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

An integrated analysis of intracellular metabolites and virulence gene expression during biofilm development of a clinical isolate of Candida tropicalis on distinct surfaces

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidi-asis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis con-sider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections

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

Temperature and force dependence of nanoscale electron transport via the Cu protein Azurin

The mechanisms of solid-state electron transport (ETp) via a monolayer of immobilized Azurin (Az) was examined by conducting probe atomic force microscopy (CP-AFM), both as function of temperature (248 - 373K) and of applied tip force (6-12 nN). By varying both temperature and force in CP-AFM, we find that the ETp mechanism can alter with a change in the force applied via the tip to the proteins. As the applied force increases, ETp via Az changes from temperature-independent to thermally activated at high temperatures. This is in contrast to the Cu-depleted form of Az (apo-Az), where increasing the applied force causes only small quantitative effects, that fit with a decrease in electrode spacing. At low force ETp via holo-Az is temperature-independent and thermally activated via apo-Az. This observation agrees with macroscopic-scale measurements, thus confirming that the difference in ETp dependence on temperature between holo- and apo-Az is an inherent one that may reflect a difference in rigidity between the two forms. An important implication of these results, which depend on CP-AFM measurements over a significant temperature range, is that for ETp measurements on floppy systems, such as proteins, the stress applied to the sample should be kept constant or, at least controlled during measurement.Comment: 24 pages, 6 figures, plus Supporting Information with 4 pages and 2 figure

Fungal endophytes from arid areas of Andalusia: high potential sources for antifungal and antitumoral agents

Native plant communities from arid areas present distinctive characteristics to survive in extreme conditions. The large number of poorly studied endemic plants represents a unique potential source for the discovery of novel fungal symbionts as well as host-specific endophytes not yet described. The addition of adsorptive polymeric resins in fungal fermentations has been seen to promote the production of new secondary metabolites and is a tool used consistently to generate new compounds with potential biological activities. A total of 349 fungal strains isolated from 63 selected plant species from arid ecosystems located in the southeast of the Iberian Peninsula, were characterized morphologically as well as based on their ITS/28S ribosomal gene sequences. The fungal community isolated was distributed among 19 orders including Basidiomycetes and Ascomycetes, being Pleosporales the most abundant order. In total, 107 different genera were identified being Neocamarosporium the genus most frequently isolated from these plants, followed by Preussia and Alternaria. Strains were grown in four different media in presence and absence of selected resins to promote chemical diversity generation of new secondary metabolites. Fermentation extracts were evaluated, looking for new antifungal activities against plant and human fungal pathogens, as well as, cytotoxic activities against the human liver cancer cell line HepG2. From the 349 isolates tested, 126 (36%) exhibited significant bioactivities including 58 strains with exclusive antifungal properties and 33 strains with exclusive activity against the HepG2 hepatocellular carcinoma cell line. After LCMS analysis, 68 known bioactive secondary metabolites could be identified as produced by 96 strains, and 12 likely unknown compounds were found in a subset of 14 fungal endophytes. The chemical profiles of the differential expression of induced activities were compared. As proof of concept, ten active secondary metabolites only produced in the presence of resins were purified and identified. The structures of three of these compounds were new and herein are elucidated.This work was supported by Fundación MEDINA and the Andalusian Government grant RNM-7987 ‘Sustainable use of plants and their fungal parasites from arid regions of Andalucía for new molecules useful for antifungals and neuroprotectors’