An Investigation into Clinically Relevant Determinants of Azole Resistance in Candida albicans

Candida albicans is a commensal organism commonly colonizing the human gut and skin. As an opportunistic pathogen, it can cause persistent and serious infections in individuals with compromised immune systems, including the very young and elderly. Moreover, C. albicans can cause a wide spectrum of diseases ranging from superficial mucosal infections to life-threatening invasions of the organs and bloodstream. Candida species are the most common cause of invasive fungal disease, which is associated with high mortality and imposes a heavy toll on the healthcare system. Over the last 30 years, the azole antifungals have been a mainstay of antifungal therapy, being effective in a wide variety of fungal infections and serving as the primary oral treatment option. However, increased use, inappropriate dosing, and prolonged treatments have given rise to azole-resistant Candida albicans and other Candida species. Resistance in C. albicans results from a combination of different mechanisms. Increased expression of the efflux pump encoding genes CDR1, CDR2, and MDR1 as well as increased expression of ERG11, encoding the azole target (14α-lanosterol demethylase, also known as CYP51) are all primary mechanisms of azole resistance that arise in azole-resistant clinical isolates. These changes are known to be mediated through gain-of-function mutations in the genes of a fungal-specific transcription factor family known as the zinc cluster transcription factors. Furthermore, genetic changes in the ergosterol biosynthesis genes, ERG11 and ERG3, encoding a C-5 sterol desaturase, also contribute to clinical azole resistance in C. albicans. The interplay of these mechanisms can result in azole-resistance, treatment failure, and ultimately, poorer outcomes in patients. Therefore, to improve healthcare outcomes, understanding resistance development and the mechanisms that drive them in C. albicans is crucial. Within a collection of predominantly fluconazole-resistant clinical isolates of C. albicans, our lab had previously characterized most known mechanisms of azole resistance present in each isolate. Increased CDR1 expression in isolates lacking TAC1 gain-of-function mutations coupled with recent literature suggesting a role of the Mrr2 zinc cluster transcription factor in azole resistance lead us to sequence and test mutations in the MRR2 gene in across this collection. By placing mutant MRR2 alleles in azole- susceptible backgrounds, we hoped to measure the contribution of MRR2 mutations to azole resistance through changes in CDR1 expression and fluconazole minimum inhibitory concentrations (MICs). Counter to what has been recorded in the literature, we found no evidence that mutations in MRR2 impact either CDR1 expression or azole susceptibility in C. albicans. This is a novel finding correcting a previous mistaken paradigm of a clinically relevant mechanism driving resistance in C. albicans. Next we more closely examined the role of ERG11 mutations found in clinical isolates. Though the contributory effects of ERG11 mutations to azole resistance had been quantified, the specific biochemical impact of these mutations on enzyme function and ligand-binding interaction have only recently come to light. Here we introduced additional CaCYP51 amino acid substitution mutants (D278N and Y132H) in C. albicans vi and tested our entire collection of CaCYP51 mutant strains to determine their in vitro azole susceptibilities in the context of these findings. In general, we observed differences in the fluconazole and voriconazole MICs between CaCYP51 amino acid substitutions. In contrast, MICs to itraconazole showed a small, fairly consistent increase in MIC across tested CaCYP51 strains and MICs to posaconazole did not increase at all over the wild type except for the G448E substitution, suggesting posaconazole possesses the best in vitro activity against these CaCYP51 mutants. Overall, we also revealed that not all ERG11 mutations confer azole resistance through decreased binding interactions with the target and the azole drug, suggesting that CaCYP51 amino acid substitutions may instead interact with other associated proteins to confer resistance. Furthermore, it was discovered that many ERG11 mutations from clinical isolates result in low catalytic turnover of the enzyme, which is crucial to normal rates of ergosterol production in a healthy cell. Though preliminary results of growth in CaCYP51 mutant strains does not support attenuated fitness in competitive assay, the findings here prove that some clinical ERG11 mutations result in diminished enzyme function. We also sequenced the collection of clinical isolates and discovered an A351V Erg3 amino acid substitution in our azole-resistant isolates and predominantly in those with multiple ERG11 mutations. This suggested a possible connection between CaCYP51 mutants and amino acid substitutions in Erg3. As the proteins encoded for by the ERG11 and ERG3 genes are involved in the same ergosterol biosynthesis pathway, defects in Erg11 enzyme function might be expected to impact accumulation of substrates of Erg3, specifically, precursors of the toxic sterol metabolite 14α-methylergosta- 8,24(28)-dien-3β, 6α-diol. By testing ERG11 mutant strains with and without the ERG3A351V allele in growth competition experiments, we hoped to observe a conferred fitness benefit by the ERG3 mutation. Interestingly, we were unable to generate one of our selected ERG11 mutants with the poorest catalytic turnover in the absence of the A351V amino acid change in Erg3. Future investigation by other lab members is needed to determine if ERG3 mutations can indirectly influence azole susceptibility through permissive mutation. Lastly, we tested susceptibility of our clinical collection to the new tetrazole antifungals VT-1161 and VT-1598, which have been reported to exhibit potent activity against azole-resistant C. albicans and a host of other fungal species. We additionally investigated determinants of resistance to the two new agents by obtaining susceptibilities to C. albicans strains containing individual known mechanisms of azole resistance. While susceptibility to VT-1161 was reduced when CDR1 and MDR1 were overexpressed, VT-1598 seemed unaffected by any tested resistance mechanism. Importantly, both retained activity against a significant portion of mutant ERG11 strains. VT-1598 MICs were not affected by any single mechanism of resistance. However, screening of our azole-resistant clinical isolates identified five isolates with greatly elevated MICs to all tested agents. While one of these isolates possesses an ERG3 nonsense mutation that likely explains its pan-azole resistant profile, the other four isolates do not uniquely overexpress known resistance genes or possess known gene mutations that might explain their resistance. This finding suggests that there are determinants of azole resistance that are as yet undiscovered in C. albicans

Phase diagram of the one-dimensional half-filled extended Hubbard model

We study the ground state of the one-dimensional half-filled Hubbard model with on-site (nearest-neighbor) repulsive interaction $U$ ($V$) and nearest-neighbor hopping $t$. In order to obtain an accurate phase diagram, we consider various physical quantities such as the charge gap, spin gap, Luttinger-liquid exponents, and bond-order-wave (BOW) order parameter using the density-matrix renormalization group technique. We confirm that the BOW phase appears in a substantial region between the charge-density-wave (CDW) and spin-density-wave phases. Each phase boundary is determined by multiple means and it allows us to do a cross-check to demonstrate the validity of our estimations. Thus, our results agree quantitatively with the renormalization group results in the weak-coupling regime ($U \lesssim 2t$), with the perturbation results in the strong-coupling regime ($U \gtrsim 6t$), and with the quantum Monte Carlo results in the intermediate-coupling regime. We also find that the BOW-CDW transition changes from continuous to first order at the tricritical point $(U_{\rm t}, V_{\rm t}) \approx (5.89t, 3.10t)$ and the BOW phase vanishes at the critical end point $(U_{\rm c}, V_{\rm c}) \approx (9.25t, 4.76t)$.Comment: 4 pages, 5 figure

Electron correlations and bond-length fluctuations in copper oxides: from Zhang--Rice singlets to correlation bags

We perform first principles, multiconfiguration calculations on clusters including several CuO$_6$ octahedra and study the ground-state electron distribution and electron--lattice couplings when holes are added to the undoped $d^9 p^6$ configuration. We find that the so-called Zhang--Rice state on a single CuO$_4$ plaquette is nearly degenerate with a state whose leading configuration is of the form Cu $d^9$-- O $p^5$-- Cu $d^9$. A strong coupling between the electronic and nuclear motion gives rise to large inter-site charge transfer effects for half-breathing displacements of the oxygen ions. Under the assumption of charge segregation into alternating hole-free and hole-rich stripes of Goodenough \cite{jbg_02,jbg_03}, our results seem to support the vibronic mechanism and the traveling charge-density wave model from Refs.\cite{jbg_02,jbg_03} for the superconductivity in copper oxides.Comment: submitted to Phys. Rev.

Study of Apollo water impact. Volume 8 - Unsymmetric shells of revolution analysis Final report

Numerical analysis of static, and dynamic shell response to water impact load

Renormalization of the quasiparticle hopping integrals by spin interactions in layered copper oxides

Holes doped within the square CuO2 network specific to the cuprate superconducting materials have oxygen 2p character. We investigate the basic properties of such oxygen holes by wavefunction-based quantum chemical calculations on large embedded clusters. We find that a 2p hole induces ferromagnetic correlations among the nearest-neighbor Cu 3d spins. When moving through the antiferromagnetic background the hole must bring along this spin polarization cloud at nearby Cu sites, which gives rise to a substantial reduction of the effective hopping parameters. Such interactions can explain the relatively low values inferred for the effective hoppings by fitting the angle-resolved photoemission data. The effect of the background antiferromagnetic couplings of renormalizing the effective nearest-neighbor hopping is also confirmed by density-matrix renormalization-group model Hamiltonian calculations for chains and ladders of CuO4 plaquettes

Design of an automobile turbocharger gas turbine engine

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2003.Includes bibliographical references (leaf 24).The turbocharger gas turbine engine was designed with the intent of being built as a demonstration for the Massachusetts Institute of Technology Department of Mechanical Engineering courses 2.005 and 2.006 to supplement material covered. A gas turbine operates on an open version of the Brayton cycle and consists of a compressor, a combustion chamber and a turbine. An automobile turbocharger was chosen because it contains a compressor and turbine on a common shaft. Designs for the combustion chamber, oil system, fuel system, and ignition system were created based on research of similar projects. Many of the necessary parts were also specified.by Keane T. Nishimoto.S.B

Basic properties of three-leg Heisenberg tube

We study three-leg antiferromagnetic Heisenberg model with the periodic boundary conditions in the rung direction. Since the rungs form regular triangles, spin frustration is induced. We use the density-matrix renormalization group method to investigate the ground state. We find that the spin excitations are always gapped to remove the spin frustration as long as the rung coupling is nonzero. We also visibly confirm spin-Peierls dimerization order in the leg direction. Both the spin gap and the dimerization order are basically enhanced as the rung coupling increases.Comment: 4 pages, 2 figure

A Model Study of the Low-Energy Charge Dynamics of NaV_2O_5

An exact-diagonalization technique on small clusters is used to calculate the dynamical density correlation functions of the dimerized t-J chain and coupled anisotropic t-J ladders (trellis lattice) at quarter filling, i.e., the systems regarded as a network of pairs (dimers or rungs) of sites coupled weakly via the hopping and exchange interactions. We thereby demonstrate that the intersite Coulomb repulsions between the pairs induce a low-energy collective mode in the charge excitations of the systems where the internal charge degrees of freedom of the pairs play an essential role. Implications to the electronic states of NaV_2O_5, i.e., fluctuations of the valence state of V ions and phase transition as a charge ordering, are discussed.Comment: 4 pages, 4 gif figures. Hardcopies of figures (or the entire manuscript) can be obtained by e-mail request to [email protected]

Optical conductivity in the CuO double chains of PrBa_2Cu_4O_8: Consequences of charge fluctuation

We calculate the optical conductivity of the CuO double chains of PrBa$_2$Cu$_4$O$_8$ by the mean-field approximation for the coupled two-chain Hubbard model around quarter filling. We show that the $\sim$40 meV peak structure, spectral shape, and small Drude weight observed in experiment are reproduced well by the present calculation provided that the stripe-type charge ordering presents. We argue that the observed anomalous optical response may be due to the presence of stripe-type fluctuations of charge carriers in the CuO double chains; the fast time scale of the optical measurement should enable one to detect slowly fluctuating order parameters as virtually a long-range order.Comment: 7 pages, 5 eps figure