CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-ß mimic.

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-ß mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-ß receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type-specific potency of TGF-ß signaling in mammalian cells.

Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Convergent evolution of a parasite-encoded complement control protein-scaffold to mimic binding of mammalian TGF-ß to its receptors, TßRI and TßRII.

The mouse intestinal helminth Heligmosomoides polygyrus modulates host immune responses by secreting a transforming growth factor (TGF)-ß mimic (TGM), to expand the population of Foxp3+ Tregs. TGM comprises five complement control protein (CCP)-like domains, designated D1-D5. Though lacking homology to TGF-ß, TGM binds directly to the TGF-ß receptors TßRI and TßRII and stimulates the differentiation of naïve T-cells into Tregs. However, the molecular determinants of binding are unclear. Here, we used surface plasmon resonance, isothermal calorimetry, NMR spectroscopy, and mutagenesis to investigate how TGM binds the TGF-ß receptors. We demonstrate that binding is modular, with D1-D2 binding to TßRI and D3 binding to TßRII. D1-D2 and D3 were further shown to compete with TGF-ß(TßRII)2 and TGF-ß for binding to TßRI and TßRII, respectively. The solution structure of TGM-D3 revealed that TGM adopts a CCP-like fold but is also modified to allow the C-terminal strand to diverge, leading to an expansion of the domain and opening potential interaction surfaces. TGM-D3 also incorporates a long structurally ordered hypervariable loop, adding further potential interaction sites. Through NMR shift perturbations and binding studies of TGM-D3 and TßRII variants, TGM-D3 was shown to occupy the same site of TßRII as bound by TGF-ß using both a novel interaction surface and the hypervariable loop. These results, together with the identification of other secreted CCP-like proteins with immunomodulatory activity in H. polygyrus, suggest that TGM is part of a larger family of evolutionarily plastic parasite effector molecules that mediate novel interactions with their host.

Modulation of haematopoiesis by protozoal and helminth parasites.

During inflammation, haematopoietic stem cells (HSCs) in the bone marrow (BM) and periphery rapidly expand and preferentially differentiate into myeloid cells that mediate innate immune responses. HSCs can be directed into quiescence or differentiation by sensing alterations to the haematopoietic niche, including cytokines, chemokines, and pathogen-derived products. Most studies attempting to identify the mechanisms of haematopoiesis have focused on bacterial and viral infections. From intracellular protozoan infections to large multicellular worms, parasites are a global health burden and represent major immunological challenges that remain poorly defined in the context of haematopoiesis. Immune responses to parasites vary drastically, and parasites have developed sophisticated immunomodulatory mechanisms that allow development of chronic infections. Recent advances in imaging, genomic sequencing, and mouse models have shed new light on how parasites induce unique forms of emergency haematopoiesis. In addition, parasites can modify the haematopoiesis in the BM and periphery to improve their survival in the host. Parasites can also induce long-lasting modifications to HSCs, altering future immune responses to infection, inflammation or transplantation, a term sometimes referred to as central trained immunity. In this review, we highlight the current understanding of parasite-induced haematopoiesis and how parasites target this process to promote chronic infections.

Helminth Imprinting of Hematopoietic Stem Cells Sustains Anti-Inflammatory Trained Innate Immunity That Attenuates Autoimmune Disease.

Certain proinflammatory stimuli can metabolically and epigenetically modify monocytes/macrophages or NK cells to be more responsive to secondary stimuli, a process known as trained innate immunity. However, the longevity of trained innate immunity is unclear. In this study, we report that Fasciola hepatica excretory-secretory products (FHES) can imprint an anti-inflammatory phenotype on long-term hematopoietic stem cells (HSCs) and monocyte precursor populations, enhancing their proliferation and differentiation into anti-inflammatory Ly6Clow monocytes. These monocytes expand and populate multiple compartments within mice, conferring hyporesponsiveness to proinflammatory stimuli and reduced susceptibility to induction of experimental autoimmune encephalomyelitis. Mice treated with FHES had enhanced alternatively activated macrophages, reduced Th1 and Th17 responses, and attenuating effects on autoimmunity that persisted for 8 mo. Furthermore, transplantation of HSCs from FHES-treated mice transferred the anti-inflammatory phenotype to naive mice. Our findings demonstrate that helminth products can modulate HSCs to promote development of anti-inflammatory myeloid cells that attenuate T cell-mediated autoimmune disease.

IL-33-Stimulated Murine Mast Cells Polarize Alternatively Activated Macrophages, Which Suppress T Cells That Mediate Experimental Autoimmune Encephalomyelitis.

IL-33 is known to promote type 2 immune responses through ST2, a component of the IL-33R complex, expressed primarily on mast cells, Th2 cells, group 2 innate lymphoid cells and regulatory T cells, and to a lesser extent, on NK cells and Th1 cells. Consistent with previous studies, we found that IL-33 polarized alternatively activated macrophages (AAMΦ) in vivo. However, in vitro stimulation of murine bone marrow-derived or peritoneal macrophages with IL-33 failed to promote arginase activity or expression of YM-1 or Retnla, markers of AAMΦ. Furthermore, macrophages have low/no basal expression of ST2. This suggested that alternative activation of macrophages may involve an IL-33-responsive third-party cell. Because mast cells have the highest expression of ST2 relative to other leukocytes, we focused on this cell type. Coculture experiments showed that IL-33-stimulated mast cells polarized AAMΦ through production of soluble factors. IL-33-stimulated mast cells produced a range of cytokines, including IL-6 and IL-13. Mast cell-derived IL-13 was required for induction of AAMΦ, whereas mast cell-derived IL-6 enhanced macrophage responsiveness to IL-13 via upregulation of the IL-4Rα receptor. Furthermore, we found that AAMΦ polarized by IL-33-stimulated mast cells could suppress proliferation and IL-17 and IFN-γ production by T cells. Finally, we show that AAMΦ polarized by IL-33-stimulated mast cells attenuated the encephalitogenic function of T cells in the experimental autoimmune encephalomyelitis model. Our findings reveal that IL-33 can promote immunosuppressive responses by polarizing AAMΦ via mast cell-derived IL-6 and IL-13.

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.

Predictive Accuracy of Quick Sequential Organ Failure Assessment for Hospital Mortality Decreases With Increasing Comorbidity Burden Among Patients Admitted for Suspected Infection.

OBJECTIVES: Evaluate the accuracy of the quick Sequential Organ Failure Assessment tool to predict mortality across increasing levels of comorbidity burden. DESIGN: Retrospective observational cohort study. SETTING: Twelve acute care hospitals in the Southeastern United States. PATIENTS: A total of 52,187 patients with suspected infection presenting to the Emergency Department between January 2014 and September 2017. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was hospital mortality. We used electronic health record data to calculate quick Sequential Organ Failure Assessment risk scores from vital signs and laboratory values documented during the first 24 hours. We calculated Charlson Comorbidity Index scores to quantify comorbidity burden. We constructed logistic regression models to evaluate differences in the performance of quick Sequential Organ Failure Assessment greater than or equal to 2 to predict hospital mortality in patients with no documented (Charlson Comorbidity Index = 0), low (Charlson Comorbidity Index = 1-2), moderate (Charlson Comorbidity Index = 3-4), or high (Charlson Comorbidity Index ≥ 5) comorbidity burden. Among the cohort, 2,030 patients died in the hospital (4%). No comorbidities were documented for 5,038 patients (10%), 9,235 patients (18%) had low comorbidity burden, 12,649 patients (24%) had moderate comorbidity burden, and 25,265 patients (48%) had high comorbidity burden. Overall model discrimination for quick Sequential Organ Failure Assessment greater than or equal to 2 was the area under the receiver operating characteristic curve of 0.71 (95% CI, 0.69-0.72). A model including both quick Sequential Organ Failure Assessment and Charlson Comorbidity Index had improved discrimination compared with Charlson Comorbidity Index alone (area under the receiver operating characteristic curve, 0.77; 95% CI, 0.76-0.78 vs area under the curve, 0.61; 95% CI, 0.59-0.62). Discrimination was highest among patients with no documented comorbidities (quick Sequential Organ Failure Assessment area under the receiver operating characteristic curve, 0.84; 95% CI; 0.79-0.89) and lowest among high comorbidity patients (quick Sequential Organ Failure Assessment area under the receiver operating characteristic curve, 0.67; 95% CI, 0.65-0.68). The strength of association between quick Sequential Organ Failure Assessment and mortality ranged from 30.5-fold increased likelihood in patients with no comorbidities to 4.7-fold increased likelihood in patients with high comorbidity. CONCLUSIONS: The accuracy of quick Sequential Organ Failure Assessment to predict hospital mortality diminishes with increasing comorbidity burden. Patients with comorbidities may have baseline abnormalities in quick Sequential Organ Failure Assessment variables that reduce predictive accuracy. Additional research is needed to better understand quick Sequential Organ Failure Assessment performance across different comorbid conditions with modification that incorporates the context of changes to baseline variables.

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.

We are what we eat: macrophages and efferocytosis.

Liebold et al. recently revealed how the identity of dying cells drives distinct changes to the macrophages which engulf and clear them, a process known as efferocytosis. During infection with the helminth Schistosoma mansoni, liver macrophages recapitulate these phenotypes, mediated by Axl/MerTK receptors and regulating egg burdens.

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.

Eastern Association for the Surgery of Trauma system wellness white paper: An evaluation of wellness from a systems perspective.

ABSTRACT: Providers are charged with responsibility to maintain their own health and wellness; however, well-being is multifactorial and the construct lacks clarity. In the current state of health care, burnout is on the rise with increasing demands for clinical productivity and strained system resources. The health care industry has recognized wellness's patient safety and financial impact, recently applying research and resources to identify sustainable solutions. We reviewed the wellness literature with a focus on systems to provide a framework for consensus building for a quality acute care surgery system. Our review revealed several areas within system wellness for consideration: (1) provider wellness, (2) culture of safety, (3) learning health systems, and (4) organizational perspectives. We provide specific system recommendations for the acute care surgery practice to preserve our workforce by creating a system that works for its providers.

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.

Extracorporeal Membrane Oxygenation During Surgical Stabilization of Rib Fractures in Successful Management of Severe Blunt Chest Trauma.

Early surgical stabilization of rib fracture (SSRF) improves outcomes in patients with flail physiology and severely displaced fractures. We present two cases of patients with severe chest injury and large flail segment who underwent SSRF while on veno-venous extracorporeal membrane oxygenation (VV-ECMO). The patients developed respiratory failure within 24 hours of admission requiring VV-ECMO. The extent of their chest wall injury limited pulmonary mechanics prohibiting transition off VV-ECMO. Therefore, SSRF was performed on hospital days 2 and 3 and while on VV-ECMO support. Stabilizing the chest wall allowed for improved ventilation and successful decannulation from VV-ECMO on postoperative days 3 and 4. Ultimately, both achieved a functional recovery and were discharged home. These cases demonstrate a unique thoracic damage control strategy wherein SSRF is performed while on VV-ECMO. Improving chest stability and pulmonary mechanics with SSRF allowed for safe transition off VV-ECMO and achieved a favorable long-term outcome.

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.

Surgical stabilization of rib fractures in the geriatric trauma population is associated with equivalent outcomes to a younger cohort: A propensity matched analysis.

BACKGROUND: Surgical stabilization of rib fractures (SSRF) improves outcomes in chest wall trauma. Geriatric patients are particularly vulnerable to poor outcomes; yet, this population is often excluded from SSRF studies. Further delineating patient outcomes by age is necessary to optimize care for the aging trauma population. METHODS: A retrospective cohort study was conducted examining outcomes among patients aged 40+ for whom an SSRF consult was placed between 2017 and 2022 at a level 1 trauma center. Patients were categorized into geriatric (65+) and adult (40-64), as well as 80 years and older (80+) and 79 and younger (40-79). Patient outcomes were assessed comparing non-operative and operative management of chest wall trauma. Propensity matched analysis was performed to evaluate mortality differences between adult and geriatric patients who did and did not undergo SSRF. RESULTS: A total of 543 patients had an SSRF consult. Of these, 227 were 65+, and 73 were 80+. A total of 129 patients underwent SSRF (24 %). The percentage of patients undergoing SSRF did not vary between 40 and 64 and 65+ (23.7 % and 23.6 %, respectively, p = 0.97) or 40-79 and 80+ (24.0 vs 21.9, p = 0.69). Patients undergoing SSRF had higher chest injury burden and were more likely to require mechanical ventilation and ICU level care on admission. Overall, in-hospital mortality rate was 4.6 %. Among patients who underwent SSRF, mortality rate did not significantly differ between 65+ and 40-64 (7.8% vs 2.7 %, p = 0.18) or 80+ and 40-79 (6.3% vs 4.6 %, p = 0.77). This remained true in propensity matched analysis. CONCLUSION: Geriatric and octogenarian patients with rib fractures underwent SSRF at similar rates and achieved equivalent outcomes to their younger counterparts. SSRF did not differentially affect mortality outcomes based on age group in propensity matched analysis. SSRF is safe for geriatric patients including octogenarians.