Erratum: Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains.

[This corrects the article DOI: 10.1093/ve/vey027.][This corrects the article DOI: 10.1093/ve/vey027.].

Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains.

The respiratory syncytial virus (RSV) group A variant with the 72-nucleotide duplication in the G gene, genotype ON1, was first detected in Kilifi in 2012 and has almost completely replaced circulating genotype GA2 strains. This replacement suggests some fitness advantage of ON1 over the GA2 viruses in Kilifi, and might be accompanied by important genomic substitutions in ON1 viruses. Close observation of such a new virus genotype introduction over time provides an opportunity to better understand the transmission and evolutionary dynamics of the pathogen. We have generated and analysed 184 RSV-A whole-genome sequences (WGSs) from Kilifi (Kenya) collected between 2011 and 2016, the first ON1 genomes from Africa and the largest collection globally from a single location. Phylogenetic analysis indicates that RSV-A circulation in this coastal Kenya location is characterized by multiple introductions of viral lineages from diverse origins but with varied success in local transmission. We identified signature amino acid substitutions between ON1 and GA2 viruses' surface proteins (G and F), polymerase (L), and matrix M2-1 proteins, some of which were positively selected, and thereby provide an enhanced picture of RSV-A diversity. Furthermore, five of the eleven RSV open reading frames (ORFs) (G, F, L, N, and P) formed distinct phylogenetic clusters for the two genotypes. This might suggest that coding regions outside of the most frequently studied G ORF also play a role in the adaptation of RSV to host populations, with the alternative possibility that some of the substitutions are neutral and provide no selective advantage. Our analysis provides insight into the epidemiological processes that define RSV spread, highlights the genetic substitutions that characterize emerging strains, and demonstrates the utility of large-scale WGS in molecular epidemiological studies.

Tranexamic acid decreases risk of haematomas but not pain after hip arthroplasty.

BACKGROUND: Tranexamic acid decreases total blood loss after total hip arthroplasty (THA). Total blood loss is the sum of external bleeding and bleeding into tissues, i.e., haematomas. Haematomas may cause acute or even chronic postoperative pain. HYPOTHESIS: Tranexamic acid decreases haematomas, thereby diminishing postoperative pain after THA. METHODS: In a retrospective matched case-control study, patients receiving tranexamic acid (15 mg/kg, before the incision and again at skin closure) were compared to controls not given tranexamic acid. Matching was on sex, surgeon, and peri-operative analgesics (ketamine, ketoprofen, pregabalin, and nefopam). Standardised protocols were used for anaesthesia, analgesia, and blood sparing. Haematoma volume was computed as the difference between total blood loss (estimated from the erythrocyte counts on days -1 and +5) and measured external blood loss. Patients were monitored from D0 to D7 then interviewed by telephone on D30, D90, and D180. To detect a 30%-decrease in the morphine dose at H24 (criterion 1) and D7 (criterion 2) and a 20% decrease in haematoma volume on D5 (criterion 3), the required numbers of patients were 90, 90, and 77, respectively; therefore, 95 patients were included. RESULTS: Tranexamic acid decreased haematoma volume by 30% (351±254 mL versus 247±189 mL erythrocytes, P=0.002), had no effect on morphine consumption at H24 (12±11 mg versus 14±12 mg, P=0.346), increased morphine consumption on D7 (26±24 mg versus 35±36 mg, P=0.029), and had no effect on long-term pain. DISCUSSION: After THA, tranexamic acid decreases haematoma volume without improving analgesia. LEVEL OF EVIDENCE: 3 (case-control study).

Rescuing the neonatal brain from hypoxic injury with autologous cord blood.

Brain injury resulting from perinatal hypoxic-ischemic encephalopathy (HIE) is a major cause of acute mortality in infants and chronic neurologic disability in surviving children. Recent multicenter clinical trials demonstrated the effectiveness of hypothermia initiated within the first 6 postnatal hours to reduce the risk of death or major neurological disabilities among neonates with HIE. However, in these trials, approximately 40% of cooled infants died or survived with significant impairments. Therefore, adjunct therapies are required to improve the outcome in neonates with HIE. Cord blood (CB) is a rich source of stem cells. Administration of human CB cells in animal models of HIE has generally resulted in improved outcomes and multiple mechanisms have been suggested including anti-inflammation, release of neurotrophic factors and stimulation of endogenous neurogenesis. Investigators at Duke are conducting studies of autologous CB infusion in neonates with HIE and in children with cerebral palsy. These pilot studies indicate no added risk from the regimens used, but results of ongoing placebo-controlled trials are needed to assess efficacy. Meanwhile, further investigations are warranted to determine the best strategies, that is, timing, dosing, route of delivery, choice of stem cells and ex vivo modulations, to attain long-term benefits of CB stem cell therapy.

Application of a novel, rapid, and sensitive oligonucleotide ligation assay for detection of cancer-predicting mutations in the precore and basal core promoter of hepatitis B virus.

Hepatocellular carcinoma (HCC) and cirrhosis are important causes of mortality worldwide. Persistent hepatitis B virus (HBV) infection is a major cause of these diseases. Double mutations in the basal core promoter (BCP) (A1762T and G1764A) and precore (pre-C) (G1896A) regions of the virus are associated with progression to HCC. The current study is aimed at developing a simple method for screening and detecting BCP and pre-C mutations in HBV carriers. We have developed and validated an oligonucleotide ligation assay (OLA) to detect point mutations in the HBV core gene. We have applied OLA methods to samples from HBV-infected carriers recruited from the Gambia Liver Cancer Study (GLCS) comprising asymptomatic HBsAg carriers, patients with cirrhosis, and patients with HCC. We observed an 89.3% and 95.8% concordance between the OLA and DNA sequencing for BCP and pre-C mutations, respectively. OLA detected the mutations in single-strain infections and in infections with mixtures of wild-type and mutant viruses under conditions where sequencing detected only the single dominant strains. BCP mutations were detected in 75.7% of patients with advanced liver disease (cirrhosis/HCC) compared to 47.6% of asymptomatic carriers, while pre-C mutations were detected in 34.5% of advanced liver disease patients and in 47.6% of asymptomatic HBsAg carriers. There was a significant association between the presence of BCP mutations and advanced liver disease. In conclusion, OLA is a simple, economical, and reliable assay for detection of pre-C and BCP mutations. Its application can lead to improvement in diagnosis and clinical care in regions where HBV is endemic.

Noncontact dipole effects on channel permeation. IV. Kinetic model of 5F-Trp(13) gramicidin A currents.

Nonlinear least squares fitting was used to assign rate constants for the three-barrier, two-site, double-occupancy, single-filing kinetic model for previously reported current-voltage relations of (5F-Indole)Trp(13) gramicidin A and gramicidin A channels (, 75:2830-2844). By judicious coupling of parameters, it was possible to reduce the parameter space from 64 parameters to 24, and a reasonable fit consistent with other experimental data was obtained. The main features of the fit were that fluorination increased the rate constant for translocation by a factor of 2.33, consistent with a free energy change in the translocation barrier of -0.50 kcal/mol, and increased first-ion binding affinity by a factor of 1.13, primarily by decreasing the first-ion exit rate constant. The translocation rate constant was 5.62 times slower in diphytanoyl phosphatidylcholine (DPhPC) bilayers than in monoolein (GMO) bilayers (coupled for the four combinations of peptide and salt), suggesting a 44.2-mV difference in the projection of the interfacial dipole into the channel. Thus fluorination caused increased currents in DPhPC bilayers, where a high interfacial dipole potential makes translocation more rate limiting because the translocation barrier was reduced, and decreased currents in GMO bilayers, where ion exit or entry is rate limiting because these barriers were increased.

MHC class II presentation of endogenously expressed antigens by transfected dendritic cells.

Dendritic cells (DC) present immunogenic epitopes of antigens in the context of MHC class I and class II molecules in association with costimulatory molecules, and efficiently activate both cytotoxic T cells and T helper cells. Gene modified DC expressing antigen encoding cDNA represent a particularly attractive approach for the immunotherapy of disease. We previously described a gene delivery system for DC based on receptor-mediated endocytosis of ligand/polyethylenimine (PEI) DNA transfer complexes that target cell surface receptors which are abundantly expressed on DC. Employing this gene delivery system, DC were generated that express chicken ovalbumin (OVA) cDNA as a model antigen and introduce antigen into the MHC class I presentation pathway. We demonstrate here that modification of OVA cDNA as transferrin receptor (TfR) or invariant chain (Ii) fusions effectively generate MHC class II specific immune responses in addition to MHC class I responses. TfR-OVA contains the membrane anchoring region of transferrin receptor and represents a membrane-bound form of OVA for access to the MHC class II compartment. Ii-OVA fusions directly target the MHC class II processing pathway. Thus, modification of antigen encoding cDNA represents a convenient and effective means to direct antigens to MHC class II presentation and thus to generate T cell help.

Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells.

Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

Microtubule-independent motility and nuclear targeting of adenoviruses with fluorescently labeled genomes.

A novel adenovirus system for analyzing the adenovirus entry pathway has been developed that contains green fluorescent protein bound to the encapsidated viral DNA (AdLite viruses). AdLite viruses enter host cells and accumulate around the nuclei and near the microtubule organizing centers (MTOC). In live cells, individual AdLite particles were observed trafficking both toward and away from the nucleus. Depolymerization of microtubules during infection prevented AdLite accumulation around the MTOC; however, it did not abolish perinuclear localization of AdLite particles. Furthermore, depolymerization of microtubules did not affect AdLite motility and did not affect gene expression from wild-type adenovirus and adenovirus-derived vectors. These data revealed that adenovirus intracellular motility and nuclear targeting can be supported by a mechanism that does not rely on the microtubule network.

The science of neonatal high-frequency ventilation.

Despite improvements in respiratory care, ventilator-induced lung injury remains an important cause of morbidity and mortality in neonates who require assisted ventilation. Animal data clearly demonstrate that high-frequency ventilation can be used successfully to reduce lung injury in experimental models of acute lung injury. These models and human research show that the efficacy of high-frequency ventilation is dependent on optimizing functional residual capacity and avoiding lung overinflation. When used with a strategy that promotes lung recruitment, high-frequency ventilation effectively reduces the occurrence of chronic lung disease and is not associated with significant brain injury. When used with a strategy that allows the lung to collapse or is associated with hyperventilation, however, high-frequency ventilation does not reduce lung injury and is associated with significant brain injury. Like every tool we use to support critically ill neonates, high-frequency ventilation needs a careful carpenter. As therapies and health care strategies evolve, there remains nothing more important than the health care team at the bedside. Critical evaluation of the patient and his or her response to the therapy being offered is essential to promotion of the patient health outcome.

Preparation of adenovirus-polylysine-DNA complexes.

This unit describes preparation of adenovirus-polylysine-DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin-polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols describes preparation of streptavidin-polylysine and transferrin-polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct polylysine-virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires polylysine-modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.

Activation of heat-shock response by an adenovirus is essential for virus replication.

Successful viral infection requires viruses to redirect host biochemistry to replicate the viral genome, and produce and assemble progeny virions. Cellular heat-shock responses, which are characterized as elevation and relocalization of heat-shock proteins, occur during replication of many viruses. Such responses might be host reactions to the synthesis of foreign protein, or might be irrelevant consequences of the viral need to activate transcription. Alternatively, as heat-shock proteins can facilitate protein folding, activating a heat-shock response might be a specific virus function ensuring proper synthesis of viral proteins and virions. It is not possible to determine whether heat-shock response is essential for virus replication, because the implicated viral genes (such as Ad5 EIA, ref. 10) also control other essential replication steps. Here we report that expression of Gam1, a protein encoded by the avian virus CELO (ref. 11), elevates and relocalizes hsp70 and hsp40. Gam1-negative CELO is replication-defective; however, Gam1 function can be partially replaced by either heat shock or forced hsp40 expression. Thus, an essential function of Gam1 during virus replication is to activate host heat-shock responses with hsp40 as a primary target.

Inter- and intramolecular distance measurements by solid-state MAS NMR: determination of gramicidin A channel dimer structure in hydrated phospholipid bilayers.

Distance constraints are an important complement to orientational constraints. While a high-resolution monomer structure of the ion channel forming polypeptide, gramicidin A, has been solved with 120 orientational constraints, the precise geometry of the dimer interface has not been characterized. Here, using both 13C and 15N labeled gramicidin A samples in hydrated phospholipid bilayers, both inter- and intramolecular distances have been measured with a recently developed simultaneous frequency and amplitude modulation (SFAM) solid-state NMR scheme. Using this approach 15N-13C1 residual dipolar couplings across a hydrogen bond as small as 20+/-2 Hz have been characterized. While such distances are on the order of 4.2+/-0.2 A, the spectroscopy is complicated by rapid global motion of the molecular structure about the bilayer normal and channel axis. Consequently, the nominal 40 Hz dipolar coupling is averaged depending on the orientation of the internuclear vector with respect to the motional axis. The intermolecular distance confirmed the previously described monomeric structure, while the intramolecular distance across the monomer-monomer interface defined this junction and confirmed the previous model of this interface.

Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus.

The aim of this study was to investigate the influence of cell cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation- or lipid-based transfection systems was 30- to more than 500-fold higher when transfection was performed during S or G2 phase compared with cells in G1 phase which have the lowest expression levels. In contrast, this effect was not observed with recombinant adenovirus which varied only four-fold. Our results indicate that mitotic activity enhances transfection not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown. Furthermore, low transfection success into G1 cells indicates that DNA complexes deposited in G1 cells are probably not retained long enough to take advantage of mitosis effects or that passage of transfected cells through S phase is inhibitory. Gene Therapy (2000) 7, 401-407.

Fibroblast growth factors are required for efficient tumor angiogenesis.

Although the function of vascular endothelial growth factor in the induction of tumor angiogenesis is well understood, the role of a second group of angiogenic factors, the fibroblast growth factors (FGFs), remains elusive. We used a recombinant adenovirus expressing soluble FGF receptor (AdsFGFR) to interfere with FGF function in tumor angiogenesis. AdsFGFR repressed endothelial cell proliferation in vitro and inhibited tumor angiogenesis in an ex vivo bioassay, in which endothelial cells were cocultured with angiogenic tumor biopsies in a collagen gel. Moreover, AdsFGFR repressed tumor angiogenesis and hence tumor growth in vivo in allograft transplantation experiments. Whereas adenoviral expression of a soluble form of VEGF receptor 1 (AdsFlt) predominantly affected the initiation of tumor angiogenesis, soluble FGF receptor (sFGFR) appeared to impair the maintenance of tumor angiogenesis. The combination of sFGFR and soluble Flt exhibited a synergistic effect in the repression of tumor growth. Finally, i.v. injection of AdsFGFR resulted in a dramatic repression of tumor growth in a transgenic mouse model of pancreatic beta cell carcinogenesis. Similar to control infections with AdsFlt, tumor-associated vessel density was decreased, indicating that the expression of sFGFR impaired tumor angiogenesis. These data indicate that FGFs play a critical role in tumor angiogenesis.

Correlations of structure, dynamics and function in the gramicidin channel by solid-state NMR spectroscopy.

The high resolution structure of the gramicidin A channel has been determined in a lamellar phase environment using solid-state NMR spectroscopy. While the fold is similar to previous characterizations, channel function is exquisitely dependent on structural detail. There is essentially no structural change upon cation binding and no significant change in dynamics. The cations appear to be adequately solvated in their binding site by no more than two carbonyls and no fewer than three water molecules at any one time. The relatively large number of water molecules allows for geometric flexibility and little selectivity among monovalent cations. However, the dehydration energies of cations clearly explain the selectivity for monovalent versus divalent cations. Moreover, the binding site is shown to be delocalized, resulting in a shallow potential energy well so that efficient cation conductance can be realized. The potential energy barrier at the bilayer centre has been shown to be rate limiting under certain circumstances through a correlation between conductance and the electrostatic interactions between cations at the gramicidin monomer-monomer junction and the indole dipole moments at the lipid-water interface. The dynamics are functionally important. The time-scale for carbonyl fluctuations about the C alpha-C alpha axis and kinetic rates for cation movement in the channel are the same, suggesting a correlation between molecular dynamics and kinetics. These functional correlations will be described in light of the recent K+ channel structure and the biological challenge to achieve both selectivity and efficiency.

Modulating dipoles for structure-function correlations in the gramicidin A channel.

Dipoles of the tryptophan indole side chains have a direct impact on ion conductance in the gramicidin channel. Here, fluorination of the indoles (both 5- and 6-fluoro) is used to manipulate both the orientations and the magnitudes of the dipoles. The orientations and positions with respect to the channel axis were determined using (2)H solid state NMR of uniformly aligned lipid bilayer preparations. By exchange of the remaining four protons in the indole ring for deuterium, comparison could be made to d(5)-indole spectra that have previously been recorded for each of the four indoles of gramicidin A. After making the assignments which were aided by the observation of (19)F-(2)H dipolar interactions, we found that fluorination caused only minor changes in side chain conformation. With the high-resolution structural characterization of the fluorinated indoles in position 11, 13, and 15, the electrostatic interactions with a cation at the channel and bilayer center can be predicted and the influence of the modified dipoles on ion conductance estimated. The importance of the long-range electrostatic interaction was recently documented with the observation of alpha-helical dipoles oriented toward the bilayer center on the ion conductance pathway for the Streptomyces K(+) channel. We present direct measurements of the orientation of gramicidin channel F-Trp positions for use in analysis of dipole effects on channel permeation.

Characterization of CELO virus proteins that modulate the pRb/E2F pathway.

The avian adenovirus CELO can, like the human adenoviruses, transform several mammalian cell types, yet it lacks sequence homology with the transforming, early regions of human adenoviruses. In an attempt to identify how CELO virus activates the E2F-dependent gene expression important for S phase in the host cell, we have identified two CELO virus open reading frames that cooperate in activating an E2F-inducible reporter system. The encoded proteins, GAM-1 and Orf22, were both found to interact with the retinoblastoma protein (pRb), with Orf22 binding to the pocket domain of pRb, similar to other DNA tumor virus proteins, and GAM-1 interacting with pRb regions outside the pocket domain. The motif in Orf22 responsible for the pRb interaction is essential for Orf22-mediated E2F activation, yet it is remarkably unlike the E1A LxCxD and may represent a novel form of pRb-binding peptide.

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells.

Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.

Efficient gene delivery into human dendritic cells by adenovirus polyethylenimine and mannose polyethylenimine transfection.

Gene-modified human dendritic cells (DCs) were generated by transfection with adenovirus polyethylenimine DNA (Ad/PEI/DNA) and mannose polyethylenimine DNA (ManPEI/DNA) complexes. Ad/PEI/DNA complexes have plasmid DNA bound to adenovirus particles by PEI and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yield high transduction levels and sustained expression of luciferase and green fluorescent protein reporter genes and were almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor-mediated endocytosis via mannose receptor, which is highly expressed on DCs. While gene delivery by ManPEI/DNA complexes was less efficient than by Ad/PEI transfection, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhanced transduction efficiencies and transgene expression. We also demonstrate that Ad/PEI-transfected DCs are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions, and in activating T cells from T cell receptor (TCR)-transgenic mice in an antigen-specific manner. Thus, the present study establishes the following relative order of transduction efficiencies of viral and nonviral gene delivery systems for primary human DCs: recombinant adenovirus > Ad/PEI = Ad/ManPEI > ManPEI > PEI. Ad/PEI and ManPEI transfection modes represent particularly versatile transduction systems for DCs, with ManPEI being built up exclusively of synthetic compounds.