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Article
Peer Reviewed

EFICIENTE EXTENSIÓN Y DEFENSA DE LOS DERECHOS DE PROPIEDAD INTELECTUAL

Rodríguez García, Gustavo M.

Latin American and Caribbean Law and Economics Association (ALACDE) Annual Papers (2010)

Cover page: EFICIENTE EXTENSIÓN Y DEFENSA DE LOS DERECHOS DE PROPIEDAD INTELECTUAL

Thesis
Peer Reviewed

Modulation of neutrophil inflammatory responses by iron oxide nanoparticles, DAMPs, and cytokines in health and psoriatic disease

Garcia, Gustavo
Advisor(s): Simon, Scott I

UC Davis Electronic Theses and Dissertations (2023)

Neutrophils are vital for elimination of foreign particulates and pathogens, but their inappropriate activation in circulation can lead to tissue damage upon recruitment to sites of inflammation. Thus, neutrophil activation and recruitment is tightly regulated by the synergy between mechanical forces acting on neutrophil adhesion receptors and chemotactic stimulation mediated by inflammatory mediators. However, the synergy between outside-in signaling through selectins and integrins and inside-out signaling via G-protein coupled and Tumor necrosis factor receptors can be altered by foreign materials and chronic exposure to endogenous molecules that signal inflammatory responses such as Toll-like receptors during disease. In this thesis we explored how iron oxide nanoparticles and damage associated molecular patterns DAMPs alter the neutrophil's inflammatory response to chemokines and cytokines in freshly obtained blood samples from healthy individuals and patients with psoriasis. Intravenous delivery of nanoparticles is increasingly being employed for diagnostics and therapeutics. Feraheme is an FDA approved intravenous delivered iron oxide nanoparticle for the treatment of iron deficiency anemia (IDA) in adult patients with chronic kidney failure, but research on its effect on the innate immune response of neutrophils during inflammation has not been reported. Increasing interest in use of Feraheme for novel imaging techniques and therapeutics motivates the first objective of this dissertation; to study the immunomodulatory effects of Feraheme on neutrophil adhesive capacity and responses to GPCR agonists and function. In contrast to foreign nanoparticles, DAMPS and cytokines are produced endogenously to regulate inflammatory responses of immune cells, however during chronic inflammation continuous exposure to inflammatory stimuli can lead to aberrant neutrophil function. Psoriasis is a systemic inflammatory disease that is characterized by exuberant neutrophil recruitment into skin plaques where neutrophils are primed for production of ROS and release of extracellular traps that exacerbate inflammation. Here we explored the role of neutrophil activating cytokines IL-8 and TNF-α as well as DAMPs S100A8/A9 and LL37, known to be upregulated in psoriasis, to prime circulating neutrophils for enhanced β2-integrin-mediated recruitment and effector functions. Exposure to the foreign iron oxide nanoparticles Feraheme had no effect on baseline levels of neutrophil adhesion marker expression, indicating no priming or activation. However, Feraheme treatment reduced neutrophil response to IL-8 mediated upregulation of membrane CD11b/CD18 receptors, activation of high affinity CD18, and shedding of CD62L. Feraheme uptake by neutrophils also inhibited N-formyl-Met-Leu-Phe (fMLP) induced reactive oxygen species (ROS) production. Preincubation of neutrophils with Feraheme greatly reduced capacity capture efficiency on a substrate presenting E-selectin and ICAM-1 in vascular mimetic flow channels. Lastly, Feraheme modulated calcium flux dynamics in neutrophils with accelerated clearance of cytosolic calcium following IL-8 induced flux. Ca2+ is a vital signaling molecule downstream of chemotactic signaling and integrin mechanosignaling. Thus, we conclude that inducing rapid sequestration of calcium is a likely mechanism by which Feraheme uptake inhibits neutrophil chemotactic activation and recruitment efficiency. Psoriatic neutrophils in blood display a primed phenotype characterized by elevated β2-integrin receptor number and binding affinity that correlated with plasma calprotectin levels. Psoriatic neutrophils were exquisitely sensitive to ex vivo stimulation with TNF-α that elicited secretion of calprotectin and synergistically amplified CD18 activation. In accordance with increased β2-integrin activation, psoriatic neutrophils arrest on E-selectin and ICAM-1 twice as efficiently and had greater capacity for calcium flux and production of ROS than healthy neutrophils. These studies highlight the contrasting effects that foreign and innate stimuli can have on modulating neutrophil responsiveness. We report that the Feraheme iron oxide nanoparticles limit neutrophil recruitment and inhibit inflammatory responses to GPCR simulation by accelerating clearance of calcium after stimuli induced flux. In contrast, psoriatic neutrophils were primed for increased adhesion and effector function that correlated with circulating levels of DAMPs and cytokines, particularly S100A8/A9 which is secreted more readily in response to TNF-α by psoriasis neutrophils compared to healthy control.

Cover page: Modulation of neutrophil inflammatory responses by iron oxide nanoparticles, DAMPs, and cytokines in health and psoriatic disease

Article
Peer Reviewed

Membrane hydrophobicity determines the activation free energy of passive lipid transport

UC Berkeley Previously Published Works (2021)

The collective behavior of lipids with diverse chemical and physical features determines a membrane's thermodynamic properties. Yet, the influence of lipid physicochemical properties on lipid dynamics, in particular interbilayer transport, remains underexplored. Here, we systematically investigate how the activation free energy of passive lipid transport depends on lipid chemistry and membrane phase. Through all-atom molecular dynamics simulations of 11 chemically distinct glycerophospholipids, we determine how lipid acyl chain length, unsaturation, and headgroup influence the free energy barriers for two elementary steps of lipid transport: lipid desorption, which is rate limiting, and lipid insertion into a membrane. Consistent with previous experimental measurements, we find that lipids with longer, saturated acyl chains have increased activation free energies compared to lipids with shorter, unsaturated chains. Lipids with different headgroups exhibit a range of activation free energies; however, no clear trend based solely on chemical structure can be identified, mirroring difficulties in the interpretation of previous experimental results. Compared to liquid-crystalline phase membranes, gel phase membranes exhibit substantially increased free energy barriers. Overall, we find that the activation free energy depends on a lipid's local hydrophobic environment in a membrane and that the free energy barrier for lipid insertion depends on a membrane's interfacial hydrophobicity. Both of these properties can be altered through changes in lipid acyl chain length, lipid headgroup, and membrane phase. Thus, the rate of lipid transport can be tuned through subtle changes in local membrane composition and order, suggesting an unappreciated role for nanoscale membrane domains in regulating cellular lipid dynamics.

Cover page: Membrane hydrophobicity determines the activation free energy of passive lipid transport

Article
Peer Reviewed

Bound Electron Enhanced Radiosensitisation of Nimorazole upon Charge Transfer

Chemical Sciences (2022)

This novel work reports nimorazole (NIMO) radiosensitizer reduction upon electron transfer in collisions with neutral potassium (K) atoms in the lab frame energy range of 10-400 eV. The negative ions formed in this energy range were time-of-flight mass analyzed and branching ratios were obtained. Assignment of different anions showed that more than 80% was due to the formation of the non-dissociated parent anion NIMO^•- at 226 u and nitrogen dioxide anion NO₂^- at 46 u. The rich fragmentation pattern revealed that significant collision induced the decomposition of the 4-nitroimidazole ring, as well as other complex internal reactions within the temporary negative ion formed after electron transfer to neutral NIMO. Other fragment anions were only responsible for less than 20% of the total ion yield. Additional information on the electronic state spectroscopy of nimorazole was obtained by recording a K⁺ energy loss spectrum in the forward scattering direction (θ ≈ 0°), allowing us to determine the most accessible electronic states within the temporary negative ion. Quantum chemical calculations on the electronic structure of NIMO in the presence of a potassium atom were performed to help assign the most significant lowest unoccupied molecular orbitals participating in the collision process. Electron transfer was shown to be a relevant process for nimorazole radiosensitisation through efficient and prevalent non-dissociated parent anion formation.

Cover page: Bound Electron Enhanced Radiosensitisation of Nimorazole upon Charge Transfer

Article
Peer Reviewed

Isotope Effect in D₂O Negative Ion Formation in Electron Transfer Experiments: DO-D Bond Dissociation Energy.

LBL Publications (2023)

H₂O/D₂O negative ion time-of-flight mass spectra from electron transfer processes at different collision energies with neutral potassium yield OH^-/OD^-, O^-, and H^-/D^-. The branching ratios show a relevant energy dependence with an important isotope effect in D₂O. Electronic state spectroscopy of water has been further investigated by recording potassium cation energy loss spectra in the forward scattering direction at an impact energy of 205 eV (lab frame), with quantum chemical calculations for the lowest-lying unoccupied molecular orbitals in the presence of a potassium atom supporting most of the experimental findings. The DO-D bond dissociation energy has been determined for the first time to be 5.41 ± 0.10 eV. The collision dynamics revealed the character of the singly excited (1b₂^-1) molecular orbital and doubly excited states in such K-H₂O and K-D₂O collisions.

Cover page: Isotope Effect in D<sub>2</sub>O Negative Ion Formation in Electron Transfer Experiments: DO-D Bond Dissociation Energy.

Article
Peer Reviewed

Human iPSC-Derived Cardiomyocytes Are Susceptible to SARS-CoV-2 Infection

UCLA Previously Published Works (2020)

Coronavirus disease 2019 (COVID-19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is defined by respiratory symptoms, but cardiac complications including viral myocarditis are also prevalent. Although ischemic and inflammatory responses caused by COVID-19 can detrimentally affect cardiac function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is not well understood. Here, we utilize human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model to examine the mechanisms of cardiomyocyte-specific infection by SARS-CoV-2. Microscopy and RNA sequencing demonstrate that SARS-CoV-2 can enter hiPSC-CMs via ACE2. Viral replication and cytopathic effect induce hiPSC-CM apoptosis and cessation of beating after 72 h of infection. SARS-CoV-2 infection activates innate immune response and antiviral clearance gene pathways, while inhibiting metabolic pathways and suppressing ACE2 expression. These studies show that SARS-CoV-2 can infect hiPSC-CMs in vitro, establishing a model for elucidating infection mechanisms and potentially a cardiac-specific antiviral drug screening platform.

Cover page: Human iPSC-Derived Cardiomyocytes Are Susceptible to SARS-CoV-2 Infection

Article
Peer Reviewed

Correction to “Isotope Effect in D2O Negative Ion Formation in Electron Transfer Experiments: DO–D Bond Dissociation Energy”

LBL Publications (2023)

In a recent Letter,1 a set of minor inconsistencies have been found in the main body text and in the Supporting Information files; these are now thoroughly corrected. Thus, we highlight the sections where these changes apply. ¦ MAIN MANUSCRIPT BODY TEXT Abstract: The DO-D bond dissociation energy has been reported to be 5.41 ± 0.10 eV, while considering EA(O) in the original manuscript. However, EA(OD) should be used instead, thus leading to a bond dissociation energy of 5.28 ± 0.20 eV. Page 5365, left column, second paragraph; “From the appearance...” is reorganized and reads as follows: Taking the values in Table S4 together with the BDE, the enthalpy of reaction can be obtained from ?Hr(OH-) = D(H-OH) - EA(OH) = 3.34 eV. In the charge transfer process, if we add the potassium ionization energy, OH- is expected at 7.68 eV. The reactions threshold was obtained assuming no excess energy (E#), yet momentum conservation of the dissociating partners may impact on the lighter fragment kinetic energy, thus shifting the energy to a higher value. We note a difference of ~1.1 eV for the energy loss data, which is certainly plausible given the kinetic-energy release distribution of H- in Figure S3. Thus, from the appearance energy (AE) in the H2O energy loss spectrum (Figure 2) at ?E ˜ 8.8 eV, one can obtain the HO-H bond dissociation energy (BDE) by taking the potassium ionization energy and the data from Table S4,2 i.e., D(HO-H) = AE(OH-) - IE(K) + EA(OH) - E#, yielding therefore D(HO-H) = 5.19 ± 0.20 eV which is in good agreement with the values 5.15 eV (118.81 ± 0.07 kcal/ mol)3 and 5.17 eV.4 Following the same approach for D2O, the energy loss spectrum shows a threshold feature at ?E ˜ 9.0 eV. We obtain for the first time the DO-D bond dissociation energy to be D(DO-D) = 5.28 ± 0.20 eV. In D2O the DO-D energy value is slightly higher than the O-D bond dissociation energy (5.176 eV5), which is in agreement with its analogue H2O. Page 5366, right column, last paragraph; “Electronic state spectroscopy...” reads as follows: The electronic state spectroscopy of H2O/D2O was thoroughly discussed from the experimental K+ energy loss spectra obtained, from which the DO-D bond dissociation energy has been obtained for the first time to be 5.28 ± 0.20 eV. ¦ SUPPORTING INFORMATION Enthalpies of formation (?fHg°) in reactions 2a.1-2b.2 and 4a.1-4b.2 are actually enthalpies of reaction (?Hr). Table S2 decimal places for O- and OH- from H2O have been corrected; they now read 12.08 ± 0.20 and 7.80 ± 0.20.

Cover page: Correction to “Isotope Effect in D2O Negative Ion Formation in Electron Transfer Experiments: DO–D Bond Dissociation Energy”

Creative Commons 'BY' version 4.0 license

Article
Peer Reviewed

A Designed Host Defense Peptide for the Topical Treatment of MRSA-Infected Diabetic Wounds

UC Davis Previously Published Works (2023)

Diabetes mellitus is a chronic disease characterized by metabolic dysregulation which is frequently associated with diabetic foot ulcers that result from a severely compromised innate immune system. The high levels of blood glucose characteristic of diabetes cause an increase in circulating inflammatory mediators, which accelerate cellular senescence and dampen antimicrobial activity within dermal tissue. In diabetic wounds, bacteria and fungi proliferate in a protective biofilm forming a structure that a compromised host defense system cannot easily penetrate, often resulting in chronic infections that require antimicrobial intervention to promote the healing process. The designed host defense peptide (dHDP) RP557 is a synthesized peptide whose sequence has been derived from naturally occurring antimicrobial peptides (AMPs) that provide the first line of defense against invading pathogens. AMPs possess an amphipathic α-helix or β-sheet structure and a net positive charge that enables them to incorporate into pathogen membranes and perturb the barrier function of Gram-positive and Gram-negative bacteria along with fungi. The capacity of skin to resist infections is largely dependent upon the activity of endogenous AMPs that provided the basis for the design and testing of RP557 for the resolution of wound infections. In the current study, the topical application of RP557 stopped bacterial growth in the biofilm of methicillin-resistant Staphylococcus aureus (MRSA) USA300 infected wounds on the flanks of clinically relevant diabetic TALLYHO mice. Topical application of RP557 reduced bacterial load and accelerated wound closure, while wound size in control diabetic mice continued to expand. These studies demonstrate that RP557 reduces or eliminates an infection in its biofilm and restores wound-healing capacity.

Cover page: A Designed Host Defense Peptide for the Topical Treatment of MRSA-Infected Diabetic Wounds

Article
Peer Reviewed

Sensing the ortho Positions in C6Cl6 and C6H4Cl2 from Cl2− Formation upon Molecular Reduction

Chemical Sciences (2022)

The geometrical effect of chlorine atom positions in polyatomic molecules after capturing a low-energy electron is shown to be a prevalent mechanism yielding Cl₂^-. In this work, we investigated hexachlorobenzene reduction in electron transfer experiments to determine the role of chlorine atom positions around the aromatic ring, and compared our results with those using ortho-, meta- and para-dichlorobenzene molecules. This was achieved by combining gas-phase experiments to determine the reaction threshold by means of mass spectrometry together with quantum chemical calculations. We also observed that Cl₂^- formation can only occur in 1,2-C₆H₄Cl₂, where the two closest C-Cl bonds are cleaved while the chlorine atoms are brought together within the ring framework due to excess energy dissipation. These results show that a strong coupling between electronic and C-Cl bending motion is responsible for a positional isomeric effect, where molecular recognition is a determining factor in chlorine anion formation.

Cover page: Sensing the ortho Positions in C6Cl6 and C6H4Cl2 from Cl2− Formation upon Molecular Reduction

Article
Peer Reviewed

ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling

UCLA Previously Published Works (2022)

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca²⁺ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca²⁺ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca²⁺ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca²⁺-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca²⁺ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca²⁺ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.

Cover page: ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling