Cortical Plasticity: From Young to Adult
Kexin Yuan, PhD

Synapses and the neuronal receptive fields of the cerebral cortex are plastic in both young and mature brains. Early in life, neural circuits are highly susceptible to outside influences. The organization of primary auditory cortex (AI), in particular, is governed by acoustic experience during the critical period. One important principle of synaptic organization in mature brains is the balance between excitation and inhibition, but it is unknown how and when this excitatory-inhibitory balance is established and calibrated. We used whole-cell recordings to determine the processes underlying the development of synaptic receptive fields in rat AI. We found that sensory evoked inhibitory responses were initially less selective and mismatched with excitatory responses. We further reported that the pace of cortical synaptic receptive field development is set by progressive, experience-dependent refinement of intracortical inhibition. In mature brains, cortical receptive fields can be modified in a manner that depends on the patterns of electrical activity, sensory experience, and engagement of neuromodulatory systems such as the cholinergic nucleus basalis. The relationship between synaptic and receptive field changes and perceptual plasticity, however, is poorly understood. We used in vivo whole–cell recordings and behavioral testing to explore that relationship in more detail.  We will discuss how synaptic modifications and receptive field changes are reflected in the encoding of frequency and intensity information in rat auditory cortex and demonstrate that these changes are expressed in the perceptual behavior of animals in sensory detection and classification tasks. We have found that direct modification of specific cortical inputs leads to wide-scale synaptic changes, which collectively support improved sensory perception and enhanced behavioral performance.

Underlying Cellular Mechanisms of Tinnitus
Thanos Tzounopoulos, PhD

Tinnitus is a debilitating condition for many millions of tinnitus sufferers. Despite its high prevalence, the mechanisms and molecules that underly the generation of the perception of phantom sound have remained enigmatic. We have developed an animal model of noise-induced tinnitus to investigate the molecular mechanisms underlying tinnitus generation. Our findings link changes in the biophysical properties of a single ion channel to the perception of phantom sound. Moreover, our results point to biological targets for designing novel therapeutics that may prevent the development of tinnitus in humans.


Structural Invstigations of Voltage-Gated Sodium Channels
Nieng Yan, PhD

Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle and are important drug targets. Elucidation of the structures and functional mechanisms of Nav channels will shed light on fundamental ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Nav channels, exemplified by NaChBac (Na+-selective Channel of Bacteria), provides a good model system for structure-function analysis. Here we report the crystal structure of NavRh, a NaChBac orthologue from marine bacteria alpha proteobacterium HIMB114, at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr178 and Leu179 constitute an inner site within the selectivity filter (178TLSSWE183) where a hydrated Ca2+ can bind and resides in the crystal structure. The outer mouth of the Na+ selectivity filter, defined by Ser181 and Glu183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation, with all the gating charges exposing to the extracellular side. We hypothesize that NavRh is captured in an inactivated conformation. Comparison of NavRh with NavAb reveals significant conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.


Understanding the Evolutionary Connection between Tailed Phages and Animal Viruses
James F. Conway, PhD

Structural studies in the 1990s on the icosahedrally-symmetric capsid of bacteriophage HK97 revealed a protein fold not previously encountered but which now appears likely to be common to all double-stranded DNA tailed phages. Since these viruses are abundant on the planet, the fold may be the most represented in nature and is consequently of interest to understand its function in capsid assembly and maturation. More recent studies of the herpesvirus capsid by high-resolution cryo-electron microscopy (cryo-EM) show that the lower domain that maintains subunit connectivity across the capsid structure is also consistent with the HK97 capsid protein fold, suggesting that the dsDNA tailed phages and herpesviruses share a common ancestor. This structural linkage with an important human pathogen also bolsters the significance of understanding the capsid biology of phages, which are considerably more tractable to work with. We have embarked upon structural determination and analysis of several capsids that share significant similarities with phage HK97 but differ in icosahedral geometry by forming larger capsids. Our goal is to understand how the different capsid proteins are able to form capsids of a specific geometry. Determination of high resolution cryo-EM structures of several related capsids with different sizes, including herpesvirus capsids, enables us to discover new features of these structures that appear to play roles during assembly. We expect that deeper analysis may suggest how size and shape are controlled.



Crossing Boundaries: Lymphocyte Interactions, Cell Trafficking, and the Formation of the Germinal Center
Hai Qi, PhD

Germinal center (GC) reactions support antibody affinity maturation and the formation of humoral immune memory. Follicular helper T (Tfh) cells play a key role in promoting proliferation and differentiation of antigen-specific B cells inside the follicle. A genetic deficiency in the inducible costimulator (ICOS), a classic CD28 family co-stimulatory molecule that is highly expressed by Tfh cells, causes profound defects in the GC reaction, leading to the widely held view that ICOS specifically co-stimulates T cell receptor signaling to preferentially induce the Tfh differentiation program. Unexpectedly, we have found that ICOS directly controls follicular recruitment of activated helper T cells. This effect is independent from ICOS ligand (ICOSL)-mediated co-stimulation during T cell interactions with antigen-presenting dendritic cells or cognate B cells and does not rely on Bcl6-mediated programming as an intermediate step. Instead, it requires constitutive expression of ICOSL on follicular bystander B cells, which do not present cognate antigen to helper T cells but collectively form an ICOS-engaging field. ICOS triggering in vitro without concomitant antigen receptor engagement drives coordinated pseudopod dynamics and promotes persistent migration of T cells. Intravital imaging by two-photon microscopy reveals that at the T zone-follicle border, optimal pseudopod dynamics, and persistent motility of activated T cells rely on ICOS triggering by ICOSL-expressing bystander B cells. When these B cells are deficient in ICOSL expression, otherwise competent helper T cells fail to migrate into the follicle and fail to promote the GC response normally. These results demonstrate a costimulation-independent function of the ICOS molecule in driving T cell motility and homing behaviors, uncover a key role for bystander B cells in promoting the development of follicular helper T cells , and reveal unsuspected sophistication in the biology of dynamic T cell positioning in vivo.

Organogenesis of Ectopic Tissue in Lymph Nodes
Eric Lagasse, PharmD, PhD

Cell therapy has been viewed as a promising alternative to organ transplantation. However for some patients, orthotopic cell-based therapy directed at a diseased organ may not be feasible for many reasons, ranging from a possible lack of an appropriate environment in cirrhotic and fibrotic liver during end-stage disease to the lack of a thymus in complete DiGeorge syndrome. Consequently, a critical component of cell-based therapy for these patients is to establish an optimal in vivo site for cell and tissue transplantation to restore organ functions. Here, we address the challenge of determining a suitable location and test the hypothesis that transplantation of three distinct normal epithelial cell types directly into the lymph nodes of mice would engraft and demonstrate ectopic organ functions in vivo. This approach will be beneficial to the field of regenerative medicine and provides a new concept to use the lymph note as an in vivo bioreactor in which to regenerate functional organs.



Mosquito C-Type Lectins Facilitate Flavivirus Infection
Gong Cheng, PhD

Flaviviral infection shows the importance of global public health. Flaviviruses are usually transmitted by arthropod vectors to humans; however, the vector ligand(s) that participate in infection are largely unknown. An Aedes aegypti C-type lectin, mosGCTL-1, is induced by West Nile virus (WNV), interacts with WNV in a calcium-dependent manner, and facilitates viral infection in vivo and in vitro. We further identified that the other nine mosGCTL paralogues play crucial roles in Dengue infection of mosquitos: mosGCTL-3, the most phenotypic subtype during infection, interacts with Dengue-2 virus surface E protein and virion. For arthropod-borne microbes, vector ligands that interact with pathogens are potential targets for interfering with the successful acquisition of the microbe from the vertebrate host. Treatment of the host with mosGCTL-3 antisera dramatically interferes with the ability of DENV-2 to infect the mosquito through blood feeding from the host. We propose that a humoral response against mosGCTLs in vertebrate hosts could reduce vector competence for flaviviruses and control viral dissemination in nature.


Deep Sequencing in Tracking Viral Emergence and Transmission
Elodie Ghedin, PhD

Influenza A virus populations display high genetic diversity, facilitating host adaptation and affecting disease pathogenesis. Second- and third-generation deep sequencing of within-host viral populations promise to transform our understanding of viral evolutionary dynamics, allowing the dissection of the mutational spectrum at an unprecedented level of precision. Presenting data on emergence and transmission of influenza variants in host populations, I will illustrate the clinical and epidemiological value of high-resolution mapping of virus variants by deep sequencing and how chains of transmission can be reconstructed. I will also highlight the limitations the field would need to overcome for this novel approach to reach its full potential.



From Membrane Transporter to Cancer Cell Survival
Yigong Shi, PhD

Food-borne hemorrhagic E. coli require elaborate acid resistance systems (ARs) to survive an extremely acidic environment like the stomach (pH~2). AR2 expels intracellular protons through decarboxylation of L-glutamate (Glu) in the cytoplasm and exchange of the reaction product g-aminobutyric acid (GABA) with extracellular Glu. The latter process is mediated by the Glu-GABA antiporter GadC, a membrane transporter of the amino acid/polyamine/organocation (APC) superfamily. We determined the crystal structure of GadC, which reveals a closed state with 12 transmembrane segments. GadC functions only at acidic pH.

We serendipitously discovered that L-glutamine (Gln), the most abundant food-borne free amino acid, is also efficiently transported by GadC. We speculated and proved that Gln plays a key role in bacterial acid resistance. Upon uptake into E. coli, Gln is converted to Glu by the acid-activated glutaminase YbaS, with concomitant release of gaseous ammonia. The free ammonia neutralizes protons, elevating intracellular pH under an acidic environment. YbaS and GadC, which exchange extracellular Gln with intracellular Glu, together constitute an important acid resistance system that is sufficient for E. coli survival under an extremely acidic environment.

We further speculated that Gln might play a key role in cancer cell survival through acid resistance because (1) cancer cells must cope with acidification due to the Warburg effect (greatly increased level of aerobic glycolysis with accumulation of lactic acid) and (2) Gln is essential for cancer cell growth (which is generally thought to relate to the nutritional value of Gln as a source of carbon and nitrogen).  We propose that the primary role of Gln in cancer cells is to fight acid, rather than to provide nutrition, through enzymatic deamidation. We provide preliminary experimental evidence to support this hypothesis and show that cancer cell survival is compromised by inhibition of glutaminase. Our data strongly suggest that glutaminase inhibitors, currently under clinical trials as anti-cancer agents, may work by countering the ability of cancer cells to survive in an acidic environment. Recognition and discovery of the importance of acid resistance in cancer cells may prove to be a significant aspect of cancer biology and cancer treatment.



CREPT Promotes Cyclin D1 Expression Oppositely to Its Homologue p15RS
Zhijie Chang, PhD

We have cloned a novel gene CREPT (Cell-cycle Related and Expression-elevated Protein in Tumor) based on a homology screen using p15RS, which contains a RPR domain (regulation of nuclear pre-mRNA, or CID, CTD-interacting domain). Human CREPT shares a high similarity of amino acids with p15RS. Our previous studies revealed that p15RS negatively regulates cyclin D1 expression, functioning as an intrinsic inhibitor for the canonical Wnt/b-catenin signal pathway. We found that p15RS inhibits cell growth and Wnt-targeted gene expression by blocking the interaction of b-catenin and TCF4 in the nucleus. Interestingly, when we overexpressed CREPT, we observed that cell proliferation was enhanced and the expression of cell cycle-related genes, including cyclin D1, was up-regulated. We further demonstrated that CREPT regulates cyclin D1 expression by binding to its promoter, enhancing its transcription both in vivo and in vitro and interacting with RNA polymerase II (RNAPII). Interestingly, CREPT promotes the formation of a chromatin loop and prevents RNAPII from reading through the 3’-end termination site of the gene. Finally, we observed that CREPT is highly expressed in human tumors and that positive CREPT staining is significantly correlated with shorter survival time of patients after surgery and treatment. Our findings reveal a novel mechanism by which CREPT increases cyclin D1 transcription during tumorigenesis, possibly through enhancing the recruitment of RNAPII to the promoter region as well as chromatin looping. We propose that CREPT, functioning oppositely to p15RS, may be used as a marker for tumor diagnosis and a therapeutic target.


Tumor Evolution and Heterogeneity
Adrian V. Lee, PhD

Carcinogenesis generally proceeds over a long period of time, with genetic alterations providing growth and survival advantages.  Pathologists have long recognized that tumors show intra-tumor heterogeneity of both cell types and molecular markers.  This heterogeneity is thought to evolve either from clonal selection of cancer cells and/or via alterations in cell differentiation.  Limited evidence suggests that tumors with increased intra-tumor heterogeneity may have unfavorable outcome, perhaps due to their ability to evolve and circumvent therapy.  Recent advances in the miniaturization and multiplexing of high throughput analytic platforms has allowed an unprecedented and comprehensive view of molecular changes in breast cancers.  However, nearly all studies have been performed on whole crushed tissue and, thus, represent only an average of molecular changes across the tumor.  Recent studies on molecular changes in specific regions of tumors, or on single cancer cells, reveal dramatic evolution of tumor heterogeneity.  We have measured intra-tumor heterogeneity both in cell lines in culture and in breast tumors and find an evolution of proteomic and genomic change during breast cancer progression.  This tumor heterogeneity has important implications for precision medicine, particularly as it relates to measuring prognostic/predictive markers, and the successful delivery of targeted therapies. 



Functional Roles of RNA Binding Proteins during Human Germ Cell Development
Kehkooi Kee, PhD

Germ cells are the only cell type capable of transmitting totipotency and genetic material from one generation to the next. Although model organisms, like mice, have provided an excellent platform to study germ cell development, many features of germ cell development are unique to humans. For example, DAZ genes, which are required for male fertility, exist only in male primates. Studies of human germ cell development, especially early development, however, have been hindered by inaccessibility during fetal development. We have sought to develop an in vitro system based on differentiation of human embryonic stem cells (hESCs) to elucidate the molecular and genetic mechanisms of human germ cell development.  Human germ cell identity is defined by a combination of extrinsic and intrinsic factors in contrast to other model organisms, such as Drosophila, in which existence of germ plasm dictates germ cell fate. Via differentiation from hESCs, we have demonstrated that BMPs can induce PGC formation. We have confirmed the expression of a group of RNA binding proteins, including LIN28a and PELOTA, in human testis by quantitative PCR and immunofluorescent staining. To examine whether these genes are required for human PGC formation, we are determining whether overexpression or silencing of this gene will affect the developmental process. Moreover, we are examining whether these proteins interact with DAZL or other RNA binding proteins during human germ cell formation.


Placental Function and Adaptation to Injury
Yoel Sadovsky, MD

The placenta plays an essential role in fetal growth and pregnancy health. Throughout pregnancy, the placenta is obligatory for embryonic development, growth, and the maintenance of healthy maternal-fetal communication, while preserving maternal health. At the surface of the human and mouse placentas, and in direct contact with maternal blood, are the trophoblasts, which fulfill the critical functions of maternal-fetal gas exchange, supply of nutrients, removal of waste products, endocrine regulation, and immunological defense. As the interface between the fetus and its environment, the placenta is a target for diverse intrauterine injuries, spanning genetic, epigenetic, molecular, and acquired perturbations, which are associated with common diseases of pregnancy. Substandard growth leaves its mark on the newborn, rendering it vulnerable to a host of childhood diseases as well as adult disorders, such as obesity, type-2 diabetes, and their sequelae. The repertoire of placental response to injury includes the production of common and unique microRNAs. Using high throughput miRNA screens, as well as overexpression and silencing approaches, we interrogate the function of placental microRNAs. Some of these microRNAs are members of the chromosome 19 microRNA cluster. These microRNAs are primate specific and are expressed almost exclusively in the placenta. They are packaged within trophoblast-derived exosomes and confer viral resistance to recipient cells. They also play a role in regulation of trophoblast differentiation. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of placental-specific effector microRNAs to directly communicate with target cells and regulate their immunity.



Developing New Heterocycle Chemistry for Early Drug Discovery
Yu Rao, PhD

Since joining Tsinghua in 2010, my group has been mainly working on developing new heterocycle chemistry, which is the core of modern medicinal chemistry. A high-quality small molecule library is the key for successful early drug discovery. We have been employing synthetic methods developed in our laboratory to build up highly diverse and novel small molecule libraries. In the long term, my major research interest is to discover new chemical methodologies to access various unique “privilege-structure”-based heterocycles that may serve as leading compounds for early drug discovery or tools for biological study. Through screening our compound libraries, we have been able to identify some hits that may have potential applications in the fields of antibiotics, anti-cancer stem cells, and iPS cells in the future.

Better Mitochondria through Imine Addition Chemistry
Peter Wipf, PhD


Mitochondria are key organelles that perform essential cellular functions and play pivotal roles in cell death and survival signaling.  Hence, they represent an attractive target for drugs to treat metabolic, degenerative, and hyperproliferative diseases. Targeting mitochondria with organelle-specific agents or prodrugs holds considerable promise as an effective therapeutic strategy.  More specifically, controlling the cellular reactive oxygen species (ROS) balance via selective delivery of an antioxidant “payload” into mitochondria is an elegant emerging therapeutic concept.  Based on the natural antibiotic gramicidin S, we have developed several mitochondrial targeting scaffolds, to which we have attached active agents that modulate the mitochondrial biochemistry and trap ROS generated in the OxPhos process.  A key enabling synthetic methodology for this project has been the nucleophilic imine addition with vinyl organometallics.  We have used the hydrozirconation-transmetalation-imine addition protocol to generate structurally diverse allylic amine intermediates for peptide bond isosteres, phosphatase inhibitors, and mitochondria-targeted peptide mimetics.  The gramicidin S-derived XJB-5-131 and JP4-039 and their analogs have been prepared on up to 160 g scale for preclinical studies.  These (E)-alkene peptide isosteres adopt type II′ b-turn secondary structures and display impressive biological properties, including selective reactions with ROS and prevention of apoptosis.