Bat teeth illuminate the diversification of mammalian tooth classes.

Tooth classes are an innovation that has contributed to the evolutionary success of mammals. However, our understanding of the mechanisms by which tooth classes diversified remain limited. We use the evolutionary radiation of noctilionoid bats to show how the tooth developmental program evolved during the adaptation to new diet types. Combining morphological, developmental and mathematical modeling approaches, we demonstrate that tooth classes develop through independent developmental cascades that deviate from classical models. We show that the diversification of tooth number and size is driven by jaw growth rate modulation, explaining the rapid gain/loss of teeth in this clade. Finally, we mathematically model the successive appearance of tooth buds, supporting the hypothesis that growth acts as a key driver of the evolution of tooth number and size. Our work reveal how growth, by tinkering with reaction/diffusion processes, drives the diversification of tooth classes and other repeated structure during adaptive radiations.

Polyunsaturated Fatty Acids Mend Macrophage Transcriptome, Glycome, and Phenotype in the Patients with Neurodegenerative Diseases, Including Alzheimer's Disease.

BACKGROUND: Macrophages of healthy subjects have a pro-resolution phenotype, upload amyloid-ß (Aß) into endosomes, and degrade Aß, whereas macrophages of patients with Alzheimer's disease (AD) generally have a pro-inflammatory phenotype and lack energy for brain clearance of Aß. OBJECTIVE: To clarify the pathogenesis of sporadic AD and therapeutic effects of polyunsaturated fatty acids (PUFA) with vitamins B and D and antioxidants on monocyte/macrophage (MM) migration in the AD brain, MM transcripts in energy and Aß degradation, MM glycome, and macrophage clearance of Aß. METHODS: We followed for 31.3 months (mean) ten PUFA-supplemented neurodegenerative patients: 3 with subjective cognitive impairment (SCI), 2 with mild cognitive impairment (MCI), 3 MCI/vascular cognitive impairment, 2 with dementia with Lewy bodies, and 7 non-supplemented caregivers. We examined: monocyte migration in the brain and a blood-brain barrier model by immunochemistry and electron microscopy; macrophage transcriptome by RNAseq; macrophage glycome by N-glycan profiling and LTQ-Orbitrap mass spectrometry; and macrophage phenotype and phagocytosis by immunofluorescence. RESULTS: MM invade Aß plaques, upload but do not degrade Aß, and release Aß into vessels, which develop cerebrovascular amyloid angiopathy (CAA); PUFA upregulate energy and Aß degradation enzyme transcripts in macrophages; PUFA enhance sialylated N-glycans in macrophages; PUFA reduce oxidative stress and increase pro-resolution MM phenotype, mitochondrial membrane potential, and Aß phagocytosis (p < 0.001). CONCLUSION: Macrophages of SCI, MCI, and AD patients have interrelated defects in the transcriptome, glycome, Aß phagocytosis, and Aß degradation. PUFA mend macrophage transcriptome, enrich glycome, enhance Aß clearance, and benefit the cognition of early-stage AD patients.

Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress.

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

Visualizing Pericyte Mimicry of Angiotropic Melanoma by Direct Labeling of the Angioarchitecture.

In addition to intravascular dissemination, angiotropic melanoma cells have the propensity to spread along the external surface of blood vessels in a pericytic location, or pericytic mimicry. Such continuous migration without intravasation has been termed "extravascular migratory metastasis" or EVMM. In order to visualize this mechanism of tumor propagation, we used a murine brain melanoma model utilizing green fluorescent human melanoma cells and red fluorescent lectin-tagged murine vessels. This model allows the direct microscopic visualization and mapping of the interaction of melanoma cells with the brain vasculature. In this chapter, we describe the methodology of lectin perfusion to label the entire angioarchitecture in conjunction with confocal microscopy imaging to study the pericyte mimicry of the angiotropic GFP+ melanoma cells.

Three-dimensional virtual refocusing of fluorescence microscopy images using deep learning.

We demonstrate that a deep neural network can be trained to virtually refocus a two-dimensional fluorescence image onto user-defined three-dimensional (3D) surfaces within the sample. Using this method, termed Deep-Z, we imaged the neuronal activity of a Caenorhabditis elegans worm in 3D using a time sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field by 20-fold without any axial scanning, additional hardware or a trade-off of imaging resolution and speed. Furthermore, we demonstrate that this approach can correct for sample drift, tilt and other aberrations, all digitally performed after the acquisition of a single fluorescence image. This framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. Deep-Z has the potential to improve volumetric imaging speed while reducing challenges relating to sample drift, aberration and defocusing that are associated with standard 3D fluorescence microscopy.

Human Neural Stem Cells Flown into Space Proliferate and Generate Young Neurons.

Here we demonstrate that human neural stem cells (NSCs) proliferate while in space and they express specific NSC markers after being in space. NSCs displayed both higher oxygen consumption and glycolysis than ground controls. These cells also kept their ability to become young neurons. Electrophysiological recordings of space NSC-derived neurons showed immature cell membrane properties characterized by small capacitance and very high input resistance. Current injections elicited only an incipient action potential. No spontaneous synaptic events could be detected, suggesting their immature status even though most recorded cells displayed complex morphology and numerous cell processes. Ascertaining the origin of the NSCs' increased energy requirement is of the essence in order to design effective measures to minimize health risks associated with long-duration human spaceflight missions.

Self-Sorting Microscale Compartmentalized Block Copolypeptide Hydrogels.

Multicomponent interpenetrating network hydrogels possessing enhanced mechanical stiffness compared to their individual components were prepared via physical mixing of diblock copolypeptides that assemble by either hydrophobic association or polyion complexation in aqueous media. Optical microscopy analysis of fluorescent-probe-labeled multicomponent hydrogels revealed that the diblock copolypeptide components rapidly and spontaneously self-sort to form distinct hydrogel networks that interpenetrate at micron length scales. These materials represent a class of microscale compartmentalized hydrogels composed of degradable, cell-compatible components, which possess rapid self-healing properties and independently tunable domains for downstream applications in biology and additive manufacturing.

Deep learning enables cross-modality super-resolution in fluorescence microscopy.

We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Angiotropism and extravascular migratory metastasis in cutaneous and uveal melanoma progression in a zebrafish model.

Cutaneous melanoma is a highly aggressive cancer with a propensity for distant metastasis to various organs. In contrast, melanoma arising in pigmented uveal layers of the eye metastasizes mostly in the liver. The mechanisms of these metastases, which are ultimately resistant to therapy, are still unclear. Metastasis via intravascular dissemination of tumour cells is widely accepted as a central paradigm. However, we have previously described an alternative mode of tumour dissemination, extravascular migratory metastasis, based on clinical and experimental data. This mechanism is characterised by the interaction of cancer cells with the abluminal vascular surface, which defines angiotropism. Here, we employed our 3D co-culture approach to monitor cutaneous and uveal human melanoma cells dynamics in presence of vascular tubules. Using time-lapse microscopy, we evaluated angiotropism, the migration of tumour cells along vascular tubules and the morphological changes occurring during these processes. Cutaneous and uveal melanoma cells were injected in zebrafish embryos in order to develop xenografts. Employing in vivo imaging coupled with 3D reconstruction, we monitored the interactions between cancer cells and the external surface of zebrafish vessels. Overall, our results indicate that cutaneous and uveal melanoma cells spread similarly along the abluminal vascular surfaces, in vitro and in vivo.

Intravital Imaging of Human Melanoma Cells in the Mouse Ear Skin by Two-Photon Excitation Microscopy.

Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear's skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.

Enhancing image quality in cleared tissue with adaptive optics.

Our ability to see fine detail at depth in tissues is limited by scattering and other refractive characteristics of the tissue. For fixed tissue, we can limit scattering with a variety of clearing protocols. This allows us to see deeper but not necessarily clearer. Refractive aberrations caused by the bulk index of refraction of the tissue and its variations continue to limit our ability to see fine detail. Refractive aberrations are made up of spherical and other Zernike modes, which can be significant at depth. Spherical aberration that is common across the imaging field can be corrected using an objective correcting collar, although this can require manual intervention. Other aberrations may vary across the imaging field and can only be effectively corrected using adaptive optics. Adaptive optics can also correct other aberrations simultaneously with the spherical aberration, eliminating manual intervention and speeding imaging. We use an adaptive optics two-photon microscope to examine the impact of the spherical and higher order aberrations on imaging and contrast the effect of compensating only for spherical aberration against compensating for the first 22 Zernike aberrations in two tissue types. Increase in image intensity by 1.6× and reduction of root mean square error by 3× are demonstrated.

Imaging of Angiotropism/Vascular Co-Option in a Murine Model of Brain Melanoma: Implications for Melanoma Progression along Extravascular Pathways.

Angiotropism/pericytic mimicry and vascular co-option involve tumor cell interactions with the abluminal vascular surface. These two phenomena may be closely related. However, investigations of the two processes have developed in an independent fashion and different explanations offered as to their biological nature. Angiotropism describes the propensity of tumor cells to spread distantly via continuous migration along abluminal vascular surfaces, or extravascular migratory metastasis (EVMM). Vascular co-option has been proposed as an alternative mechanism by which tumors cells may gain access to a blood supply. We have used a murine brain melanoma model to analyze the interactions of GFP human melanoma cells injected into the mouse brain with red fluorescent lectin-labeled microvascular channels. Results have shown a striking spread of melanoma cells along preexisting microvascular channels and features of both vascular co-option and angiotropism/pericytic mimicry. This study has also documented the perivascular expression of Serpin B2 by angiotropic melanoma cells in the murine brain and in human melanoma brain metastases. Our findings suggest that vascular co-option and angiotropism/pericytic mimicry are closely related if not identical processes. Further studies are needed in order to establish whether EVMM is an alternative form of cancer metastasis in addition to intravascular cancer dissemination.

Fluorescent Image-Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real Time.

PURPOSE: The inability to visualize cancer during prostatectomy contributes to positive margins, cancer recurrence, and surgical side effects. A molecularly targeted fluorescent probe offers the potential for real-time intraoperative imaging. The goal of this study was to develop a probe for image-guided prostate cancer surgery. EXPERIMENTAL DESIGN: An antibody fragment (cys-diabody, cDb) against prostate stem cell antigen (PSCA) was conjugated to a far-red fluorophore, Cy5. The integrity and binding of the probe to PSCA was confirmed by gel electrophoresis, size exclusion, and flow cytometry, respectively. Subcutaneous models of PSCA-expressing xenografts were used to assess the biodistribution and in vivo kinetics, whereas an invasive intramuscular model was utilized to explore the performance of Cy5-cDb-mediated fluorescence guidance in representative surgical scenarios. Finally, a prospective, randomized study comparing surgical resection with and without fluorescent guidance was performed to determine whether this probe could reduce the incidence of positive margins. RESULTS: Cy5-cDb demonstrated excellent purity, stability, and specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 6 hours. In mice carrying PSCA(+) and negative (-) dual xenografts, the mean fluorescence ratio of PSCA(+/-) tumors was 4.4:1. In surgical resection experiments, residual tumors <1 mm that were missed on white light surgery were identified and resected using fluorescence guidance, which reduced the incidence of positive surgical margins (0/8) compared with white light surgery alone (7/7). CONCLUSIONS: Fluorescently labeled cDb enables real-time in vivo imaging of prostate cancer xenografts in mice, and facilitates more complete tumor removal than conventional white light surgery alone.

Novel components of the Toxoplasma inner membrane complex revealed by BioID.

UNLABELLED: The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. IMPORTANCE: The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.

Angiotropism, pericytic mimicry and extravascular migratory metastasis in melanoma: an alternative to intravascular cancer dissemination.

For more than 15 years, angiotropism in melanoma has been emphasized as a marker of extravascular migration of tumor cells along the abluminal vascular surface, unveiling an alternative mechanism of tumor spread distinct from intravascular dissemination. This mechanism has been termed extravascular migratory metastasis (EVMM). During EVMM, angiotropic tumor cells migrate in a 'pericytic-like' manner (pericytic mimicry) along the external surfaces of vascular channels, without intravasation. Through this pathway, melanoma cells may spread to nearby or more distant sites. Angiotropism is a prognostic factor predicting risk for metastasis in human melanoma, and a marker of EVMM in several experimental models. Importantly, analogies of EVMM and pericytic mimicry include neural crest cell migration, vasculogenesis and angiogenesis, and recent studies have suggested that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes reminiscent of cancer migration and embryonic/stem cell state transitions. A recent work revealed that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression via angiotropism and migration along the abluminal vascular surface. Finally, recent data using imaging of melanoma cells in a murine model have shown the progression of tumor cells along the vascular surfaces. Taken together, these data provide support for the biological phenomenon of angiotropism and EVMM, which may open promising new strategies for reducing or preventing melanoma metastasis.

Mouse infection and pathogenesis by Trypanosoma brucei motility mutants.

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.

Select G-protein-coupled receptors modulate agonist-induced signaling via a ROCK, LIMK, and ß-arrestin 1 pathway.

G-protein-coupled receptors (GPCRs) are typically present in a basal, inactive state but, when bound to an agonist, activate downstream signaling cascades. In studying arrestin regulation of opioid receptors in dorsal root ganglia (DRG) neurons, we find that agonists of delta opioid receptors (δORs) activate cofilin through Rho-associated coiled-coil-containing protein kinase (ROCK), LIM domain kinase (LIMK), and ß-arrestin 1 (ß-arr1) to regulate actin polymerization. This controls receptor function, as assessed by agonist-induced inhibition of voltage-dependent Ca(2+) channels in DRGs. Agonists of opioid-receptor-like receptors (ORL1) similarly influence the function of this receptor through ROCK, LIMK, and ß-arr1. Functional evidence of this cascade was demonstrated in vivo, where the behavioral effects of δOR or ORL1 agonists were enhanced in the absence of ß-arr1 or prevented by inhibiting ROCK. This pathway allows δOR and ORL1 agonists to rapidly regulate receptor function.

Fluorescent imaging of single nanoparticles and viruses on a smart phone.

Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments.

WNT10B/ß-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer.

Wnt/ß-catenin signalling has been suggested to be active in basal-like breast cancer. However, in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of ß-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active ß-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical ß-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/ß-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.