 was standardized and plotted in the heatmap. Rows and columns underwent hierarchical clustering, considering Euclidean distance as distance metric. Heatmap plot was performed using the pheatmap R-package. Gene ontology analyses and gene networks were obtained by Ingenuity pathway analysis . Over-representation analysis was carried out using the R packages DOSE and ReactomePA [Read quality was asserted through FastQC, and quality/adapter trimming was carried out using Cutadapt. Single end reads were aligned to mouse reference mm10 using STAR aligner. Subsequently, reads were counted for genes using htseq-count  repository, under accession number GSE199136.P < 0.05), ANOVA was followed by planned post hoc comparisons (Tukey HSD test) and nested  one-way ANOVA. Nested ANOVA was performed to take into account variability within samples  as well as variability within and between groups [t-test (t-test). Minocycline experiments were analyzed by two-way ANOVA, considering group (stress) and treatment (minocycline) as main factors. For all tests, significance was set at P < 0.05. All data are expressed as mean ± standard error of the mean (s.e.m.). Statistica software Version 12.0  was used to perform the statistical analyses.All data were first checked for normality by graphical inspection of the residuals’ distribution and for homogeneity of variances with the Levene’s test. VTA and SN were analyzed separately. Analysis of the main effects of group and sex, and interactions between these variables on immunohistochemical data was performed with two-way analysis of variance (ANOVA). In case of main factor or interaction significance , sex , or group*sex  effect . Similar results were obtained for the SN . These results indicate that male and female CTR and SS mice display similar DA neurons number and VTA/SN total area.Since it has been demonstrated that dopaminergic neurons undergo a wave of physiological death/apoptosis at PD14 , and thaλ = 0.93, F3,114 = 2.90; P = 0.036), but no significant effect of sex  or group*sex interaction . Univariate results indicated a significant group effect only on aspect ratio  and roundness , but not on cell body area , but not between female CTR and SS animals , demonstrating that SS male DA neurons present an altered, elongated shape, with reduced short axis and augmented long axis, resulting in increased aspect ratio and decreased roundness compared to CTR .This effect was restricted to the VTA, as no significant main or interaction effect was detected in the SN  but not in the SN , and no main effect of group or sex in either region . Similar results were obtained for TH intensity . Post hoc comparisons of VTA data revealed significant differences between male CTR and SS DAT (P = 0.039) and TH (P = 0.038) intensity, but not between female CTR and SS , demonstrating that SS induced down-regulation of DAT and TH fluorescence intensity in male VTA compared to CTR.We performed immunofluorescence staining and confocal imaging to assess distribution and expression of dopamine transporter DAT) and tyrosine hydroxylase (TH) proteins Fig. , B, whic SS Fig. , D. ThisAT and tyN = 10 cells) displayed significant lower amplitudes of DAT dependent currents compared with CTR (N = 12 cells)  expression in CTR-saline and CTR-minocycline male VTA at PD22. TH intensity and Iba1 + cell count were comparable between minocycline- and saline-injected mice , treatment , and group*treatment interaction . Univariate results indicated a significant group*treatment effect only on roundness , but not on the aspect ratio , and no main effect of group or treatment on either index. Post hoc comparisons confirmed significant differences in the roundness only between CTR and SS untreated mice (P = 0.012), but no difference between the other groups, suggesting that roundness levels in SS-minocycline mice returned to CTR levels .Thus, we proceeded by comparing CTR, SS, CTR-minocycline, and SS-minocycline mice to evaluate group (stress) and treatment (minocycline) effects. MANOVA of somatic indices showed a significant effect of group , and no effect of the group or the treatment alone. Post hoc comparisons confirmed significant differences between CTR and SS untreated mice (P = 0.035), CTR-minocycline vs SS-minocycline (P = 0.0037), SS untreated vs SS-minocycline (P < 0.001), but no difference between CTR untreated and CTR-minocycline (P = 0.33), and in CTR untreated vs SS-minocycline (P = 0.13), indicating that minocycline treatment in SS mice restored immunoreactivity of DAT to CTR levels .Two-way ANOVA of DAT immunoreactivity showed a significant effect of group*treatment interaction  and group*treatment interaction , but no effect of treatment alone. Post hoc comparisons showed significant differences between CTR and SS untreated mice (P = 0.035), CTR-minocycline vs SS-minocycline (P < 0.001), SS untreated vs SS-minocycline (P < 0.001), and CTR untreated vs SS-minocycline (P = 0.0084), indicating that minocycline treatment in SS mice (but not CTR mice) increased immunoreactivity of TH to levels above CTR .Two-way ANOVA of TH immunoreactivity revealed significant main effect of group (padj < 0.05) modulated 81 genes (En1 and En2), specification , differentiation  and maturation . Moreover, some of these genes encode for proteins that are critical for dopamine metabolism, such as the TH and the DDC, and for dopamine activity, such as the DAT and the dopamine receptor D2, DRD2. Other highly significant downregulated genes include the GTP cyclohydrolase 1 gene (Gch1), responsible for the production of a TH cofactor, nicotinic cholinergic receptors  receptor subunit beta-3), genes associated with synaptic vesicle exocytosis such as complexin 1 and 2 genes, neuron-derived neurotrophic factor, and the peptide guanylate cyclase 2c (Gucy2c). Among the few upregulated genes, the most interesting targets were the corticotropin releasing hormone binding protein (Crhbp), the thyrotropin releasing hormone (Trh), and the neuropeptide neurokinin 2/alpha   Fig. .Fig. 4SSP < 0.05). We focused on pathways associated with dopaminergic function, developmental processes, and neuro-psychological disorders. Significant diseases or functions annotations have been grouped on the basis of IPA categories in  software was used to determine the functional annotation and to predict the biological pathways affected by SS  at the end of the stress procedure. Our transcriptome analysis confirmed massive downregulation of numerous dopaminergic markers, including TH and DAT genes, and alterations in pathways involved in the synthesis, signaling, and degradation of DA. This effect is not a consequence of reduction in dopaminergic neurons, as CTR and SS mice exhibit the same number of DA cells in the VTA, but it is probably due to transcriptional regulation processes that ultimately affect protein expression, as we demonstrated for TH and DAT. Furthermore, pathway analysis predicted alterations associated with neurodevelopmental trajectories and neurological and psychiatric disorders. Overall, transcriptome, immunohistochemical, and electrophysiological data suggest that SS interferes with developmental programs of the male VTA, possibly inducing diminished DA signaling at the end of the ELS procedure (PD22). Reduction of DA function may be an adaptive response to the repeated, inescapable stress that pups face . It has Gch1 gene, the rate-limiting enzyme in the production of BH4 [Microglia and immune pathways may be important regulators of the observed dopaminergic effects, since minocycline was able to rescue the deficits in DAT and TH induced by SS. It has been shown that immune-derived cytokines and prostaglandins cause reductions in DA synthesis, release, and DA metabolites, possibly via depletion of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase and TH –99. Thisn of BH4  whose den of BH4 . AdditioCrhbp [Tac2 and Trh genes. Tac2 is upregulated in the brain after chronic social isolation stress [Trh is involved in the regulation and release of thyroid-stimulating hormone (thyrotropin) and prolactin from the pituitary gland. In the VTA, it is possibly expressed by a sub‐group of GABA and/or glutamate neurons or coming from hypothalamic afferent fibers [Gucy2c, which encodes a transmembrane receptor for the intestinal hormone uroguanylin that takes part in the gut-brain endocrine axis regulation of feeding behavior. In the brain, it is selectively expressed by the hypothalamus and dopaminergic neurons from the VTA and SN and distributed in their afferent sites [Among the few upregulated pathways, we found hormonal and peptide signaling pathways. The related DEGs include markers that are canonically involved in hypothalamic–pituitary–adrenal axis-VTA responses to stress and drugs of abuse, such as Crhbp , and newn stress , and itsn stress . Trh is t fibers . TRH seet fibers . Moreovet fibers , 109. A nt sites . It is int sites , 112. Innt sites –116.In light of these results, we speculate that during social stress exposure, the VTA may be a station where neurotransmitters, hormones, peptides, and immune molecules signal internal homeostatic states from the brain and the periphery to influence central dopaminergic neurotransmission and establish appropriate learning and predictions , 117. ThA possible limitation of the study concerns the fact that we stopped analyzing females after we detected significant effects only in males. Including female mice in subsequent experiments  would have been advantageous to better explore the observed sex differences and to draw more reliable conclusions on the potential of minocycline to prevent SS-induced dopaminergic aberrations. Future efforts should be made to address this limitation, and to detect which brain substrates are affected by SS in female mice at different developmental time points, in order to provide better knowledge on possible sex-specific pathophysiology in chronic medical conditions.Overall, these results suggest important targets of developmental repeated social stress effects, with sex and regional specificity, and involvement of microglia and immune system in the mediation of these effects. Additional investigations are needed to evaluate whether the SS-induced developmental dopaminergic alterations described here are maintained in adulthood, or whether different adaptations will occur in the system, and the possible causal relationship with psychopathological risk. A better understanding of such pathways is critical for improving strategies that counteract aberrant brain maturation and psychopathological outcomes following adverse early experiences.Supplementary file1 (PDF 192 KB)Supplementary file2 (CSV 10 KB)Below is the link to the electronic supplementary material.


This study suggests that South Africa has an intermediate HAV seroprevalence, with rates < 90% by 10 years of age (68.6%). Increased age and informal dwellings are statistically associated with HAV seropositivity, while HIV status does not significantly influence HAV seropositivity.Hepatitis A virus (HAV) infection is one of the most important global causes of viral hepatitis. Recent reviews suggested that HAV endemicity in South Africa could shift from high to intermediate. A hospital-based HAV seroprevalence study was conducted between February 2018 and December 2019 in Pretoria, South Africa. Systematic sampling was performed on children and adolescents (1–15 years) who attended outpatient services. Participants with a known HIV status and valid HAV serology results were included. Of the 1220 participants, the median age was 7 years (IQR: 4–11), with 648 (53.11%) males and 572 (46.89%) females. Of 628 (51.48%) HIV-infected participants, most  were both immunologically and virologically controlled or had low-level viremia . Almost three-quarters  were living in formal dwellings, and just over half  had access to clean water sources inside the house. Increasing age was associated with testing HAV IgG-positive (OR 1.25; 95% CI 1.20–1.30,  Global epidemiological data estimate that 1.5 million of people are infected annually with acute HAV, with the highest HAV-associated mortality occurring in sub-Saharan Africa and Asia2. Age at natural infection with HAV and burden of disease are closely linked to the economic development of a country. Shifts in age-specific prevalence patterns that reflect a transition from high to intermediate endemicity are linked to improvements in socioeconomic factors. A decrease in age-seroprevalence rates and HAV infection incidence is strongly associated with socioeconomic factors such as household income and improved clean water sources within communities3. Countries where HAV endemicity is changing from high to intermediate have demonstrated an increase in hepatitis A infections severity, leading to more clinically severe cases and consequently in an increase in fulminant hepatic failure secondary to HAV infection2. Hence, large-scale hepatitis A vaccination is likely to be cost-effective in such circumstances, as supported by the World Health Organization (WHO) recommendation for HAV vaccination in populations with < 90% seropositivity by age 10 years. Integration of HAV vaccines into the national immunization schedules is recommended in the abovementioned scenario2.Hepatitis A virus (HAV) infection is one of the most important causes of viral hepatitis, both in South Africa and worldwide4. Although the true burden of hepatitis A disease in South Africa is not known at present, a seroprevalence review of laboratory data from 2005 to 2015 demonstrated that seroprevalence rates of more than 90% are only reached in adulthood within the South African public health sector, suggesting that South Africa could be in transition from high to intermediate endemicity5. More recent reviews performed by Patterson et al. in 2019 supported the suspicion that South Africa is most likely changing endemicity from high to intermediate6.Prior to 2005, Southern African HAV seroprevalence rates among children aged 1–4 and 5–9 years were estimated to be as high as 83 and 92%, respectively8.Human immunodeficiency virus (HIV) infection is one of the most serious health concerns in South Africa. The country has the highest number of people afflicted with HIV of any country and, according to the 2019 United Nations statistics, the fourth-highest adult HIV prevalence with a rate of 19%, with only Botswana (25%), Lesotho (25%) and Swaziland (27%) recording higher prevalence rates. According to Statistics South Africa’s mid-year population estimates for 2018, the total HIV prevalence rate for the country was 13.1%. The HIV prevalence rate for all adults aged 15–49 is 19.0%, with an estimated quarter of a million children < 15 years of age living with HIV in South Africa9. In countries with high HAV endemicity, no significant difference in HAV seroprevalence was observed between HIV-positive and HIV-negative adult cohorts10. In contrast, HIV-positive adults usually have a higher HAV seroprevalence than their HIV-negative counterparts in more developed countries, with low HAV endemicity11. HAV seroprevalence among HIV-infected children and adolescents has not been described in South Africa before. Hence, we conducted a seroprevalence surveillance study in an urban setting in healthy, HIV-infected and HIV-uninfected children and adolescents to assess current HAV endemicity.The correlation between HIV and HAV infection in adults varies according to the local epidemiologyA hospital-based cross-sectional analytical study was carried out. A probability sampling method was adopted using systematic sampling (screening and recruiting the 5th registered patient in the subsequent clinic queue) of children aged 1–15 years attending weekday outpatient department services and clinics at Kalafong Provincial Tertiary Hospital in Pretoria, South Africa. Recruitment was performed between February 2018 and December 2019. Children and adolescents with minor illnesses (i.e. illnesses not necessitating admission) and/or those attending follow-up visits were enrolled once written parental consent and patient assent (> 8 years) were obtained. Participants and their caregivers were interviewed using a standardized questionnaire, and blood samples were collected for anti-HAV IgM and IgG testing. Anti-HAV serology testing (both IgM and IgG) was performed using the Abbott ARCHITECT i2000SR immunoassay analyzer. Only participants with a known HIV status (laboratory evidence of HIV-infected or HIV-uninfected status) and valid anti-HAV IgM and IgG results were included in the study. HIV-infected participants were recruited from the paediatric HIV clinic, a weekday clinic for HIV-infected children and adolescents. All HIV-infected children and adolescents have access to age-appropriate universal antiretroviral therapy at the clinic.The sample size was calculated assuming the frequency of HAV IgG to be 50%, with power set at 80% and precision at 5%. The results of these assumptions give a sample size of 383 per age group: 1–5 yrs; 6–10 yrs; 11–15 yrs. The total sample size of the study was calculated to be 1149.p value of < 0.2. Statistical analysis was performed using R version 3.6.3.The percentages of Hepatitis A IgG- and IgM-positive results, as well as variables associated with these results, are reported as proportions of study participants. Participant age was reported as the median with interquartile range (IQR) in years. A multivariable logistic regression analysis was performed, during which odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to determine whether specific participant characteristics were associated with positive hepatitis A IgG serostatus. Ten variables were included in the model, namely, age of the participant , HIV status of participant, maternal HIV status during pregnancy with participant, participant residence , participant access to running tap water inside place of residence, pit latrine ablution facilities at place of residence, employment status of primary caregiver, participant attendance at day-care and/or school, caregiver receipt of a government support grant, and caregiver level of education. Inclusion in the multivariable model was dependent on an associated univariable logistic regression All methods were carried out in accordance with relevant guidelines and regulations. The research protocol was approved by the University of Pretoria Medical Research Ethics Committee 362/2017. Informed consent and assent (where applicable) were obtained from all subjects and/or their legal guardian(s). All patient data and laboratory results were anonymised, kept confidential and stored in a password-protected database.Among 1253 participants between the ages of 1 and 15 years enrolled in the study, 1220 were included in the final analysis , with 648 (53.11%) males and 572 (46.89%) females. There were a total of 628 (51.48%) participants who were known to be HIV-infected and receiving antiretroviral therapy. Only one (0.08%) participant indicated that they had previously received an HAV vaccine, and 5 (0.41%) had a known previous HAV infection. The majority of participants had access to a flush toilet, with 22.05% using a pit latrine. Although almost three-quarters  of households were living in formal dwellings, just over half  had access to clean water sources (taps) inside the house. A summary of variables, including indicators of socioeconomic status, is presented in Table 1, with 6+ T cell counts were available for the preceding 6-month period in 573 (91.24%). All HIV-infected participants received antiretroviral therapy at the time of enrollment. Moderate and severe immune suppression, as evidenced by CD4+ T cell counts percentages of 15–24% (moderate) and < 15% (severe) for age < 5 years and absolute values of 200–499 cells/µL (moderate) and < 200 cells/µL (severe) for children and adolescents 5 years and older, were documented in 118 of 578 (20.42%) results available, most being moderately immune suppressed . Virological suppression was documented in 367 of 574 (63.94%) participants, with HIV viremia > 1 000 copies/mL found in 113 (19.69%) participants.Of 628 HIV-infected participants, both the HIV viral load and CD4Most HIV-infected children and adolescents 329, 71.83%) were both immunologically and virologically controlled or had only a low level of viremia < 1 000 copies/mL . Among the 20 (3.49) participants who were severely immune suppressed, 14/20 (70%) had a viremia of > 1 000 copies/mL , with 19.8% of participants one year of age positive compared with 86.7% of participants 15 years of age  participants who tested anti-HAV IgG positive and 32 (2.62%) anti-HAV IgM positive, all of which were also IgG positive. Increasing age was associated with an increased likelihood of testing IgG positive  and living in an informal dwelling  were statistically significant  are reported. Increased age and informal dwellings are statistically associated with HAV seropositivity, while HIV status does not significantly influence HAV seropositivity rates when adjusted for the other variables.12. Hence, our cohort demonstrated slightly better virological control than the national average. HIV status did not have a significant impact on HAV seroprevalence; therefore, we suggest that countries with a high HIV burden should consider similar prevention strategies in HIV-infected and HIV-uninfected children and adolescents.Although HIV infection in children and adolescents and maternal HIV status showed a higher likelihood of the child/adolescent being anti-HAV IgG positive, HIV infection did not prove to be a significant indicator of HAV seropositivity in multivariate analysis. Compared to the South African national HIV data, our HIV-infected cohort demonstrated better (71.83%) immune and virological control. Virological testing yielded a 64% suppression rate, 16% low-level viremia, and 20% viremia. For South Africa, between July 2019 and June 2020, among all children and adolescents (aged 1–15 years) with an HIV viral load test, 49% were virally suppressed, 24% had low-level viremia, and 27% had viremia with VL > 1000 copies/ml13. Further to the high cost and morbidity/mortality, Bruckmann et al. showed that HAV is currently the leading cause of pediatric acute liver failure requiring transplantation in South Africa, with even further expenses and disease burden to the health sector14.Our data clearly illustrate that South Africa has now progressed to a country with an HAV intermediate endemicity rate, with HAV seroprevalence by 10 years of age only 68.6%, and > 90% seropositivity only reached beyond 15 years of age. Considerations for prevention strategies, such as HAV immunization, should include cost analyses. The cost-effectiveness of universal hepatitis A vaccination is well documented in other intermediate HAV endemicity regions, such as Argentina, Brazil, Chile, and Mexico. Patterson et al. reviewed patients presenting with hepatitis A at tertiary level hospitals Cape Town, South Africa, and calculated the total cost per hepatitis A hospitalization of $1935.41 for adult patients and $563.06 for pediatric patients. Furthermore, more than 1 in every 10 hepatitis A cases (13.3%) developed complicated hepatitis A or resulted in death15, showed that 13.1% of South African households were living in informal dwellings, 89.0% had access to an improved source of water, and 83.0% of households had improved sanitation. Our study participants were more frequently living in informal dwellings (26.7%), had less access to clean water sources inside the household (43.6%), and had less access to proper sanitation (77.9%) compared to the general South African average in 2018. This would likely have overestimated the HAV seroprevalence, making it even more evident that South Africa has reached an intermediate HAV endemicity.The study was limited by participant enrolment in a single urban centre. However, Kalafong Provincial Tertiary Hospital services a wide community of informal settlement households with poor access to clean water and sanitation. The General Household 2018 survey, released by Statistics South AfricaIn conclusion, the intermediate prevalence of total anti-HAV among children and adolescents in South Africa suggests the country’s improvement of safe water and food supply, hygiene, and sanitation. However, it leaves older children and adults vulnerable to acute HAV infection, and there is a potential for outbreaks. The public health sector should consider raising public awareness through hygiene promotion and preventive measures such as vaccination.Supplementary Information.


Morbid obesity is one of the fastest-growing subgroups of obesity and is associated with high mortality, with an estimated 2.8 million people dying from obesity each year.  This research sets out to elucidate the impact of sevoflurane (Sevo) inhalation anesthesia on the clinical outcome of morbidly obese (MO) patients undergoing laparoscopic bariatric surgery (LBS). peak), plateau pressure (Pplat), standard time out of PACU, postoperative food intake (FI), length of stay (LOS), and complication rate.  A retrospective study was conducted on 150 MO patients undergoing LBS in the Second Affiliated Hospital of Xi'an Jiaotong University from November 2019 to November 2021. According to the difference of anesthesia methods, 100 patients with Sevo anesthesia were set as group A, and 50 patients with propofol (P) anesthesia were set as group B. Intergroup comparisons were performed in terms of eye-opening time, tracheal intubation removal time, directional force recovery, heart rate (HR), mean arterial pressure (MAP), peak airway pressure (Ppeak, and Pplat. Group A was also superior to Group B in the standard time out of PACU, postoperative FI, and LOS, with a lower complication rate.  Group A had a shorter time to open eyes, remove tracheal intubation, and restore directional force than Group B, with better recovery of HR, MAP, P Sevo inhalation anesthesia is more effective and safer for MO patients undergoing LBS. In rubgroups . Morbid-ubgroups , 7. In rBariatric surgery has long been considered the most effective method to treat severe obesity and its complications, with mortality rates falling below 0.1 per cent in the past decade . Currentn = 100) and Propofol (P) anesthesia Group B (n = 50) according to the difference in inhaled anesthetics during surgery. Inclusion criteria: the participants were all MO patients undergoing LBS in our hospital, with BMI >40 kg/m2, normal psychology, and communication ability, as well as the consent and signed relevant agreements from patients and their families. Exclusion criteria: surgical contraindications; drug allergies or endocrine system diseases; history of cerebral hemorrhage and cerebral infarction within 1 year; and severe heart and brain organ lesions. This research was conducted after obtaining approval from the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University.From November, 2019 to November, 2021, 150 MO patients undergoing LBS were selected and assigned to Sevo anesthesia Group A  The positive end-expiratory pressure (PEEP) was set as 5 cm H2O when initiating the ventilation, which was increased by 10 cm H2O and 15 cm H2O after 5 times of ventilation to gradually increase the tidal volume (TV) until the peak airway pressure (Ppeak) reached 40 cm H2O; (3) TV was restored to the level before PRM by gradually reducing the PEEP to 10 cm H2O, 5 cm H2O and 0 cm H2O. Scopolamine was injected intramuscularly 30 min before anesthesia. After opening the patient's venous access, 10 ml/(kg·h) Sodium Lactate Ringer's Injection was administered intravenously, and the mask oxygen inhalation was maintained at 2–4 L/min. The patient's heart rate (HR) and mean arterial pressure (MAP) were detected by puncture and catheterization around the left artery under local anesthesia. Besides, the patient was given an iv bolus of 0.2 mg/kg atracurium and 0.4 g/kg sufentanil. Furthermore, Group B was given 3 mg/kg P, while Group A was given Sevo with an initial and a maximum inhalation concentration of 7.0% and 8.0%, respectively. The patient underwent mechanical ventilation after spontaneous breathing disappeared, with a respiratory ratio of 1 : 1.5, a frequency of 12 times/min and a TV of 8–9 ml/kg. The anesthesia concentration was adjusted according to the bispectral index scale (BIS), HR, and blood pressure (BP) of patients, and BIS was maintained at 40–50 to keep the HR stable. Urapidil was given if BIS <40 and BP was 120% above normal. Phenylephrine was administered if the patient's BP was below 70% of normal; in cases with HR > 90 beats/min, esmolol was given, while atropine was given to those with HR < 50 beats/min. The anesthetic concentration was increased once BIS >50 was observed; 5 μg of sufentanil was administered when the patient's HR was more than 90 beats/min or the BP was 120% higher than normal. Phenylephrine was given if BP was below 70% of normal. And in those with a HR < 50 beats/min, atropine was given. The inhalation of P and Sevo was gradually reduced 15 min before surgery, and withdrawn after the completion of the operation. When patients resumed spontaneous breathing, 0.5 mg atropine, and 1 mg neostigmine were injected. Airway pressures were remonitored in all patients postoperatively and the results were recorded.All patients were fasted for 10h and abstained from drinking for 6h preoperatively. The airway was established by tracheal intubation before anesthesia, and preoperative airway pressure monitoring and results recording were performed. All patients underwent LBS in the supine position. Pneumoperitoneum was established during the operation, and pulmonary recruitment maneuver (PRM) were performed every 30 min. The steps of PRM are as follows: (1) The respiration rate and pressure limit were set as 8 beats/min and 40 cm HThe times of eye-opening time (from stopping anesthetics to being awakened to open eyes), the time of tracheal intubation removal  and the time of restoring orientation  were observed and recorded.The preoperative and postoperative hemodynamics  were compared.peak, plateau pressure (Pplat)) of the two groups was compared.The pre- and posttreatment PF , postoperative food intake, and length of stay (LOS).The postoperative complications were compared. The related indicators included nausea, vomiting, labored breathing, and chills.χ2 test; the t-test was used to identify the difference of quantitative data  denoted by (s). A significance level of P < 0.05 was used in this study.SPSS19.0  and GraphPad Prism 6  were employed for comprehensive statistical analysis and visualization of data, respectively. Enumeration data , denoted by number of cases/percentage (n/%), were compared by the P > 0.05)  ( > 0.05) .P < 0.05)  .P < 0.05)  .peak and Pplat in Group A compared with Group B (P < 0.05)  .P < 0.05)  .P < 0.05)  .Morbid obesity is an extremely challenging disease that affects every aspect of patients' lives . MinimalP. Although anesthesia benefits various surgical procedures and patients, improper anesthesia puts patients at greater risk of neurocognitive impairment and even reduces their processing speed and impairs their fine motor abilities [P, another drug most commonly used in international anesthesia and intensive care, also has good pharmacokinetics and rapid and reversible sedation [P, with better and effective recovery of PF and hemodynamics. In the study on the application of Sevo in elderly patients with lung cancer, Qin et al. [P, Sevo inhalation general anesthesia can more effectively improve PF with less effect on patients' cognitive function, which is consistent with our results.From the perspective of anesthesia recovery time, Group A using Sevo had shorter eye-opening time, earlier tracheal intubation removal, and faster directional force recovery than Group B using bilities . In an abilities , 20. So,bilities , has antbilities . P, anotsedation . Howeversedation . Therefon et al.  also sugP anesthesia is not only less effective than volatile anesthetics such as Sevo but also less safe, with greater side effects [P, which explains better postoperative recovery in patients who used Sevo.In terms of postoperative complication rate and patient recovery, a lower complication rate and better recovery of various indicators were determined in Group A. It has been indicated that volatile anesthetics are safe and effective. The use of Sevo promotes early extubation and facilitates rapid transfer of patients from the operating room to the recovery room, which has a positive effect on patient recovery and restores the degree of respiratory protective reflexes and arousal as quickly as possible , 27. P a effects . TherefoThere are still many deficiencies in this study. Due to the limitations of research conditions, we were unable to detect the relevant molecular indicators of patients. Nor have we investigated patients' status during surgery and their satisfaction with the two surgical anesthesia methods. In future studies, we will supplement these tests while continuously improving the surgical procedures to facilitate patients' recovery.Collectively, Sevo inhalation anesthesia has a better anesthetic effect for MO patients undergoing LBS, with a higher safety profile, which is worthy of clinical promotion .


Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and antifungal activity against Aspergillus niger and Chaetomium globosum fungal mold species as model microorganisms. Cytotoxicity screening of the tested modified material was carried out on BALB/3T3 clone mouse fibroblasts. The SEM/EDS and FAAS tests showed good uniformity of zinc content on a large surface of the composite. The conducted research showed the possibility of using the magnetron sputtering technique as a zero-waste method for producing antimicrobial textile composites.The aim of this investigation was to evaluate the biological properties of cotton–zinc composites. A coating of zinc (Zn) on a cotton fabric was successfully obtained by a DC magnetron sputtering system using a metallic Zn target (99.9%). The new composite was characterized using scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), UV/Vis transmittance, and atomic absorption spectrometry with flame excitation (FAAS). The composite was tested for microbial activity against colonies of Gram-positive ( Cotton is a popular fabric based on cellulose ,2—the moN-halamines  was added to each well. Absorbance was read at 540 nm using a Multiskan™ GO spectrophotometer . Results were expressed as the percentage cell survival (OD of exposed vs. OD of control unexposed cells).After the 24 h incubation of BALB/3T3 clone A31 cells with the extracts, the medium was gently aspirated, and the wells were washed with 150 µL of Dulbecco’s phosphate-buffered saline (PBS) with CaThe analysis of changes in the morphological structure of fibers in cotton fabric under zinc modification was carried out using scanning electron microscopy. According to the image analysis, it can be noted that the fibers in cotton fabric were characterized by a rather smooth surface with characteristic parallel ridges and grooves. The fibers in cotton fabric with a magnetron-sputtered zinc layer were characterized by a rougher surface with visible ridges and grooves in the fibers. The zinc coating exhibited a regular distribution of the applied modifier particles.In order to confirm and verify the uniformity of the zinc coating of the fibers, an energy-dispersive X-ray spectroscopy (EDS) study was performed, which provided a chemical analysis of the tested fabric and its elemental composition. The EDS surface analysis  in the fThe study indicated a uniform distribution of zinc on the surface-modified fibers. The black areas visible in the images were the spaces between the fibers in the yarn . Due to the dense and uniform zinc coating of the fibers, carbon became less visible, which was confirmed by the quantitative analysis of the content of elements on the fiber surface  and EDS The nature of the so-called zinc–cellulose interface matter (Zn–cell–OH) still remains unclear, since the zinc atom is reactive, and cellulose possesses reactive hydroxyl functions. It is well documented that zinc reacts with alcohols with the temporary formation of zinc alcoholate intermediates, which, in an aqueous environment, rearrange into zinc oxide ,121,122.The reactions of zinc with alcohols presumably proceeded in accordance with Subsequently, zinc atoms of a metallic monolayer deposited on the cellulose surface reacted during deposition with cellulose, forming corresponding alcoholates , which c1.2O1.1) suggests a nearly quantitative character of passivation. It is worth adding that the formation of ZnO during sputtering with pure zinc has been described in a few papers using pure Zn target in an argon–oxygen [The following layers of deposited zinc atoms were attached to the lower zinc layer, forming appropriate zinc multilayers. The zinc atoms of the upper layer were oxidized to ZnO, by reaction of zinc atoms with oxygen or water. This mechanism was partly confirmed by EDS test . The resn–oxygen ,124,125 n–oxygen . Anothern–oxygen .The determination of zinc content in COT–Zn composite samples was assessed by the FAAS method, and the results are presented in (0) material bulk after modification was uniform.The zinc content in composite samples strictly depended on the applied sputtering metallization times. The process of Zn deposition on the cotton sample was practically linear (COT–Zn-5 (1 s)—9.06 g/kg, COT–Zn-10 (1 s)—20.19 g/kg, COT–Zn-10 (2 s)—41.52 g/kg), and the distribution of metal in COT–ZnS. aureus and Gram-negative E. coli. Results of the tests are illustrated in The COT–Zn composites were tested in vitro for antimicrobial activity against Gram-positive A comparison of these results (ZID) with representative data from the literature is given in E.coli and S.aureus  and C. globosum (ATCC 6205) for the cotton sample and COT–Zn composites are illustrated in The results of the antifungal activity tests (ZID) in accordance with PN-EN 14119:2005 against colonies of A. niger or C. globosum, as expressed by the strong visible fungal growth covering the entire surface of the COT samples (A. niger and C. globosum were demonstrated by COT–Zn-10 (1 s/2 s) samples modified by magnetron sputtering metallization. The results revealed visible zones of fungal growth inhibition in Petri dishes  did not inhibit the growth of  samples a. Antifui dishes b, with n2, ZnSO4, or Zn(OAc)2). Since zinc metal presents lower solubility than ZnO (1 μg/L vs. 3.6 μg/L) [2→COT + Zn2+) is much longer, and these composites should preserve their antimicrobial nature/characteristics for much longer.The zinc oxide impact on bacteria or fungi depends on its morphology (particle size and shape), concentration, exposure time, pH, etc. . This is.6 μg/L)  and much.6 μg/L) ,152,153,Cytotoxicity assays ,155,156 2+ ions. These, in turn, induce cytotoxicity by increasing the excessive intracellular Zn ion concentration [Zinc salts have proven to exert a strong biological effect, i.e., an antimicrobial effect toward several bacteria , as wellntration ,176,177.ntration , using tntration ,178.Macroscopic observations of the extracts showed no changes in transparency for unmodified cotton, while the modified cotton had a visibly darker color, brightening after sedimentation, and leaving a black residue A.. MicroThe results of the NRU assay showed no decrease in viability of fibroblasts exposed to the unmodified cotton extracts at both tested concentrations. COT–Zn extracts significantly reduced cell viability, causing almost 100% cell death, irrespective of the extract concentration . TreatmeZinc compounds also reveal strong anticancer activity e.g. ,180,181),181180,1E. coli and S. aureus bacteria and A. niger and C. globosum fungi. The in vitro determined antimicrobial properties of COT–Zn composites revealed the antibacterial and antifungal activities. In vitro studies showed also that COT–Zn composites containing merely 9 g/kg Zn were cytotoxic. The ability to adapt clean and zero-waste magnetron sputtering methods to an industrial scale provides the possibility to obtain sustainable materials for use in a variety of applications. The prepared fiber composites have great application potential as an antimicrobial material in the field of biomedical engineering ; however, due to their cytotoxicity they have limited possibilities of use as a material interacting with cells of the human body.In summary, COT–Zn composites with different Zn content were prepared by DC magnetron sputtering technology using a zinc metal target. The composite samples were characterized by SEM, EDS, and FAAS. The biological properties of the materials were verified by cytotoxicity screening and antimicrobial activity tests against colonies of


Rhododendron fortunei, we performed a comprehensive transcriptome and metabolomic analysis of the petals of two different varieties of two alpine rhododendrons: the scented R. fortunei and the unscented Rhododendron ‘Nova Zembla’.To reveal the key genes involved in the phenylpropanoid pathway, which ultimately governs the fragrance of R. fortunei but were downregulated in Rhododendron ‘Nova Zembla’. Among these genes, EGS expression was significantly positively correlated with various volatile benzene/phenylpropanoid compounds and significantly negatively correlated with the contents of various nonvolatile compounds, whereas CCoAOMT, PAL, C4H, and BALDH expression was significantly negatively correlated with the contents of various volatile benzene/phenylpropanoid compounds and significantly positively correlated with the contents of various nonvolatile compounds. CCR, CAD, 4CL, and SAMT expression was significantly negatively correlated with the contents of various benzene/phenylpropanoid compounds. The validation of RfSAMT showed that the RfSAMT gene regulates the synthesis of aromatic metabolites in R. fortunei.Our transcriptomic and qRT–PCR data showed that nine candidate genes were highly expressed in R. fortunei. This lays a foundation for further research on the molecular mechanism underlying fragrance in the genus Rhododendron.The findings of this study indicated that key candidate genes and metabolites involved in the phenylpropanoid biosynthesis pathway may govern the fragrance of The online version contains supplementary material available at 10.1186/s12870-022-04016-7. Rhododendron is the largest genus in the heath family (Ericaceae) and the largest genus of woody angiosperms found in China. Members of this genus are widely distributed throughout the Northern Hemisphere, from tropical Southeast Asia to northeastern Australia [Rhododendron genus is complex, with varieties grouped into scented and unscented types depending on their aroma. Rhododendron fortunei is a scented rhododendron in the subgenus Azaleastrum [Rhododendron fortunei is widely distributed along sunny ridges or as a secondary layer in forests at an altitude of 620–2000 m above sea level [Rhododendron ‘Nova Zembla’ is a hybrid in the subgenus Azaleastrum. It is evergreen, and its flowers are broad and funnel-shaped, with dark rose-red petals and dark maroon spots present on the uppermost petals. Rhododendron ‘Nova Zembla’ flowers are unscented. This variety can tolerate a low temperature of − 28 °C and can survive in high-altitude areas, and it is one of the best-selling varieties of alpine rhododendrons. Compared with research on other flower characteristics, such as flower colour and type, research on flower fragrance is lacking. Currently, research on synthesis pathways and key genes involved in plant floral aromatic metabolites has become a topic of interest [ustralia . Rhododeustralia , 2. Taxoleastrum  and has leastrum . Rhododeea level , 6, and interest .Barringtonia racemosa have been detected and analysed. Shi et al. [Barringtonia racemosa are terpenoids, mainly linalool, and benzene/phenylpropanoid compounds, mainly phenylacetaldehyde [Floral aroma is an important plant trait and plays a key role in plant growth, development, and evolution. Floral aroma has protective functions and attracts both pollinators and seed dispersers . It is ai et al.  summarizi et al. , 14, andi et al. . Kong eti et al.  investigi et al.  detectedaldehyde .Magnolia, of which 46.9% were terpenes, 38.9% were volatile esters, and 5.2% were benzenes/phenylpropanoids, all of which are related to floral compounds. Moreover, the expression levels of most of the individual synthesis-related genes were found to be higher in the flowers than in the leaves.In recent years, high-throughput sequencing methods have been widely used to study the relationship between the contents of various secondary metabolites in plant organs and corresponding differentially expressed genes (DEGs) . MetabolR. fortunei at different developmental stages and screening out key candidate genes involved in the phenylpropanoid pathway, we elucidated the synthesis mechanism of benzene/phenylpropanoid floral aromatic compounds. We found that key genes involved in the phenylpropanoid pathway regulate the fragrance of R. fortunei, which lays a foundation for further research on the molecular mechanism of aromatic compound synthesis in rhododendron and provides a scientific basis for the breeding of novel scented rhododendron varieties.At present, research on the molecular mechanism underlying the synthesis of aromatic compounds in rhododendron is lacking, but this type of research is necessary for breeding new rhododendron varieties with aromatic flowers. Due to the limitations of GC–MS technology for the detection of nonvolatile metabolites, the precursors of floral aromatic compounds, such as p-hydroxycinnamic acid and methylglutaric acid, cannot be reasonably measured during the dynamic metabolic changes in organisms. However, by applying LC–MS positive (POS) and LC–MS negative (NEG) techniques, we can largely detect the accumulation of various nonvolatile metabolites. When the LC–MS POS and LC–MS NEG techniques were used, better separation was observed for compounds with different pKa values. In this study, a combined transcriptomic and metabolomic analysis was used to study differences in gene expression and metabolite accumulation in the petals of two different rhododendron varieties (a scented rhododendron and an unscented rhododendron). Focusing on the study of the differentially accumulated benzenes/phenylpropanoids of the two rhododendron varieties and the dynamic changes in benzenes/phenylpropanoids in Rhododendron ‘Nova Zembla’ (NW)   Fig. a and R. YJ) Fig. b at diffYJ) Fig. , namely,YJ) Fig. c, d, e. R. fortunei and Rhododendron ‘Nova Zembla’ at the same developmental stage  . There were significant differences in metabolites between the different varieties at the same developmental stage. For the NW1 vs. YJ1 comparison group, 481 differentially accumulated metabolites were identified by GC–TOF–MS, of which 413 and 68 of these metabolites increased and decreased in abundance, respectively Fig. a. SimilaThe metabolites of YJ also varied greatly at different developmental stages. For the YJ1 vs. YJ2 comparison group, a total of 469 differentially accumulated metabolites, including 396 and 73 that increased and decreased in abundance, respectively, were detected by GC–TOF–MS , and LC–MS (NEG) in different developmental stages and between varieties were screened, and a heatmap was constructed according to the relative quantitative values of the samples to visualize the data Fig. a, b, c. Using the DNBSEQ platform for sequencing, we obtained a total of 153.37 Gb of sequencing data after assembly and deredundancy were performed. A total of 140,527 unigenes were identified, and the total length, average length, N50 and GC content were 168,644,638 bp, 1200 bp, 1887 bp, and 44.65%, respectively. The number of unigenes with a length of 300–400 bp constituted the greatest proportion – 12,714 unigenes, accounting for 15.01%. With an increase in length, the number of unigenes decreased, and the number of unigenes longer than 3000 bp increased , cinnamate 4-hydroxylase (C4H), 4-coumaroyl-CoA ligase (4CL), salicylic acid carboxyl methyl transferase (SAMT), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), eugenol synthase (EGS), and benzaldehyde dehydrogenase (BALDH). According to the transcriptome sequencing results, RSEM was used to calculate the gene expression levels in each sample, and a heatmap was constructed to visualize the expression levels of the abovementioned genes in the petals of the different rhododendron varieties at different developmental stages to visualize the results. Figure 4CL, CCR, and CAD were expressed in in YJ at each developmental stage, and the expression of each unigene was downregulated in YJC. The expression of PAL was downregulated in YJB and YJC, that of CCoAOMT was downregulated in YJC, and that of C4H was downregulated in YJD. EGS expression was upregulated in YJB and high in YJB and YJC, SAMT expression was upregulated in YJD, and BALDH was highly expressed in YJC and YJD.According to the identification and KEGG enrichment analysis results of the DEGs, those in the NWD vs. YJD, YJB vs. YJC, and YJC vs. YJD comparison group were found to be enriched in the phenylpropanoid biosynthesis pathway. After consulting the literature on the study of floral genes involved in the phenylpropanoid pathway, we screened nine DEGs for analysis  data, we conducted qRT–PCR analyses of the nine candidate genes. The expression levels of the candidate genes in the petals of NW and YJ were measured at stages A, B, C, and D. The transcript profiles of all the selected genes were highly consistent with those detected from the RNA-seq data Fig. . The expPAL expression and significantly negatively correlated with C4H, 4CL, and BALDH expression; eugenol content was significantly positively correlated with PAL expression and significantly negatively correlated with CAD expression; benzoic acid methyl ester content was significantly negatively correlated with BALDH expression; and methyl salicylate content was significantly negatively correlated with 4CL and CAD expression and significantly positively correlated with BALDH and CCR expression , benzenemethanol, 4-trimethyl, and eugenol were significantly positively correlated with BALDH, 4CL, CAD, and EGS expression and significantly negatively correlated with CCR expression -bicyclosesquiphellandrene, methyl benzoate, salicylic acid (SA), methyl ester, α-terpineol, linalool, 3-carene, 3,5-dimethoxytoluene, sapinene, and β-bourbonene.  as a starting substrate and undergo a series of complex branch pathway reactions. The benzene/phenylpropanoid synthesis pathway is a branch of the phenylpropionic acid synthesis pathway, also known as the cinnamic acid pathway , 26. In PAL, C4H, 4CL, SAMT, CCoAOMT, CCR, CAD, EGS, and BALDH  and SA as substrates to synthesize methyl salicylate. SAMT has strong substrate specificity, and the pathway by which generate methyl salicylate is synthesized is part of the benzene/phenylpropanoid synthesis pathway; different substrates can be catalysed through the activity of enzymes in this pathway, and different products can be obtained. When the substrate is benzoic acid, SA can be formed under the catalytic activity of benzoic acid 2-hydroxylase (BA2H), and under the action of benzoic acid carboxyl methyl transferase (BAMT), the volatile methyl benzoate can be directly generated. RfSAMT expression was found to be significant negatively correlated with methyl salicylate content. It is speculated that when the expression of SAMT increases, the content of methyl salicylate increases, while the content of methyl salicylate decreases. In this study, the content of methyl salicylate decreased after transient infection of YJ compared with WT YJ. After transient overexpression, the content of methyl salicylate was 0.17 times that in the WT  and 15-year-old Rhododendron ‘Nova Zembla’ (NW) plants, which were growing in Siming Mountain National Forest Park, Ningbo city, Zhejiang Province. Voucher specimens of these rhododendron varieties are deposited in the germplasm resource garden of Siming Mountain National Forest Park, Ningbo city, Zhejiang Province, and public sampling is allowed there. The formal identification of the species was conducted by the corresponding author of this article. Ten YJ plants displaying with similar growth and 10 NW plants displaying with similar growth were randomly selected, and flower buds at different stages of opening were harvested without causing mechanical damage. Then, they were grouped into four developmental stages according to the degree of petal opening: the bud stage , first-bloom stage , bloom stage , and decay stage . The stamens were removed, after which the petals were quickly frozen in liquid nitrogen and then stored at − 80 °C for further analysis. The four samples of YJ and NW used for the metabolomic analysis were designated NW1–4 and YJ1–4, respectively, and six biological replicates were included per sample; those used for the RNA-seq analysis were designated NWA-D and YJA-D, respectively, and three biological replicates were included for sample.The test materials used were 9-year-old Rhododendron varieties (NW and YJ) at different developmental stages. A total of 100 ± 10 mg of each sample was placed into a 20 Ml headspace vial, and 10 Μl of 2-octanol  was added. The volatile metabolites in the petal samples were extracted by an SPME-PAL gas-phase solid-phase microextraction automatic sampling system for subsequent GC–TOF–MS detection and analysis. GC–TOF–MS analysis was performed using an Agilent 7890 gas chromatograph system coupled to a 5977B mass spectrometer; the system utilized DB-Wax. LC–MS was used to detect nonvolatile metabolites in the samples. An appropriate amount of sample was ground in liquid nitrogen, 50 mg of ground sample was added to an Eppendorf tube, and 1000 Μl of extraction solution  was added. Then, the samples were homogenized at 35 Hz for 4 min and sonicated for 5 min in an ice water bath; the homogenization and sonication cycles were repeated three times. Then, the samples were incubated for 1 h at − 40 °C and centrifuged at 12000 rpm for 15 min at 4 °C. The resulting supernatant was transferred to a fresh glass vial for analysis. A quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples. LC–MS analyses were performed using an UHPLC system  equipped with a UPLC HSS T3 column  coupled to a Q Exactive HFX mass spectrometer . The mobile phase consisted of 5 mmol/L ammonium acetate and 5 mmol/L acetic acid in water (A) and acetonitrile (B). The autosampler temperature was 4 °C, and the injection volume was 3 Μl. Statistical significance and fold-changes of the abundance of metabolites between samples were tested by Student’s t test. Metabolites for whose contents differed according to a P value ≤0.05 and a VIP ≥ 1 were considered significantly differentially accumulated.By applying GC–TOF–MS and LC–MS techniques, we detected metabolites in the two First, a QC analysis was performed to verify the reliability of the data. The data detected by GC–TOF–MS were analysed using Chroma TOF 4.3X software from LECO Corporation  and via information from the Nist database for raw peak extraction, filtering of data baselines, calibration of the baseline, peak alignment, deconvolution analysis, peak identification, integration and spectrum matching of the peak area. The raw data detected by LC–MS were converted to mzXML format using ProteoWizard and processed with a program written by staff at the biomedical company Shanghai Yuanxin Biomedical Technology Co., Ltd. . The program was developed using R and is based on XCMS for peak detection, extraction, alignment, and integration. Then, an in-house MS2 database (BiotreeDB) was applied for metabolite annotation. The raw data were analysed as described by Dunn et al. .Total RNA was extracted from eight groups of samples using a TRIzol reagent kit  according to the manufacturer’s instructions, and three biological replicates were included. Total RNA sample QC, library construction, and sequencing were conducted by staff at BGI Tech . The RNA concentration and quality were tested using an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) , which included determining the RNA concentration, RNA integrity number (RIN) value, 28S/18S, and fragment length distribution. The Mrna was enriched by Oligo (Dt) magnetic beads, and then the obtained RNA was fragmented with interrupt buffer. First-strand complementary DNA (Cdna) was synthesized by N6 random primers, and double-stranded cDNAs were prepared by the use of these short fragments as templates. Adapters were ligated to the short fragments using T4 DNA ligase , and after end repair and ligation of the adapters, the products were enriched by PCR to generate Cdna libraries. The libraries were subjected to sequencing with the sequencing platform DNBSEQ , and the products were considered raw reads.P value) ≤ 0.05.All the generated raw sequencing reads were filtered to remove the reads containing adapter sequences, low-quality reads, and reads comprising more than 5% ambiguous residues (N) by SOAPnuke software . After filtration, the remaining reads were considered clean reads and stored in FASTQ format. Trinity software was used to de novo assemble the clean reads, and then TGICL was used to cluster the assembled transcripts to remove redundant reads to obtain unigenes. TGICL was used again to cluster the eight samples of unigenes to remove any redundant sequences and obtain the final unigenes for follow-up analysis. All the assembled nonredundant and filtered unigenes were annotated by BLAST software against the contents within the Nr database, the Nt database, the Pfam database, the SwissProt protein database, the KOG database, the GO database and the KEGG database –48. The -ΔΔCT method was used for data analysis, SPSS 26.0 was used for one-way ANOVA, and Origin software was used for constructing the figures.Total RNA was extracted with an RNAprep Pure Plant Plus Kit , and cDNA was synthesized using a NovoScript Plus All-in-One 1st Strand cDNA Synthesis SuperMix (gDNA Purge) Reverse Transcription Kit . Primer 6 software was used to design specific primers for nine genes identified from the transcriptomic data , for sequencing verification. The bacterial solution with correct sequence was shaken, and the plasmids were extracted with an E.Z.N.A.® HP Plant DNA Kit  according to the manufacturer’s instructions. The pCAMBIA1302 vector was linearized with Ncol-HF restriction endonuclease, and the primer G-SAMT  following the manufacturer’s instructions, and the total RNA was reverse transcribed using a NovoScript Plus All-in-One 1st Strand cDNA Synthesis SuperMix (gDNA Purge) Reverse Transcription Kit  to synthesize first-strand cDNA. With cDNA as a template and the designed primer Q-SAMT  ethanesulfonic acid, 10 mM; pH 5.8). The OD 600 value was adjusted to 0.8 to obtain a sufficient Agrobacterium infection solution. Twenty unopened flower buds were randomly selected for injection with the infection solution. An additional 20 unopened flower buds displaying the same growth status were selected at the same time and injected with an Agrobacterium infection solution with the empty pCAMBIA1302 plasmid without the SAMT target gene. The solutions were incubated in the dark for 3 days in an opaque protective bag. Then, the bag was removed, and the solutions were cultured under natural conditions.After successful sequencing, samples were picked and added to 3 mL of Luria–Bertani (LB) liquid media consisting of 50 mg/L kanamycin and 50 mg/L rifampin, after which the solution was shaken at 200 r/min for 12 h at 28 °C. Then, the bacterial solution was shaken under the same culture conditions at a ratio of 1:50 between the bacterial solution and the LB liquid medium containing antibiotics. The supernatant was subsequently removed by centrifugation at 5000 rpm, and the bacterial cells were resuspended in buffer  at different varieties (NW and YJ). Figure S18. Top 20 KEGG pathways with the most significant DEG enrichment in NWAvsYJA. Figure S19. Top 20 KEGG pathways with the most significant DEG enrichment in NWBvsYJB. Figure S20. Top 20 KEGG pathways with the most significant DEG enrichment in NWCvsYJC. Figure S21. Top 20 KEGG pathways with the most significant DEG enrichment in YJAvsYCB. Figure S22. Correlation analysis of NW2 vs. YJ2 phenylpropanoid pathway candidate genes and benzene/phenylpropanoid substances. a. GC-TOFMS, b. LC-MS (POS), c. LC-MS (NEG). Figure S23. Correlation analysis of NW3 vs. YJ3 phenylpropanoid pathway candidate genes and benzene/phenylpropanoid substances. a. GC-TOFMS, b. LC-MS (POS), c. LC-MS (NEG). Figure S24 Correlation analysis of NW4 vs. YJ4 phenylpropanoid pathway candidate genes and benzene/phenylpropanoid substances. a. GC-TOFMS, b. LC-MS (POS), c. LC-MS (NEG). Figure S25. Correlation analysis of YJ1 vs. YJ2 phenylpropanoid pathway candidate genes and benzene/phenylpropanoid substances. a. GC-TOFMS, b. LC-MS (POS), c. LC-MS (NEG). Figure S26. Correlation analysis of YJ2 vs. YJ3 phenylpropanoid pathway candidate genes and benzene/phenylpropanoid substances. a. GC-TOFMS, b. LC-MS (POS), c. LC-MS (NEG). Figure S27. Correlation analysis of YJ3 vs. YJ4 phenylpropanoid pathway candidate genes and benzene/phenylpropanoid substances. a. GC-TOFMS, b. LC-MS (POS), c. LC-MS (NEG).Additional file 2: Table S1-S6.


The triglyceride-glucose (TyG) index is a reliable alternative biomarker of insulin resistance (IR). However, whether the TyG index has prognostic value in critically ill patients with coronary heart disease (CHD) remains unclear.Participants from the Medical Information Mart for Intensive Care III (MIMIC-III) were grouped into quartiles according to the TyG index. The primary outcome was in-hospital all-cause mortality. Cox proportional hazards models were constructed to examine the association between TyG index and all-cause mortality in critically ill patients with CHD. A restricted cubic splines model was used to examine the associations between the TyG index and outcomes.A total of 1,618 patients (65.14% men) were included. The hospital mortality and intensive care unit (ICU) mortality rate were 9.64% and 7.60%, respectively. Multivariable Cox proportional hazards analyses indicated that the TyG index was independently associated with an elevated risk of hospital mortality  and ICU mortality . The restricted cubic splines regression model revealed that the risk of hospital mortality and ICU mortality increased linearly with increasing TyG index (P for non-linearity = 0.467 and P for non-linearity = 0.764).The TyG index was a strong independent predictor of greater mortality in critically ill patients with CHD. Larger prospective studies are required to confirm these findings.The online version contains supplementary material available at 10.1186/s12933-023-01737-3. Coronary heart disease (CHD) remains the leading cause of disease burden globally, and an increasing number of people die prematurely because of CHD . AccordiInsulin resistance (IR), defined as a decrease in the efficiency of insulin in promoting glucose uptake and utilization, is a prominent characteristic of metabolic syndrome . The triTherefore, the aim of the present study was to evaluate potential relationships between the TyG index and all-cause mortality in critically ill patients with CHD.The present study was a retrospective observational study. Data for our analysis came from a publicly available Medical Information Mart for Intensive Care III (MIMIC-III) database. MIMIC-III is a large freely available database comprising information for patients admitted to critical care units at a large tertiary care hospital in Boston between June 1, 2001 and October 10, 2012 . One autWe included 38,511 patients (≥ 18 years of age) admitted to the ICU in MIMIC-III, whereas patients without CHD at admission were excluded. Subsequently, we further excluded patients with missing triglyceride (TG) and glucose data on the first day of admission. A total of 1,618 patients were included in the final study cohort and divided into four groups according to the quartiles of the TyG index of the first day of the ICU stay  with PostgreSQL (version 9.6) was used to extract baseline characteristics, including sex, age, body mass index (BMI), and severity at admission  score, Systemic inflammatory response syndrome (SIRS) score, Acute physiology score III (APSIII), Simplified acute physiological score II (SAPSII)), comorbidities, and laboratory variables within the first 24 h after ICU admission from the MIMIC-III database. The TyG index was calculated as ln [fasting TG (mg/dl) × fasting glucose (mg/dl)]/2 , 13. CHDVariables with missing data are common in the MIMIC-III, single imputation was used to impute missing values. Variables with missing rate over 20% were converted to dummy variables in the models to avoid possible bias caused by direct filling missing values. All screening variables contained < 25% missing value , acute coronary syndrome, ischemic heart disease, or percutaneous coronary intervention or coronary artery bypass grafting . AKI wast-test. Categorical variables are presented as frequencies and percentages, and differences between groups were performed with a Pearson chi-square test or Fisher’s exact test. The associations between the TyG index and cardiovascular risk factors were assessed with Spearman’s rank correlation test or Pearson’s correlation test. To evaluate the incidence rate of primary outcome events among groups according to different levels of the TyG index, we used Kaplan–Meier survival analysis, and discrepancies among groups were evaluated with log-rank tests. We used Cox proportional hazards models to estimate the hazard ratio (HR) and 95% confidence interval (CI) between the TyG index and primary outcomes, and adjusted for multiple models. To avoid overfitting the model because of multicollinearity among variables, we also calculated the variance inflation factor. Variables with variance inflation factor ≥ 5 were excluded. Finally, clinically relevant and prognosis-associated variables were enrolled in the multivariate model: model 1: unadjusted; model 2: adjusted for age, sex, and BMI; model 3: adjusted for age, sex, BMI, dyslipidemia, hypertension, diabetes, CKD, respiratory failure, white blood cell (WBC), red blood cell (RBC), hemoglobin, SCr, and SIRS score. We used a restricted cubic splines model to examine the associations between the TyG index and outcomes. The TyG index was entered into the models as continuous variables and categorical variables . The P values for trends were obtained through use of the quartile level as an ordinal variable. We further stratified analyses according to sex, age (≤ 65 and > 65 years), BMI (< 30 and ≥ 30 kg/m2), diabetes, hypertension, CKD, and AMI to identify the consistency of the prognostic value of the TyG index for primary outcomes. The interactions between TyG index and variables used for stratification were examined with likelihood ratio tests.Continuous variables are presented as the mean ± standard deviation or median with interquartile range, and were compared with Student’s P-value < 0.05 was considered statistically significant for all analyses.We used R 4.1.3  and SPSS 24.0  for data analysis. A two-sided A total of 1618 patients were finally enrolled in the present study. The mean age of the enrolled patients was 68.05 ± 14.05 years, and 1054 (65.14%) were men. The average TyG index value for all enrolled patients was 9.14 ± 0.71. The hospital mortality and ICU mortality rate were 9.64% and 7.60%, respectively (Table P = 0.011), hospital length of stay , ICU mortality , and hospital mortality  increased gradually.Baseline characteristics of the study patients according to the TyG index quartiles are presented in Table P < 0.05). In terms of laboratory indicators, participants with an endpoint event had higher levels of WBC, neutrophils, serum potassium, fasting blood glucose (FBG), SCr, and blood urea nitrogen, but lower levels of lymphocytes, RBC, hemoglobin, total cholesterol, low-density lipoprotein (LDL), and albumin (P < 0.05). No significant difference was observed in sex, BMI, hypertension, dyslipidemia, diabetes, AMI, TG, high-density lipoprotein (HDL), and hemoglobin A1c (HbA1c) (P > 0.05). Patients in the non-survivor group had higher SOFA scores, SIRS scores, APSIII, and SAPSII than those in the survivor group. The TyG index levels in the non-survivor group were significantly higher than those in the survivor group .Baseline characteristics between survivors and non-survivors are presented in Table As shown in Table P = 0.0058, Fig. P = 0.0058, Fig. The Kaplan–Meier survival analysis curves for incidence of primary outcomes among groups, according to the TyG index quartiles are shown in Fig. P = 0.002) and the fully adjusted model  when the TyG index was a continuous variable. Furthermore, when the TyG index was a nominal variable, it was also associated with hospital mortality in both an unadjusted model  and a fully adjusted model , and showed a tendency to increase with the TyG index   2.30 (1.54–3.44)], those without diabetes [HR (95% CI) 2.09 (1.41–3.08)], those with hypertension [HR (95% CI) 1.86 (1.09–3.20)], those without hypertension [HR (95% CI) 1.83 (1.20–2.79)], those without CKD [HR (95% CI) 1.75 (1.26–2.43)], those with AMI [HR (95% CI) 1.61 (1.10–2.36)], and those without AMI [HR (95% CI) 2.04 (1.06–3.91)] . Interestingly, the predictive value of the TyG index seemed to be more prominent in patients with BMI < 30 kg/m2  and without diabetes   cohort. The main finding of the study was that an elevated TyG index was a strong independent predictor of greater mortality in critically ill patients with CHD, and this finding persisted after adjustment for possible confounders. In addition, our study revealed that the TyG index was significantly linearly associated with the risk of all-cause mortality in critically ill patients with CHD.Despite guideline-directed therapy, CHD remains the primary cause of death globally . HyperglNumerous clinical studies have been conducted to investigate the association of the TyG index with CVD morbidity and mortality in the general population and many patient cohorts. Park et al.  have fouCurrent data about associations between TyG index and critically ill patients are limited. Recently, Zhai et al.  found th2 . In contrast, a previous study has reported that the predictive value of the TyG index is more prominent in patients with BMI > 28 kg/m2 [Intriguingly, in our study, approximately seven-tenths of the patients had AMI; however, our subgroup analysis showed that the predictive value of the TyG index was consistent in patients with and without AMI. Therefore, the predictive value of the TyG index for mortality in CHD patients remained consistent regardless of whether the patient was admitted to the ICU for AMI. However, we did not find any association between the TyG index and in-hospital all-cause mortality in participants with diabetes or CKD at baseline. This outcome might be explained by reverse causality: patients previously diagnosed with these illnesses generally might have been under treatment or might have adopted healthier habits; thus, their analytical parameters might have been well controlled despite their high risk of all-cause mortality . In addi28 kg/m2 . This diIn addition, despite the IR was not a traditional risk factor of CHD . HoweverAlthough the exact biological mechanisms accounting for the relationship between TyG index and mortality remain unclear, the possible crucial pathway may be associated with IR. IR is a state of decreased sensitivity and responsiveness to the action of insulin. Individuals with IR are predisposed to the development of several metabolic disorders, such as hyperglycemia, dyslipidemia, and hypertension, all of which are strongly associated with poor CVD outcomes . The chrThe main strength of our study was that we confirmed that an increase in the TyG index was a strong independent predictor of greater mortality in critically ill patients with CHD admitted to the ICU in a US cohort. However, this study also had several limitations. First, this was a single-center retrospective study and therefore could not definitively establish causality. Despite multivariate adjustment and subgroup analyses, residual confounding factors might have affected the prognosis. Second, we did not compare the risk of all-cause mortality between patients treated with percutaneous coronary intervention and coronary artery bypass grafting, owing to the limited number of studies. Finally, our analysis focused only on the prognostic value of the baseline TyG index in CHD. However, the TyG index might have changed during the hospital stay; hence, further research is needed to verify whether the change in the TyG index also predicts mortality.Together, our results extended the utility of the TyG index to critically ill patients with CHD, and demonstrated that the TyG index was a potential predictor of hospital and ICU mortality among these patients. Moreover, the TyG index was significantly linearly correlated with the risk of all-cause mortality in critically ill patients with CHD. Measuring the TyG index could contribute to risk stratification and prognosis prediction in patients with CHD. Further studies are required to determine whether interventions focused on the TyG index improve clinical prognosis in this population.Additional file 1: Table S1. Missing number for risk variables and outcome variables.Additional file 2: Figure S1. Hazard ratios (95% CIs) for ICU mortality according to TyG index quartiles after adjusting for age, sex, BMI, dyslipidemia, hypertension, diabetes, chronic kidney disease, respiratory failure, white blood cell, red blood cell, hemoglobin, serum creatinine, SIRS score.Additional file 3: Figure S2. Forest plots of hazard ratios for the primary endpoint in different subgroups.


The predictive value of maximum left atrial volume index (LAVI), phasic left atrial strain (LAS) and other standard echocardiographic parameters assessing left ventricular (LV) diastolic function to discriminate a future worsening of diastolic function (DD) in patients at risk is unclear. We aimed to prospectively assess and compare the clinical impact of these parameters in a randomly selected study sample of the general urban female population.p < 0.001). With an area under the curve (AUC) of 0.88 (95%CI 0.82–0.94) and 0.84 (95%CI 0.79–0.89), LASr and LAScd exhibited the highest discriminative value in predicting worsening of diastolic function, whereas LAVI was only of limited prognostic value [AUC 0.63 (95%CI 0.54–0.73)]. In logistic regression analyses, LAS remained a significant predictor for a decline of diastolic function after controlling for clinical and standard echocardiographic DD parameters, indicating its incremental predictive value.A comprehensive clinical and echocardiographic evaluation was performed in 256 participants of the Berlin Female Risk Evaluation (BEFRI) trial after a mean follow up time of 6.8 years. After an assessment of participants’ current DD status, the predictive impact of an impaired LAS on the course of DD was assessed and compared with LAVI and other DD parameters using receiver operating characteristic (ROC) curve and multivariate logistic regression analyses. Subjects with no DD (DD0) who showed a decline of diastolic function by the time of follow-up showed a reduced LA reservoir (LASr) and conduit strain (LAScd) compared to subjects who remained in the healthy range (LASr 28.0% ± 7.0 vs. 41.9% ± 8.5; LAScd −13.2% ± 5.1 vs. −25.4% ± 9.1; The analysis of phasic LAS may be useful to predict worsening of LV diastolic function in DD0 patients at risk for a future DD development. The importance of assessing the left atrium (LA) in evaluating left ventricular (LV) diastolic function is well established –3. The pThe standardization of LA strain (LAS) nomenclature and analysis by 2D STE as well as the definition of normal reference ranges have been recently published , 13 makiAccordingly, the aim of our longitudinal study was to prospectively assess the clinical value of phasic LAS alterations to predict a worsening of diastolic function over time in a randomly selected sample of study participants without clinically overt DD.The Berlin Female Risk Evaluation (BEFRI) trial comprised a randomly selected urban female population aged 25–74 years. A detailed description of the BEFRI design has been already published . In 2013We reinvited every woman who participated in the BEFRI echo study feasible for the analysis of LA structure and function for follow-up examinations. These took place between October 2019 and December 2020. Next to the assessment of demographical and clinical data, an extensive transthoracic echocardiography was performed with the focus on DD assessment as well as on LA and LV strain analysis. Sample characteristics at baseline and follow-up are presented in A comprehensive transthoracic echocardiographic examination was performed using the same system applied for baseline examinations . The predefined echocardiographic study protocol can be inspected in the 2D STE strain studies were analyzed offline using the EchoPAC v203 software . Global peak systolic longitudinal LV strain (LV GLS) was determined from apical 4-chamber, 2-chamber, and long-axis views (17 segment LV model). Phasic LAS was assessed as proposed by the recent EACVI recommendations  from an 2. Study participants who fulfilled 50% of these diagnostic criteria were graded to have signs of impaired diastolic function (DD1) while subjects meeting >50% of these criteria were assigned to the DD2 group (overt DD)  . All othApplying these criteria to data of both measurement occasions let us define a progression variable that indicates whether the DD status of the woman deteriorated  or remained the same .Descriptive statistics for baseline and follow-up measures were computed for the full sample and subgroups based on a combination of baseline DD status and DD progression.R2 (Nagelkerke) measures. A p-value <0.05 was considered statistically significant. In addition, receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic value of phasic LASr, LAScd, LAVI, and e′ velocity. All analyses were done in R  with usage of the additional package pROC , we decided to include only LASr or LAScd, respectively, as predictor. All predictors were centered at their mean value. Models were compared using a likelihood ratio test, information criteria, and Pseudo- age pROC , 24.Mean follow-up time was 6.8 years . Of 449 study subjects reinvited in written form for follow-up examinations, 332 gave written consent (response rate 73.9%). Of these 332 participants, two visits were canceled due to unexpected other medical procedures, nine patients were lost to follow-up, and 63 scheduled follow-up examinations were canceled due to restrictions in relation to the SARS-CoV-2-pandemic situation. Of the 258 participants who were included in the study, two were excluded from analysis due to the development of moderate to severe mitral regurgitation and atrial fibrillation, five because of poor acoustic window, and two due to reduced speckle tracking quality of the LA wall resulting in a total follow-up sample size of 249 women.Demographic and clinical characteristics of the total sample and subgroups depending on initial status and progression are shown in 2 – still in the normal range  and an AUC of 0.84 (95% CI 0.79–0.89), LASr and LAScd values showed a high discriminative power in predicting a decline of diastolic function over time. These LA function parameters performed significantly better than the echocardiographic standard parameter LAVI  showed an average e′ <9.0 cm/s (1). Vice versa, of the 63 participants who were classified to have DD0 and a LASr <34% at the time of baseline examinations, 31 then developed DD at follow-up (49%) compared to only 2% (3 of 157) participants with a baseline LASr ≥34%. Estimation of PA pressure and assessment of E/e′ was not helpful in discriminating patients with development of DD since PA pressure was not assessable in 157 patients, and baseline E/e′ was <14 in 248 participants.31 of 34 (91.2%) of the previously healthy women who – according to recent ASE/EACVI-recommendations – showed a decrease of diastolic function by follow-up showed a reduced baseline LASr according to our ROC analysis (LASr <34%). In contrast, only 6 of the 34 women (17.6%) featured a LAVI >34 ml/mχ2(1) = 28.9, p < 0.001), a lower AIC (181.1 vs. 154.2), and a higher Nagelkerke Index (0.35 vs. 0.49).When comparing the reference model with established measures and the extended model, including LASr improved the model as indicated by a significant Likelihood Ratio Test . The patients/participants provided their written informed consent to participate in this study.AB: funding of study, conception and design, data collection, supervision of study, analysis and interpretation, writing the manuscript, critical revision of manuscript, and funding. ER: data collection, analysis and interpretation, writing the manuscript. AW and DB-W: data collection, analysis and interpretation, critical revision of manuscript. US: funding of study, conception and design, critical revision of manuscript. CC: statistical analysis, analysis and interpretation, critical revision of manuscript. SB: analysis and interpretation, critical revision of manuscript. IM: data collection, analysis and interpretation, critical revision of manuscript. HD, KS, VR-Z, UL, and FK: conception and design, analysis and interpretation, critical revision of manuscript. VS: conception and design, analysis and interpretation, writing the manuscript, critical revision of manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the DZHK (German Center for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research). Grant number 81Z0100211.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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