Iris japonica were explored. Under drought stress, the root system, leaves, rhizomes, and terrestrial stems of plants were sequentially affected; the root system was sparse and slender; and the leaves lost their luster and gradually wilted. Among the physiological changes, the increase in the proline and soluble protein content of Iris japonica enhanced the cellular osmotic pressure and reduced the water loss. In anatomical structures, I. japonica chloroplasts were deformed after drought treatment, whereas the anatomical structures of roots did not substantially change. Plant antioxidant systems play an important role in maintaining cellular homeostasis; but, as drought stress intensified, the soluble sugar content of terrestrial stems was reduced by 55%, and the ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase (MDHAR) activities of leaves and the MDHAR activity of roots were reduced by 29%, 40%, 22%, and 77%, respectively. Overall, I. japonica was resistant to 63 days of severe drought stress and resisted drought through various physiological responses. These findings provide a basis for the application of I. japonica in water-scarce areas.Drought is among the most important abiotic stresses on plants, so research on the physiological regulation mechanisms of plants under drought stress can critically increase the economic and ecological value of plants in arid regions. In this study, the effects of drought stress on the growth status and biochemical indicators of  Drought2 to O2 . On the Acacia by measuring the growth of plants under drought conditions , as well as a series of physiological indicators such as leaf water content, photosynthetic pigments, gas exchange parameters, and leaf nutrients. Jiang et al. [Carex breviculmis to soil drought at the physiological level. Wang et al. [Plant structural changes, physio-biochemical responses, and plant drought-adaptation mechanisms under drought conditions are a recent research hotspot. For example, it was recently reported that when rice is exposed to drought stress before or during tillering, the number of tillers and panicles is reduced, and this can even favor genotypic variation . Kebbas g et al.  exploredg et al.  found thIris japonica is widely distributed in southern China and Japan [I. japonica is able to tolerate more severe droughts under these environmental conditions makes it potentially economically viable in water-scarce areas [I. japonica under drought stress remain insufficient. Therefore, the aim of this study is to investigate the effects of drought on the morphological, physiological, and biochemical characteristics of I. japonica, to provide a scientific basis for I. japonica cultivation. In addition, the research results lay the foundation for an in-depth investigation of the drought-resistance mechanism of I. japonica. At the same time, it provides an empirical basis and practical guidance for the screening and effective utilization of ornamental plants in arid areas, which is of great significance for local urban greenery planting and management.As a perennial herb, nd Japan . It readnd Japan . Under dce areas . HoweverI. japonica obtained from Wenjiang Xiyang Garden Company , specifically, three-year-old seedlings. Each pot contained five plants, with 72 pots in total , and the soil used in this study was a sandy clay loam that contained all the necessary nutrients for plant growth. The basic environmental conditions for the experimental plants included a 16 h light (25 °C)/8 h dark (22 °C) cycle, 60% relative humidity, and illumination intensity of 500 μmol m−2 s−1 photons. The plants were pre-cultured for three months to stabilize the plant roots and growth.The plant material was The abovementioned cultivation materials were randomly divided into two groups of 36 pots each, and the plants were placed in the greenhouse of Sichuan Agricultural University for experiments. During the period in which the experiment was conducted, the growth conditions (except for moisture) were the same as during the initial cultivation period. For the control group, 500 mL of water was applied every 3 days, and the excess water naturally flowed out from the bottom of the basin; for the treatment group, there was no watering. A total of four replicate groups were set up for each treatment group of the experiment. The control groups were CK1, CK2, CK3, and CK4, each with nine pots, and the same number of pots was used for the treatment groups.The drought lasted for 63 days . According to the number of drought days, the drought was divided into four stages: the early stage (0–9 d), the middle stage (18–27 d), the late stage (36–45 d), and the last stage (54–63 d). The indicators were determined as follows.(1)For morphological indicators, the plant growth status was observed, including measurements of the following traits: leaf length, leaf width, leaf weight ratio, aboveground stem weight ratio, rhizobia weight ratio, root to weight ratio, and root to shoot ratio after 63 d of drought. The determination of the anatomical structure of the leaves, aboveground stems, and roots was also conducted.(2)For physiological indicators, every 9 days, soil samples were collected to determine soil water content. Plant leaves were collected for determination of leaf cell membrane permeability and proline, soluble protein, reducing sugar, ascorbic acid (AsA), and glutathione (GSH) contents. Whole plant material was collected for determination of tissue water content. Leaves, shoots, and roots of plants were collected for determination of soluble sugar content. Roots of plants were collected for determination of root activity. After 63 d of drought, the leaves of the plants were collected for the determination of photosynthetic pigment, hydrogen peroxide, and malondialdehyde (MDA) contents and peroxidase (POD), superoxide dismutase (SOD), glutathione reductase (GR), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and ascorbic acid oxidase (AAO) activities.A combination of straightedge measurement and CAD precision measurement methods was used for the determination of plant parameters, such as leaf length and leaf width.Fresh weights of leaves, young stems, rhizomes, and roots were measured with a METTLER TOLEDO ME204 scale , with an accuracy of one part in 10,000. The leaf weight ratio, aboveground stem weight ratio, root weight ratio, root weight ratio, and root crown ratio were calculated according to the following formulas: leaf weight ratio = leaf weight/total weight; aboveground stem weight ratio = aboveground stem weight/total weight; rhizosphere weight ratio = rhizomes weight/total weight; root weight ratio = root weight/total weight; root to shoot ratio = /(leaf weight + aboveground stem weight).For anatomical structure determination, FAA fixative solution  was used to fix leaves, ground stems, and roots by immersing them in the fixing solution, after which samples were sent to Wuhan Sevier Biotechnology Co., Ltd.  to be embedded in paraffin wax. Sections were stained with saffron-fixed green, and slices were cut, observed in cross-section with a microscope, and photographed using OLYMPUS cellSens Standard software (V4.1.1).To determine the water content of leaves, aboveground stems, rhizomes, and roots, the fresh weight (W1) was first obtained, and the dry weight (W2) was obtained after drying samples at 105 °C for 15 min and 80 °C for 48 h. The dry samples were also used for subsequent sugar content determination. Water content was calculated as follows: water content = (W1 − W2)/W1 × 100%.After collecting the soil, the initial soil weight was recorded (w1), and the soil was weighed again after drying at 80 °C to a constant weight to obtain the weight of the dry soil (w2). The soil water content was calculated as follows: soil water content = (w1 − w2)/w2 × 100%.−1 phosphate that was heated and dissolved at 70 °C, cooled, and stored in a brown reagent bottle) in a boiling water bath for 15 min to produce stable red products. Then, a spectrophotometer was used to obtain the optical density of the test samples at 520 nm. Finally, the content of proline was calculated from the standard curve.The acidic ninhydrin method was used for the determination of proline content . ProlineI. japonica leaves were rinsed in water, dried, and cut into small 0.5 × 0.5 cm pieces. The leaves were divided into eight test tubes, four tubes each for the CK treatment and the drought treatment. The eight test tubes were dried under vacuum for 10 min, subsequently removed, and then kept at room temperature for 30 min, and the exudate conductivity values were determined by an electronic conductivity meter . Afterward, the tubes were placed in a boiling water bath for 5 min and then cooled to room temperature, and the exudate conductance values were determined (of which the CK measurement was recorded as C1′ and the drought group as T1′) using the following formulas:The conductivity method was used to measure the sample cell membrane permeability using an electronic conductivity meter  . Ultra-pThe determination of soluble protein was performed by the Komas Brilliant Blue G-250 staining method; the solution to be tested was reacted with quantitative Komas Brilliant Blue solution, and the absorbance value of the conjugate was measured at 595 nm, allowing the calculation of the soluble protein content . The hydExtracts for antioxidant enzymes and antioxidants were prepared following the methods described by Wu et al., with some modifications . Leaf saThe following enzyme solutions were prepared using the method described by Tan et al., with some modifications , and theLeaf samples (0.5 g) were combined with 5 mL of 5% TCA, ground into a homogenate on ice, and centrifuged at 5000 r/min for 10 min at 4 °C, and the supernatant was collected for the assay ,18. For The enzyme solution used for determination of AAO activity was the same as the enzyme solution described in −1) = (C × Vt)/(FW × 1000). Here, C is the chlorophyll concentration (mg L−1), Vt is the total volume (mL) of the extract, and FW is the fresh weight (g) of the leaves.For determination of photosynthetic pigments, 0.13 g leaf samples were cut into small pieces in the dark, added to 25 mL of 95% ethanol, and soaked for 48 h in the dark. The absorbance was measured at 665 nm, 649 nm, and 470 nm with 95% ethanol as a blank, and the concentration of each photosynthetic pigment was calculated as follows: content of each pigment  was used to conduct independent sample t-tests. Data visualization was conducted with Origin 9.0 .The soil water content of the control group was relatively stable throughout the experimental cycle, remaining above 70%. Soil water content in the treatment group decreased as the number of drought days increased and was significantly different from the control group from day 9 onwards . On day As shown in Iris japonica is a shallow-rooted plant with no obvious primary roots. After 63 days of drought, the rhizomes of both the control and treated groups appeared yellowish-brown , and vascular bundles of the aboveground stem of I. japonica can be seen from the cross-sectional anatomical view. Both control and treatment plants had a tightly packed arrangement of epidermal cells. The lateral and medial basic tissues were separated by a thick layer of mechanical tissue composed of tightly packed, multi-layered, thick-walled cells. Except for epidermal cells, thick-walled mechanical tissue layers, and vascular bundles, control and treated cell sections were all elliptical or circular and closely aligned. In the basic tissues, the control and treatment contained a lot of collenchyma that were completely or partially dyed dark red or solid green. A large number of plasmolysis phenomena occurred in the drought-treated cells but not in the control cells.I. japonica stems on day 63 of drought treatment differed from that of the control. The vascular bundle diameter of I. japonica was reduced by 35% after drought treatment, which was a significant difference. However, the number of vascular bundles in the drought-treated group increased by 45% compared with the control. The cells of the basic tissue close to the epidermis were small, and the vascular bundles were scattered throughout the basic tissue, except the epidermis. Most of the vascular bundles were distributed deeper within the stems; the vascular bundles in the outer basic tissues were smaller, while the vascular bundles in the inner basic tissues were larger.Meanwhile, from both I. japonica is a monocotyledonous plant, and the roots only have a primary structure. In the anatomy of the roots, the control and treated epidermis consisted of a tightly packed epidermal cell. The cortex within the epidermis consisted of multiple layers of parenchyma cells in which the cells were loosely arranged with intercellular spaces. The parenchyma of the control cortex was larger with a loose arrangement, while the parenchyma cells that process the cortex were more closely arranged, without much of a gap between cells. Endothelial cells and vascular bundles were more pronounced in treated plants than in controls.As can be seen in The water content of the leaves treated with drought gradually decreased as the number of drought days increased, and the difference was significant by 27 d. After 63 d, the drought treatment leaf water content was significantly lower than the control leaf water content, by 27% A. The waAs can be seen from The content of soluble sugar in drought-treated leaves decreased as the number of drought days increased. The content of reducing sugar and soluble sugar in leaves showed a significant difference from 18 d to 27 d, respectively. After 63 d of drought, the contents of leaf reducing sugar, leaf soluble sugar, and aboveground stem soluble sugar in the treated leaves decreased by 72%, 51%, and 55%, respectively C–E. HoweAs As shown in As shown in As can be seen from As shown in I. japonica became shorter, and the ratio of leaf weight to root weight decreased; however, the ratio of branch weight to root and rhizome increased. This indicated that I. japonica adapted to drought by increasing its proportion of stem, similar to the results of previous research [I. japonica tending to form new clones through rhizome cloning and reproduction, which occurs in its natural environment when I. japonica continuously elongates the apex of its rhizome and grows into new asexual plantlets. Therefore, when I. japonica is subjected to drought stress, it mitigates the stress by regulating various physiological changes in the stem.Plants subjected to drought stress first respond through changes in external morphology, including plant height, leaf size, and degree of root development . Chen etresearch . This maI. japonica were also studied. Previous studies showed that drought leads to a loss of membrane fluidity and induces structural and morphological changes in the cell wall [I. japonica leaves had no fissures and no spongy tissue but instead had well-developed aeration tissue. Anatomically, the drought-treated leaf cells were more closely arranged after 63 d and showed extensive cell lysis, indicating that I. japonica had taken measures to reduce water evaporation, but the leaf cells were unable to withstand intense long-term drought stress, as can be seen in I. japonica were too severely desiccated to be sectioned. Additionally, probably because of the reduced water content of I. japonica leaves, the plants were dehydrated, making it impossible to maintain the cells in a normal state, resulting in the mesophyll cells being irregular in shape; whereas, in terms of the root anatomy, the phenotype of the thin-walled cell gaps was large and loose, probably enabling the maintenance of good ventilation under adequate water [I. japonica to drought stress, i.e., a “numerical advantage” to partially offset the reduced efficiency of vascular water transport.In addition, changes in various cellular tissues of ell wall ,25. Thiste water ,26. Vascte water . Owing tMedicago sativa L. seedlings and found that their roots were more resistant to drought stress than their leaves or stems. Iris japonica has different growth characteristics from lucerne seedlings, and, as a perennial flower, the root and stem of I. japonica play an important role in its growth [I. japonica significantly decreased after 27 d, but the aboveground stems did not show severe drought stress until day 63. This suggested that under drought stress, I. japonica leaves withered first, protecting the stem part so that new leaves would grow after moisture might be restored. At the same time, it could be the cells of the aboveground stem being closely arranged and containing a thick-walled mechanical tissue layer that reduced the loss of water [The water content of tissues directly reflects the water content of the plant parts. Under drought stress, the reduction in water content in plants can differently respond, depending on the characteristics of the different parts of the plant . Luo et s growth ,31. The of water .Lagerstroemia indica, Glycine max, and Arabidopsis thaliana regulate their soluble sugars, proline, and other substances to alter the content of osmoregulatory substances, thereby increasing their drought tolerance [I. japonica was not obvious in the early stage of drought, but, with the continuation of drought, the proline and soluble protein content of I. japonica gradually increased, which increased the osmotic pressure of protoplasts, thereby preventing water dissipation, thus acting as a water-holding agent for protoplasts as a way to better cope with drought stress. The results are consistent with those observed in Lycium ruthenicum [Osmoregulatory substances are often used as important indicators of drought tolerance in plants. For example, olerance . In our thenicum .Oryza sativa and Triticum aestivum, produce certain small molecule metabolites, which include substances such as soluble sugars, in response to drought stress to counteract their own drought stress [I. japonica decreased as the number of drought days increased, indicating that the material accumulation of leaves and aboveground stems of I. japonica decreased, which may be the reason for the weakening of photosynthesis in I. japonica and the concomitant decrease in sugars in photosynthetic products caused by drought. Our results are similar to those observed in Sesuvium portulacastrum [Crops, such as t stress ,36. Solut stress . The conacastrum .I. japonica were reduced, which is consistent with the results of previous studies [I. japonica was weakened, which may have reduced the content of sugar (acting as an osmoregulatory substance), thus affecting the growth and development of I. japonica.Chlorophyll and carotenoids are important photosynthetic pigments. When exposed to drought stress, the chloroplast lamellar structure is damaged, and chlorophyll and carotenoid synthesis is impaired, which, in turn, affects electron transport and photosynthetic phosphorylation processes ,40. Afte studies ,42. The I. japonica, which is consistent with previous research results [Cell membrane permeability, hydrogen peroxide concentration, and MDA content can reflect the degree of damage to cell membranes. Hydrogen peroxide is a reactive oxygen species that causes strong oxidative damage to cell membrane lipids during plant metabolism . MDA is  results .Plants evolved the ability to scavenge ROS through an enzymatic scavenging system consisting of antioxidant enzymes, such as SOD and POD, over a long evolutionary period, which, in turn, inhibits membrane lipid peroxidation and enhances plant drought resistance . PreviouI. japonica. Meanwhile, the activity of MDHAR was reduced under drought, which was detrimental to AsA production. Finally, the AsA content of I. japonica decreased. This series of changes indicated that I. japonica was unable to resist the damage caused by 63 days of drought. In contrast, GSH is also an antioxidant that can effectively scavenge reactive oxygen species, and GR is one of the enzymes involved in GSH regeneration. After drought treatment, GSH content decreased with time, indicating that mild drought could indeed promote the antioxidant capacity of I. japonica, but, as the drought continued, GSH content and GR activity decreased, stress exceeded the tolerance threshold of plants, membrane lipid peroxidation increased, and normal cell metabolism was disturbed [I. japonica has a strong ability to reduce ROS accumulation to maintain membrane system stability and enhance plant drought resistance [AsA has the ability to directly scavenge various reactive oxygen species generated by stress and plays an important role in plant stress resistance . The oxiisturbed . POD is isturbed . A decreisturbed . POD andsistance .I. japonica was affected by the accumulation of harmful substances under drought conditions, mainly through the increase in POD and SOD activities to resist the cellular damage caused by ROS.Based on the above findings, the ascorbate–glutathione cycle of T. aestivum was subjected to drought stress, not only did the root length and root dry weight significantly decrease but also the root activity was also affected to some extent [I. japonica were similar to those of the abovementioned studies. The water content and root activity of I. japonica roots began to decrease at 9 d and 18 d, respectively, indicating that the response of I. japonica roots to water supply was sensitive.Root activity directly reflects plant growth. Chen et al. found that when e extent . In the I. japonica mainly physiologically adapted to drought by increasing the proportion of stems in the plant to tolerate drought stress. The increasing proline and soluble protein content of the leaves and contents of POD and SOD played important roles in allowing I. japonica to relieve drought conditions. These findings indicated the mechanism by which I. japonica is able to keep growing well under prolonged droughts. Given its excellent physiological drought response, I. japonica holds promise as an ornamental plant to be grown in urban areas that are subject to drought. While also saving municipal water, the planting of I. japonica also facilitates simplified management and reduces the maintenance cost, which can not only improve the green coverage of a city but also facilitate the construction of ecological gardens.In this study,


Background and Objectives: It is crucial to quickly identify those patients who need immediate treatment in order to avoid the various complications related to acute diverticulitis (AD). Although several studies evaluated the neutrophil-to-lymphocyte ratio (NLR) suggesting its predictive value in assessing the severity of AD, results have been inconclusive. Therefore, we aimed to assess the relationship between the neutrophil-to-lymphocyte ratio (NLR), the platelet-to-lymphocyte ratio (PLR), the monocyte-to-lymphocyte ratio (MLR), and systemic immune inflammation (SII) with the severity of AD, the ability to predict the presence or absence of complications, and the recurrence rate, based on the values of inflammatory markers. Materials and Methods: We retrospectively reviewed 147 patients diagnosed with AD between January 2012 to February 2023. Patients were divided into 2 groups, uncomplicated and complicated AD. The characteristics and full blood count between both groups were compared. Results: A total of 65 (44.22%) patients were classified as having complicated AD. The area under the ROC curve (AUROC) defining a Hinchey score ≥ 1b was as follows: SII, 0.812 , 0.73 –0.888); NLR, 0.773 ; PLR, 0.725 ; MLR: 0.665 . An SII cutoff value of > 1200 marked the highest yield for diagnosing complicated AD, with a sensitivity of 82% and a specificity of 76%. The cumulative recurrence rate was not significantly different in the groups of SII ≥ median vs. SII < median (p = 0.35), NLR ≥ median vs. NLR < median (p = 0.347), PLR ≥ median vs. PLR < median (p = 0.597), and MLR ≥ median vs. MLR < median (p = 0.651). Conclusions: Our study indicates that SII, NLR, and PLR are statistically significant and clinically useful classifying ratios to predict higher Hinchey scores. However, they cannot predict recurrences. Acute diverticulitis (AD) is defined as an inflammation of the diverticula and can be either uncomplicated or complicated. Around 5% of patients with diverticulosis will experience at least one episode of diverticulitis during their lifetime . Most caNumerous factors have been linked to a higher probability for developing AD. These factors include age , sex 4]4], genetThe triad of symptoms consisting of left-sided lower abdomen discomfort, lack of vomiting, and a C-reactive protein (CRP) levels greater than 5 mg/dl, shows a high sensitivity for detecting AD (acute diverticulitis). However, this triad does not allow differentiating between uncomplicated and complicated diverticulitis . UnfortuCirculating cells, such as lymphocytes, monocytes, neutrophils, and platelets are essential components of the inflammatory process. Their ratios, comprising the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), and systemic immune inflammation (SII), are simple and easily accessible inflammatory markers that have been studied as potential prognostic indicators in various medical conditions. Previous studies have found that high NLR and PLR values have been linked with complicated diverticulitis ,8, surgiThe development of a diagnostic and prognostic tool to identify complicated AD and assess the risk of complications in patients with uncomplicated AD can indeed be beneficial in reducing healthcare costs, unnecessary referrals, and the excessive use of CT scans. Therefore, the objective of this study was to evaluate the predictive value of NLR, PLR, MLR, and SII regarding to the severity of AD, their ability to predict the presence or absence of complications, and the recurrence rate based on inflammatory marker values.We conducted a retrospective observational study involving subjects ≥18 years old. All patients who were admitted with acute diverticulitis (AD) at the Clinical Emergency County Hospital of Cluj-Napoca, Romania between January 2012 to February 2023 were identified, and their data were collected retrospectively using electronic medical records. The patients were searched for in the hospital database, using the ICD  code K57 (diverticular disease of the intestine). All included patients in our study were diagnosed with AD using either a CT scan, colonoscopy, or medical/surgical report. Patients were excluded from the study based on the following criteria: (1) any malignancy, (2) a lack of confirmation of AD, and (3) a lack of laboratory parameters  at admission. The study was conducted according to the principles of the Declaration of Helsinki and was approved by the Ethical Committee of “Iuliu Hațieganu” University of Medicine and Pharmacy Cluj-Napoca .The research has been conducted following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines . The comThe primary outcomes were to evaluate the NLR, PLR, MLR, and SII levels in patients with complicated AD and to investigate the diagnostic and prognostic accuracy of these markers.We collected the following information from patient medical records: age, sex, body mass index (BMI), clinical symptoms at admission , laboratory parameters, diagnosis method , length of hospital stay, recurrence rate, the time interval from the first to a recurrent episode, cumulative length of stay due to AD, and initial treatment strategy . Reviewing of the records was performed by three investigators .The diagnosis of AD was based on the presence of inflammation in one or more diverticula based on CT scans, surgical reports, or colonoscopy. AD can be either uncomplicated or complicated. The classification used to define AD severity was the modified Hinchey classification based on CT findings . The modLaboratory parameters included leukocytes, neutrophils (N), lymphocytes (Ly), monocytes (M), platelets (P), red cell distribution width (RDW), platelet distribution width (PDW), urea, and creatinine. NLR (the neutrophil-to-lymphocyte ratio) and PLR (the platelet-to-lymphocyte ratio) were defined as neutrophil or platelet counts divided by the total number of lymphocytes. MLR (the monocyte to lymphocyte ratio) was defined as the absolute monocyte count, divided by the absolute lymphocyte count. The systemic immune-inflammatory index (SII) was calculated using the formula SII = (P × N)/L.The number of cases with AD diagnosis in our hospital during the study period determined the sample size. We grouped the patients into two categories of uncomplicated  vs. complicated diverticulitis (1B–4). Moreover, for a more detailed analysis, the patients were further divided into three groups according to the modified Hinchey grade. Patients with a modified Hinchey score of 0 or IA were considered the uncomplicated AD group, while patients with a modified Hinchey score of IB or II were diagnosed as having complicated AD with an abscess, and patients with modified Hinchey score of III were diagnosed as belonging to the complicated AD with perforation group.t-test. Comparisons between three independent groups concerning quantitative data that did not follow a normal distribution were made using the Kruskal–Wallis method. The discriminating qualities of inflammatory markers were assessed using receiver operating characteristic (ROC) curves, using their area under the curve , as well as by identifying the best cutoff point by maximizing the Youden index and computing its sensitivity and specificity. Comparisons between ROC curves were performed with the De Long test. The intervals between episodes were compared using Kaplan–Meier survival curves, and the Cox proportional hazards regression model was used to analyze associations of several factors such as NLR, PLR, MLR, and SII with cumulative recurrence rate. For all statistical tests, a two-tailed p-value was computed, and a value of ≤0.05 was considered to be statistically significant. All analyses were computed in the R environment for statistical computing and graphics, version 4.1.2 .Data were presented as counts and percentages when the variables were categorical, as means and standard deviations when the variables were quantitative and with a normal distribution, or as medians and interquartile ranges when the variables were quantitative and did not follow a normal distribution. The comparisons between two independent groups concerning categorical data were conducted using a chi-squared test or Fisher’s exact test. For quantitative data that did not follow a normal distribution, we used the Mann-Whitney U test. For quantitative data that followed a normal distribution, the analysis was performed using an independent n = 34) were younger than 50 years of age. According to the modified Hinchey classification ), but was different from that reported by Palacios Huatuco et al. [n = 65) vs. 9 % (n = 30) vs. (12% (n = 386)). In the complicated diverticulitis group, 29.93% (n = 44) of patients had CT findings compatible with a diagnosis of Hinchey stage III. Recurrent episodes of acute diverticulitis occurred in 10.20% (n = 15) of patients, of whom 26.66% (n = 4) presented more than one episode of acute diverticulitis. We also found that patients with complicated AD were of a similar age to those with uncomplicated AD . These findings are similar to those reported by Palacios Huatuco et al. [The prevalence of complicated AD in our study was similar to that reported by Zager et al.  , while lymphocytes were lower in this group, suggesting an inflammatory response and immune system activation. Only WBC, neutrophil, and platelet counts have a statistically significant association with complicated AD. However, we did not find any statistically significant differences related to these values in the group with perforation compared to those with abscesses, as we would have expected. This unexpected finding may indicate that other factors beyond these specific markers play a significant role in differentiating between these two types of complications in AD.Circulating cells, such as lymphocytes, monocytes, neutrophils, and platelets are essential components of the inflammatory process. In contrast to measuring each WBC component separately, measuring the divergence among them has been thought to be more accurate for predicting poor clinical outcomes . PreviouIn our study, the SII cutoff of 1200 had a sensitivity of 82% and a specificity of 76%; the NLR cutoff of 4.06 had a sensitivity of 80% and a specificity of 69.3%; and the PLR cutoff of 144.38 had a sensitivity of 80% and a specificity of 56%. The diagnostic accuracy of the SII, NLR, and PLR for complicated AD is good and is also comparable, but MLR could only predict those patients who had complicated AD with low accuracy . The SII showed the highest AUROC value of 0.812, indicating its ability to discriminate effectively between patients with and without complicated AD. This finding is particularly interesting since the SII showed a diagnostic potential similar to that of procalcitonin, a well-known biomarker for infection and inflammation . In our In a previous study conducted by Zager et al. , it was Our research has several limitations. First, the data were collected retrospectively; hence, a selection bias may exist, and also, some medical records did not contain all variables. Second, this was a single-center study conducted in Eastern Europe with a relatively small sample size, which may affect the statistical power of our findings and limit their generalizability to other populations or settings. Third, smoking status, medication use, and BMI could not be included in the analysis due to lack of data. Most patients with diverticulosis are obese and are diagnosed concurrently with metabolic dysfunction-associated steatotic liver disease (MASLD) . It has However, despite these limitations, our study also has several important strengths. It provides a valuable and comprehensive evaluation of multiple inflammatory markers and hemogram-derived ratios in complicated AD. It is the first study to evaluate the utility of SII and MLR for diagnosing complicated AD and establish their prognostic utility. Additionally, the associated complications were accurately evaluated using CT scan or surgical report. We believe that the clinical significance of this study lies in its potential to improve diagnostic accuracy, guide treatment decisions, optimize healthcare resource utilization, and contribute to the existing knowledge in the field of complicated acute diverticulitis. By evaluating multiple inflammatory markers, including SII and MLR, this study expands the range of diagnostic tools available for complicated acute diverticulitis, enhancing the accuracy and efficiency of diagnosing the severity of the disease. The early recognition of complicated AD allows for timely and targeted interventions, such as performing additional imaging studies or initiating more intensive monitoring, guiding decisions about the need for surgical consultation or aggressive medical management. Furthermore, by ruling out complicated AD in a considerable proportion of cases with good specificity, the inflammatory parameters can help avoid unnecessary CT scans and hospitalizations, thus optimizing healthcare resource utilization. Finally, it fills a gap in the literature by being the first to evaluate the utility of SII and MLR in diagnosing complicated acute diverticulitis, expanding the understanding of inflammatory markers in this specific context and opening avenues for further research and validation.Therefore, future studies with larger sample sizes, multi-center designs, and prospective data collection methods are needed to confirm our results on the predictive role of hemogram-derived ratios in complicated AD and to refine the optimized cutoff values for the assessed parameters.SII, NLR, and PLR can predict complicated AD, with an SII of >1200 having the highest yield for diagnosing complicated AD. Incorporating these hemogram-derived ratios into a comprehensive predictive model may improve accuracy and aid in clinical decision-making for patients with AD.


It is of great significance to understand the characteristics and influencing factors of vegetation coverage variation in the warm temperate zone. As a typical region of the warm temperate zone in eastern China, the mountainous and hilly region in central-south Shandong Province has fragile ecological environment and soil erosion problem. Studying on vegetation dynamics and its influencing factors in this region will help to better understand the relationship between climate change and vegetation cover change in the warm temperate zone of eastern China, and the influence of human activities on vegetation cover dynamics.Based on dendrochronology, a standard tree-ring width chronology was established in the mountainous and hilly region of central-south Shandong Province, and the vegetation coverage from 1905 to 2020 was reconstructed to reveal the dynamic change characteristics of vegetation cover in this region. Secondly, the influence of climate factors and human activities on the dynamic change of vegetation cover was discussed through correlation analysis and residual analysis.In the reconstructed sequence, 23 years had high vegetation coverage and 15 years had low vegetation coverage. After low-pass filtering, the vegetation coverage of 1911–1913, 1945–1951, 1958–1962, 1994–1996, and 2007–2011 was relatively high, while the vegetation coverage of 1925–1927, 1936–1942, 2001–2003, and 2019–2020 was relatively low. Although precipitation determined the variation of vegetation coverage in this study area, the impacts of human activities on the change of vegetation coverage in the past decades cannot be ignored. With the development of social economy and the acceleration of urbanization, the vegetation coverage declined. Since the beginning of the 21st century, ecological projects such as Grain-for-Green have increased the vegetation coverage. VegetatIn the past few decades, the global climate has undergone several unprecedented changes, and global warming has become an indisputable fact . A serieNormalized vegetation index (NDVI) can accurately reflect important information such as vegetation green dynamics, productivity, biomass, vegetation cover, and growth status, so it has been widely used to study vegetation dynamic change and its response mechanism . HoweverPlatycladus orientalis (L.) Franco is considered as a good tree species for dendrology because of its long lifespan, strong adaptability, sensitivity to climate change, and clear ring boundaries. The mountainous and hilly areas in the central south of Shandong Province as an important geographical unit is a subtropical and temperate climate transition zone and climate-sensitive area with fragile ecological environment and serious problem of soil and water loss  (The samples were collected from  (ITRDB) . The tre (ITRDB) . Using CAnd then the standard chronology was developed using ARSTAN . Negativ2.2.2http://data.cma.cn/). And the time span of them is 1952–2020. To reduce small-scale noise or random components and enable meteorological data to be representative of a wider range of regional climate conditions, we used arithmetic mean datasets from multiple weather stations for further analysis.Meteorological data, including monthly precipitation, monthly mean temperature, and relative humidity were obtained from the National Weather Science Data Center  from 2.3A maximum value composite (MVC) method was used to calculate the monthly average NDVI value of the study area . To stud33.1P. orientalis and the NDVI. From By analyzing the correlation between tree rings and summer (June–August) NDVI (Vegetation coverage is higher at this time) during their common period (1982–2015), we know that there is a certain correlation between radial growth of P. orientalis had a significant positive correlation with the monthly average precipitation in September of the previous year and the current July, and showed a significant positive correlation with the relative humidity in current July and summer by analyzing the correlation between tree-ring width and climatic factors  in the mountainous and hilly region of central-south Shandong Province. And the radial growth of trees had a certain correlation with NDVI. In addition, trees are an important form of vegetation, and their growth state represents a partial change in vegetation coverage . The transfer function is as follows:n = 34, 1982–2015 CE, r = 0.44, R2 = 0.20, R2adj=0.17, p< 0.01), being 0.006. It can be seen from 3.3Vegetation growth is affected by regional temperature variation and hydrological conditions . AccordiUnder the influence of the warm temperate monsoon climate, the precipitation in the mountainous and hilly areas in the central south of Shandong Province is concentrated in summer, and is obviously affected by ENSO events because of its location . Because2 assimilation in leaves , resulting in the destruction of the forest, and the vegetation coverage in the following years was lower than that before the fire  is the dominant factor affecting vegetation coverage in the mountainous and hilly areas in the central south of Shandong Province, but the impacts of human activities on regional vegetation change cannot be ignored.China’s climate has been warming since the 1950s, with surface air temperatures rising faster than the global average, which has significant impacts on water resources, agriculture, ecosystems, and human health . So, basn = 34, 1982–2020 CE, r = 0.61, R2 = 0.37, R2adj = 0.33, p< 0.01), In the past 115 years, the vegetation coverage in the study area showed obvious interannual and interdecadal variations. And there were 25 years with low vegetation coverage, while 23 years with high vegetation coverage. The study area experienced four periods of low vegetation coverage  and five periods of high vegetation coverage .2) Climate was the dominant factor affecting summer NDVI in the study area, especially the precipitation in spring (March–May) and summer (June–August), but the impacts of human activities on the vegetation coverage variation should not be ignored.3) In this study, the effect of human activities to vegetation cover change showed an increasing trend in the fluctuation since 1950. With the development of social economy and technology, the policy system, ecological engineering, and rapid urbanization have improved or degraded the vegetation to different degrees.Through this study, we were able to understand the relationship between climate change and vegetation cover, and the impact of human activities on vegetation cover in warm temperate regions of eastern China well. In addition, future studies should further analyze the importance degree of different types of human activities on vegetation cover.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TY, HW, PZ, and RW proposed the study and designed the experiment. TY conducted field and laboratory measurements, and analyzed the data. HW, PZ, and RW secured fundings. YiZ, YaZ, and JD helped with laboratory measurements and data analysis. JD, XL, and WY helped with data analysis. TY wrote the manuscript that was intensively edited by all authors.


To describe the trends in use of antidepressants (ADs), atypical antipsychotics (AAPs), and benzodiazepines (BZDs) among high-, middle-, and low-income countries.A cross-sectional time-series analysis by country from July 2014 to December 2019 utilizing IQVIA’s Multinational Integrated Data Analysis database was conducted. Population-controlled rates of use were calculated in number of standard units of medications per drug class per population size. The United Nations’ 2020 World Economic Situation and Prospects was used to group countries into high-, middle-, and low-income. Percent change in rates of use per drug class was calculated from July 2014 to July 2019. Linear regression analyses were conducted to assess the predictability of percent change in use utilizing a country’s baseline rate of use per drug class and economic status as predictor variables.A total of 64 countries were included: 33 high-, 6 middle-, and 25 low-income. Average baseline rates of use for ADs in high-, middle-, and low-income countries were 2.15, 0.35, and 0.38 standard units per population size, respectively. For AAPs, rates were 0.69, 0.15, and 0.13, respectively. For BZDs, rates were 1.66, 1.46, and 0.33, respectively. Average percent changes in use for ADs by economic status were 20%, 69%, and 42%, respectively. For AAPs, they were 27%, 78%, and 69%, respectively. For BZDs, they were -13%, 4%, and -5%, respectively. Some associations were found demonstrating that as a country’s economic status increases, percent change of AD (p = 0.916), AAP (p = 0.23), and BZD (p = 0.027) use decreases. Similarly, as baseline rate of use for ADs and AAPs increases, percent change in use decreases with p-values of 0.026 and 0.054, respectively. For BZDs, as baseline rate of use increases, percent change in use increases (p = 0.038).High-income countries have a higher rate of treatment utilization compared to low- and middle-income countries (LMICs) with treatment utilization increasing in all countries of interest. The rising burden of mental illness continues to be a major global concern with steep costs and unmet needs for treatment worldwide. The World Health Organization (WHO) World Mental Health (WMH) surveyed across 28 countries estimated lifetime prevalence of DSM-IV anxiety, mood, disruptive behavior, and substance disorders for individuals at approximately 18–36% [LMICs comprise over 80% of the world’s population, and despite the high prevalence and impact of anxiety, mood fluctuations, impulse control, and substance use disorders identified in the WMH surveys within LMICs, more than 75% of individuals did not receive any care which demonstrates deficiencies in treatment access –10. ThesAntidepressants (ADs), atypical antipsychotics (AAPs), and benzodiazepines (BZDs) are among the most commonly utilized mental health treatments globally –19. SincThis study utilized data from IQVIA’s Multinational Integrated Data Analysis (MIDAS) database which consists of hospital and retail medication purchasing data for 66 countries between July 2014 to September 2020. Purchasing data was organized into standard units where one unit represents one package unit of a medication. The data did not contain information about individual facilities or patients who have purchased these drugs or obtained them from other sources.We conducted a repeated cross-sectional analysis to study global trends in AD, AAP, and BZD use between high- middle- and low-income countries from July 2014 to December 2019. There are three outcomes in this study: (1) differences in baseline rates of use per drug class; (2) differences in percent change in use per drug class from 2014 to 2019 in high-, middle-, and low-income countries; and (3) the predictive quality of percent change in use per drug class using a country’s economic status and baseline rates of use as predictors.From the total 66 countries available through the MIDAS database, 64 countries were included in the analysis. This is because countries that included Venezuela and Kuwait were missing a significant percentage of greater than 50% of their total purchasing data and were excluded. Purchasing data for each of the drug classes of interest were aggregated for the remaining countries. Missing purchasing data in a country for a specific AD, AAP, or BZD were excluded in the aggregated totals of that country. Purchasing data that occurred after December 2019 were excluded due to the start of the COVID-19 pandemic and the likely increase in mental health treatment utilization at that time. In addition, medications that did not fall into the drug classes of ADs, AAPs, or BZDs were also excluded along with herbal AD products. All included medications for each of the drug classes are provided in The analysis group included 33 high-, 6 middle-, and 25 low-income countries. Countries were categorized as high-, middle-, and low-income based on the United Nations’ World Economic Situation and Prospects 2020 report’s classification criteria for developed, in-transition, and developing economies, respectively. Country population data was collected from the United Nations Department of Economic and Social Affairs. Data on average standard units purchased per drug class and per country between 2014 and 2019 were collected and included from IQVIA’s MIDAS database. Population-controlled baseline rates of use were calculated by dividing the average standard units per drug class between July 2014 to December 2019 by the country’s population between the same period. Population-controlled percent change in rates of use for the number of standard units purchased per drug class from July 2014 and July 2019 were also calculated. This was done by subtracting average population-controlled rates of use per drug class in 2014 from that of in 2019, dividing by the average population-controlled rate of use in 2014, and multiplying by 100. Lines of best fit and R-squared values were calculated per drug class using baseline rate of use on the x-axis and percent change in use on the y-axis. We conducted linear regression analyses to assess the predictability of percent change in use for ADs, AAPs, and BZDs. Linear regression analyses were conducted using Statistical Package for Social Sciences software . Each country’s economic status and baseline rate of use were used as predictor variables to assess the predictability of percent change in use per drug class.The average baseline rate of AD use across all 64 countries was 0.96 units per population. As seen in The average baseline rate of AAP use across all 64 countries was 0.32 standard units per population. Similar to ADs, The average baseline rate of BZD use across all countries was 1.15 standard units per population size. In It was determined that rates of use for all three drug classes were highest among high-income countries and lowest among LMICs. However, LMICs demonstrated higher rates of growth indicated by larger percent changes in the use of ADs and AAPs. To highlight these relationships, These results between high- and low-income countries suggest differences in mental health treatment utilization between countries of different economic status and between drug classes. Importantly, the results suggest that LMICs, with relatively low utilization rates of mental health treatments, are increasing use of ADs and AAPs to meet mental health needs. This increase in mental health treatment utilization is a potential reflection of the ongoing efforts to improve mental health treatment access globally. As reported in the 2020 WHO Mental Health Atlas, there have been improvements in increasing the number of countries with standalone policies for mental health, mental health promotion and prevention programs, and the number of mental health professionals from 2014–2020 . HoweverWhen reviewing previous publications on the utilization of ADs and AAPs, a major limitation has been the small number of LMICs included in their analyses. To improve treatment access in the most vulnerable countries, the incorporation of LMICs is essential. Previously, Lewer et al. found that country-level healthcare spending as well as sociocultural norms and attitudes towards mental health were significant in predicting AD use among 27 European countries . In addiFor BZDs, our study found that high- and low- income countries have experienced decreasing rates of use. This is expected because although BZDs have been proven effective in treating numerous conditions such as anxiety, agitation, and insomnia, their side effect profile is problematic . One of It is also important to mention that although the utilization of ADs and AAPs is increasing while the utilization of benzodiazepines is decreasing globally, this alone not does necessarily represent improvements in mental health care, but improvements in access. For example, as shown in this study, BZDs have relatively high utilization rates in high-income countries such as the U.S., France, and other European countries. However, these countries also have high rates benzodiazepine misuse such as the high utilization rates in older adults , 31–33. This study focuses on quantifying baseline trends in mental health treatment utilization prior to the COVID-19 pandemic. This is significant because the pandemic has resulted in increased anxiety and depression globally. As reported by the WHO, there was a 25% global increase in the prevalence of anxiety and depression within the first year of the COVID-19 pandemic . The U.SOur results are not without limitations. Firstly, IQVIA’s MIDAS dataset did not include information on individual facilities or patients, meaning that our analysis was not able to capture data beyond medication use at the country-level. Due to this limitation, this study could not take into account the demographic characteristics of the individuals using the medications of interests, and how differences in demographic characteristics between countries impacted the estimated utilization rates. Furthermore, due to the lack of patient characteristics, our study could not take into account indications for treatment. Given that ADs, AAPs, and BZDs can be used in the treatment of non-mental health conditions, it is possible that rates of ADs, AAPs, and BZDs use are not a reflection that these mental health medications are being used only in the setting of mental health. However, based on studies conducted in the U.S. and Canada that assessed prescription patterns by indication for each of the three drug classes have shown that, especially for ADs and AAPs, the most common indications for use are for mental health conditions –43. It iIn this study, we determined baseline rates of use and percent changes in use of ADs, AAPs, and BZDs in high-, middle-, and low-income countries to analyze levels of treatment utilization per drug class in each country. We found that high-income countries demonstrated higher rates of treatment use for all three drug classes compared to LMICs, indicating that treatment may be marginalized in both low- and middle-income countries. In addition, we also found that the rate of treatment growth is highest in LMICs for ADs and BZDs, which, over time, may lower the disparities in treatment utilization between high- and LMIC. However, as discussed, due to potential differences between countries in demographic characteristics, rational use of mental health treatment, and the potential for ADs, AAPs, and BZDs to be used in non-metal health settings, our results cannot conclude that these trends are entirely reflective of rational mental health treatment utilization. In addition, these results may not reflect trends in other countries or for other mental health medications not included in this study. Moving forward, it is important for future research to explore these trends within countries, for a specific medication, and using other indicators for drug utilization such defined daily doses. Overall, our results suggest that there is a continuous need for analyzing global treatment utilization patterns to assess disparities in mental health treatment access, especially in LMICs.S1 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Tablea The medications are listed by their active drug that can be used as an antidepressant, atypical antipsychotic, or benzodiazepine, respectively.(DOCX)Click here for additional data file.


High levels of occupational physical activity (PA) have been linked to an increased risk of dementia. We assessed the association of trajectories of occupational PA at ages 33–65 with risk of dementia and mild cognitive impairment (MCI) at ages 70+.We included 7005 participants  from the HUNT4 70+ Study. Group-based trajectory modelling was used to identify four trajectories of occupational PA based on national registry data from 1960 to 2014: stable low , increasing then decreasing , stable intermediate , and stable high . Dementia and MCI were clinically assessed in 2017–2019. We performed adjusted multinomial regression to estimate relative risk ratios (RRR) with 95% confidence intervals (CI) for dementia and MCI.902 participants were diagnosed with dementia and 2407 were diagnosed with MCI. Absolute unadjusted risks for dementia and MCI were 8.8% (95% CI: 7.6–10.0) and 27.4% (25.5–29.3), respectively, for those with a stable low PA trajectory, 8.2% (6.0–10.4) and 33.3% (29.6–37.0) for those with increasing, then decreasing PA; while they were 16.0% (14.3–17.7) and 35% (32.8–37.2) for those with stable intermediate, and 15.4% (14.0–16.8) and 40.2% (38.3–42.1) for those with stable high PA trajectories. In the adjusted model, participants with a stable high trajectory had a higher risk of dementia  and MCI , whereas participants with a stable intermediate trajectory had a higher risk of MCI  compared to the stable low trajectory. While not statistically significant, participants with increasing then decreasing occupational PA had a 24% lower risk of dementia and 18% higher risk of MCI than the stable low PA group.Consistently working in an occupation with intermediate or high occupational PA was linked to an increased risk of cognitive impairment, indicating the importance of developing strategies for individuals in physically demanding occupations to prevent cognitive impairment.10.13039/100000002National Institutes of Health (R01AG069109-01) and the 10.13039/501100005416Research Council of Norway .This work was supported by the  Existing evidence suggests that individuals working in occupations with high physical activity are at an increased risk of cognitive impairment later in life, compared to individuals working in less physically demanding occupations.We searched PubMed and individual article reference lists to find relevant studies from December 15th 2022 to April 1st 2023. The search terms included, but were not limited to, Our findings extend those from previous studies by incorporating a life-course perspective into research on occupational physical activity and cognitive impairment. Whereas previous studies have mainly focused on a single measurement of occupation, often close to retirement, we include occupational trajectories from ages 33–65 to give a broader picture of the occupational histories of the participants and how these relate to risk of cognitive impairment in later adulthood. We also show that the increased risk of dementia and MCI in individuals with high occupational physical activity persists after accounting for factors such as education and income. In addition, our use of rich national registry data on occupation provides additional strength to our findings by reducing the biases commonly associated with self-report.The observed associations of high occupational physical activity and increased risk of late-life cognitive impairment indicates that special attention must be given to workers in these occupations, warranting strategies aimed at preventing or reducing cognitive impairment.With a rapidly growing population of older adults worldwide, identifying modifiable risk factors for cognitive decline and impairment is receiving increasing attention. While there has been a focus on educational attainment, the role of other modifiable factors in the prevention of age-related cognitive decline and impairment has received less attention. However, accumulating evidence points towards the influence of occupational characteristics on cognitive health  are robustly related to lower risk of cognitive impairment,,,Prior studies on occupational PA and dementia have several limitations. Occupational PA has mainly been self-reported, i.e., participants were asked to recall and interpret the extent to which their job was physically demanding.∗NET).∗NET is a comprehensive database of the characteristics of over 900 occupations based on data from employees, occupational experts, and occupational analysts. This provides a unique possibility to use standardized as opposed to self-reported data to investigate the relationship between occupational PA and later-life cognitive impairment following a life-course perspective.In Norway, a resident's occupational history is prospectively registered and coded according to the International Standard Classification of Occupations-88 (ISCO) by Statistics Norway.Utilizing registry or administrative job task and occupational exposure matrix approaches can represent an important avenue for research into chronic disease outcomes with long latency periods or where cumulative repetitive exposures matter.We used a historical cohort design in which cognitive outcomes at age 70+ years were linked to trajectories of occupational PA at age 33–65 years. The study sample comprised 7005 participants from the 70+ sub-study of the fourth wave of the Trøndelag Health Study in Norway . The HUNT Study is a large ongoing population-based health survey performed in the Trøndelag County in Norway, where all adult residents were invited to participate in four surveys: HUNT1 (1984–1986), HUNT2 (1995–1997), HUNT3 (2006–2008), and HUNT4 (2017–2019). In the HUNT4 Survey, all county inhabitants aged 70 years and older  were invited to participate in the 70+ sub-study, and 9930 (51.2%) individuals aged 70–105 participated. In the present study, we included 7005 (72.0%) participants aged 70–105 (born in 1914–1949) from the HUNT4 70+ study for whom data from the cognitive assessment and data on occupational PA from at least two time points were available; notably, at least once before the age of 50 years, and at least once at or after the age of 50 years . This reIndividual data on occupation were obtained from administrative registers with full population coverage from Statistics Norway and linked to data on our study participants using the unique personal identification number assigned to all registered residents of Norway. Annual data were available for 1960, 1970, 1980, and the years spanning 1995–2014. The main occupation for each participant in each year was defined as the occupation which yielded the highest salary that year. Occupation was coded using four-digit occupation codes according to the Norwegian version of the ISCO-88 Standard.∗NET (2003 version)To determine occupational PA, the ISCO-88 occupational codes were linked to data from OAll participants in HUNT4 70+ were clinically evaluated for dementia and MCI using a comprehensive assessment of cognitive function, activities of daily living, neuropsychiatric symptoms, disease course, and a family caregiver interview.2 at HUNT1 and/or HUNT2.Data on age, sex, education , marital status , retirement age , and income  were obtained from national registries. Annual income commonly peaks around age 45–55. It is also less influenced by childbearing and breaks due to higher education found in earlier ages and avoids early retirement common in some occupations.Group-based trajectory modelling using the traj package in Stata 17.0,stable low , increasing then decreasing , stable intermediate , and stable high  that were used in the final statistical analyses. The decision on the number and shape of the trajectory groups was based on standard criteria,We used inverse-probability weighting (IPW) to account for non-response in HUNT4 70+ and multiple imputation (MI) with 20 iterations to impute missing values on potential covariates. Details on the IPW and MI procedures are provided in the The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.p for interaction = 0.65), age (p for interaction = 0.25), or education (p for interaction = 0.67) on risk of dementia or MCI. In the age- and sex-adjusted model (Model 1), individuals with stable high occupational PA trajectories had higher risks of dementia and MCI compared to individuals with a stable low occupational PA trajectory. A similar pattern was observed among individuals with stable intermediate occupational PA trajectories. Further, participants with an increasing then decreasing occupational PA trajectory had a higher risk of MCI, but not dementia, compared to individuals with a stable low trajectory . According to the fully adjusted estimates presented in stable low trajectory, participants with a stable high trajectory had a RRR for dementia of 1.34 (95% CI 1.04–1.73) and MCI of 1.80 (1.54–2.11), whereas participants with a stable intermediate trajectory had a RRR for MCI of 1.36 (1.15–1.61). While not statistically significant, participants with increasing then decreasing occupational PA had a RRR for dementia of 0.76 (0.53–1.11) and of MCI of 1.18 (0.96–1.46) when compared to the stable low PA group.There were no interactions between trajectories of occupational PA and sex  without IPW did not differ appreciably from the results from the multiple imputed and IPW weighted analyses, although the associations between trajectories of occupational PA and dementia risk had wider 95% CIs and were no longer statistically significant after adjustment for age, sex, education and mean annual income at ages 45–55 .Our findings extend those of prior studies,There are several plausible explanations for the observed associations between occupational PA and dementia and MCI. Higher occupational physical demands in later adulthood have previously been linked to smaller hippocampal volume and poorer memory performance.,Individuals working in physically-demanding professions may differ in terms of genetics, socioeconomic factors and environmental exposures, and confounding due to unobserved variation in these is possible. For instance, people with high occupational PA may have had lower early-life cognitive abilities,,,,,,Greater physical demands, lack of recuperation, and resulting exhaustion could imply more somatic ‘wear and tear’ and shorter recovery periods, which could collectively worsen cognition.,,intensity of PA, both at work and during leisure-time, on the brain in older ages.In line with the PA paradox demonstrating opposing effects of leisure-time PA and occupational PA on outcomes such as cardiovascular disease and mortality,The present study has several important strengths. We used a large, population-based cohort with clinically-based diagnoses of dementia and MCI, individually linked with rich Norwegian prospective registry data going back to 1960, and incorporating longitudinal information on socioeconomic, health, and admistrative data. These data enabled the construction of occupational PA trajectories to help elucidate how variation in occupational histories is associated with the risk of cognitive impairment. Further, we had available data on non-participants, which allowed us to weight the analyses for non-participation using IPW.∗NET is less vulnerable to recall bias than individual self-reported evaluations of occupational physical activity, the availability and quality of data in national registries tend to improve with time. The older individuals in our study had fewer data points as data availability is greater in more recent periods. Although fewer observations suggest lower estimation precision, point estimates are not affected when we drop those aged 90 or older from the analyses.As with most other studies, ours also has a few limitations. Although our use of registry data linked with O∗NET means that we may have missed information on how physically demanding different occupations were in earlier years, particularly in the years 1960, 1970, and 1980. In other words, our analyses did not account for temporal changes in work tasks within the same occupational category.,Further, our use of the 2003 version of OResults from this registry-based study underline the importance of occupational histories for cognitive health in later life. Our results particularly underscore the need to follow up individuals with high lifetime occupational PA, as they appear to have a greater risk of developing dementia.While leisure-time PA can lower cardiovascular risk factors such as heart rate and blood pressure, as well as lower the risk of diabetes, long durations of occupational PA may have the opposite  effects on cardiovascular disease, MCI and dementia risk. Our study and those of others undescore the need to find technologically-based solutions and ways to reduce high occupational PA across the working life, for instance, through greater work flexibility, job rotation, forced pauses and mandatory recovery periods, as well as possibly imposing a reduction of weekly or lifetime work hours. Future research should assess how occupational PA and interventions to reduce occupational PA or technological changes leading to altered occupational PA, in combination with other occupational characteristics, relate to dementia and MCI risk in older ages to further our understanding of the association between occupational histories and cognitive impairment.EZ: responsible for the original and final draft of the manuscript, data curation and formal analyses, worked with methodology, contributed to the conceptualization, drafting, and reviewing of the final manuscript.BB: data curation and formal analyses, worked with methodology, contributed to the conceptualization, drafting, and reviewing of the final manuscript.BHS: project administrator, data curation and formal analyses, acquired funding, worked with methodology, contributed to the conceptualization, drafting, and reviewing of the final manuscript.AJ: acquired funding, contributed to the conceptualization, drafting, and reviewing of the final manuscript.BLE: contributed to the conceptualization, drafting, and reviewing of the final manuscript.CB: acquired funding, contributed to the conceptualization, drafting, and reviewing of the final manuscript.GS: acquired funding, contributed to the conceptualization, drafting, and reviewing of the final manuscript.HPK: acquired funding, contributed to the conceptualization, drafting, and reviewing of the final manuscript.JRH: acquired funding, contributed to the conceptualization, drafting, and reviewing of the final manuscript.JW: contributed to the conceptualization, drafting, and reviewing of the final manuscript.SET: acquired funding, contributed to the conceptualization, drafting, and reviewing of the final manuscript.SK: contributed to the conceptualization, drafting, and reviewing of the final manuscript.TM: contributed to the conceptualization, drafting, and reviewing of the final manuscript.THE: contributed to the conceptualization, drafting, and reviewing of the final manuscript.YS: acquired funding, contributed to the conceptualization, drafting, and reviewing of the final manuscript.AKH: project administrator, acquired funding, worked with methodology, contributed to the conceptualization, drafting, and reviewing of the final manuscript.VS: project leader, contributed significantly to the original draft of the manuscript, acquired funding, worked with methodology, contributed to the conceptualization, drafting, and reviewing of the final manuscript.10.13039/100000002National Institutes of Health (grant number R01AG069109-01). It was also partly supported by the Research Council of Norway through its Centers of Excellence funding scheme . The funding agencies played no role in the design, execution, analysis or interpretation of data, or the writing of this manuscript.This work was supported by the https://www.ntnu.edu/hunt. Any research group with a Principal Investigator affiliated with a Norwegian research institute can apply for access to analyze HUNT data. This means that research groups from non-Norwegian countries must find a collaboration partner in Norway to be able to use HUNT material. Each project needs to be approved by the HUNT Data Access Committee (DAC), Regional Committee for Medical and Health Research Ethics, and in some cases also the Data Inspectorate. Due to participant confidentiality, participant data are not publicly available.The data used for this study were derived from The Trøndelag Health Study (HUNT), This study was approved by the HUNT study board of directors and the Regional Committee for Medical and Health Research Ethics. All participants in the HUNT Study gave a written informed consent upon participation.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.


Maternal stress in the postpartum period affects not only the mother but also her newborn child, who is at increased risk of developing metabolic and mental disorders later in life. The mechanisms by which stress is transmitted to the infant are not yet fully understood. Human milk (HM) is a potential candidate as maternal stress affects various components of HM, e.g., fat and immunoglobulin concentrations. To date, it is unknown whether maternal stress also affects the amino acids (AAs) in HM, even though this nutrient is of extreme importance to child health and development. This study aimed to investigate whether and how maternal stress is associated with the AA composition of HM.n = 24), and a control (CTL) group; women who gave birth to a healthy child (n = 73). HM was collected three times a day, on postpartum days 10, 17, and 24. Perceived psychological stress was measured using validated questionnaires, while biological stress measures were based on hair, saliva, and HM cortisol concentrations. HM protein-bound and free AAs were analyzed by liquid chromatography and compared between groups.In this observational cohort study , lactating women were recruited in two study groups: a high-stress (HS) group; women whose child was hospitalized (p < 0.01). The concentrations of protein-bound AAs in HM were higher in the HS group compared to the CTL group (p = 0.028) and were positively associated with HM cortisol concentrations (p = 0.024). The concentrations of free AAs did not differ between study groups and were unrelated to cortisol concentrations.Maternal perceived stress scores were higher in the HS group (Findings from this prospective cohort study suggest that maternal stress in the postpartum period is associated with an altered human milk amino acid composition, which could play a role in the transmission of maternal stress effects to her child. The physiological implications of these stress-induced changes for infant development await future research. Maternal stress in the postpartum period not only affects the mother but may also have consequences for her newborn child. It has been shown that maternal stress occurring during this sensitive developmental period is associated with the infant's risk of developing a wide range of disorders, including metabolic and mental health disorders –3. BecauHM is the optimal source of nutrition for newborn infants –8. It isIn both human and animal plasma, AAs decrease as a result of stress –20. As mThis study aimed to investigate whether maternal stress in the first month postpartum is associated with the fraction of protein-bound AA (BAA), which makes up 90–95% of the AAs, and the free AA (FAA), which comprises a relatively small fraction of the AA in HM . We furtWe studied a prospective observational cohort of lactating women who were followed over their first month postpartum, and experienced various amounts of stress. Participants were recruited during pregnancy or within the first 10 days after giving birth, via social media, flyers at midwife practices, or at the maternal or neonatal ward of the Amsterdam University Medical Center . Mothers were eligible to participate when they were 18 years of age or older, and if they had the intention to breastfeed their infant for at least the first month after birth. Exclusion criteria were as follows: (1) maternal  diabetes mellitus, as the glucocorticoid system may be regulated differently , 26, (2)To ensure the inclusion of a large enough range of stress levels among the included participants, two groups of women who delivered at term were included: a high-stress group (HS group) and a control group (CTL group). Women were included in the CTL group when they gave birth to a healthy infant at term. Women were included in the HS group when they gave birth to an infant at term who was admitted to the hospital for a minimum of 2 days. Hospitalization of the infant was considered a maternal stressor.Recruitment took place in The Netherlands between November 2017 and December 2019. Written informed consent was obtained from all participants prior to participation. This study was approved by the Ethics Committee of the Amsterdam University Medical Centre, AMC on 2 May 2017  and conducted in accordance with the Declaration of Helsinki.A strand of hair  was cut by the researcher as close to the scalp as possible at the posterior vertex position. The hair was stored in the dark at room temperature until analysis. A short questionnaire was filled out by the participants in order to correct for factors influencing hair glucocorticoid concentrations. Hair samples were analyzed for cortisol and cortisone as a baseline biological stress measurement reflecting the last trimester of pregnancy , 29.Saliva was collected two times in the morning on every collection day to measure the cortisol awakening response. Participants were instructed to chew on a swab  for 1 min. The first sample (S1) was obtained within 0–10 min after waking, and the second sample (S2) was obtained 30–45 min after waking , participants collected three HM samples in which concentrations of AAs and cortisol/cortisone were measured. HM cortisol/cortisone was measured to be able to directly correlate this with the HM AAs. To take into consideration, the circadian rhythm of HM cortisol and to make sure that circadian variation in HM AAs was represented in the samples , 32, parThe questionnaires used in the study are described below. For the analyses, the total questionnaire scores and their ranges were used. To establish the participant's lifetime stress exposure, the participants filled out two questionnaires at the start of the study.The Dutch version of the LSC-r questionnaire. This checklist is a 26-item scale to identify exposure to traumatic events or other stressful life events . Each itThe Dutch version of the JTV questionnaire (25 items) is a self-reported inventory that provides a brief and relatively non-invasive retrospective assessment of early life traumatic experiences . The JTVAfter each collection day, participants received a 24 h-recall using the digital program Compl-eat™ developed by the Department of Human Nutrition at Wageningen University (the Netherlands) . This quduring the study period was obtained by three questionnaires that the participants filled out at the end of the study, concerning the levels of stress they experienced during the past month.A measure of psychological stress levels The perceived stress scale is a validated 14-item questionnaire. The questionnaire determines the degree to which certain situations are experienced as stressful , 38. EacThe Dutch version of the well-validated EPDS is a 10-item self-inventory to assess symptoms of depression and/or anxiety in women who recently gave birth .The Dutch version of the STAI is a well-established measure of trait and state anxiety and consists of two parts. The first part of this inventory, the STAI-state (STAI-s), contains 20 items to assess anxiety at this moment, rated on a 4-point intensity scale. The second part, the STAI-trait (STAI-t), contains 20 items and assesses anxiety, in general, rated on a 4-point intensity scale .For analyses, the proximal 3-cm hair segment was used. Wash and steroid extraction procedures were performed as described by Stalder et al., with some changes being made to allow analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) . The lowCortisol and cortisone in saliva were determined using supported liquid extraction (SLE+) followed by LC-MS/MS detection. Quantification was performed using an isotope dilution, with the limit of quantification being 0.3 nmol/L. The mean intra-assay variation was 6 and 7% for cortisol and cortisone, respectively.For each HM sample, cortisol and cortisone were determined using liquid extraction followed by SLE+ and LC-MS/MS detective as described earlier by van der Voorn et al. . QuantifFor the determination of BAA in HM samples, the three milk samples from 1 collection day were mixed to have a good representation of BAA concentrations during the whole day. Thereafter, 0.20 ml of diluted hydrochloric acid containing 0.5% 2-mercaptoethenol was added to the HM sample and mixed. The present oxygen was removed by flushing the headspace of the tube with nitrogen for 60 s. The protein was hydrolyzed by heating the mixture for 20–22 h. When the mixture was cooled down, 0.20 ml of sodium hydroxide solution, 2.0 ml of demi water, and 0.2 ml of internal standard  were added and mixed. The mixture was centrifuged, and a small part of the liquid was filtered over a 0.45 mm polyvinylidene difluoride filter. The peak area of each AA was calculated using the LabSolutions software from Shimadzu and compared to the peak area of the internal standard (Sigma).The mixed HM samples were also used to determine the free AA in HM. An ultra-fast liquid chromatography (UFLC)-based protocol was used. Each 50 μl of milk sample was mixed with 1.0 ml of internal standard solution . This mixture was centrifuged, and 25 μl of supernatant was transferred into a sample vial. A pre-column derivatization process was carried out by adding 30 μl of o-phthalaldehyde (OPA) reagent to the vial and mixing three times with a mixing volume of 45 μl. One microliter of this OPA-derivatized sample was injected and analyzed in a UFLC system with fluorimetry to detect the signal.Standard AA solution Sigma AA-S-18 was used for calibration. To prepare the calibration AA solution, asparagine, and tryptophan were added in to Sigma AA-S-18 stock solution to reach a concentration of 2.5 μM/ml of each AA. Next, 0.50, 1.0, 2.0, and 5.0 ml of this solution were mixed with 1.6 ml of perchloric acid and further diluted to 50 ml. The calibration AA solution was prepared to OPA derivate as described previously and measured in an UFLC system. The calibration curve was constructed from peak areas and AA concentrations. Response factors for each AA were obtained by an extra analysis of a standard AA solution containing internal standards.t-tests , chi-square tests , Mann–Whitney U-tests , or linear mixed models (for data that were measured at multiple time points) were used. The mean of the three 24-h recalls was used to compare the dietary intake of AA between groups, as this reflects the overall intake of the participants during the study period.Sample characteristics were described as mean with standard deviation (SD), median with 25th and 75th percentiles (Q1–Q3), or frequencies. To test differences in maternal and infant characteristics , dietary AA intake over the study period, and stress measurements (questionnaires and cortisol) between both study groups, unpaired Student's * [new (standardized)] time point – real-time point) (The HM cortisol area under the curve (AUC) was calculated to provide a value that better reflects HM cortisol throughout the day, which is known to follow a circadian rhythm . Therefoe point) . Subseque point) . The core point) . The higAnalyses were performed separately for BAA and FAA. Before analysis, the HM AAs were categorized into different outcome variables: total AA, essential AA, and non-essential AA. Because AA from one precursor family can be converted into other members of this family, AAs were also grouped into precursor groups: the glutamate precursor group , the aspartate precursor group , the serine precursor group , the pyruvate precursor group , the aromatic precursor group , and the histidine precursor group (histidine). Methionine was analyzed separately .All outcome variables were checked to see whether they were normally distributed. When variables were not distributed normally, a log transformation was performed. When participants completed <1 full day of sample collection, they were excluded from the final analysis.To answer the research question whether and how maternal stress affects the BAA and FAA concentrations in HM, the AA outcomes as described above were compared between the HS and CTL groups. As all study time points were taken into account in the comparison, linear mixed models were used to analyze the group differences to control for within-person repeated measures. The analysis was corrected for factors differing between study groups. In addition, we tested whether maternal dietary AA intake during the study period differed between study groups. If the maternal dietary intake of a specific AA statistically differed between groups, the comparison of that specific HM AA was corrected for the maternal intake. As HM AA concentrations differ between the different weeks and stages of lactation, the interaction between the study time point and study group was investigated and reported.p-value of <0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics for Windows, version 27. GraphPad Prism 9 for Windows was used to display the results.To answer the research question of whether HM cortisol concentrations (cortisol AUCs) are related to HM AA concentrations, we performed a secondary analysis. The relationship between HM AA and HM cortisol AUCs was investigated independently of the study group. This relationship was only investigated for total AA, essential AA, non-essential AA, and methionine in HM. As all study time points were taken along in this secondary analysis, linear mixed models were used to control for within-person repeated measures. The analysis was corrected for potential confounding factors that have been shown to influence the AA composition of HM in the previous literature: AA dietary intake and maternal BMI , 45–47. p = 0.048); the birthweights did not differ between study groups. In the HS group, hospitalization duration ranged between 2 and 12 days, with a median of 7 days. The duration of hospitalization in this group was not associated with maternal stress scores (p = 0.282 for PSS score).In total, 86 lactating women were included in the CTL group and 30 in the HS group. Nineteen women stopped participating before the end of the study due to various reasons see . Charactp < 0.01)  and biological stress (hair cortisol) measurements were the same between study groups . Perceiv < 0.01) . There wp = 0.028]. This was also the case for essential BAAs  and non-essential BAAs  and for the concentrations of the BAA precursor groups (p < 0.035), except for the glutamate precursor group, which showed the same concentrations in both study groups. There were no interactions between the study group and study time point ; p = 0.001]. All statistical analyses were corrected for infant sex.Despite higher concentrations of BAA of the aspartate precursor family in the HS group . Protein-bound methionine was not associated with the HM cortisol AUC. Total FAA, essential FAA, non-essential FAA, and free methionine were also not associated with the HM cortisol AUC, at the separate study time points.Total BAA concentrations, as well as essential BAAs and non-essential BAAs, were positively associated with the HM cortisol AUC [5.54 ; In this study, we explored associations between maternal stress and the AA composition of HM. We demonstrated that perceived and biological maternal stresses in the first month postpartum were positively associated with the concentrations of BAAs in HM. In particular, BAA concentrations were increased in the HM of mothers with high-stress levels, with the exception of bound methionine. Methionine was, contrary to our hypothesis, not associated with maternal stress. However, while the overall concentrations of FAAs in HM did not differ between study groups, free methionine was higher in the HM of mothers with high levels of perceived stress.Our results are in line with previous animal experimental studies , 23. In The biological mechanism behind the observed associations between maternal stress and HM AA composition is not yet clear. In fact, the process behind the transport of AAs from the maternal bloodstream into the mammary gland and into the HM is complex and not yet fully understood . Both BA+ amino acid transporters, one could hypothesize that cortisol is involved in the mechanisms leading to these stress-induced changes in HM (p = 0.074).Exactly how and to what extent stress influences transport processes also remains to be elucidated. As previous research showed associations between glucocorticoid levels and the expression of certain AA transporters in the mammary gland, e.g., the system L and the system yes in HM , 48. Indes in HM . A possiWe further focused specifically on methionine, as in mice, maternal stress has led to reduced methionine in the brain and plasma of the offspring, which was associated with later-life cognitive deficits . ContrarIncreased concentrations of BAA in the HM of mothers in the HS group may be due to the fact that mothers with high levels of stress produce less milk, in general, compared to mothers with lower levels of stress –51. WhenDuring lactation, milk-specific proteases break down HM proteins into FAAs. HM further contains protease activators and protease inhibitors . Over thThe strengths of our current study are its longitudinal design and the timing and frequency of HM sample collection. The first month postpartum is a sensitive time window that has been frequently missed in earlier HM research, but since breastfed infants depend on HM during this period as their only source of nutrition these first weeks after birth represent a very important period. Furthermore, the fact that mothers were exposed to a stressor  ensured that the study population contained participants with established high levels of perceived stress. Additionally, extensive information on both maternal psychological and biological stress was collected, and the HS and CTL groups were similar at baseline with regard to lifetime stress levels or other important characteristics that could have influenced the relationship between stress and HM AA levels. The limitations of this study are, first, the relatively small sample size, especially in the HS group. Second, current perceived stress scores were only measured once, i.e., at the end of the study. Therefore, we were not able to investigate whether the different dynamics of FAA in the HS group were related to changes in the amount of stress experienced at that exact moment. Moreover, because of the exploratory nature of this study, we decided not to correct for multiple testing, even though we did perform a relatively large number of statistical tests, which increases the likelihood of a type 1 error. Therefore, our results should be interpreted with caution, and future studies should demonstrate whether these findings can be replicated. In addition, no reliable information was collected about infant feeding modes and additional feeding practices. The relatively high loss to follow-up and the fact that this was higher in the HS group compared to the CTL group  may have caused selection bias. Dropout of women who may have been more stressed, especially in the HS group, may have led to an underestimation of associations between maternal stress and AA. Finally, selection bias might have contributed to the fact that our cohort mostly consisted of healthy and highly educated women. Furthermore, the majority of our participants were of Western ethnic background. This may limit the generalizability of the results.The results of our study suggest that mothers with high-stress levels have increased concentrations of BAA in their milk. The increased concentrations of BAA that we found may partially compensate for the higher nutritional requirements of infants under stressful circumstances. However, as impaired growth and development have been observed in children of mothers with high amounts of stress in the postnatal period, this increase in AA in the HM might not be sufficient to maintain optimal growth and development. Moreover, the increase was not observed for protein-bound methionine or total HM FAA.In conclusion, findings from this unique prospective cohort study suggest that there is a relationship between maternal stress in the first month postpartum and the AA concentrations of HM during this time. Our findings emphasize the importance of the maternal psychological state during lactation. In the last few years, attention to the prevention of the detrimental consequences of stressful experiences in early life has increased. Stress reduction programs for children and parents have been developed, such as family-integrated care and relaxation therapy using relaxing music –59, withThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Amsterdam University Medical Centre, AMC. The patients/participants provided their written informed consent to participate in this study.EN, JG, SR, LS, and AK designed the research. HJ and EN conducted the research. LS, JG, and AK provided essential materials. HJ, JH, and SR analyzed the data or performed statistical analysis. JH, HJ, BK, SR, and AK accessed, verified, and interpreted the data. HJ, SR, and AK wrote the first version of the manuscript. AK had primary responsibility for the final content. All authors critically read and contributed to finalizing the manuscript, had full access to all the data in the study, and accept responsibility to submit it for publication.