Milk biosynthesis requires the Golgi cation exchanger TMEM165.

Milk is a hallmark of mammals that is critical for normal growth and development of offspring. During biosynthesis of lactose in the Golgi complex, H+ is produced as a by-product, and there is no known mechanism for maintaining luminal pH within the physiological range. Here, using conditional, tissue-specific knockout mice, immunostaining, and biochemical assays, we test whether the putative H+/Ca2+/Mn2+ exchanger known as TMEM165 (transmembrane protein 165) participates in normal milk production. We find TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and for normal growth rates of nursing pups. The milk of TMEM165-deficient mice contained elevated concentrations of fat, protein, iron, and zinc, which are likely caused by decreased osmosis-mediated dilution of the milk caused by the decreased biosynthesis of lactose. When normalized to total protein levels, only calcium and manganese levels were significantly lower in the milk from TMEM165-deficient dams than control dams. These findings suggest that TMEM165 supplies Ca2+ and Mn2+ to the Golgi complex in exchange for H+ to sustain the functions of lactose synthase and potentially other glycosyl-transferases. Our findings highlight the importance of cation and pH homeostasis in the Golgi complex of professional secretory cells and the critical role of TMEM165 in this process.

Comparing the utility of in vivo transposon mutagenesis approaches in yeast species to infer gene essentiality.

In vivo transposon mutagenesis, coupled with deep sequencing, enables large-scale genome-wide mutant screens for genes essential in different growth conditions. We analyzed six large-scale studies performed on haploid strains of three yeast species (Saccharomyces cerevisiae, Schizosaccaromyces pombe, and Candida albicans), each mutagenized with two of three different heterologous transposons (AcDs, Hermes, and PiggyBac). Using a machine-learning approach, we evaluated the ability of the data to predict gene essentiality. Important data features included sufficient numbers and distribution of independent insertion events. All transposons showed some bias in insertion site preference because of jackpot events, and preferences for specific insertion sequences and short-distance vs long-distance insertions. For PiggyBac, a stringent target sequence limited the ability to predict essentiality in genes with few or no target sequences. The machine learning approach also robustly predicted gene function in less well-studied species by leveraging cross-species orthologs. Finally, comparisons of isogenic diploid versus haploid S. cerevisiae isolates identified several genes that are haplo-insufficient, while most essential genes, as expected, were recessive. We provide recommendations for the choice of transposons and the inference of gene essentiality in genome-wide studies of eukaryotic haploid microbes such as yeasts, including species that have been less amenable to classical genetic studies.

Redefining Mild Traumatic Brain Injury (mTBI) delineates cost effective triage.

OBJECTIVES: Mild traumatic brain injury (mTBI) is defined as Glasgow Coma Score (GCS) of 14 or 15. Despite good outcomes, patients are commonly transferred to trauma centers for observation and/or neurosurgical consultation. The aim of this study is to assess the value of redefining mTBI with novel radiographic criteria to determine the appropriateness of interhospital transfer for neurosurgical evaluation. METHODS: A retrospective study of patients with blunt head injury with GCS 13-15 and CT head from Jan 2014-Dec 2016 was performed. A novel criteria of head CT findings was created at our institution to classify mTBI. Outcomes included neurosurgical intervention and transfer cost. RESULTS: A total of 2120 patients were identified with 1442 (68.0%) meeting CT criteria for mTBI and 678 (32.0%) classified high risk. Two (0.14%) patients with mTBI required neurosurgical intervention compared with 143 (21.28%) high risk TBI (p < 0.0001). Mean age (55.8 years), and anticoagulation (2.6% vs 2.8%) or antiplatelet use (2.1% vs 3.0%) was similar between groups (p > 0.05). Of patients with mTBI, 689 were transferred without receiving neurosurgical intervention. Given an average EMS transfer cost of $700 for ground and $5800 for air, we estimate an unnecessary transfer cost of $733,600. CONCLUSION: Defining mTBI with the described novel criteria clearly identifies patients who can be safely managed without transfer for neurosurgical consultation. These unnecessary transfers represent a substantial financial and resource burden to the trauma system and inconvenience to patients.

CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans.

BACKGROUND: Saccharomyces cerevisiae ScGdt1 and mammalian TMEM165 are two members of the UPF0016 membrane protein family that is likely to form a new group of Ca2+/H+ antiporter and/or a Mn2+ transporter in the Golgi apparatus. We have previously shown that Candida albicans CaGDT1 is a functional ortholog of ScGDT1 in the response of S. cerevisiae to calcium stress. However, how CaGdt1 together with the Golgi calcium pump CaPmr1 regulate calcium homeostasis and cell wall integrity in this fungal pathogen remains unknown. METHODS: Chemical sensitivity was tested by dilution assay. Cell survival was examined by measuring colony-forming units and staining with Annexin V-FITC and propidium iodide. Calcium signaling was examined by expression of downstream target gene CaUTR2, while cell wall integrity signaling was revealed by detection of phosphorylated Mkc1 and Cek1. Subcellular localization of CaGdt1 was examined through direct and indirect immunofluorescent approaches. Transcriptomic analysis was carried out with RNA sequencing. RESULTS: This study shows that Candida albicans CaGDT1 is also a functional ortholog of ScGDT1 in the response of S. cerevisiae to cell wall stress. CaGdt1 is localized in the Golgi apparatus but at distinct sites from CaPmr1 in C. albicans. Loss of CaGDT1 increases the sensitivity of cell lacking CaPMR1 to cell wall and ER stresses. Deletion of CaGDT1 and/or CaPMR1 increases calcium uptake and activates the calcium/calcineurin signaling. Transcriptomic profiling reveals that core functions shared by CaGdt1 and CaPmr1 are involved in the regulation of cellular transport of metal ions and amino acids. However, CaGdt1 has distinct functions from CaPmr1. Chitin synthase gene CHS2 is up regulated in all three mutants, while CHS3 is only up regulated in the pmr1/pmr1 and the gdt1/gdt1 pmr1/pmr1 mutants. Five genes (DIE2, STT3, OST3, PMT1 and PMT4) of glycosylation pathway and one gene (SWI4) of the cell wall integrity (CWI) pathway are upregulated due to deletion of CaGDT1 and/or CaPMR1. Consistently, deletion of either CaPMR1 or CaGDT1 activates the CaCek1-mediated CWI signaling in a cell wall stress-independent fashion. Calcineurin function is required for the integrity of the cell wall and vacuolar compartments of cells lacking both GDT1 and CaPMR1. CONCLUSIONS: CaPmr1 is the major player in the regulation of calcium homeostasis and cell wall stress, while CaGdt1 plays a compensatory role for CaPmr1 in the Golgi compartment in C. albicans.

Kch1 family proteins mediate essential responses to endoplasmic reticulum stresses in the yeasts Saccharomyces cerevisiae and Candida albicans.

The activation of a high affinity Ca(2+) influx system (HACS) in the plasma membrane is required for survival of yeast cells exposed to natural or synthetic inhibitors of essential processes (secretory protein folding or sterol biosynthesis) in the endoplasmic reticulum (ER). The mechanisms linking ER stress to HACS activation are not known. Here we show that Kch1, a recently identified low affinity K(+) transporter in the plasma membrane of Saccharomyces cerevisiae, is up-regulated in response to several ER stressors and necessary for HACS activation. The activation of HACS required extracellular K(+) and was also dependent on the high affinity K(+) transporters Trk1 and Trk2. However, a paralog of Kch1 termed Kch2 was not expressed and not necessary for HACS activation in these conditions. The pathogenic yeast Candida albicans carries only one homolog of Kch1/Kch2, and homozygous knock-out mutants were similarly deficient in the activation of HACS during the responses to tunicamycin. However, the Kch1 homolog was not necessary for HACS activation or cell survival in response to several clinical antifungals (azoles, allylamines, echinocandins) that target the ER or cell wall. Thus, Kch1 family proteins represent a conserved linkage between HACS and only certain classes of ER stress in these 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.

The ReInvigorate Study-phrenic nerve-to-diaphragm stimulation for weaning from mechanical ventilation: a protocol for a randomized pivotal clinical trial.

BACKGROUND: In the United States in 2017, there were an estimated 903,745 hospitalizations involving mechanical ventilation (MV). Complications from ventilation can result in longer hospital stays, increased risk of disability, and increased healthcare costs. It has been hypothesized that electrically pacing the diaphragm by phrenic nerve stimulation during mechanical ventilation may minimize or reverse diaphragm dysfunction, resulting in faster weaning. METHODS: The ReInvigorate Trial is a prospective, multicenter, randomized, controlled clinical trial evaluating the safety and efficacy of Stimdia's pdSTIM System for facilitating weaning from MV. The pdSTIM system employs percutaneously placed multipolar electrodes to stimulate the cervical phrenic nerves and activate contraction of the diaphragm bilaterally. Patients who were on mechanical ventilation for at least 96 h and who failed at least one weaning attempt were considered for enrollment in the study. The primary efficacy endpoint was the time to successful liberation from mechanical ventilation (treatment vs. control). Secondary endpoints will include the rapid shallow breathing index and other physiological and system characteristics. Safety will be summarized for both primary and additional analyses. All endpoints will be evaluated at 30 days or at the time of removal of mechanical ventilation, whichever is first. DISCUSSION: This pivotal study is being conducted under an investigational device exception with the U.S. Food and Drug Administration. The technology being studied could provide a first-of-kind therapy for difficult-to-wean patients on mechanical ventilation in an intensive care unit setting. TRIAL REGISTRATION: Clinicaltrials.gov, NCT05998018 , registered August 2023.

Tn-seq of the Candida glabrata reference strain CBS138 reveals epigenetic plasticity, structural variation, and intrinsic mechanisms of resistance to micafungin.

C. glabrata is an opportunistic pathogen that can resist common antifungals and rapidly acquire multidrug resistance. A large amount of genetic variation exists between isolates, which complicates generalizations. Portable Tn-seq methods can efficiently provide genome-wide information on strain differences and genetic mechanisms. Using the Hermes transposon, the CBS138 reference strain and a commonly studied derivative termed 2001 were subjected to Tn-seq in control conditions and after exposure to varying doses of the clinical antifungal micafungin. The approach revealed large differences between these strains, including a 131 kb tandem duplication and a variety of fitness differences. Additionally, both strains exhibited up to 1000-fold increased transposon accessibility in subtelomeric regions relative to the BG2 strain, indicative of open subtelomeric chromatin in these isolates and large epigenetic variation within the species. Unexpectedly, the Pdr1 transcription factor conferred resistance to micafungin through targets other than CDR1 . Other micafungin resistance pathways were also revealed including mannosyltransferase activity and biosynthesis of the lipid precursor sphingosine, the drugging of which by SDZ 90-215 or myriocin enhanced the potency of micafungin in vitro . These findings provide insights into complexity of the C. glabrata species as well as strategies for improving antifungal efficacy. Summary: Candida glabrata is an emerging pathogen with large genetic diversity and genome plasticity. The type strain CBS138 and a laboratory derivative were mutagenized with the Hermes transposon and profiled using Tn-seq. Numerous genes that regulate innate and acquired resistance to an important clinical antifungal were uncovered, including a pleiotropic drug resistance gene (PDR1) and a duplication of part of one chromosome. Compounds that target PDR1 and other genes may augment the potency of existing antifungals.

Tn-seq of the Candida glabrata reference strain CBS138 reveals epigenetic plasticity, structural variation, and intrinsic mechanisms of resistance to micafungin.

Candida glabrata (also called Nakaseomyces glabratus) is an opportunistic pathogen that can resist common antifungals and rapidly acquire multidrug resistance. A large amount of genetic variation exists between isolates, which complicates generalizations. Portable Tn-seq methods can efficiently provide genome-wide information on strain differences and genetic mechanisms. Using the Hermes transposon, the CBS138 reference strain and a commonly studied derivative termed 2001 were subjected to Tn-seq in control conditions and after exposure to varying doses of the clinical antifungal micafungin. The approach revealed large differences between these strains, including a 131 kb tandem duplication and a variety of fitness differences. Additionally, both strains exhibited up to 1000-fold increased transposon accessibility in subtelomeric regions relative to the BG2 strain, indicative of open subtelomeric chromatin in these isolates and large epigenetic variation within the species. Unexpectedly, the Pdr1 transcription factor conferred resistance to micafungin through targets other than CDR1. Other micafungin resistance pathways were also revealed including mannosyltransferase activity and biosynthesis of the lipid precursor sphingosine, the inhibition of which by SDZ 90-215 and myriocin enhanced the potency of micafungin in vitro. These findings provide insights into complexity of the C. glabrata species as well as strategies for improving antifungal efficacy.

Calcineurin-dependent contributions to fitness in the opportunistic pathogen Candida glabrata.

The protein phosphatase calcineurin is vital for the virulence of the opportunistic fungal pathogen Candida glabrata. The host-induced stresses that activate calcineurin signaling are unknown, as are the targets of calcineurin relevant to virulence. To potentially shed light on these processes, millions of transposon insertion mutants throughout the genome of C. glabrata were profiled en masse for fitness defects in the presence of FK506, a specific inhibitor of calcineurin. Eighty-seven specific gene deficiencies depended on calcineurin signaling for full viability in vitro both in wild-type and pdr1∆ null strains lacking pleiotropic drug resistance. Three genes involved in cell wall biosynthesis (FKS1, DCW1, FLC1) possess co-essential paralogs whose expression depended on calcineurin and Crz1 in response to micafungin, a clinical antifungal that interferes with cell wall biogenesis. Interestingly, 80% of the FK506-sensitive mutants were deficient in different aspects of vesicular trafficking, such as endocytosis, exocytosis, sorting, and biogenesis of secretory proteins in the endoplasmic reticulum (ER). In response to the experimental antifungal manogepix that blocks GPI-anchor biosynthesis in the ER, calcineurin signaling increased and strongly prevented cell death independent of Crz1, one of its major targets. Comparisons between manogepix, micafungin, and the ER-stressing tunicamycin reveal a correlation between the degree of calcineurin signaling and the degree of cell survival. These findings suggest that calcineurin plays major roles in mitigating stresses of vesicular trafficking. Such stresses may arise during host infection and in response to antifungal therapies.IMPORTANCECalcineurin plays critical roles in the virulence of most pathogenic fungi. This study sheds light on those roles in the opportunistic pathogen Candida glabrata using a genome-wide analysis in vitro. The findings could lead to antifungal developments that also avoid immunosuppression.

Development of a predictive algorithm for patient survival after traumatic injury using a five analyte blood panel.

Severe trauma can induce systemic inflammation but also immunosuppression, which makes understanding the immune response of trauma patients critical for therapeutic development and treatment approaches. By evaluating the levels of 59 proteins in the plasma of 50 healthy volunteers and 1000 trauma patients across five trauma centers in the United States, we identified 6 novel changes in immune proteins after traumatic injury and further new variations by sex, age, trauma type, comorbidities, and developed a new equation for prediction of patient survival. Blood was collected at the time of arrival at Level 1 trauma centers and patients were stratified based on trauma level, tissues injured, and injury types. Trauma patients had significantly upregulated proteins associated with immune activation (IL-23, MIP-5), immunosuppression (IL-10) and pleiotropic cytokines (IL-29, IL-6). A high ratio of IL-29 to IL-10 was identified as a new predictor of survival in less severe patients with ROC area of 0.933. Combining machine learning with statistical modeling we developed an equation ("VIPER") that could predict survival with ROC 0.966 in less severe patients and 0.8873 for all patients from a five analyte panel (IL-6, VEGF-A, IL-21, IL-29, and IL-10). Furthermore, we also identified three increased proteins (MIF, TRAIL, IL-29) and three decreased proteins (IL-7, TPO, IL-8) that were the most important in distinguishing a trauma blood profile. Biologic sex altered phenotype with IL-8 and MIF being lower in healthy women, but higher in female trauma patients when compared to male counterparts. This work identifies new responses to injury that may influence systemic immune dysfunction, serving as targets for therapeutics and immediate clinical benefit in identifying at-risk patients.

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.

Vacuolar H+-ATPase (V-ATPase) promotes vacuolar membrane permeabilization and nonapoptotic death in stressed yeast.

Stress in the endoplasmic reticulum caused by tunicamycin, dithiothreitol, and azole-class antifungal drugs can induce nonapoptotic cell death in yeasts that can be blocked by the action of calcineurin (Cn), a Ca(2+)-dependent serine/threonine protein phosphatase. To identify additional factors that regulate nonapoptotic cell death in yeast, a collection of gene knock-out mutants was screened for mutants exhibiting altered survival rates. The screen revealed an endocytic protein (Ede1) that can function upstream of Ca(2+)/calmodulin-dependent protein kinase 2 (Cmk2) to suppress cell death in parallel to Cn. The screen also revealed the vacuolar H(+)-ATPase (V-ATPase), which acidifies the lysosome-like vacuole. The V-ATPase performed its death-promoting functions very soon after imposition of the stress and was not required for later stages of the cell death program. Cn did not inhibit V-ATPase activities but did block vacuole membrane permeabilization (VMP), which occurred at late stages of the cell death program. All of the other nondying mutants identified in the screens blocked steps before VMP. These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons.

The Association of the Affordable Care Act With Medicaid Enrollment Status and Costs of Care at a Level I Trauma Center in a Medicaid Non-expansion State.

INTRODUCTION: The intended purpose of the Patient Protection and Affordable Care Act (ACA) was to expand access to health care insurance for all Americans. In our study, we examine the association of Medicaid enrollment status, health care outcomes, and financial outcomes for trauma patients at a level I urban trauma center in a state that did not expand Medicaid coverage under the ACA. METHODS: We retrospectively reviewed trauma admissions from 2011 to 2016, via the trauma registry (n = 36,250). A subgroup of Medicaid patients (n = 8840) was identified and compared for changes in selected variables and demographics following ACA implementation. The association of Medicaid payor status, by 3 year average pre-ACA (n = 3516) and post-ACA (n = 3324), on patient outcomes, payments collected, and accrued costs of care were analyzed. RESULTS: Three-year Medicaid median actual payments decreased 7.5% following implementation of the ACA ($4072 vs. $3767, P < .01). In contrast, the Medicaid median total cost of care increased 23% ($3964 vs. $4882, P < .01). The rate of patients insured by Medicaid decreased (24.0% vs. 16.2%, P<.001). Patients were admitted longer (1 d vs. 2 d, P < .01), and more injured (ISS 5 vs. 6, P < .01). DISCUSSION: Medicaid payor status under the ACA was associated with a decrease in actual payments and an increase in total cost of care. Moreover, the divergence in actual payments collected with the increased total cost of care warrants examination to ascertain the root cause in efforts to reduce this widening gap.

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.

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.

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.

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.