siRNA carrying an (E)-vinylphosphonate moiety at the 5' end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2

Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5'- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5'- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5'-(E)-vinylphosphonate (5'-E-VP) guide RNA at 2.5-A resolution. The structure demonstrates how the 5' binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5'-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5'-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA

Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.National Institutes of Health (U.S.) (grant R01 AI087879

Stochastic motion of test particle implies that G varies with time

The aim of this letter is to propose a new description to the time varying gravitational constant problem, which naturally implements the Dirac's large numbers hypothesis in a new proposed holographic scenario for the origin of gravity as an entropic force. We survey the effect of the Stochastic motion of the test particle in Verlinde's scenario for gravity\cite{Verlinde}. Firstly we show that we must get the equipartition values for $t\rightarrow\infty$ which leads to the usual Newtonian gravitational constant. Secondly,the stochastic (Brownian) essence of the motion of the test particle, modifies the Newton's 2'nd law. The direct result is that the Newtonian constant has been time dependence in resemblance as \cite{Running}.Comment: Accepted in International Journal of Theoretical Physic

The potential of marginal coastal nursery habitats for the conservation of a culturally important Caribbean marine species

Aim: Identifying the potential of marginal habitats for species conservation is of key importance when their core high-quality habitats are under substantial disturbances and threats. However, there is currently a knowledge gap on how useful marine marginal habitats may be for conserving endangered marine species. Here, we investigate the potential of groundwater-fed coastal areas for the conservation of the queen conch, an economically and culturally important marine gastropod. Location: The inlet of Xel-Ha, typical of groundwater-fed coastal areas widely distributed along the Yucatan Peninsula coast in Mexico and partially protected by a network of marine protected areas. Methods: We tracked 66 queen conchs (Lobatus gigas) using acoustic telemetry over a period of 3.5 years. We investigated for ontogenetic niche shift using a network analysis and by modelling their growth. Results: The queen conchs exhibited the same ontogenetic niche shift required to complete their life cycle in this marginal habitat as they do in offshore core habitats. A total of 33 individuals departed the inlet and migrated from shallow groundwater-affected nursery grounds to deeper marine habitats more suitable for breeding aggregation. Main conclusions: As the broad-scale movement behaviour of queen conch in this inlet is similar to that observed on the overfished core habitats, our findings suggest that groundwater-fed coastal areas should be included in conservation planning for an effective management of this species within a network of marine protected areas

Sodium channel slow inactivation interferes with open channel block

Mutations in the voltage-gated sodium channel Nav1.7 are linked to inherited pain syndromes such as erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). PEPD mutations impair Nav1.7 fast inactivation and increase persistent currents. PEPD mutations also increase resurgent currents, which involve the voltage-dependent release of an open channel blocker. In contrast, IEM mutations, whenever tested, leave resurgent currents unchanged. Accordingly, the IEM deletion mutation L955 (ΔL955) fails to produce resurgent currents despite enhanced persistent currents, which have hitherto been considered a prerequisite for resurgent currents. Additionally, ΔL955 exhibits a prominent enhancement of slow inactivation (SI). We introduced mutations into Nav1.7 and Nav1.6 that either enhance or impair SI in order to investigate their effects on resurgent currents. Our results show that enhanced SI is accompanied by impaired resurgent currents, which suggests that SI may interfere with open-channel block

Jejunum free flap in hypopharynx reconstruction: Case series

BACKGROUND: Surgical treatment of hypopharyngeal cancers with extension to the retrocricoid region generally requires a circumferential pharyngolaryngectomy followed by a reconstruction of the removed segment of the upper digestive tract. Historically, many techniques have been used in order to achieve a safe and functional reconstruction. Jejunum interposition is generally considered the best reconstructive technique. METHODS: This study examines the details of the surgical technique, the complications, the oncological and the functional results in a series of 29 consecutive patients submitted to circumferential pharyngoesophageal resection for advanced hypopharyngeal cancer followed by reconstruction with a free flap of jejunum. RESULTS: Three of the transplants failed because of venous thrombosis. The overall success rate was 90%. There were no general complications. A good swallowing has been preserved in all our patients. All our patients where a phonatory prosthesis was positioned (20/29) were able to achieve speech following speech therapy and all were satisfied with their own capacity to communicate. CONCLUSIONS: The prognosis of hypopharyngeal tumours (18–40% at 5 years) remains poor, but jejunum autografts are being shown to be an excellent choice for the reconstruction of the cervical hypopharyngo-oesophagus offering the patient fast rehabilitation and a reasonable quality of survival. Our experience confirm that this kind of reconstruction is safe with a good results in improving oncologic controls and restoring a good quality of life

Imidazol-1-ylethylindazole Voltage-Gated Sodium Channel Ligands Are Neuroprotective during Optic Neuritis in a Mouse Model of Multiple Sclerosis

[Image: see text] A series of imidazol-1-ylethylindazole sodium channel ligands were developed and optimized for sodium channel inhibition and in vitro neuroprotective activity. The molecules exhibited displacement of a radiolabeled sodium channel ligand and selectivity for blockade of the inactivated state of cloned neuronal Na(v) channels. Metabolically stable analogue 6 was able to protect retinal ganglion cells during optic neuritis in a mouse model of multiple sclerosis

Downregulation of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) in human hepatocellular carcinoma and their prognostic significance

<p>Abstract</p> <p>Background</p> <p>Organic cation transporters (OCT) are responsible for the uptake and intracellular inactivation of a broad spectrum of endogenous substrates and detoxification of xenobiotics and chemotherapeutics. The transporters became pharmaceutically interesting, because OCTs are determinants of the cytotoxicity of platin derivates and the transport activity has been shown to correlate with the sensitivity of tumors towards tyrosine kinase inhibitors. No data exist about the relevance of OCTs in hepatocellular carcinoma (HCC).</p> <p>Methods</p> <p>OCT1 (<it>SLC22A1</it>) and OCT3 (<it>SLC22A3</it>) mRNA expression was measured in primary human HCC and corresponding non neoplastic tumor surrounding tissue (TST) by real time PCR (n = 53). Protein expression was determined by western blot analysis and immunofluorescence. Data were correlated with the clinicopathological parameters of HCCs.</p> <p>Results</p> <p>Real time PCR showed a downregulation of <it>SLC22A1 </it>and <it>SLC22A3 </it>in HCC compared to TST (p ≤ 0.001). A low <it>SLC22A1 </it>expression was associated with a worse patient survival (p < 0.05). Downregulation was significantly associated with advanced HCC stages, indicated by a higher number of T3 tumors (p = 0.025) with a larger tumor diameter (p = 0.035), a worse differentiation (p = 0.001) and higher AFP-levels (p = 0.019). In accordance, <it>SLC22A1 </it>was less frequently downregulated in tumors with lower stages who underwent transarterial chemoembolization (p < 0.001) and liver transplantation (p = 0.001). Tumors with a low <it>SLC22A1 </it>expression (< median) showed a higher <it>SLC22A3 </it>expression compared to HCC with high <it>SLC22A1 </it>expression (p < 0.001). However, there was no significant difference in tumor characteristics according to the level of the <it>SLC22A3 </it>expression.</p> <p>In the western blot analysis we found a different protein expression pattern in tumor samples with a more diffuse staining in the immunofluorescence suggesting that especially OCT1 is not functional in advanced HCC.</p> <p>Conclusion</p> <p>The downregulation of OCT1 is associated with tumor progression and a worse patient survival.</p

Oncological outcome after free jejunal flap reconstruction for carcinoma of the hypopharynx

It has been a common practice among the oncologist to reduce the dosage of adjuvant radiotherapy for patients after free jejunal flap reconstruction. The current aims to study potential risk of radiation to the visceral flap and the subsequent oncological outcome. Between 1996 and 2010, consecutive patients with carcinoma of the hypopharynx requiring laryngectomy, circumferential pharyngectomy and post-operative irradiation were recruited. Ninety-six patients were recruited. TNM tumor staging at presentation was: stage II (40.6%), stage III (34.4%) and stage IV (25.0%). Median follow-up period after surgery was 68 months. After tumor ablation, reconstruction was performed using free jejunal flap (60.4%), pectoralis major myocutaneous (PM) flap (31.3%) and free anterolateral thigh (ALT) flap (8.3%). All patients underwent adjuvant radiotherapy within 6.4 weeks after surgery. The mean total dose of radiation given to those receiving cutaneous and jejunal flap reconstruction was 62.2 Gy and 54.8 Gy, respectively. There was no secondary ischaemia or necrosis of the flaps after radiotherapy. The 5-year actuarial loco-regional tumor control for the cutaneous flap and jejunal flap group was: stage II (61 vs. 69%, p = 0.9), stage III (36 vs. 46%, p = 0.2) and stage IV (32 vs. 14%, p = 0.04), respectively. Reduction of radiation dosage in free jejunal group adversely affects the oncological control in stage IV hypopharyngeal carcinoma. In such circumstances, tubed cutaneous flaps are the preferred reconstructive option, so that full-dose radiotherapy can be given