Order and disorder control the functional rearrangement of influenza hemagglutinin

Influenza hemagglutinin (HA), a homotrimeric glycoprotein crucial for membrane fusion, undergoes a large-scale structural rearrangement during viral invasion. X-ray crystallography has shown that the pre- and postfusion configurations of HA₂, the membrane-fusion subunit of HA, have disparate secondary, tertiary, and quaternary structures, where some regions are displaced by more than 100 Å. To explore structural dynamics during the conformational transition, we studied simulations of a minimally frustrated model based on energy landscape theory. The model combines structural information from both the pre- and postfusion crystallographic configurations of HA₂. Rather than a downhill drive toward formation of the central coiled-coil, we discovered an order-disorder transition early in the conformational change as the mechanism for the release of the fusion peptides from their burial sites in the prefusion crystal structure. This disorder quickly leads to a metastable intermediate with a broken threefold symmetry. Finally, kinetic competition between the formation of the extended coiled-coil and C-terminal melting results in two routes from this intermediate to the postfusion structure. Our study reiterates the roles that cracking and disorder can play in functional molecular motions, in contrast to the downhill mechanical interpretations of the “spring-loaded” model proposed for the HA₂ conformational transition.Hemagglutinin (HA) is a viral receptor-binding and membrane-fusion glycoprotein involved in the invasion of influenza virions into host cells (1). Structural rearrangements of HA during membrane fusion are crucial for the delivery of the viral genome. The postfusion conformation of HA shows considerable similarity to other viral fusion proteins and eukaryotic membrane receptors involved in intracellular vesicle trafficking (2), suggesting there may be common mechanisms in the function of these proteins. Therefore, HA may serve as a model system, allowing characterization of the molecular and energetic details that underlie its conformational transition to provide insights into general principles of membrane fusion (3).HA is a homotrimer consisting of two domains connected by disulfide bonds (4): a globular receptor binding domain (HA₁), and a coiled-coil membrane-fusion domain anchored to the viral membrane (HA₂). Recognized by the sialic acid receptor of a host cell, the intact virus enters the cell via endocytosis. Low pH in a late endosome then induces the dissociation of HA₁ from HA₂ (1) and an irreversible conformational transition of HA₂. Experimentally, this conformational change can be triggered by either low pH, high temperature, or urea denaturation (5).Structures of HA in pre- and postfusion pH conformations have been solved by X-ray crystallography. The structure of the prefusion ectodomain contains both HA₁ and HA₂, and was purified from influenza virions (4). A postfusion conformation of HA₁ and HA₂ were obtained from prefusion viral HA that was sequentially treated with low pH and trypsin (6, 7). Comparison of these two structures shows no structural changes in HA₁, but a major rearrangement in HA₂, including secondary, tertiary, and quaternary structural changes. The N-terminal domain of HA₂, initially adjacent to the transmembrane region in the prefusion configuration, undergoes a large movement (over 100 Å) during the transition. The C-terminal domain of HA₂ changes from a globular structure to three extended loops packed against the central coiled-coil.Although experiments have probed the fusion mechanism through mutation (8) and provided measures of fusion kinetics (9), there is a lack of structural information about how HA₂ transitions from the prefusion to postfusion conformations. Theoretical models have been suggested to describe the fusion mechanism based on the available experimental kinetic data (10–12). However, because of the large scale of the HA₂ rearrangement, only limited computational techniques, such as targeted molecular dynamics (13), have been applied to study the molecular details of the transition.In this study we applied the principles of the energy landscape theory as developed in the context of protein folding (14–16) to examine structural details of the HA₂ conformational transition. We used a structure-based model (SBM) (17, 18) built with a dual-funneled landscape (19, 20) that has both the pre- and postfusion structures as explicit minima. The HA₂ landscape has at least two competing basins of attraction, corresponding to the pre- and postfusion structures of HA₂, respectively. HA₁ dissociation sterically enables HA₂ to explore beyond the prefusion local free-energy minimum and to diffuse toward the postfusion configuration. The long-length scale and extensive shuffling of secondary and tertiary structures is reminiscent of protein folding, but distinct in that both ends of the HA₂ transition can be described by ensembles of structurally similar configurations. Just as in protein folding, there may be free-energy barriers and structural intermediates along the HA₂ transition caused by the imperfect cancellation of energy and entropy. These intermediate ensembles may be interesting candidates for drug design to inhibit HA function.Previously, a “spring-loaded” model has been applied to describe the mechanism for the HA₂ transition (21). This model suggested a downhill mechanical transition of the N-terminal region of HA₂ into an ordered helical structure that orients the fusion peptides away from the virus and toward the host membrane. Our simulations expand this view by showing that the conformational change of the N-terminal domains is associated with an entropic barrier and the unfolding of the C-terminal region is associated with the major energetic barrier during the HA₂ conformational transition. Kinetic competition between these two events creates a long-lived metastable intermediate that allows for two dominant routes. The first route (the “sequential route”) resembles the spring-loaded model, and the second route (the “cooperative route”) involves cooperative interactions between the N-terminal and C-terminal domains in forming the central coiled-coil. The presence of these distinct routes suggests multiple mechanisms for HA₂ rearrangement and membrane fusion.     

Photocatalysis of nanocomposite titania–natural silica as antibacterial against Staphylococcus aureus and Pseudomonas aeruginosa

The entries of pathogenic bacteria into the human body remain a severe problem to health that can be prevented using antibacterial agents. Meanwhile, the photocatalytic technique using semiconductor nanocomposite TiO₂–SiO₂ has great potential as an antibacterial method. In order to utilize natural resources, SiO₂ supporting materials are obtained from the extraction of beach sand due to the high silica content. Therefore, this study aims to synthesize a nanocomposite of TiO₂ with SiO₂ extracted from beach sand as an antibacterial agent against Staphylococcus aureus and Pseudomonas aeruginosa. The antibacterial activity test used the dilution and optical density method. Based on XRD analysis, the crystals of TiO₂ in the synthesized composites showed a more dominant anatase structure. Furthermore, Ti–O–Si bonds were identified from the IR spectrum, which showed the interaction between TiO₂ and SiO₂. In addition, SEM-EDX results showed agglomerated spherical particles with a TiO₂–SiO₂ nanocomposite particle size of 40–107 nm. The best antibacterial activity was demonstrated by the 1 : 0.5 TiO₂–SiO₂ nanocomposite, with inactivation percentages of S. aureus and P. aeruginosa of 98.69% and 97.44%, respectively.

TiO₂ material is composited with silica obtained from natural sand with indirect sonochemistry method. The addition of SiO₂ increase the photocatalyst activity of TiO₂ as an antibacterial against S. aureus and P. aeruginosa.     

Transforming Growth Factor β1 Regulates Tissue Inhibitor of Metalloproteinases—1 Expression in Differentiated Human Articular Chondrocytes

Objective. To investigate the role of interleukin-6 (IL-6) and transforming growth factor β1 (TGF β1) in the regulation of tissue inhibitor of metalloproteinases—1 (TIMP-1) synthesis in human articular chondrocytes. Methods. Articular cartilage was obtained from human knee joints 24 hours after death. Chondrocytes were isolated by collagenase digestion and embedded in low-gelling-temperature agarose. After stimulation by cytokines, total RNA was isolated and analyzed by Northern blotting. TIMP-1 protein levels were determined using a competitive enzyme-linked immunosorbent assay. Results. Human chondrocytes in agarose culture expressed messenger RNA (mRNA) for the IL-6 receptor (gp80) and its signal-transducing subunit gp130. In contrast to the findings in a previous study, IL-6 did not stimulate TIMP-1 expression in these cells, whereas TGF β1 was an important inducer of TIMP-1 mRNA and protein synthesis. Conclusion. Our findings suggest that TGF β1 has a protective effect on the extracellular matrix of human articular chondrocytes.     

PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction

BACKGROUND: Progressive renal disease is characterized by the induction of plasminogen activator inhibitor-1 (PAI-1), suggesting that impaired activity of the renal plasmin cascade may play a role in renal fibrosis. METHODS: To test this hypothesis, the severity of renal fibrosis caused by unilateral ureteral obstruction (UUO) was compared in PAI-1 wild-type (+/+) and PAI-1 deficient (-/-) mice. The extent of interstitial inflammation and fibrosis, renal plasminogen activator and plasmin activity, and renal expression of profibrotic genes was evaluated after 3, 7, and 14 days of UUO. RESULTS: Renal PAI-1 mRNA levels increased 8- to 16-fold in the +/+ mice after UUO surgery, and PAI-1 protein was detected in kidney homogenates. Interstitial fibrosis was significantly attenuated in -/- mice compared with +/+ mice at day 7 and day 14, based on the interstitial area stained with picrosirius red and total kidney collagen content. However, neither the mean renal plasminogen activator nor plasmin activities were increased in -/- mice compared with +/+ mice. The number of interstitial macrophages were significantly lower in the -/- mice three and seven days after UUO; interstitial myofibroblasts were significantly fewer at three days. At the same time points, this altered interstitial cellularity was associated with a significant reduction in renal mRNA levels for transforming growth factor-beta and procollagens alpha 1(I) and alpha 1(III). CONCLUSIONS: These studies establish an important fibrogenic role for PAI-1 in the renal fibrogenic response. The results demonstrate that one important fibrosis-promoting function of PAI-1 is its role in the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.     

Asymptomatic fistula from a giant aneurysmatic left anterior descending artery to the right ventricular outflow tract

Coronary artery fistulas are unusual congenital or acquired coronary artery abnormalities in which blood is shunted into a cardiac chamber, great vessel or other structure, bypassing the myocardial capillary network (Jung et al. in Cardiovasc Ultrasound 5:10, 2007). We present a young adult patient with an asymptomatic fistula from a giant aneurysmatic left anterior descending artery to the right ventricular outflow tract, first diagnosed by echocardiography examination and further confirmed by 128-slice computed tomography coronary angiography.     

Dysphagia in Head and Neck Cancer Patients Treated with Chemoradiotherapy

Dysphagia is a very common complaint of head and neck cancer patients and can exist before, during, and after chemoradiotherapy. It leads to nutritional deficiency, weight loss, and prolonged unnatural feeding and also has a major potential risk for aspiration. This has a significant negative impact on the patient’s entire quality of life. Because treatment of dysphagia in this setting is rarely effective, prevention is paramount. Several strategies have been developed to reduce dysphagia. These include swallowing exercises, treatment modification techniques such as intensity-modulated radiotherapy, selective delineation of elective nodes, reducing xerostomia by parotid-sparing radiotherapy, and adding of radioprotectors. However, more research is needed to further decrease the incidence of dysphagia and improve quality of life.     

Reproducibility of Standing Posture for X-Ray Radiography: A Feasibility Study of the <Emphasis Type="Italic">BalancAid</Emphasis> with Healthy Young Subjects

Unreliable spinal X-ray radiography measurement due to standing postural variability can be minimized by using positional supports. In this study, we introduce a balancing device, named BalancAid, to position the patients in a reproducible position during spinal X-ray radiography. This study aimed to investigate the performance of healthy young subjects’ standing posture on the BalancAid compared to standing on the ground mimicking the standard X-rays posture in producing a reproducible posture for the spinal X-ray radiography. A study on the posture reproducibility measurement was performed by taking photographs of 20 healthy young subjects with good balance control standing on the BalancAid and the ground repeatedly within two consecutive days. We analyzed nine posterior–anterior (PA) and three lateral (LA) angles between lines through body marks placed in the positions of T3, T7, T12, L4 of the spine to confirm any translocations and movements between the first and second day measurements. No body marks repositioning was performed to avoid any error. Lin’s CCC test on all angles comparing both standing postures demonstrated that seven out of nine angles in PA view, and two out of three angles in LA view gave better reproducibility for standing on the BalancAid compared to standing on the ground. The PA angles concordance is on average better than that of the LA angles.