Aerosol Exposure to Rift Valley Fever Virus Causes Earlier and More Severe Neuropathology in the Murine Model,which Has Important Implications for Therapeutic Development

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that can cause severe disease including acute-onset hepatitis, delayed-onset encephalitis, retinitis and blindness, or a hemorrhagic syndrome. Currently, no licensed vaccine or therapeutics exist to treat this potentially deadly disease. Detailed studies describing the pathogenesis of RVFV following aerosol exposure have not been completed and candidate therapeutics have not been evaluated following an aerosol exposure. These studies are important because while mosquito transmission is the primary means for human infection, it can also be transmitted by aerosol or through mucosal contact. Therefore, we directly compared the pathogenesis of RVFV following aerosol exposure to a subcutaneous (SC) exposure in the murine model by analyzing survival, clinical observations, blood chemistry, hematology, immunohistochemistry, and virus titration of tissues. Additionally, we evaluated the effectiveness of the nucleoside analog ribavirin administered prophylactically to treat mice exposed by aerosol and SC. The route of exposure did not significantly affect the survival, chemistry or hematology results of the mice. Acute hepatitis occurred despite the route of exposure. However, the development of neuropathology occurred much earlier and was more severe in mice exposed by aerosol compared to SC exposed mice. Mice treated with ribavirin and exposed SC were partially protected, whereas treated mice exposed by aerosol were not protected. Early and aggressive viral invasion of brain tissues following aerosol exposure likely played an important role in ribavirin''s failure to prevent mortality among these animals. Our results highlight the need for more candidate antivirals to treat RVFV infection, especially in the case of a potential aerosol exposure. Additionally, our study provides an account of the key pathogenetic differences in RVF disease following two potential exposure routes and provides important insights into the development and evaluation of potential vaccines and therapeutics to treat RVFV infection.     

KCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma

Background

The Ca²⁺-activated K⁺ channel K_Ca3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K_Ca3.1 modifies experimental asthma in sheep.

Methodology and Principal Findings

Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K_Ca3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.

Conclusions

These findings suggest that K_Ca3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K_Ca3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.     

Pharmacological Inhibition of polysialyltransferase ST8SiaII Modulates Tumour Cell Migration

Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated post-translational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (K _i = 10 µM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers.     

The K+ Channel KCa3.1 as a Novel Target for Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that K_Ca3.1 K⁺ channel-dependent cell processes contribute to IPF pathophysiology.

Methods

K_Ca3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of K_Ca3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct K_Ca3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).

Results

Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed K_Ca3.1 channel mRNA and protein. K_Ca3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. K_Ca3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. K_Ca3.1 was expressed strongly in IPF tissue. K_Ca3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca²⁺.

Conclusions

K_Ca3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking K_Ca3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic.     

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

Copper residues in meat from wild artiodactyls hunted with two types of rifle bullets manufactured from copper

Copper content in muscle and the presence of bullet fragments were assessed in samples from red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama) and wild boar (Sus scrofa) (total of 46 animals) which had been hunted with two types of solid copper bullets. Also, the release of copper from bullets or fragments remaining in muscle was tested. For bullet type “B”, a fragment was detected in only 1 out of 34 carcasses whereas for type “A”, fragments were detected in all 12 carcasses, with up to 24 fragments (maximum size, 5?×?7?×?2 mm). Median copper concentrations around the shot wound (0–30 mm distance) were 1.25 and 1.77 mg/kg fresh weight for bullet types B and A, respectively, and thus in the expected range for venison. Around bullet fragments that had remained in muscles, the copper content increased significantly. In roe deer longissimus muscle that had been exposed for 7 days at +7 °C to bullets of type B, up to 1,000 mg/kg copper (fresh weight) was found in a distance of 0–2 mm. However, in a distance of 10–20 mm, maximum copper contents were <10 mg/kg fresh weight. Bullet fragments can constitute physical hazards and will release copper under acidic conditions as those prevailing in meat. Removal of bullet fragments prior to culinary preparation should ensure that a recommended dietary copper intake of 1.25 mg per adult consumer per day is not exceeded. From a food hygiene viewpoint, non-fragmenting bullets seem to be preferable.     

New zeolite adsorbents for downstream processing of polyphenols from renewable resources

Commercial materials with polyvinylpolypyrrolidone and polymeric amberlites (XAD7HP, XAD16) are commonly used for the adsorptive downstream processing of polyphenols from renewable resources. In this study, beta‐zeolite‐based adsorbent systems were examined, and their properties were compared to organic resins. Batch adsorption experiments were conducted with synthetic solutions of major polyphenols. Adsorption isotherms and desorption characteristics of individual adsorbent were determined based on these results. Maximum adsorption capacities were calculated using the Langmuir model. For example, the zeolites had capacities up to 203.2 mg/g for ferulic acid. To extend these results to a complex system, additional experiments were performed on rapeseed meal and wheat seed extracts as representative renewable resources. HPLC analysis showed that with 7.5% w/v, which is regarded as the optimum amount of zeolites, zeolites A and B could bind 100% of the major polyphenols as well as release polyphenols at high yields. Additionally, regeneration experiments were performed with isopropyl alcohol at 99°C to evaluate how zeolites regenerate under mild conditions. The results showed only a negligible loss of adsorption capacity and no loss of desorption capacity. In summary, it was concluded that beta‐zeolites were promising adsorbents for developing new processes to isolate polyphenols from renewable resources.     

A New Class of Quorum Quenching Molecules from Staphylococcus Species Affects Communication and Growth of Gram-Negative Bacteria

The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community.     

The Linker Pivot in Ci-VSP: The Key to Unlock Catalysis

In the voltage-sensitive phosphatase Ci-VSP, conformational changes in the transmembrane voltage sensor domain (VSD) are transduced to the intracellular catalytic domain (CD) leading to its dephosphorylation activity against membrane-embedded phosphoinositides. The linker between both domains is proposed to be crucial for the VSD-CD coupling. With a combined approach of electrophysiological measurements on Xenopus oocytes and molecular dynamics simulations of a Ci-VSP model embedded in a lipid bilayer, we analyzed how conformational changes in the linker mediate the interaction between the CD and the activated VSD. In this way, we identified specific residues in the linker that interact with well-defined amino acids in one of the three loops forming the active site of the protein, named TI loop. With our results, we shed light into the early steps of the coupling process between the VSD and the CD, which are based on fine-tuned electrostatic and hydrophobic interactions between the linker, the membrane and the CD.     

Chorioamnionitis and Subsequent Lung Function in Preterm Infants

Objective

To explore the relationship between prematurity, gender and chorioamnionitis as determinants of early life lung function in premature infants.

Methods

Placenta and membranes were collected from preterm deliveries (<37 weeks gestational age) and evaluated for histological chorioamnionitis (HCA). Patients were followed and lung function was performed in the first year of life by Raised Volume-Rapid Thoracic Compression Technique.

Results

Ninety-five infants (43 males) born prematurely (median gestational age 34.2 weeks) were recruited. HCA was detected in 66 (69%) of the placentas, and of these 55(58%) were scored HCA Grade 1, and 11(12%) HCA Grade 2. Infants exposed to HCA Grade 1 and Grade 2, when compared to those not exposed, presented significantly lower gestational ages, higher prevalence of RDS, clinical early-onset sepsis, and the use of supplemental oxygen more than 28 days. Infants exposed to HCA also had significantly lower maximal flows. There was a significant negative trend for z-scores of lung function in relation to levels of HCA; infants had lower maximal expiratory flows with increasing level of HCA. (p = 0.012 for FEF₅₀, p = 0.014 for FEF_25–75 and p = 0.32 for FEV_0.5). Two-way ANOVA adjusted for length and gestational age indicated a significant interaction between sex and HCA in determining expiratory flows (p<0.01 for FEF₅₀, FEF_25–75 and p<0.05 for FEV_0.5). Post-hoc comparisons revealed that female preterm infants exposed to HCA Grade 1 and Grade 2 had significant lower lung function than those not exposed, and this effect was not observed among males.

Conclusions

Our findings show a sex-specific negative effect of prenatal inflammation on lung function of female preterm infants. This study confirms and expands knowledge upon the known association between chorioamnionitis and early life chronic lung disease.