Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.     

Band Structure and Local Dynamics of Excitons in Bacterial Light-harvesting Complexes Revealed by Spectrally Selective Spectroscopy

Hole-burned absorption and line-narrowed fluorescence spectra are studied at 5 K in wild type and mutant LH1 and LH2 antenna preparations from the photosynthetic purple bacterium Rhodobacter sphaeroides. Evidence was found in all samples, even in intact membranes, of the presence of a broad distribution of bacteriochlorophyll species that are unable to communicate energy between each other and to the exciton states of functional antenna complexes. The distribution maximum of these localized species determined by zero phonon hole action spectroscopy is at 783.5 nm in purified LH1 complexes and at 786.8 nm in B850-only mutant LH2 complexes. A well-resolved peak at 807 nm in LH1 complexes is assigned to the exciton band structure of functional core antenna complexes. Similar structure in LH2 complexes overlaps with the distribution of localized species. Off-diagonal (structural) disorder may be responsible for this exciton band structure. Our data also imply that pair-wise inter-chlorophyll couplings determine the resonance fluorescence lineshape of excitonic polarons.     

Cell transduction pathways of transportans

Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 mum diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportan-streptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportan-protein complexes is temperature dependent and strongly inhibited at 8-10 degrees C and blocked at 4 degrees C when only interaction with the plasma membrane takes place.     

Studying the uptake of cell-penetrating peptides

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.     

Shifts in water quality in a drinking water reservoir during and after the removal of cyprinids

Lake ülemiste, the drinking water reservoir of Estonia’s capital city Tallinn, was biomanipulated by manual removal of cyprinids in 2004–2006 and its impact on water quality in the vegetation period was studied. A total biomass of 156 tonnes corresponding to 160 kg ha⁻¹ of fish, predominantly cyprinids, were removed. A decline in the unit catches of fishing was observed. The removed fish biomass versus phosphorus concentration of the lake was considered sufficient to reduce the impact of cyprinids on water quality. The phosphorus removed within fish biomass corresponded to 38 μg l⁻¹ and 21% of the external phosphorus load of the fishing period. The mean total phosphorus concentration dropped from >50 to ≤36 μg l⁻¹. However, the densities of planktivorous young-of-the-year percids remained high and the role of zooplankton grazing in improving water quality was found non-significant or transient. The cladocerans biomass decreased and the small-sized Daphnia cucullata remained almost the only daphnid in Lake ülemiste during and after the manipulation. Predomination of filamentous cyanobacteria was replaced by a more diverse phytoplankton composition and co-domination of micro- and pico-sized colonial cyanobacteria during summer. Mean phytoplankton biomass decreased from 15 to 6 mg l⁻¹ primarily as a result of decreased in-lake TP availability. The Secchi disc transparency increased only in May 2005–2007. The effects of coincidental events, a decline of external loading of phosphorus and a simultaneous flushing induced by heavy rainfall, on lake water quality are discussed with some implications to the future management of the reservoir.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

The non-synonymous SNP, R1150W, in SCN9A is not associated with chronic widespread pain susceptibility

Background

Chronic pain conditions are characterized by significant individual variability complicating the identification of pathophysiological markers. Leukocyte telomere length (TL), a measure of cellular aging, is associated with age-related disease onset, psychosocial stress, and health-related functional decline. Psychosocial stress has been associated with the onset of chronic pain and chronic pain is experienced as a physical and psychosocial stressor. However, the utility of TL as a biological marker reflecting the burden of chronic pain and psychosocial stress has not yet been explored.

Findings

The relationship between chronic pain, stress, and TL was analyzed in 36 ethnically diverse, older adults, half of whom reported no chronic pain and the other half had chronic knee osteoarthritis (OA) pain. Subjects completed a physical exam, radiographs, health history, and psychosocial questionnaires. Blood samples were collected and TL was measured by quantitative polymerase chain reaction (qPCR). Four groups were identified characterized by pain status and the Perceived Stress Scale scores: 1) no pain/low stress, 2) no pain/high stress, chronic pain/low stress, and 4) chronic pain/high stress. TL differed between the pain/stress groups (p = 0.01), controlling for relevant covariates. Specifically, the chronic pain/high stress group had significantly shorter TL compared to the no pain/low stress group. Age was negatively correlated with TL, particularly in the chronic pain/high stress group (p = 0.03).

Conclusions

Although preliminary in nature and based on a modest sample size, these findings indicate that cellular aging may be more pronounced in older adults experiencing high levels of perceived stress and chronic pain.     

Changes in regional long-term oxidative metabolism induced by partial serotonergic denervation and chronic variable stress in rat brain

Stressful experiences and genetic predisposition have both independent and interactive contributions to the development of depression. The serotonergic system is involved in the development of depression, and administration of neurotoxins that specifically compromise its function leads to symptoms of affective disorders. In order to find out which brain regions are most affected by stress, partial serotonergic denervation and their combination, chronic variable stress (CVS) was applied for 3 week. Serotonergic denervation was elicited by parachloroampetamine (PCA, 2mg/kg), and cytochrome oxidase histochemistry was used to characterize the long-term levels of neuronal oxidative energy metabolism. PCA pretreatment blocked the increase in oxidative activity in chronically stressed rats in medial preoptic area, cortical and medial amygdala. PCA raised oxidative activity compared to control animals in substantia nigra and ventrolateral division of laterodorsal thalamus. CVS reduced the oxidative activity induced by PCA in suprachiasmatic hypothalamus, anteroventral thalamus, hippocampal CA3 region and cortical amygdala. In the dorsal part of the anterior olfactory nucleus chronic stress blocked the decrease in oxidative activity evoked by PCA. Conclusively, partial serotonergic denervation with PCA and chronic variable stress both had independent effects on long-term energy metabolism in several rat brain structures, tending to increase it. However, partial serotonergic denervation by parachloroampetamine and chronic variable stress had in many brain regions an interactive effect on energy metabolism, each factor reducing the effect of the other, which could reflect the weakening of adaptive mechanisms.     

Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes

Expression and function of creatine kinase (CK), adenylate kinase (AK) and hexokinase (HK) isoforms in relation to their roles in regulation of oxidative phosphorylation (OXPHOS) and intracellular energy transfer were assessed in beating (B) and non-beating (NB) cardiac HL-l cell lines and adult rat cardiomyocytes or myocardium. In both types of HL-1 cells, the AK2, CKB, HK1 and HK2 genes were expressed at higher levels than the CKM, CKMT2 and AK1 genes. Contrary to the saponin-permeabilized cardiomyocytes the OXPHOS was coupled to mitochondrial AK and HK but not to mitochondrial CK, and neither direct transfer of adenine nucleotides between CaMgATPases and mitochondria nor functional coupling between CK-MM and CaMgATPases was observed in permeabilized HL-1 cells. The HL-1 cells also exhibited deficient complex I of the respiratory chain. In conclusion, contrary to cardiomyocytes where mitochondria and CaMgATPases are organized into tight complexes which ensure effective energy transfer and feedback signaling between these structures via specialized pathways mediated by CK and AK isoforms and direct adenine nucleotide channeling, these complexes do not exist in HL-1 cells due to less organized energy metabolism.