There has been steadily increasing use of bilateral mastectomy (BMX) in the treatment of primary breast cancer (BC). In this study, we utilized functional magnetic resonance imaging (fMRI) to examine the influence of emotion regulation on the decision of newly diagnosed BC patients to choose BMX rather than non‐BMX treatments.We recruited 123 women with unilateral BC, 61 of whom received BMX and 62 of whom received non‐BMX treatments, and 39 healthy controls. While participants were in the fMRI scanner, we showed them BC‐related and non‐BC‐negative images. In one condition, they were instructed to watch the images naturally. In another, they were instructed to regulate their negative emotion. We compared the fMRI signal during these conditions throughout the brain.With non‐BC‐negative images as the baseline, BC patients showed greater self‐reported reactivity and neural reactivity to BC‐related images in brain regions associated with self‐reflection than did controls. Among the BC patients, the BMX group showed weaker activation in prefrontal emotion regulation brain regions during emotion regulation than did the non‐BMX group.BC patients are understandably emotionally hyper‐reactive to BC‐related stimuli and those who ultimately received BMX experience more difficulty in regulating BC‐related negative emotion than non‐BMX BC patients. These findings offer neuropsychological evidence that difficulty in managing anxiety related to the possibility of cancer recurrence is a factor in surgical treatment decision‐making and may be an intervention target with the goal of strengthening the management of cancer‐related anxiety by nonsurgical means.NCT03050463. It may be that exposure to such information has an inordinate influence on people at risk for BC, especially those with difficulty managing their emotional responses, even if they are not mutation carriers.While the use of unilateral mastectomies in the treatment of primary breast cancer (BC) has decreased, there has been steadily increasing use of bilateral mastectomy (BMX)BMX is an elective procedure for a unilateral BC, because it involves more surgery than is required for treatment. Given that BMX may have detrimental effects including surgical complications and associated costsEmotional responses are complex, coordinated phenomena that lead to behavioral, cognitive, and physiological changes, activate action tendencies, and modulate feelings.Thus, emotion dysregulation, involving anxiety, fear, and other negative emotions may be a major factor in decisions regarding BC faced by some 255,000 women in the United States every year. The desire to reduce understandable current anxiety by accepting major treatment side effects to avoid the future risk of regret may drive decisions that are inconsistent with actual risk reduction. We conducted a case–control study utilizing fMRI to better understand the influence of emotion regulation on the decision of newly diagnosed BC patients choosing BMX rather than unilateral mastectomies or breast‐conserving treatments (non‐BMX), with the ultimate goal of developing an intervention to enable the management of cancer‐related anxiety by nonsurgical means. The central hypotheses of the study are that: (1) BC patients choosing BMX rather than non‐BMX treatments would show more difficulty regulating the emotion elicited by unpleasant stimuli, in particular those relevant to BC, and, (2) BC patients would show greater emotional reactivity to stimuli with content relevant to BC than controls.22.1We recruited a patient sample of 123 women diagnosed with BC within the preceding 12 months  with stage 0‐III unilateral breast cancer. We identified 33% of potential participants from Stanford Cancer Center records, 26% from the Army of Women website, 26% from social media, and the remaining 15% from other sources. Among the BC patients, 61 had undergone or later underwent BMX, and the other 62 had undergone or later received non‐BMX treatments. The recruitment of 39 healthy controls was monitored to maintain comparability in demographics. The controls were women with no history of cancer, no first‐degree relatives or 2 or more second‐degree relatives with a BC diagnosis, or any first‐ or second‐degree relatives with ovarian cancer. We identified 57% of potential control participants from a Stanford University volunteer pool, 33% from the Army of Women website, and the remaining 10% from other sources. All women (BC and control) were English‐proficient, willing to suspend intake of benzodiazepines and to undergo brain MRI, and with no contraindications to MRI imaging . Exclusion criteria included other current or past cancers, any significant neurologic disease, current untreated psychosis or bipolar disorder, substance/alcohol abuse/dependence, current use of psychotropic medication, pregnancy, and hearing impairment. The research protocol was reviewed and approved by the Stanford Institutional Review Board (protocol number: 34959), and all subjects provided written informed consent. The details of functional and structural MRI acquisition and data preprocessing are presented in Data 2.2During the task, participants viewed a series of pictures and were instructed to either respond naturally (“WATCH” condition) or to regulate their emotional response (“RETHINK” condition). Specifically, following the “WATCH” cue, they were instructed to look at and respond naturally to the picture without attempting to change their emotion. In WATCH trials, participants viewed either a neutral picture from the International Affective Picture System (IAPS),2.3https://osf.io/sq5rg/components). In particular, we preregistered the analysis plan (https://osf.io/3ju28/wiki/home/). Specifically, we preregistered the following contrasts between task conditions to index emotional reactivity: IAPS‐watch > neutral‐watch (reactivity for IAPS), BC‐watch > neutral‐watch (reactivity for BC), BC‐watch > IAPS‐watch , and IAPS‐watch + BC‐watch >2 × neutral‐watch . Similarly, we preregistered contrasts to index emotion regulation: IAPS‐rethink > IAPS‐watch (regulation for IAPS), BC‐rethink > BC‐watch (regulation for BC), BC‐rethink > IAPS‐rethink , IAPS‐rethink + BC‐rethink > IAPS‐watch + BC‐watch . We followed this logic in analyzing both the self‐report and fMRI data with the details illustrated in the subsections below. We compared these self‐report and neural indices between (1) BMX recipients versus non‐BMX recipients, and (2) controls versus BC patients. The general linear model (GLM) analyses of the fMRI data were performed using the FEAT tool in FSL 5.0.10 (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki). All other statistical tests were performed in R 3.5.1 (https://www.r‐project.org/). See subsections below for the specific statistical tests performed on the fMRI data and self‐report data. Power was estimated based on comparisons between the two subgroups of breast cancer patients, with and without BMX. Given the absence of prior data on the relationship between the BMX decision and affect reactivity/regulation, we assumed a substantial but conservative effect of d = 0.6. Under this scenario, with the initial sample size of 120 , the estimated power to detect the association between the BMX choice and affect reactivity/regulation was 0.9. We share the fMRI and self‐report data on the OSF (https://osf.io/8tfps/).The study is preregistered on the Open Science Framework, and its detail can be found in the wiki pages of each of the components , and nuisance regressors Data . The pre2.3.2The self‐report negativity rating data of each participant were averaged across trials for each trial type. Following the preregistered analysis above  of participants with BMX had their BMXs before their fMRI assessments  119.25, 252.5); and nine (14.5%) of non‐BMX participants had their unilateral mastectomies before their fMRI assessments  Table .3.1.2Fifteen (24.6%) of participants with BMX finished chemotherapy before their fMRI assessments . Seven (11.3%) of non‐BMX participants finished chemotherapy before their fMRI assessments . Six (9.8%) of participants with BMX were receiving chemotherapy at the time of their fMRI assessments . Twelve (19.4%) of non‐BMX participants were receiving chemotherapy at the time of their fMRI assessments  Table .3.23.2.1p < 0.001) and left dlPFC (clusterwise p = 0.038) than BMX recipients  on the relationship between the self‐report and neural indices of the BC‐IAPS differential regulation, F = 3.56, p = 0.062. Specifically, this self‐report‐neural correlation was medium‐sized positive in the non‐BMX group, r = 0.37, p = 0.003, while it was close to 0 in the BMX group, r = 0.03, p = 0.784  = 0.45, p = 0.505. To account for the individual difference in the time gap between BC surgery and fMRI experiment, we also ran the BMX versus non‐BMX group comparison with the surgery‐fMRI time gap as a covariate. This analysis revealed similar results, which included the same dmPFC and left dlPFC clusters  = −2.38, p = 0.019 , posterior cingulate cortex , and a precuneus‐cuneus cluster (clusterwise p < 0.001) than did the healthy controls. Furthermore, the BC‐IAPS differential neural reactivity in the vmPFC and PCC clusters was significantly positively correlated with participants' self‐report of BC‐IAPS differential reactivity  = 2.30, p = 0.024, while controls were more emotionally reactive to the general IAPS images than the cancer images, t(38) = 5.14, p < 0.001  than did controls, 1 Figure . Among t4The women who ultimately received BMX showed less activation in prefrontal brain regions that are associated with emotion regulation than those who received non‐BMX surgeries. Particularly, the non‐BMX group showed greater activation than the BMX group in the dmPFC and dlPFC during the application of reappraisal to regulate negative emotions elicited by BC‐related pictures.Consistent with our hypothesis, the self‐reported BC‐IAPS differential reactivity was significantly higher in BC patients than controls. It suggests that with IAPS pictures as a baseline, BC patients reported stronger relative emotional reactivity to BC‐related pictures than their healthy counterparts. The fMRI data further corroborated this finding. When comparing the neural differential BC‐IAPS reactivity, BC patients showed significantly stronger activation in regions within the default mode network (DMN), including vmPFC, PCC, and precuneus. These brain regions are typically involved in self‐reflection and self‐evaluation processes.It is interesting that the BC patient‐control group difference in neural reactivity did not appear in core limbic regions such as the amygdala, but was consistently evident in several DMN regions. The amygdala typically reflects low‐level aspects of emotional reactivity. The DMN, on the contrary, is typically involved in self‐referential cognitive processes.It is of clinical importance that greater activations of dmPFC and dlPFC during emotion regulation differentiate the non‐BMX and BMX groups. These components of the Executive Control Network (ECN) are crucial to emotion regulation, so their hypoactivation during the presentation of distressing visual stimuli, for cancer‐related images in particular, is consistent with a reduced ability to modulate negative emotion.4.1There are several limitations to the study. First, some of our BC participants underwent the fMRI assessment after undergoing their surgical treatment. Although our secondary analysis showed that including surgery timing as a covariate did not change the main finding, future studies would benefit from assessing emotion reactivity/regulation in BC patients before they experienced their surgical treatment. Second, a small number of the BC patients were receiving chemotherapy at the time of their fMRI assessments. Although this happened in both the BMX and the non‐BMX groups, we cannot exclude the possibility that undergoing chemotherapy could have influenced participants' emotion reactivity and regulation. Third, we used BC‐related pictures to induce negative emotion but not necessarily cancer‐related anxiety in particular. Also, pictures presented in a lab setting may not fully simulate real‐life experience of cancer‐related negative emotions, although they likely stimulate affect related to the experience of women with breast cancer. Future research is encouraged to employ other paradigms to assess real‐life cancer‐related anxiety directly.5Employing an fMRI experiment, we found that BC patients experienced greater negative emotion in response to BC‐related stimuli than their healthy counterparts. More importantly, those who chose BMX had less activation in prefrontal regions while regulating BC‐related negative emotion than did those who chose more conservative non‐BMX treatments. These findings offer neuropsychological evidence that difficulty in managing anxiety related to cancer is a crucial factor in surgical treatment decision‐making. Difficulty in emotion regulation may serve as an intervention target with the goal of improving breast cancer treatment decision‐making by enhancing the ability to regulate cancer‐related anxiety.Jin‐Xiao Zhang: Formal analysis (lead); methodology ; visualization (lead); writing – original draft (lead); writing – review and editing . Allison Kurian: Methodology ; writing – review and editing . Booil Jo: Methodology ; writing – review and editing . Bita Nouriani: Data curation ; methodology ; writing – review and editing . Eric Neri: Data curation ; methodology ; writing – review and editing . James Gross: Conceptualization ; methodology ; writing – review and editing . David Spiegel: Conceptualization ; funding acquisition (lead); methodology ; writing – original draft (supporting); writing – review and editing .The authors declare no potential conflicts of interest.Data S1.Click here for additional data file.


Anemia is associated with increased rates of heart failure (HF)‐related mortality and hospitalization. No studies have focused on the association between the red blood cell (RBC) count and the prognosis of patients with HF with mildly reduced left ventricular ejection fraction (HFmrEF). We retrospectively analyzed the effect of the RBC count on outcome events in patients with HFmrEF.We investigated the association of the RBC count with outcome events in 1691 patients with HFmrEF  in Xiangtan Central Hospital. Using Cox proportional hazards models, the RBC count was assessed as both a continuous and categorical variable.12 cell/L increase in the RBC count was associated with a 28% lower risk of cardiovascular death in men and a 43% lower risk in women. Patients with low RBC counts had a 0.5‐fold higher risk of cardiovascular death than those with normal RBC counts. The hazard ratio for men was 1.42 , and the hazard ratio for women was 1.79 (95% CI: 1.20–2.67). The RBC count was not significantly associated with the composite endpoint of cardiovascular death and HF readmission (cardiovascular events) (p > .05).During follow‐up (median: 33 months), cardiovascular death occurred in 168 patients (114 men and 54 women). After adjusting for established risk factors, each 1.0 × 10A decreased RBC count is associated with increased cardiovascular mortality in patients with HFmrEF. Correcting a low RBC count might potentially reduce the risk of cardiovascular death in patients with HFmrEF. 12 cell/L increase in the RBC count was associated with a 28% lower risk of cardiovascular death in men and a 43% lower risk in women.We investigated the association of the RBC count with outcome events in 1691 patients with HFmrEF . During follow‐up (median: 33 months), cardiovascular death occurred in 168 patients (114 men and 54 women). A decreased RBC count is associated with increased cardiovascular mortality in patients with HFmrEF. Each 1.0 × 10 Integral to oxygen transportation, diminished counts may indicate conditions such as anemia, while elevated counts could signal disorders like polycythemia vera.Hemoglobin: This intracellular protein is responsible for transporting oxygen to tissues and returning carbon dioxide to the lungs. Hemoglobin concentrations, quantified in grams per deciliter, can reflect various health conditions, with lower levels potentially indicative of anemia and higher levels suggestive of polycythemia vera or chronic hypoxia.Red blood cell specific volume (RBCSV): This parameter represents the volume fraction of RBCs in the blood, also called hematocrit. Normal hematocrit ranges differ by sex. Deviations from normal ranges can indicate various conditions—low levels may suggest anemia, and high levels could indicate dehydration or other disorders.2.212/L and that for women is 3.5 to 5.0 × 1012/L. After excluding patients with elevated RBC counts (>5.5 × 1012/L in men and >5.0 × 1012/L in women), the patients were divided into those with reduced RBC counts (<4.0 × 1012/L in men and <3.5 × 1012/L in women) and normal RBC counts (≥4.0 × 1012/L in men and ≥3.5 × 1012/L in women). We chose to evaluate RBC count as our primary parameter rather than hemoglobin or RBC specific volume (hematocrit), because RBC count is less affected by hypoxia, dehydration, and volume status, which can be confounding clinical factors within this patient population.According to the definition of the standard RBC count in routine blood testing at our hospital, the reference range for men is 4.0 to 5.5 × 102.3ClinicalTrials.gov number NCT05240118). The OUDI‐HF study included 1691 patients with HFmrEF who were admitted to our hospital from January 1, 2015 to August 31, 2020. The inclusion criteria were HF with an LVEF of 41%–49% and a New York Heart Association HF score of II–IV. The exclusion criteria were malignancies or other non‐cardiac diseases with expected survival of less than 1 year. After excluding 52 patients with increased RBC counts, 1149 patients with normal RBC counts and 490 patients with decreased RBC counts were included in the study  and conformed to the principles outlined in the Declaration of Helsinki.2.4Demographic and procedural data were collected from the patients’ hospital charts or the hospital database. All study participants were followed up on August 31, 2021. A panel of seven experienced physicians reviewed suspected cardiovascular (CV) events by examining the information obtained from hospital records and follow‐ups, including clinical telephone interviews and community visits. The primary outcome of interest was CV death after discharge, and the secondary outcome was the composite of CV death and HF readmissions (CV events). CV death was defined as death from any CV mechanism, including acute myocardial infarction, sudden cardiac death, HF, stroke, CV surgery, CV hemorrhage, and other CV causes.2.512 cell/L increase) and as a categorical variable. We adjusted for the following baseline covariates: age, body mass index，smoking status, alcohol use, hypertension, hyperlipidemia, diabetes, coronary heart disease, atrial fibrillation, previous stroke, chronic obstructive pulmonary disease, renal insufficiency, New York Heart Association functional class, myocardial infarction, percutaneous coronary intervention, uric acid concentration, estimated glomerular filtration rate, potassium concentration and LVEF.We used Cox proportional hazards regression models stratified by cohort to examine the association of the RBC count with the rate of outcome events. We performed a sex‐stratified analysis. The RBC count was assessed as a continuous variable  and EmpowerStats .Clinical characteristics were compared between the groups using the 33.1n = 1067). The patients’ mean age was 68.5 ± 12.3 years, their mean body mass index was 25.1 ± 4.1 kg/m2, their mean LVEF was 44.4% ± 2.7%, and more than half of the patients had been hospitalized for myocardial infarction (51.8%) , not female sex   had CV deaths and 953 patients (618 men and 335 women) had CV events. A reduced RBC count was associated with a doubling of CV mortality compared with a normal RBC count . The risk of CV death was reduced by 49% in women  and by 37% in men  (Table p < .0001), including a 111% higher risk in women  and 86% higher risk in men  (Table p > .05) . The risk of CV death was reduced by 43% in women  and by 28% in men  (Table p = .0005), including a 79% higher risk in women  and a 42% higher risk in men   Table . Howeverp < .05). The two groups had similar gender, coronary heart disease, atrial fibrillation, previous stroke, and LVEF (p > .05). After propensity score matching, both groups were well‐matched in terms of parameters. All p values had been greater than .05, indicating no difference between the two groups, and were comparable. Table p = .018). However, the incidence of CV events was not significantly correlated between the two groups .Table p < .0001) and 0.99 in the Adjust II model . Similarly, each unit increase in RBCSV yielded an HR of 0.94 for males and 0.96 for females in the non‐adjusted model , 0.96 for males and 0.94 for females in the Adjust I model (p < .0001 for both), and 0.97 for males and 0.95 for females in the Adjust II model . These findings underscore the clinical significance of Hemoglobin and RBCSV as potential indicators for prognosis and management in patients with HFmrEF.Table 4This study showed that reduced RBC counts were associated with increased CV mortality in patients with HFmrEF after adjusting for covariates. However, there was little correlation between changes in RBC counts and the incidence of CV events in patients with HFmrEF.Anemia is common in patients with HFrEF or HFpEF and is associated with a poorer long‐term prognosis.Furthermore, anemia and CKD often coexist in patients with HF. One study showed that although anemia doubled the risk of death in patients with HF, the adjusted risk of death was further increased by 1.5‐fold in the presence of CKD.Severe anemia may progress to HF syndrome, which disappears when the anemia is corrected.Furthermore, studies have also shown that an increased hemoglobin concentration in patients with HFrEF increases systemic vascular resistance and decreases the LVEF.4.1This study had several limitations. First, this was designed as a retrospective study to minimize bias in patient selection; however, unobserved confounders remained. Second, we exclusively recruited patients from an isolated population at a local heart center in China, and the study population thereby lacked adequate diversity to justify the uniformity of the findings. Finally, because of the study's retrospective nature, some test results could not collected; the most important of these was the absence of data on the B‐type natriuretic peptide concentration.5Our findings suggest that reduced RBC counts are associated with increased CV mortality in patients with HFmrEF. Women with HFmrEF are more sensitive to changes in RBC counts than are men. Compared with men with HFmrEF, women with reduced RBC counts had a greater risk of CV death, and an increased RBC count was associated with a more significant reduction in the risk of CV death in women. Correcting a decreased RBC count can potentially reduce the risk of CV death in patients with HFmrEF. However, there is no clear conclusion regarding the need for medication to correct anemia in patients with HFmrEF. Further research is needed to understand the risk associated with anemia in patients with HFmrEF. A better understanding of risk factors for anemia in patients with HFmrEF may help to develop strategies to improve outcomes of patients with this severe disease.Zhican Liu, Yunlong Zhu, and Lingling Zhang: main authors of the study, established the idea to study the heart failure with mildly reduced ejection fraction in Chinese population. Writing main ideas for this research, main results and discussion of the findings. Zhican Liu: was a major contributor in writing the manuscript. Mingxin Wu, Haobo Huang, Ke Peng, and Wenjiao Zhao: interpreted statistical analysis and conducted multivariate analysis to prove the main findings of this project. Sihao Chen, Xin Peng, Na Li, Hui Zhang, Yuying Zhou, Jie Fan and Yiqun Peng: data collected and follow‐up. Jianping Zeng: corresponding author of the study, contributed on editing this manuscript and giving advice for the main authors to organise the manuscript and ideas of the project.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.


Kawasaki disease (KD) is a prevalent form of systemic vasculitis that can damage various organs and systems in children. Typical KD is not difficult to diagnose in clinical practice. In recent years, it has been shown that an increasing number of children do not satisfy the diagnostic criteria for typical KD. This condition is known as incomplete KD (IKD). It is challenging to promptly diagnose and treat such children in clinical practice.A 10‐year‐old girl was admitted to the hospital due to fever and abdominal pain. She presented with shock symptoms. An enhanced abdominal computed tomography scan revealed intestinal pneumatosis, effusion, and gallbladder enlargement, indicating intestinal obstruction. Due to the poor outcome following an emergency laparoscopic cholecystectomy, IKD was suspected. A 3‐month‐old male pediatric patient was admitted to the hospital due to a fever. Patchy, congestive rashes formed on the patient's body as KD progressed. IKD was suspected based on the clinical signs of fever, rash, and hyperemia of the lips. The two patients were then treated with human immunoglobulin and aspirin treatment. The prognosis for the two children was favorable following prompt treatment.Due to the fact that IKD is frequently misdiagnosed, it is vital to (1) improve the patient prognosis for the early identification of children with KD with prolonged fever and anti‐infection failure as the initial manifestation and (2) perform timely diagnosis and comprehensive treatment. Kawasaki disease (KD) has various clinical manifestations, which symptoms are severe and often atypical, and it is complicated with multiple organ damage. In the acute phase of KD, there may be obvious gallbladder enlargement, the key to avoiding surgery is accurate diagnosis and prompt anti‐inflammatory treatment by clinicians, which reduces the rate of misdiagnosis and improves prognosis. In patients with hypotension, the possibility of KDSS should be considered, and immediate echocardiography and dynamic evaluation should be conducted. It may occur in children aged <5 years throughout the entire year. The disease etiology and pathogenesis are currently unclear. In addition, KD is the main factor inducing coronary artery dilation and coronary aneurysm, and it has become the main cause of acquired heart disease in children.22.1A 10‐year‐old female pediatric patient was admitted to the hospital due to 3 days of fever and abdominal pain. Before consultation and hospitalization, the patient was required to undergo new coronavirus nucleic acid testing to exclude out SARS‐CoV‐2‐related multisystem inflammatory syndrome in children. The result of the test was negative. The child had a fever (with a peak of 37.7°C) 3 days before admission, accompanied by nausea, poor appetite, and abdominal pain (mostly around the umbilicus), which was alleviated by massage. The patient's symptoms were not relived by infusion of the fever‐treating medications at the community clinic, and the fever remained, with a peak of 39.5°C. The patient was consequently transported to our hospital for diagnosis and treatment. Before the onset of the disease, the patient was in good health and had no unusual personal or family medical history. The test results are summarized in Table Physical examination on admission: Clear mind, poor mental status, acute face, mild yellow staining of skin and mucosa all over the body, no rash or bleeding spots, no local swelling, slightly yellow sclera, slight conjunctival congestion, a 3 × 3 cm lymph node palpable on the right neck with partial fusion and obvious tenderness, mild tenderness in the entire abdomen (obvious in the right upper abdomen), no rebound pain, and no obvious abnormalities in other physical examination items.9/L, hemoglobin (Hb) = 106 g/L, platelet count (PLT) = 341 × 109/L, NEUT% = 95.6%, LYM% = 0.7%, CRP = 81.58 mg/L, ESR = 55.00 mm/h, and plateletcrit (PCT) = 4.10 ng/mL; (2) blood biochemistry: Na+ = 131.8 mmol/L, alanine transaminase = 83.50 U/L, aspartate aminotransferase = 68.50 U/L, glutamyl transpeptidase = 210.00 U/L, total protein = 60.60 g/L, albumin = 28.30 g/L, globulin = 32.30 g/L, total bilirubin = 77.20 μmol/L, direct bilirubin = 71.50 μmol/L, indirect bilirubin = 5.7 μmol/L, N‐terminal brain natriuretic peptide (NT‐BNP) = 220.6 pg/mL, blood amylase = 115.00 U/L, and lipase = 196.80 U/L; (3) urine routine examination: urobilinogen = 3+, bilirubin = 3+, and protein = 1+; (4) stool routine examination: normal; (5) emergency abdominal color ultrasonography after admission: segmental intestinal wall thickening of the left abdomen; and (6) color Doppler echocardiography: no obvious abnormalities.Laboratory examination on admission: (1) Blood routine examination: white blood cell (WBC) count = 23.3 × 10On the fourth day of the course of the disease, the patient was still suffering from high fever, with blood pressure (BP) measured as low as 55/35 mmHg, obvious abdominal pain, and aggravated yellow skin staining; after consultation with the Department of Pediatric Surgery, this was considered to possibly be acute obstructive jaundice and peritonitis, and septic shock could not be ruled out. Hence, the patient was transferred to the PICU.After transfer to the PICU, volume expansion, booster, and cardiac function improvement for circulation improvement were administered to correct shock. Meropenem+linezolid+ornidazole were used for the anti‐infection treatment, and a low dose of prednisolone injection was administered for anti‐inflammatory treatment.On the sixth day of the course of the disease, the patient's BP was relatively controlled; however, the high (remittent) fever continued, and the body temperature control using oral antipyretic (four times/day) did not show good results.(1) Physical examination: yellow sclera, conjunctival congestion, bilateral cervical lymph node enlargement, obvious tenderness, abdominal muscle tension, tenderness, rebound pain (+), obvious tenderness in the right upper abdomen, and Murphy's sign (+); (2) bedside abdominal color ultrasound: abdominal effusion, increased gallbladder volume, and deposits ; (3) bedside electrocardiogram (ECG): normal; and (4) chest and abdominal computed tomography (CT): abdominal and pelvic effusion, increased gallbladder volume, and pleural effusion on both sides.On the seventh day of the course of the disease, an enhanced abdominal CT showed intestinal pneumatosis and effusion; the gallbladder volume was significantly increased, and the spleen was enlarged. This was considered to be intestinal obstruction.On the eighth day of the course of the disease, emergency laparoscopic cholecystectomy was performed; however, the patient still had intermittent fever and an unstable BP, with no bacteria growth found in the multiple blood cultures. Based on the patient's history and clinical manifestations, a diagnosis of IKD was suspected.On the 10th day of the course of the disease, immunoglobulin (IVIG) (2 g/kg) was injected intravenously. After 2 days, the patient's body temperature and BP returned to normal, jaundice subsided, and the patient's spirits improved. On the 12th day, fingertip and perianal skin exfoliation occurred, and the patient took low‐dose aspirin enteric‐coated tablets and dipyridamole tablets orally.On the 15th day of the course of the disease, the color Doppler echocardiography showed a widening in the openings of the left and right coronary arteries and the inner diameter of the left anterior descending branch; a final diagnosis of KD was made. The patient was followed‐up with at 1, 3, and 6 months as well as at 1 and 1.5 years after discharge; the patient's coronary artery diameter was normal and without recurrence of KD.2.2A male pediatric patient aged 3 months was admitted to the hospital due to a fever and poor mental status lasting for 5 days as well as diarrhea and abdominal distension for 2 days. The test results were concluded in Table Physical examination on admission: body temperature = 37.4°C, pulse = 140 bpm, respiration = 30 bpm, BP = 99/51 mmHg, weight = 7.0 kg, head circumference = 42.5 cm, clear mind, poor mental state, and acute face. There was no yellow staining of the skin and mucosa over the entire body, no rash or bleeding spots, and no palpable enlargement of superficial lymph nodes in the neck, behind the ears, in the bilateral axilla, and in the inguinal area. The anterior fontanel was approx. 2 × 2 cm in size, flat, and soft.The patient had an amputated palm of the left hand, ruddy lips without chapping, congestion of the pharyngeal cavity, a high palatal arch, soft neck, distension of the abdomen, no palpebral edema, no conjunctival congestion, no waxberry tongue, unclear palpation of the liver and spleen, drum sound upon percussion, bowel sound occurring five times/min, bilateral scrotal swelling, perianal flushing and ulceration, no desquamation, no swelling at the fingertips and toe ends, no flushing at the palm, and no abnormalities found in other physical examination items.The patient was inoculated with the diphtheria vaccine 3 days before the onset of the disease and was allergic to cephalosporins and amoxicillin injections. The patient had been previously healthy, with no special personal or family medical history. Preliminary diagnosis: 1. Fever and poor spirits when examined: (1) Sepsis? (2) Intracranial infection? 2. Gastrointestinal dysfunction auxiliary examination on admission, diagnosis, and treatment.9/L, Hb = 94 g/L, PLT = 156 × 109/L, NEUT% = 89.0%, LYM% = 9.5%, CRP = 24.9 mg/L, ESR = 31 mm/h, PCT = 0.172 ng/mL, and interleukin‐6 (IL‐6) = 26 pg/mL; (2) blood biochemistry: alanine transaminase = 404 U/L, aspartate aminotransferase = 155 U/L, glutamyl transpeptidase = 542 U/L, total protein = 53.4 g/L, albumin = 31.3 g/L, globulin = 22.1 g/L, coagulation four indices = normal, and NT‐BNP = 780.01 pg/mL; (3) urine routine examination: normal; (4) stool routine examination: loose yellow stools, no red blood cells, WBCs, and phagocytes observed under the microscope; (5) occult blood test: (−). Shigella and Salmonella were not detected in the stool culture.(1) Blood routine examination: WBC count = 25.94 × 10Lumbar puncture was performed, as intracranial infection could not be ruled out at first. A routine biochemistry of cerebrospinal fluid showed normal results, and adenosine deaminase, acid‐fast staining, shaking‐off liquid, and ink staining were all negative. The culture was negative. After admission, meropenem injection was given for anti‐infection. Liver protection, fluid rehydration, intestinal probiotics, and other symptomatic supportive treatments were provided.On the afternoon of the sixth day, patchy congestive rashes appeared on the patient's body (some with an annular shape); the patient exhibited labial mucosa congestion without chapping, relieved abdominal distension compared with that on admission, and a quasi‐circular cystic mass (approx. 5 cm in diameter) palpable in the upper abdomen (liver) 1 cm beneath the rib. The spleen was not palpable beneath the rib; upon dermatological consultation, this was considered to be caused by a drug allergy. Hence, a dexamethasone injection of 0.2 mg/kg × 3 day was given intravenously.9/L, Hb = 84 g/L, PLT = 98×109/L, NEUT% = 81.9%, LYM% = 13.8%, CRP = 148.7 mg/L, PCT = 0.334 ng/mL, and IL‐6 = 90.9 pg/mL; these values were significantly higher than before.On the seventh day, the patient still had recurrent fever (with a peak of 40°C), a poor mental state, and diarrhea (the nature remained the same as before). Blood routine examination was performed again: WBC count = 40.66 × 10Empirical anti‐infection therapy with vancomycin injection was administered. On the eighth day, the patient still had recurrent fever, and the physical examination showed mild nonpitting edema on the back of hands and lower limbs. Based on the clinical manifestations of fever, rash, and hyperemia of the oral lips, a diagnosis of IKD was suspected; the patient was immediately given human IVIG injections (2 g/kg) for 3 days via intravenous infusion and was orally administered with high‐dose aspirin enteric‐coated tablets.On the ninth day, the patient's body temperature was normal, his mental status improved, abdominal distention relieved, stool frequency decreased, and scrotal swelling and perianal ulceration improved slightly; however, the rash did not subside. On the 10th day, the blood routine examination indices and three inflammation indices all decreased significantly, the WBC count began to return to normal, and no bacterial growth was found in multiple blood cultures; hence, antibiotic therapy was discontinued.(1) Head magnetic resonance imaging: 1. The bilateral frontotemporal subarachnoid space was slightly widened; 2. the cerebral cortex was slightly thinner than normal in both cerebral hemispheres; and 3. the white matter T2WI signals increased in both cerebral hemispheres ; (2) chest CT: inflammation of both lungs, with local pleural thickening on the left lung.The bedside ECG showed sinus tachycardia, and the bedside cardiac and abdominal ultrasonography showed no obvious abnormalities in heart structure and function, no coronary dilation, and an enlarged gallbladder ; this was considered to be KD complicated with gallbladder enlargement (effusion). The congenital choledochal cyst should also be taken into consideration. As the child was very young, magnetic resonance cholangiography was performed; the results indicated a significantly increased gallbladder volume, dilated cystic duct, slightly widened intrahepatic bile duct, widened upper segment of the hepatic common duct, and a presence of small round abnormal signals in the hepatic anterior lobe and hepatic right posterior lobe , the gallbladder volume was increased .As reported in literature, methotrexate, cyclosporin A, or the combined therapy of biological agents should be used in the treatment of refractory KD or its complication with shock or even macrophage activation syndrome; plasma exchange should be performed as needed, and extracorporeal membrane oxygenation should be provided as life support therapy for critically ill patients with severe cardiopulmonary failure on the basis of conventional treatment.In addition to the involvement of the cardiovascular system, extracardiac symptoms include arthritis, jaundice, hepatomegaly, and cystic effusion; neonates may experience convulsions, irritability, and diarrhea.In the present report, both patients had KD complicated with gallbladder enlargement, with Case 1 undergoing gallbladder removal surgery due to apparent abdominal symptoms and cholestasis. The ultrasonography conducted 6 weeks following the discharge of the Case 2 patient revealed that the morphology of the gallbladder had reverted to normal, ruling out the likelihood of congenital gallbladder or biliary tract lesions.Gatterre et al.In conclusion, it is crucial to identify the risk of KD in children with fever and acute abdomen as the primary symptoms, accompanied by multiple organ damage, as early as possible. In the acute phase of KD, there may be obvious gallbladder enlargement, which can be confirmed by abdominal color ultrasonography and magnetic resonance cholangiopancreatography. With standard treatment for KD, the enlarged gallbladder can return to its normal size. In patients with hypotension, the possibility of KDSS should be considered, and immediate echocardiography and dynamic evaluation should be conducted. The key to avoiding surgery is accurate diagnosis and prompt anti‐inflammatory treatment by clinicians, which reduces the rate of misdiagnosis and improves prognosis. Our study shows that it is vital to improve the patient prognosis for the early identification of children with KD with prolonged fever and anti‐infection failure as the initial manifestation and perform timely diagnosis and comprehensive treatment.Conception and design of the research: Hui‐Qiong Liu. Acquisition of data: Xiao‐Ya Shi and Man‐Man Mu. Analysis and interpretation of the data: Pei‐Sheng Jia and Yu‐Feng Huo. Statistical analysis: Yu‐Feng Huo and Lei Xie. Writing of the manuscript: Hui‐Qiong Liu. Critical revision of the manuscript for intellectual content: Huai‐Li Wang. All authors read and approved the final draft.The authors declare no conflict of interest.This study was conducted with approval from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. This study was conducted in accordance with the declaration of Helsinki. Written informed consent was obtained from the guardians of the pediatric patients.


The severe forms of influenza infection requiring intensive care unit (ICU) admission remain a medical challenge due to its high mortality. New H1N1 strains were hypothesized to increase mortality. The studies below represent a large series focusing on ICU‐admitted influenza patients over the last decade with an emphasis on factors related to death.A retrospective study of patients admitted in ICU for influenza infection over the 2010–2019 period in Réunion Island (a French overseas territory) was conducted. Demographic data, underlying conditions, and therapeutic management were recorded. A univariate analysis was performed to assess factors related to ICU mortality.Three hundred and fifty adult patients were analyzed. Overall mortality was 25.1%. Factors related to higher mortality were found to be patient age >65, cancer history, need for intubation, early intubation within 48 h after admission, invasive mechanical ventilation (MV), acute respiratory distress syndrome (ARDS), vaso‐support drugs, extracorporal oxygenation by membrane (ECMO), dialysis, bacterial coinfection, leucopenia, anemia, and thrombopenia. History of asthma and oseltamivir therapy were correlated with a lower mortality. H1N1 did not impact mortality.Patient's underlying conditions influence hospital admission and secondary ICU admission but were not found to impact ICU mortality except in patients age >65, history of cancer, and bacterial coinfections. Pulmonary involvement was often present, required MV, and often evolved toward ARDS. ICU mortality was strongly related to ARDS severity. We recommend rapid ICU admission of patients with influenza‐related pneumonia, management of bacterial coinfection, and early administration of oseltamivir. It benefits from a survey system to monitor influenza cases. Influenza has a seasonal incidence that is less pronounced than in temperate countries such as mainland France.In this retrospective analysis, the severe cases of influenza infections admitted to the ICU in Réunion Island from 2010 to2019 and the factors related to poor outcomes and death were described and analyzed.22.1Patients were admitted to the ICU of the two tertiary teaching hospitals in Réunion Island (St‐Denis and St‐Pierre). Reunion Island has a population of about 850, 000 people. The medical system and accessibility to medical care is not different to that of mainland France.The data of 379 patients admitted in ICU between January 2010 and December 2019 were retrospectively analyzed. Twenty‐seven cases were excluded because of age <18 years. Two other cases were excluded: one for false diagnosis and one for the lack of a definite diagnosis. A total of 350 adult patients met with definite diagnosis assessed by a positive real‐time polymerase chain reaction (RT‐PCR) nasopharyngeal swab or a tracheal aspirate were included in the analysis.ICU admission demographic data, underlying conditions such as diabetes mellitus, asthma, chronic obstructive pulmonary disease (COPD), obstructive sleep apnea (OSA), chronic renal failure, hypertension, coronary diseases, liver diseases, pregnancy, obesity, prior illness or treatment , alcohol abuse and smoking habits, standard biological variables, and information on vaccine status within the current year were collected.Simplified acute physiologic score II (SAPS II) was calculated at admission.The need for invasive mechanical ventilation (MV), administration of vasopressor drugs within the first 48 h after admission, dialysis requirement in the case of acute renal failure, length of MV, and intubation were recorded during ICU stay. The use of antiviral for treatment of influenza before and during ICU stay was recorded.Acute respiratory distress syndrome (ARDS) was defined according to guidelines.4/mL in bronchoalveolar lavage (BAL) or >103/mL in protected distal aspiration.Concomitant bacterial infections were defined if a significant predominant strain was found with a cut point >102.2p value < 0.05 was considered statistically significant.Prognostic factors for mortality were analyzed using the chi‐squared test with Fisher's exact test correction, if needed. A The relative risk (RR) for outcome was defined according to the following variables: age (>65 or <65 years), obesity (BMI > 30 or <30), COPD (Y/N), asthma (Y/N), OSA (Y/N), diabetes mellitus (Y/N), hypertension (Y/N), coronary disease (Y/N), liver cirrhosis (Y/N), chronic renal failure with dialysis (Y/N), alcohol abuse (>80 g/day or <80 g/day), tobacco (active or past Y/N), pregnancy for females under 41 years of age (Y/N), history of cancer (Y/N), immunosuppressive drugs including oral steroids >10 mg per day (Y/N), SPAS II score (>40 or <40), intubation during stay (Y/N), need of MV within first 48 h after admission (Y/N), MV during stay (>10 days or <10 days), vasopressor drugs (Y/N), ARDS (Y/N), ECMO (Y/N), H1N1 strains versus others (Y/N), concomitant bacterial infections (Y/N), pneumococcal strains versus all (Y/N), staphylococcus strains versus all (Y/N), oseltamivir administration (Y/N), polynuclear cells (<2.5 G/L or >2.5 G/L), hemoglobin (<8 g/dL or >8 g/dL), platelets (<50 G/L or >50 G/L), and C‐reactive protein (>100 mg/L or <100 mg/L).The study received approval from the institutional review board (IRB) and the Comité National de l'informatique et des Libertés (CNIL).3Among 350 patients, 205 (58.5%) were male. The mean of age was 58.9 +/− 15.2 years (19–88). The patient's characteristics, underlying conditions, and prior illnesses are presented in Table The mean SAPS II score was 43.8 +/− 20.3. The mean duration of ICU stay was 12.3 +/− 14.3 days (1–122).A total of 204 (58%) patients required MV, with 185 of them intubated within the first 48 h of ICU stay. The severity of patients was further documented by the use of vaso‐drug support (51.5%), dialysis (19.1%), and ECMO (7.7%) and by the onset of ARDS (40.2%).ARDS was recorded in 134 patients according to Berlin guidelines. The ARDS severity grade for 107 patients was recorded. Grade III (ratio of PaO2/FIO2 < 100) for 86 (80.4%) and grade II (ratio of PaO2/FIO2 < 200) for 17 (15.9%) patients, respectively, were found.H1N1 strains were found in 153 (43.7%) cases and were the predominant influenza type followed by B 68 (19.4%), H3Nx 66 (18.9%), and non‐defined A strains 63 (18%).p < 0.04). Data revealed pneumococcal stains in 28 (24.1%) cases and Staphylococcus aureus in 40 (34.5%) cases. Staphylococcus and streptococcus coinfection was not correlated with mortality. Other agents are listed as follows: wild strains Pseudomonas aeruginosa 9 (7.8%), Streptococcus species 7 (6%), Klebsiella pneumoniae 6 (5.2%), Moraxella catarrhalis 4 (3.4%), Enterobacter 3 (2.6%), Haemophilus influenzae 3 (2.6%), Legionella pneumophila 3 (2.6%), and others 13 (11.2%).Bacterial coinfections were observed in 116 patients and were correlated with mortality (p < 0.29).Oseltamivir was the unique antiviral drug used in this study and was administrated in 229 (66.4%) inpatients, according to hospital's attending physician's judgment. Only 16/96 (16.6%) patients were vaccinated against influenza with no impact on mortality .All conditions and care are presented in Table The univariate analysis recorded variables related to death are listed in Table Asthma and oseltamivir treatment were found to be a protective factor.4This study focuses on adult patients admitted to the ICU for severe confirmed influenza infection. The objectives of the study were, first, to describe the severity of the disease, its potential complications, and evolution during ICU stay and, second, to analyze the impact of demographics, underlying conditions, influenza vaccination and treatment, bacterial coinfection, severity, and ICU management of influenza on ICU mortality and outcome.The univariate analysis found risk factors of ICU mortality to be only patients over age 65 years and patients with a previous history of cancer and bacterial coinfections.Underlying conditions such as diabetes mellitus, smoking, COPD, cystic fibrosis, coronary diseases, overweight, hypertension, renal insufficiency, history of cancer, immunosuppression status, pregnancy, and age over 65 years are considered as recognized risk factors for developing a severe influenza disease.The characteristics of the population in Réunion Island is slightly different from those of mainland France and Western Europe. Diabetes mellitus is more frequent in Réunion Island's population with a 17.7% rate that is two times higher than in mainland France. Diabetes mellitus complications as renal insufficiency, and coronary diseases are of major concerns. Obesity is frequent as 35% of the entire population is overweight (17% in France).In the present study, three factors increasing ICU mortality were found. Age >65 years was statistically related with mortality. This result is consistent with the literature. Only the Spanish outbreak in 1918 showed an abnormal percentage of deaths in young adults.History of cancer was the other factor related with fatality in the study and was recorded in 40 patients (11.5% of study population). No information on the type of cancer was available. Fifteen patients had ongoing chemotherapy or radiotherapy at ICU admission. Due to a small number of cases, immunosuppression drug therapy (16 patients) did not reach statistical significance, opposing recent data.Few studies focused on specific ICU populations of severe influenza, and some of them included a small patient cohort. Beumer et al analyzed 45 patients admitted in ICU for severe influenza.On the other hand, asthma was found as a protective factor. The mean age of asthmatics was 55.9 years of age and was not different from the overall study population. Twenty‐four of them were over 65 years of age (38%). In other studies, asthma was considered as a pejorative factor for hospitalization.The impact of therapy based on inhaled corticosteroids (ICS) widely used in asthma patients could be the reason for our finding. Recently, attention has been drawn on a potential protective effect of ICS in asthmatic patients with SARS COV‐2 infection,Considering all the usual underlying conditions described in the study, the analysis of the literature confirmed that these factors are generally related with an increase for hospitalization in medical wards and secondary ICU admissions.Information about influenza vaccination was only available in 96 (27%) of patients. Among these patients, only 16 were vaccinated within the previous 12 months. Due to this small number, statistical analysis did not show any benefit. From a public health standpoint, influenza vaccination is therefore highly recommended in these populations.The primary objective of this large study was to analyze patient's severity at the time of ICU admission and its evolution during the ICU stay.We observed a crude mortality in the ICU of 25.1%. There are few studies analyzing the mortality in ICU‐admitted patients with severe influenza infection.An old meta‐analysis found a wide range of crude mortality from 14% to 71%.p < 0.0001, RR 4.81). Another indicator was the need of invasive MV that reached 58% of patients. The majority of patients initially had respiratory failure as demonstrated by 81 (23.1%) of them requiring noninvasive MV. Only 65 (18.5%) patients received only oxygen. An important rate of ARDS cases (40.2%) was observed with ECMO requirement for 27 patients.The severity of patients at the time of admission in the ICU was described. The SAPS II score is a valid indicator of severity in ICU. A mean SPAS II score of 43.8 +/− 20.3 was considered severe and strongly related with ICU's poor outcome. In the univariate analysis, observations revealed a strong link with mortality when patients had a SAPS II score over 40  of MV requirement,p < 0.0001). The study of Abaziou et al focused on ECMO benefit for severe influenza.ECMO is considered as the ultimate technique for refractory ARDS. Twenty‐seven patients required ECMO, and 21 (77%) died  strain on increase in hospitalization and death was recorded during the 2009 pandemic.Oseltamivir was the unique anti‐influenza drug available in the study and was administrated at hospital or ICU admission in 229 (66.4%) of patients with a benefit in mortality reduction. The favorable impact of oseltamivir in influenza was previously described in the general populationS. aureus or pneumococcus pneumoniae strains was not statistically correlated with mortality. Considering these results, it is advised to diagnose the presence of bacterial coinfections at admission in hospital and in the ICU to avoid delaying the introduction of effective antibiotic therapy.Bacterial coinfection is frequent in patients with influenza and was reported between 11% and 35%.As the study was of a retrospective nature, the limitations due to data collection are acknowledged. A lack of information is important for BMI and vaccination status. In life‐threatening situation, information on height and weight may not be systematically recorded. History of influenza vaccination is sometimes difficult to assess in ICU due to patient's condition and family questioning. For chronic diseases, compliance to daily therapy is impossible to assess. Patient's underlying conditions may be imprecise and may explain discrepancies in the results compared to literature. During hospitalization, the exact timing of starting oseltamivir was difficult to record, but the majority of patients were rapidly admitted to the ICU, and oseltamivir was early administered.5In summary, a large cohort of ICU‐admitted patients for severe influenza disease over 10 years was analyzed. The overall mortality was 25.1%. The predominant clinical presentation was a specific pulmonary involvement rapidly requiring MV and often evolving toward ARDS. Timely ICU admission for patients with severe influenza is recommended and as early as possible in the case of respiratory failure in order to enable effective intensive care.Patient's underlying conditions are important to consider. Most of them are factors that influence hospital admission and secondary ICU admission and were not found to impact specific ICU mortality. In the study of ICU‐admitted patients presenting with severe influenza, patient age >65 years, history of cancer, and bacterial coinfections were identified as the three pejorative prognostic factors. Influenza vaccination remains important to promote in the community for elderly people and patients with comorbidities.Victor Verdier collected the data and wrote the manuscript. Fabrice Paganin analyzed the data and wrote the manuscript. Francois Lilienthal, Arnaud Desvergez, Arnaud Winer, and Virgile Gazaille wrote the manuscript. All the authors contributed equally.All the authors declare that they have no conflict of interest.https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/irv.13168.The peer review history for this article is available at We declare the study to the French Comite national de l'informatique et des libertés (CNIL) and received approval on May 15, 2018 (Number 2181973 V0). All data were kept anonymous. Not applicable for this section.


Pre‐exposure prophylaxis (PrEP) has been available for young people for over a decade, yet only ∼15% of young people in the United States with indications for PrEP have a prescription for it. Next‐generation PrEP modalities may address some of the challenges of daily oral PrEP. However, preferences for these products are unknown.From October 2020 to June 2021, we conducted an online survey of 737 cisgender, young men who have sex with men (age 15–24 years) without HIV across the United States who reported same‐sex attraction or consensual sex with another man in the past 6 months. Participants completed a conjoint experiment comparing daily oral pills, event‐driven oral pills, event‐driven rectal douches, intramuscular injections, intravenous broadly neutralizing antibody (bnAb) infusions and subcutaneous implants. Participants ranked the products from most to least preferred. Exploded logit models examined the association between ranked preferences of PrEP modalities and socio‐demographic and behavioural characteristics.p = 0.058), injectable , implant , bnAb infusions  and rectal douches . There were differences in PrEP preferences across age, insurance status, sexual behaviour, PrEP use history, HIV and sexually transmitted infection (STI) testing history, and STI diagnoses (omnibus tests: p < 0.05). Participants also provided reasons for selecting their top product choice: ease of use for those who chose daily oral (99%) and daily event‐driven (98.5%); feel more protected against HIV for those who chose injectable (95.4%) and implants (100%); not worrying about forgetting to take it for those who chose bnAbs (93.8%); and being able to stop taking it when they want for those who chose rectal douche (90.9%).Participants’ mean age was 21 years (SD = 2.3), and 56% identified as White. Nineteen percent were currently taking daily oral PrEP, and another 9% had previously taken it. Participants prioritized efficacy, absence of side effects and costs in the conjoint analyses. Daily oral PrEP had the highest preference ranking, followed by event‐driven oral (OR = 0.89, Next‐generation modalities were less likely to be preferred over daily oral PrEP, with differences in the magnitude by socio‐demographic and behavioural characteristics. Given the low uptake of daily oral PrEP, end‐users’ preferences for and concerns about PrEP products must be understood to ensure high acceptability and penetration. In May Moreover, a recent meta‐analysis showed that, across studies, about one‐third of YMSM who initiated PrEP have suboptimal adherence , 7, 12. New PrEP formulations, including implants and long‐acting injectable products, may help address challenges individuals face adhering to a daily oral pill , 23, 24.Achieving consistent and correct use among the product's consumers will require researchers to develop desirable and acceptable products to end‐users within the context of clinical trials and in the real world , 28, 29.within YMSM participants via conjoint analyses and (2) across PrEP product preferences and correlates of these preferences between YMSM participants (via rank‐choice selection).We conducted an online survey with U.S. YMSM examining PrEP modality preferences to address this gap. In this study, we aim to examine the (1) preferences for features of PrEP products 22.1YMSM in the United States were recruited for an online survey from social media platforms, dating apps and targeted mailing lists between October 2020 and June 2021. Participants were eligible for this study if they were 15–24 years old, were assigned male sex at birth and currently identified as male, self‐reported being HIV negative or did not know their HIV status, reported same‐sex attractions or consensual sexual behaviours with other men in the past 6 months, lived in the United States and spoke English.Interested individuals completed an online screener to ascertain eligibility. We used automatized  and manual methods (e.g. time to complete screener) to verify the legitimacy of responses and checked phone numbers and email addresses to identify duplicate entries, as recommended by previous research , 31, 32.The Institutional Review Board at the University of Pennsylvania approved study procedures and materials. Informed consent/assent was obtained from all participants, and a waiver of parental consent was obtained for participants 15–17 years old. Participants received a USD$40 gift card for participation.2.22.2.1Definitions of PrEP products and images for the product features were reviewed by participants prior to the conjoint experiment. Definitions and images were developed through cognitive interviews with YMSM (reported previously ). Given 2.2.2Following the conjoint experiment, participants were then asked to rank each modality  from best (1) to worst (6) if they provided equal protection against HIV transmission based on their preferences and how the product would fit their lifestyles.We then asked participants to indicate on a 4‐point scale their agreement with 11 reasons they selected their top‐ranked PrEP modality. Sample reasons included, “It would be easy to use this product,” “I would have fewer concerns about side effects,” and “I feel more protected against HIV using this product.” We further dichotomized responses to each statement as agree/strongly agree versus disagree/strongly disagree.2.2.3We classified health insurance status as uninsured, covered by their parents’/guardians’ insurance plan and covered by their own insurance plan. We assessed whether participants had a primary care provider and the extent to which participants believed the healthcare services accessed met their health needs. Responses on a 5‐point scale ranging from “never” to “always” were dichotomized as “usually/always” and “never/rarely/sometimes” meet health needs.2.2.4We assessed previous HIV and other STI testing, prior diagnosis of STIs, awareness of PrEP for HIV prevention and PrEP use .2.2.5Participants were asked about the number of men with whom they had condomless anal sex (CAS) in the past month, with responses dichotomized into any versus none. Participants were also asked about any use of the following non‐prescribed substances in the past 30 days: cocaine/crack cocaine, stimulants, inhalants, sedatives, hallucinogens and opioids. Our composite measure of drug use included any use of these substances in the past 30 days. Lastly, participants were asked how frequently they had a drink containing alcohol in the past 30 days, which was dichotomized into 2+ drinks per week versus less than 2 drinks per week.2.2.6We also assessed participants’ age, race, ethnicity, sexual orientation, educational attainment and ZIP code (coded into Census regions). To measure food insecurity, we asked participants how often they or their families had to skip a meal in the past 3 months because there was not enough money for food. Responses on a 4‐point scale ranging from “almost every week” to “never” were dichotomized into any versus no food insecurity.2.3We described frequencies and descriptive statistics for socio‐demographic and behavioural characteristics.Using Qualtrics Conjoint Experiment platform, we estimated the feature importance of each attribute in the participants’ decision‐making process. We also calculated YMSM's willingness‐to‐pay  for a feature based on trade‐offs between different levels.We described ranking frequencies and mean ranking across all six PrEP modalities and frequencies of endorsement of reasons for selecting the top‐ranked PrEP modality. We used rank‐ordered regression (e.g. exploded logit regression) to model the probability of ranking each modality highest , which wRank‐ordered regression is a generalization of conditional logit models that estimates the preference for a given PrEP modality compared to an arbitrarily defined reference category based on sets of ranked items. Following the procedures outlined by Allison and Christakis , we usedp‐values <0.10 in bivariate analysis for adjusted models. STI diagnosis and alcohol use were both excluded from the adjusted models due to high collinearity with HIV testing and drug use, respectively. Given that ORs in adjusted rank‐ordered models assume reference levels of other covariates in regression models and that ORs are non‐collapsible [p‐values of adjusted ORs.We used an exploratory approach to model building, first examining bivariate associations between modality preferences and several measures of socio‐demographic characteristics, access to healthcare and HIV prevention, and sexual and drug use behaviours selected based on our previous research on acceptability and access to PrEP. We then retained variables with lapsible , the magAnalyses were done in SAS 9.4 and Stata 17.3N = 737 YMSM was 21.1 years (Standard Deviation = 2.27). Nearly, one‐quarter (24%) identified as Latino. Over half (56%) identified as White, 19% as Black, 12% as Asian and 13% as Multiracial or some other racial identity. While nearly all (95%) were aware of PrEP, only 28% had ever used PrEP.Table 3.1See Table The willingness to pay metric indicates that efficacy and side effects had the greatest cost trade‐offs in participants’ choices. YMSM were less willing to adopt a given technology in the presence of lower efficacy and certain side effects, whereas they were more forgiving of the mode of delivery, duration of HIV protection and timing of regimen use. For example, across products, participants were willing to pay >$250 more for a product with 95% efficacy compared to 50% efficacy. Additionally, a negative impact on kidney function was seen as a major trade‐off for the event‐driven products (>$200 compared to no side effects), as was weight gain for all products ($125–$176).3.2The complete ranks by each product are described in Table p = 0.058; injectable PrEP = 0.83, p = 0.005, PrEP implant = 0.48, p < 0.0001; bnAb infusion = 0.38, p < 0.0001; PrEP rectal douche = 0.24, p < 0.0001) , the odds of selecting daily oral PrEP was not significantly lower than the odds of choosing any next‐generation modality. Notably, over 90% of individuals ranking rt Table .3.3.1p < 0.05). Moreover, participants reporting CAS in the past 6 months were 33% less likely to prefer event‐driven to daily oral PrEP (p = 0.002). Finally, compared to individuals who had never used PrEP, current users were 52% less likely to prefer event‐driven over daily oral PrEP (p < 0.0001)  Table .event‐driven oral PrEP first included ease of use, ability to stop taking it, no impact on daily routine, perceived protection against HIV and lack of discomfort (Table The highest endorsed reasons for ranking rt Table .3.3.2p < 0.05). Additionally, participants who had prior HIV tests were 74% more likely to prefer injectables to daily oral compared to those who had not been tested (p < 0.001), and participants reporting recent CAS were 27% less likely to prefer injectables to daily oral PrEP (p = 0.02).Compared to individuals 15–17 and 18–20 years, participants 21–24 years old were 21% and 47% more likely to prefer injectable PrEP to daily oral PrEP (p = 0.40) . Participants reporting prior HIV tests were 74% more likely to prefer implants compared to those without prior tests (p < 0.001), and, compared to PrEP‐naïve individuals, past PrEP users were 21% more likely to choose PrEP implants over daily oral PrEP (p = 0.001).Compared to individuals 15–17 and 18–20 years, participants 21–24 years old were 51% and 57% more likely to prefer implants to daily oral PrEP  . Participants who had prior HIV test were 65% more likely to prefer bnAb to daily oral PrEP in comparison to individuals not previously tested (p < 0.01). Past PrEP users were 60% more likely to choose bnAb infusions (p = 0.03) over daily oral PrEP compared to individuals who had never used PrEP  compared to never users . Notably, while preferences for injectable PrEP were not different across PrEP use categories, compared to current PrEP users, individuals who had never used PrEP were significantly more likely to prefer event‐driven oral PrEP and rectal douches. Previous work has shown that event‐driven oral PrEP may appear appealing to some PrEP‐naïve individuals, but challenges related to planning sexual activity may make some of these individuals eventually switch to daily regimens . AdditioThese findings should be considered in light of some limitations. First, while this was a large, diverse, national survey, recruitment was primarily conducted online, and as such, the sample may not represent all YMSM in the United States. Further studies on modality preferences are needed with other key populations in the HIV epidemic, such as transgender women and people who inject drugs. Additionally, a number of these products are still in development and precise information on their efficacy, side effects and other characteristics was not available when our study was conducted, and as such were presented as hypothetical. Familiarity with next‐generation products was not measured. Although we used descriptions of the products developed and tested in cognitive interviews with YMSM , prefere5Both low rates of PrEP use and suboptimal adherence among YMSM , 10, 12 KBB received unrestricted research grants from Merck. KHM received unrestricted research grants from Gilead and Merck; on the Scientific Advisory Board: Gilead, Merck and ViiV. The authors report no other competing interests.PKV, RD and WL performed the research. KBB, KHM, LH‐W, DTdS and JB designed the research study. KBB, PKV and JB analysed the data. KBB and PKV led the writing of the paper. All authors provided edits to the paper, and have read and approved the final manuscript.This work was made possible through support from the National Institute of Child Health and Human Development (NICHD) (U19HD089881).The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.


RARS1, a gene that encodes the cytoplasmic tRNA synthetase for arginine (ArgRS), are associated with central nervous system (CNS) manifestations, such as hypomyelinating leukodystrophy‐9 and developmental and epileptic encephalopathy (DEE). This study aimed to better understand the RARS1 biallelic mutations and the associated phenotypes, particularly in patients with DEE.Biallelic variants of RARS1 biallelic mutations and functionally validated these mutations in vitro. Furthermore, we performed a review of the literature.We identified two patients with RARS1 biallelic variants (Patient 1: c.1535G>A (p.Arg512Gln) and c.1382G>A (p.Arg461His); Patient 2: homozygous variants c.5A>T ). Patient 2 had a severe clinical manifestation of DEE. A review of the literature identified 27 patients from five studies. Among the 29 patients, intellectual disability, developmental delay, and hypomyelination were the common symptoms, while 13 of them exhibited DEE and malformations of cortical development. Of the 25 variants identified, c.5A>G (p.Asp2Gly) was identified in 10 patients. ArgRS protein expression and stability were substantially reduced in the two newly identified patients.Two patients with hypomyelinating leukodystrophy were found to have RARS1 biallelic mutations frequently exhibit DEE, a severe phenotype, along with hypomyelinating leukodystrophy. Besides its effects on the white matter, this mutation also influences cortical development. Moreover, the variants c.5A>T , c.1382G>A (p.Arg461His), and c.1535G>A (p.Arg512Gln) are pathogenic and affect the expression of ArgRS by reducing the protein stability.Patients with  RARS1 biallelic variants could lead to DEE.RARS1‐related DEE more likely to have MCD.All the patients with RARS1 gene were pathogenic.The variants p.Asp2Val, p.Arg461His, and p.Arg512Gln of 1RARS1 gene, do not encode structural myelin proteins.Myelination, a process of compact myelin formation by oligodendrocytes, is crucial for the proper functioning of the central nervous system.RARS1 gene encodes the cytoplasmic tRNA synthetase for arginine (ArgRS), a component of the multienzyme aminoacyl‐tRNA synthetase complex that is essential for protein synthesis.RARS1‐related HLD  have been reported. Intellectual disability (ID) or developmental delay (DD) was identified in the vast majority of these patients while nystagmus, epilepsy, and microcephaly were also common.The RARS1 variants, this study aimed to further investigate the relationships between genotype and/or phenotype in patients with HLD‐9. Also, we reviewed the literature and present two examples of patients with HLD9 who had biallelic RARS1 mutations. Furthermore, RARS1 variants from the two patients were functionally validated to verify their pathogenicity.Since studies have shown that a broad spectrum of clinical symptoms are linked with 22.1RARS1 biallelic variants (identified by the trio‐WES) were enrolled, and their clinical and radiological data were retrospectively collected. Relevant studies were identified by two independent reviewers (LW and GY) by searching the electronic databases, including MEDLINE, PubMed, Embase, Google, and the China National Knowledge Infrastructure. The keyword search terms used were as follows: [RARS1 OR RARS OR arginine‐tRNA synthetase] AND [Hypomyelinating leukodystrophy OR Hypomyelinating leukodystrophy‐9 OR hypomyelination OR leukodystrophy] up to July 2022. We restricted the search to human studies published in English or Chinese. SPSS 26.0 statistical software was used for analysis. We used the chi‐squared and Fisher's exact tests to analyze the variables.Two unrelated children with hypomyelinating leukodystrophy carrying 2.2RARS1 variants of the two patients were analyzed using some mutation‐prediction tools, including SIFT, PolyPhen‐2, MutationTaster, PROVEAN, and CADD to evaluate their pathogenicity. Results identified these variants as variants of unknown significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) guidelines. We obtained the protein structure template of RARS1 from the UniProt database via the AlphaFold website (www.uniprot.org) as wild‐type and mapped the corresponding mutations to generate mutated PDB files. We compared the mutated forms with the wild‐type using an online comparison method (fatcat.godziklab.org)biosig.lab.uq.edu.au/dynamut)biosig.lab.uq.edu.au/dynamut2)The 2.3RARS1 (RefSeq: NM_004233.2), PCR amplification was performed using the following primer sets: phage‐RARS1‐SalI‐F, TGACGTCGACAATGGACGTACTGGTGTCTGA; phage‐RARS1‐NotI‐R, CGACGCGGCCGCTCATCCTTTGGACAGGTTTTA; RARS1‐variant1 c.5A>T‐F, TGACGTCGACAATGGTCGTACTGGTGTCTGA; RARS1‐mut1 c.1382.G>A‐F, TGACGTCGACAATGGACGTACTGGTGTCTGA; RARS1‐mut2 c.1382.G>A‐R, AGAAGATCCATGAGGTGCACTGTTTCACCCG; RARS1‐mut2 c.1535G>A‐F, ACCTTTCCCATAACCAGTTGAATGACTACAT; RARS1‐mut3 c.1535G>A‐ACCTTTCCCATAACCAGTTGAATGACTA; RARS1‐mut3 c.1535G>A‐R, ATGTAGTCATTCAACTGGTTATGGGAAAGGT. The PCR products were subjected to SalI and NotI digestion, cloned into the phage vector, and validated by sequencing. The vectors were then transfected into 293T cells using Lipo2000 .Using the cDNA sequence of 2.4RARS1 expression was validated by qPCR. Total protein was extracted from cells using the RIPA lysis buffer, and protein concentration was measured using the BCA Protein Quantification Kit . Proteins were denatured, and ArgRS expression was confirmed by Western blot using the HA‐Tag monoclonal antibody .Cells were collected 48‐h posttransfection. The TRIzol method was used to extract total RNA. After DNA digestion, cDNA synthesis was performed by reverse transcription, and 2.5Forty‐eight‐hour posttransfection, cells were treated with 500 μM cycloheximide and collected at 0, 4, 8, and 24 h after the treatment to confirm ArgRS protein expression by Western blot using the HA‐Tag monoclonal antibody .33.1Patient 1, a 12‐month‐old girl, is the first child of unrelated, healthy parents with no family history of neurological disorders. She was born at term via spontaneous delivery with normal height and weight; congenital aural atresia was diagnosed during the postnatal period. Seizure onset as epileptic spasms occurred at 5 months of age. Hypsarrhythmia was observed on the electroencephalogram, and brain magnetic resonance imaging (MRI) showed hypomyelinating and a simplified gyral pattern, leading to the diagnosis of infantile spasms. Physical examination identified nystagmus and microcephaly, although ataxia and dystonia were absent. Adrenocorticotropic hormone and multiple antiseizure medications, including vigabatrin, were initiated, but these medications failed to control the problems. Brain MRI at 12 months showed hypomyelinating leukodystrophy and a simplified gyral pattern Figure .The patient had severe developmental delay and exhibited partial head control at 3 months of age and started rolling over at 4 months of age; however, during her 12‐month follow‐up, she was still unable to sit. Her language development cannot be assessed effectively during this period.RARS1  and c.1535G>A (p.Arg512Gln) in 1 Figure  were idePatient 2, a 16‐month‐old girl, is the first child of unrelated, healthy parents with no family history of neurological disorders. She was born at term via spontaneous delivery with normal height and weight and exhibited no abnormalities during the postnatal period. Nystagmus and ataxia were diagnosed at 11 months of age. Brain MRI showed hypomyelinating leukodystrophy at 16 months of age Figure .She had severe developmental delay, exhibited partial head control at 3 months of age, started rolling over at 4 months of age, and was able to sit unassisted at 12 months of age, although she was unable to walk at the 16‐month follow‐up. In addition, her language development was delayed and speech was absent.RARS1  in 1 Figure  was iden3.2RARS1 biallelic variants (including the two newly reported in this study), 13 were female patients and 15 were male patients (one gender was not reported). Twenty‐five different variants were identified and variant c.5A>G (p.Asp2Gly) was present in 10 patients. The newly identified variants c.5A>T  and c.1382G>A (p.Arg461His) were not reported in previous studies. Among the 25 variants, missense was the major type with a frequency of 13, followed by start codon defects (4), splicing (4), and frameshift (3).We reviewed the literature and identified 27 patients from five studies. Among the 29 patients carrying the Among the 29 patients, 21 exhibited nystagmus, 27 had intellectual disability or developmental delay, 22 had language delay (17 with absent speech), 25 exhibited motor delay, 17 had microcephaly, and 24 showed hypomyelination. Symptoms observed in less than half of the patients were ataxia (12 patients), dystonia (11 patients), hypotonia (8 patients), epilepsy (13 patients), and feeding difficulties (12 patients). Only eight patients were able to walk without any support Table .RARS1‐related epilepsy were more likely to have microcephaly, cerebral atrophy, and speech absence, but less likely to be able to walk independently , 12 had hypomyelination (no results were reported for one patient), and 11 had microcephaly (no results were reported for one patient). Of the 12 patients who reported seizure outcomes, 11 developed drug‐resistant epilepsy Table . CompareP < 0.05, Table Missense mutations, which result in the substitution of a single amino acid residue only, are less likely to produce aberrant transcripts, the majority of which may arise due to frameshift mutations, splicing defects, start codon mutations, etc. There were no significant differences in symptoms between patients with or without missense mutations only  compared to those expressing wild‐type RARS1  are a heterogeneous group of disorders with both nongenetic and genetic etiologies and are characterized by early‐onset and often severe epileptic seizures and EEG abnormalities on a background of developmental impairment that tends to worsen as a consequence of epilepsy.RARS1 variant occur not only in the white matter but also in the cortex and are presented as MCD. Therefore, patients with DEE are more likely to have MCD.We found that patients with epilepsy more often had microcephaly and cerebral atrophy. Microcephaly can be primary when it is congenital and presents at the time of birth or in utero as a small head with a simple gyral pattern,RARS1 gene carrying c.5A>G (p.Asp2Gly) and c.1535G>A (p.Arg512Gln) mutations produced ArgRS protein similar to the control; however, the decay rate of the ArgRS protein produced by the c.1535G>A variant was significantly increased. These results indicate that Arg512 is crucial for ArgRS enzyme activity and stability. In contrast, the c.1367C>T variant was associated with the production of ArgRS protein with normal stability and Arg activation ability; however, the enzyme exhibited a decreased aminoacylation activity.Using Western blot analysis, Mendes et al. confirmed that ArgRS protein is expressed in fibroblasts, and its expression was decreased in all four patients.RARS1‐related disorders and two novel RARS1 variants. We confirmed that these mutations can reduce the stability of ArgRS protein and cause disease, including DEE. Furthermore, brain MRI examination in these patients with RARS1‐related DEE may indicate both hypomyelinating leukodystrophy and MCD. When a child experiences seizures during infancy and HLD is suspected based on MRI, it is necessary to consider testing for the RARS1 gene as the patient may develop DEE in the future. Due to our limited knowledge of mutation sites, even a single variation classified as VUS by the ACMG guidelines requires additional functional validation to determine its potential pathogenicity.In conclusion, we report two patients with Lin Wan, Dan Yu, and Zhichao Li wrote the first draft of the manuscript and contributed equally to this work (cofirst authors). Dan Yu, Gang Zhu, and Guang Yang performed the acquisition of data. Yan Liang, Huimin Yan, and Bo Zhang performed the data analyses. Lin Wan and Yang Guang contributed to the conception and design of the study. All authors helped to revise the manuscript regarding crucial intellectual content. All authors approved the final version for publication.This research was funded by the General project of Beijing Natural Science Foundation (Reference: 7222187), the BINC foundation of Nutrition and Care of Maternal & Child (Reference: 2021BINCMCF030), the Epilepsy research foundation of Chinese Association Against Epilepsy (Reference: CU‐B‐2021‐11), and the Special scientific research project of military family planning (Reference: 22JSZ20).No financial or nonfinancial benefits have been received or will be received from any party related directly or indirectly to the subject of this article. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.Informed consent for clinical study and molecular genetic analysis was obtained from their parents. Ethics approval for the study was granted by the Ethics Committee of the First Medical Center of the PLA General Hospital.Data S1.Click here for additional data file.


Sorghum bicolor is a relatively drought‐tolerant cereal. Functional stay‐green sorghum genotypes can maintain green leaf area and efficient grain filling during terminal post‐flowering water deprivation, a period of ~10 weeks. To obtain molecular insights into these characteristics, two drought‐tolerant genotypes, BTx642 and RTx430, were grown in replicated control and terminal post‐flowering drought field plots in California's Central Valley. Photosynthetic, photoprotective, and water dynamics traits were quantified and correlated with metabolomic data collected from leaves, stems, and roots at multiple timepoints during control and drought conditions. Physiological and metabolomic data were then compared to longitudinal RNA sequencing data collected from these two genotypes. The unique metabolic and transcriptomic response to post‐flowering drought in sorghum supports a role for the metabolite galactinol in controlling photosynthetic activity through regulating stomatal closure in post‐flowering drought. Additionally, in the functional stay‐green genotype BTx642, photoprotective responses were specifically induced in post‐flowering drought, supporting a role for photoprotection in the molecular response associated with the functional stay‐green trait. From these insights, new pathways are identified that can be targeted to maximize yields under growth conditions with limited water.Climate change is globally affecting rainfall patterns, necessitating the improvement of drought tolerance in crops.  Plots were arranged in a randomized complete block design (RCBD) with three replications of each genotype and water regime Figure . Two watAll irrigated plots received volumes of water equal to 100% of the average weekly calculated crop evapotranspiration for the 7‐day period before irrigation. Surface drip lines were used for irrigation to provide accurate water application amounts and a high level of water application uniformity. Irrigation once per week in plots is comparable with sorghum farming irrigation practices in the Western United States. Additionally, providing equal water volume to all irrigated plots prevents a scenario where genotypic differences in evapotranspiration rates lead to a difference in total water volume supplied to specific plots. For greater details on crop evapotranspiration and irrigation management, see the −1 for BTx642 and 32.82 T ha−1 for RTx430. Planting in 2019 occurred on June 10. Four sampling dates were selected, and for each date, control plots had not received water for 5 days: (1) August 20, 2019 (D1), 5 days since last watering for all plots, 70 DAP; (2) August 27, 2019 (D2), 12 days of post‐flowering drought, 77 DAP; (3) September 10, 2019 (D3), 26 days of post‐flowering drought, 91 DAP; (4) September 24, 2019 (D4), 40 days of post‐flowering drought, 105 DAP.Total final biomass was comparable between control plots for both genotypes with an average forage yield (65% moisture) of 32.17.02 T ha2.2−1 and sample [CO2] to 400 μmol mol−1. Stability variables typically converged within 60 s of clamping a leaf, then an infrared gas analyzer match was performed, and once stability variables were restored following the match, the measurement was taken. Leaves were clamped to avoid the midrib and always near the midpoint of the leaf . A multiphase flash routine was used to estimate chlorophyll fluorescence parameters  and 14:00 to 16:00 (mid‐afternoon), using LI‐COR 6400XT instruments . Given that these sampling dates all occurred post‐anthesis, all leaves had emerged, and thus, it was possible to randomly sample the uppermost three leaves including the flag leaf from plants growing in the interior of each plot on each sampling date. Each of the LI‐COR 6400XT instruments was factory calibrated the month prior to this field work, and the calibrations and instrument checks, as described in Chapter 4 of the LI‐COR 6400 manual, were performed on each sampling date. Leaves were maintained near ambient light levels and temperatures by measuring ambient PAR levels and local temperatures and re‐adjusting actinic light levels and blocking temperature prior to each set of measurements. The ratio of blue‐to‐red LED contribution to the cuvette light source was 10%/90%. Relative humidity in the measurement cuvette was maintained between 50% and 60% to maintain stomatal aperture width. Flow rate was set to 400 μmol sv/Fm and NPQ were determined. Specific to NPQ measurements, these values were measured exclusively on leaves without visible signs of leaf senescence in both control and droughted plots. This decision was made to ensure that photoprotective traits could be accurately quantified in leaves with photosynthetic machinery intact prior to the onset of leaf senescence traits. Green leaf area was determined by imaging the three uppermost leaves including the flag leaf on 10 randomly selected plants per plot. Stomatal density and guard cell length were quantified using leaf peels collected on the D4 sampling date from the abaxial leaf surface of the uppermost non‐flag leaf of the main culm  for 5 min. All samples were then flash‐frozen in liquid nitrogen within 5 min of being removed from the field. Each week, all samples were collected less than 1 h after dawn (dawn), within 1 h of the midpoint of the light period (midday), and less than 1 h before dusk (dusk).Plant samples were collected manually on the same days as physiological measurements with root systems to a depth of approximately 30 cm. Three plants from each plot were collected, and the uppermost three leaves, stems (below the peduncle and above the node for the next leaf below), and roots were harvested to create a single leaf, stem, and root sample for each plot for each timepoint. Root tissue was collected as previously described  values were calculated by subtracting total N levels by spectrophotometrically determined ammonium  and nitrate levels  were analyzed as outlined in Kyle et al.  and furthttps://omics.pnl.gov/software/pnnl-preprocessor) was used to generate new raw MS files (Agilent MassHunter “.d”) for each sample, run with all frames (ion mobility separations) summed into a single frame and applying 3‐point smoothing in the ion mobility dimension and noise filtering with a minimum intensity threshold of 20 counts. Details of the data processing and compound identification can be found in the For SPE‐IMS‐MS metabolomics, extracts were analyzed using a RapidFire 365  were removed from our datasets and attributed to either machine or user error.All statistical analyses were performed using JMP Pro 16 software  and analysis of variance (ANOVA) used to analyze the effects of treatment and genotype, appropriate for the RCBD field layout. Prior to the analysis of gas exchange values, six measurements (out of the 462 measurements taken) with physiologically impossible 2.6S. bicolor genes mapped to a common reference (S. bicolor BTx623) and accompanying metadata from the EPICON field trial described previously  and Log2‐transformation (with a pseudocount of 1). These values were subtracted from Log2‐transformed (plus a pseudocount of 1) normalized counts for each locus, genotype, day, and treatment from the EPICON dataset, to generate a control mean‐corrected dataset of gene expression for pre‐ and post‐flowering drought treatments. These values were then plotted as points, with loess‐smoothed values computed from these transformed data plotted as lines.To generate expression plots for selected gene sets, we obtained normalized counts of 33.1Sorghum plants were grown in irrigated, replicated plots with 10 rows in each plot , stomatal conductance (gs), and operating efficiency of PSII in the light (ΦPSII) revealed few statistically significant differences between control and droughted plots  at D2 , D3 , and D4  was not significantly depressed in droughted plants until D4 in the stay‐green genotype BTx642 and not until D3 in RTx430  was specifically depressed in RTx430 at the final drought timepoint, whereas phosphotidylserine lipids (PS) were induced in both genotypes Figure .An and gs values measured in parallel with sample harvesting . Given the relatively weak correlations for some of these molecules, the Normality of the residuals from the line fit was tested for Goodness‐of‐Fit. If the residuals are not Normally distributed, this supports that the correlation may be superficially caused by outliers or only a small subset of the data. Indeed, the residuals for maleate, fumarate, and α‐ketoglutarate were, in fact, non‐Normally distributed when assessed for Goodness‐of‐Fit to a Normal distribution (Table p < .05). Thus, out of all the metabolites and lipids measured, galactinol was the sole metabolite to have a robust correlation with gs activity, as well as a statistically significant drought‐induced change in abundance in both genotypes even after correcting for multiple sampling bias , and GolS2 (Sobic.002G423600) Figure . In both3.3An, gs, and percent green leaf each had strong treatment and genotype × treatment effects  content, often associated with a loss in green leaf area, were observed in both genotypes, but there was not a significant genotype × treatment effect for either of these parameters , Sobic.010G207900 (VTE2‐2), and Sobic.006G260800 (VTE5), in droughted BTx642 relative to droughted RTx430  throughout the grain filling period despite terminal water deprivation for 40 days  between the two genotypes.The role of photoprotection in preventing drought‐induced leaf senescence is well‐established (Challabathula et al., 0 Figure , Table 10 Figure . Both lers Table . In contPSBS during post‐flowering drought or by supporting a more de‐epoxidized xanthophyll cycle pool for NPQ (Głowacka et al., The BTx642 genotype more strongly induced a suite of photoprotective responses, supporting the hypothesis that the stay‐green BTx642 would have a stronger photoprotective response in post‐flowering drought Figures  and S2. As climate change constrains agricultural productivity in the coming decades, conferring functional post‐flowering drought tolerance to drought‐susceptible genotypes can improve yields with limited water inputs. Here, we have applied metabolomic, transcriptomic, and physiological measurements across time to post‐flowering droughted, field grown sorghum plants in a RCBD. By bringing together these different techniques, we were able to discover an unanticipated correlation between foliar galactinol levels and stomatal conductance in post‐flowering drought. This finding is further supported by features of our transcriptomic data, as well as previously published findings, as discussed above. We also find stronger induction of photoprotective mechanisms in the stay‐green BTx642 genotype, providing a new type of explanation for the mechanisms underlying the functional stay‐green phenotype. Given the importance that improved post‐flowering drought tolerance may have for agriculture in a water‐scarce world, we hope that future work will test these findings, currently resting on correlative data, through generating mutants that alter foliar galactinol levels and manipulating photoprotective responses to directly test their role in the functional stay‐green phenotype.CRB, KKN, PGL, JD, RBH, and KK designed and planned the research. CRB and DP conducted experiments, collected field samples, and analyzed data. LGC, OD, and AK conducted experiments and collected/processed field samples. RBH, BJC, AB, JYL, YMK, JEK, KJB, and VP conducted experiments and analyzed data. CA, JP, and JS collected/processed field samples. CRB wrote the manuscript with contributions from BJC, AB, JYL, and YMK. The manuscript was edited by KKN, JD, PGL, DP, and AK. OD and AK contributed equally to this work. CA, JP, and JS contributed equally to this work. AB, JYL, and YMK contributed equally to this work.The authors declare that they have no conflict of interest, financial or otherwise, that influenced this manuscript.The peer review history for this article is available in the Data S1. Peer Review.Click here for additional data file.