Post-Synthetic Nucleobase Modification of Oligodeoxynucleotides by Sonogashira Coupling and Influence of Alkynyl Modifications on the Duplex-Forming Ability.

Recently, it was reported that the alkynyl modification of nucleobases mitigates the toxicity of antisense oligonucleotides (ASO) while maintaining the efficacy. However, the general effect of alkynyl modifications on the duplex-forming ability of oligonucleotides (ONs) is unclear. In this study, post-synthetic nucleobase modification by Sonogashira coupling in aqueous medium was carried out to efficiently evaluate the physiological properties of various ONs with alkynyl-modified nucleobases. Although several undesired reactions, including nucleobase cyclization, were observed, various types of alkynyl-modified ONs were successfully obtained via Sonogashira coupling of ONs containing iodinated nucleobases. Evaluation of the stability of the duplex formed by the synthesized alkynyl-modified ONs showed that the alkynyl modification of pyrimidine was less tolerated than that of purine, although both the modifications occurred in the major groove of the duplex. These results can be attributed to the bond angle of the alkyne on the pyrimidine and the close proximity of the alkynyl substituents to the phosphodiester backbone. The synthetic method developed in this study may contribute to the screening of the optimal chemical modification of ASO because various alkynyl-modified ONs that are effective in reducing the toxicity of ASO can be easily synthesized by this method.

Impaired TRIM16-Mediated Lysophagy in Chronic Obstructive Pulmonary Disease Pathogenesis.

Insufficient autophagic degradation has been implicated in accelerated cellular senescence during chronic obstructive pulmonary disease (COPD) pathogenesis. Aging-linked and cigarette smoke (CS)-induced functional deterioration of lysosomes may be associated with impaired autophagy. Lysosomal membrane permeabilization (LMP) is indicative of damaged lysosomes. Galectin-3 and tripartite motif protein (TRIM) 16 play a cooperative role in recognizing LMP and inducing lysophagy, a lysosome-selective autophagy, to maintain lysosome function. In this study, we sought to examine the role of TRIM16-mediated lysophagy in regulating CS-induced LMP and cellular senescence during COPD pathogenesis by using human bronchial epithelial cells and lung tissues. CS extract (CSE) induced lysosomal damage via LMP, as detected by galectin-3 accumulation. Autophagy was responsible for modulating LMP and lysosome function during CSE exposure. TRIM16 was involved in CSE-induced lysophagy, with impaired lysophagy associated with lysosomal dysfunction and accelerated cellular senescence. Airway epithelial cells in COPD lungs showed an increase in lipofuscin, aggresome and galectin-3 puncta, reflecting accumulation of lysosomal damage with concomitantly reduced TRIM16 expression levels. Human bronchial epithelial cells isolated from COPD patients showed reduced TRIM16 but increased galectin-3, and a negative correlation between TRIM16 and galectin-3 protein levels was demonstrated. Damaged lysosomes with LMP are accumulated in epithelial cells in COPD lungs, which can be at least partly attributed to impaired TRIM16-mediated lysophagy. Increased LMP in lung epithelial cells may be responsible for COPD pathogenesis through the enhancement of cellular senescence.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-ß-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.

Although 2'-deoxy-2'-α-F-2'-ß-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

Chaperone-Mediated Autophagy Suppresses Apoptosis via Regulation of the Unfolded Protein Response during Chronic Obstructive Pulmonary Disease Pathogenesis.

Cigarette smoke (CS) induces accumulation of misfolded proteins with concomitantly enhanced unfolded protein response (UPR). Increased apoptosis linked to UPR has been demonstrated in chronic obstructive pulmonary disease (COPD) pathogenesis. Chaperone-mediated autophagy (CMA) is a type of selective autophagy for lysosomal degradation of proteins with the KFERQ peptide motif. CMA has been implicated in not only maintaining nutritional homeostasis but also adapting the cell to stressed conditions. Although recent papers have shown functional cross-talk between UPR and CMA, mechanistic implications for CMA in COPD pathogenesis, especially in association with CS-evoked UPR, remain obscure. In this study, we sought to examine the role of CMA in regulating CS-induced apoptosis linked to UPR during COPD pathogenesis using human bronchial epithelial cells (HBEC) and lung tissues. CS extract (CSE) induced LAMP2A expression and CMA activation through a Nrf2-dependent manner in HBEC. LAMP2A knockdown and the subsequent CMA inhibition enhanced UPR, including CHOP expression, and was accompanied by increased apoptosis during CSE exposure, which was reversed by LAMP2A overexpression. Immunohistochemistry showed that Nrf2 and LAMP2A levels were reduced in small airway epithelial cells in COPD compared with non-COPD lungs. Both Nrf2 and LAMP2A levels were significantly reduced in HBEC isolated from COPD, whereas LAMP2A levels in HBEC were positively correlated with pulmonary function tests. These findings suggest the existence of functional cross-talk between CMA and UPR during CSE exposure and also that impaired CMA may be causally associated with COPD pathogenesis through enhanced UPR-mediated apoptosis in epithelial cells.

Azobenzene-modified DNA aptamers evolved by capillary electrophoresis (CE)-SELEX method.

Chemically modified aptamers have recently emerged as important materials for nucleic acid based therapeutics and diagnostic tools. Here, we report in vitro evolution of azobenzene-modified DNA aptamers by capillary electrophoresis (CE)-SELEX method. Azobenzene has been considered to be a fascinating functional group due to its trans-cis photo-isomerization property. We harnessed C5-azobenzene-modified 2'-deoxyuridine (dUAz) as a azobenzene-tethered unit and subjected it to CE-SELEX with human thrombin. The obtained dUAz-modified aptamer showed strong binding affinity toward human thrombin and could be reversibly photo-isomerized by different wavelengths of light. This work demonstrates that CE-SELEX is a powerful method to obtain chemically modified aptamers and dUAz is an excellent photo-responsive nucleoside for nucleic acid photo-switches.

Involvement of GPx4-Regulated Lipid Peroxidation in Idiopathic Pulmonary Fibrosis Pathogenesis.

The imbalanced redox status in lung has been widely implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis. To regulate redox status, hydrogen peroxide must be adequately reduced to water by glutathione peroxidases (GPx). Among GPx isoforms, GPx4 is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide. Increased lipid peroxidation products have been demonstrated in IPF lungs, suggesting the participation of imbalanced lipid peroxidation in IPF pathogenesis, which can be modulated by GPx4. In this study, we sought to examine the involvement of GPx4-modulated lipid peroxidation in regulating TGF-ß-induced myofibroblast differentiation. Bleomycin-induced lung fibrosis development in mouse models with genetic manipulation of GPx4 were examined. Immunohistochemical evaluations for GPx4 and lipid peroxidation were performed in IPF lung tissues. Immunohistochemical evaluations showed reduced GPx4 expression levels accompanied by increased 4-hydroxy-2-nonenal in fibroblastic focus in IPF lungs. TGF-ß-induced myofibroblast differentiation was enhanced by GPx4 knockdown with concomitantly enhanced lipid peroxidation and SMAD2/SMAD3 signaling. Heterozygous GPx4-deficient mice showed enhancement of bleomycin-induced lung fibrosis, which was attenuated in GPx4-transgenic mice in association with lipid peroxidation and SMAD signaling. Regulating lipid peroxidation by Trolox showed efficient attenuation of bleomycin-induced lung fibrosis development. These findings suggest that increased lipid peroxidation resulting from reduced GPx4 expression levels may be causally associated with lung fibrosis development through enhanced TGF-ß signaling linked to myofibroblast accumulation of fibroblastic focus formation during IPF pathogenesis. It is likely that regulating lipid peroxidation caused by reduced GPx4 can be a promising target for an antifibrotic modality of treatment for IPF.

[A Case of Advanced Rectal Cancer with Left Hydronephrosis Treated with Left Ureter-Sparing Laparoscopic High Anterior Resection after Chemotherapy].

A 47-year-old woman with a complaint of weight loss for the past 5 months was referred to our hospital. Colonoscopy revealed advanced rectal cancer 20 cm from the anal verge. The patient had left hydronephrosis caused by ureteral invasion. Firstly, we performed transverse colostomy and left nephrostomy. After 8 courses of capecitabine, oxaliplatin plus bevacizumab( CAPOX plus Bmab)therapy, colonoscopy and computed tomography revealed shrinkage of both the primary and metastatic lesions. Laparoscopic high anterior resection was performed, and the left ureter was successfully preserved. The patient received chemotherapy after surgery. Neither local recurrence nor enlargement of metastases has been observed 8 months after surgery.

[A Case of Synchronous Multiple Colorectal Cancer with Rectal Neuroendocrine Tumor and Ascending Colon Cancer].

A 43-year-old man who had no previous medical history or family history had positive fecal occult blood test in a local physician. Colonoscopy revealed a type 2 tumor of the ascending colon and a 10 mm submucosal tumor(SMT)of the lower rectum. Biopsy indicated moderately-differentiated adenocarcinoma of the ascending colon and neuroendocrine tumor (NET)of the lower rectum. No metastasis was detected by computed tomography. Therefore, the rectal SMT was resected first by endoscopic submucosal resection. Histopathologically, the lesion was localized in the submucosa and no lymphovascular invasion was found. Vertical margin was also negative. We decided not to perform additional intestinal resection for rectal NET. Thereafter, the patient underwent laparoscopic right hemicolectomy for ascending colon cancer. The histopathological findings were pT3, pN1, pM0, pStage Ⅲb. The patient received adjuvant chemotherapy. No relapse was found 18 months after surgery. We reported a rare case of a lower rectal NET with concomitant ascending colon cancer.

Synthesis and Evaluation of Artificial Nucleic Acid Bearing an Oxanorbornane Scaffold.

Natural oligonucleotides have many rotatable single bonds, and thus their structures are inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form a duplex with single-stranded DNA or RNA. An effective approach to reduce such entropic loss in the duplex-formation is the conformational restriction of the flexible phosphodiester linkage and/or sugar moiety. We here report the synthesis and biophysical properties of a novel artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA), where the adamant oxanorbornane was expected to rigidify the structures of both the linkage and sugar parts of nucleic acid. OxNorNA phosphoramidite with a uracil (U) nucleobase was successfully synthesized over 15 steps from a known sugar-derived cyclopentene. Thereafter, the given phosphoramidite was incorporated into the designed oligonucleotides. Thermal denaturation experiments revealed that oligonucleotides modified with the conformationally restricted OxNorNA-U properly form a duplex with the complementally DNA or RNA strands, although the Tm values of OxNorNA-U-modified oligonucleotides were lower than those of the corresponding natural oligonucleotides. As we had designed, entropic loss during the duplex-formation was reduced by the OxNorNA modification. Moreover, the OxNorNA-U-modified oligonucleotide was confirmed to have extremely high stability against 3'-exonuclease activity, and its stability was even higher than those of the phosphorothioate-modified counterparts (Sp and Rp). With the overall biophysical properties of OxNorNA-U, we expect that OxNorNA could be used for specialized applications, such as conformational fixation and/or bio-stability enhancement of therapeutic oligonucleotides (e.g., aptamers).

Synthesis and biophysical properties of 5'-thio-2',4'-BNA/LNA oligonucleotide.

Phosphorothioate modification of oligonucleotides is one of the most promising chemical modifications in nucleic acid therapeutics. Structurally similar 5'-thio or phosphorothiolate-modified nucleotides, in which the 5'-bridging oxygen atom is replaced with a sulfur atom, are attracting attention and gaining importance in oligonucleotide-based research. In our present study, we synthesized 5'-thio-2',4'-BNA/LNA monomers bearing thymine or 5-methylcytosine nucleobase. The 5'-thio-2',4'-BNA/LNA monomers were successfully incorporated into target oligonucleotides, and their nuclease stability and binding affinity with complementary strands were evaluated.

Comparison of oncological results for early- and advanced-stage thymomas: thoracoscopic thymectomy versus open thymectomy.

BACKGROUND: This study evaluated the feasibility of thoracoscopic thymectomy (TT) for the treatment of early- and advanced-stage thymoma and compared patient outcomes with those following open thymectomy (OT). METHODS: A retrospective review was conducted for 140 patients who underwent TT or OT for Masaoka stage I-IV thymoma between 1996 and 2014. RESULTS: TT was performed in 88 patients and OT in 52 patients. The postoperative hospital stay was significantly shorter in the TT group than in the OT group (4 and 13 days, respectively; P < 0.0001). WHO types B3-C were identified in Masaoka stage III-IV disease with high frequency. There was a significant relationship between Masaoka stage and WHO type (P < 0.05); the numbers of advanced-stage thymoma progressively increased in WHO type B3-C. Eight patients in each group had recurrent disease, with greater recurrence for WHO types B3-C and stage III-IV tumors. Five-year disease-free survival (DFS) was not different between groups (P = 0.3906); however, survival for patients with stage III-IV thymomas (47 %) was significantly worse than that for patients with stage I and II tumors (97.5 and 94.1 %, respectively; P < 0.0001). Based on multivariate analysis, both Masaoka stage and WHO type were significant predictors of thymoma patient survival. CONCLUSIONS: These results demonstrate the safety and substantially decreased invasiveness of TT for thymoma. The oncological results were comparable between the TT and OT groups. Furthermore, Masaoka stage III-IV and WHO B3-C were revealed as independent prognostic factors for DFS.

Histology is a Prognostic Indicator After Pulmonary Metastasectomy from Renal Cell Carcinoma.

OBJECTIVES: There are only a few detailed reports concerning the prognosticators following surgical resection of pulmonary metastases (PMs) from renal cell carcinoma (RCC). We investigated the prognosis of patients with RCC PMs undergoing pulmonary metastasectomy and identified prognostic factors in a multi-institutional retrospective study. METHODS: We retrospectively evaluated 84 patients who underwent resection of PMs from RCC between 1993 and 2014. We assessed the clinicopathological characteristics, focusing on the histological findings of PMs. We classified the histology into three types: pure clear cell carcinoma (N = 68), clear cell carcinoma combined with other histology type (N = 8), and non-clear cell carcinoma (N = 8). We examined the relationship between these histological types and the prognosis of patients with PMs from RCC. RESULTS: Complete resection was achieved in 78 patients (93%). The 5-year overall survival rate after metastasectomy was 59.7%. In multivariate analysis, three factors were found to be independent favorable prognostic factors of overall survival after lung metastasectomy [tumor size <2 cm, hazard ratio (HR) = 0.31, 95% confidence interval (CI) 0.13-0.78, P = 0.012; clear cell type, HR = 0.37, 95% CI 0.16-0.83, P = 0.025; and complete resection, HR = 0.27, 95% CI 0.10-0.78, P = 0.015]. CONCLUSIONS: This study indicates that a histological finding of the clear cell type is a significant favorable prognostic factor in addition to complete resection and a tumor size <2 cm. Histological evaluation of PM lesions is important for predicting survival after metastasectomy.

High expression of programmed cell death 1 ligand 1 in lung adenocarcinoma is a poor prognostic factor particularly in smokers and wild-type epidermal growth-factor receptor cases.

A clinical implication of programmed cell death 1 ligand 1 (PD-L1) expression in lung adenocarcinoma has not been well established. We evaluated PD-L1 expression immunohistochemically on 296 surgically resected lung adenocarcinomas to investigate a clinical implication of PD-L1 expression especially in terms of smoking history and epidermal growth-factor receptor (EGFR) mutation status. Patients were classified into high- and low-PD-L1 expression groups. The high-expression group (n = 107) showed a significantly higher proportion of smokers and poor differentiation compared with the low-expression group (n = 189). Survival analysis showed that the prognosis of the high-expression group was worse in overall survival than that of the low-expression group (3-year overall survival 85 vs. 94%, P = 0.005). Stratified survival analyses showed that the prognoses of the high-expression group were worse than those of the low-expression group in both strata of smokers and wild-type EGFR (P = 0.009 and P = 0.007, respectively). We found that high PD-L1 expression was a poor prognostic factor in the smokers or the patients with wild-type EGFR, whereas it was not the case in those who never smoked or those with EGFR mutation, implying the importance of adenocarcinoma driver mutations and etiology.

Decreased choroidal blood flow velocity in the pathogenesis of multiple evanescent white dot syndrome.

PURPOSE: On the basis of angiographic features, it is suggested that choroidal circulation disturbance may be involved in the pathogenesis of multiple evanescent white dot syndrome (MEWDS). The aim of this study is to quantitatively evaluate changes in choroidal circulation hemodynamics using laser speckle flowgraphy (LSFG) in patients with MEWDS. METHODS: Twelve eyes of 12 patients with MEWDS and 12 unaffected fellow eyes as controls were included. The macular mean blur rate (MBR), a quantitative index of relative blood flow velocity in the choroid, was measured by LSFG. Sequential changes in the average MBR values at the macula with granular changes and the lesion area with white dots were analysed. Moreover, correlations between the MRR changing rate and initial visual functions were examined. RESULTS: Visual functions significantly improved 3 months after initial visit with accompanying improvements in outer retinal morphology. When compared with the baseline measurements, the MBR significantly increased at the macula of the affected eyes by 20.2 % and 13.0 % at 1 and 3 months respectively (P < 0.01 for both), while no significant change was detected in fellow eyes. Similarly, the MBR increased at the lesion area by 17.8 % and 12.0 % at 1 and 3 months respectively (P < 0.05 for both). Notably, the macular MBR elevation at 1 month was negatively correlated with both initial best-corrected visual acuity and the average threshold at the macula on Humphrey perimetry at baseline (R = -0.76, P = 0.003; R = -0.60, P = 0.03, respectively), suggesting a close link between initially reduced choroidal blood flow and functional abnormalities at the onset of MEWDS. CONCLUSIONS: These results, in concert with angiographic findings, are likely to reinforce the concept of choroidal circulation impairment as a predisposing factor for MEWDS.

Photoinduced changes in hydrogen bonding patterns of 8-thiopurine nucleobase analogues in a DNA strand.

Hydrogen bonds (H-bonds) formed between nucleobases play an important role in the construction of various nucleic acid structures. The H-donor and H-acceptor pattern of a nucleobase is responsible for selective and correct base pair formation. Herein, we describe an 8-thioadenine nucleobase analogue and an 8-thiohypoxanthine nucleobase analogue with a photolabile 6-nitroveratryl (NV) group on the sulfur atom (SA(NV) and SH(NV), respectively). Light-triggered removal of the NV group causes tautomerization and a change in the H-bonding pattern of SA(NV) and SH(NV). This change in the H-bonding pattern has a strong effect on base recognition by 8-thiopurine nucleobase analogues. In particular, base recognition by SH(NV) is clearly shifted from guanine to adenine upon photoirradiation. These results show that a photoinduced change in the H-bonding pattern is a unique strategy for manipulating nucleic acid assembly with spatiotemporal control.

C5-azobenzene-substituted 2'-deoxyuridine-containing oligodeoxynucleotides for photo-switching hybridization.

A new photoisomeric nucleoside dUAz bearing an azobenzene group at the C5-position of 2'-deoxyuridine was designed and synthesized. Photoisomerization of dUAz in oligodeoxynucleotides can be achieved rapidly and selectively with 365 nm (forward) and 450 nm (backward) irradiation. Thermal denaturation experiments revealed that dUAz stabilized the duplex in the cis-form and destabilized it in the trans-form with mismatch discrimination ability comparable to thymidine. These results indicate that dUAz could be a powerful material for reversibly manipulating nucleic acid hybridization with spatiotemporal control.

Estimation error in speed of sound caused by rotation of measured cross-section from short-axis plane of blood vessels: a preliminary study.

PURPOSE: Estimating the speed of sound (SoS) in ultrasound propagation media is important for improving the quality of B-mode images and for quantitative tissue characterization. We have been studying a method for estimating the SoS by measuring the reception time distribution of waves scattered from a scatterer at the elements in a probe. Previously, the measurement cross section was assumed to be perpendicular to the long axis of the blood vessel. In this study, we experimentally investigated the relationship between rotation angle [Formula: see text] of the probe relative to the short-axis plane of the blood vessel and the estimated SoS, [Formula: see text]. METHODS: Water tank and phantom experiments were conducted to investigate the characteristics of [Formula: see text] and element signals when the probe was rotated. RESULTS: The received signal powers at the elements around both edges greatly decreased as [Formula: see text] increased. We introduced a parameter representing the decrease in power, [Formula: see text], in the received signal at the elements at both edges relative to the center element. [Formula: see text] was estimated to be larger as [Formula: see text] increased, especially for [Formula: see text]. [Formula: see text] also increased as [Formula: see text] increased. An approximately proportional relationship existed between the errors in [Formula: see text] and [Formula: see text]. CONCLUSION: Based on these results, we can distinguish between the presence and the absence of SoS misestimations using the difference in power among the elements in the received signal. In the absence of misestimation, we can obtain the true SoS, even if the target has a non-negligible size, by applying our previously proposed methods.

A novel ultrasonic method for measuring minute sinusoidal displacement by network analyzer.

We developed a method for generating continuous sinusoidal displacements of an object to estimate viscoelastic parameters. However, the amplitude of the displacement caused by the ultrasonic excitation force under safe guidelines was small (a few micrometers), and it was difficult to stably measure the displacement. Therefore, to stably measure the amplitude of sinusoidal displacement as small as the order of micrometers, we proposed a novel method using a network analyzer. Ultrasonic waves were irradiated using an ultrasonic transducer on an object vibrating sinusoidally. The S parameter of the first reflected wave received from the surface of the object was measured using a network analyzer. The S parameter and the inverse Fourier transform were formulated theoretically, and the amplitude of the sinusoidal displacement of the object was estimated from the amplitude characteristics of the inverse Fourier-transformed signal. The proposed method was applied to measure sinusoidal displacements on the order of micrometers from 10 to 300 Hz on an object using a water tank experiment. The obtained sinusoidal displacement agreed well with the reference values measured using a laser displacement meter. The proposed method can accurately measure minute sinusoidal displacements that occur on an object.

Influence of Power-Weighted Center of Echo Signal Within Window Function on Local Strain Rate Distribution in Left Ventricular Wall.

OBJECTIVE: The deviation of the power-weighted center of the echo signal from the geometric center within the velocity estimation window for calculating strain rate (SR) causes an estimation error. This study aimed to confirm whether an erroneous multilayer pattern in the SR distribution of the left ventricular wall could be corrected by considering the power-weighted center of the echo signal. METHODS: The SR distributions were measured locally in the transmural direction around the pre-ejection and early diastolic phases in healthy volunteers. The estimation error related to the power-weighted center of the echo signal was corrected using a previously proposed method, and the effectiveness of the correction was confirmed based on the accuracy of the estimated myocardial displacement. RESULTS: The SR distribution in early diastole was observed as multilayers of low- and high-amplitude negative SRs. However, this multilayer pattern disappeared after correction. In the pre-ejection phase, multilayers of positive and negative SRs were observed in the SR distributions with and without correction. This correction was sufficiently effective in accurately tracking the local peak of the echo signal. CONCLUSION: The multilayer pattern of low- and high-amplitude positive or negative SRs is caused by estimation errors related to the power-weighted center of the echo signal. The multilayer pattern of positive and negative SRs might not be caused by these errors and might relate to the actual change in myocardial thickness because the estimation errors do not convert the negative (positive) SR to positive (negative) in a homogeneous negative (positive) SR distribution.

Optimizing irradiation conditions for low-intensity pulsed ultrasound to upregulate endothelial nitric oxide synthase.

PURPOSE: Here we aimed to develop a minimally invasive treatment for ischemic heart disease and demonstrate that low-intensity pulsed ultrasound (LIPUS) therapy improves myocardial ischemia by promoting myocardial angiogenesis in a porcine model of chronic myocardial ischemia. Studies to date determined the optimal treatment conditions within the range of settings available with existing ultrasound equipment and did not investigate a wider range of conditions. METHODS: We investigated a broad range of five parameters associated with ultrasound irradiation conditions that promote expression of endothelial nitric oxide synthase (eNOS), a key molecule that promotes angiogenesis in human coronary artery endothelial cells (HCAEC). RESULTS: Suboptimal irradiation conditions included 1-MHz ultrasound frequency, 500-kPa sound pressure, 20-min total irradiation time, 32-48-[Formula: see text] pulse duration, and 320-[Formula: see text] pulse repetition time. Furthermore, a proposed index, [Formula: see text], calculated as the product of power and the total number of irradiation cycles applied to cells using LIPUS, uniformly revealed the experimental eNOS expression associated with the various values of five parameters under different irradiation conditions. CONCLUSION: We determined the suboptimal ultrasound irradiation conditions for promoting eNOS expression in HCAEC.