A Multicenter, Retrospective Outcome Analysis of Vancomycin Area Under the Curve versus Trough-Based Dosing Strategies in Patients with Burn OR Inhalational Injuries (MONITOR).

Vancomycin is a glycopeptide antibiotic that requires close therapeutic monitoring. Prolonged exposure to elevated concentrations increases risk for serious adverse effects such as nephrotoxicity. However, sub-therapeutic concentrations may lead to bacterial resistance and clinical failure or death. The most recent Infectious Diseases Society of America (IDSA) publication regarding therapeutic monitoring of vancomycin recommends utilizing area under the curve (AUC)-based monitoring to maximize clinical success. Despite the guideline recommendation for AUC-guided dosing, many institutions still use trough-only monitoring in their practices, including those caring for patients with acute burn injuries. Following burn injury, patients are at a higher risk for infections, multi-organ failure, and pharmacokinetic alterations. The primary objective of this multi-center retrospective study is to determine optimal therapeutic monitoring of vancomycin by comparing clinical success between AUC vs. trough-based monitoring in burn patients. MONITOR was a multicenter, retrospective study of patients with thermal or inhalation injury admitted to one of 13 burn centers from 1/1/17 to 8/31/22 who received vancomycin. Demographic and clinical course data, including acute kidney injury (AKI) incidence and clinical success were obtained. Patients were evaluated for clinical success and grouped according to method of monitoring and adjusting doses: AUC vs. trough-based monitoring. Clinical success was a composite definition and lack of meeting any 1 of 5 criteria: 1) persistent infection, 2) relapse, 3) antibiotic failure (clinical worsening), 4) AKI, 5) death. Five-hundred seventeen vancomycin courses were assessed from 485 patients. There was no difference in the rate of clinical success between AUC monitored and the trough-only monitored groups. Incidence of AKI was higher in the trough-only group; however, was not statistically significant after controlling for renal function on admission, past medical history of chronic kidney disease (CKD), and concomitant nephrotoxins.

Effect of Germination on Seed Protein Quality and Secondary Metabolites and Potential Modulation by Pulsed Electric Field Treatment.

Plant-based foods are being increasingly favored to feed the ever-growing population, but these need to exhibit improved nutritional value in terms of protein quality and digestibility to be considered a useful alternative to animal-based foods. Germination is essential for plant growth and represents a viable method through which the protein quality of plants can be further improved. However, it will be a challenge to maintain efficient rates of germination in a changing climate when seeds are sown. In the context of the indoor germination of seeds for food, consumption, or processing purposes, a more efficient and sustainable process is desired. Therefore, novel techniques to facilitate seed germination are required. Pulsed electric fields (PEF) treatment of seeds results in the permeabilization of the cell membrane, allowing water to be taken up more quickly and triggering biochemical changes to the macromolecules in the seed during germination. Therefore, PEF could be a chemical-free approach to induce a stress response in seeds, leading to the production of secondary metabolites known to exert beneficial effects on human health. However, this application of PEF, though promising, requires further research to optimize its impact on the protein and bioactive compounds in germinating seeds.

Post-COVID-19 approach to teaching an undergraduate laboratory class focused on experimental design and data interpretation.

To best prepare students for the real-world research environment, key skills, including experimental design, data analysis, communication of results, and critical thinking, should be key components of undergraduate science courses. Furthermore, the impact of the COVID-19 pandemic on in-person teaching has resulted in a need to develop courses that enable flexible learning. This paper details the laboratory component of a senior-level toxicology class that was developed to emphasize all these skills and allow for flexible learning. The aim of the laboratory class was for students to determine how curcumin protected against acetaminophen-induced hepatoxicity. To stimulate critical thinking, students were required to choose a maximum of four experiments from the six on offer. Before conducting an experiment, students stated a hypothesis and selected the appropriate treatment groups. Once an experiment was completed, students were given access to a complete dataset, on which they performed statistical analysis and drew conclusions. Students who were unable to attend the laboratory session in person were able to complete the required pre-lab work and access the dataset. Following each experiment, students could write a lab summary, and receive thorough feedback. The final assessment was a written manuscript of their findings as well as a chance to respond to reviewer comments. This teaching approach prioritized the critical thinking, analysis, and experimental design aspects of scientific research. Overall, this structure was well received by students and it could easily be adapted for use on other life science courses.

Characterization of marine-derived halogenated indoles as ligands of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor thought to mediate a number of physiological roles in the body, is becoming a target of interest for the development of new therapeutics. However, previous research has demonstrated that the downstream effects of AhR ligands cannot be predicted based simply on whether a ligand acts as an agonist or antagonist and the persistence of AhR signaling is thought to be a key determining feature. The current study investigated the AhR activity of four halogenated indoles isolated from the New Zealand red alga, Rhodophyllis membranacea: 4,7-dibromo-2,3-dichloroindole (4DBDCI), 7-bromo-2,3-dichloro-6-iodoindole (BDCII), 6,7-dibromo-2,3-dichloroindole (6DBDCI) and 2,6,7-tribromo-3-chloroindole (TBCI). Their ability to activate AhR signaling, measured as CYP1A1 activity via the ethoxyresorufin O-deethylase (EROD) assay, was determined in human HepG2, mouse Hepa1c1c7 and rat H4IIE liver cancer cells. All four compounds induced CYP1A1 activity in HepG2 cells, suggesting they all acted as AhR agonizts. 4DBDCI was particularly efficacious, inducing an 11-fold increase. Hepa1c1c7 and H4IIE cells, however, were generally less responsive to the halogenated indoles. All four compounds were persistent AhR agonizts, inducing peak CYP1A1 activity after 72 h. Moreover, the 2,3,6,7-substituted BDCII, 6DBDCI and TBCI, but not 4DBDCI, competed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for AhR binding as observed by the inhibition of TCDD-induced CYP1A1 activity. Overall, the current study has characterized four previously untested AhR ligands, highlighting differences in species sensitivity and persistence of signaling to provide a framework for their potential future use.

Disconnected perspectives: Patient and care provider's experiences of substance use in pregnancy.

OBJECTIVE: Generations of colonialism, abuse, racism, and systemic trauma have contributed to Indigenous women in Canada bearing the greatest burden of substance use in pregnancy. Stigma associated with substance use in pregnancy translates into multiple barriers to women engaging in care. Care providers have key interactions that can act as a bridge or a barrier to care. METHODS: Patient journey maps were created for women living with substance use (n = 3) and semi-structured interviews (n = 20) were performed to understand perceptions of maternity-care providers around women with substance use in pregnancy at a regional hospital in northern British Columbia. RESULTS: Patient journey maps showed overall emotions of hurt, loss, judgment, and anger at their interface with health care during pregnancy. Providers described gaps in knowledge of substance use in pregnancy and harm reduction. Although care providers overall perceived themselves to be providing compassionate care without bias, the patient journey maps suggested profound judgment on behalf of providers. CONCLUSION: Ongoing cultural humility and trauma-informed care training along the continuum of care is critical to impacting discrepancies between perceived lack of bias and harm in patient interactions. Acknowledgment of systemic racism's impact on provision of maternity care is critical for health system change.

X-linked inhibitor of apoptosis regulates lung fibroblast resistance to Fas-mediated apoptosis.

The accumulation of apoptosis-resistant fibroblasts within fibroblastic foci is a characteristic feature of idiopathic pulmonary fibrosis (IPF), but the mechanisms underlying apoptosis resistance remain unclear. A role for the inhibitor of apoptosis (IAP) protein family member X-linked inhibitor of apoptosis (XIAP) has been suggested by prior studies showing that (1) XIAP is localized to fibroblastic foci in IPF tissue and (2) prostaglandin E2 suppresses XIAP expression while increasing fibroblast susceptibility to apoptosis. Based on these observations, we hypothesized that XIAP would be regulated by the profibrotic mediators transforming growth factor (TGF)ß-1 and endothelin (ET)-1 and that increased XIAP would contribute to apoptosis resistance in IPF fibroblasts. To address these hypotheses, we examined XIAP expression in normal and IPF fibroblasts at baseline and in normal fibroblasts after treatment with TGF-ß1 or ET-1. The role of XIAP in the regulation of fibroblast susceptibility to Fas-mediated apoptosis was examined using functional XIAP antagonists and siRNA silencing. In concordance with prior reports, fibroblasts from IPF lung tissue had increased resistance to apoptosis compared with normal lung fibroblasts. Compared with normal fibroblasts, IPF fibroblasts had significantly but heterogeneously increased basal XIAP expression. Additionally, TGF-ß1 and ET-1 induced XIAP protein expression in normal fibroblasts. Inhibition or silencing of XIAP enhanced the sensitivity of lung fibroblasts to Fas-mediated apoptosis without causing apoptosis in the absence of Fas activation. Collectively, these findings support a mechanistic role for XIAP in the apoptosis-resistant phenotype of IPF fibroblasts.

Increased survivin expression contributes to apoptosis-resistance in IPF fibroblasts.

Fibroblasts perform critical functions during the normal host response to tissue injury, but the inappropriate accumulation and persistent activation of these cells results in the development of tissue fibrosis. The mechanisms accounting for the aberrant accumulation of fibroblasts during fibrotic repair are poorly understood, although evidence supports a role for fibroblast resistance to apoptosis as a contributing factor. We have shown that TGF-ß1 and endothelin-1 (ET-1), soluble mediators implicated in fibrogenesis, promote fibroblast resistance to apoptosis. Moreover, we recently found that ET-1 induced apoptosis resistance in normal lung fibroblasts through the upregulation of survivin, a member of the Inhibitor of Apoptosis (IAP) protein family. In the current study, we sought to determine the role of survivin in the apoptosis resistance of primary fibroblasts isolated from the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF), a fibrotic lung disease of unclear etiology for which there is no definitive therapy. First, we examined survivin expression in lung tissue from patients with IPF and found that there is robust expression in the fibroblasts residing within fibroblastic foci (the "active" lesions in IPF which correlate with mortality). Next, we show that survivin expression is increased in fibroblasts isolated from IPF lung tissue compared to cells from normal lung tissue. Consistent with a role in fibrogenesis, we demonstrate that TGF-ß1 increases survivin expression in normal lung fibroblasts. Finally, we show that inhibition of survivin enhances susceptibility of a subset of IPF fibroblasts to apoptosis. Collectively, these findings suggest that increased survivin expression represents one mechanism contributing an apoptosis-resistant phenotype in IPF fibroblasts.

Mechanisms of HIV-tat-induced phosphorylation of N-methyl-D-aspartate receptor subunit 2A in human primary neurons: implications for neuroAIDS pathogenesis.

HIV infection of the central nervous system results in neurological dysfunction in a large number of individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV-infected neurons. Neuronal damage and dropout must therefore be due to indirect effects of HIV infection of other central nervous system cells through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivator, tat. We previously demonstrated that HIV-tat-induced apoptosis in human primary neurons is dependent on N-methyl-D-aspartate receptor (NMDAR) activity. NMDAR activity is regulated by various mechanisms including NMDAR phosphorylation, which may lead to neuronal dysfunction and apoptosis in pathological conditions. We now demonstrate that tat treatment of human neurons results in tyrosine (Y) phosphorylation of the NMDAR subunit 2A (NR2A) in a src kinase-dependent manner. In vitro kinase assays and in vivo data indicated that NR2A Y1184, Y1325, and Y1425 are phosphorylated. Tat treatment of neuronal cultures enhanced phosphorylation of NR2A Y1325, indicating that this site is tat sensitive. Human brain tissue sections from HIV-infected individuals with encephalitis showed an increased phosphorylation of NR2A Y1325 in neurons as compared with uninfected and HIV-infected individuals without encephalitis. These findings suggest new avenues of treatment for HIV-associated cognitive impairment.

HIV-tat induces formation of an LRP-PSD-95- NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes.

HIV infection of the central nervous system can result in neurologic dysfunction with devastating consequences in AIDS patients. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence that HIV can infect neurons. Here we show that the HIV-encoded protein tat triggers formation of a macromolecular complex involving the low-density lipoprotein receptor-related protein (LRP), postsynaptic density protein-95 (PSD-95), N-methyl-d-aspartic acid (NMDA) receptors, and neuronal nitric oxide synthase (nNOS) at the neuronal plasma membrane, and that this complex leads to apoptosis in neurons negative as well as positive for NMDA receptors and also in astrocytes. Blockade of LRP-mediated tat uptake, NMDA receptor activation, or neuronal nitric oxide synthase significantly reduces ensuing neuronal apoptosis, suggesting that formation of this complex is an early step in tat toxicity. We also show that the inflammatory chemokine, CCL2, protects against tat toxicity and inhibits formation of the complex. These findings implicate the complex in HIV-induced neuronal apoptosis and suggest therapeutic targets for intervention in the pathogenesis of NeuroAIDS.

mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.

mTOR/RAFT1/FRAP is the target of the immunosuppressive drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth. We report that mTOR forms a stoichiometric complex with raptor, an evolutionarily conserved protein with at least two roles in the mTOR pathway. Raptor has a positive role in nutrient-stimulated signaling to the downstream effector S6K1, maintenance of cell size, and mTOR protein expression. The association of raptor with mTOR also negatively regulates the mTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the mTOR-raptor association and inhibit mTOR kinase activity. We propose that raptor is a missing component of the mTOR pathway that through its association with mTOR regulates cell size in response to nutrient levels.

Enumeration of the simian virus 40 early region elements necessary for human cell transformation.

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.