Handoffs and transitions of care: A systematic review, meta-analysis, and practice management guideline from the Eastern Association for the Surgery of Trauma.

BACKGROUND: The Joint Commission reports that at least half of communication breakdowns occur during handovers or transitions of care. There is no consensus on how best to approach the transfer of care within acute care surgery (ACS). We conduct a systematic review and meta-analysis of the current data on handoffs and transitions of care in ACS patients and evaluate the impact of standardization and formalized communication processes. METHODS: Clinically relevant questions regarding handoffs and transitions of care with clearly defined patient Population(s), Intervention(s), Comparison(s), and appropriately selected Outcomes were determined. These centered around specific transitions of care within the setting of ACS, specifically perioperative interactions, emergency medical services and trauma team interactions, and intra/interfloor and intensive care unit (ICU) interactions. A systematic literature review and meta-analysis were conducted using the Grading of Recommendations Assessment, Development, and Evaluation methodology. RESULTS: A total of 10 studies were identified for analysis. These included 5,113 patients in the standardized handoff group and 5,293 in the current process group. Standardized handoffs reduced handover errors for perioperative interactions and preventable adverse events for intra/interfloor and ICU interactions. There were insufficient data to evaluate outcomes of clinical complications and medical errors. CONCLUSION: We conditionally recommend a standardized handoff in the field of ACS, including perioperative interactions, emergency medical services and trauma team interactions, and intra/interfloor and ICU interactions. LEVEL OF EVIDENCE: Systematic Review/Meta-analysis; Level III.

Early warning: End-tidal carbon dioxide is associated with central venous oxygenation under continuous cardiorespiratory monitoring in a porcine model of hemorrhagic shock and resuscitation.

BACKGROUND: End-tidal carbon dioxide (ETCO2) has previously shown promise as a predictor of shock severity and mortality in trauma. ETCO2 monitoring is non-invasive, real-time, and readily available in prehospital settings, but the temporal relationship of ETCO2 to systemic oxygen transport has not been thoroughly investigated in the context of hemorrhagic shock. METHODS: A validated porcine model of hemorrhagic shock and resuscitation was used in male Yorkshire swine (N â€‹= â€‹7). Both ETCO2 and central venous oxygenation (SCVO2) were monitored and recorded continuously in addition to other traditional hemodynamic variables. RESULTS: Linear regression analysis showed that ETCO2 was associated with ScvO2 both throughout the experiment (ߠ​= â€‹1.783, 95% confidence interval (CI) [1.552-2.014], p â€‹< â€‹0.001) and during the period of most rapid hemorrhage (ߠ​= â€‹4.896, 95% CI [2.416-7.377], p â€‹< â€‹0.001) when there was a marked decrease in ETCO2. CONCLUSIONS: ETCO2 and ScvO2 were closely associated during rapid hemorrhage and continued to be temporally associated throughout shock and resuscitation.

Redefining pleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Redefining Pleiotropic Drug Resistance in a Pathogenic Yeast: Pdr1 Functions as a Sensor of Cellular Stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 ( CYB5 , SSK1 , SSK2 , HOG1 , TRP1 ). A bZIP transcription repressor of mitochondrial function ( CIN5 ) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata . Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. Importance: Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1 - a key determinant of fluconazole resistance - is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Timing of venous thromboembolism chemoprophylaxis using objective hemoglobin criteria in blunt solid organ injury.

BACKGROUND: The purpose of this study was to evaluate the safety and efficacy of early venous thromboembolism (VTE) chemoprophylaxis following blunt solid organ injury. METHODS: A retrospective review of patients was performed for patients with blunt solid organ injury between 2009-2019. Enoxaparin was initiated when patients had <1g/dl Hemoglobin decline over a 24 h period. These patients were then categorized by initiation: ≤48 h and >48 h. RESULTS: There were 653 patients: 328 (50.2%) <48 h and 325 (49.8%) ≥48 h. Twenty-nine (4.4%) developed VTE. Patients in ≥48 h group suffered more frequent VTE events (6.5% vs 2.4%, p = 0.021). Non-operative failure occurred in 6 patients (1.9%) in ≥48 h group, and 5 patients (1.5%) < 48 h group. Blood transfusion following chemophrophylaxis initiation was required in 69 (21.3%) in ≥48 h group, and 46 (14.0%) in < 48 h group, occurring similarly between groups (p=0.021). CONCLUSION: Stable hemoglobin in the first 24 h is an efficacious, objective measure that allows early initiation of VTE chemoprophylaxis in solid organ injury. This practice is associated with earlier initiation of and fewer VTE events.

Atrial Cannulation During Resuscitative Clamshell Thoracotomy.

BACKGROUND: Resuscitative thoracotomy and clamshell thoracotomy are performed in the setting of traumatic arrest with the intent of controlling hemorrhage, relieving tamponade, and providing open chest cardiopulmonary resuscitation. Historically, return of spontaneous circulation rates for penetrating traumatic arrest as well as out of hospital survival have been reported as low as 40% and 10%. Vascular access can be challenging in patients who have undergone a traumatic arrest and can be a limiting step to effective resuscitation. Atrial cannulation is a well-established surgical technique in cardiac surgery. Herein, we present a case series detailing our application of this technique in the context of acute trauma resuscitation during clamshell thoracotomy for traumatic arrest in the emergency department. METHODS: A retrospective case series of atrial cannulation during traumatic arrest was conducted in Charlotte, NC at Carolinas Medical Center an urban level 1 trauma center. RESULTS: The mean rate of return of spontaneous circulation in our series, 60%, was greater than previously published upper limit of return of spontaneous circulation for penetrating causes of traumatic arrest. DISCUSSION: Intravenous access can be difficult to establish in the hypovolemic and exsanguinating patient. Traditional methods of vascular access may be insufficient in the setting of central vascular injury. Atrial appendage cannulation during atrial cannulation is a quick and reliable technique to achieve vascular access that employs common methods from cardiac surgery to improve resuscitation of traumatic arrest.

Emergency General Surgery Regionalization: Retrospective Cohort Study of Emergency General Surgery Patients at a Tertiary Care Center.

BACKGROUND: Emergency general surgery (EGS) patients presenting at tertiary care hospitals may bypass local hospitals with adequate resources. However, many tertiary care hospitals frequently operate at capacity. We hypothesized that understanding patient geographic origin could identify opportunities for enhanced system triage and optimization and be an important first step for EGS regionalization and care coordination that could potentially lead to improved utilization of resources. METHODS: We analyzed patient zip code and categorized EGS patients who were cared for at our tertiary care hospital as potentially divertible if the southern region hospital was geographically closer to their home, regional hospital admission (RHA) patients, or local admission (LA) patients if the tertiary care facility was closer. Baseline characteristics and outcomes were compared for RHA and LA patients. RESULTS: Of 14 714 EGS patients presenting to the tertiary care hospital, 30.2% were categorized as RHA patients. Overall, 1526 (10.4%) patients required an operation including 527 (34.5%) patients who were potentially divertible. Appendectomy and cholecystectomy comprised 66% of the operations for potentially divertible patients. Length of stay was not significantly different (P = .06) for RHA patients, but they did have lower measured short-term and long-term mortality when compared to their LA counterparts (P < .05). CONCLUSIONS: EGS diagnoses and patient geocode analysis can identify opportunities to optimize regional operating room and bed utilization. Understanding where EGS patients are cared for and factors that influenced care facility will be critical for next steps in developing EGS regionalization within our system.

Emergencies do not shut down during a pandemic: COVID pandemic impact on Acute Care Surgery volume and mortality at a level I trauma center.

BACKGROUND: The aim of this study was to evaluate the impact of the COVID-19 pandemic on volume and outcomes of Acute Care Surgery patients, and we hypothesized that inpatient mortality would increase due to COVID+ and resource constraints. METHODS: An American College of Surgeons verified Level I Trauma Center's trauma and operative emergency general surgery (EGS) registries were queried for all patients from Jan. 2019 to Dec. 2020. April 1st, 2020, was the demarcation date for pre- and during COVID pandemic. Primary outcome was inpatient mortality. RESULTS: There were 14,460 trauma and 3091 EGS patients, and month-over-month volumes of both remained similar (p > 0.05). Blunt trauma decreased by 7.4% and penetrating increased by 31%, with a concomitant 25% increase in initial operative management (p < 0.001). Despite this, trauma (3.7%) and EGS (2.9-3.0%) mortality rates remained stable which was confirmed on multivariate analysis; p > 0.05. COVID + mortality was 8.8% and 3.7% in trauma and EGS patients, respectively. CONCLUSION: Acute Care Surgeons provided high quality care to trauma and EGS patients during the pandemic without allowing excess mortality despite many hardships and resource constraints.

Attention to detail: A dedicated rib fracture consultation service leads to earlier operation and improved clinical outcomes.

BACKGROUND: Surgical stabilization of rib fractures (SSRF) has been correlated with improved outcomes, including decreased length of stay (LOS). We hypothesized that an SSRF consultation service would increase the frequency of SSRF and improve outcomes. METHODS: A prospective observational study was performed to compare outcomes before and after implementing an SSRF service. Primary outcome was time from admission to surgery; secondary outcomes included LOS, mortality and morphine milligram equivalents (MME) prescribed at discharge. RESULTS: 1865 patients met consultation criteria and 128 patients underwent SSRF. Mortality decreased (6.3% vs. 3%) and patients were prescribed fewer MME at discharge (328 MME vs. 124 MME) following implementation. For the operative cohort, time from admission to surgery decreased by 1.72 days and ICU LOS decreased by 2.6 days. CONCLUSION: Establishment of an SSRF service provides a mechanism to maximize capture and evaluation of operative candidates, provide earlier intervention, and improve patient outcomes. Additional study to determine which elements and techniques are most beneficial is warranted. LEVEL OF EVIDENCE: III.

PATHway to success: Implementation of a multiprofessional acute trauma health care team decreased length of stay and cost in patients with neurological injury requiring tracheostomy.

BACKGROUND: The aim of this study was to determine whether the implementation of a dedicated multiprofessional acute trauma health care (mPATH) team would decrease length of stay without adversely impacting outcomes of patients with severe traumatic brain and spinal cord injuries. The mPATH team was comprised of a physical, occupational, speech, and respiratory therapist, nurse navigator, social worker, advanced care provider, and physician who performed rounds on the subset of trauma patients with these injuries from the intensive care unit to discharge. METHODS: Following the formation and implementation of the mPATH team at our Level I trauma center, a retrospective cohort study was performed comparing patients in the year immediately prior to the introduction of the mPATH team (n = 60) to those in the first full year following implementation (n = 70). Demographics were collected for both groups. Inclusion criteria were Glasgow Coma Scale score less than 8 on postinjury Day 2, all paraplegic and quadriplegic patients, and patients older than 55 years with central cord syndrome who underwent tracheostomy. The primary endpoint was length of stay; secondary endpoints were time to tracheostomy, days to evaluation by occupational, physical, and speech therapy, 30-day readmission, and 30-day mortality. RESULTS: The median time to evaluation by occupational, physical, and speech therapy was universally decreased. Injury Severity Score was 27 in both cohorts. Time to tracheostomy and length of stay were both decreased. Thirty-day readmission and mortality rates remained unchanged. A cost savings of US $11,238 per index hospitalization was observed. CONCLUSION: In the year following the initiation of the mPATH team, we observed earlier time to occupational, physical, and speech therapist evaluation, decreased length of stay, and cost savings in severe traumatic brain and spinal cord injury patients requiring tracheostomy compared with our historical control. These benefits were observed without adversely impacting 30-day readmission or mortality. LEVEL OF EVIDENCE: Therapeutic/care management, Level III.

Not a Routine Case, Why Expect the Routine Outcome? Quantifying the Infectious Burden of Emergency General Surgery Using the NSQIP.

Emergent surgeries have different causes and physiologic patient responses than the same elective surgery, many of which are due to infectious etiologies. Therefore, we hypothesized that emergency cases have a higher risk of postoperative SSI than their elective counterparts. The ACS NSQIP database was queried from 2005 to 2016 for all cholecystectomies, ventral hernia repairs, and partial colectomies to examine common emergency and elective general surgery operations. Thirty-day outcomes were compared by emergent status. Any SSI was the primary outcome. There were 863,164 surgeries: 416,497 cholecystectomies, 220,815 ventral hernia repairs, and 225,852 partial colectomies. SSIs developed in 38,865 (4.5%) patients. SSIs increased with emergencies (5.3% vs 3.6% for any SSI). Postoperative sepsis (5.8% vs 1.5%), septic shock (4.7% vs 0.6%), length of stay (8.1 vs 2.9 days), and mortality (3.6% vs 0.4%) were increased in emergent surgery; P < 0.001 for all. When controlling for age, gender, BMI, diabetes, smoking, wound classification, comorbidities, functional status, and procedure on multivariate analysis, emergency surgery (odds ratio 1.15, 95% confidence interval 1.11-1.19) was independently associated with the development of SSI. Patients undergoing emergency general surgery experience increased rates of SSI. Patients and their families should be appropriately counseled regarding these elevated risks when consenting for emergency surgery.

Filled to the brim: The characteristics of over-triage at a level I trauma center.

BACKGROUND: Interfacility transfers are necessary and valuable for the trauma system, but despite regional guidelines, many patients are inappropriately transferred. We evaluated over-triage at our Level I center and identified risk factors for over-triage. METHODS: Retrospective analysis at our Level I urban trauma center assessed patients transferred from regional facilities during 2017. Over-triage was defined as patients discharged <48 h without procedures. Exclusion criteria were leaving against medical advice or no outside records. RESULTS: Overall, 2352 patients met criteria. Nine hundred thirty (39.5%) with complete hospital records were discharged in <48 h; 498 (53.5%) received no procedural intervention and 909 (97.7%) were ultimately discharged home. CONCLUSION: Many patients are inappropriately transferred to tertiary care centers without a definitive need for advanced services. Studies are needed to improve triage criteria without increasing under-triage.

Infection after penetrating brain injury-An Eastern Association for the Surgery of Trauma multicenter study oral presentation at the 32nd annual meeting of the Eastern Association for the Surgery of Trauma, January 15-19, 2019, in Austin, Texas.

BACKGROUND: Fatality rates following penetrating traumatic brain injury (pTBI) are extremely high and survivors are often left with significant disability. Infection following pTBI is associated with worse morbidity. The modern rates of central nervous system infections (INF) in civilian survivors are unknown. This study sought to determine the rate of and risk factors for INF following pTBI and to determine the impact of antibiotic prophylaxis. METHODS: Seventeen institutions submitted adult patients with pTBI and survival of more than 72 hours from 2006 to 2016. Patients were stratified by the presence or absence of infection and the use or omission of prophylactic antibiotics. Study was powered at 85% to detect a difference in infection rate of 5%. Primary endpoint was the impact of prophylactic antibiotics on INF. Mantel-Haenszel χ and Wilcoxon's rank-sum tests were used to compare categorical and nonparametric variables. Significance greater than p = 0.2 was included in a logistic regression adjusted for center. RESULTS: Seven hundred sixty-three patients with pTBI were identified over 11 years. 7% (n = 51) of patients developed an INF. Sixty-six percent of INF patients received prophylactic antibiotics. Sixty-two percent of all patients received one dose or greater of prophylactic antibiotics and 50% of patients received extended antibiotics. Degree of dural penetration did not appear to impact the incidence of INF (p = 0.8) nor did trajectory through the oropharynx (p = 0.18). Controlling for other variables, there was no statistically significant difference in INF with the use of prophylactic antibiotics (p = 0.5). Infection was higher in patients with intracerebral pressure monitors (4% vs. 12%; p = <0.001) and in patients with surgical intervention (10% vs. 3%; p < 0.001). CONCLUSION: There is no reduction in INF with prophylactic antibiotics in pTBI. Surgical intervention and invasive intracerebral pressure monitoring appear to be risk factors for INF regardless of prophylactic use. LEVEL OF EVIDENCE: Therapeutic, level IV.

Advancing the Use of Laparoscopy in Trauma: Repair of Intraperitoneal Bladder Injuries.

Traumatic intraperitoneal bladder rupture (IBR) requires surgical repair. Traditionally performed via laparotomy, experience with laparoscopic bladder repair (LBR) after blunt trauma is limited. Benefits of laparoscopy include decreased length of stay (LOS), less pain, early return to work, fewer adhesions, and lower risk of incisional hernia. The aim of this series is to demonstrate the potential superiority of LBR in select trauma patients. This is a retrospective review performed of all IBR patients from 2008 to 2016. Demographics, clinical management, outcomes, and follow-up were compared between LBR and open bladder repair (OBR) patients. Twenty patients underwent OBR, and seven underwent LBR. There was no significant difference in gender, age, or Injury Severity Score. There were no deaths or reoperations in either group. Average hospital length of stay and ICU days were similar between groups. There was one patient with UTI and one with readmission in each group. There were two incisional hernias and two bowel obstructions in the OBR group, with one patient requiring operative intervention. No such complications occurred in the LBR group. LBR for traumatic IBR can be safely performed in select patients, even in those with multiple extra-abdominal injuries.

Returning from the acidotic abyss: Mortality in trauma patients with a pH < 7.0.

INTRODUCTION: We hypothesized that a pH of <7.0 on presentation would correlate with almost universal mortality in trauma patients. METHODS: A retrospective cohort study was performed on a Level I trauma center registry from 2013 to 2014. Hospital mortality was the primary outcome, which was compared by pH cohort (<7.0 or ≥7.0) using standard univariate statistics and multivariate logistic regression. RESULTS: There were 593 patients included in the analysis: 66 in <7.0, 527 in ≥7.0. Mortality was 3× higher in the <7.0 pH cohort (62.1 vs. 20.3%; p < 0.0001), however there was no threshold for a pH below which there was 100% mortality. After controlling for these confounding variables, initial pH was found to be an independent predictor of inpatient mortality: pH < 7.0 (OR 6.33, 3.29-12.19; p < 0.0001). CONCLUSION: This data indicates that while patients with severe acidosis are at increased risk for mortality, a pH < 7.0 is still recoverable in select cases.

H+ and Pi Byproducts of Glycosylation Affect Ca2+ Homeostasis and Are Retrieved from the Golgi Complex by Homologs of TMEM165 and XPR1.

Glycosylation reactions in the Golgi complex and the endoplasmic reticulum utilize nucleotide sugars as donors and produce inorganic phosphate (Pi) and acid (H+) as byproducts. Here we show that homologs of mammalian XPR1 and TMEM165 (termed Erd1 and Gdt1) recycle luminal Pi and exchange luminal H+ for cytoplasmic Ca2+, respectively, thereby promoting growth of yeast cells in low Pi and low Ca2+ environments. As expected for reversible H+/Ca2+ exchangers, Gdt1 also promoted growth in high Ca2+ environments when the Golgi-localized V-ATPase was operational but had the opposite effect when the V-ATPase was eliminated. Gdt1 activities were negatively regulated by calcineurin signaling and by Erd1, which recycled the Pi byproduct of glycosylation reactions and prevented the loss of this nutrient to the environment via exocytosis. Thus, Erd1 transports Pi in the opposite direction from XPR1 and other EXS family proteins and facilitates byproduct removal from the Golgi complex together with Gdt1.

A LAPF/phafin1-like protein regulates TORC1 and lysosomal membrane permeabilization in response to endoplasmic reticulum membrane stress.

Lysosomal membrane permeabilization (LMP) is a poorly understood regulator of programmed cell death that involves leakage of luminal lysosomal or vacuolar hydrolases into the cytoplasm. In Saccharomyces cerevisiae, LMP can be induced by antifungals and endoplasmic reticulum stressors when calcineurin also has been inactivated. A genome-wide screen revealed Pib2, a relative of LAPF/phafin1 that regulates LMP in mammals, as a pro-LMP protein in yeast. Pib2 associated with vacuolar and endosomal limiting membranes in unstressed cells in a manner that depended on its FYVE domain and on phosphatidylinositol 3-phosphate (PI(3)P) biosynthesis. Genetic experiments suggest that Pib2 stimulates the activity of TORC1, a vacuole-associated protein kinase that is sensitive to rapamycin, in a pathway parallel to the Ragulator/EGO complex containing the GTPases Gtr1 and Gtr2. A hyperactivating mutation in the catalytic subunit of TORC1 restored LMP to the gtr1∆ and pib2∆ mutants and also prevented the synthetic lethality of the double mutants. These findings show novel roles of PI(3)P and Pib2 in the regulation of TORC1, which in turn promoted LMP and nonapoptotic death of stressed cells. Rapamycin prevented the death of the pathogenic yeast Candida albicans during exposure to fluconazole plus a calcineurin inhibitor, suggesting that TORC1 broadly promotes sensitivity to fungistats in yeasts.

Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters.

In the budding yeast Saccharomyces cerevisiae, mating pheromones activate a high-affinity Ca(2+) influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K(+) and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K(+) but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K(+) environments, suggesting they promote K(+) uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K(+) currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K(+) currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K(+) influx and that HACS may electrochemically respond to K(+) influx in much the same way as the homologous voltage-gated Ca(2+) channels in most animal cell types.

Multiple signaling pathways regulate yeast cell death during the response to mating pheromones.

Mating pheromones promote cellular differentiation and fusion of yeast cells with those of the opposite mating type. In the absence of a suitable partner, high concentrations of mating pheromones induced rapid cell death in approximately 25% of the population of clonal cultures independent of cell age. Rapid cell death required Fig1, a transmembrane protein homologous to PMP-22/EMP/MP20/Claudin proteins, but did not require its Ca2+ influx activity. Rapid cell death also required cell wall degradation, which was inhibited in some surviving cells by the activation of a negative feedback loop involving the MAP kinase Slt2/Mpk1. Mutants lacking Slt2/Mpk1 or its upstream regulators also underwent a second slower wave of cell death that was independent of Fig1 and dependent on much lower concentrations of pheromones. A third wave of cell death that was independent of Fig1 and Slt2/Mpk1 was observed in mutants and conditions that eliminate calcineurin signaling. All three waves of cell death appeared independent of the caspase-like protein Mca1 and lacked certain "hallmarks" of apoptosis. Though all three waves of cell death were preceded by accumulation of reactive oxygen species, mitochondrial respiration was only required for the slowest wave in calcineurin-deficient cells. These findings suggest that yeast cells can die by necrosis-like mechanisms during the response to mating pheromones if essential response pathways are lacking or if mating is attempted in the absence of a partner.