Structural atlas of human primary microRNAs generated by SHAPE-MaP.

MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

Multifunctional synthetic nano-chaperone for peptide folding and intracellular delivery.

Artificial, synthetic chaperones have attracted much attention in biomedical research due to their ability to control the folding of proteins and peptides. Here, we report bio-inspired multifunctional porous nanoparticles to modulate proper folding and intracellular delivery of therapeutic α-helical peptide. The Synthetic Nano-Chaperone for Peptide (SNCP) based on porous nanoparticles provides an internal hydrophobic environment which contributes in stabilizing secondary structure of encapsulated α-helical peptides due to the hydrophobic internal environments. In addition, SNCP with optimized inner surface modification not only improves thermal stability for α-helical peptide but also supports the peptide stapling methods in situ, serving as a nanoreactor. Then, SNCP subsequently delivers the stabilized therapeutic α-helical peptides into cancer cells, resulting in high therapeutic efficacy. SNCP improves cellular uptake and bioavailability of the anti-cancer peptide, so the cancer growth is effectively inhibited in vivo. These data indicate that the bio-inspired SNCP system combining nanoreactor and delivery carrier could provide a strategy to expedite the development of peptide therapeutics by overcoming existing drawbacks of α-helical peptides as drug candidates.

High-throughput chemical screening to discover new modulators of microRNA expression in living cells by using graphene-based biosensor.

MicroRNAs (miRNAs) are important regulatory RNAs that control gene expression in various biological processes. Therefore, control over the disease-related miRNA expression is important both for basic research and for a new class of therapeutic modality to treat serious diseases such as cancer. Here, we present a high-throughput screening strategy to identify small molecules that modulate miRNA expression in living cells. The screen enables simultaneous monitoring of the phenotypic cellular changes associated with the miRNA expression by measuring quantitative fluorescent signals corresponding to target miRNA level in living cells based on a novel biosensor composed of peptide nucleic acid and nano-sized graphene oxide. In this study, the biosensor based cellular screening of 967 compounds (including FDA-approved drugs, enzyme inhibitors, agonists, and antagonists) in cells identified four different classes of small molecules consisting of (i) 70 compounds that suppress both miRNA-21 (miR-21) expression and cell proliferation, (ii) 65 compounds that enhance miR-21 expression and reduce cell proliferation, (iii) 2 compounds that suppress miR-21 expression and increase cell proliferation, and (iv) 21 compounds that enhance both miR-21 expression and cell proliferation. We further investigated the hit compounds to correlate cell morphology changes and cell migration ability with decreased expression of miR-21.

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors.

We present a new platform for multiplexed protein kinase activity assay using TiO2 decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening. On the basis of the strong affinity of TiO2 for the phosphate group and the fluorescence quenching capability of GO, phosphorylation of substrates by protein kinases was quantitatively measured in a short time.

BSA as additive: A simple strategy for practical applications of PNA in bioanalysis.

Application of peptide nucleic acid (PNA) in bioanalysis has been limited due to its nonspecific adsorption onto hydrophobic surface in spite of favorable properties such as higher chemical/biological stability, specificity and binding affinity towards target nucleic acids compared to natural nucleic acid probes. Herein, we employed BSA in PNA application to enhance the stability of PNA in hydrophobic containers and improve the sensing performance of the DNA sensor based on graphene oxide (GO) and PNA. Addition of 0.01% BSA in a PNA solution effectively prevented the adsorption of PNA on hydrophobic surface and increased the portion of the effective PNA strands for target binding without interfering duplex formation with a complementary target sequence. In the GO based biosensor using PNA, BSA interrupted the unfavorable adsorption of PNA/DNA duplex on GO surface, while allowing the adsorption of ssPNA, resulting in improvement of the performance of the DNA sensor system by reducing the detection limit by 90-folds.

Direct, sequence-specific detection of dsDNA based on peptide nucleic acid and graphene oxide without requiring denaturation.

Sequence-specific detection of double stranded DNA (dsDNA) is important in various research fields. In general, denaturation of dsDNA into single strands is necessary for the sequence-specific recognition of probes to target DNA, posing several drawbacks which decrease the efficiency as a DNA sensor. Herein, we report a direct, sequence-specific dsDNA detection system without requiring any thermal denaturing step. Our strategy utilizes peptide nucleic acid (PNA) and graphene oxide (GO) as a probe and as a fluorescence quencher, respectively. The PNA first binds to the end of dsDNA strand due to the relatively easily dissociable terminal base pairs of DNA duplex. Next, superior binding affinity of PNA towards complementary DNA induces branch migration for gradual strand replacement, resulting in the formation of PNA/DNA duplex. Unlike other dsDNA sensors based on complementary DNA probes, PNA in combination with GO enabled hybridization with the target sequence hidden as a duplex form without denaturing step and thus, the formation of PNA/DNA duplex was translated into selective fluorescence signal. Moreover, it provided tighter turn-on signal control with very low background signal and high sensitivity and sequence selectivity even in the presence of serum proteins.

A graphene oxide-based platform for the assay of RNA synthesis by RNA polymerase using a fluorescent peptide nucleic acid probe.

We report a simple, direct fluorometric assay based on graphene oxide (GO) for RNA polymerase-mediated RNA synthesis. In principle, fluorescent peptide nucleic acid (PNA) probes were designed, and annealed with RNA products and the resultant RNA-PNA hybrids induced the recovery of fluorescence intensity of the PNA probes adsorbed onto the GO surface.

Robot-assisted total preservation of the pelvic autonomic nerve with extended systematic lymphadenectomy as part of nerve-sparing radical hysterectomy for cervical cancer.

OBJECTIVE: To evaluate our short-term clinical outcomes of robot-assisted autonomic nerve-sparing extended systematic lymphadenectomy as part of nerve-sparing radical hysterectomy. METHODS: Between March 2011 and June 2012, we observed prospectively 28 consecutive patients who underwent robot-assisted autonomic nerve-sparing extended systematic lymphadenectomy, including the superior and inferior gluteal, presacral (subaortic), common iliac, and lower para-aortic nodes. RESULTS: The predominant International Federation of Gynecology and Obstetrics stage was IB1 (15 patients), followed by IB2 (5 patients), IA2 (3 patients), IIA1 (3 patients), and IIA2 (2 patients). The mean ± SD total operating time was 308.8 ± 54.9 minutes, and the mean ± SD console time was 280.0 ± 46.0 minutes. The mean ± SD blood loss was 102.7 ± 153.8 mL. The mean ± SD acquired pelvic lymph node was 27.1 ± 9.3, the mean ± SD extended lymph node was 19.2 ± 9.6, and the mean ± SD total lymph node was 46.3 ± 14.5. A total of 10 patients (35.7%) had nodal metastasis; among them, 6 patients (21.4%) had single pelvic nodal metastasis, 3 patients (10.7%) had concurrent pelvic and extended nodal metastasis, and one patient (3.6%) had single extended nodal metastasis. No intraoperative complications that required treatment occurred; however, ureterovaginal fistula was identified in 4 patients (14.3%) and ureter stricture in 4 patients (14.3%) after radiotherapy. After a median follow-up of 10 months (range, 1-16 months), there was no pelvic recurrence; however, one patient had recurrence at transposition site of ovary. CONCLUSIONS: With the advantage of delicate movement of robot instrument, robot-assisted systematic extended lymphadenectomy with total preservation of pelvic autonomic nerves did not compromise the radicality, and its surgical technique was feasible and safe. By using this approach, we could harvest more lymph nodes and have a high rate of metastatic nodes without disturbing voiding function; however, there was increased rate of urological complications. Moreover, long-term survival benefit after an extended systematic lymphadenectomy must be evaluated.

Inverted Y incision and trans-sacral approach in retroperitoneal aggressive angiomyxoma: a case report.

INTRODUCTION: Aggressive angiomyxoma is a rare myxedematous mesenchymal tumor that mainly occurs in the female pelvis and perineum. The principle of treatment for aggressive angiomyxoma is surgical excision. The tumor can be removed by local excision alone when it occurs locally on the perineum. However, it cannot be completely excised by a perineal approach alone when it passes through the perineum and pelvic bone to extend into the retroperitoneal space. CASE PRESENTATION: A 34-year-old Asian woman presented with a rapidly growing left perineal mass and swelling in the left gluteal region. The swelling was associated with a mild, dull pain in the left gluteal region. In the present case of bulky aggressive angiomyxoma extending to the perineum and retroperitoneal space, the authors made an inverted Y incision through the buttock, removed the coccyx and lower portion of the sacrum, and excised the retroperitoneal mass and perineal lesion through a perineal approach. CONCLUSION: The inverted Y incision and trans-sacral approach can provide easy access to deep retroperitoneal aggressive angiomyxoma and reduce damage to neighboring organs.

Robot versus laparoscopic nerve-sparing radical hysterectomy for cervical cancer: a comparison of the intraoperative and perioperative results of a single surgeon's initial experience.

OBJECTIVE: The aim of the study was to compare the initial surgical outcomes and learning curve of nerve-sparing robotic radical hysterectomy (RRH) with nerve-sparing total laparoscopic radical hysterectomy (TLRH) for the treatment of early-stage cervical cancer in the first 50 cases. METHODS: Between January 2008 and March 2012, 50 consecutive patients underwent nerve-sparing RRH. These patients were compared with a historic cohort of the first 50 consecutive patients who underwent nerve-sparing TLRH. RESULTS: Both groups were similar with respect to patients and tumor characteristics. The mean operating time in the RRH group was significantly longer than that in the TLRH group (230.1 ± 35.8 vs 211.2 ± 46.7 minutes; P = 0.025). The mean blood loss for the robotic group was significantly lower compared with the laparoscopic group (54.9 ± 31.5 vs 201.9 ± 148.4 mL; P < 0.001). There was no significant difference in the mean pelvic lymph nodes between the 2 groups (25.0 ± 9.9 vs 23.1 ± 10.4; P = 0.361). The mean days to normal residual urine were 9.6 ± 6.4 in RRH and 11.0 ± 6.2 in TLRH (P = 0.291). The incidence of intraoperative complication was profoundly lower in RRH compared with that of TLRH (0% vs 8%; P = 0.041). Moreover, no intraoperative transfusion was required in RRH, whereas 4 (8%) were required in TLRH (P = 0.041). In both groups, we found no evidence of a learning effect during the first 50 cases. CONCLUSIONS: During the first 50 cases, surgical outcomes and complication rates of nerve-sparing RRH were found to be comparable to those of nerve-sparing TLRH. Moreover, the mean blood loss and intraoperative complication rate in the robotic group were significantly lower than those in the laparoscopic group. Surgical skills for nerve-sparing TLRH easily and safely translated to nerve-sparing RRH in case of experienced laparoscopic surgeon.

Multivalent nanofibers of a controlled length: regulation of bacterial cell agglutination.

Control of the size and shape of molecular assemblies on the nanometer scale in aqueous solutions is very important for the regulation of biological functions. Among the well-defined supramolecular structures of organic amphiphiles, one-dimensional nanofibers have attracted much attention because of their potential applications in biocompatible materials. Although much progress has been made in the field of self-assembled nanofibers, the ability to control the fiber length remains limited. The approach for control of the fiber length presented herein overcomes this limitation through the coassembly of amphiphilic rod-coil molecules in which the crystallinity of the aromatic segment can be regulated by π-π stacking interactions. The introduction of carbohydrate segments into the fiber exterior endows the nanofibers with the ability to adhere to bacterial cells. Notably, the fiber length systematically regulates the agglutination and proliferation of bacterial cells exposed to these fibers.

Smart hydrogels from laterally-grafted peptide assembly.

Small peptides carrying laterally-grafted azobenzene units self-assemble into photo-responsive hydrogels which are applied as a smart matrix for controlling the dye molecules release. We demonstrate that a delicate balance among peptides interactions plays a pivotal role in the photo-responsive gel-sol transition.

Control of peptide assembly through directional interactions.

We demonstrate the self-assembly of tripeptide amphiphiles into spherical hollow capsules from linear nanoribbons via control of the molecular packing. We achieved a transition of arrangement from anisotropic to isotropic by an elaborate design of the molecular architecture.

Clinicopathologic characteristics of granulosa cell tumors of the ovary: a multicenter retrospective study.

OBJECTIVE: To evaluate the clinicopathologic characteristics and prognostic factors of ovarian granulosa cell tumors. METHODS: Medical records of 113 patients presenting between January 1995 and December 2007 were retrospectively reviewed. RESULTS: One-hundred two patients had adult type disease, with a mean age of 46.2 years (range, 18 to 83 years) and a mean follow-up period of 54.7 months (range, 1 to 155 months). The distribution of FIGO stages was 86 patients at stage I, 11 at stage II, and 5 at stage III. During follow-up, ten patients recurred at a mean time of 48 months (range, 4 to 109 months). Among them, three patients died after a mean of 57 months (range, 25 to 103 months). In recurrence analysis, advanced stage (p=0.032) and presence of residual disease (p=0.012) were statistically significant, and age<40 years, premenopause and positive washing cytology were marginally significant (p<0.1). In multivariate analysis, stage was the only factor associated with recurrence; adjuvant chemotherapy and fertility-sparing surgery were not statistically significant. Among 36 patients with fertility-sparing operations, eight patients had nine pregnancies and delivered seven babies. Eleven patients had juvenile type tumors; the mean age was 20.0 years (range, 8 to 45 years) and the mean follow-up period was 69.8 months (range, 20 to 156 months). The distribution of FIGO stage was nine patients at stage I and two at stage III. There were no recurrences or deaths reported. Four patients had seven pregnancies and delivered six babies. CONCLUSION: Stage is the only factor associated with disease-free survival, and fertility-sparing surgery may be a treatment option for women with early-stage disease who want to retain fertility.

Prenatal diagnosis of scalp cyst with spontaneous regression.

Fetal scalp cysts are rare, and they may be easily misdiagnosed as meningoceles or encephaloceles. A 38-year-old pregnant woman was referred to our hospital for ultrasound examination, which revealed a small cyst above the skull with no defect in the skull. The cyst could not be seen on follow-up ultrasound examinations. After delivery, a three-dimensional CT scan of the head revealed no defect in skull and scalp. Fetal scalp cysts usually require surgical excision after birth, but can regress without any treatment.

Laparoscopic second look operation for ovarian cancer: single center experiences.

We compared the clinical outcomes of laparoscopic surgery with those of laparotomy in second-look operations for ovarian cancer. We retrospectively reviewed the medical records of 35 patients treated between January 2000 and December 2005. They were categorized into two groups: Laparoscopy versus laparotomy group. Among 35 patients, 18 patients (51.4%) were treated with laparoscopy, and 17 patients (48.6%) were treated with laparotomy. There were no statistically significant differences except for hospital stay (laparoscopy vs. laparotomy five days vs. nine days, p < 0.05). Eight patients (44.4%) in the laparoscopy group and six (35.3%) in the laparotomy group were found to be pathologically positive after the second look operation. There were two recurrences (20.0%) in the laparoscopy group and four (36.4%) in the laparotomy group pathologically negative in the second-look operation (p = 0.557). There were no differences in disease-free survival rates (p = 0.705) between the two groups. The second look operation is an ideal method for histologic comfirmation of recurrent or persistent ovarian cancer. Laparoscopic surgery seems a safe and accurate method for the second-look examination, and can also reduce patient discomfort, hospital stay and morbidity associated with laparotomy.

Oncologic results and surgical morbidity of laparoscopic nerve-sparing radical hysterectomy in the treatment of FIGO stage IB cervical cancer: long-term follow-up.

OBJECTIVES: The aim of this study was to evaluate a long-term follow-up data for oncologic results and surgical morbidity of a laparoscopic nerve-sparing radical hysterectomy (NSRH) in the treatment of FIGO stage IB cervical cancer. METHODS: This was a retrospective study that comprised consecutive 125 patients with cervical cancer stage IB1 (n = 105) and IB2 (n = 20) who underwent a laparoscopic NSRH (Piver type III) by a gynecologic oncologist without selecting patients from January 1999 to December 2007. RESULTS: In regression analysis, the operating time (R linear = 0.311, P < 0.001) and estimated blood loss (R linear = 0.261, P < 0.001) were decreased, whereas the number of harvested pelvic lymph nodes (R linear = 0.250, P < 0.001) was increased. Seventeen patients (13.6%, 17/125) were found to have pelvic node metastasis. Para-aortic node metastasis had occurred in 2 patients (5.1%, 2/39). There were high urological complications (13/125, 10.4%) related to radical surgery. Forty-one patients (33%) needed transfusions. Positive surgical margins did not exist. Patients were able to self-void at a mean of 10.3 days postoperatively. The return rates to normal voiding function at postoperative 14 and 21 days were 92.0% and 95.2%, respectively. Thirteen patients (IB1 n = 9, IB2 n = 4) experienced a recurrence postoperatively. Six patients (IB1 n = 3, IB2 n = 3) died of recurrent disease. Five-year disease-free survival rates of cervical cancer IB1 and IB2 were 92% and 78%, respectively (P = 0.1772). Five-year overall survival rates of cervical cancer IB1 and IB2 were 96% and 83%, respectively (P = 0.0437). CONCLUSIONS: A laparoscopic NSRH for FIGO stage IB cervical cancer was comparable to open NSRH in terms of early recovery of bladder function. It did not compromise surgical radicality, but revealed high urological complications, long operating time, and much blood loss, compared with conventional radical hysterectomy. However, these surgical morbidities were corrected with increase in experiences.

Robotic uterine artery preservation and nerve-sparing radical trachelectomy with bilateral pelvic lymphadenectomy in early-stage cervical cancer.

OBJECTIVE: The aim of the study was to evaluate the safety and feasibility of robotic uterine artery preservation and nerve-sparing radical trachelectomy with pelvic lymphadenectomy using the da Vinci surgical system. METHODS: Three patients who were diagnosed with early-stage cervical cancer underwent robotic uterine artery preservation and nerve-sparing radical trachelectomy with bilateral lymphadenectomy from January 2010 to March 2010. The data were compared with those of 4 cases of total laparoscopic nerve-sparing radical trachelectomy that were performed from July 2004 to May 2005 and were previously reported. RESULTS: In the robotic group, the mean console time was 275 minutes (range, 240-305 minutes). The mean postoperative hemoglobin change was 0.4 g/dL (range, 0.2-0.6 g/dL). The mean estimated blood loss was 23 mL (range, 15-40 mL), which is less than that of the laparoscopic group. There were no metastases detected in any of the cases, and the resection margins were negative in both groups. CONCLUSIONS: The robotic uterine artery preservation and nerve-sparing radical trachelectomy with pelvic lymphadenectomy were efficient in reducing blood loss and feasible methods such as other approaches.

Amino-terminal pro-brain natriuretic peptide levels in hypertensive disorders complicating pregnancy.

OBJECTIVE: Amino-terminal pro-brain natriuretic peptide (NT-proBNP) is synthesized in cardiac ventricles in response to volume expansion. This study evaluated NT-proBNP levels to determine the clinical correlation with the severity of hypertensive disorders complicating pregnancy. METHODS: NT-proBNP levels of 95 pregnant women (severe preeclampsia [n = 26], mild preeclampsia [n = 15], gestational hypertension [n = 9], and healthy controls [n = 45]) were determined using an electrochemiluminescence immunoassay. RESULTS: Comparisons of the mean values of NT-proBNP levels in the different groups were significantly different, as follows: 1766.43 ± 4197.39 pg/mL (median, 339.8 pg/mL) in severe preeclampsia, 214.97 ± 226.35 pg/mL (median, 152.3 pg/mL) in mild preeclampsia, 39.75 ± 24.85 pg/mL (median, 34.09 pg/mL) in gestational hypertension, and 78.78 ± 81.56 pg/mL (median, 48.54 pg/mL) in the healthy controls. The NT-proBNP levels of the patients with mild and severe preeclampsia were significantly higher than in the patients with gestational hypertension and the healthy control patients. There was no significant difference in NT-proBNP levels between patients with mild and severe preeclampsia (p = 0.17). CONCLUSION: In patients with mild and severe preeclampsia, NT-proBNP levels were elevated. This may reflect ventricular stress and/or subclinical cardiac dysfunction associated with preeclampsia.

Designer nanorings with functional cavities from self-assembling ß-sheet peptides.

ß-Barrel proteins that take the shape of a ring are common in many types of water-soluble enzymes and water-insoluble transmembrane pore-forming proteins. Since ß-barrel proteins perform diverse functions in the cell, it would be a great step towards developing artificial proteins if we can control the polarity of artificial ß-barrel proteins at will. Here, we describe a rational approach to construct ß-barrel protein mimics from the self-assembly of peptide-based building blocks. With this approach, the direction of the self-assembly process toward the formation of water-soluble ß-barrel nanorings or water-insoluble transmembrane ß-barrel pores could be controlled by the simple but versatile molecular manipulation of supramolecular building blocks. This study not only delineates the basic driving force that underlies the folding of ß-barrel proteins, but also lays the foundation for the facile fabrication of ß-barrel protein mimics, which can be developed as nanoreactors, ion- and small-molecule-selective pores, and novel antibiotics.