BRIAN AGUADO, UNIVERSITY OF COLORADO, BOULDER
Precision biomaterial platforms to probe valvular fibroblast activation
Aortic valve stenosis (AVS) is a progressive disease characterized by aberrant stiffening of the aortic valve, leading to inadequate blood flow from the left ventricle and eventual heart failure. AVS is currently treated exclusively with valve replacement surgeries, which may be avoided if effective small molecule therapeutics could be identified to slow or reverse AVS progression. During AVS progression, a large fraction of valvular interstitial cells (VICs) differentiate into pathogenically activated myofibroblasts, which contribute to excessive matrix deposition and eventual valve leaflet stiffening. Effective small molecule therapeutics intended to reverse myofibroblast activation remain elusive, owing to the inherent heterogeneity of the cellular microenvironment from patient to patient. For example, male patients show increased calcification and female patients show increased tissue fibrosis in the aortic valve microenvironment, leading to differential drug responses. Considering these clinical observations, we seek to engineer precision biomaterial microenvironments to recapitulate patient-specific AVS progression in vitro and identify molecular mechanisms that mediate reversal of myofibroblast activation. Precision biomaterials are defined as engineered environments that enable the evaluation of how patient-specific variables may influence disease progression. As a strategy to evaluate patient-specific AVS progression, we utilize poly(ethylene glycol) (PEG) hydrogels as precision in vitro platforms to probe various biochemical and mechanical cues that activate VICs to a myofibroblast state, as well as cues that reverse activation. Our work demonstrates how engineered hydrogel matrices enable an increased understanding of the molecular mechanisms guiding myofibroblast activation and reversal, which may provide a critical bridge toward patient-specific small molecule AVS therapies.
ISMAIL AHMED, UNIVERSITY OF PENNSYLVANIA
Development of a Blue Fluorescent Amino Acid for Biological Spectroscopy and Microscopy
The discovery of green fluorescent protein (GFP) ushered in a transformative era in biological spectroscopy and imaging, allowing for a wide range of biochemical and biophysical processes and interactions to be studied at the cellular and molecular level. However, several factors limit a broader use of GFP (and its derivatives), which include (1) the fluorophore requires molecular oxygen to form; (2) the fluorophore needs a relatively long maturation time (2-4 hours in vivo); (3) GFP and its derivatives are relatively large; and (4) the fluorescence lifetime of GFP and its derivatives is relatively short (1-3 ns). Hence, the development of a smaller, ideally amino acid-based fluorophore that does not require a specific environment and time to mature, has a large fluorescence quantum yield, long fluorescence lifetime, with good photostability, and an emission spectrum in the visible range, is the new frontier of fluorescent probes that would revolutionize in vitro and in vivo spectroscopic and microscopic measurements. Herein, we show that a tryptophan derivative meets these specifications and therefore has great potential as a complementary tool to open up new avenues of biological research where GFP is not ideal. Moreover, we also demonstrate its use in application of both Förster resonance energy transfer and photo-induced electron transfer in biological systems.
CHRISTOPHER BARNES, CALIFORNIA INSTITUTE OF TECHNOLOGY
Structures of HIV-1 envelope trimers define antibody-mediated neutralization of HIV-1 and the mechanism of viral entry
The human immunodeficiency virus type-1 (HIV-1) targets cells in the immune system increasing the likelihood of susceptibility to infections and cancers. Upon infection, the trimeric HIV-1 envelope glycoprotein (Env), a trimer of gp120-gp41 heterodimers, initiates fusion of viral and host cell membranes through interactions with the CD4 receptor and a co-receptor (CCR5 or CXCR4) on the surface of the host-cell. Since Env-mediated fusion represents the first step in HIV-1 infection, targeted therapies or vaccines to disrupt the mechanism of viral entry would be ideal. To this end, we investigate broadly neutralizing antibodies (bNAbs) isolated from HIV-1 infected individuals that target Env to inform HIV-1 vaccine design efforts. Developing bNAbs with increased efficacy requires understanding how antibodies interact with the native oligomannose and complex-type N-glycan shield that hides most protein epitopes on HIV-1 Env. Using a combination of X-ray crystallography and single particle cryo-EM structural techniques, we have determined structures of trimeric Env bound to bNAbs targeting conserved epitopes, including the most potent V3/N332gp120 glycan-targeting bNAb reported to date. Furthermore, we have solved sub-4Å cryo-EM structures of partially-open Env trimers bound to CD4, revealing conformational changes relevant for co-receptor binding and fusion. These results increase our understanding of bNAb-glycan interactions critical for immunogen design, as well as illuminate potential receptor-induced Env changes that may facilitate therapeutic design efforts to disrupt viral entry.
KIAH BUTLER, MERCK
Discovery of peptidase inhibitor 16 (PI-16) as an immunomodulatory ligand
Immunomodualtory receptors on myeloid and T cells are essential in preventing tissue damage following immune activation against pathogens. Dysregulation of this communication results in hyper- or hypo-inflammatory environments as observed in autoimmunity and cancer, respectively. In a recent screen of ligands with the potential to engage Immunoreceptor tyrosine-based inhibitory motif (ITIM) receptors, protease inihibitor-16 (PI-16) was identified as a T cell-specific ligand for an inhibitory receptor expressed by myeloid cell. PI-16 was previously identified as a soluble protein in prostate cancer patients and an activation marker of regulatory T cells. Our work revealed a novel role for PI16 in mediating myeloid cell suppression as well as a T cell activation marker in the circulation and tumors from patient samples. Additionally, utilizing a mouse tumor model system, we have demonstrated that blockade with aPI-16 antibodies can drive activation of the immune system and potent anti-tumor immunity. These data reveal PI-16 as a novel and biologically relevant target for tumor immunotherapy.
MARÍA M. CORONEL, GEORGIA INSTITUTE OF TECHNOLOGY
Synthetic Immunomodulatory Biomaterials to Improve Islet Graft Survival
Type 1 diabetes (T1D) is a chronic condition in which patients produce little to no insulin due to the progressive loss of beta-cells. While the disease can be managed via external insulin delivery, patients still experience a high incidence of mortality and development of chronic debilitating comorbidities. Islet transplantation has emerged as a potential strategy to restore a patient’s glycemic control, yet long-term rejection and the need for chronic immunosuppression present a major barrier. Because of the potential of PD-L1 to negatively regulate allo-immunity and initiate the activation of tolerogenic responses, we have engineered bio-functionalized synthetic hydrogel particles for the delivery of PD-L1, in a localized and spatially controlled manner, for the long-term induction of tolerance to extrahepatic pancreatic islet allografts. PD-L1 delivery via synthetic PEG microgels, developed via flow focusing microfluidics, is achieved via surface biotin modification for the binding and presentation of a streptavidin-PD-L1 chimera (SA-PD-L1). These bioactive microgels are co-delivered with unmanipulated pancreatic islets, with the objective of shifting the immune balance towards a tolerogenic environment to promote islet survival in the absence of chronic immunosuppression. This bioactive platform can provide an active interface to instruct local immune responses, which can have a broad impact for cell-based therapies by establishing a targeted biomaterial-mediated immunotherapy for inducing a tolerizing response to allogeneic cells.
DANIELLE D. DESOUZA, STANFORD UNIVERSITY
Structural correlates of theta-burst stimulation treatment response in major depressive disorder
Repetitive transcranial magnetic stimulation (rTMS) is an effective, noninvasive treatment for medication-refractory major depressive disorder (MDD); however, mechanisms underlying its efficacy remain poorly understood. In recent years, functional and neurochemical changes at both the rTMS target site and remote brain regions as detected by neuroimaging methods have been reported, but few studies have examined structural correlates of treatment response. Magnetic resonance imaging (MRI) methods, such as cortical thickness analysis (CTA), provide an automated means to measure thickness of the cortex with submillimeter accuracy and demonstrate subtle changes following effective treatment for chronic pain. In one study, we evaluated cortical thickness as a potential biomarker of intermittent theta-burst stimulation (iTBS) applied to the left dorsolateral prefrontal cortex in patients with MDD. iTBS is a modified form of rTMS and modeled after endogenous hippocampal discharge patterns and has been shown to be more efficient than standard rTMS in modifying cortical excitability. Whole-brain CTA analyses corrected for multiple comparisons revealed that treatment response, as measured by the Hamilton Rating Scale for Depression-17 Item (HAMD-17) scale, was significantly correlated with cortical thickness change in the insula. In summary, we provide evidence that structural MRI can be used to noninvasively identify potentially clinically useful biomarkers that provide an objective measure of treatment response. Future studies will combine multimodal structural and functional neuroimaging methods to provide further insight into the pathogenesis of and treatment of MDD and additionally to other patient populations including those with chronic pain.
ANTONIA DOMINGUEZ, STANFORD UNIVERSITY
Enhanced CRISPRa by the use of both Transcription and Epigenetic Activators
The ability to regulate endogenous gene expression in a sequence-specific manner offers great promises in deepening our understanding of human health and disease. Beyond gene editing, the CRISPR-Cas9 technology offers a gene regulation tool by using the nuclease-deficient dCs9, which does not cleve, but precisely binds to DNA when guided by a single guide RNA (sgRNA). CRISPR activation (CRISPRa) uses dCas9 fusion proteins to recruit transcriptional activators for targeted endogenous gene activation. Strategies to recruit multiple activators to a gene locus have been implemented, such as dCas9-VPR (VP64, p65,Rt1), and have increased the ability to efficiently activate endogenous genes with one sgRNA. Additionally, fusion of the catalytic domain of histone acetyltrnsferase p300 (p300core) to dCas9 serves as an epigenetic activator at promoters and enhancers. Here, we implement a strategy that combine both transcription and epigenetic activation by pairing them together, expressing them sequentially or on orthogonal dCas9 proteins. WE have tested the activation of multiple endogenous genes in HEK293T cells and demonstrate that our combined activation system outperforms the individual systems. This combined system allows for a wider dynamic range of activation that can then be modulated by sgRNA selection. Additionally we have engineered these components into an inducible single-vector based CRISPR system that contains selection, fluorescence and utilizes the Piggybac transposon system for integration.
CRISTINA E. FERNANDEZ, DUKE UNIVERSITY
Therapeutic Biopolymers for Targeted Cardiac Recovery after Injury
Heart failure is an epidemic that is impacting a growing number of adults around the world. Heart failure results from the loss of functional myocardium, which, untreated, leads to progressive disease that can be fatal. Progressive heart disease is denoted by chamber dilation and thinning of the vascular wall in an effort to maintain cardiac output while compensating for loss of contractile function. This phenomenon leads to additional myocardial damage and tissue fibrosis, which contributes to further progression of heart failure. Injecting biopolymers into the injury site has demonstrated improved cardiac function in several animal models. Our goal is to develop an injectable biopolymer to provide mechanical support and promote regeneration within the failing heart. I am using a recently developed class of novel biopolymers based on elastin-like polypeptide (ELP), in which ordered polyalanine repeats have been introduced into the disordered ELP amino acid structure. These ELP-based biopolymers meet three parameters for the ideal biopolymer for heart regeneration: they are injectable, they may be modified to be genetically fused or conjugated to regenerative compounds, and they are able to form a stable, solid matrix in vivo to support new tissue growth. I am optimizing the composition and delivery of these biopolymers to promote integration and mechanical support within an experimental mouse model of myocardial infarction (MI). In addition, I have performed a large-scale CRISPR-Cas9 based genetic screen to identify novel regulators of endothelial cell proliferation and angiogenesis. I will use the results of this screen to highlight novel angiogenic compounds, with the long-term goal of creating an injectable biopolymer system that can be directly injected into the injured heart to promote regeneration through increased angiogenesis and cardiomyocyte proliferation.
GALO GARCIA III, UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
How is the cell’s antenna built? Super-resolution Detection of Ciliary Architecture
Cilia are signaling hubs protruding from the surface of most human cell types, where they detect optical and molecular signals outside of the cell. Despite being specialized for the reception of signals, primary cilia are often found buried in the cell partially hidden from the external environment. These buried cilia are enshrouded by a membrane invagination called the ciliary pocket. Ciliary membrane compartmentalization is essential for proper ciliary signaling. However, it is unclear what role compartmentalization of the ciliary pocket membrane plays in ciliary signaling. We are investigating the cytoskeleton and other membrane remodeling proteins as mediators of ciliary pocket formation and function. By tracking the location of the phosphoinositide PI(4,5)P2 in the cell by live and super-resolution imaging we have found that the ciliary pocket membrane is PI(4,5)P2-rich. The absence of PI(4,5)P2 in the ciliary membrane and the presence of the phosphoinositide in the contiguous ciliary pocket membrane suggests the existence of an effective lipid barrier between the two membrane surfaces. We are employing an inducible CRISPR-Cas9 knockout system to identify molecular determinants of ciliary pocket formation, as well as mediators of membrane compartmentalization and membrane protein trafficking.
NYDIARIS HERNANDEZ-SANTOS, UNIVERSITY OF WISCONSIN, MADISON
Lung Epithelial Cell Immunity to Pulmonary Fungal Pathogens
Lung epithelial cells (LEC) reside at the interface between the airway and the underlying lung tissue. This locale strategically positions LEC to serve as a physical barrier between the tissue
and the environment, and to actively participate in the immune response against pathogens that enter the body through the airways. In fact, LEC express pattern recognition receptors and
inflammatory mediators that equip them for recognition of microbial moieties and antimicrobial defense1, 2. However, despite their prime location and immunological endowment, their role is often concealed by the activities of “professional” immune cells, particularly in the context of pulmonary fungal infections. It is estimated that up to 10 fungal spores are inhaled with every breath. Upon arrival into the airway, LEC are the spores’ first point of contact. Therefore, understanding the mechanisms of LEC-mediated immunity to fungal pathogens is of paramount importance. To better understand LEC-fungus interactions, I have chosen the fungal pathogen Blastomyces dermatitidis, which is endemic to Wisconsin and the Ohio and Mississippi river valleys and causes pneumonia in 50% of exposed persons regardless of immune competence and age4. One of the largest blastomycosis outbreaks reported occurred in a group of children that played at a beaver dam in Eagle River, Wisconsin. I recently reported that LEC are key elements of the immune response to B. dermatitidis. LEC orchestrate innate antifungal immunity via the actions of innate lymphocyte populations (nTh17 and TCRγδ cells) that produce interleukin 17A (IL-17A) and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), which are known to be crucial in the immune response against B. dermatitidis. These IL-17- and GM-CSF-producing lymphocytes equip neutrophils to kill B. dermatitidis. Future experiments aim to elucidate the mechanisms by which LEC “sense” B. dermatitidis, and the LEC products influencing the function of nTh17 and TCRγδ cells.
JASMINE KWASA, BOSTON UNIVERSITY
Neural Measures of ADHD: Probing Brain Networks of Auditory and Visual Selective Attention
Selective auditory attention is the human ability to volitionally pay attention to a single acoustic source (like a friend’s voice) within a complex listening environment (like a loud cocktail party). Individuals vary in their abilities to successfully perform this cognitively demanding task, even in the absence of hearing problems of the auditory periphery. We have previously shown that selective attention requires precisely coordinated communication within and between brain regions responsible for encoding space and time, including prefrontal, auditory, and visual cortices. My thesis work asks the following question: what happens to this brain network when cognitive control is diminished due to Attention Deficit Hyperactivity Disorder (ADHD)?
I will discuss my ongoing thesis work in which we recruited young adults with ADHD to perform auditory and visual psychophysics tasks while undergoing simultaneous high-density electroencephalography (EEG). Using signal processing and machine learning, we seek to explain differences in performance on these tasks—and clinical psychiatric measures—in terms of quantifiable neural correlates: evoked event-related potentials (ERPs) and induced neural oscillations. Behavioral results suggest that adults with ADHD have a modality-independent deficit in spatial selective attention that modestly improves with the use of stimulant medications. Additionally, neural measures from the auditory task correlate with performance, indicating that this paradigm could be a promising probe of an individual’s inherent attention abilities.
HIRUY MEHARENA, MIT
Dysfunctional Interplay between the Transcriptome and Chromatin Architecture in Down Syndrome
Down syndrome is a neurodevelopmental disorder characterized by abnormal brain development and intellectual disability caused by triplication of chromosome 21. Even though DS is a caused by triplication of chromosome 21 (T21), recent studies have demonstrated genome-wide disruption of gene-expression beyond the genes on chromosome 21. Currently the consequences of T21 on the chromatin state and 3D-genome as well as the interplay between the epigenome and transcriptome remains unknown.
DIANA MONSIVAIS, BAYLOR COLLEGE OF MEDICINE
Molecular cues controlling embryo implantation and early pregnancy loss
The establishment and outcome of a successful pregnancy is dictated by effective communication between the implanting embryo and its mother. At the cellular level this communication consists of physical interactions between various ligand/receptor complexes and by subsequent intracellular responses initiated by cytokines, growth factors and hormones. It was previously determined that maternal signals mediated by the bone morphogenetic proteins (BMPs) and their cognate cell surface receptors are critical for early and mid-gestation embryo development. Despite the crucial roles of the BMP ligands and receptors during pregnancy, little is known about the global genomic networks that are activated or repressed downstream of BMPs by the SMAD1/5 transcription factors. Global inactivation of SMAD1 and SMAD5 results in embryonic lethality precluding its study in reproductive function, therefore, we generated mice with conditional inactivation of SMAD1 and SMAD5 using progesterone receptor cre (Smad1flox/flox;Smad5flox/flox;Pgr-cre+/–, or “Smad1/5 cKO”). A six-month fertility trial identified that the female Smad1/5 cKO mice are sterile, and did not generate any pups over the course of the study. Histological and molecular analyses determined that sterility was the result of impaired uterine receptivity. At 4.5 days post-coitum (dpc) implantation sites were not visualized by Chicago blue vascular dye injection, yet fertilized unattached blastocysts were recovered from the lumen of Smad1/5 cKO mice, indicating failed embryo attachment to the luminal epithelium. During the window of implantation, uteri of Smad1/5 cKO mice responded abnormally to estradiol and to progesterone and showed elevated expression of the estradiol-regulated genes and decreased expression of progesterone-responsive genes. Further analyses determined that the blunted uterine progesterone response was the result of decreased levels of progesterone receptor gene expression in the Smad1/5 cKO mice. Uteri of Smad1/5 cKO mice did not respond to an artificial decidual stimulus and the stroma failed to differentiate. At 12 weeks of age Smad1/5cKO mice presented enlarged cystic uterine glands that developed into hemorrhagic lesions as the mice aged. 3-dimensional analysis of the uterus by optical projection tomography also demonstrated abnormalities in uterine glandular orientation and coiling of the Smad1/5 cKO mice during early pregnancy. Decidualization defects in the absence of SMAD1 and SMAD5 were also observed in human endometrial stromal cells. Cumulatively, these results demonstrate that the SMAD1/5 transcription factors have crucial roles in uterine homeostasis, are crucial for female fertility, and control the uterine response to hormones by modulating the expression of the progesterone receptor.
DERRICK J. MORTON, EMORY UNIVERSITY
Using CRISPR/Cas9-mediated Genome Editing to Model Human Neurological Disease in Drosophila
In the era of genome editing, one powerful approach is to employ CRISPR/Cas to generate models of human disease. I am using this approach to model human diseases that arise from recently identified mutations in genes that encode subunits of the evolutionarily conserved RNA exosome complex. The RNA exosome is an evolutionary-conserved, 3’-5’ riboexonuclease 10-subunit complex critically important for both precise processing and complete degradation of a variety of cellular RNAs. One of the most critical cellular functions of the RNA exosome is the production of mature, properly trimmed rRNAs required within the ribosome. Given, the crucial role of the RNA exosome in post-transcriptional regulation of RNA, it is not surprising that the complex is essential in systems examined thus far. The recent discovery that mutations in genes encoding structural exosome subunits cause tissue-specific diseases makes defining the role of the exosome within specific tissues critically important to understand the basis of these diseases. The Drosophila system provides ideal tools to examine tissue-specific gene function. Mutations in the RNA exosome subunit 3 gene (EXOSC3) cause Pontocerebellar Hypoplasia type 1b (PCH1b), which is an autosomal recessive neurodegenerative disease, while mutations in RNA exosome subunit 2 (EXOSC2) cause a variety of distinct tissue-specific phenotypes including mild intellectual disability. The disease-causing mutations identified are not null mutations but rather amino acid changes in evolutionarily-conserved residues. The tissue-specific defects these changes cause are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC2 and EXOSC3 cause disease by using genome editing to explore the functional consequences of these changes in the Drosophila model. To achieve this goal, I developed a novel model of RNA exosome-linked disease in Drosophila utilizing CRISPR/Cas9 technology to create flies that model the patient mutations. The Drosophila model system allows for true modeling of both the homozygous and heterozygous genotypes found in patients. My preliminary data examining disease-linked amino acid substitutions in EXOSC3 reveal reduced viability and behavioral phenotypes that align with the disease severity observed in patients. We are performing RNA-Seq analysis to identify the spectrum of RNA targets effected. Taken together, my data provide evidence that the Drosophila model can be used to provide insight into tissue-specific function of the RNA exosome in vivo and explore the functional consequences of amino acid substitution linked to distinct disease phenotypes.
SHAMSIDEEN OJELADE, BAYLOR COLLEGE OF MEDICINE
Functional Validation of the Alzheimer’s disease Susceptibility Gene CD2AP
Alzheimer’s disease (AD) is the most common form of neurodegenerative disorder worldwide and it’s projected to affect about 14 million people by 2050. Our ability to find better treatments and a cure for AD requires identifying the risk genes that makes an individual more predisposed to AD. To that end, studies have identified more than 20 novel genes that might increase AD risk. Unfortunately, our understanding of how these genes contribute to AD pathogenesis is still not known. Taking advantage of the simple but powerful experimental model system, the Fruit fly, we show that loss of CD2-associated Protein (CD2AP), one of the 20 risk genes, enhances AD pathology in flies. CD2AP is expressed in the nervous system, and flies lacking CD2AP show reduced lifespan and impairments in neuronal function. We report that loss of CD2AP affects the ubiquitin-proteasome system, which consequently alters protein turnover and calcium levels at the synapse. Studies of CD2AP null mice support a conserved function in synaptic proteostasis, and CD2AP protein levels are inversely related to synaptic protein abundance in human postmortem brains. In summary, our cross-species experimental dissection of CD2AP reveals a conserved synapse function, and highlights potential interactions with Tau-induced proteinopathy, calcium dyshomeostasis, and synaptotoxicity in AD.
KANDY T. VELÁZQUEZ-FIGUEROA, UNIVERSITY OF SOUTH CAROLINA SCHOOL OF MEDICINE
The effects of Ojeok-san on cancer-induced visceral nociception
Objective: Visceral pain can develop as result of a detrimental disease such as colon cancer. Despite the adverse effects of opioid-derived drugs in the gastrointestinal system, these analgesics are still the preferred treatment for cancer pain. Thus, it is necessary that novel analgesic therapies are developed in order to treat cancer-induced visceral pain. Ojeok-san is an herbal formula consisting of seventeen herbs. This herbal formula has been shown to possess anti-inflammatory, immunoregulatory, and analgesic properties. In this study, we examined the potential beneficial effects of Ojeok-san in a preclinical model of colon cancer-induced visceral pain.
Study Design and Outcomes: Male and female wild-type C57BL/6 mice were exposed to the carcinogen, azoxymethane (AOM, 10 mg/kg) and a chemical inflammatory driver, dextran sulfate sodium (DSS, 1-2%) to promote tumorigenesis in the colorectum. Ojeok-san was given orally (2000 mg/kg) a month after carcinogen exposure to determine its influence on disease index, tumorigenesis, and somatic and visceral nociception.
Methods: Body weight loss, fecal consistency, and blood in the stool was scored weekly to determine disease activity index. Referred somatic hyperalgesia was assessed using von Frey filament (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6). Colorectal visceromotor response to distension was calculated using intra-balloon pressure changes. Ascending phasic distension protocol (10, 25, 40, 65, 80 mmHg) was used to assess visceral pain-related to colon cancer.
Results: We found that AOM/DSS mice, regardless of treatment, presented similar disease activity and tumor burden. In relation to nociception, we found that exposure to AOM/DSS promoted referred somatic hyperalgesia and visceromotor response in mice. Meanwhile, 2000 mg/kg of Ojeok-san was able to mitigate mechanical hyperalgesia and visceral nociception.
Conclusions: Taken together, these data suggest that the herbal formula Ojeok-san might provide analgesia in cancer models.
Acknowledgments: National Center for Complementary and Integrative Health K99AT009206.
MAX VILLA, DUKE UNIVERSITY
Massively parallel culture of the human gut microbiome reveals differences in the prevalence of fiber-degrading microbes across individuals
In a relationship that evolved over millennia, microbes contribute dramatically to human health. For instance, bacteria in the gut modulate the human immune system and unlock energy from our diet that was previously inaccessible. Thus, there is interest in controlling microbiota to improve human health. Diet in particular is a major driver of gut community makeup, yet variation between gut microbial community response from person to person makes it extremely difficult to apply therapeutic microbiome-mediated dietary interventions. One hypothesis regarding inter-individual variation in gut community response to diet is that the presence or absence of taxa that degrade complex dietary polysaccharides drives inter-individual variation. However, determining which bacteria out of hundreds have the capability to degrade a given polysaccharide for even a single human host remains technically challenging. To overcome this challenge, we developed a droplet-based approach to polysaccharide screening across hundreds of gut bacteria and multiple people. As expected, we found strong inter-individual variation in gut community response. Surprisingly, this data revealed that multiple degraders of a given polysaccharide were present for all subjects examined, suggesting a degree of functional redundancy. By contrast, significant differences were observed in the number, alpha diversity, and stool abundance of polysaccharide degraders across individuals. This suggests that other factors than presence/absence such as richness, diversity, and prevalence of degraders in the gut may play a more dominant role in inter-individual variation in response to diet. Furthermore, this platform could be used as a diagnostic tool to determine which individuals might benefit the most from a given diet, enabling more precise manipulation of human microbiota to improve health and ameliorate disease.
TARSHA WARD, HARVARD MEDICAL SCHOOL
Modeling the Genetics of Congenital Heart Disease
Congenital heart disease (CHD) is a birth defect most likely caused by genetic variations in cardiac developmental genes. CHD causing genes encode for transcription factors that are heterozygous for a loss-of function (LOF) allele. How this 50% reduction of genes affect CHD remains unexplained. Increasing evidence has demonstrated that SMAD2, a transcription factor of the Nodal/TGF-Beta signaling pathway, is genetically altered in CHD patients (Homsy et al. 2015). The transcriptional targeting of this gene and how it relates to CHD has not been investigated. Here we report findings of 2 heterozygous LOF mutations and 4 heterozygous missense mutations in SMAD2 predicted to have damaging effects in CHD patients. We utilize a genetically engineered cardiac IPS stem cell assay to evaluate the damaging effects of a heterozygous LOF mutation and 2 heterozygote missense (R120T and W244C) mutations. Cardiac differentiation analysis reveals the heterozygous LOF and heterozygous W244C missense mutation progress to cardiomyocytes, in contrast to a heterozygous R120T missense mutation that fail to differentiate. Analyses of ATAC-seq and transcriptional profiling show altered SMAD2 binding affinities that coincide with differential gene expression amongst the SMAD2 IPS mutated cell lines. Together, these findings conclude that using a cardiac IPS stem cell model could elucidate downstream effects of heterozygous missense mutations found in CHD patients.
YVON L. WOAPPI, HARVARD MEDICAL SCHOOL/BRIGHAM AND WOMEN’S HOSPITAL
Multifunctional in vivo Gene-editing Platforms for Skin Cancer Research
Non-melanoma skin cancer (NMSC) is the most common form of cancer in humans, resulting in a heterogenous disease sub-divided into two distinct pathologies: Basal Cell Carcinoma (BCC) and Squamous Cell Carcinoma (SCC). BCC is mostly benign, but SCC can often develop into metastatic disease, claiming approximately 9,000 lives each year. While both BCC and SCC are thought to develop from skin keratinocytes, it is unclear which keratinocyte subpopulations determine these histological subtypes or which molecular factors drive the differences in incidence and clinical outcome. We hypothesize that the location of a keratinocyte within a particular skin compartment can influence tumor incidence, while distinct transcriptional programs expressed in these respective compartments may govern tumor maintenance. Regrettably, there doesn’t exist any skin cancer models currently able to assess the sum of these queries. My research aims to develop polyfunctional pre-clinical NMSC models that reflect the alterations found in human skin cancer. We are engineering novel CRISPR/Cas9 systems capable of keratinocyte-specific gene deactivation and epidermal- specific transcriptional program deregulation. This technology presents a high-throughput in vivo platform for genetic screening and will enable us to better define specific transcriptional programs and cell populations driving NMSC maintenance.
Validating thermal simulations with an Anthropomorphic Head Phantom using MRI
MRI is a noninvasive human imaging modality used to image various parts of the body. The FDA recently approved Siemens 7 Tesla (T) MRI machine for clinical studies and the developments within MRI will become more focused on ultra-high field MRI (7T and greater). Various technical developments must occur over the next few decades to ensure more aspects of 7T machines are clinical. One focus will be the development of radiofrequency (RF) coils that are used within the scanner to image the body. To properly characterize an RF head coil for neuroimaging, Wood et al.1 fabricated a realistic head phantom that can be used for a wide-variety of MR purposes and builds on the work of Graedel et al.2. The head phantom was simulated 3D printed with an SLA resin material similar to the averaged electromagnetic properties of the fat, bone, and skin. Work 3 has proved that using an anthropomorphic phantom is vital for applications at higher field strengths. This talk will demonstrate the importance of using an anthropomorphic heterogeneous head phantom instead of a basic phantom through RF coil characterization methods (i.e. scattering parameters, B1 mapping, SAR, RF heating) using numerical and experimental studies. This talk will further elaborate the benefit of this technology in advancing neurological brain detection, disease or damage.