Out‐of‐pocket costs (OOPC) associated with treatment have significant implications on quality of life and survival in cancer patients. Head and neck cancer patients face unique treatment‐related challenges, but to date OOPC have been understudied in this population.This study aims to identify and measure OOPC for patients with head and neck cancer (HNC) in Ontario.HNC patients between 2015 and 2018 at Princess Margaret Cancer Centre in Toronto were recruited. Participants completed OOPC questionnaires and lost income questions during radiation, post‐surgery, and 3, 6, 12, and 24 months after completion of treatment. Associations between OOPC and treatment modality and disease site were tested with multivariable hurdle regression.p < .001; 539% higher, p < .001; 370% higher, p < .001 respectively) among patients with non‐zero OOPC. Participants with non‐zero OOPC in the laryngeal cancer group paid 49% lower OOPC than those with oropharyngeal cancers in adjusted analysis (p = .025).A total of 1545 questionnaires were completed by 657 patients. Median estimated OOPC for the total duration of treatment for participants undergoing chemoradiation was $1452 [$0–14 616], for surgery with adjuvant radiation or chemoradiation (C/RT) was $1626, for radiation therapy alone was $635, and for surgery alone was $360. The major expenses for participants at the mid‐treatment time‐point was travel  and meals, parking, and accommodations . In multivariable analysis, chemoradiation, surgery with C/RT, and radiation were associated with significantly higher OOPC than surgery alone during treatment (791% higher, Patients undergoing treatment for HNC pay significant OOPC. These costs are highest during treatment and gradually decrease over time. OOPC vary by patient demographics, clinical factors, and, in particular, treatment modality. However, many costs are still incurred by patients including travel and accommodation costs, prescription medications, and many allied health care services. Some of these costs may be offset for patients who have private insurance coverage. Despite having universal healthcare coverage, patients may suffer a significant financial burden for cancer care.Recent studies have shown that cancer patients are more likely to forego medical care or medications due to financial concerns than other patients.22.1From 2015 to 2018, adult (>18) patients with HNCs  treated with curative intent were approached to participate in a prospective study at the Princess Margaret Cancer Centre in Toronto, Ontario, Canada. Ethics approval was obtained for this study from the University Health Network (UHN) Research Ethics Board (REB), REB#07‐0521. Informed consent was obtained from each participant. Participants' OOPC were assessed immediately after surgery ; at mid‐radiation treatment (in patients receiving radiation treatment); and at 3, 6, 12, and 24 months after completion of treatment. The questionnaire was administered prospectively to all potentially interested patients. Patients were enrolled from all timepoints simultaneously. As such, not all patients were followed from treatment initiation to the last follow‐up; some were enrolled well after their treatment. Demographic data and household income were collected around the time of diagnosis; patient‐reported lost wages due to cancer was collected at 12 months post‐treatment.The OOPC questionnaire used in this study Figure  was deveDollar values were reported for OOPC related to travel, meals, parking, accommodations, homecare, child care, and domestic assistance. Separate questionnaire modules were included for patients to report individual item costs for medical supplemental expenses, as well as ancillary care costs not covered by governmental or private health insurance . All costs were reported in Canadian dollars (CAD).All reported OOPC were summed to create a single estimated dollar value for OOPC incurred during the month prior to the administration of the questionnaire. For items where no dollar value was reported, OOPC was assumed to be $0 for that item. For patients undergoing surgery and adjuvant radiation or chemoradiation (C/RT), the costs from the post‐operative administration as well as the mid‐radiation therapy questionnaire were summed as an estimate of “on treatment OOPC.” For surgically treated patients, the duration of treatment was considered to be 1 month, to account for recovery time. To estimate OOPC over the duration of radiation therapy with or without concurrent chemotherapy, the OOPC reported during the mid‐treatment administration were multiplied by the total duration of treatment in months. For example, if a patient had surgery plus 6 weeks of adjuvant radiation, then that patient's total estimated mid‐treatment cost is the sum of their post‐surgery OOPC in the past month  plus their reported mid‐radiation OOPC in the past month . Nasopharyngeal cancer patients who continued to be treated with adjuvant chemotherapy after radiation/chemoradiation were removed from estimated treatment cost analyses, as costs during chemotherapy were not captured.Household income and lost income due to cancer were categorized into income ranges by dollar values in $40 000 increments from $0 to $39 999, then $40 000 to $79 999, and over $80 000, all in Canadian dollars. Relative lost household income was computed by dividing lost household income by baseline pre‐treatment household income, to indicate the percent of baseline income lost due to cancer.2.22.2.1We tested the following a priori hypotheses: (1) the number of reported trips to the hospital should correlate with OOPC, travel costs, and travel‐related costs , and (2) patients reporting car and taxi travel will have higher travel costs. These hypotheses were testing using Spearman rank correlations. Mild correlations were defined by rho = ±0.15–0.39, moderate by rho = ±0.4–0.59, and strong correlations by rho ±0.6–1.0.2.2.2The distribution of OOPC across time‐points was reported descriptively. Baseline household income and lost income due to cancer are reported using descriptive statistics. Spearman correlations were computed between OOPC and baseline income, as well as OOPC and lost income due to cancer. Kruskal–Wallis tests were used to determine associations between treatment modality and lost income due to cancer. The association between lost income and OOPC was assessed by Spearman correlation.The trajectory of OOPC over time in each treatment group was plotted using a line graph, with error bars to represent 95% confidence intervals on the mean OOPC in each treatment group, at each time‐point. Mean OOPC at the “Mid‐treatment” time‐point was taken as the mean estimated cost over the course of treatment (combining post‐surgery and mid‐radiation for patients treated with multi‐model therapy); OOPC at all other time‐points are reported for the prior month.p < .1 in the logistic or linear regression steps.Univariable and multivariable regression analyses were used to test associations between OOPC and clinico‐demographic patient characteristics. Regression analysis was conducted for both mid‐treatment costs  and follow‐up costs at 3 months post‐treatment. As is often the case with studies measuring cost data, the distribution of total OOPC was right‐skewed and zero‐inflated (a significant proportion of patients reported zero costs). We therefore applied a hurdle model, a two‐part mixture model that accounts for two separate stochastic processes occurring within the data. First, the probability of patients reporting non‐zero OOPC was modeled using logistic regression. Then, among patients reporting non‐zero costs, OOPC were log‐transformed and modeled using linear regression. This log‐linear regression was used to find the percent increase in OOPC attributable to group membership for each clinico‐demographic factor. Variable selection for multivariable models was conducted by keeping any variable with 33.1N = 636, 97%) and half of the patients had extended health insurance . The majority of patients  had an additional drug plan.A total of 657 patients completed a total of 1545 questionnaires included in the study. Demographic information is reported in Table 3.2p < .001), travel costs , and total meals, parking, and accommodation costs . Patients who reported using a car or taxi to travel to the hospital had higher travel costs than those who did not , while patients who used public transit had lower travel costs than those who did not .Questionnaire validation was performed on all patients across time points. Number of self‐reported trips to the cancer center were mildly correlated with OOPC in the past month , for those undergoing radiation therapy alone was $635 [$0–8099] (median duration 48 days), and for those undergoing surgery alone was $360 [$0–3700] (median duration 30 days). Mean OOPC in each treatment group from treatment to 12‐month follow‐up are presented in Figure N = 4 with OOPC >$0; median $112.50, IQR $90–143.75).OOPC were highest during treatment in each group and decreased over the course of post‐treatment follow‐up. The most commonly reported medical supplemental cost during treatment was prescription or over‐the‐counter medication. One hundred and twenty‐eight patients (28.1%) reported medical supplemental expenditures greater than zero, with median medical supplemental costs of $80 (IQR $50–150). After surgery, 31 patients (7.2%) spent greater than $0 on medication . The most commonly reported ancillary care expense was dental visits; 101 radiation patients (22.2%) spent greater than $0 on dental care with a median OOPC of $300 (IQR $100–485). Few surgical patients reported dental expenses after surgery (N = 98) was $25 000 CAD (IQR $5000–45 000). Relative to baseline pre‐cancer household income, patients under age 65 reported having lost a median of 20% of their income due to cancer at 12‐month follow‐up (IQR: 6–45% of baseline income). For patients age 65 years and over (N = 40), median lost income was $5000 (IQR $5000–17 500) or 11% of baseline income (IQR 5–28%). Among patient under 65 years, lost income at 12 months was not significantly correlated with treatment modality (p = .33) or OOPC during treatment (p = .08).The distribution of individual costs over the past month for each time‐point is shown in Figure 3.4p < .001). The group with surgery plus C/RT paid 539% more (p < .001), and the chemoradiation group paid 791% more (P < .001). Participants with non‐zero OOPC in the laryngeal cancer group paid 49% lower OOPC than those with oropharyngeal cancers in adjusted analysis (p = .025).Results of univariable and multivariable regression analysis on estimated OOPC during treatment are presented in Table p = .001).Three hundred and eighty‐four patients contributed OOPC at 3‐months post‐treatment; of those, 85% of patients reported costs greater than zero. Univariable and multivariable analysis is presented in Table Due to the high proportion of participants who did not report household income, we performed a sensitivity analysis while including those who did not indicate an income. In one analysis, this group had a higher likelihood of non‐zero OOPC than those with income <$40 000, however this was not statistically significant. After including those who did not indicate an income the results of multivariable analysis were in keeping with the original analysis and the predictors of OOPC in the original analysis were maintained.Subgroup analysis was performed to examine the differences between disease sites. A higher proportion of larynx cancer patients were treated with radiation alone (80.8 vs. 42.1%) and had early stage 0‐II disease (62.7 vs. 5.9%) compared to the reference oropharynx group. When we assessed specific costs by disease site Figure  orophary4A diagnosis of cancer is often unexpected, and while patients attempt to cope with the emotional aspects of their diagnosis as well as their own mortality, they may be underprepared for the financial repercussions of cancer treatment and side‐effects. Out‐of‐pocket cancer‐related costs have been understudied in HNCs, particularly in single‐payer healthcare systems where major costs of treatment are often assumed to be covered by government insurance. Therefore, it may be difficult for health care providers to recognize and refer patients who are at risk of significant financial burden. HNCs are associated with the most significant symptom burden following treatment of all common cancers and these toxicities may add additional financial burden for patients such as the need for additional prescription medications or supplies; consultations for services for which the patient may bear some or all of the costs such as outpatient dentistry, physiotherapy, or swallowing therapy; and costs for travel for health care.As expected, patients undergoing chemoradiotherapy or surgery with C/RT, and those with advanced‐stage disease, incurred the greatest OOPC during treatment. Patients treated with surgery alone had the lowest OOPC; this may be due to reduced need for frequent travel, and fewer additional medical expenses associated with prolonged treatment. Our findings were consistent with those of another Canadian study by Longo et al, which showed that cancer patients receiving chemotherapy had higher overall OOPC than those without chemotherapy.In our study, the largest expense incurred by patients, regardless of treatment modality, was travel and travel‐related costs . Patients undergoing radiation therapy, whether alone or in conjunction with other treatment modalities, incur significant travel and accommodation expenses due to the need for daily treatment visits during the week. It should be noted that the delivery of radiotherapy for HNC patients is centralized in Ontario due to the need for highly specialized resources. Due to this specialization, treatment usually requires travel to higher volume centers often located in busy cities.Patients' costs tended to be highest during treatment and decrease as they completed treatment and began surveillance. An increase in the number of trips needed for cancer related services may be a contributing factor to increased OOPC. Recommendations for recurrence screening for HNC patients requires less frequent appointments as time passes, requiring less travel and OOPC on the part of the patient.This study has several limitations. First, as participants are asked to respond to a questionnaire, the data is susceptible to recall bias. However, this questionnaire was designed to capture costs only over the previous month, to minimize recall bias as much as possible. Despite our attempts to control for recall bias, it did appear to factor into the mild correlation with pre‐selected variables in our de novo questionnaire. We do note, however, that unlike traditional patient reported outcomes and quality of life instruments, our questionnaire does not lend itself well to validation testing due to its quantitative nature. Second, results may be subject to volunteer bias: participants who agreed to participate in the study may be systematically different than those that did not as patients' ability and interest in participating may correlate with demographic or clinical characteristics. Third, assumptions used to model the calculated average OOPC at each time point might be incorrect: missing responses to individual cost questions may not have been intended by patients to indicate zero cost; and OOPC do not necessarily remain constant over the course of treatment. Fourth, patients treated in Ontario, Canada, may have a different OOPC experience than those in jurisdictions without universal health care. While other factors including younger age,In conclusion, this study found that Canadian HNC patients pay significant OOPC toward their cancer care, which tend to be highest during treatment and decrease over time. These costs vary by patient demographics, clinical factors, and, in particular, treatment modality.The authors declare there is no conflict of interest.Study conceptulaization and Formal Analysis, M.N.K., K.H., W.X., and J.D.A.; Funding Acquisition, Methodology, J.D.A.; M.M.K., M.A., L.E., G.L., and C.J.L.; Data Aquisition, M.M. and K.K.; Data Curation, C.S.; Data Validation, K.H. and J.D.A.; Writing of the Original Manuscript, M.N.K.; Review and Editing, M.M.K. and M.A.; Tables and Figures, M.N.K., M.M.K. and K.H.; Supervisors, C.J.L., D.P.G., J.R., R.M., and A.R.H.Ethics approval was obtained for this study from University Health Network (UHN) Research Ethics Board (REB), REB#07‐0521. Informed consent was obtained from each participant.Figure S1 Out‐of‐Pocket Cost QuestionnaireClick here for additional data file.Figure S2 Individual Out of Pocket Costs by Disease SiteClick here for additional data file.Figure S3 Mean and Median Out‐of‐Pocket Costs over TimeClick here for additional data file.


The aim of this study is to explore the clinical characteristics in chronic rhinosinusitis with nasal polyps (CRSwNP) patients with different serum specific IgE (SIgE) and eosinophilic granulocyte infiltration status.p < .001), endoscopic score (p = .014), computerized tomography (CT) score (p < .00) and the sino‐nasal outcome Test‐22 (SNOT‐22) score (p < .001) were significantly different among patients with SIgE‐positive combined eosinophilic chronic rhinosinusitis (ECRS), patients with SIgE‐positive combined non‐ECRS, patients with SIgE‐negative combined ECRS, and patients with SIgE‐negative combined non‐ECRS.This retrospective observational study included patients diagnosed with CRSwNP and underwent functional endoscopic sinus surgery at the Yinchuan First People's Hospital between June 2019 and June 2021. A total of 192 patients with CRSwNP were included . The patients were 41.7 ± 15.1 years old. The disease course ranged 4.6–18.2 months. The visual analog scale (VAS) score (p < .0083). The VAS score, endoscopic score, and CT score might be higher in CRSwNP patients with SIgE‐positive combined ECRS.In pairwise comparison, the VAS score , endoscopic score , CT score , and SNOT‐22 score  was significantly higher in patients with SIgE‐positive combined ECRS compared to patients with SIgE‐positive combined non‐ECRS, patients with SIgE‐positive combined non‐ECRS  Patients with CRSwNP were included in the study, including 100 males and 92 females. Positive SIgE was found in 105 cases. Among the 105 SIgE‐positive patients, 41 had ECRS, and 64 had non‐ECRS CRSwNP. Among the 87 SIgE‐negative patients, 33 had ECRS, and 54 had non‐ECRS CRSwNP. There were no significant differences (p=.290) between the two groups (A). There was no significant difference in age, sex, endoscopic score, CT score, serum eosinophil count between the two groups (p > .05), but the VAS score was significantly higher in the SIgE‐positive group compared with the SIgE‐negative group (■p < .001) (B).F I G U R E 1 (p < .001), endoscopic score (p = .014), computerized tomography (CT) score (p < .001) and SNOT‐22 score (p < .001) were significantly different among groups. In pairwise comparison, the VAS score, endoscopic score, CT score, and SNOT‐22 score was significantly higher in patients with SIgE‐positive combined ECRS compared to patients with SIgE‐positive combined non‐ECRS, patients with SIgE‐positive combined non‐ECRS (p < .0083). And patients with SIgE‐negative combined ECRS have significantly higher CT score compared to patient with SIgE‐positive combined non‐ECRS and SIgE‐negative combined non‐ECRS.F I G U R E 2 Four groups were defined according to ECRS and SIgE: SIgE‐positive combined ECRS, SIgE‐positive combined non‐ECRS, SIgE‐negative combined ECRS, and SIgE‐negative combined non‐ECRS. VAS score ( Previous case‐control studies suggested a possible role of food allergy in CRSTherefore, this retrospective observational study aimed to explore the clinical characteristics in CRSwNP patients with different SIgE and eosinophilic granulocyte infiltration status.22.1wNP and underwent functional endoscopic sinus surgery at the ENT & HN Surgery Department of the Yinchuan First People's Hospital between June 2019 and June 2021. This study was approved by the Ethics Committee of the First People's Hospital of Yinchuan City. The requirement for individual consent was waived by the committee because of the retrospective nature of the study.This retrospective observational study included patients diagnosed with CRSThe diagnostic criteria were in line with the Chinese Guidelines for the Diagnosis and Treatment of Chronic Rhinosinusitis (2018).wNP, (3) underwent functional endoscopic sinus surgery, and (4) available SIgE data. The exclusion criteria were (1) used systemic glucocorticoids 2 weeks before the surgery, (2) history of nasal or sinus tumors, (3) orally taking antihistamines, (4) fungal sinusitis, or (5) combined with rheumatoid disease.The inclusion criteria for the study were (1) >18 years of age, (2) diagnosed with CRS2.2The demographic information (including age and sex) and clinical characteristic  were collected.The SIgE test results before surgery were collected. The semi‐quantitative detection unit of specific allergen IgE was IU/mL, and the grades were classified into: grade 0, normal, <0.35; grade 1, mild, 0.35–0.7; grade 2, moderate, 0.7–3.5; grade 3, severe, >3.5. Grade 0 was considered negative, and grades 1–3 were considered positive.The subjective symptoms of rhinosinusitis  of all the patients before surgery were scored using a VAS score. The range from 0 to 10 represented symptoms gradually aggravating.wNP.The eosinophilic granulocytes in the nasal polyp tissue of the enrolled patients after surgery were counted. If the count was ≥10/HPF, it was defined as ECRS, and if the count was <10/HPF, it was defined as non‐ECRS CRSThe Lund‐Mackay CT scoreThe Lund‐Kennedy scoreSNOT‐202.3p < .05 were considered statistically significant.SPSS 14.0 (SPSS Inc.) was used for statistical analysis. The continuous data was tested for normal distribution by Kolmogorov–Smirnov test and tested for homogeneity of variance by Levene's test. All continuous data confirmed to normal distribution and are expressed as means ± standard deviation and were compared by one‐way analysis of variance test, and Bonferroni's post hoc test was used for pairwise comparisons between groups. Two‐sided 3wNP were included in the study, including 100 males and 92 females. The patients were 18–64 years old, with an average age of 41.7 ± 15.1 years, and the courses of disease ranged from 4.6 to 18.2 months. Positive SIgE was found in 105 cases (54.7%). There was no significant difference in age, sex, endoscopic score, CT score, serum eosinophil count, and ECRS between the two groups (p > .05), but the VAS score and SNOT‐22 score was significantly higher in the SIgE‐positive group compared with the SIgE‐negative group   between the two groups , endoscopic score (p = .014), CT score (p < .001) and SNOT‐22 score (p < .001) were significantly different among groups. In pairwise comparison, the VAS score , endoscopic score , CT score , and SNOT‐22 score  was significantly higher in patients with SIgE‐positive combined ECRS compared to patients with SIgE‐positive combined non‐ECRS, patients with SIgE‐positive combined non‐ECRS . And patients with SIgE‐negative combined ECRS have significantly higher CT score compared to patient with SIgE‐positive combined non‐ECRS and SIgE‐negative combined non‐ECRS  are aggravated even in the absence of significant differences in CT and endoscopic scores. Szucs et al.wNP and the difficulty in treatment are positively related to the infiltration of eosinophilic granulocyte in the tissue but not related to the patient's allergic constitution, which contradicts the present study. On the other hand, Collins et al.At present, it is generally believed that the detection of SIgE or laboratory‐specific IgE is safer and more reliable than the skin‐prick test and is the best indicator for the diagnosis of allergy or allergy‐related diseases.wNP patients cannot provide a conclusion about the allergy status. It is currently believed that the increased number and degree of infiltration and aggregation of eosinophilic granulocyte is related to the eosinophilic chemokines released by basophilic granulocyte or mastocyte at the site of allergy.wNP patients with allergy and without allergy. Snidvongs et al.wNP patients have allergic reactions. It suggests that the relationship between the general allergic reaction status and the degree of infiltration and activation of eosinophilic granulocyte in local tissues is uncertain.wNP patients does not induce an inflammatory response of eosinophilic granulocyte in the nasal polyps, but there is an independent pathophysiological mechanism of inflammation maintenance.Snidvongs et al.wNP patients, the combination of allergic reaction and ECRS could aggravate the subjective symptoms and the objective physical signs of CRSwNP, thus Reducing the quality of life of patients with of CRSwNP. Kowalik et al..In this study, patients in the SIgE‐positive group with ECRS had significant differences compared with the other groups in terms of the subjective score of VAS symptoms and the objective scores of Lund‐Mackay CT, Lund‐Kennedy physical signs, and SNOT‐22 score indicating that in CRSFor the treatment of CRSwNP, at present, it mainly depends on nasal endoscopic surgery and local or systemic drug treatment, such as glucocorticoids and macrolides; CRSwNP is associated with Th2 type inflammation and immune hypersensitivity comorbiditieswNP. The relationship between local tissue IgE and eosinophilic granulocyte has not been established. Third, the impact mechanism of allergy on CRS needs to be further explored.This study is a retrospective study and has some limitations. First, the sample size was relatively small. Second, various prediction methods have advantages and disadvantages, and there is a lack of mature prediction methods for CRSIn conclusion, the VAS score, endoscopic score, and CT score might be higher in CRSwNP patients with SIgE‐positive combined ECRS.Conception and design: Jingmin Hu; data curation: Jingmin Hu; formal analysis and visualization: Le Wang; investigation: Le Wang; writing, original draft: Jingmin Hu and Ruixia Ma; All authors read and approved the final manuscript.The authors declare no conflicts of interest.This study protocol was reviewed and approved by the First People's Hospital of Yinchuan City . The requirement for individual consent was waived by the committee because of the retrospective nature of the study.


Deoxycholic acid (DCA) is a secondary bile acid that may promote vascular calcification in experimental settings. Higher DCA levels were associated with prevalent coronary artery calcification (CAC) in a small group of individuals with advanced chronic kidney disease. Whether DCA levels are associated with CAC prevalence, incidence, and progression in a large and diverse population of individuals with chronic kidney disease stages 2 to 4 is unknown.In the CRIC  study, we evaluated cross‐sectional (n=1057) and longitudinal (n=672) associations between fasting serum DCA levels and computed tomographic CAC using multivariable‐adjusted regression models. The mean age was 57±12 years, 47% were women, and 41% were Black. At baseline, 64% had CAC (CAC score >0 Agatston units). In cross‐sectional analyses, models adjusted for demographics and clinical factors showed no association between DCA levels and CAC >0 compared with no CAC . DCA was not associated with incident CAC  or CAC progression .Among CRIC study participants, DCA was not associated with prevalent, incident, or progression of CAC. CRICChronic Renal Insufficiency CohortCYP7A1cholesterol 7‐alpha‐hydroxylaseDCAdeoxycholic acidThis is the first analysis of a large, diverse cohort of individuals with chronic kidney disease that aimed to determine whether circulating levels of the bile acid, deoxycholic acid, were associated with vascular calcification.Despite preclinical data suggesting that deoxycholic acid is directly toxic to vascular smooth muscle cells causing vascular calcification, and a small cross‐sectional observational study demonstrating a significant association between higher circulating deoxycholic acid levels and vascular calcification, we found no association between higher deoxycholic acid levels and prevalence, incidence, and progression of coronary artery calcification among participants in the CRIC  study.The bile acid, deoxycholic acid, should not be used as a predictor of vascular calcification among individuals with chronic kidney disease stages 2 to 4.Further preclinical and clinical research is required to more precisely determine the role deoxycholic acid and other bile acids play in vascular calcification and adverse outcomes in chronic kidney disease.Vascular calcification is common in chronic kidney disease (CKD).Bile acids are produced in hepatocytes by CYP7A1  and conjugated with either taurine or glycine. Conjugated bile acids are then secreted into the bile canaliculi and stored in the gallbladder. From the gallbladder, bile acids are excreted into the intestinal lumen where their main function is to emulsify dietary lipids. Once in the intestinal lumen, primary bile acids undergo bacteria‐induced transformation to secondary bile acids.2).Deoxycholic acid (DCA) is a secondary bile acid derived from gut bacterial transformation of the primary bile acid, cholic acid. Among individuals with CKD, DCA levels are higher compared with those with normal kidney function.The CRIC study is a longitudinal observational study and includes a large, diverse sample of participants with CKD stages 2 to 4. We tested the hypothesis that higher circulating DCA levels would be associated with greater CAC prevalence, incidence, and progression.https://repository.niddk.nih.gov/studies/cric/.Anonymized data and materials have been made publicly available at the CRIC National Institute of Diabetes and Digestive and Kidney Diseases biorepository and can be accessed at 2). A total of 3939 participants were enrolled from 7 centers in the United States between May 2003 and August 2008.The CRIC study is a prospective cohort study of a racially and ethnically diverse group of men and women aged 21 to 74 years with mild to moderate CKD  measurements. Of those with no history of coronary artery revascularization, 1142 participants were randomly selected and stratified by age, sex, race and ethnicity, diabetes status, and eGFR for electron‐beam or multidetector CT. In addition, all eligible participants from 3 centers were scanned as part of an ancillary study, yielding 1964 total participants scanned within the first 3 years of the original baseline examination. Of these participants, 1057 had fasting DCA levels measured at the same study visit as their first CT scan  as part of an ancillary study. Repeat CT was performed among 1123 participants an average±SD of 3.2±0.6 years later, 672 of whom had DCA data  to quantify calcification according to the Agatston score.Fasting serum samples stored at −80 °C were shipped with sufficient dry ice to the University of Colorado for measurement of DCA using liquid chromatography–tandem mass spectrometry, as previously described.We obtained covariate data from the same study visit as the first CT scan or the most recent previous annual visit if missing. Self‐reported sociodemographic characteristics, medical history, and current medications were obtained using a questionnaire. Body weight, height, and blood pressure were measured using standard protocols.Glucose, cholesterol, phosphate, calcium, magnesium, serum albumin, and total parathyroid hormone were measured using standard laboratory methods. The 24‐hour urinary protein was measured using the turbidometric method with benzethonium chloride. FGF23 (fibroblast growth factor 23) was measured using a second‐generation carboxy‐terminal assay (Immutopics). hs‐CRP (high‐sensitivity C‐reactive protein) and IL‐6 (interleukin‐6) were measured at the original baseline examination using the particle‐enhanced immunonephelometry method. We calculated eGFR using the Chronic Kidney Disease Epidemiology Collaboration equation.2 tests for categorical variables. We evaluated the cross‐sectional association of circulating DCA level with CAC score using a 2‐part model.We summarized baseline participant characteristics as mean±SD or median with interquartile range for continuous variables and percentages for categorical variables by DCA tertile. Statistical differences between tertiles were tested using ANOVA for continuous variables with normal distributions, Wilcoxon‐Mann‐Whitney test for continuous variables with skewed distributions, and χWe evaluated the longitudinal association of DCA with CAC stratifying by the presence of baseline CAC.We included covariates in sequential regression models based on prior clinical knowledge. In addition to unadjusted analyses, 4 multivariable‐adjusted models were used: (1) adjusted for age, sex, race, ethnicity, and clinical site; (2) adjusted for variables in model 1 plus eGFR, 24‐hour urinary protein, diabetes, systolic blood pressure, number of antihypertensive medications, current smoking, history of cardiovascular disease, total cholesterol level, and use of statin medications; (3) adjusted for variables in model 2 plus hs‐CRP and IL‐6; and (4) adjusted for variables in model 3 plus albumin, calcium, phosphate, magnesium, parathyroid hormone, and FGF23. Because the onset of end‐stage kidney disease may increase the risk for calcification, we conducted sensitivity analyses excluding those with end‐stage kidney disease at baseline  and during follow‐up .P<0.05.We tested for effect modification by including DCA‐by‐subgroup interaction terms  in logistic regression and Poisson regression models. All analyses were conducted using SAS version 9.4 (SAS Institute). All tests were 2‐sided, and statistical significance was defined as 2. Median (interquartile range) DCA was 65 ng/mL (23–137 ng/mL). As shown in Table P=0.001), had higher eGFR (P=0.007) and lower urinary protein excretion (P=0.03), and lower circulating FGF23 levels (P=0.008) compared with individuals with lower DCA levels.Among 1057 CRIC participants with data for DCA and CAC obtained by CT, the mean age was 57±12 years, 47% were women, 45% had diabetes, 28% had self‐reported history of cardiovascular disease, and mean eGFR was 43±17 mL/min per 1.73 mP=0.30).Figure Table Of 676 participants with prevalent CAC (CAC >0 Agatston units), 405 (60%) had CAC ≥100 Agatston units and 236 (35%) had severe CAC defined as CAC ≥400 Agatston units. In unadjusted analyses, there was no association between DCA in the highest tertile compared with the lowest tertile and CAC ≥100 Agatston units , nor was there any association in the multivariable‐adjusted model  (Table Table P=0.045) and a statistically significant interaction between diabetes and DCA among participants (n=29) with an increase in CAC of ≥200 Agatston units per year (P=0.004). We did not explore these interactions further because of the borderline significant P value and small number of participants, which limited power. Furthermore, we did not detect any statistically significant interaction between DCA and age, sex, race and ethnicity, diabetes, statin use, eGFR, or cholesterol level in the other analyses . Sensitivity analyses excluding participants with end‐stage kidney disease did not affect results . Mechanisms underlying possible bile acid metabolism dysregulation in kidney disease require further investigation.Bile acid handling in CKD may be dysregulated. Various small observational studies suggest circulating bile acid levels are elevated in CKD.Bile acid metabolism is implicated in vascular disease, including vascular calcification.2) and found an independent association between higher log‐transformed DCA and greater CAC volume score.2 in CRIC versus mean eGFR 31.5±8.7 mL/min per 1.73 m2 in the prior report), and a lower burden of vascular calcification . In addition, this work was supported in part by the Perelman School of Medicine at the University of Pennsylvania Clinical and Translational Science Award NIH/NCATS UL1TR000003, Johns Hopkins University UL1 TR‐000424, University of Maryland GCRC M01 RR‐16500, Clinical and Translational Science Collaborative of Cleveland, UL1TR000439 from the National Center for Advancing Translational Sciences (NCATS) component of the NIH and NIH roadmap for Medical Research, Michigan Institute for Clinical and Health Research (MICHR) UL1TR000433, University of Illinois at Chicago CTSA UL1RR029879, Tulane COBRE for Clinical and Translational Research in Cardiometabolic Diseases P20 GM109036, Kaiser Permanente NIH/NCRR UCSF‐CTSI UL1 RR‐024131, Department of Internal Medicine, University of New Mexico School of Medicine Albuquerque, NM R01DK119199.This work was supported by the George M. O’Brien Kidney Research Center at Northwestern University  from the National Institutes of Health (NIH)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). A.J. is supported bAmerican Journal of Kidney Disease, coaches for InMed Physicians, and works for DLA Piper LLP for Essure Litigation. Dr Isakova reports receiving consulting fees from Akebia Therapeutics. Dr Chonchol reports grants from Otsuka, Sanofi, and Kadmon. The remaining authors have no disclosures to report.Dr Dobre reports receiving consultancy fees from Tricia and Relypsa. Dr Shafi reports receiving an honorarium from Siemens for a continuing medical education lecture. Dr Feldman reports consulting fees from Kyowa Hakko Kirini Co. Ltd, is the Editor‐in‐Chief of the Tables S1–S2Click here for additional data file.


Neurologic complications of SARS‐CoV‐2 infection have been reported commonly. Peripheric facial nerve palsy is one of the most reported neurologic problems. However, idiopathic bilateral facial palsy is a very rare complication of SARS‐CoV‐2 infection. Herein, we present a case of a COVID‐19 35‐year‐old man, which developed bilateral facial palsy. Idiopathic bilateral facial palsy  is a very rare complication of SARS‐CoV‐2 infection. It has some important differential diagnosis that should rule out. Although the catastrophic days of the pandemic are over, COVID‐19 is still considered a significant health crisis.The condition known as Bell's palsy is a type of facial nerve palsy that can be idiopathic or of suspected viral etiology. However, herpes simplex virus (HSV) activation is the likely cause of Bell's palsy in most cases, although there is no well‐established, widely available method of confirming a viral mechanism in clinical practice. Inflammation starts at the geniculate ganglion, extending proximally and distally, causing facial palsy due to neural edema inside the narrow facial canal. Laboratory tests are usually not indicated. There are several differential diagnoses, including Lyme disease, neoplasia, Ramsay Hunt Syndrome, and sarcoidosis.Herein, we present a case of isolated BFP due to COVID‐19 without other neurologic complications or any underlying diseases.2A 35‐year‐old man was admitted with a chief complaint of fever and loss of consciousness. He had a history of acute watery diarrhea 5 days before admission, and since last night he had developed a high‐grade fever (105.8 °F) and a significant decrease in his consciousness. He had no history of a similar condition and any underlying diseases. His family mentioned a history of contact with COVID‐19 patients (his brother and mother) for about a week. He had injected his last dose of Sinopharm COVID‐19 vaccine (second dose) 8 months ago. He had no history of allergy to any specific drug or food and even no history of trauma.On admission, his blood pressure was 100/60 mmHg, pulse rate was 90/min, respiratory rate was 18/min, the temperature was 98.6 °F, and O2 saturation was 95%. He was not conscious, but he withdrew from pain, and open his eyes. His pupils were reactive and normal size, and the gag reflex was normal. The Glasgow Coma Scale (GSC) was 10/15 at the time of admission . Bilateral bronchial breathing sounds and the abdominal examination were normal. There was no other noteworthy finding in the physical examination. His laboratory results on admission were serum Na, 117 mEq/L; K, 3.1 mEq/L; BUN, 11 mg/dL; creatinine 0.9 mg/dL; INR, 1.3; hemoglobin, 13.8 g/dL; WBC, 6900; platelets, 132,000. On the other hand, his SARS‐CoV‐2 PCR was positive, but he had no respiratory signs, and his chest high‐resolution computed tomography (HRCT) was normal.His consciousness improved after an increase in the serum Na level 137 mEq/L). On the following day, although his blood test indicated normal metabolic and electrolyte profiles and his consciousness has improved , in the neurologic examination, a bilateral facial nerve weakness, reduced eyelid closure, drooling from both sides of the mouth, and slurred speech was detected, moreover, he could not raise his eyebrows, puff his cheeks, and keep food in his mouth (House‐Brackmann grade V on both sides), thus a nasogastric tube was inserted for him. The rest of the neurologic examinations including other cranial nerves, deep tendon reflexes, sensory examinations, and gait were normal. He moved his lower and upper limbs symmetrically, and there was no sign of focal neurologic deficit and his lower and upper limbs muscle strength test was normal. The brain MRI and brain CT (1 day after BFP onset) were normal, but unfortunately, the patient refused to undergo MRI with contrast. VII‐VIII nerves are demonstrated in Figure 37 mEq/L.According to the normal neurologic examinations  on one hand, and no progression in the neurologic disorders during the hospitalization period, on the other hand, the possibility of Guillain–Barre syndrome (GBS) is low. However, our patient refused to undergo electromyography, in order to definitely reject the possibility of GBS. The serology tests for different possible infections such as human immunodeficiency virus (HIV), Epstein–Barr virus (EBV), syphilis, HSV, and cytomegalovirus were negative. There was no sign of neoplasm, local infections, or any other causes of BFP in the ear–nose–throat examination. His rheumatological tests including antinuclear antibody (ANA), anti‐neutrophil cytoplasmic antibodies (ANCA), and complement components 3 and 4 (C3 and C4) were negative. Thus, all the possible causes had been ruled out, and our final diagnosis was idiopathic BFP  due to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection.Before LP was done, due to the probability of encephalitis and meningitis, corticosteroids (Prednisolone 50 mg per day for 1 week), Remdesivir, Acyclovir, Vancomycin, and Ceftriaxone were initiated. But after the negative result of LP for HSV, SARS‐CoV‐2, and bacterial contamination, we discontinue mentioned drugs and taper the corticosteroid course with Prednisolone 10 mg per day. His facial nerve examination improved during hospitalization. The nasogastric tube was removed after 2 weeks, and he could swallow with no problem. He was discharged after 21 days of BFP initiation, with normal facial nerve function (House‐Brackmann grade I).3The exact pathogenesis of facial palsy due to COVID‐19 has remained unclear, but the angiotensin‐converting enzyme 2 (ACE2) receptor entranceway is the most probable mechanism. ACE2 receptors are widely expressed in various tissues, especially neurons, which make them at risk of SARS‐CoV‐2 invasion.BFP is an extremely rare condition. About 0.3% of patients with facial palsy, have BFP. Up to date, various differential diagnoses have been reported for BFP, which are presented in Table According to a systematic search of available literature in PubMed, Scopus, and Embase databases using keywords including Bell's palsy, facial nerve palsy, COVID‐19, and SARS‐CoV‐2, we find four published case reports of isolated BFP  after COVID‐19.Although our patient had no sign of underlying diseases .In conclusion, medical professionals should be aware of the possible neurologic complications in COVID‐19 patients and their top differential diagnosis.Majid Dezfouli: Conceptualization; project administration; supervision; writing – review and editing. Firouzeh Moeinzadeh: Project administration; supervision; writing – review and editing. Farzin Ghiasi: Project administration; supervision; writing – review and editing. Neda Abdeyazdan: Data curation; writing – original draft; writing – review and editing. Sadegh Mazaheri‐Tehrani: Conceptualization; data curation; investigation; writing – original draft; writing – review and editing.The authors would like to express that we have no funding received from any organization.The authors declare that they have no conflict of interest.This study was approved by the institutional review board.Written informed consent was obtained from the patient for the publication of this case report.


This study was designed to investigate whether berbamine (BA)‐induced cardioprotective effects were related to 5′ adenosine monophosphate‐activated protein kinase (AMPK)/nuclear factor erythroid 2‐related factor (Nrf2) signaling and changes in the mitochondria in myocardial ischemia/reperfusion (I/R) injury. C57/BL6 mice were exposed to BA (10 mg/kg/d), with or without administration of the AMPK specific inhibitor compound C (5 mg/kg/d) or the Nrf2 specific inhibitor ML‐385 (30 mg/kg/d), and then subjected to a myocardial I/R operation. As expected, BA significantly improved post‐ischemic cardiac function, reduced infarct size and apoptotic cell death, decreased oxidative stress, and improved the mitochondrial state. Furthermore, BA markedly increased AMPK activation, Nrf2 nuclear translocation, and the levels of NAD(P)H quinone dehydrogenase and heme oxygenase‐1. Nevertheless, these BA‐induced changes were abrogated by compound C. In addition, ML‐385 also canceled the cardioprotective effects of BA but had little effect on AMPK activation. Our results demonstrate that BA alleviates myocardial I/R injury and the mitochondrial state by inhibiting apoptosis and oxidative stress via the AMPK/Nrf2 signaling pathway. ACCacetyl‐CoA carboxylaseAMPK5′ adenosine monophosphate‐activated protein kinaseBAberbamineCKcreatine kinaseELISAenzyme‐linked immunosorbent assayGAPDHglyceraldehyde 3‐phosphate dehydrogenaseHO‐1heme oxygenase‐1I/Rischemia/reperfusionLDHlactate dehydrogenaseLVEFleft ventricular ejection fractionLVFSleft ventricular fraction shorteningNQO‐1NAD(P)H quinone dehydrogenaseNrf2nuclear factor erythroid 2‐related factorROSreactive oxygen speciesSEMstandard error of the meanSODsuperoxide dismutaseTUNELterminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling1Ischemic heart disease is the leading cause of morbidity and mortality in the United States and other parts of the world.5′Adenosine monophosphate‐activated protein kinase (AMPK), a serine/threonine kinase, has been identified as a pivotal energy sensor and regulator of cellular metabolism that has an important role in the regulation of energy homeostasis and of the reduction in ROS production under normal and ischemic conditions.Berbamine (BA) is a natural compound of Berberis and its derivatives, which have been proved to have an antitumor effect. BA‐induced apoptosis exists mainly in a wide range of cancer cells and tumors. The potential mechanism of BA‐induced cell apoptosis and the activity of cancer cells may be related to the downregulation of bcr/abl gene expression and change in the mitochondrial membrane. BA derivatives can improve cell proliferation of ROS and target calcium/calmodulin‐dependent death protein kinase II. Therefore, BA may play a potential protective role in antitumor and cardiovascular protection.22.1Male C57/BL6 mice  were provided by the Laboratory Animal Center at the Air Force Medical University and housed in a controlled environment . All procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Academy of Sciences and published by the National Institutes of Health . All experimental protocols were reviewed and approved by the Research Commission on Ethics of the Air Force Medical University.2.2phox, histone H3, and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology. Goat anti‐rabbit and goat anti‐mouse secondary antibodies were purchased from the Zhongshan Company. Dihydroethidium, which detects ROS generation in cardiac tissues, was purchased from Invitrogen. Compound C and ML385 were purchased from Selleckchem.Berbamine, Evans blue, triphenyltetrazolium chloride, and 4′,6‐diamidino‐2‐phenylindole dihydrochloride were purchased from Sigma‐. Enzyme‐linked immunosorbent assay (ELISA) kits for detecting lactate dehydrogenase (LDH), SOD, and creatine kinase (CK) activities and malondialdehyde (MDA) content were purchased from the Institute of Nanjing Jiancheng Bioengineering Institute. The terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) assay kit was purchased from Roche Biochemicals. Primary antibodies against AMPK, phospho‐AMPK, acetyl‐CoA carboxylase (ACC), phospho‐ACC, Nrf2, heme oxygenase‐1 (HO‐1), and NAD(P)H quinone dehydrogenase (NQO‐1) were purchased from Abcam. A primary antibody against SOD2 (sc‐30080) was obtained from Santa Cruz Biotechnology. Primary antibodies against cytochrome c, cleaved caspase‐3, gp912.3The myocardial I/R injury model was used as described previously.2.4As described in a previous study,2.5As described previously,2.6Measurement of ROS was performed as described previously.2.7Myocardial apoptosis was analyzed by TUNEL staining using an in situ cell death detection kit as described previously.2.8The activities of serum LDH and CK were used to estimate myocardial necrosis using ELISA kits, following the manufacturer's instructions. The activity of SOD and the content of MDA in cardiac tissues were determined using ELISA kits from the Institute of Nanjing Jiancheng Bioengineering Institute.2.9g for 20 min, and the supernatants were collected. Protein concentrations were determined using the bicinchoninic assay. Denatured samples were subjected to either 8% or 12% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies against phophso‐AMPK (1:1000), AMPK (1:1000), phophso‐ACC (1:1000), ACC (1:1000), Nrf2 (1:1000), NQO‐1 (1:1000), HO‐1 (1:1000), gp91phox (1:1000), cytochrome c (1:1000), cleaved caspase‐3 (1:1000), histone H3 (1:1000), and GAPDH (1:5000), followed by horseradish peroxidase‐conjugated secondary antibody for 1 h at room temperature. After that, the membranes were exposed to the chemiluminescence reaction and imaged with ChemiDoc XRS (Bio‐Rad). Equal protein loading was confirmed by GAPDH probing of membranes. Relative densitometry was analyzed using the Image Lab (Bio‐Rad Laboratories), as described previously.Left ventricular tissues were snap‐frozen immediately and stored at −80°C until analyzed. To determine the protein of cytochrome c, a cytosolic fraction was prepared with lysis buffer containing 50 mM Tris‐HCl (pH 7.3), 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail. For others, additional 1% triton X‐100 was added to the lysis buffer to extract the total proteins. After homogenization, the samples were centrifuged at 12,0002.103 section of left ventricular myocardial tissue was cut, quickly put into 2.5% glutaraldehyde solution, and fixed overnight at 4°C. The fixed tissue was sent to the electron microscope room to make specimens. The changes in the mitochondria in the myocardial tissue from each group were observed using a transmission electron microscope and photographed for preservation.According to the requirements of electron microscope specimen preparation and sampling, for each specimen, a 1‐mm2.11t test between two groups or one‐way analysis of variance followed by Bonferroni's multiple comparison for a post hoc t‐test. A value of p < 0.05 was considered to be statistically significant.All data were expressed as mean ± standard error of the mean. Statistical analyses were performed using GraphPad Prism . The statistical significance of the differences was determined by a Student 33.1To explore the role of BA in myocardial I/R and to determine whether the AMPK pathway is involved in the cardioprotective effects of BA, compound C, the AMPK specific inhibitor, was used in this experiment, and cardiac function and myocardial injury were subsequently evaluated. As revealed in Figure 3.2It is well known that apoptosis is the main characteristic of myocardial I/R, which prompted us to assess the antiapoptotic effects of BA on cardiomyocytes during I/R. As illustrated in Figure 3.3phox, ROS, MDA, and SOD activity were detected. As shown in Figure phox, an important oxidative stress marker, was remarkably increased in the I/R group compared to the sham group. The levels of MDA and ROS were also significantly increased in the I/R group whereas the SOD activity was decreased (Figure Oxidative stress has been demonstrated to play a vital role in myocardial I/R injury. To explore the effects of BA on oxidative stress in myocardial I/R, the levels of gp91d Figure , which sd Figure , suggestd Figure . Transmid Figure .3.4The AMPK/Nrf2 pathway is described as an important mechanism in protecting against oxidative stress‐induced injury. To investigate the molecular mechanism underlying the antioxidative effects of BA, western blot was used to evaluate the protein levels of p‐AMPK, p‐ACC, NQO‐1, HO‐1, cytoplasmic Nrf2, and intranuclear Nrf2. As shown in Figure 3.5phox and increased the levels of MDA and ROS; it also led to an increase in the apoptotic index. Conversely, treatment with BA significantly reduced the expressions of cleaved caspase‐3, cytosolic cytochrome c, gp91phox, and the levels of MDA and ROS; it also led to a decrease in the apoptotic index. However, the results showed that ML‐385, a specific inhibitor of Nrf2, inhibited antiapoptotic and antioxidative effects of BA in myocardial I/R, suggesting that the antiapoptotic and antioxidative effects of BA are related to Nrf2 activation.To further confirm whether the antiapoptotic and antioxidative effects of BA are associated with Nrf2 in myocardial I/R, ML‐385, a specific Nrf2 inhibitor, was used. As shown in Figures 3.6To investigate whether Nrf2 inhibition could abolish the cardioprotective effects of BA in myocardial I/R, ML‐385 was used in these experiments and then cardiac function and myocardial injury were detected. As shown in Figure 3.7To further illustrate the correlation between AMPK and Nrf2, we performed immunoblots of the proteins p‐AMPK, p‐ACC, intranuclear Nrf2, cytoplasmic Nrf2, NQO‐1, and HO‐1. As shown in Figure 4In this study, we demonstrated that BA could alleviate myocardial I/R injury by improving cardiac function and reducing apoptotic cell death. These findings are consistent with the results of previous studies. Furthermore, the cardioprotective effects of BA against myocardial I/R injury were associated with inhibiting apoptosis and oxidative stress. More importantly, compound C, the AMPK specific inhibitor, blocked AMPK activation, and Nrf2 nuclear translocation and inhibited the antiapoptotic and antioxidative effects of BA, suggesting that AMPK/Nrf2 signaling activation was essential for BA‐alleviated myocardial I/R injury. In addition, BA protected against myocardial I/R injury by activating AMPK/Nrf2 as further demonstrated by the evidence that the specific Nrf2 inhibitor ML‐385 abolished Nrf2 nuclear translocation, the antiapoptotic and antioxidative effects, and cardioprotection induced by BA but had little effect on AMPK activation. These results demonstrate that BA protects against myocardial I/R injury by inhibiting apoptosis and oxidative stress through activating the AMPK/Nrf2 pathway.Despite timely reperfusion using primary percutaneous coronary intervention, thrombolytic therapy is the most effective treatment for patients with ST‐segment elevation myocardial infarction. Morbidity and mortality remain extremely high as evidenced by death reported in 7% and heart failure in 22% of patients at 1 year after the event.Berberis amurensis, a traditional Chinese herbal medicine in the berberiaceae family. It can promote hematopoietic function and increase the quantity of blood cells. It is used to prevent and treat leukopenia caused by radiotherapy and chemotherapy, benzene poisoning, radioactive substances, and drugs in patients with tumors. Berbamine has a curative effect on silicosis, leukopenia, thrombocytopenia, essential hypertension, and so forth. It can also be used as a muscle relaxant for traditional Chinese medicine anesthesia.Berbamine is an extract from the roots, root bark, and stem bark of phox, ROS, and MDA significantly increased in the I/R group whereas SOD activity decreased compared to the sham group. At the same time, mitochondrial morphological and functional damage was found in the I/R group compared to the sham group. As expected, I/R treatment with BA decreased the levels of gp91phox, ROS, and MDA and improved SOD activity. Nevertheless, compound C and ML‐385 could obstruct BA‐induced antioxidative effects in myocardial I/R injury, suggesting that the antioxidative effects of BA and of mitochondrial morphological and functional damage were related to AMPK and Nrf2.Oxidative stress is a prominent pathological feature and plays an important role in the pathogenesis of myocardial I/R injury.AMPK, a master energy‐sensing serine/threonine kinase, functions as a cardiac energy sensor and maintains energy homeostasis in physiological and pathological conditions.Nrf2, a ubiquitously expressed transcription factor, regulates the transcription of a range of target genes in response to oxidative stress in basal and pathological conditions.5Our study results have demonstrated that BA alleviates myocardial I/R injury by improving mitochondrial morphology and by reducing apoptosis and oxidative stress. More importantly, our results also indicate that the AMPK/Nrf2 signaling pathway plays a pivotal role in mediating the cardioprotective effects of BA in myocardial I/R injury and improving mitochondrial morphology and state. Therefore, our results suggest that BA is a promising therapeutic intervention for patients with ischemic heart disease.The authors have no conflict of interests to declare.Chennian Xu and Yang Liu designed the research. Chennian Xu, Yang Liu, and Ping Jin conducted the experiments. Mengen Zhai, Zhenge Fan, and Yang Liu analyzed and interpreted the data. Chennian Xu and Jian Yang wrote the manuscript. The study was supervised by Jian Yang and Lifang Yang. All authors read and approved the final manuscript.The experimental procedures involving animals were performed in accordance with the guidelines of the Xijing ethics committee and local laws and policies. All of the procedures were approved by the Food and Veterinary Service, China.


Lumbar interbody fusion(LIF) is the leading way to treat Lumbar Degenerative Diseases(LDD). At present, there is a lack of research on the influencing factors of hidden blood loss in minimally invasive hybrid lumbar interbody fusion. This study comprehensively explores the definite factors affecting the hidden blood loss in minimally invasive hybrid lumbar interbody fusion.One hundred patients with Lumbar degenerative diseases who underwent minimally invasive hybrid lumbar interbody fusion in our center were included. Demographics, laboratory data, surgical data, and radiographic data were collected. The Gross equation and Sehat equation were used to calculate the estimated value of hidden blood loss. Multi-factor linear regression analysis was used to determine the influencing factors of hidden blood loss.p < 0.05), hypertension (p < 0.05), psoas thickness (p < 0.05) and dorsal extensor group thickness (p < 0.05) were potential risk factors for postoperative hidden blood loss.We reviewed and collected 100 patients who underwent minimally invasive hybrid approach, mean age 65 ± 10 years, male: female 37:63; 17 patients of diabetes and 83 patients of non-diabetes; Total blood loss was 645.59 ± 376.37 ml, hidden blood loss was 421.39 ± 337.45 ml, the hidden blood loss percentage was 57 ± 26%. Results from the multi-factorial linear regression model: Diabetes (Although minimally invasive hybrid approach is minimally invasive surgery, there is still a significant amount of hidden blood loss. There is a greater risk of blood loss in diabetes, hypertension and preoperative MRI assessment of thickness of the psoas, thickness of the dorsal extensor group. Lumbar Degenerative Diseases (LDD) are the most frequent diseases in spinal surgery, and the frequency of lumbar degenerative diseases is increasing due to societal development and changes in lifestyle . IntervePrevious research has linked minimally invasive spinal methods to considerable hidden blood loss, and there are numerous possible risk factors for hidden blood loss –13. A neThe clinical data of LDD patients who underwent Minimally invasive hybird approach in the Second Affiliated Hospital of Dalian Medical University from November 2020 to April 2022 were retrospectively collected and analyzed. This study was approved by the Ethics Review Committee of the Second Affiliated Hospital of Dalian Medical University and obtained the unique identification number of research registration (the research registration number is 2022055). Each patient signed a written informed consent form.The inclusion criteria include: ① Patients with clinically diagnosed degenerative lumbar disease. ② Multi-segment LDD  ③Minimally invasive hybird approach was performed.The exclusion criteria include: ①Lack of information including demographics, Laboratory data, surgical data, radiographic data. ②Patients with severe hematological disorders and severe cardiovascular disease. ③with lumbar infections or tumors. ④with previous lumbar surgery. ⑤with autologous and allogeneic blood transfusion.A total of 124 patients were performed by Minimally invasive hybird approach during the study period. 100 patients were enrolled after applying the inclusion and exclusion criteria. Eight patients with lumbar infections or tumors,6 patients with previous lumbar surgery,5 patients with autologous and allogeneic blood transfusion,3 patients with Incomplete information,2 patients with coagulopathy were excluded.All the operations were performed by the same team in our hospital. All patients were given general anesthesia and were performed by Minimally invasive hybird approach.Surgery sequence: ALIF → OLIF → PPSF. In our experience, ALIF procedure is first performed to open the L5-S1 gap, especially in patients with high iliac, to facilitate the L4–5 segment surgery.①ALIF: The anterior approach was midline of L5-S1 level, and the cartilage of endplate was removed by conventional methods after discectomy. The endplate was prepared with a straight curette. For narrow disc space, the distraction was performed using a parallel distractor. Proper implant size was determined using the trial implants under fluoroscopy. The cage filled with the bone grafting material was inserted into the intervertebral space using fluoroscopy. Once the position of the cage was optimal, anterior fixation was performed on CAGE and L5-S1 vertebral body under fluoroscopy .②OLIF: For L2-L5 levels, OLIF surgery is performed in accordance with standard procedure. The patient was placed in the right lateral position and X-rays were used to identify the degenerative segment. .4-6 cm skin incision was made 6–10 cm anterior to the mid-portion of the marked disc . The surgeon approached the retroperitoneal space by blunt dissection and anterior displacement of the peritoneum to expose psoas. After the discectomy, endplate cartilage was removed and inserted an intervertebral cage filled with homogeneous bone.③PPSF: PPSP was performed in the last step, with anterior fixation of L5 vertebrae, L5 vertebrae are usually not screwed in the absence of significant osteoporosis. For the L2-S1 levels, PPSF was often performed on L2, L4 and S1; For the L3-S1 levels, PPSF was often performed on L3, L4 and S1; For the L4-S1 levels, PPSF was often performed on L4 and S1.On the first day and third day after the operation, blood routine examination was repeated. Methylprednisolone, omeprazole, parecoxib, and rivaroxaban were used for conventional treatment 3 days after operation. Lumbar spine x-rays are repeated 1–2 days after surgery, with the duration postoperative activity determined by the patient’s status. Most patients were able to walk after surgery day 1–3, protected by a lumbar brace.A total of 100 cases were included between November 2020 and April 2022. Demographics , body mass index (BMI) were converted to qualitative data at a cutoff of 24, laboratory data (preoperative Hematocrit (HCT), postoperative HCT), surgical data , and radiographic data, Hypertension, Diabetes were collected.Preoperative magnetic resonance imaging (MRI) was used to determine thickness of the psoas, thickness of the dorsal extensor group, and thickness of the subcutaneous fat. These measurements were made at the L4 level using image J Fig. .Fig. 1Dipre − Hctpost)/Hctave, where Hctpre is the preoperative Hct, Hctpost is the second or third postoperative Hct, and Hctave is the average of Hctpre and the Hctpost.③according to the formula of Sehat: HBL = TBL - visible blood loss (VBL). Without post-operative drainage, the VBL is approximately equal to the intraoperative bleeding.Calculation of hidden blood loss:①according to the formula of Nadler: PBV = k1 × height(m)3 + k2 × weight (kg) + k3 .②according to the Gross formula: total blood loss (TBL) = PBV  were reviewed retrospectively. And distinguish them based on quantitative or qualitative data, with quantitative data shown as means standard deviation. The patient demographic and clinical data (including blood loss results) are summarized in Tables P < 0.001;HBL: 465.61 ± 337.54 ml VS 205.51 ± 247.08 ml, P = 0.001) (Table p = 0.240),as shown in Table Post-operative Hct change and hidden blood loss were higher in diabetes patients than in non-diabetic patients , hypertension (p < 0.05), postoperative HCT (p < 0.05), psoas thickness (p < 0.05), and thickness of the dorsal extensor group (p < 0.05). Psoas thickness and dorsal extensor group thickness were favorably correlated with HBL. However, no link was found between age, BMI, fusion segment, operation duration, and subcutaneous fat thickness.Based on the result of Spearman relative analysis combined with clinical analysis, 12 variables were included in the Multi-factor linear regression Table . We idenWhile minimally invasive spine surgery has had some positive results, the hidden blood loss was also noticeable. In our study, the mean total blood loss was 645.59 ± 376.37 ml and the mean hidden blood loss was 421.39 ± 337.45 ml (representing 57 ± 26% of TBL). To further detect some differences in HBL between the different groups, we have compared this study with previous studies (see Table As shown in Table p = 0.002) and Diabetes(p < 0.001) were a positive influencing factor of hidden blood loss, which were contrary to the findings of Man Hu et al. [Hypertension was a risk factor in his study . Our meap = 0. 483). In contrast, Cai found that time to operation was an independent risk factor in elderly patients undergoing ACDF [Operation duration was illuminated in our study, which was not a positive influencing factor of hidden blood loss , which is consistent with the outcome of the research by Yoji Ogura et al. [By segmental comparison , we found that the amount of hidden blood loss increased with the increase in segment, but the difference between the groups was not statistically significant  ⑥ the lack of drainage ⑦Bleeding from other sources, such as from the gastrointestinal tract. Above are factors that may cause hidden blood loss, and further studies are necessary to verify these.Our study has limitations, such as the small amount of patient data collected and the need for a larger randomized controlled trial to further validate the trial. Additionally,measuring the thickness of the patient’s psoas major muscle and the dorsal extensor group using MRI axial slices, while measured by three different observers, inevitably results in systemic errors in manual measurements.With the development of spinal minimally invasive techniques, the amount of visible blood loss is gradually decreasing, but the existence of hidden blood loss cannot be ignored. In our study, the amount of hidden blood loss in the minimally invasive hybrid approach was 421.39 ± 337.45 ml, representing 57 ± 26% of total blood loss. The management of perioperative fluids should not be neglected, especially in patients with diabetes, hypertension and preoperative MRI revealing greater thickness of the psoas major and dorsal extensor muscles, which are often associated with greater hidden blood loss. For surgeons, a reasonable preoperative assessment of these risk factors is essential for the patient’s rapid postoperative recovery and reduction of the risk of adverse events.


In this report, Recombinant Tissue Plasminogen Activator (r‐tPA) has been nebulized to 3 critically ill COVID‐19 patients in order to resolve the deposited fibrin while avoiding the risk of bleeding. Based on these observations, nebulization of r‐tPA may be a potential therapeutic approach and new area of research for future studies.Fibrin deposition in the alveolar spaces during pulmonary involvement of COVID‐19 impairs the O Fibrin cast depositions play an important role in COVID‐19‐induced ARDS. Systemic administration of fibrinolytic may accompany with risk of bleeding. Nebulization of r‐tPA (Recombinant Tissue Plasminogen Activator) can temporally associated with transient improvements in oxygenation in COVID‐19‐induced ARDS. Fibrinolytic nebulization would minimize bleeding risk‐associated systemic administration.  Nevertheless, mortality of SARS‐CoV‐2 infection arises mainly through the development of viral pneumonia‐induced acute respiratory distress syndrome (ARDS), sepsis, and multi‐organ failure.The exact pathophysiological process associated with COVID‐19 ARDS remains unclear, endothelial dysfunction, cytokine storm, and hypercoagulability state have been defined as the main hyper inflammatory and coagulation dysregulation.In this study, we assessed the pulmonary physiologic markers of three patients with COVID‐19 after nebulizing r‐tPA individually.22/FiO2 (PF ratio) less than 100 enrolled for this assessment.This assessment was approved by ethics committee of Tehran University of Medical Science (IR.TUMS.VCR.REC.1399.025) and was performed in Shariati Hospital, Tehran, Iran. Patients with diagnosis of COVID‐19 based on Chest computed tomography (CT) and positive polymerase chain reaction (PCR) and PaO2) and fraction of oxygen inspired (FiO2) recorded every 4 h. Next dose administered at 24‐h interval.They received all of common treatments based on our institutional protocol. Jet nebulizer in all  was supplied from Saramad Teb Parayeh Company and r‐tPA Actilyse® treatment set, Batch number 003202, were used during this study. Fifty mg of Actilyse® dissolved in 50 ml of the solvent (1 mg/ml solution) and nebulized by jet nebulizer in divided volumes over 2 h. Oxygen saturation  61 fl, CRP 80 mg/L, erythrocyte sedimentation rate (ESR) 31 mm/h, and Lactic acid dehydrogenase (LDH) 725 U/L abnormal.Forty‐seven‐year‐old woman presented to emergency department (ED) with complaint of dry cough, asthenia, fever, and myalgia for 7 days. Her past medical history was positive for hypertension, and she was taking losartan 25 mg twice daily. At presentation, general physical examination revealed a blood pressure (BP) 110/80 mmHg, respiratory rate (RR) 24 resp/min, pulse rate (PR) 105 bpm, SpO2, she received 250 mg methyl prednisolone after intubation. The nebulizing r‐tPA was ordered on the sixth day after admission. Before r‐tPA administration, SpO2 88% on FiO2 60% was recorded. One dose was nebulized based on study protocol and oxygenation trend  was recorded every 4 h . Fever continued and imipenem 1 g three times a day substituted for cefepime. Hemoglobin begins to fall without any distinct source of bleeding. After 2 days, her oxygen saturation falls to 60% on 15 L reservoir bag mask, she became agitated and medical team decided to intubate her. Due to low SpOUnfortunately, at the time of the next dose, manifestation of seizure and upward gaze was detected in the patient; therefore, treatment was stopped, imipenem discontinued and antiepileptic agents started. Considering the clinical situation of the patient brain CT was not feasible. From 1 day after nebulizing, oxygen saturation began to decline. Seizure and seizure‐like events was not noticed again. Finally, 9 days later, patient expired due to asystole after 45 min of CPR.3.22 78%, RR 25 resp/min, PR 105 bpm, BP 160/90 mmHg, and oral temperature of 37.5°C. Chest CT findings were compatible with COVID‐19 pneumonia. Out of range laboratory data noticed for lymphocyte count 693 cells/mm3, CRP 87 mg/L and ESR 82 mm/h.A 78‐year‐old man with past medical history of diabetes mellitus  and hypertension admitted to ED, complaining dry cough, fever, and dyspnea for 1 week. His vital signs at presentation were SpO2 60% on reservoir bag mask, PR 160 bpm), intubation was inevitable. At day 18th of admission, r‐tPA nebulization was ordered because of low oxygen saturation (80%) despite 100% FiO2. Three doses were administered in three consecutive days according to our study protocol. As it evident in the Figure 2 and similar pattern of changes with SpO2. Even though all the efforts were made by medical team, patient expired on 38th day of admission after 45 min of CPR.Pressure support ventilation, scheduled prophylactic doses of heparin, methyl prednisolone 125 mg daily for 3 days, hydroxychloroquine 400 mg PO stat then 200 mg twice daily, and lopinavir/ritonavir 200/50 mg 2 tablets twice daily started for him. However, oxygenation progressively exacerbated, and 14 days after admission due to severe respiratory distress . He did not tolerate CPAP and oxygen saturations continued to decline. Methyl prednisolone 125 mg plus 100 mg thiamin and 6 g of vitamin C ordered for 3 days. On 13th day of admission r‐tPA nebulization started. Oxygenation trend presented on Figure Demographic data and the impact of treatment are summarized in the Table 4In all three cases, we have seen improvement in SF ratio as an oxygenation index. However, the effect of treatment was short lasting and after stopping r‐tPA nebulization these effects vanished. In the first case, nebulization led to +9% changes at the end of the day. The second case, showed +10%, +22%, and 13% improvement compared with baseline after each dose, respectively. Continuous improvement was seen in the third case, as +4.7%, +8.2%, and +15.2% variation occurred after every nebulization. However, by discontinuing the treatment, fall in SF ratio in all cases was detected.Fibrin deposition in the alveolar spaces and pulmonary parenchyma and micro‐thrombi formation in the lung vessels is the main histopathologic findings in post‐mortem studies of patients with ARDS. These findings have been observed among patients with COVID‐19‐induced ARDS. Besides coagulopathy manifestation of ARDS, COVID‐19 could lead to endothelial disruption, microangiopathy with thrombosis of capillary vessels and angiogenesis.Based on animal models of ARDS, TPA administration leads to dissolve fibrin deposition and reduction of inflammation by preventing neutrophil activationIn populations other than ARDS such as plastic bronchitis which fibrin depositions have remarkable role in disease pathophysiology, nebulization of r‐tPA has been evaluated. Instillation of 3.5 mg r‐tPA on fibrin casts during bronchoscopy degraded fibrin deposition in one patientData regarding efficacy and safety of different routes of administration is sparing. Intrathecal and intravenous routes are more effective than nebulization in animal models of acute lung injury; however, bleeding risk is much lower with the latter and there are no reports of bleeding tendency by nebulization.Based on our observation, in Case 1, hemoglobin level reduction had happened prior to r‐tPA administration and after convulsions, she was suspicious to intracranial hemorrhage, but it was not confirmed by imaging. Further, she received imipenem which is known to be neurotoxic and epileptogenic; this could be another cause of seizure‐like episode.PLATyPuS trial (NCT02315898) is the only randomized clinical trial of r‐tPA nebulization on pediatric plastic bronchitis and we should wait for the results of this trial about the safety of this therapeutic modality.Despite previous evidence to address the benefit of r‐tPA in patients with ARDS, some challenges should be considered including the optimal time, route, duration, and interval of administration. Whether the early treatment or more doses can lead to an increased and constant response is unclear. However, it seems that the nebulizing of r‐tPA is an attractive delivery of fibrinolytic medications. Well‐designed clinical trials of thrombolytic therapy would further perform to show the efficacy and utility of this therapy.Jet nebulizer is less affected than ultrasonic nebulizer by the physiochemical characteristics of the nebulizing solution. Particle size and flow with jet nebulizer is more suitable for drug delivery to alveoli. Also, less physical and thermal stress is imposed to the solution, so the risk of inactivation or degradation of the medical ingredient would greatly decrease. This is particularly important for protein structures like fibrinolytics.Although interpretation of achieved data based on three cases without control arm is not practicable, we observed transient improvement in oxygenation after r‐tPA nebulization in this study. This benefit was short lasting and depended on treatment continuation. Considering the few number of patients, no placebo arm and lack of physiochemical study on r‐tPA nebulization solution, further well‐designed clinical trials are needed.Main idea for this treatment modality proposed by Mohammad Vasei. Rasoul Aliannejad was the physician in‐charge for treatment of these patients. Mohammad Poorabbas was responsible for clinical monitoring and data gathering. Shahideh Amini analyzed and interpreted the achieved data and Zohre Labbani‐Motlagh was a major contributor in writing the manuscript. All authors read and approved the final manuscript.This work was supported by The Tehran University of Medical Sciences, Grant No: 47125‐101‐199.The authors have no conflicts of interest to declare.This assessment was approved by ethics committee of Tehran University of Medical Science (IR.TUMS.VCR.REC.1399.025).Written consent form was taken from patients or their first‐degree relatives before enrollment.


The endosperm, a tissue that nourishes the embryo in the seeds of flowering plants, is often disrupted in inviable hybrid seeds of closely related species. A key question is whether parental conflict is a major driver of this common form of reproductive isolation.Mimulus caespitosa, Mimulus tilingii, and Mimulus guttatus). The severity of hybrid seed inviability varies among these crosses, which we inferred to be due to species divergence in effective ploidy. By performing a time series experiment of seed development, we discovered parent‐of‐origin phenotypes that provide strong evidence for parental conflict in shaping endosperm evolution.Here, we performed reciprocal crosses between pairs of three monkeyflower species (Mimulus species that differ in effective ploidy. These parent‐of‐origin effects suggest that the chalazal haustorium might act as a mediator of parental conflict, potentially by controlling sucrose movement from the maternal parent into the endosperm.We found that the chalazal haustorium, a tissue within the endosperm that is found at the maternal–filial boundary, shows pronounced differences between reciprocal hybrid seeds formed from Our study suggests that parental conflict in the endosperm may function as a driver of speciation by targeting regions and developmental stages critical for resource allocation and thus proper seed development. This task has been a challenge for intrinsic postzygotic isolation, which arises when hybrids inherit novel combinations of incompatible alleles that cause inviability or sterility  viable seeds in either direction of the cross . The same inbred lines were used in previous studies of hybrid seed inviability in M. tilingii and M. guttatus  for each focal species  and six interspecific crosses . To generate diploid, experimental plants, we sowed 20–30 seeds for each inbred line on damp paper towels in Petri dishes sealed with parafilm and cold‐stratified them for 7 d to disrupt seed dormancy. After cold stratification, we transferred the Petri dishes to a growth chamber with a 16 h : 8 h, 23°C : 16°C, light : dark photoperiod. We transplanted seedlings into 3.5 × 3.5 × 3.5‐inch pots with moist Fafard 4P Growing Mix  and placed the pots in the same growth chamber. Once plants began flowering, we randomly crossed within and between individuals . For all crosses, we emasculated the maternal plant 1–3 d before each cross to prevent contamination from self‐pollination.In this study, we considered three intraspecific crosses . To generate synthetic tetraploid individuals, we treated 100–200 seeds of TWN36 and LVR1 with 0.1% or 0.2% colchicine and stored them in the dark for 24 h (16 h at 23°C and 8 h at 16°C). The next day, we planted seeds onto Fafard 4P potting soil using a pipette and placed pots inside the growth chamber under typical light and temperature conditions . Once the seeds germinated, we transplanted the seedlings into 2.5 × 2.5 × 2.5‐inch pots. After sufficient growth, we prepared samples for flow cytometry using a protocol adapted from Lu et al.  together in a single well. To extract nuclei, we chopped 100 mg of leaf tissue  in 1 ml of a pre‐chilled lysis buffer . We stained the nuclei with 4,6‐diamidino‐2‐phenylindole (DAPI), filtered them for debris using a 40 μm Flowmi™ cell strainer, and aliquoted them into a single well of a 96‐well polypropylene plate. We assessed the ploidy of each sample using a CytoFLEX  flow cytometer. We calculated total DNA content using the following equation:To investigate species divergence in effective ploidy, we performed several interspecific, interploidy crosses: Cu et al. . Brieflyx4, 2C = 2.69 ± 0.09 pg; LVR1, 2C = 1.26 pg; LVR1x4, 2C = 2.64 ± 0.05 pg). In some cases, we discovered that plants initially identified as tetraploid via flow cytometry were actually mixoploids. To ensure the crosses we performed were indeed interploidy, we determined the ploidy of the resulting progeny. From each interploidy cross, we planted 5–10 seeds per fruit, isolated nuclei from the resulting plants, and assessed 2C content using a flow cytometer for a few offspring as described in the previous paragraph . We included data from interploidy crosses only when their progenies were confirmed to be triploids, or, in the case of 4x M. caespitosa, if we were using a confirmed stable polyploid line (i.e. self‐fertilized at least one generation with polyploidy confirmed in the progeny).We generated three synthetic polyploids for TWN36 and six for LVR1. For each synthetic polyploid, 2C DNA content was nearly doubled compared to corresponding diploid lines , for a total of 150 seeds per cross . Seed area was measured using Mimulus species and their hybrids . For fruits generated from interploidy crosses and fruits that produced < 100 seeds, we stained 32–63 seeds. We immersed the seeds in a scarification solution  and placed them on a shaker for 15 min. After scarification, we washed the seeds five times with water and incubated them with 1% Tetrazolium at 30°C. Two days later, we scored the number of seeds that stained dark red (viable) vs pink or white (inviable). As noted in the confirmation of triploids paragraph above, we also planted a subset of the seeds from interploidy crosses to assess ploidy, and germination rates generally reflected both seed viability measurements (data not shown).Using these same fruits, as well as fruits from interploidy crosses , we assessed seed viability using two different methods. First, we performed visual assessments of mature seeds, looking for irregular phenotypes  known to be highly correlated with germination rates in these To assess whether aberrant endosperm development contributes to seed defects in interspecific crosses, we attempted to rescue seed viability with a sucrose‐rich medium. We collected three fruits 8–12 d after pollination (DAP) from each intra‐ and interspecific cross (not including interploidy crosses), with each fruit collected from a distinct plant. Of the three fruits per cross, at least one fruit was collected 8 DAP (to maximize the chance of rescue). On average, we dissected 40 whole immature seeds per fruit (range = 25–57) and placed them on Petri dishes with Murashige & Skoog medium containing 4% sucrose. We sealed the Petri dishes with parafilm and placed them under constant light at 23°C for 14 d before scoring germination.To compare trajectories of seed development, we performed intra‐ and interspecific crosses, and we collected fruits 3, 4, 5, 6, 8, and 10 DAP. For consistency, we performed crosses and collected fruits at the same time of day.n = 1–2 fruits per DAP per cross) and prepared them for clearing with Hoyer's solution. We placed developing fruits in a nine parts ethanol alcohol (EtOH) : one part acetic acid fixative overnight. The following day, we washed the fruits twice in 90% EtOH for 30 min per wash. We dissected immature seeds directly from each fruit and placed them onto a microscope slide with 100 μL of three parts Hoyer's solution : one part 10% gum arabic and sealed the slide with a glass cover slip. We stored the microscope slides containing cleared, immature seeds at 4°C overnight. The next day, we imaged the slides on a Leica DMRB microscope  using the differential interference contrast (DIC) setting with the ×20 objective. For each fruit, we scored the number of developing seeds with and without an intact chalazal haustorium ; only seeds with visible embryos were scored. Additionally, we imaged an average of 11 seeds per fruit  to assess size differences in the endosperm and chalazal haustorium at 3 and 4 DAP. For the interploidy Tx4×G cross, we imaged on average 18 seeds per fruit . We outlined and measured the endosperm in all seeds and the chalazal haustorium, when present, using fiji . To measure the chalazal haustorium, we began the outline near the epidermis of the seed (not including the hypostase cells) and extended it toward the micropylar region following the method described by Guilford & Fisk  (see thec. 40°C) water bath and positioned them on a microscope slide. We placed the slides on a slide warmer overnight to adhere the sections completely to the glass. In a staining series, we first used xylene as a clearing agent and performed several washes with increasing concentrations of EtOH to effectively stain the nuclei and cytoplasm . We further washed the stained slides with EtOH and finished the series with xylene. We sealed the slides with a glass coverslip using Acrytol as the mounting medium.To visualize later seed development (after 4 DAP when the seed coat is too thick to clear with Hoyer's solution), we collected whole fruits at 5, 6, 8, and 10 DAP and stored them in a formaldehyde : alcohol : acetic acid fixative (10% : 50% : 5% + 35% water) for a minimum of 48 h. After fixation, we dehydrated the developing fruits with increasing concentrations of tert‐Butyl alcohol. Next, we washed the fruits three times for 2 h each with paraffin wax at 65°C before embedding them into a wax block. We sectioned the wax blocks containing whole fruits into ribbons using a Model 45 rotary microtome . Fruits collected at 5 and 6 DAP were sectioned into 12‐μm ribbons for better visualization of micropylar and chalazal domains, and fruits collected at 8 and 12 DAP were sectioned into 8‐μm ribbons. Next, we gently placed the ribbons into a warm . We imaged at least five consecutive sections of each seed through the embryo. For all seeds imaged at 5 and 6 DAP, we scored the presence of the chalazal haustorium. Additionally, we categorized embryo development at 6, 8, and 10 DAP into four different stages: before globular to globular, late‐globular to transition, early‐heart to late‐heart, and torpedo.et al., We performed several statistical analyses to determine the effect of each cross on seed area, seed viability, germination success on sucrose, and area of the endosperm filled by the chalazal haustorium. For each seed phenotype, we used the R software package , supplying them with sucrose revealed clear reciprocal differences in viability  and reciprocal differences are subtle or absent Fig. . HoweverM. caespitosa and M. tilingii, the volume of endosperm devoted to the chalazal haustorium at 4 DAP is nearly twice that of viable seeds , arrests at the globular stage , patterns of gene expression in chalazal tissues – or in analogous endosperm transfer cells – also point to their role in nutrient transfer, with upregulation of genes involved in sugar transport and metabolism , which moves to the peripheral endosperm and initiates cellularization  and paternally expressed genes (PEGs) spar over the distribution of maternally supplied resources to the developing seeds, the chalazal endosperm should play a key role  complex, which act to epigenetically silence the maternal alleles of PEGs , Mimulus tilingii (T), and Mimulus guttatus (G).Fig. S2 Total seed area (mm2) of a subset of seeds per fruit from crosses within and between M. caespitosa (C), M. tilingii (T), and M. guttatus (G).Fig. S3 Developing seeds cleared with Hoyer's solution 3 and 4 d after pollination (DAP) in crosses among M. caespitosa (C), M. tilingii (T), and M. guttatus (G).Fig. S4 Histological sections of whole fruits from intra‐ and interspecific crosses among M. caespitosa (C), M. tilingii (T), and M. guttatus (G) at 5, 6, 8, and 10 DAP.Methods S1 Data analysis.Table S1 The effect of intra‐ and interspecific crosses among M. caespitosa (C), M. tilingii (T), and M. guttatus (G) on the proportion of fully‐developed seeds per fruit (scored by eye) as determined by generalized linear mixed models.Table S2 The effect of intra‐ and interspecific crosses among M. caespitosa (C), M. tilingii (T), and M. guttatus (G) on the proportion of a subset of seeds per fruit stained dark red by tetrazolium (i.e. viable seeds) as determined by generalized linear mixed models.Table S3 The effect of intra‐ and interspecific crosses among M. caespitosa (C), M. tilingii (T), and M. guttatus (G) on the proportion of a subset of immature seeds per fruit that germinated on sucrose rich media as determined by generalized linear mixed models.Table S4 The effect of intra‐ and interspecific crosses among M. caespitosa (C), M. tilingii (T), and M. guttatus (G) on seed area (mm2) of a subset of seeds per fruit as determined by linear mixed models.Table S5 The effect of intra‐ and interspecific crosses among Mimulus caespitosa (C), M. tilingii (T), and M. guttatus (G), days after pollination (DAP), and their interaction on the area of the endosperm filled by a chalazal haustorium (shown as a proportion) as determined by linear models.New Phytologist Central Office.Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. 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