We assessed osteocyte lacunar morphometric parameters in trabecular and cortical bone regions using micro‐computed tomography (micro‐CT) at a nominal resolution of 1.2 μm voxel size. The cortical osteocyte lacunar volume (Lc.V) was 7.7% larger (p = 0.05) and more spherical  in the T2D + MVD group. Using linear regression, we found that lacunar density (Lc.N/BV) in trabecular but not cortical bone was associated with HbA1c  independently of MVD. Furthermore, Lc.V was larger and Lc.Sr higher in the center than in the periphery of the trabecular and cortical bone regions (p < 0.05). In conclusion, these data imply that MVD may impair skeletal integrity, possibly contributing to increased skeletal fragility in T2D complicated by MVD. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Clinical studies indicate that microvascular disease (MVD) affects bone microstructure and decreases bone strength in type 2 diabetes mellitus (T2D). Osteocytes are housed in small voids within the bone matrix and lacunae and act as sensors of mechanical forces in bone. These cells regulate osteoclastic bone resorption and osteoblastic bone formation as well as osteocytic perilacunar remodeling. We hypothesized that MVD changes morphometric osteocyte lacunar parameters in individuals with T2D. We collected iliac crest bone biopsies from 35 individuals  with T2D with MVD (15%) or without MVD (21%) with a median age of 67 years (interquartile range [IQR] 62–72 years). The participants were included based on c‐peptide levels >700 pmol L In this article, we are using micro‐CT imaging to demonstrate a larger cortical osteocyte lacunar volume in patients with type 2 diabetes mellitus (T2D) and microvascular disease (MVD) compared with patients without MVD. Furthermore, we observe an association of glucose control and trabecular lacunar density independently of MVD. Finally, our data indicate a general low cortical lacunar volume in T2D when compared with previous published articles with healthy participants. Insulin insensitivity and inadequate insulin secretion in T2D cause hyperglycemia, which may lead to microvascular disease (MVD), including neuropathy, nephropathy, and retinopathy. In addition, fragility fractures are more commonly reported in T2D compared with individuals without diabetes, despite normal or elevated bone mass measured by dual energy X‐ray absorptiometry (DXA). Fracture risk is not homogeneously distributed across individuals with T2D. In a recent Swedish registry study, fracture risk was higher in patients who were on insulin treatment, with low physical activity, a body mass index (BMI) below 25 kg m−2, and long‐standing disease duration but lower in most (55%) individuals with T2D than in age‐ and sex‐matched background population. Other factors are also considered to increase fracture risk in T2D, including increased risk of falls, poor glycemic control, and diabetes complications, including MVD.Type 2 diabetes mellitus (T2D) affects more than 450 million individuals and is predicted to affect an increasing number of people in the years ahead.1 Insu which appears to be most pronounced in T2D patients with MVD. Reduced skeletal blood flow resulting from peripheral vascular disease is associated with higher cortical porosity in individuals with T2D, supporting that vascular dysfunction may influence bone structure. The mechanisms underlining higher cortical porosity in T2D complicated by MVD remain unknown and may include several factors, such as impaired recruitment of osteogenic cell types, tissue hypoxia, or restricted availability of nutrients in the cortical bone.Clinical studies based on high‐resolution peripheral quantitative computed tomography (HR‐pQCT) scans have shown an increased cortical porosity in T2D,10 whi In the process of bone remodeling, osteoclasts resorb bone, which is followed by osteoblastic bone formation. A fraction of the osteoblasts become embedded in the bone matrix, where they terminally differentiate to osteocytes. The mature osteocytes develop processes that span the entire bone matrix and connect osteocytes, cells on the bone surface, and the lumen of the vasculature to form the lacunocanalicular network. Through this network, mechanical strains are directed by fluid pressure to the osteocytes, which orchestrate bone remodeling. After mechanical unloading, osteocytes secrete sclerostin, which inhibits bone formation by blocking Wnt signaling. Although serum sclerostin levels and insulin sensitivity were not associated in healthy and prediabetic men, sclerostin levels are increased in overt T2D. This indicates that hyperglycemia changes the function of osteocytes and the lacunocanalicular network, which may lead to lower bone remodeling and higher cortical porosity in T2D. Importantly, glucose increases SOST expression and sclerostin content in cultured osteocyte‐like IDG‐SW3 cells, which was validated in vivo and ex vivo in streptozotocin‐induced hyperglycemic rats. Further, it was demonstrated that sclerostin directly promotes the resorption of the perilacunar matrix, a process called osteocytic osteolysis. These findings support that T2D may influence osteocyte function and subsequently bone remodeling.Histological investigations have demonstrated lower bone remodeling in T2D.13 In  which may be changed in pathological conditions such as T2D and MVD. In mice, lactation induces osteocytic osteolysis, which increases the sensitivity of osteocytes due to a larger osteocyte lacunar volume (Lc.V). With age, Lc.V decreases and lacunar sphericity (Lc.Sr) increases, which may lead to a reduced ability to sense mechanical loading. The consequences of the development of MVD in T2D to osteocyte lacunae morphology have not been explored. We speculate that impaired blood flow may lead to hypoxia and subsequently a metabolic shift of the osteocyte toward anerobic glycolysis, which could result in acidification of the lacunae and enhanced osteocytic osteolysis, resulting in larger lacunae. We further separately evaluated peripheral lacunae close to bone surface (<20 μm) and central lacunae (>20 μm) to account for possible differences in age of the lacunae, nutrient availability, mechanical loading, and proximity to the vasculature.Besides bone remodeling through osteoclasts and osteoblasts, osteocytes can also change the lacunar morphology through perilacunar remodeling, eg, to adapt to mechanical forces,21 whi We hypothesize that MVD as observed in T2D changes iliac crest morphometric osteocyte lacunar parameters in T2D patients. Therefore, the aim of this study was to investigate cortical and trabecular osteocyte lacunar morphology in T2D individuals with and without MVD using high‐resolution micro‐CT.Previous studies have demonstrated increased cortical porosity in T2D; however, these data are based on HR‐pQCT scans, which cannot capture osteocyte lacunar size and shape.26 We −1 at the time of enrollment, or if insulin treatment was instigated within the first 2 years after the diagnosis. In addition, candidates with known metabolic bone or calcium disorders, chronic kidney disease (creatinine >90 μMol), or liver dysfunction (aspartate transaminase >3× upper limit) were excluded. Candidates were excluded if they were on any medical treatment known to influence bone metabolism, eg, systemic corticosteroids. Information on fracture history including trauma mechanism (low‐ or high‐energy trauma) were registered at time of inclusion. Furthermore, at enrollment it was assessed whether the participants had MVD, which was defined by the presence of a clinical diagnosis of either nephropathy, neuropathy, retinopathy, or a combination of the aforementioned. According to the MVD status, participants were grouped into the T2D group or the type 2 diabetes complicated by MVD group (T2D + MVD).This is a cross‐sectional study in adult individuals with T2D with and without MVD. Individuals were prospectively included in the study between May 2017 and March 2020 at the endocrine outpatient clinics at Odense University Hospital (OUH) and Kolding hospital, Denmark. All procedures were conducted in accordance with the Declaration of Helsinki and were approved by local ethics committee (ID: 21/21773). Written informed consent was obtained from all participants. Men and postmenopausal women with a history of T2D >5 years were included. The inclusion criteria also covered plasma c‐peptide levels >700 pMol and the absence of antibodies against glutamic acid decarboxylase (anti‐GAD65) at any time point after the T2D diagnosis. Candidates for this study were excluded in case of monogenic or secondary diabetes, if glycated hemoglobin (HbA1c) was above 75 mmol mol1 to L4, lumbar spine BMD), hip , and nondominant hip region  were measured using DXA scans . The coefficient of variation (CV) is 1.5% at both the spine and the hip at our bone unit . Female reference ranges were used to calculate T‐scores in both men and women to test osteoporosis status. In addition, trans‐iliac crest bone biopsies were collected using a modified Bordier trephine (inner diameter: 8 mm). The biopsies were subsequently dehydrated in ethanol and embedded in poly methyl methacrylate (PMMA) at −20°C.Areal bone mineral density (BMD) at the lumbar spine  to a rectangular block and turned with a conventional lathe  to obtain a cylinder with a diameter of 3.8 mm ± 0.05 mm. This cylinder was placed into a custom‐made sample holder and scanned at 6 μm nominal resolution, followed by two high‐resolution (HR‐micro‐CT) scans with 1.2 μm nominal resolution covering a cortical and a trabecular bone region. The 3D image stacks were acquired using 72 μA current, 4 W power, 55 kVp energy, 1.5‐second integration time, level 6 data averaging, and 1500 X‐ray projections  used in combination with the software XamFlow  as previously described in Goff and colleagues. By this, the following lacunar morphometric parameters were assessed: bone volume/tissue volume (BV/TV), cortical thickness (Ct.Th), osteocyte lacunar number (Lc.N) and density (Lc.N/BV), osteocyte Lc.V, osteocyte lacunar oblateness (Lc.O), and osteocyte lacunar sphericity (Lc.Sr). For the lacunar parameters, local lacunar parameters were used, meaning that apart from Lc.N, the data were presented as means of each bone biopsy, which results in an equal power per sample but varying impact of each lacuna due to different Lc.N per sample. Additionally, Lc.V and Lc.Sr were assessed individually for peripheral (<20 μm from bone surface) and the central (>20 μm from bone surface) regions of the of the bone compartments.We scanned the specimens using micro‐computed tomography . A previously validated cutting and image segmentation process was used to identify osteocyte lacuna characteristics.27 In p ≤ 0.05.We applied linear regression models to test whether osteocyte lacunae characteristics were different between the T2D and T2D + MVD groups and to determine their relationship with clinical characteristics such as MVD, HbA1c, and T2D disease duration. Regression models were adjusted for age and sex of the individuals. Comparisons between peripheral and central osteocyte lacunar parameters of the same sample were tested using Wilcoxon tests. Data distribution was assessed using histograms, and non‐normally distributed data was log10 transformed. All analyses were performed using R (Version 3.5.1), and results were considered significant for n = 15, 5 female and 10 male) had MVD. The median age of the participants was 67.0 years (interquartile range [IQR] 63.8–72.0), and the mean HbA1c was 56 mmol mol−1 (SD: 9.48) or 7.3% (SD: 0.87). All participants were diagnosed with T2D for at least 5 years, with a mean of 16.09 years (SD: 7.67). Overall, clinical differences between participants with T2D with and without MVD were not observed. BMD at the lumbar spine, femoral neck, and total hip were similar in both groups (Table T‐score of −2.5 or lower (2 in the T2D group and 2 in the T2D + MVD group) or an osteoporotic vertebral or hip fracture (2 in the T2D + MVD group).Thirty‐five individuals were included  of which 43% . We did not observe group differences in Lc.V in the trabecular bone . Furthermore, cortical Lc.Sr was significantly higher in the T2D + MVD group compared with the T2D group . There were no group differences between T2D + MVD and T2D in trabecular Lc.N/BV  and Lc.O  or cortical Lc.N/BV  and Lc.O   and a lower trabecular Lc.N/BV in men   were used for cortical and 35 biopsies  for trabecular analyses. Micro‐CT scans showed that cortical and trabecular BV/TV, Ct.Th, and cortical porosity (Ct.Po) were not significantly different in the T2D and T2D + MVD groups . The linear regression showed an increase of 87 Lc.N/BV for every mmol mol−1 (0.09%) increase of HbA1c  but not in the peripheral region   and the remaining lacunae in trabecular and cortical bone . Central cortical Lc.V and Lc.Sr were significantly larger in the T2D + MVD group versus the T2D group   and 197 μm3 (IQR 185–221), respectively. In both studies, lacunae volumes in the cortical bone specimens were larger with median Lc.Vs of 223 μm3 (IQR 189–245) and 282 μm3 (IQR 228–303), respectively. In postmenopausal women, Akhter and colleagues reported trabecular Lc.Vs of 188 μm3 (IQR 172–215) and cortical Lc.Vs of 245 μm3 (IQR 211–275). Although we observed similar median trabecular Lc.V of 172 μm3 (IQR 160–193) for the T2D group and 178 (IQR 163–203) for the T2D + MVD group, cortical Lc.V were smaller in this study with 151 μm3 (IQR 146–263) for the T2D group and 163 μm3 (IQR 152–178) for the T2D + MVD group. Although direct comparisons are challenging, these data indicate that cortical Lc.V may be smaller in individuals with T2D. Importantly, this study included elderly men and postmenopausal women, suggesting that dissimilarities in Lc.V measured in the present study and those conducted in pre‐ and postmenopausal women could also be explained by age and sex in addition to the presence of T2D. Although there were no statistically significant sex‐specific differences in any of the outcomes, cortical Lc.V and Lc.Sr tended to be higher in men. The MVD status was not considered in these analyses because of a low number of samples in each group.Two studies using the same technique to assess iliac crest bone tissue from healthy premenopausal women reported similar lacunae volumes in trabecular bone with median Lc.V of 178 μmectively.29 In 3 (SD ± 31) in women and 523 μm3 (SD ± 39), whereas trabecular Lc.V was not reported. Bach‐Gansmo and colleagues did not find an association of morphometric lacunar parameters with age but a decrease of lacunar density with age. Furthermore, diseases and their treatments may also influence the lacunae morphology. Increased Lc.V have been observed in rodents on parathyroid hormone (PTH) treatment, in patients with vitamin D deficiency, and during lactation, suggesting that different mechanisms may influence Lc.V.Although lacunae volumes have been reported in other studies, comparisons are particularly challenging because of differences in imaging techniques, resolution, sample acquisition and segmentation, and sample sites. For example, one large study based on iliac crest bone using synchrotron micro‐CT reported mean cortical Lc.Vs of 538 μmwith age.30 Fur but not all previous reports. Akhter and colleagues reported higher Lc.N/BV in trabecular bone compared with cortical bone in iliac crest biopsies, whereas we observed an almost 2× higher Lc.N/BV in cortical than in trabecular bone in our study population. Although Lc.N/BV appeared not to be affected by MVD in T2D, we did observe an association between HbA1c and trabecular but not cortical Lc.N/BV, which might be due to a higher bone turnover and a higher connection of the bone surface to the bone marrow of trabecular bone than cortical bone. The higher bone turnover may drive a faster change in Lc.N/BV potentially as a compensation of hyperglycemia‐induced loss of mechanosensitivity.The osteocyte lacunar densities identified in this investigation are similar to some36 but In normal conditions, mechanical loading results in a drop of sclerostin expression to favor osteoblastogenesis and thereby bone formation. In a mouse experiment, it was demonstrated that an increase in Lc.V is associated with a higher suppression of SOST in bone after mechanical loading. This indicates that osteocytes may utilize osteocytic osteolysis to increase the mechanosensitivity. It is possible that osteocyte lacunae in the central part of the bone experience different mechanical loading than those in the peripheral regions of the bone, which may lead to changes in osteocyte Lc.V to compensate for different mechanical stimuli. Larger Lc.V in the T2D + MVD group than in the T2D group without the complication was found in the central but not in the peripheral part of the cortical bone region, indicating that group differences in the entire cortical region were driven by the central lacunae.Independent of MVD, we also observed higher Lc.V and Lc.Sr in central than in peripheral bone regions of the cortical and trabecular region. A key role of osteocytes is the inhibition of bone formation during mechanical unloading, which is achieved by the expression of sclerostin, a strong inhibitor of the Wnt signaling pathway that promotes bone formation.15 In  Therefore, changes in vasculature may be transferred to the osteocytes and may contribute to perilacunar remodeling. Although the mechanisms leading to larger Lc.V in T2D complicated by MVD remain to be determined, it may be that increased blood glucose causes the previously reported elevated sclerostin levels in T2D, which could induce osteocytic osteolysis, resulting in larger Lc.V. In murine and human cultured osteocyte‐like cells, it was demonstrated that sclerostin directly upregulates key enzymes for resorption. This finding was further validated in vivo in mouse bone by enlarged Lc.V after elevating sclerostin levels. Further, metabolic changes due to hypoxia and acidification may drive Lc.V enlargement in MVD. Long‐standing hyperglycemia is a risk factor for MVD in T2D, and larger Lc.V may also serve as an adaption to a cellular loss of mechanosensing abilities caused by elevated glucose levels in the T2D + MVD group.The vasculature is closely connected to the lacunocanalicular network with canaliculi connecting both microenvironments, the vascular system and the lacunocanalicular network.14 The(10) Using the same technique, Shanbhogue and colleagues showed that individuals with T2D complicated by MVD had a higher cortical porosity at the radius and tended to have a higher cortical porosity at the tibia than healthy controls and T2D patients without MVD. Although these studies indicate that mechanical loading may prevent expansion of cortical porosity at the tibia, the latter finding suggests that MVD affects cortical porosity even at weight‐bearing sites.A recent meta‐analysis identified and increased cortical thickness in the distal radius and tibia, as well as increased cortical porosity in the radius but not in the tibia in adult T2D patients assessed using HR‐pQCT at a resolution of 82 μm.olleagues11 sho Using micro‐CT scans with a resolution of 6 μm, we did not observe an increase in cortical porosity in iliac crest biopsies in T2D patients with MVD. Importantly, this does not preclude presence of higher cortical porosity in MVD based on pores with a size above approx. 160 μm. We speculate that MVD impairs angiogenesis, resulting in a lower number of newly formed blood vessels and basal membrane thickening that expands blood vessels. Although purely speculative, the absence of higher cortical porosity in the T2D + MVD group in the present study could be explained by higher density of small blood vessels in the T2D group and larger blood vessels in the T2D + MVD group, resulting in a similar degree of cortical porosity using micro‐CT scans but larger cortical porosity in a MVD group if assessed using HR‐pQCT scans.This is supported by higher cortical porosity in the distal tibia in T2D patients with peripheral vascular disease.12 Usi In our study, cortical Lc.Sr was higher in the T2D + MVD group, potentially indicating a loss of mechanosensitivity of the osteocyte and a counter‐regulatory perilacunar remodeling to increase the impact of mechanical stimuli. Importantly, our investigation was based on bone tissue from a non‐weight‐bearing site. It remains to be investigated if MVD in T2D affects osteocyte sphericity in bone that is subjected to larger mechanical loading than in the iliac crest.Further analyses of the osteocyte lacunar morphometry revealed an increased Lc.Sr in the T2D + MVD group. Because Lc.Sr is defined by the lacunar surface area (Lc.SA) and the Lc.V, Lc.Sr may influence Lc.V or vice versa. Lc.Sr is considered to be an indicator for the mechanosensing ability of osteocytes, especially in weight‐bearing bone. Thus, the osteocyte may reshape the osteocyte lacuna to decrease or increase the impact of mechanical loading on the osteocyte. In weight‐bearing bone, this may lead to an elongation of the lacunae along the applied load direction to decrease fluid pressure.40 In This study has several limitations. The present study included a cohort of 35 iliac crest biopsies from well‐characterized adult men and women with T2D. However, because of the absence of healthy matched controls, we were limited to comparing our results to published data sets. Furthermore, the cross‐sectional study design precluded assessment of changes in factors that may influence osteocyte lacunar morphology such as glucose control. As stated above, inverse relationships between vitamin D levels and lacunae volumes have been reported. Vitamin D levels were not available in the present investigation; therefore, we cannot exclude the possibility that vitamin D deficiency may have influenced the results. Although the imaging technique used allowed for an in‐depth assessment of the osteocyte lacunae, we were unable to test the impact of T2D and MVD on the osteocyte canaliculi or perilacunar matrix composition, which should be the focus of future investigations. Because of the lack of actual assessment of bone vasculature, we were unable to determine if MVD is present in the bone microenvironment or represents a marker for long‐standing uncontrolled blood glucose. Finally, we should mention that MVD is a composite of three clinical findings, and more investigations are needed to determine if the impact of MVD on bone is driven by neuropathy, nephropathy, changes in the vasculature, or a combination. Jointly, these investigations suggest that diabetic MVD may affect osteocyte function, which could contribute to diabetic bone fragility.In conclusion, our investigation demonstrated a larger Lc.V and a higher Lc.Sr in adults with T2D + MVD than in individuals with T2D without overt MVD. Furthermore, we observed an association of HbA1c with higher Lc.N/BV in trabecular bone, which together indicate that diabetes may change morphometric lacunar parameters and lacunar density. Finally, we report smaller cortical Lc.V in our study population, which is also reported with aging.41 JoiSebastian Zanner: Data curation; formal analysis; investigation; methodology; project administration; visualization; writing – original draft; writing – review and editing. Elliott Goff: Methodology; software; supervision; writing – review and editing. Eva Maria Wölfel: Supervision; writing – review and editing. Samuel Ghatan: Data curation; formal analysis; writing – review and editing. Charlotte Ejersted: Resources; writing – review and editing. Gisela Kuhn: Project administration; writing – review and editing. Ralph Mueller: Funding acquisition; project administration; writing – review and editing. Morten Frost: Conceptualization; funding acquisition; project administration; writing – original draft; writing – review and editing.The authors do not have any conflicts of interest to declare.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/jbm4.10832.The peer review history for this article is available at FIGURE S1. Sex stratified lacunar parameters for cort (A–F) and trab (G–L) regions. Data presented for male (m) and female (m) independently of MVD status  and separated for T2D and T2D complicated by MVD . A: Lacunar volume (Lc.V): P = 0.0.09, B: Lacunar sphericity (Lc.Sr): p = 0.51, C: Lacunar density (Lc.N/BV): p = 0.51, D: Lc.V: f: p = 0.201, M: p = 0.084, E: Lc.Sr: f: p = 0.686, M: p = 0.0016, F: Lc.N/BV: f: p = 0.803, m: 0.169, G: Lc.V: p = 0.38, H: Lc.Sr: p = 0.68, I: Lc.N/BV: p = 0.08, J: Lc.V: f: p = 0.201, m: p = 0.972, K: Lc.Sr: f: p = 0.686, m: p = 0.728, L: Lc.N/BV: f: p = 0.803, m: p = 0.803. N for cortical analysis: nine women (4 T2D + MVD), 24 men (9 T2D + MVD), for trabecular analysis: 10 women (5 T2D + MVD), 25 men .Click here for additional data file.TABLE S1. Sex stratified iliac crest cortical and trabecular morphometric osteocyte lacunar parameters. Data presented in median and IQR. p‐values were determined using Wilcoxon tests and linear regression models controlled for age.Click here for additional data file.


The present study aimed to identify the possible protective effect of diacerein (DIA) on gentamicin (GNT)‐induced parotid toxicity in rats. DIA was administered in the presence and absence of GNT. Thirty‐two Wistar adult male rats were randomly arranged into four groups: control, DIA (50 mg/kg/day), GNT (100 mg/kg) and GNT+DIA groups for 8 days. Parotid oxidative stress parameters, besides inflammatory and apoptotic biomarkers, were evaluated. Salivary flow rate, transient receptor potential canonical 1 (TRCP1), and C/EBP homologous protein (CHOP) in parotid tissue were measured. A parotid histopathological examination and an interleukin‐1 beta (IL‐1β) immunohistochemical study were also performed. GNT significantly increased parotid oxidative stress, inflammatory, apoptotic and CHOP biomarkers with decreased salivary flow rate and TRCP1 level. A histopathological picture of parotid damage and high IL‐1β immunoexpression were detected. DIA significantly normalized the distributed oxidative, inflammatory and apoptotic indicators, CHOP and TRCP1, with a prompt improvement in the histopathological picture and a decrease in IL‐1β immunoexpression. These results reported that DIA protects against GNT‐induced parotid toxicity via modulation of TLR4/NF‐κB/IL‐1β and TRPC1/CHOP signalling pathways. It is operative against gram‐negative and inadequate gram‐positive organisms. It is among the earliest and best‐known remedies for a number of bacterial diseases—such as bone, chest and urinary tract infections—pelvic inflammatory diseases, meningitis and sepsis. GNT is regarded by the WHO as being of vital importance for human medicine and is listed as one of the essential medicines.It functions by impeding the bacterial capacity to produce proteins, which usually results in bacterial death. Before being excreted in the kidneys via glomerular filtration, GNT is transported basically unaltered throughout the extracellular space instead of being metabolized. Potentially harmful side effects, most frequently ototoxicityIn humans, there are 10 members of the large family of transmembrane recognition proteins known as Toll‐like receptors (TLR). It is crucial to the detection of pathogens and the activation of innate immunity. They are capable of identifying the pathogen‐associated molecular patterns that are expressed on infectious pathogens and facilitating the synthesis of the cytokines required for the maturation of functional immunity. TLR4 has recently been shown to be crucial for triggering the inflammatory response. When it is activated, an intracellular signalling pathway of proinflammatory cytokines, such as nuclear factor‐kappa B (NF‐κB), tumour necrosis factor (TNF), and interleukin‐1 (IL‐1), is stimulated.2+ influx pathway or depolarizing the membrane potential, they are an important factor in the control of intracellular calcium.The human transient receptor potential canonical or classical (TRPC1) protein comprises functional nonselective cationic channels with high calcium permeability. These channels are widely expressed in different human tissues and cell types. By supplying the Ca2+ level drops, salivary gland cells die and C/EBP homologous protein (CHOP) expression rises.The acinar and ductal cells of the salivary gland are thought to secrete fluid mostly in response to TRPC1. When TRPC1 is damaged, the endoplasmic reticulum (ER) CaDiacerein (DIA), a novel analgesic, antipyretic and anti‐inflammatory medication created specifically for the treatment of osteoarthritis, is a derivative of anthraquinone. DIA works by preventing human monocytes from producing IL‐1. By triggering NF‐κB and mitogen‐activated protein kinase signalling, interleukin‐1, a pro‐inflammatory and pro‐apoptotic substance, stimulates the production of cytokines. DIA has been shown to significantly reduce inflammatory damage by downregulating the interleukin‐1 beta (IL‐1β) receptor,Salivary glands have become a valuable research tool to investigate some fundamental issues in physiology, pathology and pharmacology, including protein synthesis, salt and water transport and autonomic nerves and receptor pharmacology.22.1Rats' processing and therapy were done in accordance with the Institutional Ethical Committee's  guidelines for the care of experimental animals, as well as the National Institutes of Health's (NIH) Guide for the care and use of laboratory animals .2.2DIA was purchased from EVA pharma, GNT, a pharmaceutical ampoules (20 mg) preparation, was purchased from Memphis Pharmaceutical & Chemical Industries, TRPC1 and CHOP ELISA kits were obtained from Wuhan Fine Biological Technology Co., Catalogue No. EH4284 and MyBioSource Co., Catalogue No. MBS3808179, respectively. Tumour necrosis factor alpha (TNF‐α)  ELISA kits were obtained from Thermo Fisher Scientific Inc. NF‐κB and Myd88 ELISA kits were purchased from ELISA Genie Co. Catalogue No: SKU: RTFI00988 and SKU: RTFI01303, respectively. Caspase‐3 ELISA kits from MyBioSource, Catalogue No. MBS261814. Total antioxidant capacity (TAC) commercial kits from Biodiagnostic, Catalogue No. TA 25 13.2.3Thirty‐two male Wistar rats weighing 220–250 g, aged 7–10 weeks, were acquired from the National Research Center. Rats were left for acclimatization in their cages (4 rats/cage) in a normal light–dark cycle with free access to tap water and a normal diet (El‐Nile Company) for 10 days before initiating the experiment.Animals were assigned into four experimental groups (8 rats/ group):Group 1 (Control group): received vehicles.Group 2 (DIA group): received diacerein 50 mg/kg/day for 8 days p.o.Group 3 (GNT group): received 100 mg/kg, i.p. for 8 days.Group 4 (GNT+DIA group): received 100 mg/kg, i.p., with diacerein 50 mg/kg/day, p.o for 8 days.DIA dissolved in 1% carboxymethylcellulose (1% CMC) and freshly prepared before use.2.4g for 15 min (Janetzki T30 centrifuge). Then, sera were kept at −80°C for biochemical analysis. The parotid glands were excised and rinsed with saline to eliminate any blood. A part was fixed for histological studies. The other part was homogenized in ice‐cold phosphate buffer  with a glass homogenizer on ice (tissue weight (g): phosphate buffer (mL) volume = 1:5). The homogenate was centrifuged for 15 min at 5000 rpm, and the supernatant was stored at −80°C to measure the biochemical parameters.At the end of the experiment, the salivary flow rate was determined. Under the effect of anaesthesia with urethane hydrochloride (1 g/kg i.p.), salivation was stimulated by pilocarpine , which was dissolved in distilled water, and the saliva that was produced was dripped in plastic tubes and maintained on ice during all the saliva collection period (40 min).2.52.5.1Malondialdehyde (MDA) is an index of lipid peroxidation. It was measured using the thiobarbituric acid method, and the sample absorption was determined at 535 nm.According to the previously prescribed method of Marklund and Marklund,TAC was estimated by colorimetric kits according to the manufacturer's instructions.2.5.2Parotid TRCP1 and CHOP levels were measured using ELISA kits, following the manufacturer's instructions. In CHOP, the colour of the tubes changed from blue to yellow, and the optical density was read at 450 nm within 15 min. In TRCP1, the colour turned yellow immediately, and the optical density was read at 450 nm after adding the stop solution.2.5.3TNF‐, NF‐B, and caspase‐3 levels were measured using ELISA kits as directed by the manufacturer.2.6Regarding western blotting, parotid gland homogenates  were heated for 5 min with loading buffer, then placed on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) which was then exposed to running at 100 V for 2 h. Separated proteins were carried to polyvinylidene fluoride (PVDF) membranes and subjected to blocking for 1 h in a Tris‐buffered saline (TBS‐T) blocking solution. Incubation with primary antibodies (1:1000) for rabbit anti‐TLR4 and anti‐C caspase‐3 antibodies  and β‐actin  overnight at 4°C then secondary antibody of polyclonal goat anti‐rabbit immunoglobulin  was used at a dilution of 1:5000 in blocking buffer. Bands were seen by chemiluminescence and quantified using a luminescent image analyser . Bands of separated proteins were quantified after being normalized to β‐actin relative to the normal control group using ImageJ software.2.7Specimens from the parotid gland were fixed, then dehydrated, cleared and embedded in paraffin. Sections with a thickness of 5 μm were cut and stained. Sections were examined blindly. The inflammation was scored from 0 to 3. A score of 0 indicates no inflammation; a score of 1 indicates mild inflammation with no foci; a score of 2 indicates moderate inflammation and one focus. A score of 3 indicates severe inflammation and more than one focus.2.82O2. Then sections were washed and incubated with monoclonal anti‐IL‐1β; Catalogue No. YMA1156 (1: 200) (Chongqing Biospes Co.). After washing with PBS, sections were incubated with the secondary antibody HRP Envision Kit (DAKO) 20 min, incubated with diaminobenzidine (DAB) for 10 min, and counterstained with haematoxylin. The mean area fraction for expression was measured using the image J‐22 program.Immunohistochemistry for IL‐1β was performed on paraffin‐embedded tissue according to the manufacturer's protocol. Endogenous peroxidases were quenched by using 3% H2.9anova) for multiple comparisons, followed by the Tukey–Kramer test as a post‐anova test, was used for data analysis. The results were considered statistically significant if p < 0.05.All values were expressed as mean ± SEM. The statistical analyses were conducted using GraphPad Prism (version 5.0). Analysis of variance  Figure .Immunohistochemical staining for Il‐1β in both the control and DIA groups showed negative expression of the acini, ducts and blood vessels. Only scattered connective tissue cells in between the acini showed positive expression. In the GNT group, positive cytoplasmic expression was noticed in the acini, ducts and endothelium of the blood vessels. Meanwhile, in the GNT+DIA group, a faint expression was observed in some acini and in the endothelium lining the blood vessels, while the ducts appeared negative. Morphometric studies and statistical analysis of the mean area fraction of IL‐1β immune‐positive cells in the studied groups were expressed as mean ± SEM (8 rats/group) Figure .4Micromonospora purpurea and is used in the treatment of many types of bacterial infections, particularly those caused by Gram‐negative organisms.An antibiotic is a type of antimicrobial substance that is active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of such infections. Gentamycin is an aminoglycoside antibiotic derived from The findings of this study revealed that GNT caused parotid toxicity through a variety of mechanisms. Damage to the salivary gland is caused by an imbalance between oxidative stress, inflammation, and apoptosis,−2) and ROS were produced more readily by GNT, which led to injury to the salivary glands. ROS damage any molecules, primarily the polyenoic fatty acids of cell membranes, through a series of events involving membrane lipid peroxidation and necrosis.Superoxide anion  are activated and pro‐apoptotic proteins are upregulated during apoptosis, which is also influenced by intracellular CaIn the current study, DIA decreased TNF‐α, NF‐κB and caspase‐3 levels significantly, matching with Refaie et al.Moreover, the present result showed a significant increase in parotid TLR4 expression in the GNT group. This finding is in accordance with Pakfetrat et al.Furthermore, the current study showed that GNT triggers an inflammatory response by upregulating IL‐1β immunoexpression. Our results reported positive cytoplasmic expression of IL‐1β in the acini, ducts and endothelium of the blood vessels of the GNT group. IL‐1β is a key player in the chain of inflammatory events during reperfusion. Leucocytes are drawn to inflammatory regions by chemoattractants like IL‐1β.Besides, DIA treatment, in this study, suppressed the activation of the TLR4/NF‐κB mediated signalling pathway with consequent reduction of the inflammatory cytokines production of TNF‐α and IL‐1β in parotid tissue and these results are consistent with Oliveira et al.2+ ion is the common second messenger that controls a wide range of crucial processes in practically all cell types, including gene expression and cellular homeostasis. A channel known as TRPC1 controls the salivary gland's function. It has a role in controlling Ca2+ homeostasis. Damage to TRPC1 lowers the ER Ca2+ level, which leads to the loss of salivary gland cells and a rise in the expression of CHOP.Normal cell processes are critically dependent on the transport of ions across the cell membrane. The Ca2+ entry necessary for TMEM16a (CaCC) activation, which is essential to govern Cl− efflux, to control the Ca2+‐activated chloride (Cl−) channels (CaCC) in the salivary gland cells. Furthermore, ER and Ca2+ homeostasis disorders coexist with illnesses and injuries to the salivary glands. With this damage, CHOP was activated, which decreased TRPC1 expression and decreased autophagy and apoptosis, which led to cell death.It has been reported that TRPC1 controlled the sustained Ca2+ homeostasis, which in turn resulted in the deactivation of CHOP and an increase in TRPC1 levels. These results have been confirmed histologically, which revealed a quite normal appearance of parotid gland tissue in the DIA‐treated group.In addition to the antioxidant, anti‐inflammatory and anti‐apoptotic activity of DIA, our results revealed that DIA dramatically elevated parotid TRPC1 level and lowered parotid CHOP level as compared to the GNT group. This was accomplished by reducing the abnormalities in ER and CaIn conclusion, the results of the present research revealed the efficacy of DIA in reducing oxidative stress and the inflammatory response induced by GNT. DIA downregulated the TLR4/NF‐κB mediated signalling pathway, suppressed IL‐1β expression, and restored parotid gland tissue. Through the antioxidant, anti‐inflammatory and anti‐apoptotic functions of DIA, it ameliorated the signalling mechanisms and saved the parotid salivary gland from GNT‐induced harm.Dalia Mohamed Ali: Investigation ; methodology . Rehab Ahmed Rifaai: Methodology ; resources . Michael Atef Fawzy: Data curation (lead); investigation ; methodology . Medhat Atta: Investigation ; methodology . Nermeen N. Welson: Formal analysis ; writing – review and editing (lead). Gaber El‐Saber Bathiha: Funding acquisition ; project administration (lead). Athanasios Alexiou: Funding acquisition . Marios Papadakis: Funding acquisition . Walaa Yehia Abdelzaher: Formal analysis ; writing – original draft (lead). Mohamed Mahmoud: Funding acquisition .This work was supported by the University of Witten‐Herdecke Germany.The authors have no conflict of interest to declare.Not applicable.Not applicable.Not applicable.


Participants with SSD reported a significantly higher number of total SLEs compared to controls . Positive symptom severity was positively associated with the total number of SLEs . iSLEs  and dSLEs  showed similar association with positive symptoms (p = 0.16) suggesting stress-diathesis effects. Negative symptom severity was negatively associated with the number of SLEs  and dSLEs  but not iSLEs , suggesting stress generation effects. Depressive symptom severity was positively associated with SLEs , and the association was not statistically stronger for dSLEs  than iSLEs , p = 0.085, suggesting stress-diathesis effects. The SLE – symptom relationships in SSD may be attributed to stress generation or stress-diathesis, depending on symptom domain. Findings call for a domain-specific approach to clinical intervention for SLEs in SSD.We evaluated two models to link stressful life events (SLEs) with the psychopathology of schizophrenia spectrum disorders (SSD). We separated SLEs into independent  and dependent . Stress-diathesis and stress generation models were evaluated for the relationship between total, i- and d- SLEs and the severity of positive, negative, and depressive symptoms in participants with SSD. Participants with SSD ( Stressful life events (SLEs) have been implicated in the etiology and symptomatology of schizophrenia spectrum disorders (SSD) since its initial conceptualization –4. Two cWe propose to evaluate stress-diathesis and stress generation models by separating SLEs into independent and dependent life events based on the heuristics summarized in Fig. Patients with SSD report positive, negative, and affective symptoms –18. PosiIn this study, we examined the relationships between total, i- and d- SLEs and the severity of positive, negative, and depressive symptoms in SSD. We hypothesized that both iSLEs and dSLEs would be positively associated with positive symptom severity, supporting stress-diathesis but not stress-generation models Fig. . We hypoTo further establish and clarify the relationship between SLEs and symptoms of SSD should ultimately facilitate more effective prevention and intervention strategies. Conceptualizing certain symptoms of SSD as responses to SLEs may facilitate the integration of ongoing stress assessment, trauma-informed care, and stress management in SSD treatment. Providers and researchers may draw parallels between SSD and stress-related disorders and borrow from these fields. On the other hand, understanding that some symptoms of SSD may alter patients’ likelihood of experiencing SLEs may facilitate the transition from a symptom-reduction to a recovery-oriented approach, where patients and their treatment teams work to diminish the impact of symptoms on real-life experiences.n = 286; 196 males; 37.5 ± 13.5 years) and community controls  completed self-report on major negative stressful life events (SLEs) at the Maryland Psychiatric Research Center (MPRC) from 2010 to 2022 . Ten event types were surveyed: (1) death of a spouse, father, mother, child, sibling, or significant other; (2) suffered from a serious illness, serious injury, or traffic accident; 3) a significant other suffered from a serious illness or a serious injury; (4) divorce or break-up of a relationship (you or your parents); (5) unusual, extremely stressful work or school; (6) experienced violence, a sexual assault, or a robbery; (7) lost a primary job, or substantial financial loss (you or your parent); 8) experienced a legal dispute or disputes; (9) had a hospitalization due to mental illness or substance use problems; and (10) other serious life event not listed above. For each event, participants indicated whether they have never experienced the event, experienced it over 10 years ago, within 1–10 years, within 1 year, or within the last 6 months. They could also write down their age at the time of the event. We defined total SLEs as the number of SLE types that a participant endorsed as ever experienced.The distinction between independent and dependent life events is ideally made through interview , 24. HowBased on convention in the literature, we categorized likely independent SLEs (iSLEs) and potentially dependent SLEs (dSLEs) as follows. Events categorized as likely iSLEs included: (1) death of a spouse, father, mother, child, sibling, or significant other; (2) a significant other suffered from a serious illness or a serious injury; (3) divorce or break-up of a relationship (you or your parents) when happened before age 18; (4) experienced violence, a sexual assault, or a robbery; and (5) lost a primary job, or substantial financial loss (you or your parent) when happened before age 18. Events categorized as likely dSLEs included: (1) divorce or break-up of a relationship (you or your parents) when happened at or after age 18; (2) unusual, extremely stressful work or school; (3) lost a primary job, or substantial financial loss (you or your parent) when happened at or after age 18; (4) experienced a legal dispute or disputes; and (5) had a hospitalization due to mental illness or substance use problems. One event, “suffered from a serious illness, serious injury, or traffic accident”, was deemed uncategorizable without more details and was excluded from the categorization. Total iSLEs or dSLEs was the number of likely iSLE or dSLE types that a participant endorsed as ever experienced.Somewhat arbitrarily, we used age 18 years old to separate “divorce or break-up of a relationship”: events that took place when participant was a minor (before 18) were assumed to be parental divorce or break-up thus iSLEs, whereas events that happened when participant was an adult (at or after 18) dSLEs. The same applied to “lost a primary job, or substantial financial loss”. To avoid undue influence of this decision on our findings, we alternatively defined iSLEs and dSLEs which did not include these two types of events. These findings are presented in In participants with SSD, we measured positive symptoms using the positive symptom subscale of the 20-item version of the Brief Psychiatric Rating Scale , which iBecause we measured lifetime SLEs rather than SLEs within a timeframe before the assessment, we focused on the relationship between SLEs and depressive symptoms throughout adulthood . We reported results for state depression in Among the 286 participants with SSD, 27 were on both typical and atypical antipsychotics, 35 on typical antipsychotics only, 187 on atypical antipsychotics only, 24 not on any antipsychotics, and 13 deemed to not have reliable medication information. Chlorpromazine (CPZ) equivalents were calculated in 218 (81.3%) patient participants who had usable medication and dosage information.t or χ2 tests. Next, in participants with SSD, we examined the association between total SLEs and positive, negative, and depressive symptoms by regressing each symptom onto total SLEs and total SLEs × sex interaction in linear regression models when controlling for age. SLEs × sex interaction was removed if not significant and a covariate of sex was included instead. We then examined the respective association of total iSLEs and dSLEs with clinical symptoms using the same approach. To determine if there was a significant difference in the strengths of association between total iSLEs and dSLEs with the same symptom, we entered total iSLEs and dSLEs in the same model to explain the symptoms and tested if there was a significant difference in their standardized coefficients (H0: βiSLEs – βdSLEs = 0) using the linearHypothesis function in the car package (car-3.1–0) , t(405) = 4.57, p = 6.4 × 10–6), as well as significantly more dSLEs  = 4.62, p = 5.3 × 10–6) but not iSLEs  = 0.93, p = 0.35). Given the significant group differences in total SLEs and dSLEs, similar iSLEs between the groups provided some support that the iSLE categorization may indeed capture largely independent events.Table ; divorce or break-up of a relationship before 18 (presumably in parents); unusual, extremely stressful work or school; and hospitalization due to mental illness or substance use problem. On the other hand, community controls were significantly more likely to report divorce or break-up of a relationship at or after 18 (presumably in self). Prevalence of other types of SLEs was not significantly different.Supplementary Table No significant SLEs × sex interaction was found for any symptom domain and SLEs × sex interaction was dropped from regression models. The relationships of SLEs with positive and negative symptoms as well as trait depression when adjusting for age and sex are shown in Table βSLEs = 0.20, 95% CI = , t(255) = 3.30, p = 0.001). When broken down by independence, positive symptoms were significantly associated with dSLEs  = 3.47, p = 0.0006), but the association with iSLEs did not reach significance  = 1.70, p = 0.09). However, statistical tests suggested no significant difference between βdSLEs and βiSLEs (p = 0.16).Positive symptoms were significantly and positively associated with total SLEs  = –2.97, p  = 0.003). This relationship held for dSLEs  = –3.24, p = 0.001) but not iSLEs  = –0.64, p = 0.52). Statistical tests showed that βdSLEs was significantly different from βiSLEs (p = 0.039).Negative symptoms were significantly and negatively associated with total SLEs  = 5.92, p = 1.0 × 10–8), as well as both iSLEs  = 3.47, p = 0.0006) and dSLEs  = 5.74, p = 2.7 × 10–8). The difference between βdSLEs and βiSLEs did not reach significance (p = 0.085).Trait depression was significantly and positive associated with total SLEs  = 2.02, p = 0.044). CPZ equivalents were not significantly associated with negative symptoms (β = 0.06), trait depression (β = –0.06), state depression (β = –0.02), SLEs (β = –0.06), iSLEs (β = –0.07), or dSLEs (β = –0.04), all p’s > 0.30. Additionally controlling for CPZ equivalent did not change the statistical significance of most findings .In the 218 participants with available information, CPZ equivalents had a significant positive association with positive symptoms when controlling for age and sex  = 2.97, p = 0.003), whereas this association did not reach significance with the original definition.Using alternatively defined iSLEs and dSLEs (excluding events 4 and 7) yielded largely the same conclusions  and symptoms experienced by participants with schizophrenia spectrum disorders (SSD). Participants with SSD reported a significantly higher number of total SLEs compared to community controls. We separated SLEs into likely independent (iSLEs) and potentially dependent SLEs (dSLEs) and probed different symptom domains. Positive symptom severity was significantly and positively associated with total SLEs; iSLEs and dSLEs showed equal association with symptom severity supporting the stress-diathesis hypothesis. Negative symptom severity was significantly but negatively associated with total SLEs and dSLEs but not iSLEs. This is consistent with the stress generation effect in the negative direction. Depressive symptom severity was significantly and positively associated with total SLEs. The strength of association with dSLEs was not statistically stronger than that with iSLEs, suggesting stress-diathesis but not stress generation effects. This study highlights the importance of a domain-specific approach to characterizing the relationship between life events and major clinical symptoms in schizophrenia. Different underlying processes may govern the relationship between SLEs and symptom severity and call for different intervention strategies.Participants with SSD reported significantly more total SLEs and dSLEs than community controls with similar age, sex, and racial backgrounds. They were more likely to report suffering from a serious illness, serious injury, or traffic accident; unusual or stressful work or school; and having a hospitalization due to mental illness or substance use problems. Since we did not assess whether these events took place before or after illness onset, these findings cannot be taken as evidence that these events are risk factors for SSD. Events such as hospitalization due to mental illness or substance use problems are likely related to having SSD and underscore that individuals with SSD are vulnerable to having stressful experiences due to their illness. Conversely, individuals with SSD reported fewer divorces or breakup of personal relationships, presumably because they form fewer personal relationships. Lastly, individuals with SSD were more likely to report parental divorce or breakup before they reached age 18, consistent with the literature on adverse childhood experiences as a risk factor for mental illness –49.βdSLEs and βiSLEs) were both positive. Although only βdSLEs reached statistical significance, we did not find statistically significant difference between βdSLEs and βiSLEs. Our failure to replicate the previously reported positive relationship between independent life events and positive symptom severity [βiSLEs and βdSLEs, led to the same conclusion as longitudinal studies provides further support for the validity of the heuristics used in this study.The total number of SLEs was significantly and positively associated with the severity of positive symptoms in SSD. The beta coefficients for dependent and independent events  and current psychological wellbeing, prevention of and timely intervention for SLEs may constitute an effective strategy for promoting mental health in individuals with susceptibility to schizophrenia.Supplementary Results


Chronic obstructive pulmonary disease (COPD) is the most prevalent chronic respiratory disease. This study investigated the global, regional and country burden of COPD based on gender, age and socio‐demographic indices (SDIs) in the last 30‐year period from 1990 to 2019.The COPD data, including incidence, mortality and disability‐adjusted life years (DALYs), were obtained from the 2019 Global Burden of Disease Study. If age‐standardized incidence rate (ASIR) or death rate (ASDR) remains almost constant or decreases, the number of cases will still increase as the global population increases substantially. Estimated annual percentage change (EAPC) was calculated to assess incidence, mortality and DALY trends.The incidence of COPD increased by 85.89% from 8,722,966 cases in 1990 to 16,214,828 cases in 2019, and the ASIR decreased from 216.48/100,000 persons in 1990  to 200.49 per 100,000 persons  in 2019. The ASIR increased  in the low SDI region, was stable in the high SDI region, and fell in the other three SDI regions. Men had a higher ASIR than women over the past 30 years, and there were differences in the incidence rates for different age groups. Male mortality and DALYs were higher than female mortality. ASDR decreased by 2.13%  per year and the annual age‐standardized DALY rate decreased by 1.97% .The ASIR, ASDR and age‐standardized DALY rate of COPD declined overall in the last 30 years, and were highest in the low‐middle SDI region. Over the last 30 years, the absolute number of COPD‐associated deaths and DALYs increased significantly worldwide, but the ASIR, ASDR and age‐standardized DALY rate of COPD declined. Health authorities and policymakers must take these differences into account to make better use of limited resources.See relatedEditorial The World Health Organization (WHO) stated on 22 June 2021 that chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally, causing 3.23 million deaths in 2019.COPD is a preventable and treatable disease characterized by incomplete reversible airflow obstruction. As the population ages, COPD will become more prevalent.The DALY value can be estimated by adding years of disabled life (YLDs) and years of life lost (YLLs).This study aimed to investigate the incidence, mortality, DALYs and the corresponding trends of COPD in the last 30 years according to sex, age, socio‐demographic index (SDI) and country. Furthermore, the relativity of risk factors on COPD mortality and DALYs were investigated.http://ghdx.healthdata.org/gbd-results-tool). The GBD 2019 study includes data on 369 diseases and injuries in 204 countries and territories,Information on the annual incidence rate, death rate, DALY and risk factors of COPD was obtained from the GBD 2019 study . Differences in the COPD incidence rate, death rate, DALY rate and corresponding trends in 204 countries and territories were calculated from 1990 to 2019.The SDI is used as a generalized measure to reflect the development of each region in relation to health outcomes in the GBD study. It is the geometric average of the total fertility rate of the population under 25 years old, the average education level of the population over 15 years old and the distribution of per capita income. Based on the SDI values in 2019, 204 countries were divided into five quintiles: high, high‐middle, middle, low‐middle and low,Trends in COPD incidence and mortality were assessed using age‐standardized incidence rate (ASIR), age‐standardized death rate (ASDR), age‐standardized DALY and the corresponding EAPCs. ASIR/ASDR can be obtained by comparing the age structure of different age groups or the age structure of the same population over time. The purpose of using ASIR/ASDR is to exclude the age component from affecting the incidence or mortality of the population, as a result of which the incidence or mortality of disease varies greatly at different ages.ai) in age group i and the number (or weight) (wi) of the selected reference standard population group i divided by the sum of number (or weight) of the standard population, that is, ASR=∑i=1Aaiwi∑i=1Awi×100,000.y = α + βx + ε, where y is ln (ASR), x represented the calendar year. EAPC = 100 × (exp(β) − 1), and its 95%CI was also obtained from this model. If the ASIR or ASDR of a disease is almost constant or decreasing, the number of cases will still increase as the global population increases year by year. This is because the global population has increased far more than the rate has changed. In addition, to observe the relationship between EAPC and ASR/SDI, the Pearson's correlation coefficientASR  is equal to the sum of the product of the specific age ratio  incidences of COPD in 2019 and 8,722,966  incidences in 1990. Additionally, the annual incidence increased significantly and almost doubled Table . There wρ = −0.55, p < 0.01, Figure As shown in Table The ASIR showed an upward trend in most regions and countries  deaths in 2019 and 2,520,219  deaths in 1990 Table . The majρ = −0.29, p < 0.01, Figure As shown in Table The proportion of annual deaths under 80 years old decreased year by year, whereas the proportion of annual incidence cases over 80 years old increased year‐by‐year  COPD‐associated DALYs in 2019 and 59,241,939  DALYs in 1990 Table . The majρ = −0.33, p < 0.01, Figure Regarding SDI level analysis, as shown in Figure The age‐standardized DALY rate showed a declining trend in most regions and countries ; formal analysis ; investigation ; methodology ; resources ; software ; validation ; writing – original draft . Teng‐Yu Gao: Data curation ; formal analysis ; methodology ; resources ; software ; validation . Wei Fang: Data curation ; formal analysis ; methodology ; project administration ; software ; supervision . Chen‐Yang Xian‐Yu: Data curation ; formal analysis . Nian‐Jia Deng: Data curation ; formal analysis . Chao Zhang: Conceptualization (lead); investigation ; project administration ; resources ; software ; supervision ; visualization ; writing – review and editing . Yu‐Ming Niu: Conceptualization ; data curation ; investigation ; methodology ; project administration ; visualization ; writing – original draft .None declared.Not applicable.Supporting InformationClick here for additional data file.


The study determined to construct a novel predictive nomogram to access the prognosis of pancreatic cancer patients with liver metastases (PCLM).Medical records included clinical and laboratory variables were collected. The patients were randomly divided into training and validation cohort. First, in the training cohort, the optimal cutoff value of SII, PNI, NLR, PLR were obtained. Then the survival analysis evaluated the effects of above indices on OS. Next, univariate and multivariate analyses were used to identify the independent factors of OS. Moreover, a nomogram was constructed based on LASSO cox analysis. Additionally, the predictive efficacy of the nomogram was evaluated by ROC curve and calibration curve in the training and validation cohort. Finally, a risk stratification system based on the nomogram was performed.A total of 472 PCLM patients were enrolled in the study. The optimal cutoff values of SII, PNI, PLR and NLR were 372, 43.6, 285.7143 and 1.48, respectively. By combing SII and PNI, named coSII‐PNI, we divided the patients into three groups. The Kaplan–Meier curves demonstrated above indices were correlated with OS. Univariate and multivariate analyses found the independent prognostic factors of OS. Through LASSO cox analysis, coSII‐PNI, PNI, NLR, CA199, CEA, chemotherapy and gender were used to construct the nomogram. Lastly, the ROC curve and calibration curve demonstrated that the nomogram can predict prognosis of PCLM patients. Significant differences were observed between high and low groups.The nomogram based on immune, inflammation, nutritional status and other clinical factors can accurately predict OS of PCLM patients. A novel nomogram and risk classification system for predicting prognosis of pancreatic cancer patients with liver metastases. The inclusion criteria included: (1) pathologically or cytologically confirmed pancreatic cancer with liver metastases, (2) patient undergoing ablation, and (3) complete clinical data. The exclusion criteria included: (1) patients without liver metastasis and (2) patients without complete clinical information. Four hundred and seventy two patients that met our inclusion and exclusion criteria, with clinicopathological data collected from medical records, were included in our study.2.2According to the characteristics of PCLM, the following clinical information and laboratory data were collected from the results of routing text: age, gender, follow‐up time and status, tumor‐related markers such as carbohydrate antigen 199 (CA199), carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA), liver function‐related indicators such as alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin level (TBIL). Laboratory data during routine tests before the first ablation were obtained to ensure uniform baseline data.2.39/L); NLR = N/L; PLR = P/L, respectively. Then the above‐mentioned indices were divided into groups based on the optimal cutoff value defined by R software. Then coSII‐PNI was defined by combing SII and PNI. High SII and low PNI scored 0 point was defined as group 1, high SII and high PNI or low SII and low PNI scored 1 point was defined as group 2, and low SII and high PNI scored 2 points was defined as group 3.Systemic immune‐inflammation index, prognostic nutritional index, neutrophil/lymphocyte ratio, and platelet/lymphocyte counts ratio were calculated through the following formula: SII = P × N/L; PNI = ALB (g/L) + 5 × L  cox analysis in training cohort to screen variables based on the results of univariate and multivariate analyses. Furthermore, predictive performance of the nomogram was evaluated by receiver operating characteristic curve (ROC) and calibration curve.2.6A risk stratification system was developed based on the individual total score calculated from the nomogram. Using the X‐tile software, the optimal cutoff values for the total points of the training cohort were determined. The differences between different subgroups of OS were estimated and compared by Kaplan–Meier method and the log‐rank test.2.7p < 0.05 was considered statistically significant.The data were analyzed statistically using R4.0.3, in which median values and ranges were defined as the quantitative variables, while frequency and percentages were defined as the categorical variables. Training and validation cohorts were obtained through the smaple function in R software. ROC was utilized to determine the best cutoff values of SII, PNI, PLR, and NLR. The differences between groups were accessed by the Kaplan–Meier method and log‐rank test. ROC was drawn based on cox model and LASSO analysis to analyze the predictive value of the model for the prognosis of pancreatic cancer patients with liver metastasis. The area under the curve (AUC) is used to evaluate the predictive performance of the model. The clinical use of nomogram was evaluated by decision curve analysis (DCA). Univariate and multivariate cox analyses were performed to evaluate the effects of different indicators on patient prognosis, while hazard ratio (HR) and 95% confidence interval (CI) were calculated. 33.1A total of 472 patients who meet our criterion were included in our study. The first ablation treatment of all the included patients was operated at Fudan University Shanghai Cancer Center. Among them, 140 (29.7%) were female, and 332 (70.3%) were male with a median age of 59 . The tumor‐related markers and liver function‐related indicators were shown in Table 3.2The optimal cutoff values of the above‐mentioned parameters in training cohort were acquired. The optimal cutoff values of SII, PNI, PLR, and NLR were 372, 43.6, 285.7143, and 1.48, respectively.n = 100); high SII ; low PNI ; and high PNI ; low PLR ; high PLR ; low NLR ; and high NLR .On the basis of the optimal cutoff value, the patients were divided into different groups: low SII , SII (p < 0.001), PNI (p < 0.001), NLR (p < 0.001), PLR (p < 0.001), CA199 (p < 0.001), CA125 (p < 0.001), CEA (p < 0.001), ALP (p < 0.001), chemotherapy (p < 0.001), and distant metastases (p < 0.001) were significantly associated with OS. Moreover, the results of multivariate analyses , SII (p = 0.005), PNI (p = 0.005), NLR (p < 0.001), gender (p < 0.001), CA199 (p < 0.001), CEA (p < 0.001), distant metastases (p = 0.006), and chemotherapy (p < 0.001) were independent prognostic factor with OS. The results were shown in Table To probe prognostic predictors for PCLM patients, univariate and multivariate cox regression analyses were performed. On the univariate analysis Figure , coSII‐Ps Figure  showed t3.5The patients were randomly divided into training and validation cohorts in a ratio of 7:3 Table . Based oAs shown in the ROC Figure , the AUC3.6In addition to precisely managing PCLM patients individually, it is essential to classify patients based on their mortality risk. Therefore, a risk stratification system was established to further verify the stability and performance of the nomogram from different dimensions. Specifically, patients' total points were calculated. The optimal cutoff values for the total points were 126 obtained through X‐tile software Table . Then su4The onset of pancreatic cancer is insidious, and patients often tend to exist liver metastases, which leads to the limitation of treatment.The tumor‐associated immune elements in the tumor microenvironment including neutrophils, platelets, and other cells promote tumor progression, invasion, and drug resistance. In addition, these immunoinflammatory cells also contribute to tumor cell extravasation, survival in peripheral blood, and subsequent distant metastasis of tumor cells. Neutrophils can not only enhance the proliferation and spread of cancer cells but also help tumor cells evade surveillance.CA199 and CEA were usually abnormally elevated in PCLM patients, which presents a worse OS.According to related reports, nomograms have been applied to predict the survival of pancreatic cancer patients with liver metastases, and most of them applied SEER database data to construct nomogram.We identified coSII‐PNI, PNI, NLR, CA199, CEA, gender, and chemotherapy that were significantly correlated with OS. After that, we constructed the nomogram based on the above‐mentioned factors. In addition, ROC and calibration curve showed the nomogram possessed good predictive performance and an agreement between predictions and actual observations. We also develop a mortality risk stratification system to divide patients into high‐ and low‐risk subgroups, enabling clinicians to achieve patient risk stratification management and targeted therapy. The low‐ and high‐risk subgroups demonstrated significant difference in OS. However, the study exists some limitations. Firstly, the sample size of this study is limited, so we will include more patients in the future. Secondly, this is a retrospective study, and there may be a selection bias.5In this retrospective study, we have identified prognostic factors related with OS in PCLM patients before ablation. By combining immune, inflammatory, nutritional status, and three tumor markers, liver function‐related index can provide a more comprehensive assessment of PCLM patients' overall physical status. The nomogram emerged superior predictive performance and was able to accurately distinguish low‐ and high‐risk subgroups.Linjia Peng: Data curation (lead); formal analysis (lead); validation (lead); writing – original draft (lead). Hao Chen: Conceptualization ; project administration ; writing – review and editing .This study was supported by the National Natural Science Foundation of China (81973616).The authors declare that they have no conflicts of interest.The study was approved by the ethics committee of Fudan University Shanghai Cancer Center (number 2212267–7). All procedures were in accordance with the ethical standards of the Institutional Research Council and the 1964 Declaration of Helsinki and its later amendments or similar ethical standards. Written informed consent was obtained from patients prior to treatment.Each participant provided informed consent prior to treatment.Table S1Click here for additional data file.Table S2Click here for additional data file.


The use of cetyltrimethylammonium bromide (CTAB) is an effective and inexpensive method of extracting DNA from plants. The CTAB protocol is frequently modified to optimize DNA extractions, but experimental approaches rarely perturb a single variable at a time to systematically infer their effect on DNA quantity and quality.We investigated how chemical additives, incubation temperature, and lysis duration affected DNA quantity and quality. Altering those parameters influenced DNA concentrations and fragment lengths, but only extractant purity was significantly affected. CTAB and CTAB plus polyvinylpyrrolidone buffers produced the highest DNA quality and quantity. Extractions from silica gel–preserved tissues had significantly higher DNA yield, longer DNA fragments, and purer extractants compared to herbarium‐preserved tissues.We recommend DNA extractions of silica gel–preserved tissues that include a shorter and cooler lysis step, which results in purer extractions compared to a longer and hotter lysis step, while preventing fragmentation and reducing time. Efficiently extracting large quantities of high‐quality DNA from a diversity of species is crucial to the study of genetics and evolution. DNA is most commonly extracted from plant tissues using cetyltrimethylammonium bromide (CTAB), a detergent used to lyse cells and release genomic DNA from the protoplast. The Doyle and Doyle  CTAB proDue to the unique composition of secondary metabolites across plant species, the universal success of a single CTAB protocol for all species is unlikely  Link (Asparagaceae), Paronychia sessiliflora Nutt. (Caryophyllaceae), Mentzelia decapetala (Pursh) Urb. (Loasaceae), and Vicia villosa Roth (Fabaceae) (Appendix Paronychia argyrocoma (Michx.) Nutt. replaced P. sessiliflora because of tissue limitations in the latter species. Herbarium tissues were 16–60 (mean: 46.5) years old and silica‐dried tissues were 0–18 (mean: 4.75) years old  might reduce oxidation  were weighed according to the amount of material available, which was less for herbarium (~5.0 mg) than silica‐dried (8.0–10.0 mg) specimens. Dry material was ground in a Fisherbrand Bead Mill 24 Homogenizer  for 2–4 min, with the longer grinding times used as needed to assure that the leaf material was finely ground. After homogenization, replicated lysates rested at room temperature for 15–20 min with the tube caps loosened to reduce foam and maximize the usable liquid volume. Lysates from the 12 replicates per species were pooled and mixed by pipetting to standardize DNA concentrations of starting material per replicate, then 400 µL of the pooled homogenate was aliquoted to 12 1.5‐mL microcentrifuge tubes. Replicates were randomly assigned to one of the four incubation treatments  and three duration treatments . The temperatures and durations were based on commonly applied values in the literature as assessed by Schenk et al.  contamination. We tested for significant differences in treatments with an analysis of covariance in R  were measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific). DNA fragment sizes between 100–60,000 bp were quantified using the Agilent TapeStation . DNA extractant quality was estimated using the NanoDrop (Thermo Fisher Scientific), and its purity assessed by the AM. decapetala because it experienced considerable oxidation compared to the other three species. We still followed the CTAB DNA extraction protocol as detailed in Schenk et al. . For the SDS, PVP‐10, and CTAB‐only treatments, the replicates were ground in a CTAB and β‐mercaptoethanol solution, pooled, and 360 μL of homogenate were aliquoted to two 1.5‐mL microcentrifuge tubes. Four microliters each of 10% SDS, 0.2% PVP‐10, and distilled water  were added to their respective treatments. For the sorbitol treatment . Cooler temperatures resulted in significantly less secondary metabolite carryover as measured with the A260/A230 ratios , with sorbitol being significantly lower than CTAB, PVP, and SDS; in A260/A230 ratios , with sorbitol having higher relative secondary metabolites than CTAB, PVP, and SDS; and in A260/A280 ratios , with PVP having more relative protein carryover than SDS and sorbitol. However, average fragment lengths were not significantly different .The additive experiment determined that the addition of PVP‐10 produced the highest yields of DNA  had less fragmentation than others at the same temperatures and durations . Third, we recommend that recent silica‐dried tissue be used instead of tissue from herbarium specimens whenever possible. DNA extracted from silica‐dried tissue on average had significantly higher DNA concentrations, longer average DNA fragment lengths, and higher A260/A230 and A260/A280 ratios than DNA extracted from herbarium specimens. Although no single DNA extraction protocol can be universally successful, the results of comparative studies, such as ours, can reveal best practices for addressing issues that are known to compromise extraction success.We conclude with several recommendations based on the results of this study. First, we recommend that incubation duration does not exceed one hour. Longer incubation times on average resulted in lower yield, smaller DNA fragments, and greater contamination, and although these differences were not significant, they demonstrate that longer incubations are no more efficient than the 1‐h incubation. Second, we recommend that incubation temperatures be kept around 50–55°C and not exceed 60°C. Hotter temperatures resulted in shorter DNA fragments when comparing means , whereas cooler temperatures resulted in higher yields and higher AThis project was envisioned and designed by L.E.B. and J.J.S., experiments were conducted by S.J.C. and P.P.F., and the manuscript was written by S.J.C., J.J.S., L.E.B., and P.P.F. All authors approved the final version of the manuscript.Appendix S1. Lysate from additive experiment of Mentzelia decapetala (Loasaceae) that has oxidized. Two replicates of each treatment are included and have been photographed at the completion of the lysis step. PVP = polyvinylpyrrolidone, SDS = sodium dodecyl sulphate, CTAB = cetyltrimethylammonium bromide.Click here for additional data file.Appendix S2. Protocol for optimizing the lysis step in CTAB DNA extractions of silica‐dried and herbarium leaf tissues, based on Schenk et al. (2023).Click here for additional data file.Appendix S3. Results from the incubation experiment.Click here for additional data file.Appendix S4. Effect of differences in incubation duration for each of the four taxonomic groups on the (A) A260/A230 ratio, (B) A260/A280 ratio, (C) log‐transformed average fragment length, and (D) log‐transformed total amount of DNA given the input leaf tissue weight.Click here for additional data file.Appendix S5. Effect of differences in incubation temperature for each of the four taxonomic groups on the (A) A260/A230 ratio, (B) A260/A280 ratio, (C) log‐transformed average fragment length, and (D) log‐transformed total amount of DNA given the input leaf tissue weight.Click here for additional data file.


The use of light‐emitting diode (LED) as an adjustable light source represents a promising approach to improve plant growth, metabolism, and function. The objective of this study was to assess the impact of different light spectra , blue, and white) with an emission peak of around 656, 656, 450, and 449 nm, respectively, under various replacement methods of nutrient solution (complete replacement (CR), EC‐based replacement (ECBR), and replacing based on plant needs (RBPN)), on biomass, physiological traits, and macro‐ and micronutrient contents of two best‐known lettuce varieties, Lollo Rossa (LR) and Lollo Bionda (LB), in the nutrient film technique (NFT) hydroponic system. The results indicated that mix of red and blue LED spectra under RBPN method is the most effective treatment to enhance fresh and dry weights of lettuce plants. In addition, red LED spectrum under RBPN, and red and blue light under ECBR nutrient solution significantly increased leaf stomatal conductance, net photosynthesis and transpiration rate, and intercellular CO2 concentration of LR variety. Phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mn) content in LR variety, and iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) content in both varieties increased upon exposure to blue and red LED light spectrum with RBPN method. Our results suggest that exposure to combination of red and blue light along with feeding plants using RBPN and ECBR methods can increase absorption of macro‐ and micronutrient elements and improve photosynthetic properties, and eventually increase lettuce yield with lower nitrate accumulation.To enhance  Exposure to combination of red and blue light along with feeding plants using RBPN and ECBR methods can increase absorption of macro‐ and micronutrient elements and improve photosynthetic properties, and eventually increase lettuce yield with lower nitrate accumulation. According to Miao et al.  cultivated under combined red and blue light (R/B = 1: 4), the amount of Ca, Mg, P, S, B, Cu, Fe, Mn, Mo, and Zn were increased , results showed that red light increased absorption of Ca, P, and Mn, and blue light improved N content. Lettuce grown under recycled hydroponic subjected to combination of light quality (red + blue) increased the amount of nitrate (NO3−) and nitrite (NO2−) contents  caused a significant increase in leaf biomass but prevented the accumulation of K and Mg elements in the leaf of lettuce plant  cultivars, while blue LED light was more effective on P content  have been cultivated with hydroponic in a closed growth chamber with a new type of light source such as light‐emitting diode (LED) called plant factory with artificial lighting (PFAL) or vertical farms with artificial lighting (VFALs)  Qi, . Xu (200 L.) Qi,  reportedo et al. , red ligThe growth, productivity, and physiological traits of plant are related to light because these parameters are regulated by the intensity, spectra, and time regime of light , blue, and white) with peaks at 656, 656, 450, and 449 nm, respectively, and different nutrient solution replacement methods (complete replacement (CR), EC‐based replacement (ECBR), and replacing based on plant needs (RBPN)) on yield, physiological traits, and macro‐ and micronutrient contents of two lettuce varieties in the nutrient film technique (NFT) hydroponic system.22.1−1, pH: 7) formulated for lettuce was used 2, 2 mM MgSO4, 1 mM KH2PO4, 7 μM MnCl2, 0.7 μM ZnSO4, 0.8 μM CuSO4, 0.8 μM Na2MoO4, 25 μM Fe‐EDDHA, and 2 μM H3BO3) was applied to the seedlings immediately after transplanting. Deionized water was used for nutrient preparation. After transplanting (seedling in four leaves stage) of lettuce varieties, nutrient solutions were replaced by three different replacement methods  for 40 days.This experiment was conducted in a greenhouse of Vali‐e‐Asr University in 2020. The seeds of lettuce varieties (Lollo Rossa (LR) and Lollo Bionda (LB)) were purchased from Sepahan Rooyesh Co, Isfahan (Iran). The seeds were sown into the seed tray filled with fine perlite medium. Normally, after the four‐leaf stage, the seedlings were transplanted inside the small plastic net pot containing horticultural grade perlite medium. These small pots were situated into holes in the NFT plastic channels. Seedlings were transferred to three NFT systems after 21 days of growth using two polyethylene gullies . Then, each of the two gullies was connected to a vertical gully containing a channel for efficient transfer of nutrient solution to the reservoir tank (capacity: 50 L). On each channel, there were 12 holes with a distance of 20 cm from each other. After transferring the plants to the NFT systems, the Resh nutrient solution  were reduced by half and added every 2 days based on the amount of water added to the plastic container in which the plants were cultivated. Environmental condition in greenhouse was a temperature of 25/15 (day/night), a photoperiod of 16/8 h (day/night), and a relative humidity of 50 ± 5%.In complete nutrient solution replacement treatment, the nutrient solution was replaced weekly. For partial replacement according to EC, the EC of the nutrient solution was adjusted to 2.2 dS m2.2−2 s−1 for all employed treatments. Details on the characteristics of LEDs used in the present study are given in Table Lettuce plants were cultivated under LED lamps with a power of 24 W  at different spectral ranges . The LED lamps were installed at an appropriate height and distance (30 cm) from the plant canopy to make the optimum and same photosynthetically active radiation (PAR) which was about 215 ± 5.5 μmol m , the plants were harvested from each NFT channel and divided into shoots and roots. Dry mass (DM) was obtained when samples were dried in an oven for 72 h at 70°C.2.4The aboveground parts of the studied plants were collected for chemical analyses. Collected materials were dried at 45–50°C and then ground to obtain a homogenous size. For determination of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sodium (Na), plant materials were mineralized in concentrated sulfuric acid, while for analysis of total iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu), a mixture of nitric and perchloric acids  was used  was placed in a sterile centrifuge tube, 0.5 mL of hydrogen peroxide (H2O2) was added, and the tube was capped and mixed well by shaking after each addition. All samples were centrifuged at 3500 rpm at 25°C for 3 min. The supernatant was then separated and filtered through a filter paper (Watchman's no. 1), and NO3− concentration in the filtrate was measured colorimetrically by a flow injection analysis technique .For NO2.6−2 s−1 RGB LED array, or up to 2500 m−2 s−1 by white LED array; CO2 concentration: 400 ppm; chamber temperature: 20 ± 3 and precision thermistor ±0.2°C accuracy; H2O: 30 mbar and 0.1 mbar resolution; two laser‐trimmed, fast response water vapor sensors: Direct leaf temperature: 25°C self‐positioning microchip thermistor/energy balance/manually positioned thermistor; flow rate to leaf chamber: 68 to 340 μmol m−2 s−1; and gas connections: 3 mm barbed) following the procedure used by Afridi et al. , stomatal conductance , transpiration rate , intercellular CO2 concentration , and intrinsic water‐use efficiency (PN/gs) (μmol CO2 mol H2O−1) were noted.Photosynthetic and gas exchange parameters were determined using an Infra‐Red Gas Analyzer System  . SAS software version 9.4 was used to analyze the obtained data . A two‐way ANOVA model was used to test the statistical analysis of data. Duncan's multiple‐range test (DMRT) was used as a post hoc test to determine the specific differences between group means. Differences were considered significant at p ≤ .05. Multivariate analyses of variance were performed using XLSTAT software . The results were expressed as mean values ± standard errors (SE) of the means.This study was arranged in a three factors factorial experiment based on completely randomized design (CRD) with three replications (33.1p ≤ .01) were found for shoot dry mass values between lettuce varieties, replacement methods of nutrient solution, light spectrum, and the interaction of three experimental factors . Also, LR showed the minimum amount of N when exposed to blue‐light spectra under RBPN method, while in LB, all of the experimental treatments caused a decrease in N content over the control (white light and CR method) (Table Foliar N content was significantly influenced by the employed treatments (d) Table .3.4p ≤ .01, Table K by 18.5% and 17%, respectively, compared to white light spectra, but in LB, white light spectra under treatment with RBPN showed the highest K content  influenced by the employed treatments  on foliar NO3− content  influenced by the employed treatments  affected by the employed treatments  influenced by light quality and varieties  was significantly (p ≤ .01) affected by the employed treatments  were obtained for the employed treatments. As shown in Figure Based on cluster analysis, treatment is classified into three groups. The results showed that there are 35% and 65% variations within and between clusters. Except for a treatment (Rossa‐RB‐ Plant), all treatments containing nutrient solution replacement based on plant needs were located in the second group Figure .PCA analysis revealed that first two PCAs describe 72% of variability, while the first PCA solely describes 57% of variations and contains all mineral nutrients, nitrate, and net photosynthetic rate. Biplot showed the importance of mineral nutrients in classifying treatments   of the nutrient solution due to the addition of minerals causes a decrease in growth characteristics and a decrease in the concentration of nutrients in tomato plants  has been previously reported  under CR method increased the amount of foliar NO3− compared to the RBPN and ECBR methods of applying nutrient solution to lettuce varieties. According to Jing et al. , then into glutamine or glutamate acid by the enzymes glutamine synthetase (E.C. 6.3.1.2) or glutamate dehydrogenase (EC.1.4.1.2), and finally, into amino acid, protein, and other organic biological N compounds  index  generated during photosynthesis are affected by light exposure  and leaf stomatal conductance (gs) in lettuce plants could be ascribed to the closure of stomata with normalized relative expression levels and the number of stomata per leaf  Table . It was on Table . Stomata52 concentration of leaves, intrinsic water‐use efficiency and absorption, and transfer of various essential elements including macro‐ and micronutrient that play a critical role in the growth and development processes. Due to the timely and required adjustment of elements in the nutrient solution, RBPN and ECBR techniques provide better control of water and nutrient elements consumption than CR method to achieve high production efficiency with lower cost and without harming the environment. Also, such replacing methods can be easily used in all regions of the world that face a wide array of environmental challenges.This research was conducted in order to investigate the effects of different LED spectrums on morpho‐physiological characteristics and nutritional values of two lettuce varieties fed based on different replacement methods of nutrient solution. Results showed that manipulation of LED spectrums along with plant nourishment based on the plant needs in both varieties positively affects net photosynthesis rate, stomatal conductance, transpiration rate, intercellular COHamid Reza Soufi: Conceptualization ; data curation ; formal analysis ; writing – review and editing . Hamid Reza Roosta: Data curation ; funding acquisition ; methodology ; supervision ; validation ; writing – review and editing . Foad Fatehi: Data curation ; formal analysis ; visualization ; writing – original draft . Mansour Ghorbanpour: Formal analysis ; methodology ; validation ; writing – review and editing .The authors declare no conflict of interest.This material is the authors’ own original work, which has not been previously published elsewhere. Also, the manuscript is not currently being considered for publication elsewhere. This manuscript reflects the authors' own research and analysis in a truthful and complete manner.All authors have been personally and actively involved in substantial work leading to the manuscript and will take public responsibility for its content.Food Science & Nutrition.All authors have agreed to submit the manuscript in its current form for consideration and possible publication in