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Implementation of the cognitive apprenticeship model for enhancement of advanced searching skills in a pharmacy academia rotation: Appendices 1-4
Get PDFIdentification of Effectors of Synergistic Lethality in Candida albicans-Staphylococcus aureus Polymicrobial Intra-abdominal Infection
Get PDFCandida albicans, an opportunistic fungal pathogen, and Staphylococcus aureus, a ubiquitous pathogenic bacterium, are among the most prevalent causes of nosocomial infections and cause severe morbidity and mortality. Moreover, they are frequently coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Relatively little is known about the complex interactions and signaling events that occur between microbes and even less so how microbial “cross-talk” shapes human health and disease.
Using a murine model of polymicrobial intra-abdominal infection (IAI), we have previously shown that coinfection with C. albicans and S. aureus leads to synergistic lethality whereas monomicrobial infection is nonlethal. Therefore, we aimed to identify staphylococcal virulence determinants that drive lethal synergism in polymicrobial IAI. Using the toxigenic S. aureus strain JE2, we observed that co-infection with C. albicans led to a striking 80-100% mortality rate within 20 h p.i while monomicrobial infections were non-lethal. Use of a GFP-P3 promoter S. aureus reporter strain revealed enhanced activation of the staphylococcal agr quorum sensing system during in vitro polymicrobial versus monomicrobial growth. Analyses by qPCR, Western blot, and toxin functional assays confirmed enhanced agr-associated gene transcription and increases in secreted α- and δ-toxins. C. albicans-mediated elevated toxin production and hemolytic activity was determined to be agrA-dependent and genetic knockout and complementation of hla identified ⍺-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono- and polymicrobial infection 8 h p.i. demonstrated equivalent bacterial burden in the peritoneal cavity, but significantly elevated levels of α-toxin (3-fold) and the eicosanoid PGE2 (4-fold) during co-infection. Importantly, prophylactic passive vaccination using the monoclonal anti-⍺-toxin antibody MEDI4893* led to significantly improved survival rates as compared to treatment with isotype control antibody. Collectively, these results define α-toxin as an essential virulence determinant during C. albicans-S. aureus IAI and describe a novel mechanism by which a human pathogenic fungus can augment the virulence of a highly pathogenic bacterium in vivo.
We next sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal ⍺-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced ⍺-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and ⍺-toxin production. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and ⍺-toxin production during coculture. Overall, we demonstrated that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system.
Finally, we correlated ⍺-toxin production with significant increases in biomarkers of liver and kidney damage during coinfection and determined that functional toxin was required for morbidity and mortality. We next sought to determine the candidal effector(s) mediating this enhanced virulence by employing an unbiased screening approach. C. albicans transcription factor mutants were evaluated for their ability to induce S. aureus agr activation in polymicrobial culture. Incredibly, we identified several mutants that displayed defects in augmenting S. aureus agr activity in vitro. Two of the mutants failed to completely synergize with S. aureus in vivo and further analysis revealed the necessity of the uncharacterized C. albicans transcription factor, ZCF13, in driving enhanced toxin production both in vitro and in vivo. Collectively, we identified a novel effector by which C. albicans augments S. aureus virulence and identified a potential mechanism of fungal-bacterial lethal synergism
Combating HIV-1 by Targeting Drug Efflux Transporters on the Macrophage Reservoir
Get PDFIntroduction. HIV-1 eradication has not been achieved so far due to the existence of the cellular reservoir in which the virus can reside and replicate even under antiretroviral drug therapy (ART). Infected macrophages, which represent a long-term viral reservoir have been shown to lead to viral rebound independently. In response to the environmental stimuli, macrophages can be polarized into different phenotypes: the pro-inflammatory M1 and the anti-inflammatory M2. Tobacco smoking and alcohol drinking, which are prevalent among people who are living with HIV-1, have been shown to promote HIV-1 progression and decrease the efficacy of antiretroviral drugs. A commonly used macrolide antibiotic azithromycin (AZM) has been shown to shift macrophage polarization in a murine macrophage cell line. In the previous research, we found that drug efflux transporters expressed differently between macrophage phenotypes. In this dissertation, we examined the effects of CSC, ethanol exposure, and AZM on the expression and function of clinically relevant drug efflux transporters, viral suppression of antiretroviral drugs, and macrophage polarization.
Methods. The human monocytic cell lines U937 and the U1 cell line, which is derived from HIV-1 infected U937, were used and polarized to the M1 and M2 macrophages. Cells with the treatment of CSC, ethanol, and AZM were harvested for downstream analysis including macrophage polarization, oxidative stress, cytokine production, transporter expression and function, and viral suppression. Cells treated with IKK-16, an inhibitor of the NF-κB signaling pathway, were harvested for the analysis of transporter expression and function. Protease inhibitor lopinavir (LPV) was used to suppress viral replication and the intracellular LPV was measured using LC-MS/MS.
Results. Cigarette smoke condensate (CSC) and AZM shifted M1 macrophage polarization to M2 while having minimal effects on the M2 macrophage polarization. Inhibiting macrophage subset-specific transporters significantly increased intracellular antiretroviral drug (ARV) concentrations and drug efficacy. Neither CSC nor ethanol had any effect on the transporter inhibition-mediated viral reduction. AZM modulated the expression of major drug efflux transporters in both macrophage subsets and increased intracellular ARV concentration in M2 macrophages. NF-κB and JNK are involved in the M1 macrophage polarization shift and NF-κB was also shown to regulate major transporter expression.
Conclusion. Modulating the expression and function of macrophage subset-specific transporter expression can increase intracellular ARV concentration and drug efficacy of viral suppression. Targeting subset-specific transporter may be an effective way to increase intracellular ARV concentration in the macrophage reservoir
Genomic Characterization of Sickle Cell Mouse Models for Therapeutic Genome Editing Applications
Get PDFSickle cell disease (SCD) is caused by a mutation of the β-globin gene (HBB), resulting in abnormal hemoglobin molecules that polymerize when deoxygenated, forming “sickle” shaped red blood cells (RBCs). Sickle RBCs lead to anemia, multi-organ damage and pain crises, beginning the first year of life. The onset of symptoms coincides with the developmental switch of β-like globin gene expression from fetal stage γ-globin to adult stage β-globin, resulting in a shift from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2β2). Some individuals harbor rare genetic variants in the extended β-globin gene cluster that cause constitutively elevated postnatal HbF, a benign condition known as hereditary persistence of fetal hemoglobin (HPFH) which alleviates symptoms of co-inherited SCD. Previously, we showed that CRISPR-Cas9-mediated genome editing can recreate a naturally occurring HPFH variant in the γ-globin (HBG1 and HBG2) promoters. Disruption of a TGACC nucleotide motif within this region by Cas9-mediated non-homologous end joining in human erythroid cells or their progenitors caused induction of HbF by interfering with recruitment of the transcriptional repressor, BCL11A. This strategy results in potent HbF induction in human cells and is a promising therapeutic strategy. However, the efficiency of genome editing and the level of HbF induction required to arrest or reverse the pathologies of SCD are unknown.
In this work, we investigated the utility of humanized mouse models for SCD to answer this question. We further characterized the genomic configurations of two models: Berkeley mice, which harbor multiple tandem copies of three separate transgenes encoding human α-globin, sickle β-globin (βS) and a segment of the locus control region (LCR) a powerful enhancer that drives high-level erythroid-specific expression of linked genes; and Townes mice, in which the endogenous α-globin gene is replaced by the homologous human gene and the endogenous β-globin gene is replaced by human γ-globin (γA) and βS-globin genes. Genome editing of human γ-globin promoter in the Berkeley mouse induced a massive DNA damage response and cell death caused by the accumulation of multiple double-stranded DNA breaks (DSB) within the highly repetitive human transgene. In contrast, it was possible to achieve high-level editing of the single copy human γ-globin gene in the Townes model. However, induction of HbF was approximately 10-fold less that what occurred after generating the same edits in human cells, possibly because the mouse model lacks essential non-coding DNA regulatory sequences. Together, these limitations rule out the Berkeley mouse for DSB-inducing gene-editing purposes and the Townes mouse for HbF induction by regulatory element targeting.
Despite these limitations, we determined the Townes model to be a good candidate for a base editing strategy to directly alter the SCD mutation. This work sought to edit the sickle T to a G, resulting in the Hb G-Makassar variant suspected to be benign and non-sickling. Recipient mice transplanted with successfully edited (55-60%) Townes HbSS Lin- cells show marked improvement in blood count values and splenomegaly. This Hb G-Makassar strategy allows for a better understanding of the levels of hematopoietic stem cell editing required to correct the SCD phenotype
Reducing Delirium in Patients with COVID-19
Get PDFThe purpose of our critically appraised topic is to synthesize the best current evidence regarding interventions that would be effective in reducing delirium in patients with COVID-19 in the acute care setting. The final portfolio contains a total of five research articles. Study designs include three randomized controlled trials, one non randomized controlled trial, and one quasi-experimental quantitative design. All studies relate directly to interventions within the OT scope of practice and were implemented in the acute care setting. Due to limited research on the novel coronavirus, these findings apply to critically ill patients but are not specific to patients with COVID-19. Early and intensive OT intervention in combination with multicomponent intervention strategies were found to significantly decrease delirium. Promising evidence can be used to draft new practice guidelines for decreasing delirium in critically ill patients within the acute care setting. In addition, modified implementation of slow tempo music and family participation/visitation interventions are recommended for patients with COVID-19. Due to the minimal time for therapy interventions within the acute care setting, implementation of these interventions as frequently as possible is recommended
Therapeutic Potential of TRP Channels in the Targeting of Rheumatoid Arthritis Synovial Fibroblasts
Get PDFRheumatoid arthritis is a chronic inflammatory disease primarily affecting the synovium, articular cartilage, and bone within a joint, but it is a unique form of arthritis wherein effects are systemic. The cause of this autoimmune disease remains unknown, but there are many environmental and genetic factors that play into susceptibility. Research is still far from drug-free remission despite great advancements over the past few decades. The majority of therapies developed rely on immunosuppressant or immunomodulator molecules and come with risk of infection, high costs, and toxic, uncontrolled side effects. Those diagnosed maintain a significant unmet need for targeted therapies.
There is increasing evidence towards non-immune cell types in the joint as the culprit for the changes in anatomy of the joint at disease onset. A thin lining called the synovium covers the joint cartilage and acts as a barrier which secretes synovial fluid that lubricates the joint. Synovial fibroblasts, also called fibroblast-like synoviocytes, are responsible for this secretion of lubricating components hyaluronic acid and lubricin that allow for ease of movement. Together with macrophages, they make up the synovial lining and sub-lining in roughly equal proportion. Proinflammatory cytokine production in the inflamed joint leads to synovial fibroblast proliferation and transforms these cells into a “tumor-like” phenotype with the capacity to degrade cartilage and bone. Synovial fibroblasts perpetuate the destruction of articular cartilage by producing matrix-degrading enzymes, cytokines, and increasing production of adhesion molecules to attach and build on to cartilage. The synovium thickens and the cartilage and bone in the joint is broken down, and synovial fibroblasts recruit more immune cells to the joint to further exacerbate joint destruction. This positive feedback loop makes synovial fibroblasts a desirable target for anti-rheumatic drugs
An abundance of research implicating TRP channels in rheumatoid arthritis synovial fibroblasts pathogenic phenotype has accumulated over the past decade. Studies of the rheumatoid synovium demonstrate the expression of several of these channels including TRPV1, TRPV2, TRPV4, TRPA1, TRPM7, TRPM8, and more. The channels’ direct implication in synovial fibroblast aggressive phenotype is becoming better understood and shows promise for TRP channels as therapeutic targets. My master’s thesis will focus on TRP channel involvement in mechanisms by which synovial fibroblasts evade apoptosis, proliferate, degrade the joint, and migrate to unaffected joints in order to understand these biological sensors as potential rheumatoid arthritis therapeutic candidates
The Heme-Regulated Inhibitor Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics
Get PDFAnti-apoptotic MCL1 is one of the most frequently amplified genes in human cancers and its elevated expression confers resistance to many therapeutics including the BH3-mimetic agents ABT-199 and ABT-263. The anti-malarial, dihydroartemisinin (DHA) translationally represses MCL-1 and synergizes with BH3-mimetics. To explore how DHA represses MCL-1, a genome-wide CRISPR screen identified that loss of genes in the heme synthesis pathway renders mouse BCR-ABL+ B-ALL cells resistant to DHA-induced death. Mechanistically, DHA disrupts the interaction between heme and the eIF2α kinase heme regulated inhibitor (HRI) triggering the integrated stress response. Genetic ablation of Eif2ak1, which encodes HRI, blocks MCL-1 repression in response to DHA treatment and represses the synergistic killing of DHA and BH3-mimetics compared to wild-type leukemia. Furthermore, BTdCPU, a small-molecule activator of HRI, similarly triggers MCL-1 repression and synergizes with BH3-mimetics in mouse and human leukemia including both Ph+ and Ph-like B-ALL. Lastly, combinatorial treatment of leukemia bearing mice with both BTdCPU and a BH3-mimetic extended survival and repressed MCL-1 in vivo. These findings reveal that the HRI-dependent cellular heme-sensing pathway can modulate apoptosis in leukemic cells by repressing MCL-1 and increasing their responsiveness to BH3-mimetics. This signaling pathway could represent a generalizable mechanism for repressing MCL-1 expression in malignant cells and sensitizing them to available therapeutics
Examination of Antiviral Resistance in Venezuelan Equine Encephalitis Virus
Get PDFVenezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that causes Venezuelan equine encephalitis (VEE), which is characterized by a febrile illness that can progress to neurological disease and death. While no major outbreaks of VEE have occurred since 1995, VEEV is a virus of concern as, in addition to its spread through mosquitos, it can be aerosolized and used as a bioweapon. Unfortunately, there are currently no FDA-approved vaccines or antivirals against VEEV. Efforts have been made to discover small molecules with an inhibitory effect on VEEV, but the potential for emergence of antiviral resistance to these compounds will remain a concern because VEEV is an RNA virus with a high mutation rate and grows to high titers. To examine the evolutionary trajectory of antiviral resistance in VEEV, we developed a next-generation sequencing pipeline to examine single-nucleotide polymorphisms that emerged after repeated passaging of the virus with increasing concentrations of antiviral compounds. In addition, we examined the effect of the microenvironment on the evolution of antiviral resistance, both in cell culture and mouse models. We found that VEEV evolves resistance to the compound ML336 and its derivatives through mutations in the nsP2 and nsP4 genes, but the number, timing of emergence, and the extent of penetrance of these SNPs depend on the compound. These mutations emerged more slowly when infecting an astrocyte cell line. We also found that neurons in the mouse brain did not impose a selective pressure on VEEV during an infection. These results demonstrate how the population dynamics of RNA viruses can be tracked over time and the extent to which they are affected by selective pressures, as well as opening questions about how viruses can mutate and adapt at the molecular level
Hyperglycemia During the First Three Days of Life Increases the Risk of Retinopathy of Prematurity in Extremely Low Birth Weight Infants
Get PDFBackground: The association between hyperglycemia and ROP has been inconsistent in previous studies. Extremely low birth weight (ELBW) infants are at high risk for ROP. They also experience hypoglycemia initially and hyperglycemia while receiving IV glucose infusion. The effect of initial hypoglycemia on hyperglycemia associated ROP risk is unknown.
Objective: To study the effect of initial hypoglycemia and subsequent hyperglycemia during the first three days of life on the incidence of ROP and severe ROP in ELBW (birth weight ≤1000g) infants.
Methods: Clinical and demographic data were collected from 227 ELBW infants born during the years 2017-2019 at the Regional One Health NICU, Memphis, TN. All blood glucose determinations done during the first 72 hours were collected from these infants along with maternal and neonatal demographic and clinical information. The infants were divided into four groups based on hypoglycemia at birth and subsequent hyperglycemia during the first 72 hours; group I: all the blood glucose levels were between 47-125mg/dl (euglycemia group); group II: initial hypoglycemia (\u3c47mg/dl) and with treatment became euglycemic; group III: initial hypoglycemia and at least one episode of hyperglycemia (\u3e125mg/dl) later on; group IV: initial euglycemia followed by at least one episode of hyperglycemia. Incidence of ROP and severe ROP (stage III or greater) was compared between the groups after adjusting for gestational age.
Results: Clinical and outcomes data are presented in the table. The mean blood sugar levels during each day for the first 72 hours are presented in the figure. ROP incidence was lowest in infants who were euglycemic throughout the first 72 hours of life and no severe ROP was seen in this group. Infants who were hypoglycemic initially without experiencing hyperglycemia later appear to have higher incidence of ROP though it was not statistically different after adjusting for gestational age. Infants who were euglycemic at birth and became hyperglycemic later had increased incidence of ROP even after adjusting for gestational age; they also had the highest incidence of severe ROP. Mortality did not statistically differ between the groups. Comparing combined groups 1 &2 vs. 3 &4 showed no difference in ROP [aOR 1.5 (082-2.84)].
Conclusions: Hyperglycemia during the first three days of life without hypoglycemia at birth increases the risk for ROP. Hypoglycemia at birth appears to confound the risk of hyperglycemia associated ROP in ELBW infants
Opioid Free Anesthesia to Prevent Post Operative Nausea/Vomiting
Get PDFPurpose The purpose of this study is to compare the incidence of post-operative nausea and vomiting (PONV) in opioid-utilizing and opioid-free general anesthesia. Background PONV is an extremely common, potentially dangerous side effect of general anesthesia. PONV is caused by a collection of anesthetic and surgical interventions. Current practice to prevent PONV is to use 1-2 antiemetics during surgery, identify high risk patients and utilize tracheal intubation over laryngeal airways when indicated. Current research suggests minimizing the use of volatile anesthetics and opioids can reduce the incidence of PONV, but this does not reflect current practice.
Methods In this scoping review, the MeSH search terms used to collect data were “anesthesia”, “postoperative nausea and vomiting”, “morbidity”, “retrospective studies”, “anesthesia, general”, “analgesics, opioid”, “pain postoperative”, “pain management” and “anesthesia, intravenous”. The Discovery Search engine, AccessMedicine and UpToDate were the search engines used to research this data. Filters were applied to these searches to ensure all the literature was peer-reviewed, full-text and preferably from academic journals.
Results Opioid free anesthesia was found to decrease PONV by 69%. PONV incidence was overwhelming decreased with opioid free anesthesia in every study that was reviewed. Implications The future direction of opioid-free anesthesia and PONV prevention are broad topics to discuss, due to the nature of anesthesia. Administration of TIVA, esmolol and ketamine, as well as the decision to withhold opioids, are solely up to the anesthesia provider’s discretion. Increasing research and education in the importance of opioid-free anesthesia to decrease the incidence of PONV will be necessary to ensure anesthesia providers choose this protocol in their practice