Strategy for designing specific antisense oligonucleotide sequences

Antisense compounds, various forms of nucleotides or their analogs, inhibit gene function both in vitro and in vivo. Although antisense compounds have been used extensively not only as a basic research tool but also as therapeutics for various diseases, one of the major problems is the difficulty of obtaining optimal sequences to inhibit specific gene functions. Although the terms “sequence-specificity” or “sequence-nonspecificity” are often used, there is no consensus as to how to define and quantitate such sequence specificity. In this review, we introduced hybridization simulation for designing optimal antisense sequences. Each candidate antisense oligonucleotide is assessed by calculating its hybridization energy against potential hybridization sites within the specified database (including Genbank) using a realistic nearest-neighbor thermodynamic model, taking into account mismatches. The specificity of each oligonucleotide is then quantitated by the number of potential cross-hybridizable genes and their degree of cross-hybridization. Further-more, if antisense sequences exhibit a high potential for hairpin formation, they are not recommended even if they are highly specific. Therefore, to select antisense sequences, one should calculate all the potential factors for each candidate oligonucleotide such as length, location, specificity, hairpin potential, mRNA secondary structure, and dimer formation.     

A case of pulmonary infection caused by Mycobacterium szulgai

We report a case of pulmonary non-tuberculous mycobacteriosis caused by Mycobacterium szulgai. A thirty-nine-year-old man with no relevant significant past history underwent an annual medical check. His chest X-ray and CT scan showed an infiltrative shadow with a cavity in the right upper lobe. As it was suggestive of pulmonary tuberculosis, he was referred to our hospital. Smear tests of his sputum, gastric fluid, and transbronchial fluid showed no mycobacterial organisms, but culture of the samples revealed growth of mycobacteria. The organism was identified as M. szulgai using a DNA-DNA hybridization method, and the case was diagnosed as pulmonary non-tuberculous mycobacteriosis caused by M. szulgai. By anti-mycobacterial drug treatment with isoniazid, rifampicin, and ethambutol, the infiltrative shadow on chest roentogenogram and CT showed improvement. Culture of his sputum and gastric fluid showed no growth of mycobacteria after starting treatment.     

Mechanical stress-dependent secretion of interleukin 6 by endothelial cells after portal vein embolization: clinical and experimental studies

BACKGROUND/AIMS: Interleukin-6 (IL-6) is an essential early signal in liver regeneration, however, little is known about what triggers IL-6 release. Changes in portal hemodynamics after portal vein embolization (PVE) may contribute to IL-6 release, leading to regeneration of non-embolized lobe.METHODS: In 22 patients who underwent right PVE, the diameters of the left portal branches, liver volumes, and serum concentrations of IL-6, tumor necrosis factor-alpha (TNF-alpha), and hepatocyte growth factor (HGF) were measured. We then studied endothelial cells cultured on an elastic silicone membrane and subjected to continuous uni-axial stretch. Supernatant cytokine concentrations were measured.RESULTS: The diameters of the portal branches increased by 150% after PVE. Serum IL-6 concentrations increased within 3h after PVE. The concentrations of TNF-alpha and HGF remained unchanged. The left lobe volume increased 2 weeks after PVE. The IL-6 concentrations in the supernatant of endothelial cells with stretch stress were higher than that in the non-stretched control group.CONCLUSIONS: These findings indicate that PVE dilates the portal branches in the non-embolized lobe, exposing hepatic vasculature to stretch stress. This hemodynamic change may act as a trigger for IL-6 release from endothelial cells and contribute to the activation of regenerative cascade in the non-embolized lobes.     

Bradykinin improves left ventricular diastolic function under long-term angiotensin-converting enzyme inhibition in heart failure

Both systolic and diastolic cardiac dysfunction coexist in various degrees in the majority of patients with heart failure. Although ACE inhibitors are useful in the treatment of heart failure, the roles of bradykinin in the systolic and diastolic properties of left ventricular function under long-term treatment of ACE inhibitor have not been fully elucidated. We therefore evaluated the changes in left ventricular function, histomorphometry, and the expression of several failing heart related genes, by use of an orally active specific bradykinin type 2 receptor antagonist, FR173657 (0.3 mg/kg per day), with an ACE inhibitor, enalapril (1 mg/kg per day), in dogs with tachycardia-induced heart failure (270 ppm, 22 days) and compared the effects to enalapril alone. Although there were no differences observed in blood pressure, left ventricular dimension, and percentage of fractional shortening, FR173657 significantly increased left ventricular filling pressure (P<0.01), prolonged the time constant of relaxation (P<0.05), and suppressed the expression of endothelial NO synthase and sarcoplasmic reticulum Ca(2+)-ATPase mRNA (P<0.05). FR173657 also upregulated collagen type I and III mRNA (P<0.05) and increased the total amount of cardiac collagen deposits (P<0.05) in left ventricle compared with that in the enalapril-treated group. In conclusion, endogenous bradykinin contributes to the cardioprotective effect of ACE inhibitor, improving left ventricular diastolic dysfunction rather than systolic dysfunction, via modification of NO release and Ca(2+) handling and suppression of collagen accumulation.     

Outcomes of redo surgery for failed laparoscopic fundoplication

Purpose

The number of operations for laparoscopic antireflux surgery in Japan is much less than that in Western countries. This study’s aim was to evaluate outcome measures for redo antireflux surgery (redo-ARS) in Japanese patients.

Methods

Subjects consisted of 11 patients (2.3 %) who required redo-ARS, from an original group of 474 patients who had a primary ARS between December 1994 and January 2015. The mean age of the subjects was 57.7 years, and six of 11 patients were women (55 %). Clinical data were collected in a prospective manner, and were then reviewed retrospectively.

Results

The most common cause of failed primary ARS was dislocation of the wrap (6/11 or 54 %). Of the 11 patients, 10 (91 %) were approached laparoscopically, with one requiring conversion to open surgery. Eight (73 %) underwent redo fundoplication, and the others had hiatal hernia repair alone. Mean operation time and blood loss were 202 min and 56 mL, respectively. A perioperative gastric wall injury occurred in three patients. The postoperative course was uneventful in majority patients. Three (27 %) were required to take proton pump inhibitor (PPI), and two (18 %) had a recurrence of hiatal hernia. A postoperative questionnaire was answered by seven of 11 (63 %), and these all reported a high level of satisfaction with their surgery.

Conclusions

Redo-ARS can be performed safely under laparoscopy. There was no recurrence rate in almost 80 %, and more than 70 % of patients were withdrawn from PPI treatment postoperatively.

     

Idiopathic chronic calcifying pancreatitis with diabetes mellitus

Summary We present a case of a 27-year-old female suffering from chronic calcifying pancreatitis with diabetes mellitus. Radiographic examinations and exocrine pancreatic function tests revealed considerable dilatation of pancreatic ducts with large intraductal calculi and exocrine pancreatic insufficiency, respectively. Recent literature indicates that a decrease in the activity of pancreatic stone protein (PSP), which inhibits CaCO₃ crystal formation in pancreatic juice, is closely related to the development of chronic calcifying pancreatitis. The patient had no apparent cause or family history of pancreatitis. We therefore investigated the possibility that alterations in the PSP gene might explain the chronic pancreatitis seen in this patient. Six exons of the PSP gene amplified by polymerase chain reaction were directly sequenced, but there was no apparent base mutation observed. Furthermore, Southern blot analysis revealed neither rearrangement nor deletion of the PSP gene in the genomic DNA of this case. However, this genetic approach will be useful for future study of the etiology of hereditary pancreatitis.     

The effect of tumor necrosis factor-alpha on tissue specificity and selectivity to insulin signaling.

Recent studies have indicated that tumor necrosis factor (TNF)-alpha plays a significant role in insulin resistance. It has been proposed that selective impairment of insulin signaling in glucose metabolism is related to the development of atherosclerosis, although the mechanisms are not clear. The aim of this study was to elucidate the effect of TNF-alpha on tissue specificity and selectivity to insulin signaling. L6 myotubes and rat aortic vascular smooth muscle cells (VSMC) were cultured. Cells were stimulated with insulin pretreated with or without TNF-alpha. The protein extracts were used for electrophoresis and immunoblotting studies to examine phosphorylation of insulin receptor (IR)-beta, insulin receptor substrate (IRS)-1 and extracellular signal-regulated kinase (ERK). IR-beta phosphorylation was not affected by TNF-alpha in L6 or in VSMC. TNF-alpha significantly (p<0.05) inhibited IRS-1 phosphorylation by insulin but had no effect on ERK in L6. TNF-alpha had no effect on either IRS-1 phosphorylation or ERK in VSMC. Insulin induced ERK phosphorylation in a dose-dependent manner in VSMC. These results suggests that TNF-alpha plays a significant role in the tissue specificity and signal selectivity of insulin resistance. The pathway related to glucose metabolism is selectively impaired by TNF-alpha in skeletal muscle, and this impairment may induce compensatory hyperinsulinemia, which in turn would stimulate the pathway related to the cell proliferation in vascular tissues and possibly enhance the progression of atherosclerosis.