Assembly of the Auditory Circuitry by a Hox Genetic Network in the Mouse Brainstem

Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem

AFM macro-probes to investigate whole 3D cardiac spheroids

In its many applications, the Atomic Force Microscope (AFM) is a promising tool in cardiac mechanobiology because it can unravel the viscoelastic and mechano-dynamic properties of individual cardiomyocytes. However, the biophysical investigation of more accurate 3D models is hampered by commercial probes, which typically operate at the cell sub-compartmental resolution. We have previously shown how flat macro-probes can overcome these limitations by extending the AFM mechanical measurements to multicellular aggregates. Such macro-probes are fabricated by standard micromachining and carry a flat polymeric wedge to offset the AFM mounting tilt. Therefore, the AFM is upgraded to a micro-parallel plate rheometer with unmatched force range and sensitivity. In this article, we show how these macro-probes can be applied to reveal the global rheology of primary cardiomyocytes spheroids, by performing stress-relaxation tests. More importantly, we demonstrate that these macro-probes can be used as passive sensors capable of monitoring the spheroid beating force and beating pattern, and to perform a “micro-CPR” on the spheroid itself

Effects of Temperature, Light and Pre-Chilling on Seed Germination of Stevia rebaudiana (Bertoni) Bertoni Accessions

Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the Asteraceae family native to Paraguay and Brazil where it has been used for several years as a sweetener. It is a short-day species, with a critical light requirement for flowering of roughly 13 hours. In plants whose biological cycle is strongly photoperiod-dependent, latitude is one of the major factors influencing reproduction. Late flowering may adversely affect seed production if this occurs during a season that is unfavorable to pollination. At Italian latitudes, this species often gives scanty seed production, with low germination rate and poor germination energy of seeds produced. In 2001 four accessions have been grown for seed production in a field plot experiment in Central Italy. The various accessions were found to exhibit noticeably different photoperiod requirements, which affected flowering time (from late August to the end of September) and consequently also the conditions of achene filling and ripening. Late flowering and seed ripening occurred during autumn season, unfavourable to complete seed formation, leading to an increase in the empty seed percentage recorded for each accession. Detailed germination trials were therefore undertaken using seeds collected from plants of the different accessions in order to assess the quality of the seeds produced. Various germination methods have been tested in a controlled environment adopting four different temperatures (20° C, 25° C constant temperature and 15/25° C, 20/30° C (16/8h) alternating temperature) in light or darkness with or without pre-chilling. Germination rates varied over an extensive range (germination percentage from 9 to 83%), mainly due to the divergent specific characteristics of the material examined and the different treatments studied. At all temperatures tested, the most earlier accession, showed the higher germination percentages (54-83%) while the latest accession was among those with the lowest germination values (9-44%). Overall, at the constant temperatures assayed, pre-chilling gave a higher germination percentage as compared to no pre-chilling, while at alternating temperatures higher germination rates were obtained in the absence of pre-chilling. The great variability observed between accessions for photoperiodic requirement as well as in seed germinative characteristics was probably due to the fact that this species was not subjected to any genetic improvement program

Antiviral Property of the Fungal Metabolite 3-O-Methylfunicone in Bovine Herpesvirus 1 Infection

Bovine herpesvirus type-1 (BoHV-1) is a widespread pathogen that provokes infectious rhinotracheitis and polymicrobial infections in cattle, resulting in serious economic losses to the farm animal industry and trade restrictions. To date, non-toxic active drugs against BoHV-1 are not available. The exploitation of bioactive properties of microbial products is of great pharmaceutical interest. In fact, fungi are a promising source of novel drugs with a broad spectrum of activities and functions, including antiviral properties. Hence, the potential antiviral properties of 3-O-methylfunicone (OMF), a secondary metabolite produced by Talaromyces pinophilus, were evaluated on BoHV-1. In this study, during BoHV-1 infection in bovine cells (MDBK), the non-toxic concentration of 5 µM OMF considerably reduced signs of cell death and increased cell proliferation. Furthermore, OMF significantly decreased the virus titer as well as the cytopathic effect and strongly inhibited the expression of bICP0, the major regulatory protein in the BoHV-1 lytic cycle. These findings were accompanied by a considerable up-regulation in the expression of the aryl hydrocarbon receptor (AhR), a multifunctional transcription factor also linked to the host’s response to a herpesvirus infection. Overall, our results suggest that by involving AhR, OMF shows potential against a BoHV-1 infection

Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics

In Vitro Evaluation of Antiviral Activities of Funicone-like Compounds Vermistatin and Penisimplicissin against Canine Coronavirus Infection

Recent studies have demonstrated that 3-O-methylfunicone (OMF), a fungal secondary metabolite from Talaromyces pinophilus belonging to the class of funicone-like compounds, has antiviral activity against canine coronaviruses (CCoV), which causes enteritis in dogs. Herein, we selected two additional funicone-like compounds named vermistatin (VER) and penisimplicissin (PS) and investigated their inhibitory activity towards CCoV infection. Thus, both compounds have been tested for their cytotoxicity and for antiviral activity against CCoV in A72 cells, a fibrosarcoma cell line suitable for investigating CCoV. Our findings showed an increase in cell viability, with an improvement of morphological features in CCoV-infected cells at the non-toxic doses of 1 μM for VER and 0.5 μM for PS. In addition, we observed that these compounds caused a strong inhibition in the expression of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which is activated during CCoV infection. Our results also showed the alkalinization of lysosomes in the presence of VER or PS, which may be involved in the observed antiviral activities

Planar AFM macro-probes to study the biomechanical properties of large cells and 3D cell spheroids

The ability to measure mechanical response of cells under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Living cells have been mechanically investigated by several approaches. Among them, atomic force microscopy (AFM) is widely used thanks to its high versatility and sensitivity. In the case of large cells or 3D multicellular aggregates, standard AFM probes may not be appropriate to investigate the mechanical properties of the whole biological system. Owing to their size, standard AFM probes can compress only a single somatic cell or part of it. To fill this gap, we have designed and fabricated planar AFM macro-probes compatible with commercial AFM instruments. The probes are constituted of a large flat compression plate, connected to the chip by two flexible arms, whose mechanical characteristics are tuned for specific biological applications. As proof of concept, we have used the macro-probes to measure the viscoelasticity of large spherical biological systems, which have a diameter above 100 \u3bcm: human oocytes and 3D cell spheroids. Compression experiments are combined with visual inspection, using a side-view configuration imaging, which allows us to monitor the sample morphology during the compression and to correlate it with the viscoelastic parameters. Our measurements provide a quantitative estimate of the relaxation times of such biological systems, which are discussed in relation to data present in literature. The broad applicability of the AFM macro-probes can be relevant to study the biomechanical features in any biological process involving large soft materials. Statement of Significance: The understanding of the role of physical factors in defining cell and tissue functions requires to develop new methods or improve the existing ones to accurately measure the biomechanical properties. AFM is a sensitive and versatile tool to measure the mechanical features from single proteins to single cells. When cells or cell aggregates exceed few tens of microns, AFM suffers from limitations. On these biological systems the control of the contact area and the application of a precise uniform compression becomes crucial. A modification of the standard cantilevers fabrication allowed us obtaining AFM macro-probes, having large planar contact area and spring constant suitable for biological investigations. They were demonstrated valuable to characterize the mechanical properties of large hierarchical biological systems

Positive selection in Europeans and East-Asians at the ABCA12 gene

Natural selection acts on genetic variants by increasing the frequency of alleles responsible for a cellular function that is favorable in a certain environment. In a previous genome-wide scan for positive selection in contemporary humans, we identified a signal of positive selection in European and Asians at the genetic variant rs10180970. The variant is located in the second intron of the ABCA12 gene, which is implicated in the lipid barrier formation and down-regulated by UVB radiation. We studied the signal of selection in the genomic region surrounding rs10180970 in a larger dataset that includes DNA sequences from ancient samples. We also investigated the functional consequences of gene expression of the alleles of rs10180970 and another genetic variant in its proximity in healthy volunteers exposed to similar UV radiation. We confirmed the selection signal and refine its location that extends over 35 kb and includes the first intron, the first two exons and the transcription starting site of ABCA12. We found no obvious effect of rs10180970 alleles on ABCA12 gene expression. We reconstructed the trajectory of the T allele over the last 80,000 years to discover that it was specific to H. sapiens and present in non-Africans 45,000 years ago

Imaging cervical cytology with scanning near-field optical microscopy (SNOM) coupled with an IR-FEL

Cervical cancer remains a major cause of morbidity and mortality among women, especially in the developing world. Increased synthesis of proteins, lipids and nucleic acids is a pre-condition for the rapid proliferation of cancer cells. We show that scanning near-field optical microscopy, in combination with an infrared free electron laser (SNOM-IR-FEL), is able to distinguish between normal and squamous low-grade and high-grade dyskaryosis, and between normal and mixed squamous/glandular pre-invasive and adenocarcinoma cervical lesions, at designated wavelengths associated with DNA, Amide I/II and lipids. These findings evidence the promise of the SNOM-IR-FEL technique in obtaining chemical information relevant to the detection of cervical cell abnormalities and cancer diagnosis at spatial resolutions below the diffraction limit (?0.2 \ensuremathμm). We compare these results with analyses following attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy; although this latter approach has been demonstrated to detect underlying cervical atypia missed by conventional cytology, it is limited by a spatial resolution of ~3 \ensuremathμm to 30 \ensuremathμm due to the optical diffraction limit