Rhodium-Catalyzed Formal C═O Bond Insertion and Sequential Acyl 1,4-N-to-O Migratory Rearrangement.

We present here a rhodium-catalyzed reaction between N-acyl pyridazinones and diazoacetates, leading to pyridazine derivatives in good yield under mild reaction conditions. This whole sequence probably proceeds through a carbene insertion into a C═O bond and an unprecedented 1,4-N-to-O acyl rearrangement reaction.

MiR-539 inhibits the malignant behavior of breast cancer cells by targeting SP1.

The aberrant expression of microRNAs (miRNAs) is involved in the initiation and progression of human cancers. In our study, we found that miR-539 was down-regulated in breast cancer tissues and cell lines. Decreased expression of miR-539 was significantly associated with lymph node metastasis in patients with breast cancer. Overexpression of miR-539 inhibited the proliferation and promoted apoptosis of breast cancer cells. Moreover, highly expressed miR-539 significantly suppressed the epithelial-mesenchymal transition (EMT) and sensitized cells to cisplatin treatment. Mechanistically, miR-539 was found to target the specificity protein 1 (SP1) and down-regulated the expression of SP1 in breast cancer cells. Knockdown of miR-539 consistently increased the expression of SP1. The expression of miR-539 in breast cancer tissues was negatively correlated with the expression of SP1. Restoration of SP1 significantly attenuated the inhibitory effect of miR-539 on the proliferation of breast cancer cells. Taken together, our results indicate that miR-539 has a tumor suppressive role in breast cancer via targeting SP1, suggesting miR-539 as a promising target for the diagnosis of breast cancer.

Crystal structures of rice hexokinase 6 with a series of substrates shed light on its enzymatic mechanism.

Hexokinases (HXKs) have determined to be multifaceted proteins, and they are the only ones able to phosphorylate glucose in plants. However, the binding mode for ATP to plant HXKs remains unclear. Here, we report the crystal structures of rice hexokinase 6 (OsHXK6) in four different forms: (i) apo-form, (ii) binary complex with D-Glc, (iii) quaternary complex with ADP, PO4 and Mg2+, and (iv) pentanary complex with D-Glc, ADP, PO4, and Mg2+. The apo form is in the open state conformation, and the three others are in the closed state, indicating that glucose and ADP-PO4 binding induces a large conformational change by domain rearrangement. The quaternary complex is a novel intermediate during the catalytic reaction we trapped for the first time, which provides a new evidence for the enzymatic mechanism of HXKs. In addition, the latter two complexes reveal the binding mode for ADP-PO4 to plant HXKs, which provide the structural explanation for the dual-function of OsHXK6. In addition, we identified that residues Gly112, Thr261, Gly262, and Gly450 are essential to the binding between ADP-PO4 and OsHXK6 by a series of single mutations and enzymatic assays. Our study provide structural basis for the other functional studies of OsHXK6 in rice.

Completely Stereoselective Synthesis of Sulfonated 1,3-Dihydroisobenzofurans via Radical Multicomponent Reactions.

Two types of new oxidant-free radical multicomponent reactions of ß-alkynyl ketones, aryldiazonium salts, and DABCO·(SO2)2 (DABSO) were established, leading to the tunable generation of two class of sulfonated 1,3-dihydroisobenzofurans with moderate to good yields and complete stereoselectivity under the mild conditions. The radical-induced scission/recombination of the C(sp3)-C(sp3) bond enabled direct 1,8-halosulfonylation of ß-alkynyl ketones, giving 1,3-dimethylene-substituted (1Z,3Z)-1,3-dihydroisobenzofurans with substituent diversity by p-nitrobenzyl bromide (PNBB) or p-nitrobenzyl chloride (PNBC) as the halo source. Fine-tuning substituents to strong electron-withdrawing ones, such as nitro, cyano, and trifluoromethyl, linked to aryldiazonium tetrafluoroborates allowed a different annulation/1,5-azosulfonylation process to access sulfonated (Z)-1,3-dihydroisobenzofurans with one quaternary carbon-amino functionality.

PGK1 is a Potential Survival Biomarker and Invasion Promoter by Regulating the HIF-1α-Mediated Epithelial-Mesenchymal Transition Process in Breast Cancer.

BACKGROUND/AIMS: Glycolysis, a multi-step enzymatic reaction, is considered to be the root of cancer development and progression. The aim of this study is to figure out which glycolysis enzyme participates in the progression of breast cancer and its possible mechanisms. MATERIALS: We firstly screened out PGK1 by performing an RT-PCR array of glycolysis-related genes in three paired breast cancer samples, and further investigated PGK1 using TCGA and our own database. The effect and mechanism of PGK1 on cell invasion was further explored both in vitro and using patient samples. RESULTS: PGK1 was most upregulated in T3N0 with distant metastases compared to those with no metastases. In the TCGA database, high PGK1 expression predicted poor overall survival (OS) in breast cancer and some other cancers (P< 0.001). In the validation cohort, high PGK1 expression was significantly correlated with larger tumor size (P=0.011) and advanced TNM stage (P=0.033), and PGK1 expression was an independent prognostic factor for OS and disease free survival (DFS) in both univariate and multivariate regression analyses (P< 0.05). Functional studies indicated that knockdown of PGK1 expression significantly inhibited invasion and reversed the epithelial-mesenchymal transition process in breast cancer cells (P< 0.05). Mechanistically, PGK1 increased HRE luciferase activity in a dose-dependent manner, while silencing PGK1 expression decreased HRE activity. CONCLUSION: High PGK1 expression was associated with poor prognosis in breast cancer, because PGK1 and HIF-1α formed a positive feed-forward loop and thus stimulated breast cancer progression and metastases. Based on these results, PGK1 may serve as a promising biomarker and target therapy for breast cancer.

Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility.

Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3-5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice.

Mitochondrial ORFH79 is Essential for Drought and Salt Tolerance in Rice.

The mitochondrion is deemed to be one of the most important organelles, and plays an essential role in various biological processes. Nonetheless, the role of mitochondria in response to abiotic stress remains unclear. Here, we report that accumulation of the cytoplasmic male sterility (CMS) protein ORFH79 in the vegetative tissues resulted in the dysfunction of mitochondria with decreased enzymatic activities of respiratory chain complexes, reduced ATP content and even a morphological change of the mitochondria. However, the suppression of orfH79 by overexpressing a fertility restorer gene Rf5, which is targeted to mitochondria and induced an endonucleolytic cleavage on the atp6-orfH79 transcripts, could recover the function of mitochondria and further significantly improved the tolerance to drought and salt stress. The above evidence suggests that the mitochondrion plays a pivotal role in tolerance to drought and salt stress in rice.

Breast metastasis of gastric signet-ring cell carcinoma: a case report and literature review.

BACKGROUND: Cases of primary gastric adenocarcinoma with metastasis to the breast are extremely rare. Till now, only 38 cases have been reported in PubMed since 1908. CASE PRESENTATION: We herein reported a race case of gastric adenocarcinoma with metastasis to the right breast. Breast biopsy showed invasive signet-ring cell breast carcinoma in the right breast. She was given a TEC regimen (docetaxel 75 mg/m(2), epirubicin 75 mg/m(2), and cyclophosphamide 600 mg/m(2) every 3 weeks) for one cycle but showed no objective response. Upper gastrointestinal endoscopy demonstrated an ulcerative mass in the gastric body. Biopsy demonstrated low-grade gastric adenocarcinoma with signet-ring features. In immunohistochemistry, mammary malignant cells were positive for cytokeratin 7, cytokeratin 20, villin, and ErbB2/HER2, but negative for gross cystic disease fluid protein-15, estrogen receptor, and progesterone receptor. The diagnosis of metastatic poorly differentiated signet-ring cell adenocarcinoma of the right breast identical to gastric primary was confirmed finally. CONCLUSIONS: Gastric cancer with metastasis to the breast can be diagnosed by clinical history, histological findings, and immunohistochemical markers.

Protoplast: a more efficient system to study nucleo-cytoplasmic interactions.

Agrobacterium tumefaciens-mediated genetic transformation is a powerful tool for plant research, but it can be labor-intensive and time-consuming. Here, we report a protoplast-based approach to study nucleo-cytoplasmic interactions, such as cytoplasmic male sterility/fertility restoration (CMS/Rf) and organellar RNA editing. To test the system, we transfected the fertility restorer gene Rf5, which is involved in the rice HL-CMS/Rf system, into rice protoplasts prepared from the HL-CMS line. As the Rf5 protein accumulated in the transformed protoplasts, the CMS-associated transcripts were endonucleolytically cleaved. There were much lower levels of the CMS-associated protein ORFH79 in the transfected protoplasts than in the mock-transfected protoplasts. Next, we used a dsRNA-mediated gene silencing approach to down-regulate the pentatricopeptide protein gene MPR25, which participates in RNA editing of the organellar transcript nad5. The editing efficiency of mitochondrial transcripts of nad5 at nucleotide 1580 was much lower in the transfected protoplasts than in the mock-transfected protoplasts. Together, these results show that protoplast is a simple and efficient system to study interactions between the nucleus and organelles.

Identification of rice Di19 family reveals OsDi19-4 involved in drought resistance.

KEY MESSAGE: The OsDi19 proteins functioned as transcription factors and played crucial roles in response to abiotic stress. Overexpression of OsDi19-4 in rice increased drought tolerance by enhancing ROS-scavenging activity. Many transcription factors play crucial roles in plant responses to abiotic stress. Here, comprehensive sequence analysis suggested that the drought-induced 19 (Di19) gene family in rice genome contain seven members, and these proteins contained a well-conserved zinc-finger Di19 domain. Most OsDi19 proteins were mainly targeted to the nucleus and have transactivation activity in yeast. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that most OsDi19 proteins could form protein dimers. Expression analysis demonstrated that the OsDi19 genes were differentially and abundantly expressed in vegetative tissues, but expressed little in reproductive tissues and some of the OsDi19 genes were markedly induced by abiotic stresses and hormones in qRT-PCR analysis and microarray data. Overexpression of one stress-responsive gene, OsDi19-4, in rice resulted in significantly increased tolerance to drought stress compared with the wild type plants. Moreover, obviously increased ROS-scavenging ability was detected in the OsDi19-4-overexpressing plants under normal and drought stress conditions. These results suggested that the increased stress tolerance of OsDi19-4-overexpressing plants may be attributable to the enhanced ROS-scavenging activity. Taken together, these studies provide a detailed overview of the rice Di19 gene family, and suggest that the OsDi19 family may play crucial roles in the plant response to abiotic stress.

[Surface-enhanced Raman spectra analysis of trace degradation products from goat horn].

Nano-silver colloid was synthesized by using microwave method on the mixtures of sodium citrate solution and silver nitrate solution. The method has advantages of fast heating speed, uniform temperature distribution and easily controlled reaction conditions. The sizes and size distributions of the silver particles were characterized by means of quasi-elastic laser scattering (QLS). The average particles size was (53.27 +/- 2.65) nm and the size of the particles was mainly distributed around 56 nm. Surface-enhanced Raman spectra of the degradation products from goat horn were obtained with silver colloid as active substrate. It was observed that the Raman signal of SERS was enhanced significantly compared with that of regular Raman spectrum, especially at the Raman bands of 659, 830, 850, 929, 999, 1 028, 1 280, 1 439 and 1 599 cm(-1) which reflect the biochemical components in degradation products. The characteristic Raman bands of degradation products from goat horn were preliminary assigned. The assignments showed that the main constituents of the degradation products from goat horn were amino acids and polypeptides. It was for the first time that Surface-enhanced Raman spectroscopy was used to detect trace degradation products from the horns. Raman signal enhancement can be obtained with high sensitivity for the trace concentrations as low as ppm level. It is concluded that surface-enhanced Raman spectroscopy can provide a fast, direct and precise detecting method for the detection of trace degradation solution from horns.

Iridium-Catalyzed Diastereo- and Enantioselective [4 + 1] Cycloaddition of Hydroxyallyl Anilines with Sulfoxonium Ylides.

We present here an iridium-catalyzed diastereo- and enantioselective [4 + 1] cycloaddition reaction of hydroxyallyl anilines with sulfoxonium ylides under mild reaction conditions, leading to 3-vinyl indolines in moderate to good yields with excellent enantioselectivities. Control experiments disclosed a plausible reaction mechanism.

[Dynamic regulation using a quorum-sensing circuit enhances the production of tyrosol by Escherichia coli].

Tyrosol is a natural polyphenolic product that is widely used in chemical, pharmaceutical and food industries. Currently, the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield. Therefore, the phenylpyruvate decarboxylase mutant ARO10F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression, and the optimal ARO10F138L/D218G-L-YahK produced 1.09 g/L tyrosol in shake flasks. In order to further improve tyrosol production, feaB, a key gene in the competing pathway of 4-hydroxyphenylacetic acid, was knocked out, and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15% compared to that of the control. To overcome the low cell density in tyrosol fermentation, the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway, so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition. Using this strategy, the yield of tyrosol was increased to 1.74 g/L, a 33.82% increase. In a 2 L fermenter, the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD600 of 42.88. Compared with those in the engineered strain TRF5 statically regulated by induced expression, the yield was increased by 38.58% and the OD600 was enhanced by 43.62%. The combination of blocking the competing pathway using gene knockout technology, and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol. This study may facilitate the biosynthesis of other chemicals with high toxicity.

Oily sludge treatment in subcritical and supercritical water: A review.

Oily sludge, an inherent byproduct of the petroleum industry, presents dual characteristics of petroleum resources and hazardous waste. Owing to the unique physicochemical properties of sub-/supercritical water, hydrothermal technologies have been increasingly used for oily sludge treatment. This review is the first to focus on oily sludge treatment using sub-/supercritical water. Eight hydrothermal technologies used for different purposes are summarized herein: pressurized hot water extraction (PHWE) for hydrocarbon separation, thermal hydrolysis (TH) for dewaterability improvement, hydrothermal carbonization (HTC) for hydrochar production, wet air oxidation (WAO) for biodegradability improvement, hydrothermal liquefaction (HTL) for bio-oil production, supercritical water upgrading (SCWU) for light oil production, supercritical water oxidation (SCWO) for complete degradation, and supercritical water gasification (SCWG) for H2-rich syngas production. Moreover, a general reaction pathway for sub-/supercritical water treatment of oily sludge is presented, with a particular focus on the chemical mechanism at temperatures above 350 °C. Lastly, two reaction maps are included to illustrate the reaction pathways of two groups of identifiable model compounds in oily sludge: aliphatic and aromatic hydrocarbons. This review provides detailed information that can promote a better understanding of various hydrothermal technologies, a guideline for selecting the suitable hydrothermal process for a particular oily sludge, and recommendations for further researches.

Versatile on-stage microfluidic system for long term cell culture, micromanipulation and time lapse assays.

We report here a versatile on-stage microfluidic cell culture and assay system which is compatible with different microscopes and sensors, can simultaneously perform steps of long term cell culture, high throughput time lapse cell assays/imaging, and cell micromanipulations. With the system, we cultured a variety of cells for different periods of time and monitored their cell morphology, migration and division. We also performed a series non-invasive real time in situ time lapse assays and micromanipulations on different cells. They include: the first time lapse imaging and measurements on the instantaneous variations of morphology, biomechanical properties and the intracellular protein of human red blood cells in responding to pH fluctuation, drug action and electromagnetic radiation; the first continuous time lapse Raman micro-spectroscopy on a CHO cell in different phases of its entire life cycles; the micro-transfection of GFP into B16 cells and the follow up observation of the cell's morphology and expressed GFP fluorescence varying with incubation time and cell generations. The performance of these experiments not only demonstrated the capability of the system, but also proposed a variety of novel methods for obtaining time- and spatially-resolved information about the cellular and molecular heterogeneity and transformation during development or stimulations.

Stereoselective synthesis of sulfonated 1-indenones via radical-triggered multi-component cyclization of ß-alkynyl propenones.

New radical-triggered multi-component cyclizations of ß-alkynyl propenones have been developed, leading to 50 examples of sulfonated 1-indenones with generally good yields and high levels of stereoselectivity. The oxidant-free azosulfonylation of ß-alkynyl propenones with aryldiazonium salts and DABSO was realized under the neutral-redox conditions where TBHP enabled the direct selenosulfonylation of ß-alkynyl propenones by combining sulfinic acids and diphenyl diselenide. This protocol features a broad substrate scope, high functional group tolerance and mild reaction conditions.

Supercritical water oxidation of oil-based drill cuttings.

Oil-based drill cuttings (OBDC) are a typical hazardous solid waste that arises from drilling operations in oil and gas fields. The supercritical water oxidation (SCWO) of OBDC was comprehensively investigated in a batch reactor under the conditions of various oxygen coefficients (OC, 1.5-3.5), temperatures (T, 400-500°C) and reaction times (t, 0.5-10min). Preheating experiments indicated that most of the organic compounds in the initial OBDC sample were distributed within gaseous, oil, aqueous and solid phases, with no more than 9.8% of organic compounds converted into inorganic carbon. All tested variables, i.e., OC, T and t, positively affect the transformation of carbon compounds from the oil and solid phases to the aqueous phase and, ultimately, to CO2. Carbon monoxide is the primary stable intermediate. The total organic carbon (TOC) removal efficiency can reach up to 89.2% within 10min at 500°C. Analysis of the reaction pathways suggests both homogeneous and heterogeneous reactions exist in the reactor. The homogeneous reaction is a typical SCWO reaction that is governed by a free radical mechanism, and the heterogeneous reaction is dominated by mass transfer. The information obtained in this study is useful for further investigation and development of hydrothermal treatment procedures for OBDC.

Sox17 promoter methylation in plasma DNA is associated with poor survival and can be used as a prognostic factor in breast cancer.

Aberrant DNA methylation that leads to the inactivation of tumor suppressor genes is known to play an important role in the development and progression of breast cancer. Methylation status of cancer-related genes is considered to be a promising biomarker for the early diagnosis and prognosis of tumors. This study investigated the methylation status of the Sox17 gene in breast cancer tissue and its corresponding plasma DNA to evaluate the association of methylation levels with clinicopathological parameters and prognosis.The methylation status of the Sox17 gene promoter was evaluated with methylation-specific polymerase chain reaction (MSP) in 155 paired breast cancer tissue and plasma samples and in 60 paired normal breast tissue and plasma samples. Association of Sox17 methylation status with clinicopathological parameters was analyzed by χ tests. Overall and disease-free survival (DFS) curves were calculated using Kaplan-Meier analysis, and the differences between curves were analyzed by log-rank tests.The frequency of Sox17 gene methylation was 72.9% (113/155) in breast cancer tissues and 58.1% (90/155) in plasma DNA. Sox17 gene methylation was not found in normal breast tissues or in their paired plasma DNA. There was a significant correlation of Sox17 methylation between corresponding tumor tissues and paired plasma DNA (r = 0.688, P < 0.001). Aberrant Sox17 methylation in cancer tissues and in plasma DNA was significantly associated with the tumor node metastasis stage (P = 0.035 and P = 0.001, respectively) and with lymph node metastasis (P < 0.001 and P = 0.001, respectively). Kaplan-Meier survival curves showed that aberrant Sox17 promoter methylation in cancer tissues and plasma DNA was associated with poor DFS (P < 0.005) and overall survival (OS) (P < 0.005). Multivariate analysis showed that Sox17 methylation in plasma DNA was an independent prognostic factor in breast cancer for both DFS (P = 0.020; hazard ratio [HR] = 2.142; 95% confidence interval [CI]: 1.128-4.067) and for OS (P = 0.001; HR = 4.737; 95% CI: 2.088-10.747).Sox17 gene promoter methylation may play an important role in breast cancer progression and could be used as a prognostic biomarker to identify patients at risk of developing metastasis or recurrence after mastectomy.

A case of congenital giant gallbladder with massive hydrops mimicking celiac cyst.

We present a case of a 55-year-old female who suffered from a mass in the right upper abdomen, which had been present for over six months. Pre-operative blood examinations, including tumor markers, were normal. Initially, the admitting diagnosis was a giant celiac cyst, due to its liquid cystic appearance and large size (approximately 30.0×18.0 cm), visible in the hepatic region under ultrasound. Following the discovery of a cystic duct during surgery, the diagnosis was corrected to be a giant gallbladder. As no obstructive matter was observed, the giant gallbladder was considered to be congenital.