Background. The first cases of avian influenza A (H5N1) in humans in Vietnam were detected in early 2004, and Vietnam has reported the second highest number of cases globally. Methods. We obtained ...retrospective clinical data through review of medical records for laboratory confirmed cases of influenza A (H5N1) infection diagnosed in Vietnam from January 2004 through December 2006. Standard data was abstracted regarding clinical and laboratory features, treatment, and outcome. Results. Data were obtained for 67 (72%) of 93 cases diagnosed in Vietnam over the study period. Patients presented to the hospital after a median duration of illness of 6 days with fever (75%), cough (89%), and dyspnea (81%). Diarrhea and mucosal bleeding at presentation were more common in fatal than in nonfatal cases. Common findings were bilateral pulmonary infiltrates on chest radiograph (72%), lymphopenia (73%), and increased serum transaminase levels (aspartate aminotransferase, 69%; alanine aminotransferase, 61%). Twenty-six patients died (case fatality rate, 39%; 95% confidence interval, 27%–51%) and the most reliable predictor of a fatal outcome was the presence of both neutropenia and raised alanine aminotransferase level at admission, which correctly predicted 91% of deaths and 82% of survivals. The risk of death was higher among persons aged ⩽16 years, compared with older persons (P<.001), and the risk of death was higher among patients who did not receive oseltamivir treatment (P=.048). The benefit of oseltamivir treatment remained after controlling for potential confounding by 1 measure of severity (odds ratio, 0.15; 95% confidence interval, 0.026–0.893; P=.034). Conclusion. In cases of infection with Influenza A (H5N1), the presence of both neutropenia and raised serum transaminase levels predicts a poor outcome. Oseltamivir treatment shows benefit, but treatment with corticosteroids is associated with an increased risk of death.
Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb ...and index finger domains, which are each assembled from paired β strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.
Background: Wnt proteins are rich in cysteines, but their functional significance has not been systematically examined.
Results: Mutagenesis of cysteines in Wnt3a uncovers their requirements for Wnt secretion and/or receptor binding.
Conclusion: Behaviors of the Wnt3a cysteine mutants are consistent with the Wnt structure model.
Significance: This study provides insights into pathogenesis associated with WNT mutations and new tool sets for WNT research.
Nanoparticle size, surface charge and material composition are known to affect the uptake of nanoparticles by cells. However, whether nanoparticle shape affects transport across various barriers ...inside the cell remains unclear. Here we used pair correlation microscopy to show that polymeric nanoparticles with different shapes but identical surface chemistries moved across the various cellular barriers at different rates, ultimately defining the site of drug release. We measured how micelles, vesicles, rods and worms entered the cell and whether they escaped from the endosomal system and had access to the nucleus via the nuclear pore complex. Rods and worms, but not micelles and vesicles, entered the nucleus by passive diffusion. Improving nuclear access, for example with a nuclear localization signal, resulted in more doxorubicin release inside the nucleus and correlated with greater cytotoxicity. Our results therefore demonstrate that drug delivery across the major cellular barrier, the nuclear envelope, is important for doxorubicin efficiency and can be achieved with appropriately shaped nanoparticles.
Sediment scour at downstream of hydraulic structures is one of the main reasons threatening its stability. Several soil properties and initial input data have been studied to investigate its ...influence on scour hole geometry by both physical and numerical models. However, parameters of resistance affecting sedimentation and erosion phenomena have not been carried out in the literature. Besides, the auxiliary-like wing walls prevalently used in many real applications have been rarely addressed for their effect on morphological change. In this study, a 3D Computational Fluid Dynamics model is utilized to calibrate the hydraulic characteristics of steady flow going through the culvert by comparison with experimental data, showing good agreement between water depth, velocity, and pressure profiles at the bottom of the boxed culvert. The results show that a grid cell of 0.015 m gave minimum NRMSE and MAE values in test cases. Another approach is numerical testing sediment scour at a meander flume outlet with a variety of roughness/d.sub.50 ratio (c.sub.s) and diversion wall types. The findings include the following: c.sub.s = 2.5 indicates the close agreement between the numerical and analytical results of maximum scour depth after the culvert; the influence of four types of wing wall on the geometrical deformation including erosion at the concave bank and deposition at the convex bank of the meander flume outlet; and two short headwalls represent the best solution that accounts for small morphological changes. The influence of the roughness parameter of soil material and headwall types on sediment scour at the meander exit channel of hydraulic structure can be estimated by the numerical model.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We describe the synthesis of iron oxide nanoparticles (IONPs) with excellent colloidal stability in both water and serum, imparted by carefully designed grafted polymer shells. The polymer shells ...were built with attached aldehyde functionality to enable the reversible attachment of doxorubicin (DOX) via imine bonds, providing a controlled release mechanism for DOX in acidic environments. The IONPs were shown to be readily taken up by cell lines (MCF-7 breast cancer cells and H1299 lung cancer cells), and intracellular release of DOX was proven using in vitro fluorescence lifetime imaging microscopy (FLIM) measurements. Using the fluorescence lifetime difference exhibited by native DOX (∼1 ns) compared to conjugated DOX (∼4.6 ns), the intracellular release of conjugated DOX was in situ monitored in H1299 and was estimated using phasor plot representation, showing a clear increase of native DOX with time. The results obtained from FLIM were corroborated using confocal microscopy, clearly showing DOX accumulation in the nuclei. The IONPs were also assessed as MRI negative contrast agents. We observed a significant change in the transverse relaxivity properties of the IONPs, going from 220 to 390 mM–1 s–1, in the presence or absence of conjugated DOX. This dependence of MRI signal on IONP-DOX/water interactions may be exploited in future theranostic applications. The in vitro studies were then extended to monitor cell uptake of the DOX loaded IONPs (IONP@P(HBA)-b-P(OEGA) + DOX) into two 3D multicellular tumor spheroids (MCS) grown from two independent cell lines (MCF-7 and H1299) using multiphoton excitation microscopy.
Biofilms are increasingly recognized as playing a major role in human infectious diseases, as they can form on both living tissues and abiotic surfaces, with serious implications for applications ...that rely on prolonged exposure to the body such as implantable biomedical devices or catheters. Therefore, there is an urgent need to develop improved therapeutics to effectively eradicate unwanted biofilms. Recently, the biological signaling molecule nitric oxide (NO) was identified as a key regulator of dispersal events in biofilms. In this paper, we report a new class of core cross-linked star polymers designed to store and release nitric oxide, in a controlled way, for the dispersion of biofilms. First, core cross-linked star polymers were prepared by reversible addition–fragmentation chain transfer polymerization (RAFT) via an arm first approach. Poly(oligoethylene methoxy acrylate) chains were synthesized by RAFT polymerization, and then chain extended in the presence of 2-vinyl-4,4-dimethyl-5-oxazolone monomer (VDM) with N ,N-methylenebis(acrylamide) employed as a cross-linker to yield functional core cross-linked star polymers. Spermine was successfully attached to the star core by reaction with VDM. Finally, the secondary amine groups were reacted with NO gas to yield NO-core cross-linked star polymers. The core cross-linked star polymers were found to release NO in a controlled, slow delivery in bacterial cultures showing great efficacy in preventing both cell attachment and biofilm formation in Pseudomonas aeruginosa over time via a nontoxic mechanism, confining bacterial growth to the suspended liquid.
Surface modification to obtain high dispersion stability and biocompatibility is a key factor for bio-application of upconversion nanoparticles (UCNPs). A systematic study of UCNPs modified with four ...hydrophilic molecules separately, comparing their dispersion stability in biological buffers and cellular biocompatibility is reported here. The results show that carboxyl-functionalized UCNPs (modified by 3,4-dihydrocinnamic acid (DHCA) or poly(monoacryloxyethyl phosphate (MAEP)) with negative surface charge have superior even-distribution in biological buffers compared to amino-functionalized UCNPs (modified by (aminomethyl)phosphonic (AMPA) or (3-Aminopropyl)triethoxysilane (APTES)) with positive surface charge. Subsequent investigation of cellular interactions revealed high levels of non-targeted cellular uptake of the particles modified with either of the three small molecules (AMPA, APTES, DHCA) and high levels of cytotoxicity when used at high concentrations. The particles were seen to be trapped as particle-aggregates within the cellular cytoplasm, leading to reduced cell viability and cell proliferation, along with dysregulation of the cell cycle as assessed by DNA content measurements. The dramatically reduced proportion of cells in G1 phase and the slightly increased proportion in G2 phase indicates inhibition of M phase, and the appearance of sub-G1 phase reflects cell necrosis. In contrast, MAEP-modified UCNPs are bio-friendly with increased dispersion stability in biological buffers, are non-cytotoxic, with negligible levels of non-specific cellular uptake and no effect on the cell cycle at both low and high concentrations. MAEP-modified UCNPs were further functionalized with streptavidin for intracellular microtubule imaging, and showed clear cytoskeletal structures via their upconversion luminescence.
Upconversion nanoparticles (UCNP) are an exciting potential nanomaterial for bio-applications. Their anti-Stokes luminescence makes them especially attractive to be used as imaging probes and thermal therapeutic reagents. Surface modification is the key to achieving stable and compatible hydrophilic-UCNPs. However, the lack of criteria to assess molecular ligands used for ligand exchange of nanoparticles has hampered the development of surface modification, and further limits UCNP’s bio-application. Herein, we report a systematic comparative study of modified-UCNPs with four distinct hydrophilic molecules, assessing each particles’ colloidal stability in biological buffers and their cellular biocompatibility. The protocol established here can serve as a potential guide for the surface modification of UCNPs in bio-applications.
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In this study, a novel technique was used for the reversible attachment of folic acid on the surface of polymeric micelles for a tumor-specific drug delivery system. The reversible conjugation is ...based on the interaction between phenylboronic acid (PBA) and dopamine to form a borate ester. The conjugation is fast and efficient and in vitro experiments via confocal fluorescent microscopy show that the linker is stable in for several hours. Reversible addition–fragmentation chain transfer (RAFT) polymerization was used to synthesize two various sized water-soluble block copolymer of oligoethylene glycol methylether methacylate and methyl acrylic acid (POEGMEMA35-b-PMAA200 and POEGMEMA26-b-PMAA90). The platinum drug, oxoplatin, was then subsequently attached to the polymer via ester formation leading to platinum loading of 12 wt % as determined by TGA. The platinum-induced amphiphilic block copolymers that consequently led to the formation of micelles of sizes 150 and 20 nm in an aqueous environment with the longer PMAA block forming larger micelles. The small micelles were in addition cross-linked using 1,8-diaminooctane to further stabilize their structure. The targeting ability of folate conjugated polymeric micelles was investigated against two types of tumor cell lines: A549 (-FR) and OVCAR-3 (+FR). The cell line growth inhibitory efficacy of material synthesized was evaluated by using SRB method. The results revealed that folate conjugated micelles showed higher activity in FR + OVCAR-3 cells but not in FR – A549 cells. Similar results were obtained for both small and large micelles without the conjugation of folate. Comparing large and small micelles it can be observed that larger micelles are more efficient, which has been attributed to the lower stability of the smaller micelles. Micelle stabilization via cross-linking could indeed increase the toxicity of the drug carrier.
Acetyl-hexapeptide 3 (AHP-3) has good efficacy and safety profile as an anti-wrinkle small peptide. However, its skin permeation is poor due to its hydrophilicity and large molecular weight. 3D ...printing of personalised microneedles (MN), that contour to the skin surface, offers an attractive alternative for delivery for AHP-3. However, commercially available photocurable resin for 3D printing are not suitable for fabrication of drug loaded delivery systems. In this study, two liquid monomers, namely, polyethylene glycol diacrylate (PEGDA) and vinyl pyrrolidone (VP), were investigated at various proportions, for critical parameters such as mechanical strength of final polymer, rate of polymerisation, rate of swelling of final polymer, 3D printing resolution and safety profile of final polymer. The optimal resin, based on the above parameters, was that of ratio 7 VP: 3 PEGDA in weight. Drug loading into the optimal resin demonstrated that AHP-3 remained stable throughout the fabrication process and there was no effect on the physical properties of final polymer. Using a 3D scanned face model, a personalised MN patch was designed using computer aided design (CAD) software and subsequently fabricated using a Digital Light Processing (DLP) 3D printer, with the optimal resin. In vitro characterisation of fabricated MN patch demonstrated the ability to penetrate human cadaver dermatomed skin and the MN remained intact after compression. The final polymer also had minimal cytotoxicity to human dermal fibroblast. Therefore, personalised MN patch fabricated using the photopolymer can potentially be a novel approach to augment transdermal delivery of AHP-3 for effective wrinkle management.
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