Generation of mouse oocytes defective in cAMP synthesis and degradation: Endogenous cyclic AMP is essential for meiotic arrest

AbstractAlthough it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a−/− mice with Gpr3−/− mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3−/− mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3−/− phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte

apoptotic pathways of u937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry

Infrared microspectroscopy and flow cytometry were used to study apoptosis in starved and CCCP-treated U937 monocyte cells

Surface analysis of an eagle talon from Krapina

This work is licensed under a Creative Commons Attribution 4.0 International License.The Krapina white-tailed eagle talons represent a kind of jewelry worn by Krapina Neandertals some 130,000 years ago. New inspection of one Krapina talon (386.1) revealed a fiber, sealed by a thin silicate coating, adhering to the surface within a wide cut mark, as well as concentrated traces of occasional spots of red and yellow pigment and some black stains. We analyzed the fiber and small portions of pigmented areas by non-invasive, infrared synchrotron beam. Different areas were targeted, revealing the protein nature of the fiber, identified as of animal origin. Targeted areas revealed intra- and inter-strand aggregation indicating the fiber to be collagen losing its original triple α-helix conformation, further confirming the diagenetic decay of the original collagen structure and the antiquity of the fiber. It is possible that the fiber is a remnant of the leather or sinew string binding the talons together. Spectroscopic analysis of the pigments in two isolated areas confirmed two types of ochre and that the dark spots are charcoal remnants. Applying novel non-invasive technologies provides new possibilities to further test the hypothesis of using prehistoric objects for symbolic purposes

Infrared nanospectroscopy depth-dependent study of modern materials: morpho-chemical analysis of polyurethane/fibroin binary meshes

Infrared scattering-type scanning near-field optical microscopy (IR s-SNOM) and imaging is here exploited together with attenuated total reflection (ATR) IR imaging and scanning electron microscopy (SEM) to depict the chemical composition of fibers in hybrid electrospun meshes. The focus is on a recently developed bio-hybrid material for vascular tissue engineering applications, named Silkothane & REG;, obtained in the form of nanofibrous matrices from the processing of a silk fibroin-polyurethane (SFPU) blend via electrospinning. Morphology and chemistry of single fibers, at both surface and subsurface level, have been successfully characterized with nanoscale resolution, taking advantage of the IR s-SNOM capability to portray the nanoscale depth profile of this modern material working at diverse harmonics of the signal. The applied methodology allowed to describe the superficial characteristics of the mesh up to a depth of about 100 nm, showing that SF and PU do not tend to co-aggregate to form hybrid fibers, at least at the length scale of hundreds of nanometers, and that subdomains other than the fibrillar ones can be present. More generally, in the present contribution, the depth profiling capabilities of IR s-SNOM, so far theoretically predicted and experimentally proven only on model systems, have been corroborated on a real material in its natural conditions with respect to production, opening the room for the exploitation of IR s-SNOM as valuable technique to support the production and the engineering of nanostructured materials by the precise understanding of their chemistry at the interface with the environment

Subcellular elements responsive to the biomechanical activity of triple-negative breast cancer-derived small extracellular vesicles

Abstract Triple-negative breast cancer (TNBC) stands out for its aggressive, fast spread, and highly metastatic behavior and for being unresponsive to the classical hormonal therapy. It is considered a disease with a poor prognosis and limited treatment options. Among the mechanisms that contribute to TNBC spreading, attention has been recently paid to small extracellular vesicles (sEVs), nano-sized vesicles that by transferring bioactive molecules to recipient cells play a crucial role in the intercellular communication among cancer, healthy cells, and tumor microenvironment. In particular, TNBC-derived sEVs have been shown to alter proliferation, metastasis, drug resistance, and biomechanical properties of target cells. To shed light on the molecular mechanisms involved in sEVs mediation of cell biomechanics, we investigated the effects of sEVs on the main subcellular players, i.e., cell membrane, cytoskeleton, and nuclear chromatin organization. Our results unveiled that TNBC-derived sEVs are able to promote the formation and elongation of cellular protrusions, soften the cell body, and induce chromatin decondensation in recipient cells. In particular, our data suggest that chromatin decondensation is the main cause of the global cell softening. The present study added new details and unveiled a novel mechanism of activity of the TNBC-derived sEVs, providing information for the efficient translation of sEVs to cancer theranostics

Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis

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Comparative investigation of chemical and structural properties of charred fir wood samples by Raman and FTIR spectroscopy as well as X-ray-micro-CT technology

Wood surface charring is a treatment method commonly employed to enhance weather protection and aesthetic appearance of building exteriors. This study aims to investigate the differences between two wood surface charring processes: the traditional Japanese method known as Yakisugi and an alternative charring technique industrially manufactured with a gas burner. The objective of the study was to assess whether a thicker layer after Yakisugi treatment has any advantages over a thinner layer after the alternative process. Vibrational spectroscopy techniques including UV resonance Raman (UVRR) and Fourier transform infrared (FTIR) spectroscopy, were utilized in conjunction with X-ray-micro-CT analysis. The findings revealed that ATR-FTIR spectroscopy detected the degradation of carbohydrates and changes in lignin within the charred surface, although both processes exhibited similar vibrational contributions. In contrast, UVRR spectroscopy provided insights into the carbonized layers, revealing spectral differences indicating variations in temperature during the charring processes. X-ray micro-CT analysis visually highlighted significant differences in the coal layers, suggesting distinct combustion profiles. Remarkably, the macrostructure of wood treated with Yakisugi remained intact despite a thicker charred layer compared to the alternative charring techniques. However, further investigations are required to assess the weather stability of the alternative charring method for a comprehensive understanding.O

Cytotoxic Effects of 5-Azacytidine on Primary Tumour Cells and Cancer Stem Cells from Oral Squamous Cell Carcinoma: An In Vitro FTIRM Analysis

In the present study, the cytotoxic effects of 5-azacytidine on primary Oral Squamous Cell Carcinoma cells (OSCCs) from human biopsies, and on Cancer Stem Cells (CSCs) from the same samples, were investigated by an in vitro Fourier Transform InfraRed Microscospectroscopy (FTIRM) approach coupled with multivariate analysis. OSCC is an aggressive tumoral lesion of the epithelium, accounting for ~90% of all oral cancers. It is usually diagnosed in advanced stages, and this causes a poor prognosis with low success rates of surgical, as well as radiation and chemotherapy treatments. OSCC is frequently characterised by recurrence after chemotherapy and by the development of a refractoriness to some employed drugs, which is probably ascribable to the presence of CSCs niches, responsible for cancer growth, chemoresistance and metastasis. The spectral information from FTIRM was correlated with the outcomes of cytotoxicity tests and image-based cytometry, and specific spectral signatures attributable to 5-azacytidine treatment were identified, allowing us to hypothesise the demethylation of DNA and, hence, an increase in the transcriptional activity, together with a conformational transition of DNA, and a triggering of cell death by an apoptosis mechanism. Moreover, a different mechanism of action between OSSC and CSC cells was highlighted, probably due to possible differences between OSCCs and CSCs response

X-ray and FTIR \u3bc-CTs for morphological and chemical characterization of eco-sustainable insulating foams

Here it is reported a multidisciplinary approach based on tomography and infrared techniques applied to the characterization of tannin porous rigid foams, potentially usable as new insulating materials in green building technology. With conventional x-ray tomography it was possible to preliminary evaluate the homogeneity of the samples at low resolution, while then, thanks to the synchrotron source, it was possible to obtain more detailed information at a micro-scale level. At the same time chemical characterization was done through Fourier Transform infrared (FTIR) imaging. Conventionally, FTIR imaging is limited to a planar projection, not considering the 3D structure of the material. To avoid this limitation, a FTIR 3D-tomography setup was built and the foams characterized by a chemical point of view. The idea is to directly correlate these data with the 3D-structural information obtained with the x-ray computed tomography exploiting the synchrotron radiation as source, allowing a complete characterization of the material morphology and chemistry at the microscale