Faculty Bibliography

BACKGROUND:In vivo assessment of the embryonic mouse placenta and vasculature is challenging, and ultrasound remains the most commonly used imaging modality. However, even with contrast agents, conventional preclinical ultrasound lacks the temporal resolution required to resolve complex vascular networks. Ultrafast Doppler methods offer a contrast-agent-free approach for visualizing embryonic placental and vascular flow. METHODS:A 15-MHz linear array was used to acquire 0.5 sec of ultrafast ultrasound data at mouse embryonic day 13.5 (E13.5) using a semi-invasive, non-destructive preparation in which a single embryo within the uterine horn was imaged in vivo. Adjacent image planes were acquired at 0.25-mm spacing to generate a 3D dataset. The data were post-processed to produce power Doppler, color Doppler, and Doppler waveforms. Image data were also segmented to facilitate visualization of distinct organ systems, including the feto-placental vascular tree. RESULTS:Ultrafast Doppler ultrasound provided greater detail of vascular flow than a 30-MHz preclinical ultrasound system. Power Doppler maps delineated the feto-placental vascular tree and the umbilical, vitelline, and yolk sac circulations. Color Doppler enabled visualization of pulsatile flow over nearly two cardiac cycles in vessels such as the vitelline artery. Following segmentation, flow within the aorta, heart, placental labyrinth, and umbilical vessels could be clearly distinguished. CONCLUSION/CONCLUSIONS:Ultrafast Doppler ultrasound enables in vivo contrast-agent-free visualization of embryonic feto-placental circulation in the mouse. The method provides high-resolution vascular structural and dynamic flow information across the placenta, embryo, and uterus. This approach is well suited for longitudinal studies of embryonic and placental development.

Ceramide transporter syndrome (CerTra syndrome) is a rare neurodevelopmental disorder caused by pathogenic variants in CERT1 gene encoding ceramide transporter (CERT). These variants disrupt ceramide transport and sphingolipid homeostasis, leading to a clinical phenotype that includes developmental delay, movement abnormalities, and structural brain anomalies. Despite growing recognition of this condition, detailed neuroimaging and neuropathological characterization remain limited. Here, we present a 12-year-old girl with a pathogenic CERT1 variant complicated by sudden unexplained death, who presented with unreported neuroimaging abnormalities in choroid plexus (ChP) and perivascular space (PVS). Clinical Magnetic Resonance Imaging (MRI) obtained approximately 10 years prior to death, as well as postmortem MRI, revealed bilateral cystic enlargement of the ChP and prominent PVS filled with abundant proteinaceous material in white matter. Neuropathological examination demonstrated marked ChP epithelial disorganization, reduced aquaporin-1 (AQP1) expression, cyst formation, and focal calcifications, which may be associated with disturbances in cerebrospinal fluid (CSF) dynamics. These findings raise the possibility that CERT1 variants may be associated with ChP architectural changes and altered perivascular clearance.

Human retrotransposon insertions are often associated with diseases. In the case of the neurodegenerative X-Linked Dystonia-Parkinsonism disease, a human-specific SINE-VNTR-Alu subfamily F retrotransposon was inserted in intron 32 of the TAF1 gene. Here, we genomically rewrote a portion of the mouse Taf1 allele with the corresponding 78-kb XDP patient derived TAF1 allele. In mESCs, the presence of the intronic SVAs-rather than the hybrid gene structure-reduces hyTAF1 levels. This leads to transcriptional downregulation of genes with TATA box enriched in their promoters and triggering apoptosis. Chromatin and transcriptome profiling revealed that intronic SVAs are actively transcribed, forming barriers that likely impede transcription elongation. In mice, neuronal lineage TAF1 humanization resulted lethality of male progeny within two months. XDP male mice had severe atrophy centered on the striatum-the same affected brain region in XDP patients. Lastly, CRISPRa-mediated activation of hyTAF1 restored mESC viability, suggesting boosting TAF1 transcription as a therapeutic approach.

Reprogramming of macrophages into the inflammatory state (also known as M1) is currently considered as an effective way of eliminating cancer cells, but systemic deployment of this strategy is likely to induce dangerous autoimmune reactions. Consequently, converting immunosuppressive M2-type macrophages into M1 systemically is not a safe and effective therapeutic approach against cancer. Through cleavable covalent linking of curcumin to the chemotherapeutic agent Paclitaxel (Taxol), we have created a novel prodrug (STO-1) that, upon intravenous delivery, selectively reprograms tumor-associated microglia and macrophages (TAMs) and eliminates glioblastoma (GBM) without triggering autoimmunity. Demonstrating its therapeutic efficacy, prolonged treatment of six orthotopic GBM-bearing mice with STO-1 resulted in 67% long-term survival, with three surviving mice exhibiting complete tumor clearance and one displaying minimal residual disease, as confirmed by high-resolution ex vivo T2-weighted MRI 85 days after tumor inoculation. In contrast, the vehicle-treated mice displayed extensive intracranial tumors with edema and hemorrhage. Mechanistically, scRNA-seq analysis indicated induction of multiple M1-associated transcripts (ccrl2, cxcl9, ccr2, ccl5) consistent with robust TAMs reprogramming. In striking contrast to the M2⟶M1 reprogramming of TAMs, M1-type macrophages were suppressed in the spleens of STO-1-treated cancer-free mice. Therefore, STO-1 induces selective anti-tumor immunity and GBM elimination without triggering systemic autoimmune reactions.

Protein-based nanocarriers bear highly desirable properties such as biodegradability and ability to facilitate passage through biological barriers such as the blood-brain-barrier. Using modular protein engineering, we develop a strategy for iteratively improving the delivery efficacy of hydrophobic small molecules for the treatment of glioblastoma multiforme (GBM). By increasing the multiplicity m of the coiled-coil and RGD peptide targeting regions from 1 to 2, we can increase both the hydrodynamic micellar size and drug loading capacity of targeted multidomain protein assembly (TMPA) relative to its predecessor thermoresponsive assembled protein (TRAP). An upper limit of m is likely determined by steric interactions. TMPA shows a 1.7-fold increased encapsulation of doxorubicin (Dox) compared to TRAP and demonstrates improved uptake by U87 human GBM cells. Near-infrared (NIR) dye-labelled TMPA (NIR-TMPA) is injected into mice orthotopically implanted with GBM cells and fit to a 2-compartment pharmacokinetic model. NIR-TMPA demonstrates prolonged short-phase half-life in tumor-bearing mice compared to control with similar slow-phase half-life, leading to an increased area-under-the-curve clearance and pointing to its sequestration in the tumor site. Endpoint fluorescence analysis of mouse organs ex vivo also supports this conclusion. TMPA and its future derivatives therefore bear potential for targeted treatment of GBM.

Embryonic development in many species, including case reports in humans, can be temporarily halted before implantation during a process called diapause. Facultative diapause occurs under conditions of maternal metabolic stress such as nursing. While molecular mechanisms of diapause have been studied, a natural inducing factor has yet to be identified. Here, we show that oxytocin induces embryonic diapause in mice. We show that gestational delays were triggered during nursing or optogenetic stimulation of oxytocin neurons simulating nursing patterns. Mouse blastocysts express oxytocin receptors, and oxytocin induced delayed implantation-like dispersion in cultured embryos. Last, oxytocin receptor-knockout embryos transferred into wild-type surrogates had low survival rates during diapause. Our results indicate that oxytocin coordinates timing of embryonic development with uterine progression through pregnancy, providing an evolutionarily conserved mechanism for ensuring successful reproduction.

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding - thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. STATEMENT OF SIGNIFICANCE: : Direct molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. Compared with that in control mice, the protein in these mice clears from the serum faster and accumulates significantly more in fibrotic livers. This work advances the development of targeted protein probes for in vivo fibrosis imaging.

The mouse is the mammalian model of choice for investigating cardiovascular biology, given our ability to manipulate it by genetic, pharmacologic, mechanical, and environmental means. Imaging is an important approach to phenotyping both function and structure of cardiac and vascular components. This review details commonly used imaging approaches, with a focus on echocardiography and magnetic resonance imaging, with brief overviews of other imaging modalities. In this update, we also emphasize the importance of rigor and reproducibility in imaging approaches, experimental design, and documentation. Finally, we briefly outline emerging imaging approaches but caution that reliability and validity data may be lacking. © 2024 Wiley Periodicals LLC.

The development of targeted anti-cancer therapeutics offers the potential for increased efficacy of drugs and diagnostics. Utilizing modalities agnostic to tumor type, such as the hypoxic tumor microenvironment (TME), may assist in the development of universal tumor targeting agents. The hypoxia-inducible factor (HIF), in particular HIF1, plays a key role in tumor adaptation to hypoxia, and inhibiting its interaction with p300 has been shown to provide therapeutic potential. Using a multivalent assembled protein (MAP) approach based on the self-assembly of the cartilage oligomeric matrix protein coiled-coil (COMPcc) domain fused to the critical residues of the C-terminal transactivation domain (C-TAD) of the α subunit of HIF1 (HIF1α), we generate HIF1α-MAP (H-MAP). The resulting H-MAP demonstrates picomolar binding affinity to p300, the ability to downregulate hypoxia-inducible genes, and in vivo tumor targeting capability.

Triple-negative breast cancer (TNBC) lacks expressed protein targets, making therapy development challenging. Hydrogels offer a promising new route in this regard by improving the chemotherapeutic efficacy through increased solubility and sustained release. Moreover, subcutaneous hydrogel administration reduces patient burden by requiring less therapy and shorter treatment times. We recently established the design principles for the supramolecular assembly of single-domain coiled-coils into hydrogels. Using a modified computational design algorithm, we designed Q8, a hydrogel with rapid assembly for faster therapeutic hydrogel preparation. Q8 encapsulates and releases doxorubicin (Dox), enabling localized sustained release via subcutaneous injection. Remarkably, a single subcutaneous injection of Dox-laden Q8 (Q8•Dox) significantly suppresses tumors within just 1 week. This work showcases the bottom-up engineering of a fully protein-based drug delivery vehicle for improved TBNC treatment via noninvasive localized therapy.