Mechanical and morphological responses of osteoblast-like cells to two-photon polymerized microgrooved surfaces.

Microgrooved surfaces are recognized as an important strategy of tissue engineering to promote the alignment of bone cells. In this work, we have investigated the mechanical and morphological aspects of osteoblasts cells after interaction with different micro-structured polymeric surfaces. Femtosecond laser writing technique was used for the construction of circular and parallel microgrooved patterns in biocompatible polymeric surfaces based on pentaerythritol triacrylate. Additionally, we have studied the influence of the biocompatible TiO2 nanocrystals (NCs) related to the cell behavior, when incorporated to the photoresin. The atomic force microscopy technique was used to investigate the biomechanical reaction of the human osteoblast-like MG-63 cells for the different microgroove. It was demonstrated that osteoblasts grown on circular microgrooved surfaces exhibited significantly larger Young's modulus compared to cells sown on flat films. Furthermore, we could observe that TiO2 NCs improved the circular microgrooves effects, resulting in more populated sites, 34% more elongated cells, and increasing the cell stiffness by almost 160%. These results can guide the design and construction of effective scaffold surfaces with circular microgrooves for tissue engineering and bone regeneration.

Krüppel-like factor 6 participates in extravillous trophoblast cell differentiation and its expression is reduced in abnormally invasive placenta.

Trophoblast cell differentiation is of paramount importance for successful pregnancy. Krüppel-like factor 6 (KLF6), a transcription factor with diverse roles in cell physiology and tumor biology, is required for trophoblast differentiation through the syncytial pathway. Herein, we demonstrate that extravillous trophoblast (EVT) cell migration and mesenchymal phenotype are increased upon KLF6 downregulation or the expression of a deletion mutant lacking its transcriptional regulatory domain (KΔac). Raman spectroscopy revealed molecular modifications compatible with increased differentiation in cells stably expressing the KΔac mutant. Moreover, abnormally invasive placenta showed lower KLF6 immunostaining compared with the normal placenta. Thus, impaired KLF6 expression or function stimulates EVT migration and differentiation in vitro and may contribute to the physiopathology of the abnormally invasive placenta.

Tuning the photoluminescence by engineering surface states/size of S, N co-doped carbon dots for cellular imaging applications.

In this research, we have synthesized carbon dots (CDs) co-doped with nitrogen and sulfur by facile hydrothermal method, using citric acid and cysteine as carbon source. The effect of solid-state thermic treatment (STT) at 303-453 K on the size, surface, fluorescence and cellular cytotoxicity of the CDs were systematically investigated. Through a simple STT, it was possible to tune surface states and the average size of the CDs, causing a permanent red shift. Initially, CDs showed a decrease in cell viability with increasing concentration. However, after STT, its viability remained constant with an increase in concentration. Here, we show the possibility to label the cells cytoplasm according to the CDs fluorescence emission before (blue emission) and after STT (red emission). The CDs studied in this paper show selective luminescence properties, which are fundamental for any cell imaging application.

Krüppel-like factor 6 (KLF6) requires its amino terminal domain to promote villous trophoblast cell fusion.

INTRODUCTION: Villous cytotrophoblast (vCTB) cells fuse to generate and maintain the syncytiotrophoblast layer required for placental development and function. Krüppel-like factor 6 (KLF6) is a ubiquitous transcription factor with an N-terminal acidic transactivation domain and a C-terminal zinc finger DNA-binding domain. KLF6 is highly expressed in placenta, and it is required for proper placental development. We have demonstrated that KLF6 is necessary for cell fusion in human primary vCTBs, and in the BeWo cell line. MATERIALS AND METHODS: Full length KLF6 or a mutant lacking its N-terminal domain were expressed in BeWo cells or in primary vCTB cells isolated from human term placentas. Cell fusion, gene and protein expression, and cell proliferation were analyzed. Moreover, Raman spectroscopy and atomic force microscopy (AFM) were used to identify biochemical, topography, and elasticity cellular modifications. RESULTS: The increase in KLF6, but not the expression of its deleted mutant, is sufficient to trigger cell fusion and to raise the expression of ß-hCG, syncytin-1, the chaperone protein 78 regulated by glucose (GRP78), the ATP Binding Cassette Subfamily G Member 2 (ABCG2), and Galectin-1 (Gal-1), all molecules involved in vCTB differentiation. Raman and AFM analysis revealed that KLF6 reduces NADH level and increases cell Young's modulus. KLF6-induced differentiation correlates with p21 upregulation and decreased cell proliferation. Remarkable, p21 silencing reduces cell fusion triggered by KLF6 and the KLF6 mutant impairs syncytialization and decreases syncytin-1 and ß-hCG expression. DISCUSSION: KLF6 induces syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner.

Serum From Preeclamptic Women Triggers Endoplasmic Reticulum Stress Pathway and Expression of Angiogenic Factors in Trophoblast Cells.

Preeclampsia (PE) is a hypertensive disease of pregnancy-associated with placental cell death and endoplasmic reticulum (ER) stress. It is unknown whether systemic factors aggravate placental dysfunction. We investigated whether serum factors in pregnant women with PE activate ER stress and unfolded protein responses (UPRs) in placental explants and trophoblast cells lineage. We cultured placental explants from third-trimester term placentas from control non-preeclamptic (NPE) pregnant women with serum from women with PE or controls (NPE). In PE-treated explants, there was a significant increase in gene expression of GADD34, CHOP, and SDF2. At the protein level, GRP78, SDF2, p-eIF2α, and p-eIF2α/eIF2α ratio were also augmented in treated explants. Assays were also performed in HTR8/SV-neo trophoblast cell line to characterize the putative participation of trophoblast cells. In PE serum-treated protein levels of p-eIF2a and the ratio p-elF2 α/elF2α increased after 12 h of treatment, while the gene expression of GADD34, ATF4, and CHOP was greater than control. Increased expression of SDF2 was also detected after 24 h-cultured HTR8/SV-neo cells. PE serum increased sFLT1 gene expression and decreased PlGF gene expression in placental explants. Morphologically, PE serum increased the number of syncytial knots and reduced placental cell metabolism and viability. Analysis of the serum of pregnant women with PE through Raman spectroscopy showed changes in amino acids, carotenoids, lipids, and DNA/RNA, which may be associated with the induction of ER stress found in chorionic villi treated with this serum. In conclusion, this study provides evidence that the serum of pregnant women with PE may impact placental villi changing its morphology, viability, and secreted functional factors while triggers ER stress and an UPR. The differences between PE and control sera include molecules acting as inducing factors in these processes. In summary, the results obtained in our assays suggest that after the development of PE, the serum profile of pregnant women may be an additional factor that feeds a continuous imbalance of placental homeostasis. In addition, this study may expand the possibilities for understanding the pathogenesis of this disorder.

Temperature-dependence on the optical properties of chitosan carbon dots in the solid state.

We report the synthesis of chitosan-derived aminated carbon dots with dual fluorescence bands and their influence on the morphology, absorption and emission spectral profiles as well as on the band gap energy in relation to thermal treatment after synthesis. To unravel these changes, we performed spectroscopic measurements in the solid state on two stages at temperatures ranging from 303 to 453 K. For the first heating stage, the emission spectrum showed a 20 nm red shift and a new absorption band at 350 nm, possibly related to new bonds and/or nitrogenous molecular fractions. For the second heating stage in the same temperature range, no displacements in the emission spectrum were observed and both the energy gap and bandwidths for the two emission bands are practically constant, indicating a change nitrogen moiety exposed on the surface. Furthermore, through atomic force microscopy it was noted that the morphology and size of the CDs were not significantly affected by the increase in temperature. It is noteworthy that the values of the Huang-Rhys factor, respectively, 2.584 × 10-10 and 2.315 × 10-9 for band I and II emission after the second heating indicate a mechanism of weak electron-phonon interactions. This work may open a novel perspective for the development of new surface modulation strategies for carbon dots subjected to thermal treatment in the solid state.

Sorting of spatially incoherent optical vortex modes.

Coherent optical vortices have promising applications in quantum and classical optical communication. They add new degrees of freedom to code information. In this context, to implement a tool enabling sorting of spatially multiplexed vortex states is fundamental. By other hand, spatially incoherent vortices can be more robust in propagation through noise media, such as turbulent atmosphere or obstacles that block part of the light. Therefore, in this work we propose directly applying a high-resolution sorting scheme to spatially incoherent vortex states.

Flavonoids induce cell death in Leishmania amazonensis: in vitro characterization by flow cytometry and Raman spectroscopy.

Leishmaniasis comprises a group of infectious diseases with worldwide distribution, of which both the visceral and cutaneous forms are caused by Leishmania parasites. In the absence of vaccines, efficacious chemotherapy remains the basis for leishmaniasis control. The available drugs are expensive and associated with several secondary adverse effects. Due to these limitations, the development of new antileishmanial compounds is imperative, and plants offer various perspectives in this regard. The present study evaluated the in vitro leishmanicidal activity of flavonoids isolated from Solanum paludosum Moric. and investigated the mechanisms of cell death induced by them. These compounds were evaluated in vitro for their antileishmanial activity against Leishmania amazonensis promastigotes and they showed prominent leishmanicidal activity. The EtOAc fraction, gossypetin 3,7,8,4'-tetra-O-methyl ether (1), and kaempferol 3,7-di-O-methyl ether (3) were selected to be used in an in vitro assay against L. amazonensis amastigotes and cell death assays. The flavonoids (1) and (3) presented significant activity against L. amazonensis amastigotes, exhibiting the IC50 values of 23.3 ± 4.5 µM, 34.0 ± 9.6 µM, and 10.5 ± 2.5 µM for the EtOAc fraction, (1), and (3), respectively, without toxic effects to the host cells. Moreover, (1) and (3) induced blocked cell cycle progression at the G1/S transition, ultimately leading to G1/G0 arrest. Flavonoid (3) also induced autophagy. Using Raman spectroscopy in conjunction with principal component analysis, the biochemical changes in the cellular components induced by flavonoids (1) and (3) were presented. The obtained results indicated that the mechanisms of action of (1) and (3) occurred through different routes. The results support that the flavonoids derived from S. paludosum can become lead molecules for the design of antileishmanial prototypes.

Method to define non-diffracting optical beams mimicking the shape of simple plane curves.

We theoretically and experimentally demonstrate a method to define non-diffracting beams with different geometries. Our findings constitute an alternative to current methods for finding non-diffracting beams, which rely on the solution of the wave equation in a given coordinate system that has a limited number of possibilities or uses a complicated and time-consuming optimization algorithm. Therefore, the method is easier to follow, because it does not require optimization and allows one to obtain non-diffracting beams mimicking the geometry of simple plane curves. The method could find applications in manipulation of matter with optical waves, such as colloidal and living particles, and in quantum, nonlinear, and atom optics.

Talbot effect with partially coherent interfering Bessel beams.

We studied the free-space propagation of interfering partially coherent Bessel beams. The partially coherent superimposed Bessel beams were generated by diffracting a spatially incoherent light by two concentric circular slits. We observed a Talbot effect in the random intensity pattern and in the intensity correlation. We showed that the Talbot length depends only on the radii of the circular slits. We explained this effect on the basis of the plane-wave decomposition of spatially random fields.

Three-Dimensional Speckle Light Self-Healing-Based Imaging System.

Recently new methodologies for imaging have been achieved making use of multiple light scattering. Here we present the self-healing effect using a speckled light field. We present an experiment that constitutes a useful application for a three-dimensional light sheet-based imaging system through an inhomogeneous medium. Each layer can be imaged independently of the others. The axial resolution basically depends on the coherence length, which can be sub-wavelength and controllable. This allows for a simple and direct technique for imaging through scattering layers with axial resolution improvement. Our results may find applications not only in bio-microscopy systems but also in data transmission.

Talbot effect in optical lattices with topological charge.

We studied the interference resulting from the superposition of optical lattices, which are non-diffracting fields propagating in free space, and showed a Talbot self-imaging effect. These lattices are formed by spatially Fourier transforming a "quasi"-orbital angular momentum (OAM) state. We experimentally observed that although the Talbot images change, the Talbot length is insensitive to the topological charge of the "quasi"-OAM state. Our findings can be useful for laser-written photonic lattices.

Robustness of a coherence vortex.

We study, experimentally and theoretically, the behavior of a coherence vortex after its transmission through obstacles. Notably, we find that such a vortex survives and preserves its effective topological charge. Despite suffering changes on the modulus of the coherence function, these changes disappear during propagation.

Characterizing coherence vortices through geometry.

We produce coherence vortices experimentally and numerically due to the orbital angular momentum of light beams and study the dependence of their bright ring area and dark region on their different orders. This is a linear dependence with a slope proportional to the bright ring area or dark area. We show that it is possible to estimate any order of coherence vortices, including fractional orders, just by calculating the bright ring area or dark area of the vortices for some specific parameters of the incident beam.

Self-reconfiguration of a speckle pattern.

It is well known that coherent Bessel beam, a nondiffracting class of beam, possesses the ability of self-reconstructing or self-healing in the presence of obstacles. Here, we generated partially coherent Bessel and Gaussian beams using a spatial light modulator and studied the speckle pattern intensity in propagation after some speckles were blocked. We demonstrated that these partially coherent beams are unexpectedly robust against scattering by objects, overcoming the coherent Bessel beam and remaining independent of any special class of partially coherent beams.

Macrophage adhesion on fibronectin evokes an increase in the elastic property of the cell membrane and cytoskeleton: an atomic force microscopy study.

Interactions between cells and microenvironments are essential to cellular functions such as survival, exocytosis and differentiation. Cell adhesion to the extracellular matrix (ECM) evokes a variety of biophysical changes in cellular organization, including modification of the cytoskeleton and plasma membrane. In fact, the cytoskeleton and plasma membrane are structures that mediate adherent contacts with the ECM; therefore, they are closely correlated. Considering that the mechanical properties of the cell could be affected by cell adhesion-induced changes in the cytoskeleton, the purpose of this study was to investigate the influence of the ECM on the elastic properties of fixed macrophage cells using atomic force microscopy. The results showed that there was an increase (~50%) in the Young's modulus of macrophages adhered to an ECM-coated substrate as compared with an uncoated glass substrate. In addition, cytochalasin D-treated cells had a 1.8-fold reduction of the Young's modulus of the cells, indicating the contribution of the actin cytoskeleton to the elastic properties of the cell. Our findings show that cell adhesion influences the mechanical properties of the plasma membrane, providing new information toward understanding the influence of the ECM on elastic alterations of macrophage cell membranes.

Unveiling square and triangular optical lattices: a comparative study.

We study square and triangular optical lattice formation using a diffraction technique with light-possessing orbital angular momentum (OAM). We demonstrate that it is possible to use Fraunhofer diffraction of light by a square aperture to unveil OAM about two times bigger than would be possible with a triangular aperture. We notice that the pattern remains truncated until a topological charge (TC) equal to 20 with good precision. Even though a square pattern cannot be used to determine the TC sign, it is possible to measure high order of the modulus and sign of the TC up to 20, combining patterns of the triangular and square apertures.

Study of the birth of a vortex at Fraunhofer zone.

We analytically and experimentally study the Fraunhofer diffraction of an optical vortex beam possessing noninteger values of the azimuthal index. We show that the Fraunhofer diffraction of this beam presents the birth of a vortex at α=n+ε, where n is an integer number and ε is a small fraction. We discuss this behavior on the basis of the born vortex movement from a position of low intensity to high intensity when α is increased of an integer number in fractional steps of ε.

Engineering a square truncated lattice with light's orbital angular momentum.

We engineer an intensity square lattice using the Fraunhofer diffraction of a Laguerre-Gauss beam by a square aperture. We verify numerically and experimentally that a perfect optical intensity lattice takes place only for even values of the topological charge. We explain the origin of this behavior based on the decomposition of the patterns. We also study the evolution of the lattice formation by observing the transition from one order to the next of the orbital angular momentum varying the topological charge in fractional steps.

Fraunhofer diffraction of light with orbital angular momentum by a slit.

We study the Fraunhofer diffraction problem while taking into account the orbital angular momentum of light. In this case, the phase singularity of the light beam is incident on the slit in two different cases: in one, it is incident slightly above the slit, and in the other it is centered on the slit. We observed that the symmetry and the fringe formation in the interference pattern strongly depend on the amount of orbital angular momentum and the slit position in relation to the beam.