Sequentially addressable dielectrophoretic array for high-throughput sorting of large-volume biological compartments.

Droplet microfluidics has become a powerful tool in precision medicine, green biotechnology, and cell therapy for single-cell analysis and selection by virtue of its ability to effectively confine cells. However, there remains a fundamental trade-off between droplet volume and sorting throughput, limiting the advantages of droplet microfluidics to small droplets (<10 pl) that are incompatible with long-term maintenance and growth of most cells. We present a sequentially addressable dielectrophoretic array (SADA) sorter to overcome this problem. The SADA sorter uses an on-chip array of electrodes activated and deactivated in a sequence synchronized to the speed and position of a passing target droplet to deliver an accumulated dielectrophoretic force and gently pull it in the direction of sorting in a high-speed flow. We use it to demonstrate large-droplet sorting with ~20-fold higher throughputs than conventional techniques and apply it to long-term single-cell analysis of Saccharomyces cerevisiae based on their growth rate.

A genome-wide association study of coping behaviors suggests FBXO45 is associated with emotional expression.

Individuals use coping behaviors to deal with unpleasant daily events. Such behaviors can moderate or mediate the pathway between psychosocial stress and health-related outcomes. However, few studies have examined the associations between coping behaviors and genetic variants. We conducted a genome-wide association study (GWAS) on coping behaviors in 14088 participants aged 35 to 69 years as part of the Japan Multi-Institutional Collaborative Cohort Study. Five coping behaviors (emotional expression, emotional support seeking, positive reappraisal, problem solving and disengagement) were measured and analyzed. A GWAS analysis was performed using a mixed linear model adjusted for study area, age and sex. Variants with suggestive significance in the discovery phase (N = 6403) were further examined in the replication phase (N = 7685). We then combined variant-level association evidence into gene-level evidence using a gene-based analysis. The results showed a significant genetic contribution to emotional expression and disengagement, with an estimation that the 19.5% and 6.6% variance in the liability-scale was explained by common variants. In the discovery phase, 12 variants met suggestive significance (P < 1 × 10-6 ) for association with the coping behaviors and perceived stress. However, none of these associations were confirmed in the replication stage. In gene-based analysis, FBXO45, a gene with regulatory roles in synapse maturation, was significantly associated with emotional expression after multiple corrections (P < 3.1 × 10-6 ). In conclusion, our results showed the existence of up to 20% genetic contribution to coping behaviors. Moreover, our gene-based analysis using GWAS data suggests that genetic variations in FBXO45 are associated with emotional expression.

Experiences of high-dose mizoribine as antimetabolite immunosuppressants for kidney transplantation.

We have used low doses of mizoribine (MZ) or mycophenolate mofetil (MMF) as induction and maintenance immunosuppressants, but since 2009 have employed a high dose of MZ. We reviewed the efficacy and side effects of MZ compared with MMF. It is difficult to compare graft survivals between these periods because of different patient demographics, though the high dose of MZ cohort showed no significant difference from MMF. High doses of MZ serum to prevent acute rejection episodes as the induction and maintenance therapy. MZ controlled with blood concentrations showed less side effects, suggesting that high MZ doses could be safely used for an induction and maintenance antimetabolite.

Increased expression of TDP-43 in the skin of amyotrophic lateral sclerosis.

OBJECTIVES: Transactivation-responsive DNA-binding protein-43 (TDP-43) was identified as a major component of the ubiquitin-positive inclusions in sporadic amyotrophic lateral sclerosis (ALS). However, there has been no study of TDP-43 in ALS skin. The present study investigates TDP-43 in ALS skin. MATERIALS AND METHODS: We made a quantitative immunohistochemical study of the expression of TDP-43 in the skin from 15 patients with ALS and 15 control subjects. RESULTS: The proportion of TDP-43-positive (TDP-43+) cells in the epidermis in ALS patients was significantly higher (P < 0.001) than in controls. There was a significant positive relationship (r = 0.62, P < 0.02) between the proportion and duration of illness in ALS patients. The optical density of TDP-43+ cells in the epidermis in ALS patients is markedly stronger (P < 0.001) than in controls. There was a significant positive relation (r = 0.72, P < 0.01) between the immunoreactivity and duration of illness in ALS patients. CONCLUSIONS: These data suggest that changes of TDP-43 in ALS skin are likely to be related to the disease process and that metabolic alterations of TDP-43 may take place in the skin of patients with ALS.

Involvement of organic anion transporting polypeptides in the toxicity of hydrophilic pravastatin and lipophilic fluvastatin in rat skeletal myofibres.

BACKGROUND AND PURPOSE: There is a discrepancy in the adverse effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, statins between the clinical reports and the studies using skeletal muscle cell models. In the clinical reports, both hydrophilic and lipophilic statins induce myotoxicity, whereas in in vitro experiments using cell lines of myoblasts, lipophilic, but not hydrophilic, statins exert myotoxicity. We investigated the cause of this discrepancy. EXPERIMENTAL APPROACH: Skeletal myofibres, fibroblasts and satellite cells were isolated from rat flexor digitorum brevis (FDB) muscles. Using these primary cultured cells as well as the L6 myoblast cell line, we compared the toxicity of hydrophilic pravastatin and lipophilic fluvastatin. The mRNA expression levels of possible drug transporters for statins were also examined in these cells using reverse transcriptase-PCR. KEY RESULTS: In the skeletal myofibres, both pravastatin and fluvastatin induced vacuolation and cell death, whereas in the mononuclear cells only fluvastatin, but not pravastatin, was toxic. mRNA of the organic anion transporting polypeptides (Oatp) 1a4 and Oatp2b1 were expressed in the skeletal myofibres, but not in mononucleate cells. Estrone-3-sulphate, a substrate for Oatps, attenuated the effects of pravastatin and fluvastatin in skeletal myofibres; p-aminohippuric acid, a substrate for the organic anion transporters (Oats), but not Oatps, failed to do so. CONCLUSIONS AND IMPLICATIONS: The statin transporters Oatp1a4 and Oatp2b1 are expressed in rat skeletal myofibres, but not in satellite cells, fibroblasts or in L6 myoblasts. This is probably why hydrophilic pravastatin affects skeletal muscle, but not skeletal myoblasts.

Laparoscopic resection of gastrointestinal mesenchymal tumors located in the upper stomach.

BACKGROUND: The treatment strategy for a gastrointestinal mesenchymal tumor located close to the esophagogastric junction remains controversial. The authors evaluate the criteria indicating that a gastrointestinal mesenchymal tumor is suitable for laparoscopic resection and assess the surgical techniques on the basis of clinical outcomes. METHODS: The criteria specified a tumor more than 2 cm in diameter or a tendency for it to increase in size during the follow-up period. For eight patients in whom the tumor was located within 3 cm of the esophagogastric junction, an intragastric laparoscopic approach was used, whereas for seven patients in whom the tumor was further from the esophagogastric junction, an exogastric approach was used. RESULTS: In all 15 cases, the laparoscopic resection was successful, with no complications. The intragastric group had a mean maximal tumor size of 2.9 cm, a mean operation time of 168 min, and a mean postoperative hospital stay of 8.8 days, whereas these values in the exogastric group, were respectively, 3.9 cm, 121 min (p = 0.0442), and 9.6 days. There were no recurrences in either group during the follow-up period. CONCLUSION: The good clinical outcomes suggest hat the criteria used as an indication for laparoscopic resection and the surgical techniques applied were appropriate for the resection of gastrointestinal mesenchymal tumors.

Laparoscopic intragastric stapled resection of gastric submucosal tumors located near the esophagogastric junction.

BACKGROUND: Laparoscopic resection cannot be applied easily to tumors located near the esophagogastric junction or the pyloric ring. We evaluated our laparoscopic intragastric surgical technique for gastric submucosal tumors located near the esophagogastric junction and the results of a clinical study. MATERIALS AND METHODS: We performed our technique in six patients: one man and five woman with a mean age of 61 years. Using the laparoscopic procedure, after inflation of the stomach, we inserted two or three balloon-type ports into the stomach through the abdominal wall. RESULTS: A stapled resection of gastric submucosal tumors using a laparoscopic linear stapler was performed successfully in all the patients. Without exception, stapled resections were successfully performed. The mean operation time was 168 min, and the blood loss was minimal There were no intra- or postoperative complications. The mean postoperative hospital stay was 9.8 days. The mean maximal diameter size of the resected specimens was 2.4 cm. Histopathologic diagnoses were gastrointestinal stromal tumors in five cases and enterogenous cyst in one. There were no recurrences during a mean follow-up period of 27 +/- 11.6 months. CONCLUSION: Although we need to evaluate the long-term outcomes, our procedure is considered technically feasible, safe, and useful for the resection of gastric submucosal tumors located near the esophagogastric junction.

Differences in mechanisms between weight loss-sensitive and -resistant blood pressure reduction in obese subjects.

This study was conducted to clarify the mechanisms involved in the sensitivity for blood pressure (BP) reduction in response to weight loss. In particular, we focused on the contributions of sympathetic nervous system activity and fasting plasma leptin and insulin levels to BP levels during weight loss in obese subjects with weight loss-sensitive and -resistant BP reduction. Sixty-one young, obese untreated hypertensive men (HT) and 52 obese normotensive men (NT) were enrolled in a weight loss program consisting of a low caloric diet and aerobic exercise over a 24-week period. At entry and at week 24, body mass index (BMI), BP, plasma norepinephrine (NE), leptin and insulin were measured. Successful weight loss and BP reduction were respectively defined as a more than a 10% reduction in BMI or mean BP from baseline at week 24. More than 60% of subjects in either group successfully achieved weight loss by this definition. The percentage of subjects who successfully achieved BP reduction was higher (64%) among those subjects who achieved weight loss than among those who did not (22%). Plasma NE level at entry in subjects who failed to achieve BP reduction despite weight loss was significantly higher than that in subjects who succeeded in BP reduction. Plasma leptin and insulin levels were similar between subjects with and without BP reduction. In addition, the absolute decrement and percent decrement in plasma NE in subjects who succeeded in BP reduction were significantly greater than those in subjects who failed to reduce their BP. Absolute and percent decrements in plasma leptin and insulin were similar in both groups. These results suggest that individuals who are resistant to weight loss-induced BP reduction have more sympathetic overactivity both at the outset of and during weight loss.

Weight reduction and pharmacologic treatment in obese hypertensives.

This study was conducted to evaluate the mechanisms of weight loss-induced blood pressure (BP) reduction focusing, in particular, on the contributions of sympathetic nervous system activity, fasting plasma insulin, and leptin to BP levels, and to delineate the additional influence of antihypertensive drug therapy. Each of five groups of obese hypertensives were treated with the long-acting calcium channel blocker (CCB) amlodipine, the angiotensin converting enzyme (ACE) inhibitor enalapril with or without a weight reduction program, or a weight reduction program alone. The goal BP was less than 140/90 mm Hg for the pharmacologic treatment groups. The weight reduction program groups with or without pharmacologic treatment were divided into two groups; weight loss groups who succeeded in weight reduction (> or = 10%) and nonweight loss groups who failed in weight reduction (<10%) in the first 6 months. The final dose of CCB and ACE inhibitor were less in the combined pharmacologic and weight loss groups than in the pharmacologic treatment alone groups or in the pharmacologic and nonweight loss groups. In the weight reduction groups regardless of pharmacologic treatment, the percent reductions from baseline in plasma insulin, leptin, and norepinephrine (NE) were greater in the weight loss groups (> or = 10%) than in the nonweight loss groups (<10%). The reductions in plasma NE, insulin, and leptin were significantly greater and earlier in combined pharmacologic and weight loss groups than in the pharmacologic treatment alone groups. In ACE inhibitor groups, the reductions in plasma NE, in insulin, and especially in leptin were greater than the other groups. In the CCB alone group, reductions in insulin and leptin occurred, but there was no change in plasma NE. Reductions in insulin and leptin in CCB groups were less and occurred later than in the ACE inhibitor groups or the weight reduction alone group. These results show that weight loss associated with favorable metabolic improvements and these improvements are amplified when combined with pharmacologic treatment. Therefore, weight loss should be regarded as an essential component of any treatment program for obesity-related hypertension. A novel finding from this study is that ACE inhibition had a striking effect to lower plasma leptin. Suppression of sympathetic activity, insulinemia, and leptinemia appeared to play a role in the BP reduction accompanying weight loss.

Effect of M2 and M3 muscarinic receptors on airway responsiveness to carbachol in bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

The expression balance of M2 and M3 muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M2 and M3 muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (VT, Raw, and Cdyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M2 muscarinic receptor blockade were less but the effects of M3 muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M3 muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M2 muscarinic receptors mRNA was less but that of M3 muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M2 muscarinic autoreceptors and the excessive effect of M3 muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.

Effect of acetylcholinesterase activity on pathogenesis of airway hyperresponsiveness in guinea pigs.

To clarify the effect of acetylcholinesterase (AChE) on the pathogenesis of airway hyperresponsiveness, AChE activities in tracheal smooth muscle and lung tissue from congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs were compared. For this purpose, AChE activities were determined by measuring the rate of absorbance of tissue homogenate. Relative amounts of AChE mRNA were also evaluated by the RT-PCR method. In both tracheal smooth muscle and lung tissue from BHS, the AChE activity and the relative amount of AChE mRNA were less than those in BHR. These results suggest that the reduced AChE activity is at least a candidate for inducing airway hyperresponsiveness.

The difference in citric acid-induced cough in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

Cough elicitation and major physiological factors influencing cough occurrence were investigated in congenitally bronchial-hypersensitive (BHS) and -hyposensitive (BHR) guinea pigs exposed to citric acid (0.3 M) aerosol for 10 min. The number of cough in BHS was significantly larger than in BHR, while the latency to cough in BHS was significantly shorter than in BHR. Pretreatment with atropine (0.2%), lidocaine (2%) or salbutamol (0.1%) aerosol and desensitization of C-fibers with capsaicin (100 mg/kg) decreased the cough numbers in both BHS and BHR. The salbutamol, atropine and capsaicin pretreatments prolonged the cough latency in BHS, but only salbutamol prolonged the latency in BHR. After salbutamol pretreatment all BHR guinea pigs exhibited cough, while 66.7% of BHS guinea pigs exhibited it. Vagal blocking by atropine suppressed coughing in both BHS and BHR. Only a small number (33.3%) of BHR guinea pigs and no BHR guinea pigs exhibited a cough response after capsaicin and lidocaine pretreatment whereas many BHS guinea pigs still produced cough after such pretreatment. The present study demonstrated that the cough responsiveness to citric acid aerosol was significantly higher in BHS than in BHR. It was revealed that airway smooth muscle contraction and functional and/or morphological development of airway nervous receptors, especially C-fiber endings, contributed to aggravation of coughing in BHS.

The use of needlescopic instruments in laparoscopic ventral hernia repair.

A successful laparoscopic hernia repair requires complete covering of the hernia defect, adequate tension of the prosthesis, and secure stapling with a stapler. We describe herein our technique of performing laparoscopic hernia repair using a needlescopic instrument which results in minimal damage to the abdominal wall and has significant cosmetic benefits. Our technique is easy to perform and useful for achieving initial anchoring of the prosthesis before fixation to the abdominal wall with a laparoscopic stapler.

[The use factor of the long-term care insurance after enforcement half a year].

PURPOSE: The purpose of this study was to examine the factors involved in the low usage of home care services in Japan half a year after their enforcement. METHOD: Questionnaires were mailed to the 1,500 senior citizens residing in H city Osaka who were using the home care services covered by the long-term care insurance system. Of the 1,500, 888 responded (59.2%). RESULTS: Of the 888 respondents, 70.7% were women, with a mean age of 82.0 +/- 7.2 years; 33.2% were living alone and 19.3% were living with their spouse. The rate of use was higher than the national average. Use amount of money. Then, when yen 10,000-15,000 were exceeded that the number of users is halved. Of the service control factor, 40% were economical burden, 32% were no-necessary of service, 27% were feeling of resistance to staff and shortage of information, 7% were complicated proceduring and without service. The rate of use and significant relation were seen, "senior citizen only family, short-stay service, home helper service, day care service, and the senior is dementia".

Familial obesity, sympathetic activation and blood pressure level.

This study was conducted to evaluate the relative contributions of existing obesity and a family history of obesity (FHOB) to blood pressure (BP) level, sympathetic activity, plasma leptin and insulin levels in young men without a family history of hypertension. The study was of "four-corner" design according to body mass index (BMI). A positive FHOB (FHOB+) was defined as both parents being obese (BMI >26.0 kg/m2), and a negative FHOB (FHOB-) was defined as both parents being lean (BMI <22.0 kg/ m2). The cutoff limits of BP for the subjects and their parents enrolled in present study was defined as a supine reading of <140/90 mmHg. In 12 lean young subjects with FHOB-, 9 obese young subjects with FHOB-, 8 lean young subjects with FHOB+ and 16 obese young subjects with FHOB+, BMI, BP, plasma norepinephrine (NE), insulin and leptin were measured. All subjects were men and non-diabetic. Obese subjects, irrespective of FHOB, had higher levels of BMI, BP, plasma NE, leptin and insulin compared to lean subjects. In subjects with FHOB+, regardless of their current degree of adiposity, there was a higher level of BP and plasma NE than in subjects with FHOB-. In lean subjects, FHOB+ was associated with a higher plasma NE level and BP, but similar levels of plasma leptin and insulin were found when compared with FHOB- subjects. These results suggest that existing obesity and a positive family history of obesity appear to have an association with sympathetic overactivity and BP elevation.

Control of blood pressure and lifestyle-related risk factors in elderly Japanese hypertensive subjects.

The aim of this study was to analyze the treatment of elderly hypertensive patients by Japanese physicians specializing in hypertension. We enrolled 939 patients with hypertension who were treated in the outpatient clinics of 11 hospitals in 1995; 793 of these patients (388 men and 405 women; mean age, 66.6+/-9.0 years) received follow-up examinations in 1996, and the data on these patients was used for the present analysis. Blood pressure (BP), body mass index, lifestyle, and laboratory data were analyzed in all patients. The average BP was 143+/-16/81+/-10 mmHg in 1995 and 142+/-15/80+/-10 mmHg in 1996. The patients whose baseline BP was at the level of Grade 2 or 3 in the WHO-ISH classification (n=117) were characterized by a higher women-to-men ratio, higher age, a higher serum total cholesterol concentration, and higher QRS voltage. In these patients, from 1995 to 1996, the average BP significantly decreased, whereas fasting plasma glucose, serum total cholesterol and serum creatinine concentrations showed only negligible changes. In 220 patients (28%), BP was <140/<90 mmHg at both the initial and the follow-up examinations, whereas 351 patients (44%) were hypertensive in both 1995 and 1996. Thirty-three percent of the patients were smokers. More smokers than nonsmokers had had prior cardiovascular events, diabetes mellitus, or overt proteinuria. In conclusion, the average BP level among the patients treated by Japanese physicians specializing in hypertension was somewhat higher than that recommended by WHO-ISH Guidelines (1999). Patient education to control lifestyle-related risk factors, particularly to stop smoking, should be emphasized.

Frequent cyclin D1 expression in chromate-induced lung cancers.

Ex-chromate workers are frequently afflicted with lung cancers, especially central-type squamous cell carcinomas (SCCs) of the lung. However, little is known about the molecular and cellular biologic characteristics of chromate-induced lung cancers. We investigated expression of cyclin D1, bcl-2, and p53 proteins in chromate-induced lung cancers by immunohistochemistry, compared with those in lung cancers from nonexposed individuals and those in individuals with pneumoconiosis. Of 19 chromate-induced lung cancers, 16 tumors were SCCs, including 11 central and 5 peripheral types. Eleven (69%) of 16 chromate SCCs showed cyclin D1 expression. In contrast, cyclin D1 expression was observed in only 3 (12%) of 26 SCCs from nonexposed individuals and 6 (16%) of 37 SCCs that developed in patients with pneumoconiosis, respectively. The frequency of cyclin D1 expression proved to be significantly higher in chromate-induced SCCs than in SCCs from nonexposed individuals and from those with pneumoconiosis (P < .001). When comparisons were extended to all histologic types of lung cancer, cyclin D1 expression was observed significantly more often in chromate-induced lung cancers than in lung cancers from nonexposed subjects and those from patients with pneumoconiosis (11 [58%] of 19 v 5 [10%] of 52, P < .001, and 7 [11%] of 63, P < .001, respectively). Frequencies of bcl-2 and p53 expression were not significantly different among lung cancers from ex-chromate workers, nonexposed individuals and those with pneumoconiosis. The current study suggests that cyclin D1 expression may be involved in the development of chromate-induced lung cancers, although its underlying mechanism remains to be determined.

Pneumoconiosis-related lung cancers: preferential occurrence from diffuse interstitial fibrosis-type pneumoconiosis.

It has been reported that patients with pneumoconiosis occasionally have a diffuse interstitial fibrosis (DIF) that resembles interstitial pneumonia, but little is known about the relation between pneumoconiosis-associated DIF and the risk of lung cancer. In the present study, we evaluated the incidence of DIF by chest CT and its contribution to lung cancer in 563 patients with nonasbestos pneumoconiosis. Fifty-five (10%) of the 563 patients had DIF. Pneumoconiosis with DIF had an exceedingly high concurrence of lung cancers when compared with pneumoconiosis without DIF (29 [53%] of 55 versus 78 [15%] of 508, p < 0.001). Squamous cell carcinomas (SCCs) of the lung from pneumoconiosis with DIF exclusively comprised peripheral-types, as compared with SCCs from pneumoconiosis without DIF (13 [100%] of 13 versus 33 [72%] of 46, p = 0.03). In addition, lung cancers arose frequently from the area of DIF in pneumoconiosis with DIF (20 [74%] of 27). Furthermore, our pathologic examination revealed that dysplasias from pneumoconiosis with DIF were significantly more frequently observed in peripheral bronchioli than were dysplasias from pneumoconiosis without DIF (11 [69%] of 16 versus 20 [30%] of 66, p = 0.01). p53 expression evaluated by immunohistochemistry was frequently observed in dysplasias from pneumoconiosis with DIF, although it was not significantly different compared with that in dysplasias from pneumoconiosis without DIF (5 [50%] of 10 versus 12 [38%] of 32). Taken together, these results may suggest a positive causal relationship between pneumoconiosis and peripheral-type SCCs of the lung, and further indicate a pivotal role of diffuse fibrosis for the excess incidence of lung cancers, especially peripheral-type SCCs, in DIF-type pneumoconiosis.