The Hippo-YAP/TAZ-TEAD axis promotes multipotency in bone marrow stromal cells.

The mechanisms by which bone marrow stromal cells (BMSCs) maintain multilineage potency in vitro remain elusive. To identify the transcriptional regulatory circuits that contribute to BMSC multipotency, we performed paired single-nucleus multiomics of the expansion of freshly isolated BMSCs and of BMSCs undergoing tri-lineage differentiation. By computationally reconstructing the regulatory programs associated with initial stages of differentiation and early expansion, we identified the TEAD family of transcription factors, which is inhibited by Hippo signaling, as highly active in the BMSC in vitro multipotent state. Pharmacological inhibition of TEAD enhanced BMSC osteogenic and adipogenic differentiation, whereas its activation maintained BMSCs in an undifferentiated state, supporting a model whereby isolation of BMSCs coincides with a TEAD-controlled transcriptional state linked to multipotency. Our study highlights the Hippo pathway as a pivotal regulator of BMSC multipotency, and our regulatory network inferences are a reservoir of testable hypotheses that link transcription factors and their regulons to specific aspects of BMSC behavior.

Novel substituted 5-methyl-4-acylaminoisoxazoles as antimitotic agents: Evaluation of selectivity to LNCaP cancer cells.

A series of novel antimitotic agents was designed using the replacement of heterocyclic cores in two tubulin-targeting lead molecules with the acylated 4-aminoisoxazole moiety. Target compounds were synthesized via heterocyclization of ß-aryl-substituted vinylketones by tert-butyl nitrite in the presence of water as a key step. 4-Methyl-N-[5-methyl-3-(3,4,5-trimethoxyphenyl)isoxazol-4-yl]benzamide (1aa) was found to stimulate partial depolymerization of microtubules of human lung carcinoma A549 cells at a high concentration of 100 µM and to totally inhibit cell growth (IC50 = 0.99 µM) and cell viability (IC50 = 0.271 µM) in the nanomolar to submicromolar concentration range. These data provide evidence of the multitarget profile of the cytotoxic action of compound 1aa. The SAR study demonstrated that the 3,4,5-trimethoxyphenyl residue is the key structural parameter determining the efficiency both towards tubulin and other molecular targets. The cytotoxicity of 3-methyl-N-[5-methyl-3-(3,4,5-trimethoxyphenyl)isoxazol-4-yl]benzamide (1ab) to the androgen-sensitive human prostate adenocarcinoma cancer cell line LNCaP (IC50 = 0.301 µM) was approximately one order of magnitude higher than that to the conditionally normal cells lines WI-26 VA4 (IC50 = 2.26 µM) and human umbilical vein endothelial cells (IC50 = 5.58 µM) and significantly higher than that to primary fibroblasts (IC50 > 75 µM).

Fluorescent bacterial biosensor E. coli/pTdcR-TurboYFP sensitive to terahertz radiation.

A fluorescent biosensor E. coli/pTdcR-TurboYFP sensitive to terahertz (THz) radiation was developed via transformation of Escherichia coli (E. coli) cells with plasmid, in which the promotor of the tdcR gene controls the expression of yellow fluorescent protein TurboYFP. The biosensor was exposed to THz radiation in various vessels and nutrient media. The threshold and dynamics of fluorescence were found to depend on irradiation conditions. Heat shock or chemical stress yielded the absence of fluorescence induction. The biosensor is applicable to studying influence of THz radiation on the activity of tdcR promotor that is involved in the transport and metabolism of threonine and serine in E. coli.

Bone Marrow Stromal Cell Assays: In Vitro and In Vivo.

Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") contain a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). It is now apparent that cells with similar but not identical properties can be isolated from other skeletal compartments (growth plate, periosteum). The biological properties of BMSCs, and these related stem/progenitor cells, are assessed by a variety of assays, both in vitro and in vivo. Application of these assays in an appropriate fashion provide a great deal of information on the role of BMSCs, and the subset of SSCs, in health and in disease.

The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning.

Glucan linked to proteins is a natural mega-glycoconjugate (mGC) playing the central role as a structural component of a yeast cell wall (CW). Regulation of functioning of non-covalently bound glucanosyltransglycosylases (ncGTGs) that have to remodel mGC to provide CW extension is poorly understood. We demonstrate that the main ncGTGs Bgl2 and Scw4 have phosphorylated and glutathionylated residues and are represented in CW as different pools of molecules having various firmness of attachment. Identified pools contain Bgl2 molecules with unmodified peptides, but differ from each other in the presence and combination of modified ones, as well as in the presence or absence of other CW proteins. Correlation of Bgl2 distribution among pools and its N-glycosylation was not found. Glutathione affects Bgl2 conformation, probably resulting in the mode of its attachment and enzymatic activity. Bgl2 from the pool of unmodified and monophosphorylated molecules demonstrates the ability to fibrillate after isolation from CW. Revealing of Bgl2 microcompartments and their mosaic arrangement summarized with the results obtained give the evidence that the functioning of ncGTGs in CW can be controlled by reversible post-translational modifications and facilitated due to their compact localization. The hypothetical scheme of distribution of Bgl2 inside CW is represented.

Study on the effects of terahertz radiation on gene networks of Escherichia coli by means of fluorescent biosensors.

Three novel fluorescent biosensors sensitive to terahertz (THz) radiation were developed via transformation of Escherichia coli (E. coli) cells with plasmids, in which a promotor of genes matA, safA, or chbB controls the expression of a fluorescent protein. The biosensors were exposed to THz radiation from two sources: a high-intensity pulsed short-wave free electron laser and a low-intensity continuous long-wave IMPATT-diode-based device. The threshold and dynamics of fluorescence were found to depend on radiation parameters and exposure time. Heat shock or chemical stress yielded the absence of fluorescence induction. The biosensors are evaluated to be suitable for studying influence of THz radiation on the activity of gene networks related with considered gene promoters.

Labyrinth Metasurface for Biosensing Applications: Numerical Study on the New Paradigm of Metageometries.

The use of metasurfaces operating in the terahertz regime as biosensor devices has attracted increased interest in recent years due to their enhanced sensitivity and more accurate detection capability. Typical designs are based on the replica of relatively simple unit cells, usually called metaatoms. In a previous paper, we proposed a new paradigm for ultrasensitive thin-film sensors based on complex unit cells, called generically metageometries or labyrinth metasurfaces. Here, we extend this concept towards biosensing, evaluating the performance of the labyrinth as a fungi detector. The sensing capabilities are numerically evaluated and a comparison with previous works in this field is performed, showing that metageometries improve the performance compared to metaatoms both in sensitivity and figure of merit, by a factor of more than four. In particular, we find that it is able to detect five fungi elements scattered on the unit cell, equivalent to a concentration of only 0.004/µm2.

In Vivo Formation of Stable Hyaline Cartilage by Naïve Human Bone Marrow Stromal Cells with Modified Fibrin Microbeads.

Osteoarthritic and other types of articular cartilage defects never heal on their own. Medicinal and surgical approaches are often ineffective, and the supply of autologous chondrocytes for tissue engineering is very limited. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested as an adequate cell source for cartilage reconstruction. However, the majority of studies employing BMSCs for cartilage tissue engineering have used BMSCs predifferentiated into cartilage prior to implantation. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro predifferentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In this study, naïve (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and implanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo. Stem Cells Translational Medicine 2019;8:586-592.

Nanoantenna structures for the detection of phonons in nanocrystals.

We report a study of the infrared response by localized surface plasmon resonance (LSPR) modes in gold micro- and nanoantenna arrays with various morphologies and surface-enhanced infrared absorption (SEIRA) by optical phonons of semiconductor nanocrystals (NCs) deposited on the arrays. The arrays of nano- and microantennas fabricated with nano- and photolithography reveal infrared-active LSPR modes of energy ranging from the mid to far-infrared that allow the IR response from very low concentrations of organic and inorganic materials deposited onto the arrays to be analyzed. The Langmuir-Blodgett technology was used for homogeneous deposition of CdSe, CdS, and PbS NC monolayers on the antenna arrays. The structural parameters of the arrays were confirmed by scanning electron microscopy. 3D full-wave electromagnetic simulations of the electromagnetic field distribution around the micro- and nanoantennas were employed to realize the maximal SEIRA enhancement for structural parameters of the arrays whereby the LSPR and the NC optical phonon energies coincide. The SEIRA experiments quantitatively confirmed the computational results. The maximum SEIRA enhancement was observed for linear nanoantennas with optimized structural parameters determined from the electromagnetic simulations. The frequency position of the feature's absorption seen in the SEIRA response evidences that the NC surface and transverse optical phonons are activated in the infrared spectra.

THz Sensing With Anomalous Extraordinary Optical Transmission Hole Arrays.

Subwavelength hole array (HA) metasurfaces support the so-called extraordinary optical transmission (EOT) resonance that has already been exploited for sensing. In this work, we demonstrate the superior performance of a different resonant regime of HA metasurfaces called anomalous EOT, by doing a thorough numerical and experimental study of its ability in thin-film label-free sensing applications in the terahertz (THz) band. A comprehensive analysis using both the regular and anomalous EOT resonances is done by depositing thin layers of dielectric analyte slabs of different thicknesses on the structures in different scenarios. We carry out a detailed comparison and demonstrate that the best sensing performance is achieved when the structure operates in the anomalous EOT resonance and the analyte is deposited on the non-patterned side of the metasurface, improving by a factor between 2 and 3 the results of the EOT resonance in any of the considered scenarios. This can be explained by the comparatively narrower linewidth of the anomalous EOT resonance. The results presented expand the reach of subwavelength HAs for sensing applications by considering the anomalous EOT regime that is usually overlooked in the literature.

Combinatorial cassettes to systematically evaluate tissue-engineered constructs in recipient mice.

Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (µCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs.

Novel antimitotic agents related to tubuloclustin: synthesis and biological evaluation.

Tubuloclustin [N-(7-adamant-2-yloxy-7-oxoheptanoyl)-N-deacetylcolchicine], a highly cytotoxic anti-tubulin compound is known for its ability to promote microtubule disassembly followed by the formation of tubulin clusters of unique morphology. Three series of antimitotic agents related to tubuloclustin were designed and synthesized in order to enhance the molecular diversity of "tubuloclustin-like" family of compounds. The series of compounds with modified adamantane moiety was highly potent in cytotoxic effect on human lung carcinoma A549 cells (EC50 = 6-400 nM) and was active in affecting the microtubule arrays and induction of strong tubulin clusterization. In two other sets of compounds, the colchicine moiety of tubuloclustin was replaced by podophyllotoxin or combretastatin A-4. All combretastatin A-4 derivatives displayed noticeable cytotoxic activity ([Formula: see text]) but their effect on microtubules depended on the position of the linker attachment. Podophyllotoxin derivatives were also toxic to A549 cells ([Formula: see text]) and caused both microtubule depolymerization and some tubulin clustering. The data obtained gave additional evidence that the whole panel of C7-colchicine, podophyllotoxin and combretastatin derivatives could manifest clustering effect, and the strength of this effect correlated with cytotoxic activity of the compounds.

Nanoantenna-assisted plasmonic enhancement of IR absorption of vibrational modes of organic molecules.

Nanoantenna-assisted plasmonic enhancement of IR absorption and Raman scattering was employed for studying the vibrational modes in organic molecules. Ultrathin cobalt phthalocyanine films (3 nm) were deposited on Au nanoantenna arrays with specified structural parameters. The deposited organic films reveal the enhancement of both Raman scattering and IR absorption vibrational modes. To extend the possibility of implementing surface-enhanced infrared absorption (SEIRA) for biological applications, the detection and analysis of the steroid hormone cortisol was demonstrated.

Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers.

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

Selective Pyroelectric Detection of Millimetre Waves Using Ultra-Thin Metasurface Absorbers.

Sensing infrared radiation is done inexpensively with pyroelectric detectors that generate a temporary voltage when they are heated by the incident infrared radiation. Unfortunately the performance of these detectors deteriorates for longer wavelengths, leaving the detection of, for instance, millimetre-wave radiation to expensive approaches. We propose here a simple and effective method to enhance pyroelectric detection of the millimetre-wave radiation by combining a compact commercial infrared pyro-sensor with a metasurface-enabled ultra-thin absorber, which provides spectrally- and polarization-discriminated response and is 136 times thinner than the operating wavelength. It is demonstrated that, due to the small thickness and therefore the thermal capacity of the absorber, the detector keeps the high response speed and sensitivity to millimetre waves as the original infrared pyro-sensor does against the regime of infrared detection. An in-depth electromagnetic analysis of the ultra-thin resonant absorbers along with their complex characterization by a BWO-spectroscopy technique is presented. Built upon this initial study, integrated metasurface absorber pyroelectric sensors are implemented and tested experimentally, showing high sensitivity and very fast response to millimetre-wave radiation. The proposed approach paves the way for creating highly-efficient inexpensive compact sensors for spectro-polarimetric applications in the millimetre-wave and terahertz bands.

Localized surface plasmons in structures with linear Au nanoantennas on a SiO2/Si surface.

The study of infrared absorption by linear gold nanoantennas fabricated on a Si surface with underlying SiO2 layers of various thicknesses allowed the penetration depth of localized surface plasmons into SiO2 to be determined. The value of the penetration depth derived experimentally (20 ± 10 nm) corresponds to that obtained from electromagnetic simulations (12.9-30.0 nm). Coupling between plasmonic excitations of gold nanoantennas and optical phonons in SiO2 leads to the appearance of new plasmon-phonon modes observed in the infrared transmission spectra the frequencies of which are well predicted by the simulations.

Missense mutation in the PTEN promoter of a patient with hemifacial hyperplasia.

The cellular mechanisms involved in the asymmetric facial overgrowth syndrome, hemifacial hyperplasia (HFH), are not well understood. This study was conducted to compare primary cell cultures from hyperplastic and normal HFH bone for cellular and molecular differences. Primary cultures developed from biopsies of a patient with isolated HFH showed a twofold difference in cell size and cell number between hyperplastic and normal bone. Microarray data suggested a 40% suppression of PTEN (phosphatase-tensin homolog) transcripts. Sequencing of the PTEN gene and promoter identified novel C/G missense mutation (position -1053) in the regulatory region of the PTEN promoter. Western blots of downstream pathway components showed an increase in PKBa/Akt1 phosphorylation and TOR (target of rapamcyin) signal. Sirolimus, an inhibitor of TOR, when added to overgrowth cells reversed the cell size, cell number and total protein differences between hyperplastic and normal cells. In cases of facial overgrowth, which involve PTEN/Akt/TOR dysregulation, sirolimus could be used for limiting cell overgrowth.

Human bone marrow stromal cell confluence: effects on cell characteristics and methods of assessment.

BACKGROUND AIMS: Ex vivo expansion and serial passage of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) is required to obtain sufficient quantities for clinical therapy. The BMSC confluence criteria used to determine passage and harvest timing vary widely, and the impact of confluence on BMSC properties remains controversial. The effects of confluence on BMSC properties were studied and confluence-associated markers were identified. METHODS: BMSC characteristics were analyzed as they grew from 50% to 100% confluence, including viability, population doubling time, apoptosis, colony formation, immunosuppression, surface marker expression, global gene expression and microRNA expression. In addition, culture supernatant protein, glucose, lactate and pH levels were analyzed. RESULTS: Confluence-dependent changes were detected in the expression of several cell surface markers: 39 culture supernatant proteins, 26 microRNAs and 2078 genes. Many of these surface markers, proteins, microRNAs and genes have been reported to be important in BMSC function. The pigment epithelium-derived factor/vascular endothelial growth factor ratio increased with confluence, but 80% and 100% confluent BMSCs demonstrated a similar level of immunosuppression of mixed lymphocyte reactions. In addition, changes in lactate and glucose levels correlated with BMSC density. CONCLUSIONS: BMSC characteristics change as confluence increases. 100% confluent BMSCs may have compromised pro-angiogenesis properties but may retain their immunomodulatory properties. Supernatant lactate and glucose levels can be used to estimate confluence and ensure consistency in passage and harvest timing. Flow cytometry or microRNA expression can be used to confirm that the BMSCs have been harvested at the appropriate confluence.

Molecular profile of clonal strains of human skeletal stem/progenitor cells with different potencies.

Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are fibroblastic reticular cells, a subset of which is composed of multipotent skeletal stem cells (SSCs). SSCs/BMSCs are able to recreate a bone/marrow organ in vivo. To determine differences between clonogenic multipotent SSCs and similarly clonogenic but non-multipotent BMSCs, we established single colony-derived strains (SCDSs, initiated by individual Colony Forming Unit-Fibroblasts) and determined their differentiation capacity by vivo transplantation. In this series of human SCDSs (N=24), 20.8% formed fibrous tissue (F), 66.7% formed bone (B), and 12.5% formed a bone/marrow organ, and thus were multipotent (M). RNA isolated from 12 SCDSs just prior to transplantation was analyzed by microarray. Although highly similar, there was variability from one SCDS to another, and SCDSs did not strictly segregate into the three functional groups (F, B or M) by unsupervised hierarchical clustering. We then compared 3 F-SCDSs to 3 M-SCDSs that did segregate. Genes associated with skeletogenesis, osteoblastogeneis, hematopoiesis, and extracellular matrix were over-represented in M-SCDSs compared with F-SCDSs. These results highlight the heterogeneity of SSCs/BMSCs, even between functionally similar SCDSs, but also indicate that differences can be detected that may shed light on the character of the SSC.

Planar holographic metasurfaces for terahertz focusing.

Scientists and laymen alike have always been fascinated by the ability of lenses and mirrors to control light. Now, with the advent of metamaterials and their two-dimensional counterpart metasurfaces, such components can be miniaturized and designed with additional functionalities, holding promise for system integration. To demonstrate this potential, here ultrathin reflection metasurfaces (also called metamirrors) designed for focusing terahertz radiation into a single spot and four spaced spots are proposed and experimentally investigated at the frequency of 0.35 THz. Each metasurface is designed using a computer-generated spatial distribution of the reflection phase. The phase variation within 360 deg is achieved via a topological morphing of the metasurface pattern from metallic patches to U-shaped and split-ring resonator elements, whose spectral response is derived from full-wave electromagnetic simulations. The proposed approach demonstrates a high-performance solution for creating low-cost and lightweight beam-shaping and beam-focusing devices for the terahertz band.