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Synthetic Studies on Tunicamycin Towards the Discovery of Antimetastatic Agents
No full textThe tunicamycins have been broadly applied in research of N-linked glycosylation and protein misfolding fields. The tunicamycins display antiproliferative activity against a wide range of cancer and healthy cell lines with narrow therapeutic index. Cytotoxicity of the tunicamycins is believed to be the inhibition of dolichyl-phosphate N-acetylglucosamine-phosphotransferase 1 (DPAGT1), a transmembrane protein, in all mammalian cells in a non-selective manner. We have discovered a selective DPAGT1 inhibitor, APPB based on the non-cytotoxic natural product muraymycin A1. APPB inhibits migrations of solid cancers at low concentrations and induces apoptosis when increased dose concentrations. The observed anticancer profiles of APPB are completely different from those of the tunicamycins, thus, we hypothesized that cytotoxicity of the tunicamycins is unrelated to their inhibition of DPAGT1. To advance tunicamycin into a progressable drug lead, its promiscuous toxicities need to be attenuated. In addition, the discovery of chemically and metabolically stable tunicamycin analogs are essential. To this end, we have developed a super-efficient total synthesis of tunicamycin V and have established a method to identify selective DPAGT1 inhibitors based on tunicamycin cyclitol analogs. The complex pseudo-disaccharide and nucleoside segments are assembled in a one-pot Büchner-Curtius-Schlotterbeck reaction in a wet solvent system. A series of DPAGT1 inhibitors can be synthesized in 11-12 chemical steps from the commercially available starting material with high overall yields. The current lead TN-Cy-TBPA displays antimetastatic activity against triple-negative breast cancers, eventually inducing apoptosis. Unlike tunicamycin, TN-Cy-TBPA does not cause toxicity to healthy cells (even at 50 uM) and to mice (LD50 \u3e 20 mg/kg (IV), ref. \u3c 2.0 mg/kg for tunicamycin V). TN-Cy-TBPA salts can readily be dissolved in saline (ref. \u3c 0.2 mg/mL for tunicamycin V). The pharmacokinetic data for TN-Cy-TBPA obtained using CD-1 mice revealed its drug-like properties. In this program, toxic tunicamycin V was successfully converted to potential pharmacological inhibitors of DPAGT1. Efficient and scalable syntheses of the tunicamycin cyclitol analogs are established for in vivo evaluations including toxicological studies
Identification and Characterization of Auxiliary Factors in Innate Antiviral Immunity
No full textUpon viral infections, RIG-I-like receptors (RLRs) sense cytosolic viral RNAs and initiate signaling pathways culminating in the activation of IRF3 and subsequent type I interferon (IFN) antiviral response. This intrinsic protective mechanism is orchestrated by a host of positive and negative regulators, which collectively govern the magnitude and duration of antiviral signaling. SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites) is an understudied non-receptor tyrosine kinase frequently overexpressed in breast cancers. SRMS restrains autophagy and promotes tumor growth, although its physiological function is unclear. We report that SRMS is critical for IRF3-dependent innate antiviral response in murine cells. Depletion of SRMS impaired virus- or dsRNA-induced expression of type I and III IFNs and ISGs in mouse embryonic fibroblasts (MEFs), mouse hepatoma hepa1-6 cells, and primary mouse lung fibroblasts. Mechanistically, SRMS acted downstream of the RLRs and their adaptor MAVS. SRMS loss led to diminished phosphorylations of TBK1 and IRF3, IRF3 dimerization, and nuclear translocation. The kinase activity of SRMS, however, was dispensable. Reconstituting the expression of SRMS of mouse or human origin, be it wild-type or kinase-dead protein, in SRMS-null MEFs restored antiviral signaling. Rather, SRMS forms a complex with TBK1 and IRF3, in a virus-inducible manner. These interactions promoted downstream innate antiviral response. Interestingly, antiviral gene expression via the RLRs in human U2OS, MRC5, Hacat, and MDA-MB-231 cells was not impacted by SRMS knockdown or deletion, suggesting signaling redundancy in human cells. Altogether, our data uncover a novel role of SRMS in controlling IRF3-dependent innate antiviral responses and describe a non-canonical, kinase- and autophagy-independent regulatory mechanism that operates differentially between mouse and human species. Sensing invading viral pathogens early after infection is important for eliciting the host intrinsic immune defense. MDA5 and RIG-I are two major cytosolic viral RNA sensors that recognize different viruses and initiate downstream signaling leading to the induction of type I IFNs antiviral responses in a majority of cell types. Mounting evidence suggests that human oligoadenylate synthetases-like (OASL) is also rapidly induced by viral infections through activated IRF3 directly as well as by IFN autocrine/paracrine signaling. OASL has been suggested to possess antiviral activity and to promote RIG-I signaling. Whereas much has been learned about the upstream pathways and signaling mechanisms leading to transcriptional induction of OASL, how OASL executes its antiviral function and its precise role in RLR-dependent IFN inducting pathway remain obscure. By creating OASL-deficient cell models, I found that OASL was required for induction of type I IFN response by encephalomyocarditis virus (EMCV) via MDA5 signaling, but it was dispensable for antiviral gene expression in response to Sendai virus infection, which engages and activates the RIG-I-dependent pathway. Loss of OASL specifically reduced MDA5 signaling and enhanced virus replication in human and monkey cells. Conversely, OASL overexpression enhanced MDA5-mediated IFN induction and suppressed viral propagation. OASL performed this novel function depending on its RNA-binding activity associated with a lysine at position 66. In addition, OASL was found to form a complex with MDA5, in a virus-inducible manner. This interaction promoted downstream innate antiviral responses. Collectively, our data describe a novel mode of antiviral action of human OASL in which this virus- and IFN-inducible RNA-binding protein specifically engages MDA5 and promotes host innate immune response downstream of this viral RNA sensor
Postural Control in Adults with Age-Related Hearing Loss
Full text linkThe present study investigated postural control measures in adults with age-related hearing loss (ARHL). Individuals between the age of 50 and 70 were recruited (10 with normal hearing, 32 with ARHL) and evaluated using pure-tone audiometry, vestibular function assessments, and postural control measures. Results of this study revealed significant correlations between ARHL and cervical vestibular evoked myogenic potential (VEMPs) and ocular VEMPs. As ARHL increased, amplitude of cervical VEMPs and ocular VEMPs decreased. There were no significant correlations for ARHL and video head impulse test (vHIT) gain. Single leg stance with head tilt was significantly associated with ARHL. As ARHL increased, the time for single leg stance with head tilt decreased. There were no significant findings for ARHL and single leg stance without head tilt, tandem walking with and without head tilt, or Romberg on foam with and without head tilt. Further investigation is needed to validate the use of single leg stance with head tilt as a potential screening measure for decreased postural control in individuals with ARHL
Aging Skeletal Muscle: Influence of Obesity and Role of the Exercise Responsive Nuclear Receptor NOR-1
Full text linkSkeletal muscle mass and function are critical components of overall health and quality of life. Advanced age is associated with skeletal muscle disease characterized by a loss of muscle mass, function, and quality. Indeed, low muscle mass and function is associated with poor disease outcomes and greater mortality, greater incidence of falls, and a high fiscal and societal cost. Aging contributes to a decline in skeletal muscle health through several intrinsic and extrinsic mechanisms including impaired nutrient sensing, mitochondrial dysfunction, inflammation, physical inactivity, and proteostatic imbalance. Obesity, an increasingly prominent public health concern, is becoming more common in the elderly as the obesity epidemic intersects with an aging population demographic. Additionally, preclinical research on skeletal muscle aging often uses aged rodents which exhibit elevated fat mass when compared to young lean control mice. Therefore, how aging impacts skeletal muscle health in the absence or presence of obesity is currently a gap in our understanding and represents an obstacle to the interpretation of preclinical skeletal muscle research on aging. In contrast to aging and obesity, exercise is among the most potent interventions to improve skeletal muscle mass and health. Exercise effectively prevents and reverses the disease sequalae of skeletal muscle aging by mitigating the development of sarcopenia and preserving skeletal muscle metabolic health, in part through orchestrating transcriptional changes that confer improved muscle health. Indeed, the molecular transducers of exercise exist as attractive therapeutic targets for a plethora of muscle-centric pathologies. The identification of which exercise-responsive factors can be used to ameliorate the detrimental effects of aging on skeletal muscle mass and function is a critical step in the development of novel and innovative therapeutic targets. The nuclear receptor NR4A3 (NOR-1) is among the most responsive genes to exercise and is downregulated in aged muscle; whether NOR-1 expression can improve function in aged muscle is unknown.
The purpose of this dissertation was to explore the interaction of obesity and aging on skeletal muscle mass, mitochondrial function, and gene expression. Additionally, we sought to investigate the role of NOR-1 in skeletal muscle and examine whether NOR-1 overexpression can improve muscle function in aged animals. Our hypothesis was that aged skeletal muscle would mount a unique transcriptional response to obesity and that aging muscle would present with greater muscle lipid accumulation during obesity. Furthermore, we hypothesized that deficiencies in NOR-1 would perturb metabolic and contractile gene expression in vitro and that NOR-1 overexpression would improve skeletal muscle function in vivo and enhance the expression of genes associated with muscle oxidative capacity and endurance.
Chapter 3 of this dissertation examined how the coincidence of aging and obesity impacts skeletal muscle mitochondrial function, whole body metabolism, and transcriptomic changes in skeletal muscle. We observed that obesity and aging had divergent effects on skeletal muscle mass and size as well as mitochondrial function. Furthermore, we discovered that aged muscle produced a unique skeletal muscle transcriptomic signature in response to obesity, indicating that the presence of obesity alters the genetic landscape of aged skeletal muscle when compared to young and lean aged counterparts. We also discovered that aging is associated with reduced expression of NOR-1, a nuclear receptor that is among the most responsive genes to exercise in skeletal muscle. Therefore, in chapter 4 we investigated the regulation of NOR-1 by exercise and characterized the role of NOR-1 in C2C12 myotubes, an in vitro model of skeletal muscle myofibers. We observed that NOR-1 was critical for mitochondrial health and that loss of NOR 1 impaired metabolic and contractile gene expression as well as mTORC1 signaling. We also 17 identified key genes that are regulated by NOR-1 and therefore gained crucial insight into the role of NOR-1 in exercise-associated benefits. Lastly, we also explored the potential for NOR-1 to improve muscle function in aged mice. We observed that Adeno-associated virus (AAV)- mediated expression of NOR-1 resulted in improved muscle fatigability and mitochondrial function in aged mice. Indicating that NOR-1 can enhance function in aged muscle. These changes were concomitant with enhanced expression of a subset of genes associated with muscle oxidative capacity that we identified through in vitro loss-of-function experiments.
In summary, aging-associated alterations to skeletal muscle gene expression, mitochondrial function, and mass are altered with the presence of obesity. Expression of NOR-1, which is reduced with aging but enhanced by exercise in an intensity and time-dependent manner, is crucial for metabolic and contractile gene expression. Furthermore, NOR-1 overexpression in aged muscle is a viable intervention to improve muscle fatigability; however, chronic overexpression of NOR-1 resulted in a small but statistically significant loss of muscle mass. These findings fill a critical conceptual gap in our understanding of preclinical aging studies and suggest that the body composition of aged animals is a necessary factor to consider when investigating aging skeletal muscle biology. Additionally, these studies point towards a crucial role for the nuclear receptor NOR-1 in skeletal muscle health and suggest exercise may improve muscle function, in part through the actions of NOR-1. By enhancing our understanding of the factors that can influence skeletal muscle mass, health, and function we can develop interventions to improve quality of life and health during agin
A Quantum Logic Gate in the DNA Deoxyribose Moiety
Full text linkThe deoxyribose moiety of a nucleotide in the DNA molecule can act as a quantumlogic gate, in which the enantiomeric shift between the C2-endo and C3-endo conformations of each nucleotide, occurs within a logically and thermodynamically reversible situation of electron spin qubits, that are coherently held within the topologically insulating DNA crystalline nanostructure, and that are coherently conducted along the delocalized electrons of the pi-stacked nucleotide base pairs. The enantiomeric symmetry between the C2-endo and C3-endo conformations is logically and thermodynamically reversible because it functions as a symmetry-breaking Szilard engine that is effectively built out of the physicality of the information by which it functions, and therefore does not require information erasure to maintain function. Such a quantum logic gate is analogous to a Toffoli gate which operates across an energy barrier appropriate to the Landauer limit, to roll the DNA base pair and thereby break the pi-stacking coherence along a segment of the DNA molecule, thus effecting quantum-to-classical transition of information
The Effectiveness of Dexmedetomidine in the Reduction of Postoperative Delirium in Patients Over 65: A Scoping Review
Full text linkPurpose/Background
Postoperative delirium impacts as many as 51% of surgical patients, resulting in increased mortality and morbidity, longer hospital stays, and increased healthcare costs. Adults ≥ 65 have a high risk for developing delirium. This scoping review examines the use of dexmedetomidine in the perioperative period in adults ≥ 65 years old to prevent and treat postoperative delirium.
Methods
Using relevant inclusion criteria, an extensive review of the literature was performed from September through November 2022, utilizing the Cochrane, CINAHL, ClinicalKey, PubMed, and Embase databases. Search phrases included: dexmedetomidine infusion, dexmedetomidine, intraoperative dexmedetomidine, postoperative delirium, older adults, and elderly delirium. Three thousand one hundred and fifty-six articles met the search criteria. They were systematically pared down based on quality and relevance to twenty articles, and then evaluated with a Rapid Critical Appraisal (RCA) tool and placed in a synthesis table. Eight articles were selected for inclusion as the highest quality level I and II evidence available.
Results
In seven of the eight selected studies, perioperative use of dexmedetomidine decreased the incidence of postoperative delirium in adults ≥ 65 years. ICU length of stay decreased in five analyses, and mortality rate was unchanged or decreased in five analyses. The presence of dexmedetomidine- related bradycardia was less than other common perioperative medications in two articles and had varied presence in the other reports; use of dexmedetomidine was never reported in the selected studies to increase mortality or morbidity in adults ≥ 65 years old
The Effectiveness of Dexmedetomidine as an Adjunct in Spinal Anesthesia for Pain Relief in Cesarean Patients: A Scoping Review
Full text linkFood Insecurity Screening of Families in a Level III Neonatal Intensive Care Unit
Full text linkFull Study Title: Food Insecurity Screening of Families in a Level III Neonatal Intensive Care Unit
Background: Early infancy food insecurity (FI) is linked to poor health later in life, and Neonatal Intensive Care Units (NICUs) are often overlooked. Routine FI screening can help identify and assist the families suffering from FI seen in the NICU before being discharged home.
Purpose: The aim was to identify if families were assessed for FI by social workers or if they were more likely to report FI in a brief survey.
Method: A randomized chart review of social work notes for 30 NICU patients at Regional One Health from January 2023 to June 2023 was conducted to determine if FI was screened. A three-question-FI screening tool was placed in all patient charts from October 2023 to December 2023. The NICU nurses and researcher asked admitted families to complete the survey during visits. Inclusion criteria included parents greater than or equal to 18 years old and English-speaking.
Results: The chart review identified no explicit statements surrounding FI in the social work notes. The team anticipated 50 or more returned surveys throughout the study but received only 33 completed surveys. Thirty surveys were included in the study. Around 17% of families discussed receiving food assistance with social workers, whereas 39% reported FI via the survey.
Conclusion: Screening via a brief survey was successful but not cohesive to the Regional One Health NICU. A different distribution method may offer better results
Identifying Contributors to Acute Stroke Transfer Delays from Lower to Higher Level Stroke Centers
Full text linkThe intricate process of efficiently transferring patients within the healthcare setting has proven to be complex however, it is a critical component to ensure timely access to essential medical interventions. Navigating these complexities presents a multifaceted challenge that is not fully understood. For patients suffering a stroke, these transfer delays may render them ineligible for treatment and left with disabling disability. This collective work aims to identify contributors to delays in transfer from lower to higher levels of care in the acute stroke populations.
Three papers emerged from this thesis. First, a narrative review of the literature published between January 1996 to April 2020 found that several factors are associated with improved stroke outcomes. The availability of resources such as vascular neurologists or dedicated stroke units, training ambulance personnel in the early identification of stroke symptoms and stroke management, and focusing on internal performance efficiencies in the emergency department to administer clot busing medication for the treatment of ischemic stroke have all improved outcomes. However significant gaps in knowledge of what contributes to delays exist with no objective data available on stroke transfer delays in the published literature.
Summation from the narrative review was used to develop a qualitative study. Web-based interviews with stroke coordinators at primary stroke centers were conducted where they discussed their lived experience in transferring patients from a primary stroke center to a higher level of care. The results were published in Stroke: Vascular and Interventional Neurology journal in March of 2023. Comprising the narrative review of the known causes of transfer delays and gaps in knowledge and the collective insight of stroke coordinators at primary stroke centers across the United States, a data collection instrument was designed and tested at primary stroke centers on patients that required transfer to a higher level of care. The instrument was divided into two main sections. The first section consisted of time process indicators that included information pertinent to activities associated with patient presentation, evaluation, and diagnostic work-up in the emergency department until departure to the higher level of care hospital. The second section consisted of 24-Likert scale questions designed to understand the transferring team’s perception of the quality and timeliness of the entire transfer process.
The data collection instrument allowed us to examine the contributors to door-in, door-out (DIDO) times at a granular level. The median DIDO time for the overall sample was 130 minutes which exceeds the recommended DIDO time of less than 120 minutes. The greatest single contributor to DIDO time was from transport request to actual transport departure times which alone was responsible for 38% or 49.5 minutes of the total DIDO time.
Collectively, these papers have broadened knowledge of why transfer delays exist when moving acute stroke patients from lower to higher level of hospital services. Continued work to further identify contributors to transfer delays within each category is warranted, along with development of interventions to improve efficiency across the entire system of care. In conclusion, delays in transfer continue to be one of the leading causes for stroke treatment ineligibility due to worsening irreversible brain damage. Improving stroke systems of care will improve treatment eligibility, reduce stroke related disability and death, and reduce the financial burden of post-stroke care
Physiologically-Based Pharmacokinetic (PBPK) Modeling for the Preclinical Development of Spectinamide Antibiotics
Full text linkDespite being an ancient disease caused by Mycobacterium tuberculosis (Mtb) and after decades of research, tuberculosis (TB) still affects millions of people every year worldwide. In 2018, the World Health Organization (WHO) reported that 10 million people developed tuberculosis and 1.5 million died of the disease. With the increase in the multidrug-resistant (MDR) and extensively drug-resistant (XDR) cases, the treatment for TB with the standard first and second-line therapy is becoming increasingly difficult. Therefore, there is an urgent need to find new anti-TB drugs as combination partners of existing and experimental classes of antimicrobial agents to shorten and simplify the treatment of MDR or XDR TB infection. Spectinamide 1599 and spectinamide 1810 are lead spectinamide compounds currently under preclinical development for the treatment of MDR and XDR tuberculosis. These compounds have previously been tested at various combinations of dose level, dosing frequency, and routes of administration in mouse models of Mtb infection and in healthy animals. Characterizing the disposition of a compound in the tissues of interest is important in understanding the pharmacology of novel antibiotic agents. TB is primarily a pulmonary disease, but extrapulmonary disease has been reported in 10-42% of patients. Thus, understanding the drug disposition and exposure in the tissues in which Mtb is residing is of utmost importance for any new antitubercular drug. Hence, we have used a physiologically-based pharmacokinetic (PBPK) approach for the prediction of drug pharmacokinetics in organs/tissues of interest and extrapolate their disposition across different species. In the current study, we built, qualified, and refined a minimalistic PBPK model that can describe and predict the pharmacokinetics of spectinamide antibiotics in various tissue, especially those relevant to Mtb infection. The model was expanded and qualified for multiple dose levels, dosing regimens, routes of administration, and various species. The model predictions in mice (healthy and infected) and rats were in reasonable agreement, and all predicted Area Under the Curve (AUCs) in plasma and tissues met the twofold acceptance criteria relative to observed data. To further explore tissue exposure in Mtb lesions, we also utilized the established Simcyp granuloma model to explore the distribution of spectinamide 1599 within granuloma substructures. Simulation results suggest substantial exposure in all lesion substructures, with particularly high exposure in the rim region and in macrophages of the lesion. The developed model may be leveraged as an effective tool in predicting the disposition of spectinamide antibiotics in further preclinical and clinical development to identify optimal dose levels and dosing regimens