Introduction of Virulence Markers in PB2 of Pandemic Swine-Origin Influenza Virus Does Not Result in Enhanced Virulence or Transmission

In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.The new H1N1 swine-origin influenza virus (S-OIV) recently emerged to cause the first influenza pandemic in 40 years (2). The S-OIV presumably emerged from pigs, as its genome was shown to consist of six gene segments of “triple-reassortant” swine viruses and two of “Eurasian lineage” swine viruses (9). The start of the S-OIV pandemic has been relatively mild, with a clinical spectrum ranging from mild upper respiratory tract illness to sporadic cases of severe pneumonia leading to acute respiratory distress syndrome (22). As of 15 November 2009, worldwide, more than 206 countries have reported laboratory-confirmed cases of S-OIV infection, including over 6,770 deaths (32).In previous influenza pandemics, such as the Spanish influenza pandemic of 1918 and the Hong Kong influenza pandemic of 1968, a first wave of cases of relatively mild illnesses was followed by more severe subsequent waves (29). The reason for this increased severity has remained largely unknown, but one possible explanation could be that the pandemic viruses required further adaptation to the human host, resulting in the emergence of viruses that were more virulent than those of the first wave. Such adaptive changes could occur by gene reassortment between cocirculating influenza A viruses or by mutation.In the past decade, determinants of influenza A virus virulence have been mapped using reverse genetics with a variety of pandemic, epidemic, and zoonotic influenza viruses. Mutations affecting virulence and host range have frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with sialic acids, the virus receptors on host cells (11, 18, 30). Nonstructural protein 1 (NS1) has been implicated in the virulence of highly pathogenic avian influenza (HPAI) virus H5N1 and the 1918 H1N1 virus, as the NS1 proteins of these viruses were shown to act as strong antagonists of the interferon pathways (10, 25). Furthermore, the polymerase genes, in particular the PB2 gene, have been shown to be important determinants of virulence in the HPAI H5N1 and H7N7 viruses and of transmission in the 1918 H1N1 virus (11, 21, 31). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is found generally in avian influenza viruses, while human viruses have a lysine (K), and this mutation has been described as a determinant of the host range in vitro (28). When avian viruses lacking the E627K substitution were passaged in mice, the viruses acquired the mutation spontaneously upon a single passage (15, 17). In the HPAI H5N1 and H7N7 viruses, E627K was shown to be the prime determinant of pathogenesis in mice (11, 21, 23). Given that all human and many zoonotic influenza viruses of the last century contained 627K (1), it was surprising that the S-OIV had 627E.Additionally, the aspartate (D)-to-asparagine (N) mutation at position 701 of PB2, which was shown to compensate for the absence of E627K, has also not been detected in S-OIV (27). This D701N mutation has previously been shown to expand the host range of avian H5N1 to mice and humans (3, 15) and to increase virus transmission in guinea pigs (27). Thus, S-OIV was the first known human pandemic virus with 627E and 701D, and it has been speculated that S-OIV could mutate into a more virulent form by acquiring one of these mutations, or both.On 8 May 2009, the detection of another mutation in the PB2 gene of S-OIV, an E-to-glycine (G) mutation at position 667, was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090508.1722). It has previously been suggested that the E667G substitution in PB2 of HPAI H5N1 virus was under positive selection and possibly played a role in sustainable transmission in humans (14).On 28 September 2009, detection of the E627K mutation in PB2 of S-OIVs of two individuals in the Netherlands was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090928.3394) and raised concern about the possible enhanced replication of the S-OIV in humans, possibly associated with increased virulence. To date, the D701N mutation in PB2 has not been reported in any of the S-OIVs sequenced, and additional viruses with mutation E627K have not been recorded, either. In contrast, viruses with E677G have been reported from the United States, Canada, Germany, the United Kingdom, Norway, and France, according to the public sequence databases.Here, the effects of the E627K, D701N, and E677G mutations in the PB2 genes of S-OIVs was investigated using genetically engineered influenza viruses based on a prototype S-OIV, A/Netherlands/602/2009. Polymerase activity was measured in minigenome assays in human 293T cells, virus replication was analyzed in Madin-Darby Canine kidney (MDCK) cells, virulence was tested in mouse and ferret models, and transmission by aerosols or respiratory droplets was tested in ferrets. In contrast to the earlier assumptions based on experience with other influenza A viruses, S-OIVs with E627K, D701N, or E677G in PB2 did not show a marked increase in virulence or transmission compared to the wild-type virus.     

Regulatory factors and cell populations involved in skeletal muscle regeneration

Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new multinucleated myofibers or fuse to damaged myofibers. The specific microenvironment of the satellite cells, the niche, controls their behavior. The niche contains several components that maintain satellite cells quiescence until they are activated. In addition, a great diversity of stimulatory and inhibitory growth factors such as IGF‐1 and TGF‐β1 regulate their activity. Donor‐derived satellite cells are able to improve muscle regeneration, but their migration through the muscle tissue and across endothelial layers is limited. Less than 1% of their progeny, the myoblasts, survive the first days upon intra‐muscular injection. However, a range of other multipotent muscle‐ and non‐muscle‐derived stem cells are involved in skeletal muscle regeneration. These stem cells can occupy the satellite cell niche and show great potential for the treatment of skeletal muscle injuries and diseases. The aim of this review is to discuss the niche factors, growth factors, and other stem cells, which are involved in skeletal muscle regeneration. Knowledge about the factors regulating satellite cell activity and skeletal muscle regeneration can be used to improve the treatment of muscle injuries and diseases. J. Cell. Physiol. 224:7–16, 2010 © 2010 Wiley‐Liss, Inc.     

Effects of ATP-induced leg vasodilation on VO2 peak and leg O2 extraction during maximal exercise in humans

During maximal whole body exercise VO2 peak is limited by O2 delivery. In turn, it is though that blood flow at near-maximal exercise must be restrained by the sympathetic nervous system to maintain mean arterial pressure. To determine whether enhancing vasodilation across the leg results in higher O2 delivery and leg VO2 during near-maximal and maximal exercise in humans, seven men performed two maximal incremental exercise tests on the cycle ergometer. In random order, one test was performed with and one without (control exercise) infusion of ATP (8 mg in 1 ml of isotonic saline solution) into the right femoral artery at a rate of 80 microg.kg body mass-1.min-1. During near-maximal exercise (92% of VO2 peak), the infusion of ATP increased leg vascular conductance (+43%, P<0.05), leg blood flow (+20%, 1.7 l/min, P<0.05), and leg O2 delivery (+20%, 0.3 l/min, P<0.05). No effects were observed on leg or systemic VO2. Leg O2 fractional extraction was decreased from 85+/-3 (control) to 78+/-4% (ATP) in the infused leg (P<0.05), while it remained unchanged in the left leg (84+/-2 and 83+/-2%; control and ATP; n=3). ATP infusion at maximal exercise increased leg vascular conductance by 17% (P<0.05), while leg blood flow tended to be elevated by 0.8 l/min (P=0.08). However, neither systemic nor leg peak VO2 values where enhanced due to a reduction of O2 extraction from 84+/-4 to 76+/-4%, in the control and ATP conditions, respectively (P<0.05). In summary, the VO2 of the skeletal muscles of the lower extremities is not enhanced by limb vasodilation at near-maximal or maximal exercise in humans. The fact that ATP infusion resulted in a reduction of O2 extraction across the exercising leg suggests a vasodilating effect of ATP on less-active muscle fibers and other noncontracting tissues and that under normal conditions these regions are under high vasoconstrictor influence to ensure the most efficient flow distribution of the available cardiac output to the most active muscle fibers of the exercising limb.     

The human use of humanoid beings: chimeras and patent law

As biotechnology advances, the day may soon come for the creation of a self-aware, human-nonhuman chimera. The USPTO has ruled on whether a patent may issue on such an organism, but Congress must still legislate a dividing line between human and non-human patentable subject matter.     

Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.     

Polar auxin transport: an early invention

In higher plants, cell-to-cell polar auxin transport (PAT) of the phytohormone auxin, indole-3-acetic acid (IAA), generates maxima and minima that direct growth and development. Although IAA is present in all plant phyla, PAT has only been detected in land plants, the earliest being the Bryophytes. Charophyta, a group of freshwater green algae, are among the first multicellular algae with a land plant-like phenotype and are ancestors to land plants. IAA has been detected in members of Charophyta, but its developmental role and the occurrence of PAT are unknown. We show that naphthylphthalamic acid (NPA)-sensitive PAT occurs in internodal cells of Chara corallina. The relatively high velocity (at least 4-5 cm/h) of auxin transport through the giant (3-5 cm) Chara cells does not occur by simple diffusion and is not sensitive to a specific cytoplasmic streaming inhibitor. The results demonstrate that PAT evolved early in multicellular plant life. The giant Chara cells provide a unique new model system to study PAT, as Chara allows the combining of real-time measurements and mathematical modelling with molecular, developmental, cellular, and electrophysiological studies.     

The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.     

Influence of ovariectomy on cardiac oxidative stress in a renovascular hypertension model

The aim of this study was to evaluate the potential influence of endogenous ovarian hormones on cardiac oxidative stress in renovascular hypertension. Female Wistar rats (N = 10 per group) were divided among 4 groups: (i) normotensive control; (ii) hypertensive control; (iii) normotensive ovariectomized; and (iv) hypertensive ovariectomized rats. To induce hypertension, 2-kidney 1-clip (2K1C) Goldblatt's method was followed. Blood pressure (BP) was enhanced (25%) in 2K1C and it was not further altered in hypertensive ovariectomized animals. Lipid peroxidation (measured by thiobarbituric acid reactive substances; TBARS) increased in heart homogenates after ovariectomy (253%) and was additionally augmented when associated with hypertension (by 28%). Superoxide dismutase and catalase activities were similar in both hypertensive groups. Hypertension enhanced glutathione peroxidase activity (75%), but the association with ovariectomy prevented this change. Total radical trapping antioxidant potential (TRAP) decreased in hypertensive rats (34%) and was recovered when associated with ovariectomy. However, this adaptation seems not to be sufficient to avoid the increased oxidative damage in ovariectomized hypertensive animals. These results suggest a protective role for physiological ovarian hormones in the cardiac oxidative stress induced by 2K1C hypertension.