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Background: Heart disease and chronic kidney disease are often comorbid conditions owing to shared risk factors, including diabetes and hypertension. However, the effect of congestive heart failure (CHF) on arteriovenous fistula (AVF) and AV graft (AVG) patency rates is poorly understood. We hypothesize preexisting HF may diminish blood flow to the developing AVF and worsen patency., Methods: We conducted a single-institution retrospective review of 412 patients with end-stage renal disease who underwent hemodialysis access creation from 2015 to 2021. Patients were stratified based on presence of preexisting CHF, defined as clinical symptoms plus evidence of reduced left ventricular ejection fraction (EF) (<50%) or diastolic dysfunction on preoperative echocardiography. Baseline demographics, preoperative measures of cardiac function, and dialysis access-related surgical history were collected. Kaplan-Meier time-to-event analyses were performed for primary patency, primary-assisted patency, and secondary patency using standard definitions for patency from the literature. We assessed differences in patency for patients with CHF vs patients without CHF, patients with a reduced vs a normal EF, and AVG vs AVF in patients with CHF., Results: We included 204 patients (50%) with preexisting CHF with confirmatory echocardiography. Patients with CHF were more likely to be male and have comorbidities including, diabetes, chronic obstructive pulmonary disease, hypertension, and a history of cerebrovascular accident. The groups were not significantly different in terms of prior fistula history (P = .99), body mass index (P = .74), or type of hemodialysis access created (P = .54). There was no statistically significant difference in primary patency, primary-assisted patency, or secondary patency over time in the CHF vs non-CHF group (log-rank P > .05 for all three patency measures). When stratified by preoperative left ventricular EF, patients with an EF of <50% had lower primary (38% vs 51% at 1 year), primary-assisted (76% vs 82% at 1 year), and secondary patency (86% vs 93% at 1 year) rates than those with a normal EF. Difference reached significance for secondary patency only (log-rank P = .029). AVG patency was compared against AVF patency within the CHF subgroup, with significantly lower primary-assisted (39% vs 87% at 1 year) and secondary (62% vs 95%) patency rates for AVG (P < .0001 for both)., Conclusions: In this 7-year experience of hemodialysis access creation, reduced EF is associated with lower secondary patency. Preoperative CHF (including HF with reduced EF and HF with preserved EF together) is not associated with significant differences in overall hemodialysis access patency rates over time, but patients with CHF who receive AVG have markedly worse patency than those who receive AVF. For patients with end-stage renal disease and CHF, the risks and benefits must be carefully weighed, particularly for those with low EF or lack of a suitable vein for fistula creation., Competing Interests: Disclosures None., (Copyright © 2023 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Aortoiliac occlusive disease, or peripheral artery disease affecting the suprainguinal vessels, can lead to a range of clinical symptoms from claudication to more severe, chronic limb-threatening ischemia. Although open surgical reconstruction has traditionally been the reference standard, endovascular options have become significantly more robust in recent years, owing to both improved devices and increasing experience with advanced techniques. This review will discuss the demographics, presentation, and evaluation of chronic aortoiliac occlusive disease, as well as explore the options, both open and endovascular, for revascularization., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Small case series have suggested that selective nonoperative management (NOM) of penetrating internal jugular vein (IJV) injuries is safe and feasible in select patients lacking "hard signs" mandating exploration. Therefore, we sought to compare NOM to operative management (OM) of penetrating IJV injury, hypothesizing that both strategies have similar patient outcomes and mortality when patients are appropriately selected., Methods: The Trauma Quality Improvement Program (2013-2016) was queried for patients with penetrating IJV injury with an abbreviated injury scale score of the neck ≥3. Demographics and patient outcomes were compared between patients undergoing NOM and patients undergoing OM, followed by a multivariable logistic regression model to analyze the risk of mortality., Results: A penetrating IJV injury was identified in 188 (0.01%) patients meeting inclusion criteria, and OM was performed in 124 (66.0%) patients, whereas 64 (34.0%) patients underwent NOM. Although the OM group had a higher rate of pneumothorax (8.9% vs. 0.0%, P = 0.01), there was no difference in any other concomitant injuries or demographic data (all P > 0.05). The OM group had a higher rate of ventilator days (3 vs. 2 days, P = 0.01) but no other significant differences in morbidity or mortality (P > 0.05). After controlling for covariates, OM was associated with similar risk of mortality compared with NOM of patients with penetrating IJV injury (odds ratio 1.05, confidence interval 0.23-4.83, P = 0.95)., Conclusions: The NOM of penetrating IJV injuries is associated with similar risk of morbidity and mortality compared with OM, suggesting that NOM may be used in appropriately selected patients. Future research is needed to determine the ideal patients suited for NOM and to identify risk factors and outcomes associated with failure of NOM., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: To determine whether, similar to adults, early tracheostomy in pediatric patients with severe traumatic brain injury (TBI) improves inhospital outcomes including ventilator days, intensive care unit (ICU) length of stay (LOS), and total hospital LOS when compared to late tracheostomy., Design: Retrospective cohort analysis., Setting: The Pediatric Trauma Quality Improvement Program (TQIP) database., Patients: One hundred twenty-seven pediatric patients <16 years old with severe (>3) abbreviated injury scale TBI who underwent early (days 1-6) or late (day ≥7) tracheostomy between 2014 and 2016., Interventions: Not applicable., Measurements and Main Results: The Pediatric TQIP database was queried for patients <16 years old with severe TBI, who underwent tracheostomy. Patient demographics and outcomes of early versus late tracheostomy were compared using Student t test, Mann-Whitney U test, and χ2 analysis. Sixteen patients underwent early tracheostomy while 111 underwent late tracheostomy. The groups had similar distributions of age, gender, mechanism of injury, and mean injury severity scores (P > .05). Early tracheostomy was associated with decreased ICU LOS (early: 17 vs late: 32 days, P < .05) and ventilator days (early: 9.7 vs late: 27.1 days, P < .05). There was no difference in total LOS (early: 26.7 vs late: 41.3 days, P = .06), the incidence of acute respiratory distress syndrome (early: 6.3% vs late: 2.7%, P = .45), pneumonia (early: 12.5% vs late: 29.7%, P = .15), or mortality (early: 0% vs late: 2%, P = .588) between the 2 groups., Conclusion: Similar to adults, early tracheostomy in pediatric patients with severe TBI is associated with decreased ICU LOS and ventilator days. Future prospective trials are needed to confirm these findings., Article Tweet: Early tracheostomy in pediatric patients with severe TBI is associated with decreased ICU LOS and ventilator days.

Objectives: Penetrating abdominal aortic injury (PAAI) is a highly acute injury requiring prompt surgical management. When compared to surgeons at level-II trauma centers, surgeons at level-I trauma centers are more likely to take in-house call, and may more often be available within 15 minutes of patient arrival. Thus, we hypothesized that level-I trauma centers would have a lower mortality rate than level-II trauma centers in patients with PAAI., Methods: We queried the Trauma Quality Improvement Program database for patients with PAAI, and compared patients treated at American College of Surgeons (ACS)-verified level-I centers to those treated at ACS level-II centers., Results: PAAI was identified in 292 patients treated at level-I centers and 86 patients treated at level-II centers. Patients treated at the 2 center types had similar median age, injury severity scores and prevalence of diabetes, hypertension, and smoking (p > 0.05). There was no difference in the frequency of additional intra-abdominal vascular injuries (p > 0.05). Median time to hemorrhage control (level-I: 40.8 vs level-II: 49.2 minutes, p = 0.21) was similar between hospitals at the 2 trauma center levels. We found no difference in the total hospital length of stay or post-operative complications (p > 0.05). When controlling for covariates, we found no difference in the risk of mortality between ACS verified level-I and level-II trauma centers (OR:1.01, CI:0.28-2.64, p = 0.99)., Conclusion: Though the majority of PAAIs are treated at level-I trauma centers, we found no difference in the time to hemorrhage control, or the risk of mortality in those treated at level-I centers when compared to those treated at level-II trauma centers. This finding reinforces the ACS-verification process, which strives to achieve similar outcomes between level-I and level-II centers.

Background: Popliteal artery injury (PAI) is a rare occurrence in pediatric patients with significant consequences. Delays in diagnosis lead to severe complications such as lifelong disability and limb loss. We sought to identify outcomes and clinical predictors of PAI in the pediatric trauma population., Methods: The Pediatric Trauma Quality Improvement Program (2014-2016) was queried for patients ≤17 years old with PAI. Patient demographics and outcomes were characterized. A comparison of patients sustaining blunt versus penetrating PAI was performed. A multivariable logistic regression analysis was used to identify predictors of PAI., Results: From 119,132 patients, 58 (<0.1%) sustained a PAI with 74.1% from blunt trauma. Most of the patients were male (75.9%) with a median age of 15 and median Injury Severity Score of 9. A majority of the patients were treated with open repair (62.1%) in comparison to endovascular repair (10.3%) and nonoperative management (36.2%). The rates of open and endovascular repair and nonoperative management were similar between blunt and penetrating PAI patients (P = not significant). Concomitant injuries included popliteal vein injury (PVI) (12.1%), posterior tibial nerve injury (3.4%), peroneal nerve injury (3.4%), and closed fracture/dislocation of the femur (22.4%), patella (25.9%), and tibia/fibula (29.3%). Overall complications included compartment syndrome (8.6%), below-knee amputation (6.9%), and above-knee amputation (3.4%). The overall mortality was 3.4%. Patients with PAI secondary to penetrating trauma had a higher rate of concomitant PVI (26.7% vs. 7%, P = 0.04) and posterior tibial nerve injury (13.3% vs. 0%, P = 0.02) but a lower rate of closed fracture/dislocation of the patella (0% vs. 34.9%, P = 0.008) and tibia/fibula (0% vs. 39.5%, P = 0.004) compared to patients with PAI from blunt trauma. Predictors for PAI included PVI (odds ratio [OR] 296.57, confidence interval [CI] = 59.21-1,485.47, P < 0.001), closed patella fracture/dislocation (OR 50.0, CI = 24.22-103.23, P < 0.001), open femur fracture/dislocation (OR 9.05, CI = 3.56-22.99, P < 0.001), closed tibia/fibula fracture/dislocation (OR 7.44, CI = 3.81-14.55, P < 0.001), and open tibia/fibula fracture/dislocation (OR 4.57, CI = 1.80-11.59, P < 0.001). PVI had the highest association with PAI in penetrating trauma (OR 84.62, CI = 13.22-541.70, P < 0.001) while closed patella fracture/dislocation had the highest association in blunt trauma (OR 52.01, CI = 24.50-110.31, P < 0.001)., Conclusions: A higher index of suspicion should be present for PAI in pediatric trauma patients presenting with a closed patella fracture/dislocation after blunt trauma. PVI is most strongly associated with PAI in penetrating trauma. Prompt recognition of PAI is crucial as there is a greater than 10% amputation rate in the pediatric population., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Blunt abdominal aortic injury (BAAI) occurs in less than 0.1% of blunt traumas. A previous multi-institutional study found an associated mortality rate of 39%. We sought to identify risk factors for BAAI and risk factors for mortality in patients with BAAI using a large national database. We hypothesized that an Injury Severity Score of 25 or greater, and thoracic trauma would both increase the risk of mortality in patients with BAAI., Methods: The Trauma Quality Improvement Program (2010-2016) was queried for individuals with blunt trauma. Patients with and without BAAI were compared. Covariates were included in a multivariable logistic regression model to determine mechanisms of injury, examination findings, and concomitant injuries associated with increased risk for BAAI. An additional multivariable analysis was performed for mortality in patients with BAAI., Results: From 1,056,633 blunt trauma admissions, 1012 (0.1%) had BAAI. The most common mechanism of injury was motor vehicle accident (MVA; 57.5%). More than one-half the patients had at least one rib fracture (54.0%), or a spine fracture (53.9%), whereas 20.8% had hypotension on admission and 7.8% had a trunk abrasion. The average length of stay was 13.4 days and 24.6% required laparotomy, with 6.6% receiving an endovascular repair and 2.9% an open repair. The risk of death in those treated with endovascular vs open repair was similar (P = .28). On multivariable analysis, MVA was the mechanism associated with the highest risk of BAAI (odds ratio [OR], 4.68; 95% confidence interval [CI], 3.87-5.65; P < .001) followed by pedestrian struck (OR, 4.54; 95% CI, 3.47-5.92; P < .001). Other factors associated with BAAI included hypotension on admission (OR, 3.87; 95% CI, 3.21-4.66; P < .001), hemopneumothorax (OR, 3.67; 95% CI, 1.16-11.58; P < .001), abrasion to the trunk (OR, 1.49; 95% CI, 1.15-1.94; P = .003), and rib fracture (OR, 1.46; 95% CI, 1.25-1.70; P < .001). The overall mortality rate was 28.0%. Of the variables examined, the strongest risk factor associated with mortality in patients with BAAI was hemopneumothorax (OR, 12.49; 95% CI, 1.25-124.84; P = .03) followed by inferior vena cava (IVC) injury (OR, 12.05; 95% CI, 2.80-51.80; P < .001)., Conclusions: In the largest nationwide series to date, BAAI continues to have a high mortality rate with hemopneumothorax and IVC injury associated with the highest risk for mortality. The mechanism most strongly associated with BAAI is MVA followed by pedestrian struck. Other risk factors for BAAI include rib fracture and trunk abrasion. Providers must maintain a high suspicion of injury for BAAI when these mechanisms of injury, physical examination or imaging findings are encountered., (Copyright © 2019 Society for Vascular Surgery. All rights reserved.)

Background: Hiatal Hernia Repairs (HHR) are performed by both general surgeons (GS) and thoracic surgeons (TS). However, there are limited literature with respect to outcomes of HHR based on specialty training. The objective of this study was to compare the utilization, perioperative outcomes, and cost for HHR performed by GS versus TS., Methods: The Vizient database was used to identify patients who underwent elective laparoscopic HHR between October 2014 and June 2018. Patients were grouped according to surgeon's specialty (GS vs. TS). Patient demographics and outcomes including in-hospital mortality were compared between groups., Results: During the study period 13,764 patients underwent HHR by either GS or TS. GS performed 9930 (72%) cases while TS performed 3834 (28%) cases. There was no significant difference between GS versus TS with regard to serious morbidity (1.28% vs. 1.30%, p = 0.97) or mortality (0.10% vs. 0.21%, p = 0.19). The mortality index was 0.24 for GS versus 0.45 for TS. Compared to TS, laparoscopic HHR performed by GS was associated with a shorter LOS (2.57 days vs. 2.72 days, p < 0.001) and lower mean hospital costs ($7139 vs. $8032, p < 0.0001)., Conclusions: Within the context of academic centers, laparoscopic HHRs are mostly performed by GS with comparable outcome between general versus thoracic surgeons.

Background: We performed a systematic review of the literature on antibiotic prophylaxis practices in open reduction, and internal fixation of, facial fracture(s) (ORIFfx). We hypothesized that prolonged antibiotic prophylaxis (PAP) would not decrease the rate of surgical site infections (SSIs). Methods: We performed a systematic review of four databases: PubMed, CENTRAL, EMBase, and Web of Science, from inception through January 15, 2017. Three independent reviewers extracted fracture location (orbital, mid-face, mandible), antibiotic use, SSI incidence, and time from injury to surgery. Mantel-Haenszel and generalized estimating equations were carried out independently for each fracture zone. Results: Of the 587 articles identified, 54 underwent full-text review, yielding 27 studies that met our inclusion criteria. Of these, 16 studies (n = 2,316 patients) provided data for mandible fractures, four studies (n = 439) for mid-face fractures, and six studies (n = 377) for orbital fractures. Pooled analysis of each fracture type's SSI rate showed no statistically significant association with the odds ratio (OR) of developing an SSI. For mandible fractures treated with ORIFfx, the OR for an SSI after 24-72 hours of prophylaxis relative to <24 hours was 0.85 (95% confidence interval [CI] 0.62-1.17), whereas for >72 hours compared with <24 hours, the OR was 1.42 (95% CI) 0.96-2.11). For mid-face fractures, there was no improvement in SSI rate from PAP (OR 1.05; 95% CI 0.20-5.63). Conclusions: We did not demonstrate a lower rate of SSI associated with PAP for any ORIFfx repair. Post-operative antibiotics for >72 hours paradoxically may increase the SSI risk after mandible fracture repairs.

Objectives: Although traumatic injuries to the superior mesenteric vein (SMV), portal vein (PV), and hepatic vein (HV) are rare, their impact is significant. Small single center reports estimate mortality rates ranging from 29% to 100%. Our aim is to elucidate the incidence and outcomes associated with each injury due to unique anatomic positioning and varied tolerance of ligation. We hypothesize that SMV injury is associated with a lower risk of mortality compared to HV and PV injury in adult trauma patients., Methods: The Trauma Quality Improvement Program database (2010-2016) was queried for patients with injury to either the SMV, PV, or HV. A multivariable logistic regression model was used for analysis., Results: From 1,403,466 patients, 966 (0.07%) had a single major hepatoportal venous injury with 460 (47.6%) involving the SMV, 281 (29.1%) involving the PV, and 225 (23.3%) involving the HV. There was no difference in the percentage of patients undergoing repair or ligation between SMV, PV, and HV injuries ( P > .05). Compared to those with PV and HV injuries, patients with SMV injury had a higher rate of concurrent bowel resection (38.5% vs 12.1% vs 7.6%, P < .001) and lower mortality (33.3% vs 45.9% vs 49.3%, P < .01). After controlling for covariates, traumatic SMV injury increased the risk of mortality (odds ratio [OR] 1.59, confidence interval [CI] = 1.00-2.54, P = .05) in adult trauma patients; however, this was less than PV injury (OR = 2.77, CI = 1.56-4.93, P = .001) and HV injury (OR = 2.70, CI = 1.46-4.99, P = .002)., Conclusion: Traumatic SMV injury had a lower rate of mortality compared to injuries of the HV and PV. SMV injury increased the risk of mortality by 60% in adult trauma patients, whereas PV and HV injuries nearly tripled the risk of mortality.

Traumatic esophageal injury is a highly lethal but rare injury with minimal data in the trauma population. We sought to provide a descriptive analysis of esophageal trauma (ET) to identify the incidence, associated injuries, interventions, and outcomes. We hypothesized that blunt trauma is associated with higher risk of death than penetrating trauma. The Trauma Quality Improvement Program (2010-2016) was queried for patients with ET. Patients with blunt and penetrating trauma were compared using chi-square and Mann-Whitney U tests. A multivariable logistic regression model was used to determine risk of mortality. Of 1,403,466 adult patients, 651 (<0.01%) presented with ET. The most common associated thoracic injuries were rib fractures (38.7%) and pneumothorax (26.7%). More patients with a penetrating mechanism underwent open repair of the esophagus than those with blunt mechanism (46.2% vs 11.7%, P < 0.001). After controlling for covariates, there was no difference in risk of mortality between blunt and penetrating trauma ( P = 0.65). The mortality rate for patients with esophageal injury surviving greater than 24 hours was 7.5 per cent. In this large national database analysis, ET was rare and most commonly associated with rib fractures and pneumothorax. Contrary to our hypothesis, the risk of mortality was equivalent between blunt and penetrating ET.

Diabetes mellitus (DM) during pregnancy has long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that endothelial colony forming cells (ECFCs) from DM human pregnancies have decreased vasculogenic potential. Here, we evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). In human umbilical vein endothelial cells, MEOX2 upregulates cyclin-dependent kinase inhibitor expression, resulting in increased senescence and decreased proliferation. We hypothesized that dysregulated MEOX2 expression in neonatal ECFCs from DM pregnancies decreases network formation through increased senescence and altered cell cycle progression. Our studies show that nuclear MEOX2 is increased in ECFCs from DM pregnancies. To determine if MEOX2 is sufficient and/or required to induce impaired network formation, MEOX2 was overexpressed and depleted in ECFCs from control and DM pregnancies, respectively. Surprisingly, MEOX2 overexpression in control ECFCs resulted in increased network formation, altered cell cycle progression, and increased senescence. In contrast, MEOX2 knockdown in ECFCs from DM pregnancies led to decreased network formation, while cell cycle progression and senescence were unaffected. Importantly, migration studies demonstrated that MEOX2 overexpression increased migration, while MEOX2 knockdown decreased migration. Taken together, these data suggest that altered migration may be mediating the impaired vasculogenesis of ECFCs from DM pregnancies. While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment. J. Cell. Physiol. 232: 1885-1892, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Unnecessary hospital readmissions are one source of escalating costs that may be reduced through improved care coordination, but how best to design and evaluate coordination programs is poorly understood. Measuring patient flow between service visits could support decisions for coordinating care, particularly for conditions such as congestive heart failure (CHF) which have high morbidity, costs, and hospital readmission rates., Objectives: To determine the feasibility of using network analysis to explore patterns of service delivery for patients with CHF in the context of readmissions., Methods: A retrospective cohort study used de-identified records for patients ≥18 years with an ICD-9 diagnosis code 428.0-428.9, and service visits between July 2011 and June 2012. Patients were stratified by admission outcome. Traditional and novel network analysis techniques were applied to characterize care patterns., Results: Patients transitioned between services in different order and frequency depending on admission status. Patient-to-service CoUsage networks were diffuse suggesting unstructured flow of patients with no obvious coordination hubs. In service-to-service Transition networks a specialty heart failure service was on the care path to the most other services for never admitted patients, evidence of how specialist care may prevent hospital admissions for some patients. For patients admitted once, transitions expanded for a clinic-based internal medicine service which clinical experts identified as a Patient Centered Medical Home implemented in the first month for which we obtained data., Conclusions: We detected valid patterns consistent with a targeted care initiative, which experts could understand and explain, suggesting the method has utility for understanding coordination. The analysis revealed strong but complex patterns that could not be demonstrated using traditional linear methods alone. Network analysis supports measurement of real world health care service delivery, shows how transitions vary between services based on outcome, and with further development has potential to inform coordination strategies.

Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The glutathione thiol/disulfide couple is the major redox buffer in the endoplasmic reticulum (ER); however, mechanisms by which it contributes to the tightly regulated redox environment of this intracellular organelle are poorly understood. The recent development of genetically encoded, ratiometric, single green fluorescent protein-based redox-sensitive (roGFP) sensors adjusted for more oxidative environments enables non-invasive measurement of the ER redox environment in living cells. In turn, Förster resonance energy transfer (FRET) sensors based on two fluorophore probes represent an alternative strategy for ratiometric signal acquisition. In previous work, we described the FRET-based redox sensor CY-RL7 with a relatively high midpoint redox potential of -143 mV, which is required for monitoring glutathione potentials in the comparatively high oxidative environment of the ER. Here, the efficacy of the CY-RL7 probe was ascertained in the cytosol and ER of live cells with fluorescence microscopy and flow cytometry. The sensor was found to be fully reduced at steady state in the cytosol and became fully oxidized in response to treatment with 1-chloro-2,4-dinitrobenzene, a depletor of reduced glutathione (GSH). In contrast, the probe was strongly oxidized (88%) upon expression in the ER of cultured cells. We also examined the responsiveness of the ER sensor to perturbations in cellular glutathione homeostasis. We observed that the reductive level of the FRET sensor was increased two-fold to about 28% in cells pretreated with N-acetylcysteine, a substrate for GSH synthesis. Finally, we evaluated the responsiveness of CY-RL7 and roGFP1-iL to various perturbations of cellular glutathione homeostasis to address the divergence in the specificity of these two probes. Together, the present data generated with genetically encoded green fluorescent protein (GFP)-based glutathione probes highlight the complexity of the ER redox environment and indicate that the ER glutathione pool may be more oxidized than is currently considered.