Metabuli: sensitive and specific metagenomic classification via joint analysis of amino acid and DNA.

Metagenomic taxonomic classifiers analyze either DNA or amino acid (AA) sequences. Metabuli ( https://metabuli.steineggerlab.com ), however, jointly analyzes both DNA and AA to leverage AA conservation for sensitive homology detection and DNA mutations for specific differentiation of closely related taxa. In the Critical Assessment of Metagenome Interpretation 2 plant-associated dataset, Metabuli covered 99% and 98% of classifications of state-of-the-art DNA- and AA-based classifiers, respectively.

The chromatin-binding protein PHF6 functions as an E3 ubiquitin ligase of H2BK120 via H2BK12Ac recognition for activation of trophectodermal genes.

Epigenetic regulation is important for establishing lineage-specific gene expression during early development. Although signaling pathways have been well-studied for regulation of trophectoderm reprogramming, epigenetic regulation of trophectodermal genes with histone modification dynamics have been poorly understood. Here, we identify that plant homeodomain finger protein 6 (PHF6) is a key epigenetic regulator for activation of trophectodermal genes using RNA-sequencing and ChIP assays. PHF6 acts as an E3 ubiquitin ligase for ubiquitination of H2BK120 (H2BK120ub) via its extended plant homeodomain 1 (PHD1), while the extended PHD2 of PHF6 recognizes acetylation of H2BK12 (H2BK12Ac). Intriguingly, the recognition of H2BK12Ac by PHF6 is important for exerting its E3 ubiquitin ligase activity for H2BK120ub. Together, our data provide evidence that PHF6 is crucial for epigenetic regulation of trophectodermal gene expression by linking H2BK12Ac to H2BK120ub modification.

Lysine-specific demethylase 3A is important for autophagic occurrence.

Autophagy is an essential process to maintain cell survival and homeostasis under various stress conditions. Here, we report that lysine-specific demethylase 3A (KDM3A) plays an important role in starvation-induced autophagy. Using Kdm3a knockout mice, we demonstrate that KDM3A is crucial for proper hepatic autophagy in vivo. Hepatic mRNA expression analysis and ChIP assay in WT and Kdm3a knockout mouse livers reveal that KDM3A activates autophagy genes by reducing histone H3K9me2 levels upon fasting. Together, our finding represents previously unidentified function of KDM3A as a key regulator of autophagy, implicating potential therapeutic approaches for autophagy-related diseases.

Fiber-optic plasmonic probe with nanogap-rich Au nanoislands for on-site surface-enhanced Raman spectroscopy using repeated solid-state dewetting.

We report a fiber-optic plasmonic probe with nanogap-rich gold nanoislands for on-site surface-enhanced Raman spectroscopy (SERS). The plasmonic probe features nanogap-rich Au nanoislands on the top surface of a single multimode fiber. Au nanoislands were monolithically fabricated using repeated solid-state dewetting of thermally evaporated Au thin film. The plasmonic probe shows 7.8 × 106 in SERS enhancement factor and 100 nM in limit-of-detection for crystal violet under both the excitation of laser light and the collection of SERS signals through the optical fiber. The fiber-through measurement also demonstrates the label-free SERS detection of folic acid at micromolar level. The plasmonic probe can provide a tool for on-site and in vivo SERS applications.

Lissajous Scanning Two-photon Endomicroscope for In vivo Tissue Imaging.

An endomicroscope opens new frontiers of non-invasive biopsy for in vivo imaging applications. Here we report two-photon laser scanning endomicroscope for in vivo cellular and tissue imaging using a Lissajous fiber scanner. The fiber scanner consists of a piezoelectric (PZT) tube, a single double-clad fiber (DCF) with high fluorescence collection, and a micro-tethered-silicon-oscillator (MTSO) for the separation of biaxial resonant scanning frequencies. The endomicroscopic imaging exhibits 5 frames/s with 99% in scanning density by using the selection rule of scanning frequencies. The endomicroscopic scanner was compactly packaged within a stainless tube of 2.6 mm in diameter with a high NA gradient-index (GRIN) lens, which can be easily inserted into the working channel of a conventional laparoscope. The lateral and axial resolutions of the endomicroscope are 0.70 µm and 7.6 µm, respectively. Two-photon fluorescence images of a stained kidney section and miscellaneous ex vivo and in vivo organs from wild type and green fluorescent protein transgenic (GFP-TG) mice were successfully obtained by using the endomicroscope. The endomicroscope also obtained label free images including autofluorescence and second-harmonic generation of an ear tissue of Thy1-GCaMP6 (GP5.17) mouse. The Lissajous scanning two-photon endomicroscope can provide a compact handheld platform for in vivo tissue imaging or optical biopsy applications.

Batch fabrication of functional optical elements on a fiber facet using DMD based maskless lithography.

We report a facile and direct fabrication method for integrating functional optical microstructures on the top surface of an optical fiber. A programmable maskless fabrication system was developed by using digital micromirror device (DMD), which allows rapid prototyping and low-cost fabrication without physical photomask. This maskless UV exposure system has the spatial resolution of 2.2 µm for an exposed area of 245 µm x 185 µm. Diverse optical microstructures were photolithographically defined on multimode fibers and a single mode optical fiber serially spliced with a coreless silica fiber segment. This method provides a new route for developing compact functional fiber-optic applications such as laser scanning, biosensing, or laser endomicroscopy.

Atrial septal defect with normal pulmonary arterial pressure in adult cyanotic patient.

A 22-year-old male presented with recurrent stroke, central cyanosis, and dyspnea. Transesophageal echocardiography and cardiac catheterization revealed bidirectional shunt flow through atrial septal defect (ASD) without pulmonary arterial hypertension. The orifice of inferior vena cava facing towards ASD opening led partially right to left shunt resulting in cyanosis with normal pulmonary arterial pressure.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells.

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

Focal adhesion kinase plays a role in osteoblast mechanotransduction in vitro but does not affect load-induced bone formation in vivo.

A healthy skeleton relies on bone's ability to respond to external mechanical forces. The molecular mechanisms by which bone cells sense and convert mechanical stimuli into biochemical signals, a process known as mechanotransduction, are unclear. Focal adhesions play a critical role in cell survival, migration and sensing physical force. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that controls focal adhesion dynamics and can mediate reparative bone formation in vivo and osteoblast mechanotransduction in vitro. Based on these data, we hypothesized that FAK plays a role in load-induced bone formation. To test this hypothesis, we performed in vitro fluid flow experiments and in vivo bone loading studies in FAK-/- clonal lines and conditional FAK knockout mice, respectively. FAK-/- osteoblasts showed an ablated prostaglandin E(2) (PGE(2)) response to fluid flow shear. This effect was reversed with the re-expression of wild-type FAK. Re-expression of FAK containing site-specific mutations at Tyr-397 and Tyr-925 phosphorylation sites did not rescue the phenotype, suggesting that these sites are important in osteoblast mechanotransduction. Interestingly, mice in which FAK was conditionally deleted in osteoblasts and osteocytes did not exhibit altered load-induced periosteal bone formation. Together these data suggest that although FAK is important in mechanically-induced signaling in osteoblasts in vitro, it is not required for an adaptive response in vivo, possibly due to a compensatory mechanism that does not exist in the cell culture system.

High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (µCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and µCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

Imaging pulmonary NF-kappaB activation and therapeutic effects of MLN120B and TDZD-8.

NF-κB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-κB activity in the lungs, we transfected a luciferase based NF-κB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-κB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-κB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-κB activation pathway. Interventions affecting the NF-κB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-κB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-κB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ß and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-κB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-κB regulation under various inflammatory conditions.

Beta1 integrins mediate mechanosensitive signaling pathways in osteocytes.

Integrins are cell-substrate adhesion proteins that initiate intracellular signaling and may serve as mechanosensors in bone. MLO-Y4 cells were stably transfected with a dominant negative form of the beta(1) integrin subunit (beta(1)DN) containing the transmembrane domain and cytoplasmic tail of beta(1) integrin. Cells expressing beta(1)DN had reduced vinculin localization to focal contacts but no change in intracellular actin organization. When exposed to oscillatory fluid flow, beta(1)DN cells exhibited a significant reduction in the upregulation of cyclooxygenase-2 gene expression and prostaglandin E(2) release. Similarly, the ratio of receptor activator of NF-kappaB ligand mRNA to osteoprotegerin mRNA decreased significantly after exposure to fluid flow in control cells but not in beta(1)DN cells. Interfering with integrin signaling did not affect mechanically induced intracellular calcium mobilization. These data suggest that integrins may initiate the cellular response of osteocytes to dynamic fluid flow and may serve as mechanosensitive molecules in bone.

Non-invasive detection of a small number of bioluminescent cancer cells in vivo.

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.

In vivo bioluminescent imaging of mammary tumors using IVIS spectrum.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds. The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result. Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures. Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents. Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

Focal adhesion kinase is important for fluid shear stress-induced mechanotransduction in osteoblasts.

Mechanical loading of bone is important for maintenance of bone mass and structural stability of the skeleton. When bone is mechanically loaded, movement of fluid within the spaces surrounding bone cells generates fluid shear stress (FSS) that stimulates osteoblasts, resulting in enhanced anabolic activity. The mechanisms by which osteoblasts convert the external stimulation of FSS into biochemical changes, a process known as mechanotransduction, remain poorly understood. Focal adhesions are prime candidates for transducing external stimuli. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase found in focal adhesions, may play a key role in mechanotransduction, although its function has not been directly examined in osteoblasts. We examined the role of FAK in osteoblast mechanotransduction using short interfering RNA (siRNA), overexpression of a dominant negative FAK, and FAK(-/-) osteoblasts to disrupt FAK function in calvarial osteoblasts. Osteoblasts were subjected to varying periods oscillatory fluid flow (OFF) from 5 min to 4 h, and several physiologically important readouts of mechanotransduction were analyzed including: extracellular signal-related kinase 1/2 phosphorylation, upregulation of c-fos, cyclooxygenase-2, and osteopontin, and release of prostaglandin E(2). Osteoblasts with disrupted FAK signaling exhibited severely impaired mechanical responses in all endpoints examined. These data indicate the importance of FAK for both short and long periods of FSS-induced mechanotransduction in osteoblasts.

Endochondral ossification is required for haematopoietic stem-cell niche formation.

Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

Liposomal packaging generates Wnt protein with in vivo biological activity.

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration.

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

CRBL cells: establishment, characterization and susceptibility to prion infection.

The cerebellum is involved in complex physiological functions including motor control, sensory perception, cognition, language, and emotion. Humans and animals with prion diseases are characterized clinically by ataxia, postural abnormalities and cognitive decline. Pathology in the cerebellum affected by prions includes spongiform degeneration, neuronal loss, and gliosis. To develop an in vitro model system for studying prion biology in cerebellar cells, we established and characterized an immortal cell line (CRBL) isolated from the cerebellum of mice lacking expression of a protein involved in cell cycle arrest. The characteristics of the cells include morphological heterogeneity, rapid proliferation, serum responsiveness during growth, and a change in the number of chromosomes. CRBL cells expressed both neuronal and glial cell markers as well as a considerable level of cellular prion protein, PrP(C). Upon in vitro infection, CRBL cells exhibited selective susceptibility to prions isolated from different sources. These cells chronically propagated prions from SMB cells. Strain-specific prion infection in CRBL cells was not due to instability of the cell line, allelic variance, or mutations in the PrP gene. Molecular properties of prions derived from SMB cells were maintained in the infected CRBL cells. Our results suggest that the specific interaction between a prion strain and hosts determined the selective susceptibility of CRBL cells, which reflects the conditions in vivo. In addition to the future studies revealing cellular and molecular mechanism involved in prion pathogenesis, CRBL cells will contribute to the studies dealing with prion strain properties and host susceptibilities.

Beta-catenin-dependent Wnt signaling in mandibular bone regeneration.

Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.