Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer.

MicroRNA-155 (miR-155) is frequently upregulated in various types of human cancer; however, its role in cancer angiogenesis remains unknown. Here, we demonstrate the role of miR-155 in angiogenesis through targeting von Hippel-Lindau (VHL) tumour suppressor in breast cancer. Ectopic expression of miR-155 induced whereas knockdown of miR-155 inhibited human umbilical vein endothelial cell network formation, proliferation, invasion and migration. Furthermore, mammary fat pad xenotransplantation of ectopically expressed miR-155 resulted in extensive angiogenesis, proliferation, tumour necrosis and recruitment of pro-inflammatory cells such as tumour-associated macrophages. Expression of VHL abrogated these miR-155 effects. Moreover, miR-155 expression inversely correlates with VHL expression level and is associated with late-stage, lymph node metastasis and poor prognosis, as well as triple-negative tumour in breast cancer. These findings indicate that miR-155 has a pivotal role in tumour angiogenesis by downregulation of VHL, and provide a basis for miR-155-expressing tumours to embody an aggressive malignant phenotype, and therefore miR-155 is an important therapeutic target in breast cancer.

Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells.

Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.

DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice.

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.

Operative treatment of acetabular fractures through the extensile Henry approach.

OBJECTIVE: The purpose of this study was to evaluate the previously unreported application of the extensile Henry approach to the operative treatment of acetabular fractures. METHODS: Thirty-one cases were retrospectively reviewed at an average follow-up of 18.5 months. RESULTS: There were 8 simple and 23 complex associated fracture patterns. The average operative time was 4.5 hours, and the average blood loss was 1,160 mL. Reduction was anatomic in 26 patients (84%), satisfactory in 4 patients (13%), and unsatisfactory in 1 patient (3%). Radiographic results at follow-up were 25 excellent results, 4 good results, and 2 poor results. Twenty-six patients reported no limitation of ordinary activities, whereas five patients had to modify their activities because of pain. No heterotopic ossification occurred in 24 patients (77%). In the seven patients with heterotopic ossification, only one patient had a significant decrease in hip range of motion. Additional complications were two cases of superficial wound infection, one case of hardware failure, and two cases of avascular necrosis of the femoral head. There were no iatrogenic injuries to the sciatic nerve, nor was there any development of flap necrosis. CONCLUSION: The extensile Henry approach is a versatile approach offering an excellent exposure for surgical treatment of acetabular fractures. The rate of complications is comparable with or lower than that of other surgical approaches. By providing a direct exposure of the posterior pelvis, the extensile Henry approach has the advantage of minimizing the risk of iatrogenic injury to the sciatic nerve. In addition, the incidence of clinically significant heterotopic ossification may be reduced through the use of low-dose radiation prophylaxis.

Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever.

Two flaviviruses, dengue (DEN) virus and Japanese encephalitis (JE) virus, are important because of their global distribution and the frequency of epidemics in tropical and subtropical areas. To study the B-cell epitopes of nonstructural 1 (NS1) glycoprotein and anti-NS1 antibody response in DEN infection, a series of 15-mer synthetic peptides from the predicted B-cell linear epitopes of DEN-2 NS1 protein were prepared. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze antibody responses to these peptides from sera of both DEN and JE patients. One peptide derived from DEN-2 NS1, D2 NS1-P1 (amino acids 1-15), was identified as the immunodominant epitope that reacted with sera from dengue fever (DF) patients but not JE patients. The isotype of D2 NS1-P1-specific antibodies was mainly immunoglobulin M (IgM) in all sera that tested positive. A specificity study demonstrated that sera from all four DEN types reacted with D2 NS1-P1. A dynamics study showed that specific antibodies to this peptide could be detected as early as 2 days after the onset of symptoms. We observed significant anti-D2 NS1-P1 antibody responses in 45% of patients with primary and secondary infections with DF or with dengue hemorrhagic fever. This is the first report demonstrating that significant anti-DEN NS1 antibodies can be induced in the sera of patients with primary DEN infection.

Optical bit-pattern recognition by use of dynamic gratings in erbium-doped fiber.

We present a novel optical bit-pattern-recognition technique that uses erbium-doped fiber at room temperature. Counterpropagating beams write a patterned gain-depletion grating in pumped erbium-doped fiber. This grating, recorded in the erbium gain medium, can be used for correlation with other optical bit patterns. We have demonstrated correlation of arbitrary return-to-zero bit patterns of as many as 8 bits at 1 Gbit/s . Theory suggests that scaling to higher bit rates is feasible.

Purification and characterization of a 94 KD high molecular weight allergen from house dust mite, Dermatophagoides pteronyssinus.

House dust mite allergens from Dermatophagoides pteronyssinus is an important cause of severe allergic asthma and rhinitis in many countries. Although several low to medium molecular weight allergens had been well characterized, limited studies on the high molecular weight IgE-binding components were reported. In this study, a 94 kD high molecular weight allergen from crude mite body extract of D. pteronyssinus was purified and characterized. Monoclonal antibody (mAb) affinity chromatography and high performance liquid chromatography were used to purify 94 kD allergen. Its antigenicity and allergenicity were confirmed by in vitro and in vivo studies. Two mAbs 2205-3.45 and 2220-7.25 specific to 94 kD high molecular weight component of D. pteronyssinus were generated. The epitopes recognized by these mAbs were species-specific. Enzyme-linked immunosorbent assay (ELISA) of IgE reactivity in the sera from 40 asthmatic children allergic to D. pteronyssinus showed that 37.5% of them had significantly higher optical density values (range 0.011 to 0.452) than normal (range 0.013 to 0.035). In in vivo skin test showed that 9 out of 20 (45%) asthmatic children were positive to 94 kD allergen. The results demonstrate that 94 kD high molecular weight component is an important allergen existing in house dust mite in Taiwan.

Native-like beta-structure in a trifluoroethanol-induced partially folded state of the all-beta-sheet protein tendamistat.

The effect of trifluoroethanol (TFE) on the structure of the all-beta-sheet protein tendamistat was investigated. At low concentrations TFE induces cooperative loss of the native tertiary structure leading to a partially folded state. The loss of specific side-chain interactions in the transition from the native state of the TFE-induced state is demonstrated by the disappearance of the CD bands in the aromatic region, a reduced chemical shift dispersion of the one-dimensional 1H NMR spectrum and a broad, uncooperative thermal unfolding transition of the partially folded state. An increased line-width of the NMR bands in the TFE state compared with the unfolded state suggests the presence of multiple, rapidly interconverting conformations. Hydrogen-exchange studies of amide proteins in the TFE state reveal the existence of defined hydrogen bonds at the same locations as in the native state, but with largely reduced stability. This suggests the presence of most of the native beta-sheet structure. These results are supported by Fourier transformed IR measurements, which show nearly the same amount of beta-structure in the TFE state and in the native state. Far UV CD spectroscopy suggests the induction of some alpha-helical structure upon addition of TFE, which appears to be located mainly in regions corresponding to loops or random structure in the native state and which seems to represent fluctuating conformations with preferred backbone angles rather than stable, hydrogen-bonded alpha-helices. These results show that stable non-local interactions, as they occur in beta-sheets, can form in the absence of specific side-chain interactions. The presence of a subset of the native long-range interactions and the absence of stable non-native interactions suggests that the observed partially folded state might represent an early intermediate on a hierarchical folding pathway of tendamistat.

Identification of immunodominant, group-specific and subcomplex-specific, continuous epitopes in the core regions of Japanese encephalitis virus using synthetic peptides.

Two flaviviruses, Japanese encephalitis (JE) virus and Dengue (DEN) virus which have high pathogenicity for humans, continue to pose a serious public health problem in tropical and subtropical countries of the world. In order to identify the immunodominant B-cell epitopes for diagnostic application, we have prepared a series of 15-mer synthetic peptides from JE virus core protein based on computer analysis. Four linear, immunodominant epitopes corresponding to amino acids 91-105 (P78), 1-15 (P73), 8-22 (P74), and 34-48 (P75) of JE virus core proteins were identified by employing an enzyme-linked immunosorbent assay (ELISA), using high-titered immune sera from JE-vaccinated children. P78 was found to be the most immunodominant. The sero-specificity of these peptides was tested by binding to seroconverted samples from JE and DEN-1 patients. P78 and P74 belonged to group-specific epitopes which reacted with both JE and DEN-1 patient sera. P73 and P75 belonged to subcomplex-specific epitopes which reacted only with JE but not with DEN-1 patient sera. The study suggests that these peptides corresponding to the immunodominant epitopes of JE virus core protein might have the potential to be used as peptide-based diagnostic reagents for the detection and differentiation of JE and DEN antibody responses.

Redislocation with a bony bankart lesion after arthroscopic Bankart repair.

This case report presents a traumatic dislocation after an arthroscopic Bankart repair in which the repair site was intact and failure occurred through a new bony interval. The new bony Bankart lesion was identified 3 years after the initial arthroscopic repair. This highlights the issue of whether a traumatic redislocation after a surgical repair for recurrent shoulder instability represents a failure of repair or a reinjury.

Localization and quantification of endoplasmic reticulum Ca(2+)-ATPase isoform transcripts.

The Ca(2+)-adenosinetriphosphatase pump of the sarcoplasmic or endoplasmic reticulum (SERCA) plays a critical role in Ca2+ signaling and homeostasis in all cells and is encoded by a family of homologous and alternatively spliced genes. To understand more clearly the role the different isoforms play in cell physiology, we have undertaken a quantitative and qualitative assessment of the tissue distribution of transcripts encoding each SERCA isoform. SERCA1 expression is restricted to fast-twitch striated muscles, SERCA2a to cardiac and slow-twitch striated muscles, whereas SERCA2b is ubiquitously expressed. SERCA3 is expressed most abundantly in large and small intestine, thymus, and cerebellum and at lower levels in spleen, lymph node, and lung. In situ hybridization analyses revealed SERCA3 transcripts in cells of the intestinal crypt, the thymic cortex, and Purkinje cells in cerebellum. In addition, SERCA3 was expressed abundantly in isolated rat spleen lymphocytes, in various murine lymphoid cell lines, and in primary cultured microvascular endothelial cells. This analysis demonstrates that SERCA3 is expressed selectively in cells in which Ca2+ signaling plays a critical and sensitive role in regulating physiological processes.

Optical clock recovery from a data stream of an arbitrary bit rate by use of stimulated Brillouin scattering.

Using a fiber-optic stimulated-Brillouin-scattering amplifier as an active filter, we have demonstrated optical clock recovery from 5-Gbit/s return-to-zero-format optical data. Definite patterns and pseudorandom bit sequences were tested. This scheme requires no prior knowledge of the clock frequency and is well suited for operation at higher data rates.

Evaluation of viral membrane fusion assays. Comparison of the octadecylrhodamine dequenching assay with the pyrene excimer assay.

Membrane fusion, in particular the fusion of enveloped viruses, is often measured with an assay based on octadecylrhodamine (R18) fluorescence dequenching. We have studied the association of R18 with membranes and used the R18 assay to measure virus fusion in model systems and in cultured cells. The results were compared with those of an assay based on the decrease in excimer fluorescence of pyrene-labeled phospholipids. For liposomes made from premixed R18 and phosphatidylcholine (PC), R18 fluorescence quenching was proportional to the concentration of the probe up to about 4 mol %. No quenching was found at very low concentrations of R18. However, various artificial and biological membranes labeled by the addition of R18 from an ethanolic solution showed significant quenching at such low R18 concentrations. Thus, some of the R18 was not randomly distributed but likely was associated with the surface of the membranes in the form of highly quenched clusters or micelles. Moreover, in influenza virus membranes, R18 appeared highly quenched at very low concentrations, indicative of the probe interacting with viral proteins. In contrast, pyrene-labeled PC incorporated in either liposomes or reconstituted viral membranes (virosomes) showed an excimer/monomer fluorescence ratio proportional to the concentration of probe. When intracellular membrane fusion was investigated with R18-labeled influenza virus or Semliki Forest virus (SFV), fluorescence dequenching was observed in the absence of fusion, most likely due to spontaneous probe exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of cholecystokinin and secretin by sodium oleate in dogs: molecular form and bioactivity.

The release of cholecystokinin (CCK) and secretin into both circulation and duodenal lumen, after intraduodenal perfusion with sodium oleate or oral ingestion of fat, was studied in anesthetized and conscious dogs, respectively. Intraduodenal infusion with sodium oleate (4 mmol.kg.-1.h-1, pH 9.5) in anesthetized dogs with diversion of bile and pancreatic juice stimulated the release of both CCK and secretin not only into the circulation but also into the duodenal lumen. The concentration of CCK and secretin in the luminal perfusate increased from 0.2 +/- 0.1 to 2.1 +/- 0.4 nM and 0.34 +/- 0.16 to 2.59 +/- 0.63 nM, respectively. Intraduodenal infusion of NaHCO3 solution at pH 9.5 did not result in release of either hormone. Luminal release of both hormones was also observed by intraduodenal infusion of sodium oleate in the dogs without diversion of bile and pancreatic juice, albeit at lower concentrations than those released in the dogs with diversion. Analysis of the molecular form of luminal secretin by gel filtration, ion-exchange chromatography, and high-performance liquid chromatography showed only a single form of secretin with molecular size, hydrophobicity, and charge similar to those of natural porcine secretin. In contrast, multiple forms of CCK were released into both circulation and duodenal lumen with CCK-58 as the predominant form. In conscious dogs, CCK-58 was also found to be the predominant form of CCK released into the circulation after oral ingestion of fat.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane interaction of 'peptide P' derived from the repeating motif of properdin.

A 24 amino acid residue peptide corresponding to the central part of the 'thrombospondin-repeat' motif of the human serum protein properdin was synthesized. The peptide, termed 'peptide P', contains three tryptophans near the N-terminus and an arginine cluster close to the C-terminus. Its sequence closely matches a consensus sequence which has been claimed to characterize a sulfatide binding motif. Membrane binding of peptide P was analyzed using changes in its tryptophan emission upon adding small unilamellar vesicles. The peptide bound to the membranes in a way suggesting simple water/membrane partitioning. Analysis of electrostatic effects at different ionic strengths indicated small electrostatic contributions upon interaction with zwitterionic lipid, despite the large charge number (z = +4) of the peptide. Membrane affinity was increased by one order of magnitude if the bilayers contained 20% of negatively charged lipid. No difference could be detected whether the charged lipid was sulfatide or phosphatidylglycerol. Strong and rapid vesicle aggregation was evident as the peptide associated with the negatively charged vesicles. In addition, a fluorescent energy transfer assay with vesicles and internal total reflection fluorescence microscopy on supported bilayers were used to study membrane interaction of whole human properdin. No sulfatide specificity could be detected.

Psychostimulants for depression in the medically ill.

Medically ill patients who show signs of depression may have problems with traditional antidepressant therapy, because of the side effect profile and the delayed onset of action of these agents. Psychostimulants such as methylphenidate and dextroamphetamine are another treatment option. The beneficial effects of these drugs are usually noted within 36 hours, and drug habituation is generally not a problem. The primary obstacle to the use of these agents for depression in medically ill patients is the hesitancy of physicians to prescribe them.

Dysregulation of atrial natriuretic factor in hypertension-prone man.

To evaluate the hypothesis of an atrial natriuretic factor (ANF) deficiency in hypertension-prone humans, we investigated plasma ANF and other variables in 116 white offspring of normotensive parents (ONorm) or essential hypertensive parents (OHyp). Ten ONorm and 10 OHyp, all men matched for age and body habitus, were studied after 4 days of low (70 mmol/day) and high (350 mmol/day) dietary sodium intake. After mild sodium restriction, plasma ANF did not differ between ONorm and OHyp (9.7 +/- 0.7 vs. 9.0 +/- 1.3 fmol/L). On high sodium intake, plasma ANF increased in ONorm, but not in OHyp (to 18.3 +/- 1.7 vs. 11.7 +/- 1.7 fmol/L; P less than 0.001). On the other hand, acute responses of plasma immunoreactive ANF (irANF) to saline loading or a norepinephrine-induced rise in blood pressure did not differ significantly between 8 ONorm and 8 OHyp. Fifty-one additional ONorm and 45 OHyp were evaluated during liberal sodium intake. Groups were further subdivided according to whether 24-h urinary sodium excretion was 91 mmol/m2 or less (modest salt intake) or more than 91 mmol/m2 (high salt intake). Twenty-four-hour urinary sodium was similar in the 26 ONorm and 21 OHyp on a modest salt intake (121 +/- 6 vs. 116 +/- 9 mmol) and in the 25 ONorm and the 24 OHyp on a high salt intake (226 +/- 10 vs. 221 +/- 9 mmol). However, compared with ONorm, plasma irANF in OHyp was slightly lower on modest sodium intake (7.7 +/- 0.7 vs. 5.3 +/- 0.7 fmol/L; P less than 0.05) and markedly reduced on high sodium intake (15.0 +/- 1.3 vs. 8.0 +/- 1.3 fmol/L; P less than 0.001). Moreover, the slope of the relationship between plasma irANF and 24-h urinary sodium was flatter in OHyp than in ONorm (z test = 2.4). We postulate a new endocrine syndrome characterized by a relative plasma ANF deficiency during high sodium intake in some hypertension-prone humans. This functional defect becomes apparent during chronic, rather than acute, stimulation of ANF release. It occurs as a familial disturbance and may potentially predispose to the development of hypertension.

Modification of water-soluble coal-derived products by dibenzothiophene-degrading microorganisms.

To study mechanisms by which microorganisms oxidize thiophenic sulfur in coal, we tested bacterial cultures for the ability to degrade dibenzothiophene (DBT), DBT-5-oxide, and DBT-sulfone and to modify water-soluble coal products derived from Illinois no. 6 and Ugljevik coals. In yeast extract medium, the majority of selected isolates degraded DBT and accumulated DBT-5-oxide in culture fluids; all but one of the cultures degraded DBT-5-oxide, and none of them degraded DBT-sulfone. Elemental analysis data indicated that the microbial cultures were able to decrease the amount of sulfur in soluble coal products derived from Illinois no. 6 and Ugljevik coals. However, these data suggested that microbially mediated sulfur removal from soluble Ugljevik coal occurred by nonspecific mechanisms. That is, extensive degradation of the carbon structure was concurrent with the loss of sulfur. This conclusion was supported by X-ray photoelectron spectroscopic data which indicated that the reduced sulfur forms in the soluble Ugljevik coal product was not oxidized by microbial treatment.

Ulnar nerve injury with open-heart surgery.

The effects of different arm positions during open-heart surgery on the incidence of ulnar nerve injury were studied prospectively in 35 patients. It was concluded that ulnar nerve damage was common and was due to nerve compression at the elbow. A significant reduction in the incidence of nerve injury can be effected by placing the forearms above the head rather than at the patient's side.