OBJECTIVE

To examine the analgesic effect of preoperative administration of flurbiprofen axetil and that of postoperative administration of a combination of flurbiprofen axetil and fentanyl, as well as perioperative plasma β-endorphin (β-EP) levels in patients undergoing esophagectomy.

METHODS

Forty-five patients were randomly divided into three groups: group A: 100 mg flurbiprofen axetil preoperative, 10 μg/kg fentanyl + 10 ml placebo postoperative; group B: 100 mg flurbiprofen axetil preoperative, 10 μg/kg fentanyl + 100 mg flurbiprofen axetil postoperative; group C: 10 ml placebo preoperative, 10 μg/kg fentanyl + 10 ml placebo postoperative. Postoperative analgesia was achieved by intravenous infusion containing flurbiprofen axetil and/or fentanyl at 2.0 ml/h (total volume, 100 ml) using infusion pumps. The β-EP was measured at preanesthesia (T(1)), the end of surgery (T(2)), 24 h (T(3)), and 48 h (T(4)) after surgery. Visual analog scale scores (VAS) at T3, T4 (at rest), and rescue analgesic tramadol requirement was recorded.

RESULTS

The VAS of group B was significantly lower than group A and C (P < 0.01) at T(3) and T(4). The β-EP levels at T(2)-T(4) in group A did not differ significantly from those at T(1) (P>> 0.05); however, the β-EP levels in group B at T(3)-T(4) increased significantly (P < 0.05), while those in group C increased at T(2) and decreased at T(4) (P < 0.05). The β-EP levels in group B at T(3) and T(4) were the highest as compared to its levels in groups A and C (P < 0.01). Tramadol consumption in group B was significantly lower than in groups A and C (P < 0.01).

CONCLUSIONS

These results show that flurbiprofen axetil enhances the analgesic effect of fentanyl associated with increase in β-EP levels.

The present study was undertaken to examine the effect of age and pedalling frequency on metabolic internal power (MPint) and delta efficiency (deltaE), defined as the ratio of the change in external work accomplished to the change in energy expended, during sub-maximal exercise on a cycle ergometer. A group of II children [mean age (SD)][10.6 (1.0) years] and 12 adults [23.6 (3.0) years], all cyclists, performed two incremental tests at 60 rpm and 90 rpm in a randomised order. External power (EP) was measured as the product of friction load and pedalling frequency. Oxygen consumption (VO2) was measured using the Douglas bag method and an energy equivalent of 20.6 kJ x lO2(-1) was used to convert VO2 into metabolic power (MP). Linear relationships were drawn between MP and EP (MP = aEP + b) to enable the calculation of deltaE (I/a) and MPint (b). All coefficients of determination were greater than 0.97. The results showed that children and adults increased their deltaE with the increase in pedalling rate [27 (6)% to 36 (5)%, P < 0.05 in children and 27 (3)% to 30 (2)%, P < 0.05 in adults]. Likewise, net MPint (MPint minus basal metabolism) expressed relative to total leg volume was higher at 90 rpm compared to 60 rpm [16.5 (5.0) W x l(-1) and 4.2 (2.0) W x l(-1), P<0.05, respectively, in children and 7.4 (3.0) W x l(-1) and 5.0 (2.5) W x l(-1), NS, respectively, in adults]. At 60 rpm, children and adults showed the same deltaE and net MPint values. At 90 rpm, children showed significantly higher deltaE and net MPint compared to adults. This study demonstrated that deltaE and net MPint are equally influenced by increasing pedalling rate in children and adults. Furthermore, it was hypothesized that differences between children and adults at 90 rpm could be related to different anthropometric characteristics.

Prostaglandin E(2), which exerts its functions by binding to four G protein-coupled receptors (<em>EP</em>1-4), is implicated in tumorigenesis. Among the four E-prostanoid (<em>EP</em>) receptors, <em>EP</em>3 is unique in that it exists as alternatively spliced variants, characterized by differences in the cytoplasmic C-terminal tail. Although three <em>EP</em>3 variants, alpha, <em>beta</em>, and gamma, have been described in mice, their functional significance in regulating tumorigenesis is unknown. In this study we provide evidence that expressing murine <em>EP</em>3 alpha, <em>beta</em>, and gamma receptor variants in tumor cells reduces to the same degree their tumorigenic potential in vivo. In addition, activation of each of the three m<em>EP</em>3 variants induces enhanced cell-cell contact and reduces cell proliferation in vitro in a Rho-dependent manner. Finally, we demonstrate that <em>EP</em>3-mediated RhoA activation requires the engagement of the heterotrimeric G protein G(12). Thus, our study provides strong evidence that selective activation of each of the three variants of the <em>EP</em>3 receptor suppresses tumor cell function by activating a G(12)-RhoA pathway.

BACKGROUND

Multiparameter DNA flow cytometry using a one-laser bench-top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four-color analysis. Therefore, we optimized and extended our three-color method (Corver et al., 1994, Corver et al. 1996) to a four-color analysis of phenotypic intra-tumor heterogeneity using a bench-top flow cytometer.

METHODS

First, the effect of a range of different propidium iodide (PI) and TO-PRO-3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde-fixed lysolecithin-permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells. Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti-keratin antibodies. Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3-W vs. FL3-A). The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity. Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell-adhesion molecule (Ep-CAM; APC fluorescence), (b) keratin (R-phycoerythrin [RPE] fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein-isothiocyanate [FITC] fluorescence) to label stromal cells.

RESULTS

Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50-100 microM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25-2.0 microM. Average CVs were 2. 58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter- or intra-DNA stem line heterogeneity of Ep-CAM expression was observed in the keratin-positive fractions. Vimentin-positive, keratin-negative cells were restricted to the DNA diploid fraction.

CONCLUSIONS

PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four-color high-resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench-top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories.

Microdialysis relative recovery (RR) enhancement using different water-soluble, epichlorohydrin-based cyclodextrin polymers (CD-EPS) was studied in vitro for different analytes, amitryptiline, carbamazepine, hydroquinone, ibuprofen, and 4-nitrophenol. When compared to the native CDs (alpha, beta, and gamma) on a per mole basis, the CD-EPS enhanced microdialysis RR was either statistically greater or the same. beta-CD-EPS was more highly retained than native beta-CD by a 20 000 Da molecular weight cutoff (MWCO) polycarbonate membrane, but showed no statistical difference for loss across a 100 000 Da MWCO polyethersulfone membrane (PES). When the same weight percent of beta-CD or beta-CD-EPS was included in the microdialysis perfusion fluid, the beta-CD-EPS produced a higher microdialysis RR than native beta-CD for all analytes across the PES membrane. However, enhancements for the PC membrane were statistically insignificant when beta-CD and beta-CD-EPS were compared on a per mole basis. These results suggest that CD-EPS may be used as effective enhancement agents during microdialysis sampling and for some membranes provide the additional advantage of being retained more than native CDs.

The purpose of this best practice is to briefly define what has now been accepted regarding encapsulating peritoneal sclerosis (EPS), highlighting the latest developments and outlining future lines of research. The medical therapy that can be proposed (to be discussed individually, verifying the individual features of the patient) appears to include steroids, tamoxifen, and sirolimus or everolimus, with blood levels maintained at reference values for post-transplantation therapy. In view of the high incidence of relapse also in responders, it appears appropriate to continue therapy for prolonged periods, at least for 6 months. Moreover, a surgical assessment is indicated, especially for patients with intestinal symptoms including subocclusion status. To date the prevention of EPS is an unresolved issue. The recommended measures include the accurate prevention and best treatment of acute peritonitis, the use of biocompatible dialysis fluids (there is no consensus on their exact definition) and the monitoring of ultrafiltration characteristics and peritoneal membrane transport. Other recommended measures are the extensive use of renin-angiotensin- aldosterone axis inhibitors for the treatment of arterial hypertension in PD and the exclusion of beta- blockers. Other suggested strategies are tamoxifen prophylaxis in cases at risk and to adopt personalized immunosuppressive protocols for patients with PD who undergo renal transplantation.

The exopolysaccharide produced by a ropy strain of Lactobacillus spp. G-77 in a semi-defined medium, was found to be a mixture of two homopolymers composed of D-Glc. The two poly-saccharides were separated and, on the basis of monosaccharide and methylation analyses, 1H, 13C, 1D and 2D NMR experiments, one of the polysaccharides was shown to be a 2-substituted-(1-3)-beta-D-glucan, identical to that described for the EPS from Pediococcus damnosus 2.6 (M.T. Dueñas-Chasco, M.A. Rodríguez-Carvajal, P. Tejero-Mateo, G. Franco-Rodríguez, J. L. Espartero, A. Irastorza-Iribar, and A.M. Gil-Serrano, Carbohydr. Res., 303 (1997) 453-458), and the other polysaccharide was shown to consist of repeating units with the following structure [formula: see text]

In eukaryotic cells, when exposed to certain types of stress including hypoxia, eIF2α is phosphorylated by several kinases including protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK). Subsequently, protein translation is stopped and stress granules (SGs) are formed. Cancer cells form SGs under hypoxia. SGs accumulate apoptosis-related molecules and play anti-apoptotic roles. Thus, hypoxia-induced SG formation contributes to drug resistance in cancer cells. For this reason, inhibition of SG formation is expected to be beneficial in cancer therapy. To prove this concept, chemical reagents that inhibit SG formation are required as experimental tools. We searched for chemical compounds that suppress SG formation and identified that β-estradiol, progesterone, and stanolone (hereafter described as EPS) inhibit SG formation in human cervical cancer HeLa cells. As it turned out, EPS block PKR but not PERK, thus fail to suppress SG formation in most cancer cells, where SGs are formed via PERK. Nevertheless, in this study, we used HeLa cells as a model and demonstrated that EPS block hypoxia-induced SG formation in HeLa cells and consequently reduce drug resistance that HeLa cells acquire under hypoxia. Our findings support that inhibition of SG formation is a useful method to control cancers.

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.

Bacteroides is among the most abundant microorganism inhabiting the human intestine. They are saccharolytic bacteria able to use dietary or host-derived glycans as energy sources. Some Bacteroides fragilis strains contribute to the maturation of the immune system but it is also an opportunistic pathogen. The intestine is the habitat of most Bifidobacterium species, some of whose strains are considered probiotics. Bifidobacteria can synthesize exopolysaccharides (EPSs), which are complex carbohydrates that may be available in the intestinal environment. We studied the metabolism of B. fragilis when an EPS preparation from bifidobacteria was added to the growth medium compared to its behavior with added glucose. 2D-DIGE coupled with the identification by MALDI-TOF/TOF evidenced proteins that were differentially produced when EPS was added. The results were supported by RT-qPCR gene expression analysis. The intracellular and extracellular pattern of certain amino acids, the redox balance and the α-glucosidase activity were differently affected in EPS with respect to glucose. These results allowed us to hypothesize that three general main events, namely the activation of amino acids catabolism, enhancement of the transketolase reaction from the pentose-phosphate cycle, and activation of the succinate-propionate pathway, promote a shift of bacterial metabolism rendering more reducing power and optimizing the energetic yield in the form of ATP when Bacteroides grow with added EPSs. Our results expand the knowledge about the capacity of B. fragilis for adapting to complex carbohydrates and amino acids present in the intestinal environment.

We have previously found in human renal proximal tubular HK-2 cells that hypoxia- and all-trans retinoic acid-induced hypoxia-inducible factor-1α up-regulation is accompanied by retinoic acid receptor-β up-regulation. Here we first investigated whether hypoxia-inducible factor-1α expression is dependent on retinoic acid receptor-β and our results confirmed it since (i) hypoxia-inducible factor-1α-inducing agents hypoxia, hypoxia-mimetic agent desferrioxamine, all-trans retinoic acid and interleukin-1β increased retinoic acid receptor-β expression, (ii) hypoxia-inducible factor-1α up-regulation was prevented by retinoic acid receptor-β antagonist LE-135 or siRNA retinoic acid receptor-β and (iii) there was direct binding of retinoic acid receptor-β to the retinoic acid response element in hypoxia-inducible factor-1α promoter upon treatment with all-trans retinoic acid and 16,16-dimethyl-prostaglandin E(2). Since intracellular prostaglandin E(2) mediates hypoxia-inducible factor-1α up-regulation in normoxia in HK-2 cells, we next investigated and confirmed, its role in the up-regulation of retinoic acid receptor-β in normoxia by hypoxia-inducible factor-1α-inducing agents all-trans retinoic acid, interleukin-1β and 16,16-dimethyl-prostaglandin E(2) by inhibiting cyclooxygenases, prostaglandin influx transporter or EP receptors. Interestingly, the hypoxia-induced increase in retinoic acid receptor-β expression and accumulation of hypoxia-inducible factor-1α was also blocked by the inhibitors tested. This is the first time, to our knowledge, that retinoic acid receptor-β signalling is involved in the control of the expression of transcription factor hypoxia-inducible factor-1α in both normoxia and hypoxia and that retinoic acid receptor-β expression is found to be strictly regulated by intracellular prostaglandin E(2). Given the relevance of hypoxia-inducible factor-1α in the kidney in terms of tumorigenesis, progressive renal failure, production of erythropoietin and protection in several models of renal disease, our results open new therapeutic opportunities on the control of hypoxia-inducible factor-1α based upon the pharmacological modulation of retinoic acid receptor-β, either directly or through the control of intracellular prostaglandin E(2) levels/signalling.

BACKGROUND

There is growing evidence to suggest increased arterial stiffness in patients with a history of Kawasaki disease (KD). Pulse-wave velocity (PWV) is the most validated measure of arterial stiffness. The aim of this study was to determine if aortic PWV is increased in children with KD.

METHODS

This was a retrospective cohort study. The study cohort was composed of 42 patients with KD (mean age, 9.7 ± 2.0 years) and 44 age-matched control subjects. The primary measure was aortic PWV. Secondary measures included characteristic impedance (Zc), input impedance (Zi), elastic pressure-strain modulus (Ep), and β stiffness index and the following measures of left ventricular size and function: end-diastolic and end-systolic dimensions, wall thickness in diastole and systole, mass, shortening and ejection fractions, mean velocity of circumferential fiber shortening, and stress at peak systole. The appropriate measures were indexed to body surface area. The aortic stiffness and impedance indexes were derived using an echocardiography-Doppler method.

RESULTS

Height, weight, body mass index, and body surface area were similar between the groups. PWV was higher in patients with KD compared with controls (495 vs 370 cm/sec, P = .0008). Zc, Ep, and β stiffness index were higher in patients with KD, but the difference was not statistically significant. Left ventricular dimensions were all within normal limits, with no differences between the groups. Patients with KD had lower stress at peak systole compared with controls (55 vs 64 g/cm(2), P = .01). There was a significant association between the length of time between the initial diagnosis and testing with PWV (r = 0.32, P = .04) and Zi (r = -0.38, P = .01) in patients with KD. There was no significant association between the arterial stiffness indexes (PWV, Zi, Zc, Ep, and β stiffness index) and length of fever, age at KD diagnosis, or heart rate. Logistic regression analysis revealed no association between coronary artery lesion classification and length of fever, day of illness at first treatment, age at KD diagnosis, or any of the arterial stiffness indexes. In the control group, there were significant associations between age and heart rate (r = -0.48, P = .001), Zi (r = -0.55, P < .0001), Zc (r = -0.66, P < .0001), and β stiffness index (r = -0.31, P = .04). There was an association between heart rate and Zc (r = 0.44, P = .003) but no association between heart rate and PWV, Zi, Ep, or β stiffness index.

CONCLUSIONS

Arterial stiffness was increased in children with KD. There was no association between acute-phase KD coronary involvement and PWV. This implies that patients with KD may be at increased cardiovascular risk in the future.

Renal prostaglandin (PG) E2 regulates salt and water transport, and affects disease processes via <em>EP</em>1-4 receptors, but its role in the proximal tubule (PT) is unknown. Our study investigates the effects of PGE2 on mouse PT fluid reabsorption, and its role in growth, sodium transporter expression, fibrosis, and oxidative stress in a mouse PT cell line (MCT). To determine which PGE2 <em>EP</em> receptors are expressed in MCT, qPCR for <em>EP</em>1-4 was performed on cells stimulated for 24 h with PGE2 or transforming growth factor <em>beta</em> (TGFβ), a known mediator of PT injury in kidney disease. <em>EP</em>1 and <em>EP</em>4 were detected in MCT, but <em>EP</em>2 and <em>EP</em>3 are not expressed. <em>EP</em>1 was increased by PGE2 and TGFβ, but <em>EP</em>4 was unchanged. To confirm the involvement of <em>EP</em>1 and <em>EP</em>4, sulprostone (SLP, <em>EP</em>1/3 agonist), ONO8711 (<em>EP</em>1 antagonist), and <em>EP</em>1 and <em>EP</em>4 siRNA were used. We first show that PGE2, SLP, and TGFβ reduced H(3)-thymidine and H(3)-leucine incorporation. The effects on cell-cycle regulators were examined by western blot. PGE2 increased p27 via <em>EP</em>1 and <em>EP</em>4, but TGFβ increased p21; PGE2-induced p27 was attenuated by TGFβ. PGE2 and SLP reduced cyclinE, while TGFβ increased cyclinD1, an effect attenuated by PGE2 administration. Na-K-ATPase α1 (NaK) was increased by PGE2 via <em>EP</em>1 and <em>EP</em>4. TGFβ had no effect on NaK. Additionally, PGE2 and TGFβ increased fibronectin levels, reaching 12-fold upon co-stimulation. <em>EP</em>1 siRNA abrogated PGE2-fibronectin. PGE2 also increased ROS generation, and ONO-8711 blocked PGE2-ROS. Finally, PGE2 significantly increased fluid reabsorption by 31 and 46% in isolated perfused mouse PT from C57BL/6 and FVB mice, respectively, and this was attenuated in FVB-<em>EP</em>1 null mice. Altogether PGE2 acting on <em>EP</em>1 and <em>EP</em>4 receptors may prove to be important mediators of PT injury, and salt and water transport.

A polysaccharide named EP-1 was found by screening cultured mycelium of Hericium erinaceus, which was extracted and subjected to precipitation with ethanol, hollow-fiber ultrafiltration and ion-exchange chromatography. The polysaccharide has a molecular weight of approximately 3100Da and is composed of glucose, mannose and galactose, thus being a heteroglycan. EP-1 has a backbone of α-d-Glc(1→3) and β-d-Glc(1→3). The β-d-Glc(1→3) and α-d-Gal-(1→3) were regarded as branches attached to the C-4 position. The α-d-Man was regarded as a terminal residue. The anti-CAG activity was evaluated in experimental systems using a cell model for identification. The polysaccharide significantly inhibited the growth of MC cells obtained from human gastric mucosa epithelium (GES-1) cells transformed by MNNG, which were used as a chronic atrophic gastritis cell model. It also interfered with the MC cells by inducing cell cycle arrest. Thus, EP-1 shows potential for the development of new functional foods and drugs.

It has been demonstrated that neonatal administration of monosodium glutamate (MSG) results in a clearly defined lesion of the arcuate nucleus (AN) of the hypothalamus. The present study shows that fat was accumulated in the abdomen of male rats treated with MSG; weights of the body, pituitary and testis were lower; beta-EP content in hypothalamus decreased while L.EnK content increased; serum LH, FSH, TSH, GH and TS levels all decreased in varying degrees while serum PRL level significantly increased. The cAMP content lowered in pituitary, but nor in testes; clear histological changes occurred in testicular tissue; Se-GSH-Px activity in both testis and adrenal gland lowered while LPO level significantly increased. Both Se-GSH-Px activity and LPO level in liver increased. These results indicate that MSG is harmful to the function of the hypothalamus-pituitary-target system of neonatal rats.

Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (<em>EP</em>) receptors localized to the placenta. The activation of the COX-2/PGE2/<em>EP</em> signal pathway can alter the expression of the ATP-binding cassette (A<em>B</em>C) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: A<em>B</em>C<em>B</em>1], and breast cancer resistance protein (<em>B</em>CRP; gene: A<em>B</em>CG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in A<em>B</em>C transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and <em>B</em>CRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and <em>B</em>CRP in human placental cells. The treatment of placental cells with PGE2 up-regulated <em>B</em>CRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the <em>EP</em>1 and <em>EP</em>3 receptors with specific antagonists attenuated the increase in <em>B</em>CRP. <em>EP</em> receptor signaling results in activation of transcription factors, which can affect <em>B</em>CRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated <em>B</em> activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt <em>B</em>CRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental <em>B</em>CRP expression via <em>EP</em>1 and <em>EP</em>3 receptor signaling cascades.

<A<em>b</em>stractText>Human immunodeficiency virus, hepatitis <em>B</em> virus and hepatitis C virus are among the greatest threats to <em>b</em>lood safety for the recipient. They are also the leading cause of death, chronic and life-threatening a<em>b</em>normalities. Therefore, this study was aimed to assess the Sero-prevalence of HIV, Hepatitis <em>B</em> and C virus among <em>b</em>lood donors at the University of Gondar Comprehensive Specialized Hospital.</A<em>b</em>stractText><A<em>b</em>stractText>A retrospective cross-sectional study was used to estimate the seroprevalence of HIV, Hepatitis <em>B</em> and C virus among <em>b</em>lood donors at the University of Gondar Comprehensive Specialized Hospital from May-July 2018. Screening of HIV, H<em>B</em>V, and HCV was done <em>b</em>y using the Enzyme-Linked ImmunoSor<em>b</em>ent Assay. Records of 5983 first time <em>b</em>lood donors were collected and reviewed <em>b</em>y using a checklist from registration <em>b</em>ook. Data was entered in statistical package <em>EP</em> Info version 3.5.1, and data cleaned and analyzed using the statistical package SPSS version 16.0.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Of 5983 <em>b</em>lood donors, 85.5% (5118/5983) donors were males and 14.5% (865/5983) were females. The median age was 27 years and the highest <em>b</em>lood donations age category was <em>b</em>etween 20 to 51.2% (29/5983) followed <em>b</em>y 30 to 39 years of age, 21.6% (1295/5983). The prevalence of HIV, H<em>B</em>V and HCV infections were 2.5% (95% CI: 1.07-2.398), 4.1% (95% CI: 0.461-1.053) and 1.6% (95% CI: 0.845-3.354), respectively. HIV infection was significantly associated with gender (p = 0.021, x^{2 =} 5.358) and HCV infection with age group (p = 0.003, x² = 17.673). Of all donated <em>b</em>lood, 8.2% (489/5983) had serological evidence for at least one of the screened pathogens and 58 (0.96%) of them had multiple infections.</p><A<em>b</em>stractText>This study showed a significant prevalence of HIV, H<em>B</em>V, and HCV among <em>b</em>lood donors, 2.5% (147/5983), 4.1% (244/5983) and 1.6% (98/5983), respectively. Therefore, strict selection of <em>b</em>lood donors with an emphasis on getting voluntary <em>b</em>lood donors, and highly sensitive and specific tests for screening of <em>b</em>lood donors for HIV, H<em>B</em>V, and HCV using standard methods are highly recommended to ensure the safety of <em>b</em>lood for the recipient.</A<em>b</em>stractText>

Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [3H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His6 protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of beta-structure; however, alpha-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.

This is a cross-sectional molecular epidemiological study on equine piroplasmosis (EP) affecting horses and donkeys in the Sudan. The study evaluated 499 samples from geographically distinct regions in eastern, central and western parts of the country. PCR amplification of the 18S rRNA gene of both Thelieria equi and Babesia caballi was carried out. Horses from all sampled areas were found positive to T. equi DNA but no B. caballi was detected. Absence of B. caballi infection was confirmed by another PCR targeting the B. caballi 48-kDa merozoite antigen. The overall prevalence was found to be 35.95%. The highest prevalence was detected in Showak 13 (81.3%) and the lowest was in Shearia locality in South Darfur 1 (5.6%). In another experiment, capillary electrophoresis was used to detect and differentiate between T. equi and B. caballi using one set of primers designed to amplify the 18S rRNA gene in a single PCR. Capillary electrophoresis method was found to be powerful in detecting mixed infections in artificially mixed controls samples. The data obtained in this study would contribute to the development of a national control strategy of EP in the Sudan.

This study aimed to improve the production of polysaccharide by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of α-phosphoglucomutase (PGM) gene. PGM is responsible for the linkage between sugar catabolism and sugar anabolism. The effects of PGM gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including PGM, UDP-glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the PGM gene were 23.67 mg/100 mg dry weight and 1.76 g/L, respectively, which were higher by 40.5 and 44.3% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were upregulated by 4.77-, 1.51- and 1.53-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

OBJECTIVE

To evaluate two different iodine concentrations of contrast material for detecting hypervascular hepatocellular carcinomas (HCCs) in cirrhotic liver by multi-detector row helical CT (MDCT) when a fixed contrast material volume and injection rate is used.

METHODS

Institutional Review Board approval was obtained, and informed consent was obtained from all patients. In this prospective study, 105 patients were randomly assigned a group A (an iodine concentration of 300 mg I/mL), and a group B (an iodine concentration of 370 mg I/mL). In both groups the volume of contrast material was 100 mL and the injection rate was 4 mL/s. Fifty-two patients had 122 hypervascular HCCs. The diagnosis of HCCs was established histopathologically (n=24) and by imaging findings (n=98). Three readers independently analyzed four image sets: an arterial phase (AP), a portal phase (PP), an equilibrium phase (EP), and combined all three phase images set. Sensitivity, specificity, and diagnostic accuracy were calculated by receiver operating characteristic (ROC) analysis.

RESULTS

The mean sensitivity for detecting hypervascular HCCs of the AP set, EP set, and combination set in group B (0.94, 0.81, and 0.93) was significantly higher than in group A (0.84, 0.69, and 0.80). Area under the ROC curve of the AP set and the combination set in group B (0.974 and 0.981) was significantly higher than in group A (0.939 and 0.958).

CONCLUSIONS

At the same contrast material volume and injection rate, higher iodine concentration of contrast material was effective for detecting hypervascular HCCs by MDCT.

Rhizosphere colonization <em>b</em>y plant growth-promoting rhizo<em>b</em>acteria (PGPR) along plant roots facilitates the a<em>b</em>ility of PGPR to promote plant growth and health. Thus, an understanding of the molecular mechanisms of the root colonization process <em>b</em>y plant-<em>b</em>eneficial <i><em>B</em>acillus</i> strains is essential for the use of these strains in agriculture. Here, we o<em>b</em>served that an <i>sfp</i> gene mutant of the plant growth-promoting rhizo<em>b</em>acterium <i><em>B</em>acillus velezensis</i> SQR9 was una<em>b</em>le to form normal <em>b</em>iofilm architecture, and differential protein expression was o<em>b</em>served <em>b</em>y proteomic analysis. A minor wall teichoic acid (WTA) <em>b</em>iosynthetic protein, GgaA, was decreased over 4-fold in the Δ<i>sfp</i> mutant, and impairment of the <i>ggaA</i> gene postponed <em>b</em>iofilm formation and decreased cucum<em>b</em>er root colonization capa<em>b</em>ilities. In addition, we provide evidence that the major WTA <em>b</em>iosynthetic enzyme Gta<em>B</em> is involved in <em>b</em>oth <em>b</em>iofilm formation and root colonization. The deficiency in <em>b</em>iofilm formation of the Δ<i>gta<em>B</em></i> mutant may <em>b</em>e due to an a<em>b</em>sence of UDP-glucose, which is necessary for the synthesis of <em>b</em>iofilm matrix exopolysaccharides (<em>EPS</em>). These o<em>b</em>servations provide insights into the root colonization process <em>b</em>y a plant-<em>b</em>eneficial <i><em>B</em>acillus</i> strain, which will help improve its application as a <em>b</em>iofertilizer.(<em>b</em>)IMPORTANCE</<em>b</em>)<i><em>B</em>acillus velezensis</i> is a Gram-positive plant-<em>b</em>eneficial <em>b</em>acterium which is widely used in agriculture. Additionally, <i><em>B</em>acillus</i> spp. are some of the model organisms used in the study of <em>b</em>iofilms, and as such, the molecular networks and regulation systems of <em>b</em>iofilm formation are well characterized. However, the molecular processes involved in root colonization <em>b</em>y plant-<em>b</em>eneficial <i><em>B</em>acillus</i> strains remain largely unknown. Here, we showed that WTAs play important roles in the plant root colonization process. The loss of the <i>gta<em>B</em></i> gene affects the a<em>b</em>ility of <i><em>B</em>. velezensis</i> SQR9 to sense plant polysaccharides, which are important environmental cues that trigger <em>b</em>iofilm formation and colonization in the rhizosphere. This knowledge provides new insights into the <i><em>B</em>acillus</i> root colonization process and can help improve our understanding of plant-rhizo<em>b</em>acterium interactions.

METHODS

Basic science Introduction: Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3 Figure 1 Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)-Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time Objective: The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics.

METHODS

Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5). Figure 2 Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: BoneFigure 3 Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc spaceFigure 4 Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a-c H&E (overall tissue staining for light micrsocopy) d-f Alcian blue (proteoglycans) g-i Picrosirius red (collagen I and II)Figure 5 Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body boneFigure 6 MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NPFigure 7 7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP) Results: The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months.

CONCLUSIONS

This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d-f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants.