Anorectal dysfunction in patients with mid-low rectal cancer after surgery: A pilot study with three-dimensional high-resolution manometry.

BACKGROUND: The quality of life in patients who develop low anterior resection syndrome (LARS) after surgery for mid-low rectal cancer is seriously impaired. The underlying pathophysiological mechanism of LARS has not been fully investigated. AIM: To assess anorectal function of mid-low rectal cancer patients developing LARS perioperatively. METHODS: Patients diagnosed with mid-low rectal cancer were included. The LARS score was used to evaluate defecation symptoms 3 and 6 mo after anterior resection or a stoma reversal procedure. Anorectal functions were assessed by three-dimensional high resolution anorectal manometry preoperatively and 3-6 mo after surgery. RESULTS: The study population consisted of 24 patients. The total LARS score was decreased at 6 mo compared with 3 mo after surgery (P < 0.05), but 58.3% (14/24) lasted as major LARS at 6 mo after surgery. The length of the high-pressure zone of the anal sphincter was significantly shorter, the mean resting pressure and maximal squeeze pressure of the anus were significantly lower than those before surgery in all patients (P < 0.05), especially in the neoadjuvant therapy group after surgery (n = 18). The focal pressure defects of the anal canal were detected in 70.8% of patients, and those patients had higher LARS scores at 3 mo postoperatively than those without focal pressure defects (P < 0.05). Spastic peristaltic contractions from the new rectum to anus were detected in 45.8% of patients, which were associated with a higher LARS score at 3 mo postoperatively (P < 0.05). CONCLUSION: The LARS score decreases over time after surgery in the majority of patients with mid-low rectal cancer. Anorectal dysfunctions, especially focal pressure defects of the anal canal and spastic peristaltic contractions from the new rectum to anus postoperatively, might be the major pathophysiological mechanisms of LARS.

Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development.

Msh homeobox (Msx) is a subclass of homeobox transcriptional regulators that control cell lineage development, including the early stage of vertebrate limb development, although the underlying mechanisms are not clear. Here, we demonstrate that Msx1 promotes the proliferation of myoblasts and mesenchymal stem cells (MSCs) by enhancing mitogen-activated protein kinase (MAPK) signaling. Msx1 directly binds to and upregulates the expression of fibroblast growth factor 9 (Fgf9) and Fgf18. Accordingly, knockdown or antibody neutralization of Fgf9/18 inhibits Msx1-activated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. Mechanistically, we determined that the phosphorylation of Msx1 at Ser136 is critical for enhancing Fgf9 and Fgf18 expression and cell proliferation, and cyclin-dependent kinase 1 (CDK1) is apparently responsible for Ser136 phosphorylation. Furthermore, mesenchymal deletion of Msx1/2 results in decreased Fgf9 and Fgf18 expression and Erk1/2 phosphorylation, which leads to serious defects in limb development in mice. Collectively, our findings established an important function of the Msx1-Fgf-MAPK signaling axis in promoting cell proliferation, thus providing a new mechanistic insight into limb development.

A novel role of FKN/CX3CR1 in promoting osteogenic transformation of VSMCs and atherosclerotic calcification.

Fractalkine (FKN) and its specific receptor CX3CR1 play a critical role in the pathogenesis of atherosclerosis including recruitment of vascular cells and the development of inflammation. However, its contribution to regulating the development of atherosclerotic calcification has not been well documented. Osteogenic transformation of vascular smooth muscle cells (VSMCs) is critical in the development of calcification in atherosclerotic lesions. In this study, for the first time, we evaluated the effect of FKN/CX3CR1 on the progression of VSMCs calcification and defined molecular signaling that is operative in the FKN/CX3CR1-induced osteogenic transformation of VSMCs. We found that high-fat diet induced atherosclerotic calcification in vivo was markedly inhibited in the Apolipoprotein E (ApoE) and CX3CR1 deficient (ApoE-/-/CX3CR1-/-) mice compared with their control littermates. FKN and CX3CR1 were both expressed in VSMCs and up-regulated by oxidized low-density lipoprotein (ox-LDL). FKN/CX3CR1 promoted the expression of osteogenic markers, including osteopontin (OPN), bone morphogenetic protein (BMP)-2 and alkaline phosphatase (ALP) and decreased VSMCs markers, including smooth muscle (SM) α-actin and SM22-α in a dose-dependent manner. The essential role of FKN/CX3CR1 in VSMCs calcification was further confirmed by lentivirus-mediated knockdown or overexpression of CX3CR1 blocked or accelerated osteogenic transformation of VSMCs. This response was associated with reciprocal up- and down-regulation of osteogenic factor, runt-related transcription factor 2 (RUNX2), transcription factors in osteoclast differentiation, receptor activator of nuclear factor-κB (RANK), RANK ligand (RNAKL) and osteoprotegerin (OPG), respectively. Inhibition of FKN/CX3CR1-activated Jak2/Stat3 signaling by the Jak/Stat inhibitor AG490 blocked osteogenic transformation of VSMCs and RUNX2 induction concurrently. Taken together, our data uncovered novel roles of FKN/CX3CR1 in promoting VSMC osteogenic transformation and atherosclerotic calcification by activating RUNX2 through Jak2/Stat3 signaling pathway and suppressing OPG. Our findings suggest that targeting FKN/CX3CR1 may provide new strategies for the prevention and treatment of atherosclerotic calcification.

MFN2 couples glutamate excitotoxicity and mitochondrial dysfunction in motor neurons.

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca(2+)-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.

Label-free and high-sensitive detection of human breast cancer cells by aptamer-based leaky surface acoustic wave biosensor array.

A label-free and high-sensitive sensing technology for tumor cell recognition and detection was developed based on a novel 2 × 3 model of leaky surface acoustic wave (LSAW) aptasensor array. In this methodology, every resonator crystal unit of the LSAW aptasensor array had an individual oscillator circuit to work without mutual interference, and could oscillate independently with the phase shift stability of ± 0.15° in air phase and ± 0.3° in liquid phase. The aptamer was firstly assembled to the gold electrode surface of 100 MHz LiTaO3 piezoelectric crystal, which could effectively captured target cells (MCF-7 cells) based on the specific interaction between aptamer and the overexpression of MUC1 protein on tumor cell surface. The aptamer-cell complexes increased the mass loading of LSAW aptasensor and led to phase shifts of LSAW. The plot of phase shift against the logarithm of concentration of MCF-7 cells was linear over the range from 1 × 10(2) cells mL(-1) to 1 × 10(7) cells mL(-1) with a correlation coefficient of 0.994. The detection limit as low as 32 cells mL(-1) was achieved for MCF-7 cells. The LSAW aptasensor also exhibited excellent specificity and stability. In addition, this aptasensor could be regenerated for ten times without irreversible loss of activity. Therefore, the LSAW aptasensor may offer a promising approach for tumor cell detection and have great potential in clinical applications.

Inhibition of reactive oxygen species generation attenuates TLR4-mediated proinflammatory and proliferative phenotype of vascular smooth muscle cells.

Reactive oxygen species (ROS) are associated with inflammation and vasculature dysfunction. This study aimed to investigate the potential role of the ROS on vascular Toll-like receptor 4 (TLR4)-mediated proinflammatory and proliferative phenotype of vascular smooth muscle cells (VSMCs). A wire-induced carotid injury model was used in male TLR4-deficient (TLR4(-/-)) and wild-type C57BL/6J mice to induce neointima formation. In the presence or absence of the ROS scavenger apocynin for 14 days, increased TLR4 and proinflammatory cytokines were observed in wire injury-induced carotid neointima and in platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs. The TLR4(-/-) protected the injured carotid from neointimal formation and impaired the cellular proliferation and migration in response to PDGF-BB. Apocynin attenuated intimal hyperplasia. Pre-treatment with apocynin significantly inhibited intracellular ROS generation, accompanied by a significant suppression of TLR4 and proinflammatory cytokines expression, and VSMC proliferation and migration. However, the results were not obvious in TLR4(-/-) condition. These findings highlight the importance of ROS inhibition in TLR4-mediated proinflammatory and proliferative phenotype of VSMCs, and suggest ROS as an essential therapeutic target for TLR4-associated vascular inflammation and vascular diseases.

Association between estrogen receptor alpha c.454-397T>C and c.454-351A>G and ischemic stroke risk: a systematic review and meta-analysis.

The association between estrogen receptor alpha (ESR1) c.454-397T>C and c.454-351A>G polymorphism and ischemic stroke remains controversial. The aim of this study was to perform a meta-analysis to investigate a more authentic association between c.454-397T>C and c.454-351A>G mutation and ischemic stroke. Systematic searches of electronic databases Embase, PubMed, Web of Science as well as hand-searching of the references of identified articles and the meeting abstracts were performed. Study selection, data abstraction and study quality evaluation were independently conducted in duplicate. Statistical analyses were performed using software Stata 11.0. The pooled odds ratios (ORs) with 95 % confidence intervals (95 % CIs) were performed. Different effect models were used according to the difference in heterogeneity. Publication bias was tested by Begg's funnel plot and Egger's regression test. For c.454-397T>C mutation, five studies were combined. Significant association was found in allelic model (OR = 1.12, 95 % CI = 1.01-1.25, p = 0.03), additive model (OR = 1.25, 95 % CI = 1.01-1.54, p = 0.04), and recessive model (OR = 1.23, 95 % CI = 1.02-1.49, p = 0.03), whereas no evidence of association was found for dominant model (OR = 1.10, 95 % CI = 0.85-1.42, p = 0.47). For c.454-351A>G mutation, no evidence of association was found for all genetic models. Our meta-analysis suggests that ESR1 c.454-397T>C mutation is significantly associated with increased risk of ischemic stroke, whereas no evidence of association was found for ESR1 c.454-351A>G mutation.

Improved isolation of good-quality total RNA from the optic stalk of mud crab, scylla paramamosain

An improved and efficient protocol was developed based on the TaKaRa RNAiso Plus Kit (Code: D9108A) for isolating good-quality total RNA from the optic stalk of mud crab, Scylla paramamosain. The protocol was based on the Trizol method with modifications. The carapace overlapping the optic stalk was retained with RNA in regular protocol. In order to remove the abundant deposition correlative with the carapace which makes the isolation of RNA particularly difficult, 5M potassium acetate solution (pH = 6.0) was added before the precipitation of RNA, and the temperature of RNA deposition was also decreased to -70ºC to ensure the stabilization of RNA. Good-quality total RNA from the optic stalk of S. paramamosain could be easily isolated with this modified protocol and three conventional methods were also employed to confirm the quality of RNA. This improved method would be helpful in facilitating molecular research of crabs involving RNA from the optic stalk.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkalo id accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments.

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkaloid accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

Promotion of nicotine biosynthesis in transgenic tobacco by overexpressing allene oxide cyclase from Hyoscyamus niger.

Plant secondary metabolites are a wide variety of low-molecular weight compounds whose productions are often enhanced in response to both biotic and abiotic stresses. Many of the responses are mediated by a class of hormones, named as jasmonates. In jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, EC 5.3.99.6) catalyzes the most crucial step. Here a heterologous AOC gene from Hyoscyamus niger L. (black henbane), named HnAOC (GenBank accession No. AY708383), was overexpressed in Nicotiana tabacum cv. Petit Havana to investigate the consequence on nicotine content. This study revealed that the transcription of HnAOC in tobacco resulted in overexpression of nicotine biosynthetic pathway genes and higher yield of nicotine, with the maximum of 4.8-fold over control. Therefore, it indicated that without the cost of extrinsic hormones, genetic manipulation of jasmonate biosynthetic pathway genes could be an alternative approach in metabolic engineering for the production of valuable secondary metabolites, which were induced by jasmonates.

Allene oxide cyclase from Camptotheca acuminata improves tolerance against low temperature and salt stress in tobacco and bacteria.

Allene oxide cyclase (AOC, E 5.3.99.6) is an essential enzyme in jasmonate (JA) biosynthetic pathway. An AOC gene (defined as CaAOC, Database Accession No. AY863428) had been isolated from Camptotheca acuminata in previous work. Real-time quantitative PCR analysis indicated that mRNA expression of CaAOC was induced by salt stress (120 mM NaCl) and low temperature (4 degrees C). In order to further investigate the role of AOC gene in the processes, CaAOC was introduced into tobacco via Agrobacterium tumefaciens, and the transgenic lines were subjected to the examination of tolerance against salt stress and low temperature. Under salt stress, the chlorophyll content in transgenic tobacco was higher than that of in the wild plants. The electrolyte leakage test revealed that transgenic tobacco plants were more resistant to low temperature over control. Furthermore, 5'-truncated CaAOC was inserted into pET30 and then expressed in Escherichia coli strain BL21DE3 (pLysS). Interestingly, the transformants could grow on 2YT agar containing 400 mM NaCl. Although these mechanisms are not clear yet, this study suggested that CaAOC could not only be a potential target gene in the engineering of plants and bacteria for improved endurance against salt stress, but also be quite useful in enhancing plant tolerance to cold.

Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (CaHDR) from Camptotheca acuminata and its functional identification in Escherichia coli.

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 microM methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.

Molecular cloning, expression profiling and functional analysis of a DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Camptotheca acuminata.

As the second enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis, DXP reductoisomerase (DXR, EC: 1.1.1.267) catalyzes a committed step of the MEP pathway for camptothecin (CPT) biosynthesis. In order to understand more about the role of DXR involved in the CPT biosynthesis at the molecular level, the full-length DXR cDNA sequence (designated as CaDXR) was isolated and characterized for the first time from a medicinal Nyssaceae plant species, Camptotheca acuminata. The full-length cDNA of CaDXR was 1823 bp containing a 1416 bp open reading frame (ORF) encoding a polypeptide of 472 amino acids. Comparative and bioinformatic analyses revealed that CaDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPH binding motif in its N-terminal region owned by all plant DXRs. Phylogenetic analysis indicated that CaDXR was more ancient than other plant DXRs. Tissue expression pattern analysis revealed that CaDXR expressed strongly in stem, weak in leaf and root. CaDXR was found to be an elicitor-responsive gene, which could be induced by exogenous elicitor of methyl jasmonate. The functional color complementation assay indicated that CaDXR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that DXP reductoisomerase plays an influential step in isoprenoid biosynthesis.

Molecular cloning and characterization of the yew gene encoding squalene synthase from Taxus cuspidata.

The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semiquantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.

Molecular cloning and characterization of a novel stem-specific gene from Camptotheca acuminata.

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissuespecific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.

Cloning and functional analysis of a cDNA encoding Ginkgo biloba farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.

Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast.

In plants, the first committed step in the pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34). Here we report for the first time the cloning of a full-length cDNA encoding HMGR (Tm-HMGR) from a taxol-producing gymnosperm, Taxus media Rehder. The full-length cDNA of Tm-HMGR (GenBank accession number: AY277740) was 2307 base pairs (bp), with a 1791-bp open reading frame (ORF) encoding a 596-amino-acid polypeptide. Bioinformatic analysis revealed that Tm-HMGR contained two trans-membrane domains and a catalytic domain, and showed high homology to other plant HMGRs. Phylogenetic analysis indicated that Tm-HMGR was more ancient than other plant HMGRs. The structural modelling showed that Tm-HMGR had the typical spatial structure of HMGRs whose catalytic domains could be folded and divided into three spatial domains, L-domain, N-domain and S-domain. Southern blot analysis revealed that Tm-HMGR belonged to a small HMGR gene family. Northern blot analysis showed that Tm-HMGR was expressed in roots, stems and needles, with higher expression in stems and needles than in roots. Functional complementation of Tm-HMGR in a HMGR-deficient mutant yeast demonstrated that Tm-HMGR mediated the biosynthesis of mevalonate and provided the general precursor for taxol biosynthesis.