A preliminary evaluation of the reliability of a modified functional scoring system for assessing neurologic function in ambulatory thoracolumbar myelopathy dogs.

BACKGROUND: The objective of this study was to develop and assess the reliability of a modified scoring system for evaluating the function of the two pelvic limbs separately, in ambulatory thoracolumbar myelopathy dogs. A previously established neurologic score scale for dogs with T3-L3 lesions was modified in order to provide a separate score for each pelvic limb. RESULTS: Seventeen ambulatory dogs with thoracolumbar myelopathies were evaluated. Using the new scale, two observers independently performed 22 observational gait analyses (OGAs) in ten dogs without videotape. Another 18 OGAs were performed in seven dogs by watching videotapes of them ambulating. There was poor agreement (concordance correlation coefficient, 0.87) between the two observers for all 40 OGAs. When stratified, the agreement was moderate (concordance correlation coefficient, 0.90) in the OGAs without videotaping and poor (concordance correlation coefficient, 0.80) for the OGAs based on videotapes. For the decision regarding which pelvic limb was more severely affected, a fair agreement (kappa value, 0.30) between the two observers was noted. Without videotape there was only slight agreement (kappa value, 0.05), but with videotape there was moderate agreement (kappa value, 0.56). CONCLUSIONS: The modified scoring system in this study provides moderate reliability in assessing the functional neurologic status of each pelvic limb, by OGA without videotape, in canine T3-L3 patients. Further development of this scoring system is required. However, imperfect agreement when visually quantifying neurological deficits is not unexpected.

Double Nutri (Liposomal Encapsulation) Enhances Bioavailability of Vitamin C and Extends Its Half-Life in Plasma.

Vitamin C is an important antioxidant in the human body that plays a role in many body functions. Liposomal encapsulation is a technology commonly used in food processing and medicine. This study determines whether Double Nutri (liposomal encapsulation) increases the absorption rate for vitamin C supplementation. Subjects enrolled in this study took vitamin C without liposome; then, 14 d washout was given with liposomal process A vitamin C. After 14 d washout, the subjects took liposomal process B vitamin C (Double Nutri) was given. After taking the test sample, the vitamin C concentration in venous blood was measured from the baseline (0 h), 0.5, 1, 2, 3, 4, and 8 h. Eleven healthy subjects were recruited for three tests. The vitamin C concentration for liposomal process B vitamin C (Double Nutri) group is higher (7.26±3.52, p<0.01) than that for the liposomal process A vitamin C group (6.41±3.80, p<0.05) and for vitamin C without liposome (2.21±4.07). This shows that Double Nutri has better bioavailability and can last up in the body to 8 hours. In addition, this study shows that Double Nutri increases the half-life of vitamin C in plasma and has higher bioavailability.

Upregulation of cluster of differentiation 36 mRNA expression in peripheral blood mononuclear cells correlates with frailty severity in older adults.

BACKGROUND: Aging-associated frailty has been connected to low-grade chronic inflammation and also to progressive monocytic activation. CD36 (cluster of differentiation 36, platelet glycoprotein 4 or fatty acid translocase) has been shown to induce the expression of pro-inflammatory cytokines and to activate macrophage connected inflammation. This study aims to examine whether the expression of CD36 is up-regulated among frail older adults. METHODS: The demographic data, Fried Frailty Index, metabolic and inflammatory parameters of our observational study were obtained from the comprehensive geriatric assessment programme of a hospital-based outpatient department. The mRNA isolated from the peripheral blood mononuclear cells (PBMCs) was used to determine the levels of CD36, tumour necrosis factor alpha (TNF-α), and CXC chemokine ligand-10 (CXCL10) mRNAs with real-time polymerase chain reaction (PCR). RESULTS: A total of 189 older adults (58% female) were included in the analysis, and the mean age was 77.19 ± 6.12 years. The numbers of participants who fitted in the groups of robust, pre-frail, and frail were 46, 106, and 37, respectively. Our data showed that CD36 mRNA expression levels in PBMCs were the highest in the frail group (1.25 ± 0.53 in robust, 2.13 ± 1.02 in pre-frail, and 2.78 ± 1.15 in frail group, P < 0.001). Further regression analyses revealed that CD36 mRNA levels were positively correlated with both the pre-frail and frailty status in the univariate analysis (both P's < 0.001). What might suggest something worthy of further investigation is that, with potential confounders being adjusted for, CD36 remained as an independent factor that positively correlated with the pre-frail and frailty status in the multivariable analysis (P < 0.001). CONCLUSIONS: CD36 mRNA levels in PBMCs in robust older adults are significantly lower than in pre-frail and in frail. Our findings suggest that CD36 mRNA levels in PBMCs may be considered a potential biomarker for frail severity.

The Expression of Toll-Like Receptor 4 mRNA in PBMCs Is Upregulated in Smokers and Decreases Upon Smoking Cessation.

Background: Studies have shown in vitro that cigarette smoke condensate stimulates monocytes to express toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule 1 (ICAM-1), and enhances their adhesion to the endothelium. However, the same effects of cigarette smoking have not been explored in vivo. This study is to investigate the effect of cigarette smoking and smoking cessation on their mRNA expression in human peripheral blood mononuclear cells (PBMCs). Methods: A group of 97 smokers and 62 nonsmokers were enrolled. The RNA from PBMCs was assessed with real-time polymerase chain reaction (PCR) to determine the levels of ICAM-1, TNF-α, and TLR4. The same markers in PBMCs of 87 quitters were examined before and at one week, one month, and two months after smoking cessation. Results: Of the 97 smokers, 85 (87.6%) were males, and 30 (48.4%) of the nonsmokers were males (p < 0.0001). The mean (SD) age of the smokers was 43.24 (10.89) years, which was younger than 43.45 (11.41) years of nonsmokers (p < 0.0001). The incidence of cardiovascular diseases was 13.4% in smokers, which was higher than 1.6% in nonsmokers (p < 0.05). Both ICAM-1 and TNF-α mRNA levels in PBMCs were higher among the smokers (p < 0.0001). In addition, TLR4 mRNA levels in PBMCs were statistically elevated in the smokers (p < 0.0001) comparing with those in the nonsmokers. The mRNA levels of TLR4 and TNF-α in PBMCs decreased in those who had quit smoking for 2 months (p < 0.0001). Conclusions: ICAM-1, TNF-α, and TLR4 mRNA expression levels in PBMCs increased in smokers and decreased after being on a smoking cessation program for 2 months. This finding suggested that TLR4 expression may mediate the atherogenic inflammatory process induced by smoking.

The signal sequence coding region promotes nuclear export of mRNA.

In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs.

Human DDX3 functions in translation and interacts with the translation initiation factor eIF3.

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.

Plasma vascular adhesion protein-1 levels correlate positively with frailty severity in older adults.

Geriatric frailty is associated with increased mortality and links to increased inflammatory activity. Vascular adhesion protein-1 (VAP-1) is important in inflammatory process. This study investigates the relationship between plasma VAP-1 level and frailty in older adults.The cross-sectional study recruited community dwelling older adults from a hospital-based comprehensive geriatric assessment program. The demographic data, Fried Frailty Index, metabolic and inflammatory parameters were assessed.A total of 151 participants (76 women, 50.3%) were included in the analysis, and the age (mean ±â€Š standard deviation) was 77.1 ±â€Š6.1 years. The mean plasma VAP-1 level (ng/mL) was significantly different (P = .029) among different frailty groups (346.3 ±â€Š86.5 in the robust older adults, 371.6 ±â€Š107.9 in the pre-frail older adults, and 416.6 ±â€Š141.1 in the frail older adults). Multivariate ordered logistic regression analysis also demonstrated that plasma VAP-1 levels were positively associated with frailty severity (P = .039). Analysis of the frailty components with plasma VAP-1 levels showed that the elderly who had "exhaustion" (P = .016) or "weakness" (P = .025) tended to have higher plasma VAP-1 levels.The data support that VAP-1 might represent a potential plasma biomarker of frailty.

Plasma zinc alpha2-glycoprotein levels correlate positively with frailty severity in female elders.

Frailty is a geriatric syndrome associated with adiposity. Zinc alpha2-glycoprotein (ZAG), a novel adipokine, is a modulator of body fat mass and positively correlates with age. This observational study aims to investigate the relationship between plasma ZAG levels and frailty in the elderly.We enrolled 189 elder participants from a hospital-based comprehensive geriatric assessment program in Taiwan from January 2007 to June 2008. The demographic data, body weight, body mass index, appendicular skeletal muscle mass index (ASMI), body fat mass percentage, metabolic and inflammatory parameters including plasma tumor-necrosis factor alpha, C-reactive protein and ZAG levels, were assessed. The frailty score was assessed by Fried Frailty Index.The mean age of all participants (91 [48.1%] men and 98 [51.9%] women) was 77.19 ±â€Š6.12 years. Judged by the FFI score, 46 (24.34%) elders were robust, 106 (56.08%) were pre-frail, and 37 (19.58%) were frail. Older men showed greater ASMI and lower fat mass percentage in comparison to older women (P < 0.0001). The log-transformed mean plasma ZAG (µg/mL) level of overall was 1.82 ±â€Š0.11, and it was higher in men than in women (1.85 ±â€Š0.12 vs 1.79 ±â€Š0.1, P = 0.0006). Plasma ZAG levels were different among the robust, pre-frail and frail subgroups (1.78 ±â€Š0.09, 1.83 ±â€Š0.12, 1.83 ±â€Š1.10, respectively, P = 0.028), and the differences were more significant in woman elders (P = 0.005). Further multiple linear regression analysis showed plasma ZAG levels positively correlated with frailty severity in women (P for trend = 0.0435).Plasma ZAG levels positively correlated with frailty severity in woman elders. The difference between sexes suggests certain sex-specific mechanisms may exist to affect the association between plasma ZAG levels and frailty.

The proteins PDIP3 and ZC11A associate with the human TREX complex in an ATP-dependent manner and function in mRNA export.

The conserved TREX complex, which contains UAP56, Aly, CIP29, and the multi-subunit THO complex, functions in mRNA export. Recently, several putative new components of the human TREX complex were identified by mass spectrometry. Here, we investigated the function of two of these, PDIP3 and ZC11A. Our data indicate that both of these proteins are components of a common TREX complex and function in mRNA export. Recently, we found that both CIP29 and Aly associate with the DEAD box helicase UAP56 and with the TREX complex in an ATP-dependent manner. We now show that this is also the case for PDIP3 and ZC11A. Thus, together with previous work, our data indicate that the TREX complex participates in multiple ATP-dependent interactions.

The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large ribosomal subunit in sucrose gradients and coprecipitates with 28S ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S ribosomal subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome.

Human mRNA export machinery recruited to the 5' end of mRNA.

Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.

Agarose gel separation/isolation of RNA-protein complexes.

In many methods currently used to analyze RNA-protein complexes, high salt or other stringent treatments are required for reducing nonspecific interactions and resolving the complex of interest. RNA-protein complexes often dissociate on native polyacrylamide gels and can only be detected on density gradients or by gel filtration. Agarose gel electrophoresis provides an alternative method that is simple, rapid, and can have high resolution of RNA-protein complexes. Moreover, the use of low-melting point agarose for the fractionation readily allows for the isolation of the RNA species in each complex detected on the native gel.

A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay.

In eukaryotes, a surveillance mechanism known as nonsense-mediated decay (NMD) degrades the mRNA when a premature-termination codon (PTC) is present. NMD requires translation to read the frame of the mRNA and detect the PTC. During pre-mRNA splicing, the exon-exon junction complex (EJC) is recruited to a region 20-24 nt upstream of the exon junction on the mature mRNA. The presence of a PTC upstream from the EJC elicits NMD. Eukaryotic initiation factor 4A (eIF4A) III is a nuclear protein that interacts physically or functionally with translation initiation factors eIF4G and eIF4B, respectively, and shares strikingly high identity with the initiation factors eIF4AI/II. Here we show that siRNA against eIF4AIII, but not against eIF4AI/II, inhibits NMD. Moreover, eIF4AIII, but not eIF4AI, is specifically recruited to the EJC during splicing. The observations that eIF4AIII is loaded onto the mRNA during splicing in the nucleus, has properties related to a translation initiation factor, and functions in NMD raises the possibility that eIF4AIII substitutes for eIF4AI/II during NMD.