Publications

We explore the significance of phenylalanine outside of the phosphotriesterase (PTE) dimer interface through mutagenesis studies and computational modeling. Previous studies have demonstrated that the residue-specific incorporation of para-fluorophenylalanine (pFF) into PTE improves stability, suggesting the importance of phenylalanines in stabilization of the dimer. However, this comes at a cost of decreased solubility due to pFF incorporation into other parts of the protein. Motivated by this, eight single solvent-exposed phenylalanine mutants are evaluated via ROSETTA and good correspondence between experiments and these predictions is observed. Three residues, F304, F327, and F335, appear to be important for PTE activity and stability, even though they do not reside in the dimer interface region or active site. While the remaining mutants do not significantly affect structure or activity, one variant, F306L, reveals improved activity at ambient and elevated temperatures. These studies provide further insight into role of these residues on PTE function and stability.

The Brownian motion of two particles in three dimensions serves as a model for predicting the diffusion-limited reaction rate, as first discussed by von Smoluchowski. Deutch and Felderhof extended the calculation to account for hydrodynamic interactions between the particles and the target, which results in a reduction of the rate coefficient by about half. Many chemical reactions take place in quasi-two-dimensional systems, such as on the membrane or surface of a cell. We perform a Smoluchowski-like calculation in a quasi-two-dimensional geometry, i.e., a membrane surrounded by fluid, and account for hydrodynamic interactions between the particles. We show that rate coefficients are reduced relative to the case of no interactions. The reduction is more pronounced than the three-dimensional case due to the long-range nature of two-dimensional flows.

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3′Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

In the microenvironment of a malignancy, tumor cells do not exist in isolation, but rather in a diverse ecosystem consisting not only of heterogeneous tumor-cell clones, but also normal cell types such as fibroblasts, vasculature, and an extensive pool of immune cells at numerous possible stages of activation and differentiation. This results in a complex interplay of diverse cellular signaling systems, where the immune cell component is now established to influence cancer progression and therapeutic response. It is experimentally difficult and laborious to comprehensively and systematically profile these distinct cell types from heterogeneous tumor samples in order to capitalize on potential therapeutic and biomarker discoveries. One emerging solution to address this challenge is to computationally extract cell-type specific information directly from bulk tumors. Such in silico approaches are advantageous because they can capture both the cell-type specific profiles and the tissue systems level of cell-cell interactions. Accurately and comprehensively predicting these patterns in tumors is an important challenge to overcome, not least given the success of immunotherapeutic drug treatment of several human cancers. This is especially challenging for subsets of closely related immune cell phenotypes with relatively small gene expression differences, which have critical functional distinctions. Here, we outline the existing and emerging novel bioinformatics strategies that can be used to profile the tumor immune landscape.

An engineered supercharged coiled-coil protein (CSP) and the cationic transfection reagent Lipofectamine 2000 are combined to form a lipoproteoplex for the purpose of dual delivery of siRNA and doxorubicin. CSP, bearing an external positive charge and axial hydrophobic pore, demonstrates the ability to condense siRNA and encapsulate the small-molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading relative to Lipofectamine 2000. Furthermore, it induces effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells with efficiencies comparing favorably to Lipofectamine 2000. When the lipoproteoplex is loaded with doxorubicin, the improved doxorubicin loading (∼40 μg Dox/mg CSP) results in a substantial decrease in MCF-7 cell viability.

The tumor microenvironment is now widely recognized for its role in tumor progression, treatment response, and clinical outcome. The intratumoral immunological landscape, in particular, has been shown to exert both pro-tumorigenic and anti-tumorigenic effects. Identifying immunologically active or silent tumors may be an important indication for administration of therapy, and detecting early infiltration patterns may uncover factors that contribute to early risk. Thus far, direct detailed studies of the cell composition of tumor infiltration have been limited; with some studies giving approximate quantifications using immunohistochemistry and other small studies obtaining detailed measurements by isolating cells from excised tumors and sorting them using flow cytometry. Herein we utilize a machine learning based approach to identify lymphocyte markers with which we can quantify the presence of B cells, cytotoxic T-lymphocytes, T-helper 1, and T-helper 2 cells in any gene expression data set and apply it to studies of breast tissue. By leveraging over 2,100 samples from existing large scale studies, we are able to find an inherent cell heterogeneity in clinically characterized immune infiltrates, a strong link between estrogen receptor activity and infiltration in normal and tumor tissues, changes with genomic complexity, and identify characteristic differences in lymphocyte expression among molecular groupings. With our extendable methodology for capturing cell type specific signal we systematically studied immune infiltration in breast cancer, finding an inverse correlation between beneficial lymphocyte infiltration and estrogen receptor activity in normal breast tissue and reduced infiltration in estrogen receptor negative tumors with high genomic complexity.

PURPOSE:
The aim of this study was to determine if zona pellucida thickness variation (ZPTV) is associated with implantation and if this relationship changes with use of assisted hatching (AH).

METHODS:
Day 3 embryos from single or double embryo transfers (DETs) performed between 2014 and 2016 were included. ZPTV was assessed by examining photographs taken before transfer using an automated image processing platform to segment the zona pellucida (ZP) with an active contour technique. One hundred points were obtained of ZP thickness (ZPT) of each embryo to calculate ZPTV ([maximum ZPT-mean ZPT]/mean ZPT). Logistic regression was used to calculate the odds ratio (OR) and 95% confidence intervals (CI) of implantation by tertile of ZPTV. Maternal age and AH were adjusted for a priori. Other cycle and embryo characteristics were adjusted for if they altered the continuous effect estimate by >10%.

RESULTS:
There was no statistically significant association between ZPTV and implantation across tertiles although embryos with greater ZPTV showed a trend of decreased implantation (Tertile 2 (T2) versus Tertile 1 (T1), OR = 0.80, CI = 0.50-1.28; Tertile 3 (T3) versus Tertile 1 (T3), OR = 0.75, CI = 0.47-1.20). While similar nonsignificant trends for the association between ZPTV and implantation were observed across tertiles after stratification of embryos hatched or not, embryos with the greatest ZPTV had slightly higher odds for implantation when AH was utilized (T3 vs. T1: with AH, OR = 0.89, CI = 0.49-1.62; without AH, OR = 0.61, 0.29-1.27).

CONCLUSION:
ZPTV was not associated with implantation after day 3 transfer. This finding did not vary by use of AH.

Therapies targeting inflammation reveal inconsistent results in idiopathic pulmonary fibrosis (IPF). Aerosolised interferon (IFN)-γ has been proposed as a novel therapy. Changes in the host airway microbiome are associated with the inflammatory milieu and may be associated with disease progression. Here, we evaluate whether treatment with aerosolised IFN-γ in IPF impacts either the lower airway microbiome or the host immune phenotype.

Patients with IPF who enrolled in an aerosolised IFN-γ trial underwent bronchoscopy at baseline and after 6 months. 16S rRNA sequencing of bronchoalveolar lavage fluid (BALF) was used to evaluate the lung microbiome. Biomarkers were measured by Luminex assay in plasma, BALF and BAL cell supernatant. The compPLS framework was used to evaluate associations between taxa and biomarkers.

IFN-γ treatment did not change α or β diversity of the lung microbiome and few taxonomic changes occurred. While none of the biomarkers changed in plasma, there was an increase in IFN-γ and a decrease in Fit-3 ligand, IFN-α2 and interleukin-5 in BAL cell supernatant, and a decrease in tumour necrosis factor-β in BALF. Multiple correlations between microbial taxa common to the oral mucosa and host inflammatory biomarkers were found.

These data suggest that the lung microbiome is independently associated with the host immune tone and may have a potential mechanistic role in IPF.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

We explore theoretically the aerodynamics of a recently fabricated jellyfish-like flying machine (Ristroph & Childress, J. R. Soc. Interface, vol. 11 (92), 2014, 20130992). This experimental device achieves flight and hovering by opening and closing opposing sets of wings. It displays orientational or postural flight stability without additional control surfaces or feedback control. Our model ‘machine’ consists of two mirror-symmetric massless flapping wings connected to a volumeless body with mass and moment of inertia. A vortex sheet shedding and wake model is used for the flow simulation. Use of the fast multipole method allows us to simulate for long times and resolve complex wakes. We use our model to explore the design parameters that maintain body hovering and ascent, and investigate the performance of steady ascent states. We find that ascent speed and efficiency increase as the wings are brought closer, due to a mirror-image ‘ground-effect’ between the wings. Steady ascent is approached exponentially in time, which suggests a linear relationship between the aerodynamic force and ascent speed. We investigate the orientational stability of hovering and ascent states by examining the flyer’s free response to perturbation from a transitory external torque. Our results show that bottom-heavy flyers (centre of mass below the geometric centre) are capable of recovering from large tilts, whereas the orientation of the top-heavy flyers diverges. These results are consistent with the experimental observations in Ristroph & Childress (J. R. Soc. Interface, vol. 11 (92), 2014, 20130992), and shed light upon future designs of flapping-wing micro aerial vehicles that use jet-based mechanisms.