Search

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9‐2 monoclonal antibody (mAb) which recognizes a MM‐specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9‐2 mAb‐derived CARs that included the CD28 (SKM‐28z) or 4‐1BB (SKM‐BBz) costimulatory domain. SKM‐28z CAR‐T cells showed continuous growth and enhanced Tim‐3, LAG‐3, and PD‐1 expression in vitro, which might be induced by tonic signaling caused by self‐activation; however, these phenotypes were not observed in SKM‐BBz CAR‐T cells. In addition, SKM‐BBz CAR‐T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM‐28z CAR‐T cells. More importantly, only SKM‐BBz CAR‐T cells, but not SKM‐28z CAR‐T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM‐BBz CAR‐T cells exhibited enhanced NF‐kB signaling and reduced NFAT activation. In addition, SKM‐BBz CAR‐T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro‐apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM‐BBz and SKM‐28z CAR‐T cells. In conclusion, we developed novel SKM9‐2‐derived CAR‐T cells with the 4‐1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM. [ABSTRACT FROM AUTHOR]

Scholars have argued that multiparty elections have a profound and immediate influence on mass evaluations of political support. However, what is less clear is whether the effects of elections are short lived or long lasting. Investigating dynamic effects of elections on mass perceptions of political regimes has profound implications on popular foundations of democratic consolidation in an era of democratic backsliding. This article examines electoral cycles in citizens' satisfaction with democracy (SWD)—an important dimension of political support—in multiparty regimes. First, we argue that proximity to elections enhances SWD because campaigns and elections include several processes that reduce the costs and increase the benefits of citizen engagement with the political system. This results in a bell‐shaped relationship between citizens' proximity to elections and SWD. Second, we contend that electoral cycles in SWD should vary by the quality of elections and citizens' winner/loser status. We examine these hypotheses using Afrobarometer data in 34 multiparty regimes between 1999 and 2015 finding compelling support. SWD is higher among respondents surveyed closer to elections, while electoral cycles in SWD are more prominent among winners and around low‐quality elections. [ABSTRACT FROM AUTHOR]

Intratumor bacteria modify the tumor immune microenvironment and influence outcomes of various tumors. Periodontal pathogen Fusobacterium nucleatum has been detected in pancreatic cancer tissues and is associated with poor prognosis. However, it remains unclear how F. nucleatum affects pancreatic cancer. Here, we compared clinical features with F. nucleatum colonization in pancreatic cancer tissues. F. nucleatum was detected in 15.5% (13/84) of pancreatic cancer patients. The tumor size was significantly larger in the F. nucleatum‐positive group than in the negative group. To clarify the biological effect of intratumor F. nucleatum on pancreatic cancer progression, we performed migration/invasion assays and cytokine array analysis of cancer cells cocultured with F. nucleatum. F. nucleatum promoted CXCL1 secretion from pancreatic cancer cells, leading to cancer progression through autocrine signaling. Intratumor F. nucleatum suppressed tumor‐infiltrating CD8+ T cells by recruiting myeloid‐derived suppressor cells (MDSCs) to the tumor in an F. nucleatum‐injected subcutaneous pancreatic cancer mouse model, resulting in tumor progression. Furthermore, tumor growth accelerated by F. nucleatum was suppressed by MDSC depletion or cytokine inhibitors. Intratumor F. nucleatum promoted pancreatic cancer progression through autocrine and paracrine mechanisms of the CXCL1‐CXCR2 axis. Blockade of the CXCL1‐CXCR2 axis may be a novel therapeutic approach for patients with intratumor F. nucleatum‐positive pancreatic cancer. [ABSTRACT FROM AUTHOR]

Background: Immunotherapy has become a standard‐of‐care for patients with non‐small‐cell lung cancer (NSCLC). Although several biomarkers, such as programmed cell death‐1, have been shown to be useful in selecting patients likely to benefit from immune checkpoint inhibitors (ICIs), more useful and reliable ones should be investigated. The prognostic nutritional index (PNI) is a marker of the immune and nutritional status of the host, and is derived from serum albumin level and peripheral lymphocyte count. Although several groups reported its prognostic role in patients with NSCLC receiving a single ICI, there exist no reports which have demonstrated its role in the first‐line ICI combined with or without chemotherapy. Materials and Methods: Two‐hundred and eighteen patients with NSCLC were included in the current study and received pembrolizumab alone or chemoimmunotherapy as the first‐line therapy. Cutoff value of the pretreatment PNI was set as 42.17. Results: Among 218 patients, 123 (56.4%) had a high PNI (≥42.17), while 95 (43.6%) had a low PNI (<42.17). A significant association was observed between the PNI and both the progression‐free survival (PFS; hazard ratio [HR] = 0.67, 95% confidence interval [CI]: 0.51–0.88, p = 0.0021) and overall survival (OS; HR = 0.46, 95% CI: 0.32–0.67, p < 0.0001) in the entire population, respectively. The multivariate analysis identified the pretreatment PNI as an independent prognosticator for the PFS (p = 0.0011) and OS (p < 0.0001), and in patients receiving either pembrolizumab alone or chemoimmunotherapy, the pretreatment PNI remained an independent prognostic factor for the OS (p = 0.0270 and 0.0006, respectively). Conclusion: The PNI might help clinicians appropriately identifying patients with better treatment outcomes when receiving first‐line ICI therapy. [ABSTRACT FROM AUTHOR]

Purpose: Spermatogenesis is a complex process orchestrated by several essential genes. Prominin‐1 (Prom1/PROM1) is a gene that is expressed in the testis but with a poorly understood role in spermatogenesis. Methods: We used Prom1 knockout (Prom1 KO) mice to assess the role of Prom1 in spermatogenesis. To this end, we performed immunohistochemistry, immunofluorescence, western blotting, β‐galactosidase staining, and apoptosis assay. Additionally, we analyzed the morphology of sperm and assessed litter sizes. Results: We observed that PROM1 is localized to the dividing spermatocytes in seminiferous epithelial cells, sperm, and columnar epithelium in the epididymis. In the Prom1 KO testis, an aberrant increase in apoptotic cells and a decrease in proliferating seminiferous epithelial cells were observed. Cellular FLICE‐like inhibitory protein (c‐FLIP) and extracellular signal‐regulated kinase 1/2 (ERK1/2) expression were also significantly decreased in Prom1 KO testis. In addition, a significantly increased number of epididymal spermatozoa with abnormal morphology and less motility was found in Prom1 KO mice. Conclusions: PROM1 maintains spermatogenic cell proliferation and survival via c‐FLIP expression in the testis. It is also involved in sperm motility and fertilization potential. The mechanism underlying the effect of Prom1 on sperm morphology and motility remains to be identified. [ABSTRACT FROM AUTHOR]

Acute and chronic inflammation is a basic pathological event that contributes to atherosclerosis, cancer, infectious diseases, and immune disorders. Inflammation is an adaptive process to both external and internal stimuli experienced by the human body. Although the mechanism of gene transcription is highly complicated and orchestrated in a timely and spatial manner, recent developments in next‐generation sequencing, genome‐editing, cryo‐electron microscopy, and single cell‐based technologies could provide us with insights into the roles of super enhancers (SEs). Initially, SEs were implicated in determining cell fate; subsequent studies have clarified that SEs are associated with various pathological conditions, including cancer and inflammatory diseases. Recent technological advances have unveiled the molecular mechanisms of SEs, which involve epigenetic histone modifications, chromatin three‐dimensional structures, and phase‐separated condensates. In this review, we discuss the relationship between inflammation and SEs and the therapeutic potential of SEs for inflammatory diseases. [ABSTRACT FROM AUTHOR]

Introduction: Robot‐assisted radical prostatectomy (RARP) is nowadays being performed worldwide, and inguinal hernia (IH) continues to be a common complication of radical prostatectomy. Laparoscopic repair of IH is often difficult via internal dissection because of adhesion of the preperitoneal cavity. This study aimed to categorize the intraoperative condition of, and devise a strategy for, IH after RARP. Methods: Of 577 patients who underwent RARP, 18 developed IH. These 18 patients then underwent laparoscopic IH repair using the transabdominal preperitoneal approach (TAPP) or modified intraperitoneal onlay mesh repair (mIPOM). Internal dissection was categorized into two groups according to the appearance of Cooper's ligament (exposed or not exposed). Results: After RARP, four patients had exposure of Cooper's ligament while 14 patients showed no exposure. Both patients in the exposed group underwent TAPP and all patients in the not‐exposed group underwent mIPOM. There was no significant difference between normal TAPP and RARP regarding operative factors, and IH recurrence was not observed. Conclusion: Laparoscopic repair of IH after RARP is rendered safe and efficient by using our categorization based on the exposure of Cooper's ligament and extension of preperitoneal space. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to investigate the influence of obesity and the usefulness of a preoperative weight loss program (PWLP) for obese patients undergoing laparoscopic colorectal resection (LCR). Methods: Study 1: 392 patients who underwent LCR for colorectal cancer were divided into two groups: those with a body mass index (BMI) ≥25 kg/m2 (n = 113) and those with a BMI <25 kg/m2 (n = 279). The influence of BMI on LCR was investigated. Study 2: Patients with a BMI ≥28 kg/m2 who were scheduled to undergo LCR (n = 7, mean body weight 87.0 kg, mean BMI 33.9 kg/m2) undertook a PWLP including caloric restriction and exercise for 29.6 (15–70) days. The effects of this program were evaluated. Results: Study 1: The BMI ≥25 kg/m2 group had a prolongation of operation time and hospital stay than the BMI <25 kg/m2 group. Study 2: The patients achieved a mean weight loss of 6.9% (−6.0 kg). The mean visceral fat area was significantly decreased by 18.0%, whereas the skeletal muscle mass was unaffected. The PWLP group had a significantly lower prevalence of postoperative complications compared with the BMI ≥25 kg/m2 group. Conclusion: Obesity affected the surgical outcomes in LCR. A PWLP may be useful for obese patients undergoing LCR. This article indicated that preoperative weight loss program for obese patients undergoing laparoscopic colorectal resection was important. Our program is so useful for obese patients with colorectal cancer. [ABSTRACT FROM AUTHOR]

Histone H3 lysine‐9 di‐methylation (H3K9me2) and lysine‐27 tri‐methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)‐α signaling in endothelial cells (ECs), where they are regulated by a novel TNF‐α‐responsive microRNA, miR‐3679‐5p. TNF‐α rapidly induces co‐occupancy of KDM7A and UTX at nuclear factor kappa‐B (NF‐κB)‐associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF‐κB‐dependent inflammatory genes. Chromosome conformation capture‐based methods furthermore uncover increased interactions between TNF‐α‐induced super enhancers at NF‐κB‐relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF‐κB‐dependent regulation of genes that control inflammatory responses of ECs. Synopsis: Comprehensive analysis of epigenetic responses during early inflammation in endothelial cells reveals that lysine demethylase 7A and 6A act cooperatively to rapidly erase repressive histone marks, which is required for activation of the pro‐inflammatory TNF‐α/NF‐κB‐signaling pathway. Lysine demethylase 7A (KDM7A) and 6A (UTX) cooperatively control NF‐κB‐dependent transcription in mammalian vascular endothelial cells.Demethylation of repressive histone marks by KDM7A and UTX is critical for early inflammatory responses.KDM7A and UTX are associated with TNF‐α‐induced chromatin looping of super enhancers.Pharmacological inhibition of KDM7A and UTX reduces leucocyte adhesive interactions with endothelial cells in mice. [ABSTRACT FROM AUTHOR]

Introduction: Urethral injury is one of the most important complications that can occur during transanal total mesorectal excision in male patients with rectal cancer. This report shows the usefulness of intraoperative X‐ray fluoroscopy to avoid urethral injury associated with transanal total mesorectal excision. Materials and Surgical Technique: Real‐time navigation using fluoroscopy was performed to check the distance between the urethra and the dissection line at the level of the exposed rectourethral muscle, the middle level of the divided rectourethral muscle, and the level at which the prostate was identified. Discussion: The dissection was completed transanally up to the level of peritoneal reflection on the anterior side without urethral injury. Pathological examination confirmed that the circumferential resection margin was tumor free. This novel technique using intraoperative X‐ray fluoroscopy is an easy‐to‐use approach that helps prevent urethral injury in male patient who undergo transanal total mesorectal excision for rectal cancer. [ABSTRACT FROM AUTHOR]

Aim: The purposes of this study are to present cases of emergency surgery in which gastrointestinal perforation occurred during bevacizumab administration, consider the indications for emergency surgery, and examine the safety of scheduled surgery after a washout period for bevacizumab. Methods: (a) We retrospectively investigated seven patients who underwent emergency surgery for bevacizumab‐associated intestinal perforation. (b) We investigated 104 patients with advanced colorectal cancer treated with neoadjuvant therapy who underwent surgery from 2008 to 2018, retrospectively. Results: (a) In the seven patients undergoing emergency surgery for gastrointestinal perforation, the median bevacizumab administration and washout periods were 16 weeks and 24 days, respectively. A stoma was created in all patients except in those who were not candidates. Two patients developed postoperative abdominal abscesses, and two patients died from perioperative sepsis and gastrointestinal bleeding, respectively; both of these patients had poor performance status. (b) In patients receiving bevacizumab (n = 45) and patients treated with bevacizumab‐free regimens as neoadjuvant therapy (n = 59), 31 and 52 patients received chemoradiotherapy, respectively. We found no correlation with postoperative complications with or without bevacizumab. Conclusion: The surgical indications should be considered carefully in patients with gastrointestinal perforation secondary to bevacizumab administration. Meanwhile, after appropriate cessation time, scheduled surgery following bevacizumab administration is feasible. In planned surgery, anastomosis after appropriate cessation time of bevacizumab may be acceptable. Surgical indication should be carefully considered in severe perforated cases during bevacizumab administration. [ABSTRACT FROM AUTHOR]

Aims/Introduction: The role of glucagon abnormality has recently been reported in type 2 diabetes; however, its role in gestational diabetes mellitus (GDM) is still unknown. The glucose intolerance in GDM is heterogeneous, and not all patients require insulin treatment during pregnancy. Here, we investigated whether glucagon abnormality is associated with the requirement for insulin treatment during pregnancy. Materials and Methods: A total of 49 pregnant women diagnosed with GDM were enrolled. They underwent a 75‐g oral glucose tolerance test during mid‐gestation, and we measured their plasma glucagon levels (by a new sandwich enzyme‐linked immunosorbent assay) at fasting (0 min), and at 30, 60 and 120 min after glucose load in addition to the levels of plasma glucose and serum insulin. All participants underwent another oral glucose tolerance test at postpartum. Results: Of the 49 patients, 15 required insulin treatment (Insulin group) and 34 were treated with diet therapy alone until delivery (Diet group). The early‐phase glucagon secretion after glucose load, as determined by the changes in glucagon from the baseline to 30 min, was paradoxically augmented during mid‐gestation in the Insulin group, but not in the Diet group. The impaired glucagon suppression during mid‐gestation in the Insulin group was not associated with insulin secretory/sensitivity indexes studied, and was ameliorated postpartum, although the plasma glucose levels remained higher in the Insulin group versus the Diet group. Conclusions: Impaired early‐phase suppression of glucagon could be associated with the requirement for insulin treatment during pregnancy in patients with GDM. [ABSTRACT FROM AUTHOR]

We present a case of malignant change of an ovarian mature cystic teratoma. Our patient was a 48‐year‐old woman and she visited a primary care doctor presenting with abdominal pain. At her first visit, her pelvic tumor measured 70 × 50 mm by ultrasonography. She was diagnosed as rupture of the malignant tumor occurred secondary to mature cystic teratoma and she took the surgery (abdominal total hysterectomy, bilateral oophorectomy and partial omentectomy). Pathologic diagnosis was squamous cell carcinoma (SCC) occurred secondary to mature cystic teratoma. Treatment with paclitaxel/carboplatin (TC chemotherapy) and gemcitabine hydrochloride/carboplatin (GC chemotherapy) after operation was not effective, and the refractory ileus resulting from rapid progression of the disease continued. She was died of disease progression 7 months after the diagnosis of ovarian cancer. We discuss about the clinical characteristics of malignant transformation of mature cystic teratoma and considered about the treatment of the ovarian SCC. [ABSTRACT FROM AUTHOR]

Background Objective Methods Results Conclusion Adenosinergic system has been implicated in the pathophysiology of bipolar disorder and drugs that affect adenosine neurotransmission have shown some efficacy as add‐on therapy in manic patients.Thus, the aim of the present study was to screen adenosinergic drugs for antimanic‐like effect in methylphenidate (MPH)‐induced hyperlocomotion in mice.Male and female Swiss mice received a single allopurinol (50 and 200 mg/kg, ip), dipyridamole (20 mg/kg, ip), or inosine (50 mg/kg, ip) administration before an acute MPH challenge (5 mg/kg, sc). In experiments with repeated treatment, male mice received a daily administration of allopurinol (25 and 50 mg/kg, ip), dipyridamole (20 mg/kg, ip), or inosine (50 mg/kg, ip) for 14 days. Finally, pretreatment with aminophylline (2 mg/kg, sc), an unspecific adenosine receptor antagonist, was used to evaluate a putative adenosinergic mediation. Locomotor activity was measured in the automated activity chamber for 20 min.Acute and repeated dipyridamole reduced the increase in locomotor activity induced by MPH, while allopurinol and inosine had no effect. Aminophylline blocked the effect of dipyridamole in MPH‐induced hyperlocomotion.The present results suggest that dipyridamole may have an antimanic‐like effect through adenosine receptors and reinforce the proposal that the adenosine system may be an interesting target for new antimanic drugs. [ABSTRACT FROM AUTHOR]

Recent epidemiologic studies revealed a correlation between acute kidney injury (AKI) episodes and the progression to chronic kidney disease (CKD). Although the severity and duration of the initial insult likely predict the development of CKD, information regarding tissue markers predictive of early development of renal fibrosis is limited. We investigated key markers in fibrotic kidney in rats and mice. Seven- to eight-week-old male Sprague-Dawley rats underwent bilateral ischemia-reperfusion injury (IRI). Kidney tissues were collected to determine the markers correlated with the severity of kidney fibrosis. In a separate set, a specific chemokine (C-C motif) receptor 2 (CCR2) inhibitor, RS-102895, was administered to 9-week-old male C57BL/6J mice that underwent unilateral IRI (9.2 mg/kg/day in drinking water for 17 days) to investigate whether blockade of the monocyte chemotactic protein-1 (MCP-1) signaling was sufficient to prevent fibrosis. Among candidate tissue markers, neutrophil gelatinase-associated lipocalin (NGAL) and MCP-1 mRNA expressions were correlated with kidney fibrosis. Studies on macrophage polarity showed that mRNA expression of M2, but not M1 macrophage markers, were correlated with acute-phase serum creatinine and fibrosis. Pharmacological blockade of the MCP-1-CCR2 signaling downregulated CCR2, which was insufficient to improve fibrosis in mouse unilateral IRI model, suggesting that additional, redundant pathways contribute to fibrosis. These findings suggested that tissue NGAL expression and M2 macrophage markers are promising markers that show severity of kidney fibrosis. Mechanistic involvement of these markers in CKD pathogenesis warrant additional investigation. [ABSTRACT FROM AUTHOR]

Abstract: Aim: The aim of this study was to establish the reference values for circulating pregnancy‐associated placental microRNAs in maternal plasma and clarify their clinical significance in patients with hypertensive disorder of pregnancy (HDP). Methods: Blood samples were collected from 145 women with uncomplicated pregnancies (24, 26, 31 and 32 women at 12, 23, 30 and 36 weeks of gestation, respectively, and 32 women 1 day after delivery). Plasma concentrations of pregnancy‐associated placental microRNAs (miR‐515‐3p, miR‐517a, miR‐517c and miR‐518b) were measured by quantitative real‐time reverse‐transcription polymerase chain reaction. Reference values for each microRNA were determined by the line of best fit and 95% prediction interval and are expressed as logarithmic transformation. To clarify the clinical significance of these reference values, we measured the plasma concentrations of pregnancy‐associated microRNAs in a different population comprising 33 pregnant women with HDP and 44 women with uncomplicated pregnancies. Results: Reference values for circulating pregnancy‐associated placental microRNAs on chromosome 19 miRNA clusters showed an increasing tendency as pregnancy progressed and decreased significantly 1 day after delivery (P < 0.05). The sensitivity and specificity of each reference value were 57.6% and 93.2% for miR‐515‐3p, 63.6% and 75.0% for miR‐517a, 75.8% and 79.5% for miR‐517c and 63.6% and 75.0% for miR‐518b, respectively. The positive and negative predictive values of each reference value were 86.4% and 74.5% for miR‐515‐3p, 65.6% and 73.3% for miR‐517a, 73.5% and 81.4% for miR‐517c and 65.6% and 73.3% for miR‐518b, respectively. Conclusion: Establishing the reference values for circulating pregnancy‐associated placental microRNAs in maternal plasma could be useful for the evaluation of HDP. [ABSTRACT FROM AUTHOR]

Green-to-red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave-length illumination with blue and red/near-infrared light. By modifying a confocal laser-scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide-spread use of this method. Here, we investigated whether spatially-restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473 nm and 635 nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half-width half-maximum for the axial dimension was below 5 μm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single-cell resolution. [ABSTRACT FROM AUTHOR]

Background Fbxw7 is a tumor suppressor through ubiquitination and degradation of multiple oncoproteins. Loss of Fbxw7 is frequently observed in various human cancers. In this study, we examined the role of Fbxw7 expression in colorectal liver metastasis ( CRLM) and its mechanism. Methods Fifty-six patients with CRLM who undergo curative resection were enrolled. Fbxw7 in tumor tissue was determined by immunohistochemistry. Patients were divided into two groups, the Fbxw7 high and low groups. Clinicopathological factors including miR-223 expression were compared between the high ( n = 32) and low Fbxw7 groups ( n = 24). Results Fbxw7 expression in tumor tissues was significantly lower than that in normal tissues. The disease-free survival in the low Fbxw7 group was significantly worse than that in the high Fbxw7 group, and 3 years disease-free survival of the low and high Fbxw7 groups were 12.5% and 47.0%, respectively ( P = 0.023). On multivariate analysis, loss of Fbxw7 was detected as one of the independent risk factors for recurrence of CRLM (hazard ratio: 2.390, P = 0.017). Likewise, Fbxw7 expression inversely correlated to miR-223 expression ( P = 0.017). Conclusion Loss of Fbxw7 in tumor tissues could be a reliable predictor of recurrence after hepatectomy in patients with CRLM, and miR-223 might be a possible regulator of Fbxw7. [ABSTRACT FROM AUTHOR]

Voltage-gated proton channels (Hv1/VSOP) are expressed in various cells types, including phagocytes, and are involved in diverse physiological processes. Although hvcn1, the gene encoding Hv1, has been identified across a wide range of species, most of the knowledge about its physiological function and expression profile is limited to mammals. In this study, we investigated the basic properties of DrHv1, the Hv1 ortholog in zebrafish (Danio rerio) which is an excellent animal model owing to the transparency, as well as its functional expression in native cells. Electrophysiological analysis using a heterologous expression system confirmed the properties of a voltage-gated proton channel are conserved in DrHv1 with differences in threshold and activation kinetics as compared to mouse (Mus musculus) Hv1 (mHv1). RT-PCR analysis revealed that hvcn1 is expressed in zebrafish neutrophils, as is the case in mammals. Subsequent electrophysiological analysis confirmed the functional expression of DrHv1 in zebrafish neutrophils, which suggests Hv1 function in phagocytes is conserved among vertebrates. We also found that DrHv1 is comparatively resistant to extracellular Zn2+, which is a potent inhibitor of mammalian Hv1, and this phenomenon appears to reflect variation in the Zn2+-coordinating residue (histidine) within the extracellular linker region in mammalian Hv1. Notably, the serum Zn2+ concentration is much higher in zebrafish than in mouse, raising the possibility that Zn2+ sensitivity was acquired in accordance with a change in the serum Zn2+ concentration. This study highlights the biological variation and importance of Hv1 in different animal species. [ABSTRACT FROM AUTHOR]

A majority of early colorectal cancers ( CRCs) with submucosal invasion undergo surgical operation, despite a very low incidence of lymph node metastasis. Our study aimed to identify micro RNAs (mi RNAs) specifically responsible for lymph node metastasis in submucosal CRCs. Micro RNA microarray analysis revealed that miR-100 and miR-125b expression levels were significantly lower in CRC tissues with lymph node metastases than in those without metastases. These results were validated by quantitative real-time PCR in a larger set of clinical samples. The transfection of a miR-100 or miR-125b inhibitor into colon cancer HCT116 cells significantly increased cell invasion, migration, and MMP activity. Conversely, overexpression of miR-100 or miR-125b mimics significantly attenuated all these activities but did not affect cell growth. To identify target m RNAs, we undertook a gene expression array analysis of miR-100-silenced HCT116 cells as well as negative control cells. The Ingenuity Pathway Analysis, TargetScan software analyses, and subsequent verification of m RNA expression by real-time PCR identified mammalian target of rapamycin (m TOR) and insulin-like growth factor 1 receptor ( IGF1R) as direct, and Fas and X-linked inhibitor-of-apoptosis protein ( XIAP) as indirect candidate targets for miR-100 involved in lymph node metastasis. Knockdown of each gene by si RNA significantly reduced the invasiveness of HCT116 cells. These data clearly show that downregulation of miR-100 and miR-125b is closely associated with lymph node metastasis in submucosal CRC through enhancement of invasion, motility, and MMP activity. In particular, miR-100 may promote metastasis by upregulating m TOR, IGF1R, Fas, and XIAP as targets. Thus, miR-100 and miR-125b may be novel biomarkers for lymph node metastasis of early CRCs with submucosal invasion. [ABSTRACT FROM AUTHOR]

Background and Aim Various randomized clinical studies have suggested that short- and long-term outcomes of laparoscopic surgery (LAP) for colorectal cancer are comparable with those of open surgery (OP). However, these studies were performed in high-volume hospitals. The aim of the present study was to compare the outcomes of LAP versus OP for colorectal cancer in rural hospitals. Methods This was a multicenter retrospective propensity score-matched case-control study of patients who underwent colorectal surgery from January 2004 to April 2009 in 10 hospitals in Japan. All patients underwent curative surgery for pathologically diagnosed stage II or III colorectal cancer. The primary end point was 5-year overall survival (OS). The secondary end points were disease-free survival (DFS) and postoperative complications. Results In total, 319 patients who underwent LAP and 1020 patients who underwent OP were balanced to 261 pairs. There was no significant difference in the OS and DFS between two groups. The operation time was significantly shorter for OP than for LAP. Blood loss was significantly lower in LAP than in OP. There was no difference in intraoperative morbidity between the two groups. The postoperative morbidity was significantly lower in LAP than in OP. The hospital stay was significantly shorter in LAP than in OP. There was no significant difference in 90-day postoperative mortality. Conclusions Laparoscopic surgery may be a feasible option for colorectal cancer in rural hospitals. [ABSTRACT FROM AUTHOR]

Background: Thrombospondin‐1 (THBS‐1), a glycoprotein, is an endogenous inhibitor of angiogenesis and tumor growth. In this study, we investigated the clinical role and mechanism of THBS‐1 expression in colorectal liver metastases, focusing on the relationships between its expression and tumor growth, epithelial‐mesenchymal transition (EMT), and expression of other relevant molecules. Methods: Ninety‐four patients who initially underwent curative hepatic resection were enrolled in this study and correlations between expression of THBS‐1 (THBS‐1 high [n = 35] and THBS‐1 low [n = 59]) and tumor growth, Ki‐67 labeling index (Ki‐67 LI), expression of other relevant molecules, and microvessel density (MVD) investigated. Results: THBS‐1 low expression correlated with more advanced grade of liver and lymph node metastases and significantly worse overall survival than strong THBS‐1 expression (3‐year survival: 96.7% vs. 65.4%, P < 0.01). Multivariate analysis identified THBS‐1 low expression as an independent prognostic factor (HR 2.82, 95% CI 1.21–7.71, P = 0.01). THBS‐1 low expression correlated positively with high Ki‐67 LI (P < 0.05) and inversely with E‐cadherin (P < 0.05) and hypoxia inducible factor‐1α (HIF‐1α) expression (P < 0.05); THBS‐1 expression and MVD were not significantly correlated. Conclusions: Low THBS‐1 expression may be an independent poor prognostic factor that affects tumor growth and EMT acquisition. Additionally, THBS‐1 may be regulated by the HIF‐1 pathway. Highlight Setting out to investigate the clinical role and mechanism of thrombospondin‐1 expression in colorectal liver metastases, Teraoku and colleagues revealed that low thrombospondin‐1 expression may be an independent poor prognostic factor that affects tumor growth and epithelial–mesenchymal transition acquisition and may be regulated by the hypoxia inducible factor‐1 pathway. [ABSTRACT FROM AUTHOR]

Objective Hypothalamic hamartoma ( HH) is a congenital anomalous brain tumor. Although most HHs are found without any other systemic features, HH is observed in syndromic disorders such as Pallister-Hall syndrome ( PHS) and oral-facial-digital syndrome ( OFD). Here, we explore the possible involvement of somatic mutations in HH. Methods We analyzed paired blood and hamartoma samples from 18 individuals, including three with digital anomalies, by whole-exome sequencing. Detected somatic mutations were validated by Sanger sequencing and deep sequencing of target amplicons. The effect of GLI3 mutations on its transcriptional properties was evaluated by luciferase assays using reporters containing eight copies of the GLI-binding site and a mutated control sequence disrupting GLI binding. Results We found hamartoma-specific somatic truncation mutations in GLI3 and OFD1, known regulators of sonic hedgehog (Shh) signaling, in two and three individuals, respectively. Deep sequencing of amplicons covering the mutations showed mutant allele rates of 7-54%. Somatic mutations in OFD1 at Xp22 were found only in male individuals. Potential pathogenic somatic mutations in UBR5 and ZNF263 were also identified in each individual. Germline nonsense mutations in GLI3 and OFD1 were identified in each individual with PHS and OFD type I in our series, respectively. The truncated GLI3 showed stronger repressor activity than the wild-type protein. We did not detect somatic mutations in the remaining 9 individuals. Interpretation Our data indicate that a spectrum of human disorders can be caused by lesion-specific somatic mutations, and suggest that impaired Shh signaling is one of the pathomechanisms of HH. [ABSTRACT FROM AUTHOR]

Introduction During laparoscopic gastrectomy, it is important to establish a good operative field and ensure an adequate working space. The combined liver mobilization and retraction method is used to get a safe and optimal view. Methods We retrospectively analyzed 32 consecutive patients who underwent laparoscopic total gastrectomy for gastric cancer. The patients were divided into two groups: the mobilization (+) group ( n = 12) and the mobilization (−) group ( n = 20). Hepatic function tests were performed in all patients. Results Mobilization provided a satisfactory view of the working field, especially the gastroesophageal junction and the angle of His during laparoscopic total gastrectomy, and no complications were observed during liver retraction. On postoperative hepatic function testing, there was no significant difference between the two groups on any day. Conclusions Combined liver mobilization and retraction may be helpful in laparoscopic total gastrectomy. [ABSTRACT FROM AUTHOR]

Aim To clarify the association between circulating chromosome 19 miRNA cluster (C19MC) microRNAs in maternal plasma and severe pre-eclampsia. Method Maternal blood samples (7 mL) at 27-34 weeks of gestation were obtained from 20 pregnant women with severe pre-eclampsia (sPE group) and from 20 uncomplicated pregnant women (NP group). Twenty cases of severe pre-eclampsia were classified into late onset (sPELO group; n = 14) and early onset (sPEEO group; n = 6). Plasma concentration of C19MC microRNAs (miR-518b, -1323, -516b, -516a-5p, -525-5p, -515-5p, -520 h, -520a-5p, -519d and -526b) was measured on quantitative real-time reverse transcription-polymerase chain reaction. Results The circulating levels of all 10 C19MC microRNAs in maternal plasma were significantly increased in the sPE group compared with the NP group. Plasma concentration of all 10 C19MC microRNAs tested was significantly increased in the sPEEO group compared with the NP group, while plasma concentration of nine miRNAs, except for miR-519d, was significantly increased in the sPELO group compared with the NP group. Of the 10 C19MC microRNAs measured, plasma concentration of eight miRNAs, except for miR-518b and miR-519d, was significantly increased in the sPEEO group compared with the sPELO group. Conclusions Increased levels of C19MC microRNAs in maternal plasma are a characteristic phenomenon of established severe pre-eclampsia, and it has been shown for the first time that the upregulation of C19MC miRNAs occurred as a consequence of, not in advance of, the onset of pre-eclampsia. [ABSTRACT FROM AUTHOR]

Background and Aim Bariatric surgery not only elicits weight loss but also rapidly resolves diabetes. However, the mechanisms remain unclear. The present study investigates how diabetes and liver steatosis are improved after duodenal-jejunal bypass ( DJB) compared with a glucagon-like peptide-1 ( GLP-1) analog and correlations between bile acids and GLP-1 secretion. Methods We initially determined the effects of bile acids on GLP-1 in vitro and then assigned 12 male 16-week-old Otsuka Long- Evans Tokushima Fatty rats to groups that underwent DJB, a sham operation, or were treated with the GLP-1 receptor agonist, liraglutide ( n = 4 each). Blood glucose, insulin, GLP-1, serum bile acids, liver steatosis, and the number of GLP-1 positive cells ( L cells) in the small intestine and colon were investigated in the three groups at eight weeks postoperatively. Results Levels of GLP-1m RNA were upregulated and GLP-1 secretion increased in cells incubated with bile acids in vitro. Weight gain was suppressed more in the DJB than in the sham group in vivo. Diabetes was more improved and GLP-1 levels were significantly higher in the DJB than in the sham group. Serum bile acids were significantly increased, the number of L cells in the ileum was upregulated compared with the sham group, and liver steatosis was significantly improved in the DJB compared with the other two groups. Conclusions Duodenal-jejunal bypass might improve diabetes and liver steatosis by enhancing GLP-1 secretion through increasing serum bile acids and the proliferation of L cells in the ileum, compared with liraglutide. [ABSTRACT FROM AUTHOR]

Objective This study aimed to investigate the effect of labor on plasma concentrations of cell-free, pregnancy-associated, placenta-specific microRNAs (miRNAs) before and after delivery. Method In the non-labor group (32 women), cesarean section (C/S) was performed before the beginning of labor. In the labor group (32 women), C/S was performed after the beginning of labor. Plasma concentrations of cell-free, pregnancy-associated, placenta-specific miRNAs (miR-515-3p, miR-517a, miR-517c, and miR-518b) were measured by real-time quantitative PCR. Each miRNA concentration was compared between the non-labor and labor groups. Results Before C/S, plasma concentrations of cell-free, pregnancy-associated, placenta-specific miRNAs in the labor group were significantly higher than those in the non-labor group ( P = 0.001 for 515-3p, P = 0.002 for 517a, P = 0.001 for 517c, and P = 0.003 for 518b). Twenty-four hours after delivery, plasma concentrations of cell-free, pregnancy-associated, placenta-specific miRNAs in the labor group were significantly higher than those in the non-labor group ( P = 0.002 for 515-3p, P = 0.017 for 517a, P = 0.043 for 517c, and P = 0.009 for 518b). Conclusion The presence of labor affects cell-free, pregnancy-associated, placenta-specific miRNA levels in maternal plasma. Labor also affects postpartum clearance of these miRNAs 24 h after delivery. © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Introduction Incisional ventral hernia is one of the most common surgical complications after laparotomy. Laparoscopic repair of incisional ventral hernia has been conducted recently, and the advantages of this procedure have been reported. However, in large orifice cases, the recurrence rate is increased. To improve recurrence rates in large cases, a hybrid method combining laparoscopic primary closure and mesh repair can be applied. Materials and Surgical Technique Monofilament thread was inserted into the abdominal cavity for hernia closure and pulled from the other side of the orifice. The same procedure was performed from the upper side to the lower side without closure, and all thread was placed in line. Both sides of the thread were then introduced to the midline of the incision through a subcutaneous route. This procedure was conducted with an introducer. All threads were tied, and then a mesh was placed. Discussion Hybrid techniques already combine mini-laparotomy for hernia closure and subsequent laparoscopic intraoperative onlay mesh for reinforcement, but such techniques require laparotomy. In our technique, closure of the linea alba does not require laparotomy. All procedures were performed laparoscopically. This procedure is very easy and safe, and does not require the abdominal cavity to be opened. Thus, hybrid methods are effective for treating cases of incisional hernia involving a large orifice. [ABSTRACT FROM AUTHOR]

ABSTRACT Objective This study aimed to identify a set of predominantly placental (PP) mRNAs, which are associated with later-developing twin-to-twin transfusion syndrome (TTTS). Method First, out of 50 PP mRNAs we previously reported, we select target mRNAs that are ordinarily detectable in maternal plasma. Plasma concentrations of these PP mRNAs were measured in monochorionic diamniotic twin (MCDA-T) pregnancies complicated by TTTS later ( n = 11) and in uncomplicated MCDA-T pregnancies ( n = 17). Finally, the diagnostic values of the PP mRNAs in plasma were evaluated. Results From 50 PP mRNAs, nine [human placental lactogen ( hPL); pregnancy-specific glycoproteins 2 ( PSG2); human pregnancy-specific glycoproteins 3 ( PSG3); syncytin; syncytin 2; retinoic acid-induced 14; A disintegrin and metalloproteinase domain-containing protein 12 ( ADAM12); chorionic glycoprotein hormones, alpha polypeptide; and chorionic glycoprotein hormones, and beta polypeptide] were selected as target mRNAs. Changes in six PP mRNAs [increased hPL, PSG2, and PSG3 and decreased syncytin, syncytin2, and ADAM12] in maternal plasma were detected in MCDA-T pregnant women who subsequently developed TTTS. Finally, mRNA signatures gave elevated AUCs ( hPL/ PSG2: 0.8717; hPL/ PSG3: 0.8449; hPL/ ADAM12: 0.8396) compared with single hPL mRNA. Conclusion Quantitative aberration of plural cell-free PP mRNAs in maternal plasma precedes the appearance of clinically apparent TTTS. This suggests that pathophysiological changes in the placenta are associated with morbid conditions of TTTS. © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Chronic hypoxia in the tubulointerstitium serves as a final common pathway in progressive renal disease. Circumstantial evidence suggests that hypoxia-inducible factor (HIF)-1 in the ischemic tubules may be functionally inhibited in a chronic kidney disease (CKD) milieu. In this study, we hypothesized that indoxyl sulfate (IS), a uremic toxin, impairs the cellular hypoxic response. In human kidney (HK-2) proximal tubular cells, IS reduced the hypoxic induction of HIF-1 target genes. This effect was not associated with quantitative changes in the HIF-1α protein, but with functional impairment of the HIF-1α C-terminal transactivation domain (CTAD). Among factors that impeded the recruitment of transcriptional coactivators to the HIF-1αCTAD, IS markedly up-regulated Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) through a mechanism of post-transcriptional mRNA stabilization involving the extracellular signal-regulated kinase (ERK) 1/2 pathway. In vivo, disproportionate expression of HIF target genes was demonstrated in several CKD models, which was offset by an oral adsorbent, AST-120. Furthermore, administration of indole reduced the induction of angiogenic, hypoxia-inducible genes in rats with experimental heart failure. Results of these studies reveal a novel role of IS in modulating the transcriptional response of HIF-1 and provide insight into molecular mechanisms underlying progressive nephropathies as well as cardiovascular complications. [ABSTRACT FROM AUTHOR]

ABSTRACT Objective The aim of this study was to characterize placenta-specific microRNAs in fetal growth restriction (FGR) pregnancy. Method Placenta-specific miRNAs were identified by next-generation sequencing analysis. Subsequently, quantitative real-time reverse-transcription polymerase chain reaction was used to identify FGR placenta-specific miRNAs whose level of expression was significantly decreased in FGR placenta ( n = 45) compared with uncomplicated placenta ( n = 50). FGR pregnancy-associated, placenta-specific microRNAs were identified in maternal plasma after delivery at significantly decreased concentrations, and their circulating levels in maternal plasma was compared between FGR pregnancies ( n = 10) and uncomplicated pregnancies ( n = 10). Results Out of the ten placenta-specific microRNAs that we identified, seven placenta-specific microRNAs (hsa-miR-518b, hsa-miR-1323, hsa-miR-516b, hsa-miR-515-5p, hsa-miR-520h, hsa-miR-519d, and hsa-miR-526b) from the chromosome 19 microRNA cluster were identified as FGR placenta-specific microRNAs. Four FGR placenta-specific microRNAs (hsa-miR-518b, hsa-miR-1323, hsa-miR-520h, and hsa-miR-519d) were confirmed as FGR pregnancy-associated, placenta-specific miRNAs, but their circulating levels in maternal plasma showed no significant differences between FGR pregnancy and uncomplicated pregnancy. Conclusion Our data suggest that reduced expression in placenta of certain FGR placenta-specific miRNAs is associated with FGR and that the discrepancy between expression in FGR placenta and their circulating levels in maternal plasma will be crucial to understanding how placenta-specific microRNAs are released into the maternal circulation. © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Introduction The feasibility of laparoscopic surgery for clinically staged T3 and T4 rectal cancer has not been clearly defined specifically in cases following preoperative chemoradiation therapy ( CRT). Our aim was to investigate the feasibility of laparoscopic surgery after preoperative CRT for clinically staged T3 and T4 rectal cancer. Methods Between May 2003 and June 2009, 57 patients ( T3: n = 50, T4: n = 7) who underwent preoperative CRT for rectal cancer were identified. Forty-three patients with laparoscopic surgery ( Lap group) were compared with 14 patients who underwent open surgery ( Open group). Perioperative data including postoperative morbidity were assessed between the two groups. Results All patients underwent complete laparoscopic operations, and none was converted to laparotomy. Operating time was longer in the Open group (331 vs 375 min, P < 0.01). Blood loss was lower in the Lap group (160 vs 316 mL, P < 0.01). Lymph node harvest and morbidity rate were similar in both groups. The distal tumor margin was negative in all patients. No patients had perioperative mortality associated with surgery after CRT. Conclusion Laparoscopic surgery after preoperative CRT is a feasible and a safe option for T3 and T4 rectal cancer compared to conventional open surgery. [ABSTRACT FROM AUTHOR]

Background: The developing nervous system consists of a variety of cell types. Animal models that allow the visualization of specific classes of neurons are crucial for the study of neuronal networks. Results: We performed an enhancer trap screening in zebrafish and generated a collection of transgenic lines that expressed GFP in a spatially and temporally restricted manner. Among the fish generated, we identified an insertion of the enhancer trap construct in the vicinity of the mnr2b/hlxb9lb gene encoding the mnx class of homeodomain transcription factor. The insertion gave rise to GFP expression predominantly in spinal motor neurons and abducens motor neurons. During embryogenesis, GFP expression was also detected in endodermal and mesodermal tissues, where mnr2b is known to be expressed. Conclusion: These results show that the enhancer trap construct recapitulated the expression pattern of the mnr2b gene and this transgenic line should be useful for the visualization of the spinal and abducens motor neurons in the developing nervous system. Developmental Dynamics 241:327-332, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The hypermodified nucleoside N-threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N-threonylcarbamoyl moiety is composed of -threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the -threonine-binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds -threonine more strongly than -serine and glycine. The Kd values of Sua5 for -threonine and -serine are 9.3 μ M and 2.6 m M, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and -threonine, at 1.8 Å resolution. The -threonine is bound next to AMPPNP in the same pocket of the N-terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine-specific recognition. The carboxyl group and the side-chain hydroxyl and methyl groups of -threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The -threonine is located in a suitable position to react together with ATP for the synthesis of N-threonylcarbamoyladenosine. Proteins 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The zebrafish heart provides a useful vertebrate cardiovascular model with outstanding advantages, including genetic manipulatability, optical accessibility and rapid development. In addition, an emerging topic in cardiotoxicity assay and drug discovery is its use in phenotype-based chemical screening. Here, we report a technique that allows non-invasive voltage mapping in beating heart using a genetically encoded probe for transmembrane potential. Application of the anti-allergy compound astemizole resulted in aberrant propagation of excitation, which accounted for a lack of ventricular contraction. This optical method will provide new opportunities in broad areas of physiological, developmental and pharmacological cardiovascular research. [ABSTRACT FROM AUTHOR]

The medaka fish ( Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene. [ABSTRACT FROM AUTHOR]

Zebrafish is a powerful model system for investigations of vertebrate neural development. The animal has also become an important model for studies of neuronal function. Both in developmental and functional studies, transgenic zebrafish expressing fluorescent proteins in central nervous system neurons have been playing important roles. We review here the methods for producing transgenic zebrafish. Recent advances in transposon- or bacterial artificial chromosome-based transgenesis greatly facilitate the creation of useful lines. We also present our study on alx-positive neurons to reveal how transgenic zebrafish expressing fluorescent proteins in a specific class of neurons can be used to investigate their development and function. [ABSTRACT FROM AUTHOR]

Group I introns are thought to be self-propagating mobile elements, and are distributed over a wide range of organisms through horizontal transmission. Intron invasion is initiated through cleavage of a target DNA by a homing endonuclease encoded in an open reading frame (ORF) found within the intron. The intron is likely of no benefit to the host cell and is not maintained over time, leading to the accumulation of mutations after intron invasion. Therefore, regular invasional transmission of the intron to a new species at least once before its degeneration is likely essential for its evolutionary long-term existence. In many cases, the target is in a protein-coding region which is well conserved among organisms, but contains ambiguity at the third nucleotide position of the codon. Consequently, the homing endonuclease might be adapted to overcome sequence polymorphisms at the target site. To address whether codon degeneracy affects horizontal transmission, we investigated the recognition properties of a homing enzyme, I-CsmI, that is encoded in the intronic ORF of a group I intron located in the mitochondrial COB gene of the unicellular green algaChlamydomonas smithii. We successfully expressed and purified three types of N-terminally truncated I-CsmI polypeptides, and assayed the efficiency of cleavage for 81 substrates containing single nucleotide substitutions. We found a slight but significant tendency that I-CsmI cleaves substrates containing a silent or tolerated amino acid change more efficiently than nonsilent or nontolerated ones. The published recognition properties of I-SpomI, I-ScaI, and I-SceII were reconsidered from this point of view, and we detected proficient adaptation of I-SpomI, I-ScaI, and I-SceII for target site sequence degeneracy. Based on the results described above, we propose that intronic homing enzymes are adapted to cleave sequences that might appear at the target region in various species, however, such adaptation becomes less prominent in proportion to the time elapsed after intron invasion into a new host. [ABSTRACT FROM AUTHOR]

Transfer of more than one embryo following in vitro fertilization/intracytoplasmic sperm injection cycles have increased pregnancy rate at the cost of increasing the incidence of triplets and twins. It has been proposed that prolonged culture to the blastocyst stage would automatically result in the selection of good quality embryos for transfer and minimize the incidence of triplets and twins. The objectives of the present retrospective analysis were to examine the pregnancy outcome, multiple pregnancy and related data following: (i) single blastocyst transfer (BT) and double BT; (ii) single BT in patients belonging to different age groups; and (iii) good, fair or poor quality of BT. A total of 260 BT were carried out between August 1998 and July 2002 and they are included in the current study. Sixty of the 260 BT patients received a single BT, and 41 of them received selected single good quality BT (SSBT). The implantation rate has no significant difference between following single BT (53.3%) and double BT (42.8%). No multiple pregnancy occurred following single BT, while significantly higher ( P < 0.05) multiple pregnancy rate was observed following a double BT (45.8%). The clinical pregnancy and implantation rates following a single BT were similar ( P > 0.05) in patients belonging to <30 years (62.5%), 30–34 years (57.9%) and 35–39 years old (35.8%). Selected single good quality BT maintained pregnancy and avoided multiple pregnancies. It is recommended for patients with a risk for high-order multiple pregnancy. (Reprod Med Biol 2004; 3: 13–18) [ABSTRACT FROM AUTHOR]

What's already known about this topic? Circulating cell‐free microRNA levels in maternal plasma are measurable.Circulating levels of some microRNAs are associated with pregnancy‐associated disorders. What does this study add? Cell‐free miR‐21 levels in maternal plasma are independent of fetal gender.Neonatal birth weight affects plasma cell‐free miR‐21 levels.Maternal body mass index affects plasma cell‐free miR‐21 levels. [ABSTRACT FROM AUTHOR]

The 270-MHz ¹H and 68-MHz 13C nuclear magnetic resonance spectra of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) and Met-enkephalinamide are analyzed in a variety of solvents. For the dipolar form of Met-enkephalin in (C²H3)2SO solution, significant concentration dependences are found of C-α proton chemical shifts, indicating an aromatic ring-current effect in molecular aggregates. An anomalous temperature dependence is observed of the amide proton chemical shift of the Met-5 residue. Furthermore, the chemical shifts of C-α protons of the dipolar form are found to depend appreciably on temperature. From the analyses of the temperature dependences together with concentration dependences of C-α proton resonances, the dipolar form of Met-enkephalin is found to be in an equilibrium of folded and extended conformations at low concentration in (C²H3)2SO solution. Solvent-composition dependences of the amide and C-α proton resonances and carbonyl and α-carbon resonances of the dipolar form in ²H2O/(C²H3)2SO solution are consistent with the conformation equilibrium and the association equilibrium. The folded conformation of the dipolar form in (C²H3)2SO solution is stabilized by the intramolecular attraction between the positively charged N-terminal group and negatively charged C-terminal group. The presence of the folded conformation is confirmed by the measurements of Gd(III)-induced relaxation enhancements of C-α protons. Nuclear Overhauser effects on the dipolar form are not consistent with the predominant formation of the β-turn structure with the intramolecular hydrogen bond (Gly-2) C=O · H - N (Met-5). For the dipolar form of Met-enkephalin in ²H2O solution and for the cationic form of Met-enkephalinamide in (C²H3)2SO solution and in ²H2O solution there is no evidence for the formation of folded conformations. [ABSTRACT FROM AUTHOR]

The conformation of tridecapeptide α-mating factor from yeast Saccharomyces cerevisiae in aqueous solution was analyzed, in comparison with those of active analog and inactive analog peptides. 270-MHz ¹H-NMR spectra of these peptides were observed and the spectral patterns of main-chain N-H proton resonances were classified into three groups, α-mating factor and Trp¹-bearing active peptides belong to the group A1, active des-Trp¹-peptides belong to the group A2 while the peptides of group B are inactive. The main-chain N-H proton resonances of the groups A1 and A2 and side-chain N-H proton resonances were all assigned to individual residues. The 13C-NMR analysis of α-mating factor indicates that the Lys7-Pro8 and Gln10-Pro11 peptide bonds exclusively take the trans form. From the temperature and pH dependences of chemical shifts and Gd(III)-induced relaxation enhancements of amide proton resonances, α-mating factor is found to lake partly a folded conformation in aqueous solution, with an α-helical form in the N-terminal domain and two β-turn forms in the central and C-terminal domain. The pH dependence of fluorescence intensity indicates that, in this folded conformation, the C-terminal carboxylate group lies close to the N-terminal domain. The presence of the folded form in the N-terminal domain and the β-turn form in the central domain correlates with the biological activity of α-mating factor and analog peptides. However, the folded conformation of α-mating factor is in equilibrium with predominantly unordered form, as found from the circular dichroism and NMR analyses. The N-H proton and C-a proton resonances of free α-mating factor as assigned in the present study allow the transferred nuclear Overhauser enhancement (NOE) analysis of the membrane-bound conformation that is more directly related with the activity. [ABSTRACT FROM AUTHOR]

The C-H proton resonances of α-mating factor, yeast pheromone, in 2H2O solution were assigned. The phase transition temperature of perdeuterated dipalmitoylglycerophosphocholine (suspension) was found to be 35.5°C. In the presence of vesicles of this phospholipid, the exchange broadening and transferred nuclear Overhauser effect (TRNOE) of peptide proton resonances (at 50°C) were analyzed. The mode of binding of this peptide with the phospholipid bilayer was elucidated. The N-terminal nine residues (Trp1 - Gly9) are tightly bound to the bilayer, while the C-terminal four residues (Gln10 - Tyr13) are left free in aqueous phase. This is consistent with the previous observation that the C-terminal three residues (Pro11 - Tyr13) are not essential for the activity of this pheromone [Masui, Y. et al. (1977) Biochem. Biophys. Res. Commun. 78, 534- 538]. Furthermore, from the TRNOE analyses, the conformation of the membrane-bound N-terminal part of α-mating factor was elucidated; the residues Trp1 - Gin5 form a compact helical structure while the residues Lys7 - Gly9 form an extended structure. A similar TRNOE was also observed for an active decapeptide analog Trp1 - Gln10. This confirms the previous conclusion that the physiological activities of this pheromone and analog peptides are correlated with the conformations of membrane-bound peptide molecules.