Influences of different manure N input on soil ammonia-oxidizing archaea and bacterial activity and community structure in a double-cropping rice field.

AIMS: The short-term effects of different organic manure nitrogen (N) input on soil ammonia-oxidizing archaea (AOA) and bacterial (AOB) activity and community structure at maturity stages of early rice and late rice were investigated in the present paper, in a double-cropping rice system in southern China. METHODS AND RESULTS: A field experiment was done by applying five different organic and inorganic N input treatments: (i) 100% N of chemical fertilizer (M0), (ii) 30% N of organic manure and 70% N of chemical fertilizer (M30), (iii) 50% N of organic manure and 50% N of chemical fertilizer (M50), (iv) 100% N of organic manure (M100) and (v) without N fertilizer input as control (CK). Microbial community changes were assessed using fatty acid methyl esters, and ammonia oxidizer (AO) changes were followed using quantitative PCR. The results showed that AOA were higher than that of AOB based upon amoA gene copy at maturity stages of early rice and late rice. Also, the abundance of AOB and AOA with M30, M50 and M100 treatments was significantly higher than that of CK treatment. Manure N input treatments had significant effect on AOB and AOA abundance, and a higher correlation between AOB and manure N input was observed. AOB correlated moderately with soil organic carbon content, and AOA correlated moderately with water-filled pore space. CONCLUSIONS: This study found that abundance of AOB and AOA was increased under the given organic N conditions, and the soil AOB and AOA community and diversity were changed by different short-term organic manure N input treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil microbial community and specific N-utilizing microbial groups were affected by organic manure N input practices.

Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass.

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.

An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers.

Epithelial-mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality.

Ubiquitin D is correlated with colon cancer progression and predicts recurrence for stage II-III disease after curative surgery.

BACKGROUND: Our recent study observed that the expression of ubiquitin D (UBD), a member of ubiquitin-like modifier family, was upregulated in colon cancer parenchymal cells. The present study further investigated the clinical signicance of UBD in colon cancer. METHODS: Using quantitative PCR, tissue microarray (TMA), western blot analysis and immunohistochemical stain, we evaluated UBD mRNA and protein levels in tumour tissues from patients with colon cancer at different stages and in paired adjacent normal epithelium. RESULTS: Immunohistochemical detection of UBD on a TMA containing 203 paired specimens showed that increased cytoplasmic UBD was signicantly associated with depth of cancer invasion, lymph node metastasis, distant metastasis, tumour histologic grade, advanced clinical stage and Ki-67 proliferative index. Patients with UBD-positive tumours had a significantly higher disease recurrence rate and poorer survival than patients with UBD-negative tumours after the radical surgery. Stratification analysis according to tumour stage revealed UBD as an independent predictor for tumour recurrence in patients with stage II and III tumours. CONCLUSION: UBD may contribute to the progression of colon carcinogenesis and function as a novel prognostic indicator of forecasting recurrence of stage II and III patients after curative operations.

Phospholipase C delta 1 is a novel 3p22.3 tumor suppressor involved in cytoskeleton organization, with its epigenetic silencing correlated with high-stage gastric cancer.

Located at the important tumor suppressor locus, 3p22, PLCD1 encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movements. We identified PLCD1 as a downregulated gene in aerodigestive carcinomas through expression profiling and epigenetic characterization. We found that PLCD1 was expressed in all normal adult tissues but low or silenced in 84% (16/19) gastric cancer cell lines, well correlated with its CpG island (CGI) methylation status. Methylation was further detected in 62% (61/98) gastric primary tumors, but none of normal gastric mucosa tissues. PLCD1 methylation was significantly correlated with tumor high stage. Detailed methylation analysis of 37 CpG sites at the PLCD1 CGI by bisulfite genomic sequencing confirmed its methylation. PLCD1 silencing could be reversed by pharmacological demethylation with 5-aza-2'-deoxycytidine, indicating a direct epigenetic silencing. Ectopic expression of PLCD1 in silenced gastric tumor cells dramatically inhibited their clonogenicity and migration, possibly through downregulating MMP7 expression and hampering the reorganization of cytoskeleton through cofilin inactivation by phosphorylation. Thus, epigenetic inactivation of PLCD1 is common and tumor-specific in gastric cancer, and PLCD1 acts as a functional tumor suppressor involved in gastric carcinogenesis.

Reversibility of apoptosis in cancer cells.

Apoptosis is a cell suicide programme characterised by unique cellular events such as mitochondrial fragmentation and dysfunction, nuclear condensation, cytoplasmic shrinkage and activation of apoptotic protease caspases, and these serve as the noticeable apoptotic markers for the commitment of cell demise. Here, we show that, however, the characterised apoptotic dying cancer cells can regain their normal morphology and proliferate after removal of apoptotic inducers. In addition, we demonstrate that reversibility of apoptosis occurs in various cancer cell lines, and in different apoptotic stimuli. Our findings show that cancer cells can survive after initiation of apoptosis, thereby revealing an unexpected potential escape mechanism of cancer cells from chemotherapy.

Pharmacological characteristics of Ro 115-1240, a selective alpha1A/1L-adrenoceptor partial agonist: a potential therapy for stress urinary incontinence.

OBJECTIVE: To describe the preclinical pharmacology of Ro 115-1240, a peripherally acting selective alpha1A/1L-adrenoceptor (AR) partial agonist, compared with the alpha1A/1L-AR full agonist amidephrine, as AR agonists have some utility in the treatment of stress urinary incontinence (SUI) but are limited by undesirable cardiovascular and central nervous system side-effects. RESULTS: In radioligand-binding studies Ro 115-1240 had greater affinity for alpha1A than for alpha1B and alpha1D subtypes. The potency and intrinsic activity of amidephrine and Ro 115-1240 relative to noradrenaline were determined in native and cell-based assays using human recombinant alpha1-ARs; they acted as selective alpha1A/1L-AR full and partial agonists, respectively. In anaesthetized micropigs and rabbits, amidephrine and Ro 115-1240 produced non-selective, dose-dependent increases in intraurethral and arterial blood pressures but the magnitude of the pressure increases evoked by Ro 115-1240 were about a third of those with amidephrine. In conscious micropigs both agents produced dose-dependent increases in urethral tension. Again, the magnitude of the urethral response to Ro 115-1240 was about a third of that with amidephrine. More importantly, only amidephrine produced dose-dependent increases in blood pressure and decreases in heart rate. Ro 115-1240 produced a maximum increase in urethral tension with no effect on blood pressure or heart rate. CONCLUSION: These results show that by combining selectivity for the alpha1A/1L-AR subtype with a reduction in intrinsic agonist efficacy, Ro 115-1240 has reduced haemodynamic effects while retaining to some degree the contractile effects on urethral smooth muscle. These studies indicate that Ro 115-1240 may be useful as a novel treatment for SUI.

Evidence for the ability of hippocampal neurons to develop acute tolerance to ethanol in behaving rats.

BACKGROUND: The cellular mechanisms underlying acute tolerance to alcohol are unclear. This study aimed to determine whether hippocampal neurons have the ability to develop acute tolerance to alcohol in behaving rats. METHODS: Intrahippocampal microdialysis was performed in freely behaving rats, and the firing of single neurons in the dialysis area was recorded. The control microdialysis fluid, artificial cerebrospinal fluid (ACSF), was replaced with 1 M ethanol in ACSF for a 30 min period. One hour later, the ethanol perfusion was repeated. To test the functional integrity of the microdialysis probe in situ, each microdialysis session was completed with recording the effect of a 10-20 min perfusion of 500 microM N-methyl-D-aspartate (NMDA). The extracellular concentration profile of ethanol during intrahippocampal microdialysis with 1 M ethanol was estimated in a separate study in anesthetized rats. The ethanol content was measured in tissue slices surrounding the probe with gas chromatography (GC), and the generated data were analyzed with a mathematical model for microdialysis to estimate the concentration of ethanol at the recording site. RESULTS: The predominant effect of the first intrahippocampal microdialysis with ethanol was a decrease in firing rate in both pyramidal cells and interneurons. In contrast, such firing rate decrease did not develop during the second ethanol perfusion. Subsequent NMDA perfusion still induced robust changes in the electrical activity of the neurons. The estimated extracellular ethanol concentration at the recording site was 45-70 mM. CONCLUSION: This study revealed that hippocampal neurons have the ability to develop acute tolerance to a single exposure of clinically relevant concentrations of ethanol in behaving rats, without influences from the rest of the body.

Single-cell recording from the brain of freely moving monkeys.

Single-cell recording from the brain of non-human primates has traditionally been performed in monkeys seated in a primate chair. However, this arrangement makes long-term recordings difficult, causes stress that may confound the data, and prevents the manifestation of natural behaviors. Extending our previous neurophysiological studies in non-human primates (Ludvig et al. Brain Res. Protocols 2000;5:75-85), we have developed a method for recording the electrical activity of single hippocampal neurons in freely moving squirrel monkeys (Saimiri sciureus). The recording sessions lasted for up to 6 h, during which the monkeys moved freely around on the walls and the floor of a large test chamber and collected food pellets. Stable action potential waveforms were readily kept throughout the sessions. The following factors proved to be critical in this study: (a) selecting squirrel monkeys for the experiments, (b) using a driveable bundle of microwires for the recordings, (c) using a special recording cable, (d) implanting the microwires into the brain without causing neurological deficits, and (e) running the recording sessions in a special test chamber. The described method allows long-term extracellular recordings from the brain of non-human primates, without the stress of chairing, during a wide range of natural behaviors. Using this model, new insights can be obtained into the unique firing repertoire of the neurons of the primate brain.

Cellular electrophysiological changes in the hippocampus of freely behaving rats during local microdialysis with epileptogenic concentration of N-methyl-D-aspartate.

N-methyl-D-aspartate (NMDA) receptor dysfunctions are thought to be involved in the pathophysiology of seizures of hippocampal origin. While the cellular effects of excessive NMDA receptor stimulation have been widely studied in vitro, no data are available on the sequence of cellular electrophysiological events that follow the overstimulation of hippocampal NMDA receptors in awake, behaving subjects. Therefore, the present study addressed this problem. Intrahippocampal microdialysis with 500 microM NMDA was performed in freely behaving rats, and the electrical activity of single neurons in the dialysis area were monitored. In all recorded neurons (n = 9), regardless of their type, NMDA induced a long-lasting electrical silence preceded in most cells by a brief but robust firing rate increase. During these firing rate increases, place cells lost the spatial selectivity of their discharges, and a gradual reduction in the amplitude of the action potentials was also observed. Remarkably, electroencephalographic (EEG) seizures developed exclusively after the appearance of cellular electrical silence in the recording/dialysis site. The NMDA-induced electrophysiological changes were reversible. This study demonstrates that the combined single-cell recording-intracerebral microdialysis technique can be readily used for inducing focal epileptiform events in the hippocampus and monitoring the induced cellular electrophysiological events in behaving animals.

Delivering drugs, via microdialysis, into the environment of extracellularly recorded hippocampal neurons in behaving primates.

Hippocampal neurons in primates have been extensively studied with electrophysiological and neuroanatomical methods. Much less effort has been devoted to examining these cells with contemporary pharmacological techniques. Therefore, we modified a recently developed integrative technique (N. Ludvig, P.E. Potter, S.E. Fox, Simultaneous single-cell recording and microdialysis within the same brain site in freely behaving rats: a novel neurobiological method, J. Neurosci. Methods 55 (1994) 31-40 [9] ) for cellular neuropharmacological studies in behaving monkeys. A driveable microelectrode-microdialysis probe guide assembly was implanted stereotaxically into the left hippocampus of squirrel monkeys (Saimiri sciureus) under isoflurane anesthesia. The assembly was covered with a protective cap. After 3 weeks of postsurgical recovery and behavioral training, the experimental subject was seated in a primate chair. For 4-5 h, single-cell recording and microdialysis were simultaneously performed in the hippocampal implantation site. The technique allowed the recording of both complex-spike cells and fast-firing neurons without the use of head restraint. The control microdialysis solution, artificial cerebrospinal fluid (ACSF), was replaced with either 1 M ethanol or 500 microM N-methyl-D-aspartate (NMDA) for 10-30 min intervals. The ethanol perfusions principally suppressed the firing of the neurons in the dialysis area. The NMDA perfusions initially increased the firing of local neurons, then caused electrical silence. These drug delivery/cell recording sessions were performed with 1-4 day intersession intervals over a 1-month period. The described method provides a tool to elaborate the pharmacology of primate hippocampal neurons during behavior and without the confounding effects of systemic drug administrations.

Pulpal response to prolonged dentinal exposure to sodium hypochlorite.

AIM: The aim of this study was to examine the effect of undiluted NaOCl on vital pulp tissue when applied to freshly cut dentine. METHODOLOGY: Class V cavities were prepared to a depth of 2 mm in 20 teeth in four Beagle dogs. The cavities on one side of each dog were irrigated continuously for 5 min with 5.25% NaOCl, whilst the cavities on the opposite side in each dog were irrigated with saline for the same length of time. Prior to filling each cavity with Cavit, they were again rinsed with saline and gently dried with an air stream. The dogs were sacrificed to allow for investigation of pulpal conditions under the cavities after periods of 24 h, 1 week, and 4 weeks. Histological preparations were made and stained with H & E for evaluation of the pulp subjacent to each cavity. The tissues were examined for presence of inflammatory cells and categorized as (i) no inflammation, (ii) mild, (iii) moderate, and (iv) severe inflammation. RESULTS: The six teeth in the 24 h observation group all showed mild inflammation, both in the NaOCl and the saline groups. After 1 week and 4 weeks, the pulps from all the teeth were free of inflammatory cells. CONCLUSION: Under the conditions of this experiment, the use of NaOCl in a freshly cut cavity in an intact tooth of a dog, with exposed dentinal tubules, does not appear to cause additional pulpal damage to that caused by the physical contact in cutting tooth structure.

Tissue reaction to implanted root-end filling materials in the tibia and mandible of guinea pigs.

The purpose of this study was to examine the tissue reaction to implanted mineral trioxide aggregate (MTA), amalgam, Intermediate Restorative Material, and Super-EBA in the tibias and mandibles of guinea pigs. After anesthetizing 20 guinea pigs, raising tissue flaps, and preparing bony cavities, the test materials were placed in Teflon cups and implanted in the tibias and 10 days later in the mandibles. The animals were euthanized 80 days later and the tissues prepared for histological examination. The presence of inflammation, predominant cell type, and thickness of fibrous connective tissue adjacent to each implant were recorded. The tissue reaction to MTA implantation was the most favorable observed at both sites; as in every specimen, it was free of inflammation (p < 0.01). In the tibia, MTA was the material most often observed with direct bone apposition. Based on these results, MTA seems to be a biocompatible material.

Starting thrombolytic therapy for patients with acute myocardial infarction in Accident and Emergency Department: from implementation to evaluation.

OBJECTIVE: To evaluate the effectiveness of initiating thrombolysis for patients with acute myocardial infarction (AMI) in the Accident and Emergency Department. METHODS: From January 1993 to December 1995, all AMI patients who were admitted to the United Christian Hospital and given thrombolytic therapy were studied. The patients' demographic data, time and mode of presentation, site of myocardial infarction, treatment modality and timing, and complications related to AMI or treatment were recorded prospectively in our AMI database. The frequency of thrombolysis administered in Accident and Emergency Department and Coronary Care Unit, as well as the median door-to-needle time (time interval between hospital arrival to initiation of thrombolytic therapy) were compared. Cases of inappropriate thrombolysis and complication were also analyzed. RESULTS: Over these 3 years, 257 patients received thrombolysis in the United Christian Hospital. The percentage of patients receiving thrombolysis in Accident and Emergency Department increased from 3.2% in 1993 to 12.3% in 1994, and to 39.4% in 1995. The median time interval between arrival to hospital and thrombolysis (door-to-needle time) was 25 minutes, compared with 81 minutes in the Coronary Care Unit. The door-to-needle time also improved over these 3 years: from 95 minutes in 1993 to 75 minutes in 1995 in Coronary Care Unit group, and from 35 minutes in 1993 to 20 minutes in 1995 in the Accident and Emergency Department group. Over these 3 years, 2 cases of inappropriate thrombolysis were reported but these did not result in any mortality. Four complications from thrombolytic therapy were reported, and these were managed appropriately by the staff in Accident and Emergency Department and did not result in mortality. CONCLUSIONS: Starting thrombolytic therapy in Accident and Emergency Department is safe and effectively decreases the door-to-needle time.

The relationship between the morphology of nucleus of liver cells and the liver functions and prognosis of portal hypertension due to hepatic cirrhosis.

The morphology of nucleus of liver cells from 30 patients with portal hypertension due to hepatic cirrhosis and 5 normal persons were measured using an image analyzer coupled with a computer. It was found that the diameters, perimeters, areas and form factor (FF) of the nucleus of liver cirrhosis portal hypertension patients were significantly increased as compared with those of the normal subjects (P < 0.05 or P < 0.01). There was a very significant difference in this parameters between the normal persons and patients with Child-Pugh A liver function or patients with Child-Pugh C liver function (P < 0.01 for both). Significant difference in these parameters existed between the normal persons or patients with Child-Pugh A liver function and patients with liver function of Child-Pugh B (P < 0.05). No significant difference in the parameter of optic density (OD) were found between the normal persons and patients with impairment of liver function of varying degrees (Child-Pugh Classification) (P > 0.05). Our results suggest that the hepatocytes of patients with portal hypertension due to hepatic cirrhosis became juvenile and the morphology of the hepatocytes of patients with impairment of liver function of Child-Pugh C changed obviously. The enlargement and sparsity of nucleus of hepatocytes as revealed by pathological examination is a sign of severe impairment of liver function.

Tracheal paraganglioma: a case report with review of the literature.

Only three cases of tracheal paraganglioma have been reported in the literature. This paper describes an additional case, which showed pharmacological and ultrastructural evidence of hormone secretion. It is suggested that this tumor is derived from true paraganglia located in the trachea and not from misplaced or aberrant paraganglionic tissue. Hemoptysis was the only presenting symptom in two of the four reported cases and significant bleeding occurred during biopsy in the other two cases. The long-term prognosis appears good if complete resection is possible.