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Electromagnetic (EM) losses estimation in complex scenarios lacking subsurface continuous reflectors is challenging. We present here a robust approach based on the centroid frequency shift method and Ricker wavelet as the input signal, to compute intrinsic (absorption) losses. The centroid-frequency shift is calculated using the amplitude and power spectrum of records, leading to equations that relate the centroid frequency variation in time to the loss tangent (intrinsic losses). First, the approaches are evaluated and compared by implementation to synthetic ground penetrating radar (GPR) data generated with the gprMax simulator, starting from an inhomogeneous material with spatially smooth dielectric variations. Then, they are applied to real GPR data collected on a fractured basaltic lava flow. The retrieved loss tangent values are compared to the total attenuation estimated from the signal amplitude decay, to evaluate the contribution due to scattering phenomena. The proposed analysis is particularly suitable for GPR data collected in planetary environments, since dielectric losses could be used to constrain the oxide composition of the material as well as scattering losses to determine the amount and distribution of subsurface inhomogeneities.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of “active chromatin” by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of “active chromatin” by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

It has recently been suggested that clay minerals, which are widespread on the Martian surface, could be the possible source of the basal bright reflections detected by MARSIS at Ultimi Scopuli, instead of briny water. This hypothesis is based on dielectric measurements on a wet Ca‐Montorillonite (STx‐1b) sample conducted at 230 K, which reported permittivity values (apparent permittivity of 39 at 4 MHz) compatible with the median value of 33 retrieved by MARSIS 4 MHz data inversion in the high reflectivity area. These experimental results are, however, incompatible with well‐established dielectric theory and with laboratory measurements on clays, at MARSIS frequency and Martian temperatures, reported in the literature. Here, we replicate the experiment using a setup to precisely control the rate of cooling/warming and the temperature inside and outside the clay sample. We found that the rate of cooling, the position of the temperature sensor and, consequently, the thermal equilibrium between the sample and the sensor play a fundamental role in the reliability of the measurements. Our results indicate that even for a large water content in the clay sample, at 230 K and 4 MHz, the apparent permittivity is only 8.4, dropping to 4.1 at 200 K, ruling out clays as a possible source of the bright reflections detected by MARSIS at the base of the SPLD. The recent discovery of bright basal reflections below the South polar ice cap at Ultimi Scopuli (Mars) by MARSIS has sparked an intense debate on their origin. Because bright radar reflections detected below the ice sheets in Antarctica and Greenland usually indicate the presence of basal water, and based on additional data processing methods, these anomalies were interpreted to be caused by liquid briny water. Recent laboratory measurements questioned this hypothesis and instead suggested that the presence of clay could explain the observed results. According to these measurements, the dielectric properties of wet clay at 230 K (−43°C) mimic the high reflectivity of briny water. Here, we replicate the experiment, making sure to reach thermal equilibrium during each measurement run to guarantee that the temperature is correctly evaluated when the dielectric properties of the sample are measured. This was accomplished using a dedicated setup to precisely control the thermal cycle and to carefully check the internal and external temperatures of the wet clay sample. Our experimental results clearly show that the dielectric properties of clay at 230 K are incompatible with the observed MARSIS anomalies and thus briny water remains the most plausible explanation for the bright basal reflections detected by MARSIS. Anomalous bright basal reflections have been detected by MARSIS at Ultimi Scopuli, MarsDielectric measurements of wet clays at low temperature show that they cannot generate strong basal reflectionsReliable measurements of dielectric behavior of planetary analogs require a careful control of the temperature inside the sample Anomalous bright basal reflections have been detected by MARSIS at Ultimi Scopuli, Mars Dielectric measurements of wet clays at low temperature show that they cannot generate strong basal reflections Reliable measurements of dielectric behavior of planetary analogs require a careful control of the temperature inside the sample

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell–cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli–germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin–positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ–Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell–cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli–germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin–positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ–Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.

Cardiac hypertrophy arises as an adaptive response to increased afterload. Studies in knockout mice have shown that catecholamines, but not α1-adrenergic receptors, are necessary for such an adaptation to occur. However, whether β-adrenergic receptors are critical for the development of cardiac hypertrophy in response to pressure overload is not known at this time.

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (−5.7 to −3.3 kb) which can not only drive expression of a β-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2β-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.

During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle.

ABSTRACTDuring the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle.

In this study we demonstrate that Raji cells, a CR2-positive Burkitt lymphoma-derived cell line, during cell growth, need the cross-linking of multiple OKB7 binding sites or C3d determinants to mediate signal transduction. The loss of one of these affects the cellular response. Moreover, OKB7, the anti-CR2 MoAb, recognizes C3d determinants on the cell surface and inhibits signal transduction induced by anti-C3d polyclonal antibody. Since Raji cells are always CR2 positive during cell growth, we suppose that at least another protein, along with CR2, may be involved in setting up a cell surface complex able to receive and transduce the signal triggered by OKB7. In our experimental system the protein that offers a third binding site to OKB7, may be represented by a 33 kDa protein bearing C3d determinants.

The cholinergic responses of the human tumour cell line TE671/RD were examined using digital Ca2+ imaging fluorescence microscopy and patch-clamp measurements. In response to stimulation of the muscarinic acetylcholine (ACh) receptor (mAChR), the intracellular concentration of Ca2+ ([Ca2+]i) rose about two-fold, in parallel with inositol 1,4,5-trisphosphate accumulation, measured by chromatographic techniques. By contrast, there was no increment of [Ca2+]i upon stimulation of the nicotinic ACh receptor (nAChR), nor after caffeine application. Electrophysiological experiments showed that TE671/RD cells lack functional voltage-activated Ca2+ channels. The stimulation of the nAChR induced transient whole-cell currents (IACh). Little or no current was detected in isotonic extracellular Ca2+, with Cs+ in the patch pipette. Cell pretreatment with muscarine reduced IACh by about 20%, without consistent modifications of current kinetics. Muscarine applied to the extra-patch membrane under the cell-attached configuration had no obvious effect on ACh-evoked unitary events. In conclusion, in human TE671/ RD cells, muscarinic stimulation increases [Ca2+]i, while nicotinic stimulation does not. In addition, the nAChR exhibits peculiar ion permeability properties and is not functionally regulated by the breakdown of phosphoinositides.

Experiments were addressed to investigate the mechanisms by which cholinergic stimulation is coupled to the enhancement of proliferation of small cell lung cancer cells H69. Muscarinic stimulation triggers the release of cytosolic Ca2+and of inositol(1,4,5)trisphosphate with comparable time courses. The presence of α-bungarotoxin or the absence of Ca2+in external medium suppresses enhancement of clonal growth induced by brief applications of nicotine. Here we suggest that Ca2+mobilization represents a trigger for the enhancement of small cell lung cancer cell proliferation upon cholinergic stimulation.

The induction of thermotolerance was studied in two groups of murine tumors, one able to produce heat shock proteins (HSP) and the other entirely lacking HSP expression in response to various stress inducers. Heat treatments were performed in vitro and the development of thermotolerance was then evaluated in vivo. The data obtained on the of death rate of mice inoculated with tumor cells previously conditioned at 42 °C for 1 h and then challenged at 45 °C for 30 min following 2 h of reincubation at 37 °C, show that the rate of survival is far higher in mice inoculated with HSP negative tumor cells. This indicates that a large number of cells able to increase HSP synthesis following stress escape heat killing, whereas cells unable to express HSP after adequate stimuli are less tolerant against heat challenge.

Summary M-14 human tumor cells have been subjected to two regimens of step-down heating (SDH) consisting of a conditioning treatment at 42 °C for 1 h or at 44.5 °C for 20 min, immediately followed by heating at 40 °C. Both conditioning treatments thermosensitize the cells towards the subsequent heating at 40 °C; the thermosensitization ratio is 6.4 for cells conditioned at 42 °C for 1 h and 32.3 for cells conditioned at 44.5 °C for 20 min. The overall protein synthetic activity is reduced to 32.7% or 18.4% of control values following 1 h at 42 °C and 20 min at 44.5 °C, respectively; this inhibition is followed by a full recovery of the synthetic activity during the subsequent exposure at 40 °C. SDH-treated cells synthetize four heat shock proteins, with approximate molecular weights of 28, 64, 70 and 90 kDa. The pattern of HSPs induction observed in SDH-treted cells is similar to that found in cells subjected to single hyperthermic exposures. Cells subjected to the SDH sequence 42 °C/1 h?40 °C/4 h develop thermotolerance, as indicated by a reduced sensitivity to further hyperthermic challenges.

In this study we demonstrate that Raji cells, a CR2‐positive Burkitt lymphoma‐derived cell line, during cell growth, need the cross‐linking of multiple OKB7 binding sites or C3d determinants to mediate signal transduction. The loss of one of these affects the cellular response. Moreover, OKB7, the anti‐CR2 MoAb, recognizes C3d determinants on the cell surface and inhibits signal transduction induced by anti‐C3d polyclonal antibody. Since Raji cells are always CR2 positive during cell growth, we suppose that at least another protein, along with CR2, may be involved in setting up a cell surface complex able to receive and transduce the signal triggered by OKB7. In our experimental system the protein that offers a third binding site to OKB7, may be represented by a 33 kDa protein bearing C3d determinants.

The induction of stress proteins was studied in two human and two murine melanoma cell lines. Exposure for 1 h to heat (42 °C), to ethanol (6%), to arsenate (100 μM) and to disulfiram (50 μM) induced the expression of SPs with apparent molecular weights of 100, 86, 70-72 and 24-26 Kd. Quantitation of the single SPs indicated that the basal level as well as the enhanced synthesis following the various stressors were different in each cell line. The induction of the 100 Kd species occurred in only one murine melanoma and not in the others. The 86 and in particular the 70-72 Kd species were the most prominent groups, whereas the 24-26 SPs were induced only following arsenate and disulfiram exposure in the three melanoma cell lines. In one of the murine melanomas, the expression of SPs was markedly reduced compared to the other cell lines. No definite specific patterns of SP expression could be identified in tumors of the same histologic type.

Time- and dose-dependent protein synthesis inhibition takes place following exposure to high doses of dimethylnitrosamine (DMN) or diethylnitrosamine (DENA) in isolated rat hepatocytes. The ability of DENA to depress protein synthesis is 5-fold higher than that of DMN. Cells inhibited by 60 min exposure to DMN or DENA, and then incubated in a nitrosamine-free medium, regain their initial rate of protein synthesis. This recovery is faster and more complete for DENA-treated cells.