Online Learning of Gait Models From Older Adult Data.

This paper proposes a novel approach for online, individualized gait analysis, based on an adaptive periodic model of any gait signal. The proposed method learns a model of the gait cycle during online measurement, using a continuous representation that can adapt to inter- and intra-personal variability by creating an individualized model. Once the algorithm has converged to the input signal, key gait events can be identified based on the estimated gait phase and amplitude. The approach is implemented and tested on retirement home resident 6 min walk (6MW) data using wearable accelerometers at the ankle. The proposed approach converges within approximately four gait cycles and achieves 3% error in detecting initial swing events.11 An early version of this work was presented in [1]. A more extensive description of related work and an extended method, including optimization of learning rates, were added to this paper. Further, this paper applies and evaluates the method to a new and much larger gait dataset taken from older adults who each have a variety of medical conditions. Therefore, the experimental protocol was also updated and the results are entirely novel.

Novel dengue virus inhibitor 4-HPR activates ATF4 independent of protein kinase R-like Endoplasmic Reticulum Kinase and elevates levels of eIF2α phosphorylation in virus infected cells.

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic.

Influence of dietary docosahexaenoic acid in combination with other long-chain polyunsaturated fatty acids on expression of biosynthesis genes and phospholipid fatty acid compositions in tissues of post-smolt Atlantic salmon (Salmo salar).

To investigate interactions of dietary LC-PUFA, a dose-response study with a range of docosahexaenoic acid (DHA; 22:6n-3) levels (1 g kg(-1), 5 g kg(-1), 10 g kg(-1), 15 g kg(-1) and 20 g kg(-1)) was performed with post-smolts (111 ± 2.6g; mean ± S.D.) over a nine-week feeding period. Additional diets included 10 g kg(-1) DHA in combination with 10 g kg(-1) of either eicosapentaenoic acid (EPA; 20:5n-3) or arachidonic acid (ARA; 20:4n-6), and a diet containing 5 g kg(-1) each of DHA and EPA. The liver, brain, head kidney and gill were collected at the conclusion of the trial, and lipid and fatty acid compositions were determined as well as expression of genes of LC-PUFA biosynthesis. Total lipid content and class composition were largely unaffected by changes in dietary LC-PUFA. However, phospholipid (PL) fatty acid compositions generally reflected that of the diet, although the response varied between tissues. The liver most strongly reflected diet, followed by the head kidney. In both tissues increasing dietary DHA led to significantly increased DHA in PL and inclusion of EPA or ARA led to higher levels of these fatty acids. The brain showed the most conserved composition and gene expression profile, with increased dietary LC-PUFA resulting in only minor changes in PL fatty acids. Dietary LC-PUFA significantly affected the expression of Δ6 and Δ5 desaturases, Elovl 2, 4 and 5, and SREBPs although this varied between tissues with greatest effects observed in the liver followed by the head kidney, similar to PL fatty acid compositions.

Nuclear localization of dengue virus (DENV) 1-4 non-structural protein 5; protection against all 4 DENV serotypes by the inhibitor Ivermectin.

Infection by one of the 4 distinct serotypes of dengue virus (DENV) threatens >40% of the world's population, with no efficacious vaccine or antiviral agent currently available. DENV replication through the virus-encoded nonstructural protein (NS) 5 protein occurs in the infected cell cytoplasm, but NS5 from DENV2 has thus far been shown to localize strongly in the nucleus throughout infection. Here we use specific antibodies cross-reactive with NS5 from DENV1-4 to demonstrate nuclear localization of NS5 from all DENV serotypes for the first time in both infected as well as transfected cells, although to differing extents. The small-molecule inhibitor Ivermectin was inhibitory towards both DENV 1 and 2 NS5 interaction with its nuclear transporter importin α/ß in vitro, and protected against infection from DENV1-4. Ivermectin thus has potential in the clinical setting as a dengue antiviral.

Synthesis and characterization of a novel double crosslinked hyaluronan hydrogel.

Hyaluronan has great potential in medicine as a biomaterial. However, in its native form, hyaluronan is rapidly metabolized in vivo by free radicals and enzymes such as hyaluronidase, and it is highly soluble. Various methods have been adopted therefore, to modify the physicochemical properties of hyaluronan, while maintaining biocompatibility, and thereby widen its spectrum of therapeutic applications. Hyaluronan has four reactive groups (acetamido, carboxyl, hydroxyl and the reducing end) available for crosslinking to itself or other polymers. Using a variety of crosslinking agents, researchers have developed a host of crosslinked hyaluronan derivatives with an increased in vivo residence time. This chemical modification has enabled the production of gels and films, which can be used in applications such as the prevention of post-surgical adhesions, wound healing and dermal augmentation. We have found that if hyaluronan is crosslinked to itself, or to other polymers (either synthetic or biopolymer), in two stages, then a high degree of crosslinking is achieved, conferring improved biostability. In each of the two stages, the same crosslinking agent is used, but different functional groups are bound by altering the reaction conditions. The novel process can be tailored to yield water insoluble gels and films with a broad range of physical and chemical characteristics, and greater resistance to degradation by hyaluronidase and free radicals. These derivatives are currently undergoing biocompatibility testing, and should ultimately lead to a series of innovative second-generation medical products.

Identification of binding proteins for PSP94 in human prostate adenocarcinoma cell lines LNCaP and PC-3.

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94) is one of the predominant proteins found in human seminal fluid. Limited information is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determination of the mechanism of interaction of PSP94 with potential cellular targets. METHODS: Equilibrium binding assay was employed to demonstrate specific binding of biotinylated-PSP94 to the LNCaP and PC-3 cell lines. Binding proteins were partially purified by PSP94 affinity-chromatography from LNCaP, PC-3 cells, and prostate tissues. RESULTS: Binding of biotinylated-PSP94 to LNCaP and PC-3 cells was saturable and time and temperature dependent. The binding could be specifically competitively inhibited by unlabelled PSP94. Two types of PSP94 binding sites with distinct affinity (Kd) and density (Bmax) were determined by Scatchard analysis for each of the two cell lines. For the LNCaP cells, these values were Kd 1 = 0.75 nM and Bmax1 = 300 fmol/mg protein and Kd 2 = 4.5 nM, Bmax2 = 780 fmol/mg protein, respectively. Similar affinity and density results were obtained for PC-3 cells: Kd 1 = 0.83 nM, Bmax1 = 250 fmol/mg protein, and Kd 2 = 5.0 nM, Bmax2 = 700 fmol/mg. The binding of biotinylated-PSP94 to the LNCaP cells was competitively inhibited by the partially purified proteins. Analysis of these proteins SDS-PAGE showed three main bands and the molecular weights of these three bands were approximately 180, 100 and 60 kD, respectively. CONCLUSIONS: The data showed the presence of specific binding proteins to the PSP94 in LNCaP, PC-3 cells, and prostate tissue.

A new scalable purification procedure for prostatic secretory protein (PSP94) from human seminal plasma.

A simple three-step procedure for the purification of native prostate secretory protein (PSP94) from human seminal plasma is described. The steps are ammonium sulfate fractionation followed by a Macro-Prep S support (cation) flowthrough process and the final Macro-Prep high Q support (anion exchange) chromatography using two step-gradient elution. The benefits of this procedure lie in its simplicity, speed, and relatively inexpensive materials, thus providing advantages over the previously reported schemes. The purity of the product as judged by single band on SDS-polyacrylaminde gel electrophoresis was equivalent to that attained by analytical HPLC anion exchange procedure. Yields were in the range of 18-25 mg highly pure PSP94 per 50 ml of seminal plasma. The desalted, lyophilized, purified PSP94 sample was characterized by SDS-PAGE, Western blot, and N-terminal sequencing. All parameters tested confirm its identity. Preliminary data show that this procedure is suitable for a large-scale production. The direct quantitation of PSP94 by SDS-PAGE and densitometric image analysis at various purification steps for evaluating the recovery of PSP94 is described. The results obtained show that this is an efficient strategy for preparation of highly purified native PSP94.

Recombinant PSP94 (prostate secretory protein of 94 amino acids) demonstrates similar linear epitope structure as natural PSP94 protein.

PSP94 has the potential to be a useful diagnostic marker and therapeutic agent in prostate cancer. Recently, different immunoassay systems for quantitative analysis of PSP94 in clinical samples have been developed, but the epitope structure of PSP94 protein has not been elucidated. In this study, we report an Escherichia coli expression system for recombinant GST-PSP94 fusion protein. GST-PSP94 contains antigenic determinants similar to natural PSP94 protein (determined both by Western blotting experiments and by ELISA) and can be used to study the structure of natural PSP94 antigen. Since GST-PSP94 was expressed in E. coli and purification involved a denaturing process, we propose that the epitope structure of PSP94 is linear and largely dependent on the primary amino acid sequence, rather than conformational structure. This hypothesis was supported by reciprocal competition in ELISA among natural, GST-PSP94 fusion protein, and purified recombinant PSP94 protein. The results demonstrate that the various forms of PSP94 can compete with each other in binding to rabbit PSP94 polyclonal antibody, although the natural PSP94 has a slightly higher affinity. When natural and recombinant PSP94 protein were denatured in vitro with urea and alkali, no effect on the binding to antibody was found. The epitope activity of natural PSP94 was also shown to be resistant to the treatment of detergent and reducing agent. The location of one of the linear epitopes recognized by the PSP94 antibody was determined to be in the N-terminus by using two synthetic peptides representing N- and C-terminal sequences. Competitive ELISA between the N-terminal peptide and PSP94 protein indicate that both natural and GST-PSP94 have similar immunoactive N-termini.

Sialic acid and mucus production in rainbow trout (Salmo gairdneri Richardson) in response to zinc and seawater.

1. Rainbow trout exposed to seawater or 20 ppm zinc showed hypersecretion of mucus whose sialic acid (N-acetylneuraminic acid) content 0.192 micro M g-1 mucus was not significantly different to that produced by trout in freshwater, 0.184 micro M g-1 mucus. 2. Measurement of sialic content in the water indicated its production (and therefore mucus production) was about 70 times higher in seawater and 20 ppm zinc compared to freshwater production.