A Bacterial Cytotoxin Identifies the RhoA Exchange Factor Net1 as a Key Effector in the Response to DNA Damage

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. Principal Findings: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoAdependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPKactivated protein kinase 2. Significance: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin ma

Establishment, molecular and biological characterization of HCB-514: a novel human cervical cancer cell line

Cervical cancer is the fourth most common cancer in women. Although cure rates are high for early stage disease, clinical outcomes for advanced, metastatic, or recurrent disease remain poor. To change this panorama, a deeper understanding of cervical cancer biology and novel study models are needed. Immortalized human cancer cell lines such as HeLa constitute crucial scientific tools, but there are few other cervical cancer cell lines available, limiting our understanding of a disease known for its molecular heterogeneity. This study aimed to establish novel cervical cancer cell lines derived from Brazilian patients. We successfully established one (HCB-514) out of 35 cervical tumors biopsied. We confirmed the phenotype of HCB-514 by verifying its' epithelial and tumor origin through cytokeratins, EpCAM and p16 staining. It was also HPV-16 positive. Whole-exome sequencing (WES) showed relevant somatic mutations in several genes including BRCA2, TGFBR1 and IRX2. A copy number variation (CNV) analysis by nanostring and WES revealed amplification of genes mainly related to kinases proteins involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and validated by western blot analysis. Furthermore, the HCB-514 cell line was sensitive to cisplatin. In summary, this novel Brazilian cervical cancer cell line exhibits relevant key molecular features and constitutes a new biological model for pre-clinical studies.Barretos Cancer Hospital Research Support Department (NAP) for sample collection, Barretos Cancer Hospital Biobank for sample processing, Dr. Flávia de Paula and Gabriela Fernandes for technical support of STRs and BRCA2 Sanger validation, respectively, and Dr. Laura Musselwhite (Duke University) for revising the manuscript. This study was supported by grants from the FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII- BIOPLAT - Process number 01.13.0469.00) and Barretos Cancer Hospital. PhD scholarship from FINEP (Grant numbers 384088/2014-7 and 380434/2015-6) and Barretos Cancer Hospital to MNR

Olprinone Attenuates the Acute Inflammatory Response and Apoptosis after Spinal Cord Trauma in Mice

BACKGROUND: Olprinone hydrochloride is a newly developed compound that selectively inhibits PDE type III and is characterized by several properties, including positive inotropic effects, peripheral vasodilatory effects, and a bronchodilator effect. In clinical settings, olprinone is commonly used to treat congestive cardiac failure, due to its inotropic and vasodilating effects. The mechanism of these cardiac effects is attributed to increased cellular concentrations of cAMP. The aim of the present study was to evaluate the pharmacological action of olprinone on the secondary damage in experimental spinal cord injury (SCI) in mice. METHODOLOGY/PRINCIPAL FINDINGS: Traumatic SCI is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should be preventable, no effective treatment options currently exist for patients with SCI. Spinal cord trauma was induced in mice by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, apoptosis, and locomotor disturbance. Olprinone treatment (0.2 mg/kg, i.p.) 1 and 6 h after the SCI significantly reduced: (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation, (4) pro-inflammatory cytokines, (5) NF-kappaB expression, (6) p-ERK1/2 and p38 expression and (7) apoptosis (TUNEL staining, FAS ligand, Bax and Bcl-2 expression). Moreover, olprinone significantly ameliorated the recovery of hind-limb function (evaluated by motor recovery score). CONCLUSIONS/SIGNIFICANCE: Taken together, our results clearly demonstrate that olprinone treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma

The control of alternative splicing by SRSF1 in myelinated afferents contributes to the development of neuropathic pain

Neuropathic pain results from neuroplasticity in nociceptive neuronal networks. Here we demonstrate that control of alternative pre-mRNA splicing, through the splice factor serine-arginine splice factor 1 (SRSF1), is integral to the processing of nociceptive information in the spinal cord. Neuropathic pain develops following a partial saphenous nerve ligation injury, at which time SRSF1 is activated in damaged myelinated primary afferent neurons, with minimal found in small diameter (IB4 positive) dorsal root ganglia neurons. Serine arginine protein kinase 1 (SRPK1) is the principal route of SRSF1 activation. Spinal SRPK1 inhibition attenuated SRSF1 activity, abolished neuropathic pain behaviors and suppressed central sensitization. SRSF1 was principally expressed in large diameter myelinated (NF200-rich) dorsal root ganglia sensory neurons and their excitatory central terminals (vGLUT1 + ve) within the dorsal horn of the lumbar spinal cord. Expression of pro-nociceptive VEGF-Axxxa within the spinal cord was increased after nerve injury, and this was prevented by SRPK1 inhibition. Additionally, expression of anti-nociceptive VEGF-Axxxb isoforms was elevated, and this was associated with reduced neuropathic pain behaviors. Inhibition of VEGF receptor-2 signaling in the spinal cord attenuated behavioral nociceptive responses to mechanical, heat and formalin stimuli, indicating that spinal VEGF receptor-2 activation has potent pro-nociceptive actions. Furthermore, intrathecal VEGF-A165a resulted in mechanical and heat hyperalgesia, whereas the sister inhibitory isoform VEGF-A165b resulted in anti-nociception. These results support a role for myelinated fiber pathways, and alternative pre-mRNA splicing of factors such as VEGF-A in the spinal processing of neuropathic pain. They also indicate that targeting pre-mRNA splicing at the spinal level could lead to a novel target for analgesic development

Embryonic Stem Cell-Derived L1 Overexpressing Neural Aggregates Enhance Recovery after Spinal Cord Injury in Mice

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system

PDE 7 Inhibitors: New Potential Drugs for the Therapy of Spinal Cord Injury

BACKGROUND: Primary traumatic mechanical injury to the spinal cord (SCI) causes the death of a number of neurons that to date can neither be recovered nor regenerated. During the last years our group has been involved in the design, synthesis and evaluation of PDE7 inhibitors as new innovative drugs for several neurological disorders. Our working hypothesis is based on two different facts. Firstly, neuroinflammation is modulated by cAMP levels, thus the key role for phosphodiesterases (PDEs), which hydrolyze cAMP, is undoubtedly demonstrated. On the other hand, PDE7 is expressed simultaneously on leukocytes and on the brain, highlighting the potential crucial role of PDE7 as drug target for neuroinflammation. METHODOLOGY/PRINCIPAL FINDINGS: Here we present two chemically diverse families of PDE7 inhibitors, designed using computational techniques such as virtual screening and neuronal networks. We report their biological profile and their efficacy in an experimental SCI model induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. We have selected two candidates, namely S14 and VP1.15, as PDE7 inhibitors. These compounds increase cAMP production both in macrophage and neuronal cell lines. Regarding drug-like properties, compounds were able to cross the blood brain barrier using parallel artificial membranes (PAMPA) methodology. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and production of a range of inflammatory mediators, tissue damage, and apoptosis. Treatment of the mice with S14 and VP1.15, two PDE7 inhibitors, significantly reduced the degree of spinal cord inflammation, tissue injury (histological score), and TNF-α, IL-6, COX-2 and iNOS expression. CONCLUSIONS/SIGNIFICANCE: All these data together led us to propose PDE7 inhibitors, and specifically S14 and VP1.15, as potential drug candidates to be further studied for the treatment of SCI

Nanoscale Metallic Iron for Environmental Remediation: Prospects and Limitations

The amendment of the subsurface with nanoscale metallic iron particles (nano-Fe0) has been discussed in the literature as an efficient in situ technology for groundwater remediation. However, the introduction of this technology was controversial and its efficiency has never been univocally established. This unsatisfying situation has motivated this communication whose objective was a comprehensive discussion of the intrinsic reactivity of nano-Fe0 based on the contemporary knowledge on the mechanism of contaminant removal by Fe0 and a mathematical model. It is showed that due to limitations of the mass transfer of nano-Fe0 to contaminants, available concepts cannot explain the success of nano-Fe0 injection for in situ groundwater remediation. It is recommended to test the possibility of introducing nano-Fe0 to initiate the formation of roll-fronts which propagation would induce the reductive transformation of both dissolved and adsorbed contaminants. Within a roll-front, FeII from nano-Fe0 is the reducing agent for contaminants. FeII is recycled by biotic or abiotic FeIII reduction. While the roll-front concept could explain the success of already implemented reaction zones, more research is needed for a science-based recommendation of nano- Fe0 for subsurface treatment by roll-front

Limits on Anomalous WWγWW\gamma Couplings from ppˉ→Wγ+Xp\bar{p} \to W \gamma + X Events at s=1.8\sqrt{s}=1.8 TeV

We have measured the $WW\gamma$ gauge boson coupling parameters using $p\bar{p}\to \ell\nu\gamma+X$ ($\ell=e,\mu$) events at $\sqrt{s}=1.8$ TeV. The data, corresponding to an integrated luminosity of 89.1 pb^{-1}, were collected using the D0 detector at the Fermilab Tevatron Collider. The measured cross section times branching ratio for $p\bar{p} \to W\gamma+X$ with $p_T^\gamma$ > 10 GeV/c and $R_{\ell\gamma} > 0.7$ is ${11.8}^{+1.7}_{-1.6} \pm 2.0$ pb, in agreement with the Standard Model prediction. The one degree of freedom 95% confidence level limits on individual CP-conserving parameters are $-0.98<\Delta\kappa<1.01$ and $-0.33<\lambda<0.31$. Similar limits are set on the CP}violating coupling parameters.Comment: 10 pages, including two figures. Paper submitted to Phys. Rev. Let