An in vivo model for the study of ovarian cancer and the persistence of characteristic mutations in xenografts.

OBJECTIVE: To identify factors affecting xenograft growth of epithelial ovarian cancer (EOC) cells in nude mice and to detect characteristic mutations occurring in the xenografts following serial passage. MATERIALS AND METHODS: A total of 64 human EOCs were subcutaneously inoculated in Balb/c nude mice in order to obtain a series of xenografts. Whole-exome sequencing was analyzed with Agilent SureSelect targeted enrichment capture system and Illumina Solexa Hiseq 2000 sequencing platform. Mutations were confirmed by comparison against the reference genome build 37.3. RESULTS: The tumor take rate was 50% (32/64). TP53 mutation was detected in nine often Type II tumors. BRAF and CTNNB1 were not mutated in any of the samples, and PTEN mutation occurred in only one sample. The present data indicate that advanced stage serous EOCs and early stage non-serous EOCs were easy to grow in nude mice, and xenografts maintained the characteristic mutations. CONCLUSIONS: Advanced stage serous EOCs and early stage non-serous EOCs were easy to grow in nude mice, and xenografts maintained the characteristic mutations. Xenografts in nude mice are useful in vivo models for the study of human EOCs.

The prognostic significance of APC gene mutation and miR-21 expression in advanced-stage colorectal cancer.

AIM: Colorectal cancer (CRC) is the second commonest cause of cancer death in Taiwan. Although numerous genes have been associated with tumorigenesis in colorectal cancer, only a few have been validated and used as biomarkers for predicting clinical outcome. The aim of this study was to analyse the association of APC gene mutation and miR-21 expression with clinical outcome in CRC patients. METHOD: In total, 195 colorectal cancer patients were enrolled in a single medical centre between 2003 and 2007. APC gene mutation and expression of APC and miR-21 were analysed by direct DNA sequencing and real-time reverse transcription polymerase chain reaction. The primary outcome included 5-year overall survival and univariate (Kaplan-Meier) and multivariate (Cox regression) analyses of prognostic factors. RESULTS: The results showed that 66 (33.8%) of 195 tumour tissues contained an APC mutation. The predominant APC gene variations were deletion mutations (50.0%). APC gene expression was low in CRC and negatively correlated with miR-21 expression and gene mutation. In advanced-stage cancer, patients with APC mutation/high miR-21 had poorer overall survival rates than those with APC mutation/low miR-21, APC wild-type/high miR-21 and APC wild-type/low miR-21. CONCLUSION: In Taiwan, downregulation of the APC gene in CRC correlated with gene mutation and miR-21 upregulation. APC mutation and miR-21 expression could be used to predict the clinical outcome of CRC, especially in patients with advanced disease.

Enhanced nonlinear double excitation of He in intense extreme ultraviolet laser fields.

Nonlinear, three-photon double excitation of He in intense extreme ultraviolet free-electron laser fields (∼24.1 eV, ∼5 TW/cm2) is presented. Resonances to the doubly excited states converging to the He+ N=3 level are revealed by the shot-by-shot photoelectron spectroscopy and identified by theoretical calculations based on the time-dependent Schrödinger equation for the two-electron atom under a laser field. It is shown that the three-photon double excitation is enhanced by intermediate Rydberg states below the first ionization threshold, giving a greater contribution to the photoionization yields than the two-photon process by more than 1 order of magnitude.

Development and application of isothermal amplification methods for rapid detection of F4 fimbriae producing Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the causative agent of a wide range of diseases, which are the important cause of illness and mortality in piglets. ETEC strains expressing F4 fimbriae are frequently associated with post-weaning diarrhea (PWD) and lead to great economic losses in swine production industry worldwide. The aim of this study was to establish a rapid and effective isothermal amplification method for detection of F4 fimbriae. Loop-mediated isothermal amplification (LAMP), Polymerase spiral reaction (PSR) and cross-priming ampli- fication (CPA) were used to develop and optimize the detection method first time. Subsequently, the specificity and sensitivity of these methods were evaluated, and the clinical samples were detected with these methods. All the F4-positive samples could produce ladder-like amplifica- tions products and lead the chromogenic substrate SYBR Green I produce green fluorescence, while in blank control and negative samples lack of this pattern or remained orange. The sensi- tivity of LAMP and CPA were 10 times higher than PSR method. Meanwhile, these three methods were validated with clinical samples, 7 were found positive, while 125 samples were negative, the testing results were consisted with the real-time PCR method. These findings suggested that the isothermal amplification based on the F4 fimbriae is a rapid, effective and sensitive method under resource constrains.

Upregulation of lncRNA CALML3-AS1 promotes cell proliferation and metastasis in cervical cancer via activation of the Wnt/ß-catenin pathway.

OBJECTIVE: It is well verified that lncRNA are emerging as imperative regulators in various tumors. LncRNA CALML3-AS1 (CALML3-AS1), a freshly discovered lncRNA, has been confirmed as a tumor promoter in bladder cancer. This present study aimed to explore the biological functions and molecular mechanisms of CALML3-AS1 in cervical cancer (CC). PATIENTS AND METHODS: We analyzed RNA sequencing data from The Cancer Genome Atlas (TCGA) datasets to determine dysregulated lncRNAs in CC. Real Time-Polymerase Chain Reaction (RT-PCR) was applied for the assays of CALML3-AS1 amplification in CC samples and cell lines. Kaplan-Meier analysis and multivariate assays were carried out for determination of the prognostic values. The functions of CALML3-AS1 on cell proliferation, invasion, migration, and apoptosis were determined by a series of cells experiments by knocking down CALML3-AS1. MRNA and protein expressions of signaling pathways were examined using Western blot. RESULTS: We found that CALML3-AS1 was upregulated in CC tissues and this upregulation was associated with FIGO stage, histological grade, and reduced overall survival. Multivariate assays indicated that high CALML3-AS1 expression was an independent prognostic parameter indicating poorer clinical outcome for CC patients. Functional assays suggested that knockdown of CALML3-AS1 suppressed the proliferation, migration, and invasion of CC cells, and induced apoptosis. Mechanistic investigations revealed that inhibiting the expression of CALML3-AS1 decreased the levels of ß-catenin, cyclin D1, and c-myc via Western blot. CONCLUSIONS: Our study revealed that CALML3-AS1 could be an oncogenic lncRNA promoting the growth and metastasis of CC by modulating Wnt/ß-catenin pathway, suggesting that CALML3-AS1 may be an important contributor to CC progression.

Interleukin-1 alpha polymorphism has influence on late-onset sporadic Parkinson's disease in Taiwan.

Inflammatory events may contribute to the pathogenesis of Parkinson's disease (PD) and interleukin 1 (IL-1) may exert both neurotoxic and neuroprotective effects. We conducted a case-control study in a cohort of 493 PD cases and 388 ethnically matched controls to investigate the association of IL-1alpha C-889T and IL-1beta C-511T polymorphisms with the risk of PD. No significant difference in the genotype distribution of the analyzed polymorphisms was found between PD and controls. However, after stratification by age, individuals over 70 years of age carrying IL-1alpha-889 C/T genotype demonstrated a significant decrease in risk of developing PD (OR = 0.44; 95% CI = 0.22-0.88, p = 0.021) and the decrease is strengthened by IL-1beta-511 T-carrying genotype (OR = 0.28; 95% CI = 0.11-0.71, p = 0.008). Our data suggest that IL-1alpha, acting synergistically with IL-1beta, plays role in PD susceptibility among Taiwanese people older than 70 years of age.

An in situ Al K-edge XAS investigation of the local environment of H+- and Cu+-exchanged USY and ZSM-5 zeolites.

Aluminum coordination in the framework of USY and ZSM-5 zeolites containing charge-compensating cations (NH4+, H+, or Cu+) was investigated by Al K-edge EXAFS and XANES. This work was performed using a newly developed in-situ cell designed especially for acquiring soft X-ray absorption data. Both tetrahedrally and octahedrally coordinated Al were observed for hydrated H-USY and H-ZSM-5, in good agreement with 27Al NMR analyses. Upon dehydration, water desorbed from the zeolite, and octahedrally coordinated Al was converted progressively to tetrahedrally coordinated Al. These observations confirmed the hypothesis that the interaction of water with Brønsted acid protons can lead to octahedral coordination of Al without loss of Al from the zeolite lattice. When H+ is replaced with NH4+ or Cu+, charge compensating species that absorb less water, less octahedrally coordinated Al was observed. Analysis of Al K-edge EXAFS data indicates that the Al-O bond distance for tetrahedrally coordinated Al in dehydrated USY and ZSM-5 is 1.67 angstroms. Simulation of k3chi(k) for Cu+ exchanged ZSM-5 leads to an estimated distance between Cu+ and framework Al atoms of 2.79 angstroms.

Hyperexcitability in sensory neurons of rats selected for high versus low neuropathic pain phenotype.

Selection line rats congenitally high or low for autotomy in the neuroma model of neuropathic pain (HA and LA rats) were found to be correspondingly high and low in a second type of neuropathic pain, the Chung model, which employs an alternative phenotypic endpoint, tactile allodynia. It has been proposed that both phenotypes reflect ectopic hyperexcitability in axotomized primary sensory neurons. To test this hypothesis we made in vitro recordings from sensory neurons in the L4 and 5 dorsal root ganglia. Baseline excitability was similar in HA and LA rats, and axotomy caused an increase in both lines. However, in the one neuronal subclass previously linked to neuropathic pain in these models the increase was significantly greater in HA than LA rats, and only at the time when pain scores in the two lines were diverging. Heritable differences in electrical response to axotomy in a specific afferent cell type appear to be a fundamental determinant of neuropathic pain.

Induction of spinal seizures by natural stimulation in cats.

Transection of the spinal cord of the cat at a thoracic or lumbar level results, after as short a period as 12 days, in a preparation with such altered excitability that repeated natural stimulation of the dermatome just caudal to the transection site will induce, in as short a time as 3 days, seizure discharges. The trigger zone for the seizure spreads to caudal dermatomes when these caudal regions are repeatedly stimulated. The 'typical' T4-T7 seizure is a scratch reflex followed by the tonic-clonic seizure lasting for 20-30 s and ending with a scratch afterdischarge lasting for several minutes. Lower thoracic and upper lumbar seizures consist of tonic-clonic co-contractions of the muscles of the hindlegs, followed by rhythmical stepping movements lasting less than 1 min. Partial dorsal rhizotomy or local Cobalt application to the spinal cord may reduce the threshold for induction of seizure by natural stimulation and local Penicillin application to spinal cord induces seizure discharges similar to those induced by natural stimulation. Retransection of the spinal cord caudally, with elimination of the primary trigger zone, does not abolish the secondarily acquired triggers. The findings suggest that spinal circuits possess the ability to acquire new neuronal patterns of discharge and to transfer them to other more caudal segments.

Effect of age and nerve injury on cross-excitation among sensory neurons in rat dorsal root ganglia.

Spike activity in dorsal root ganglion (DRG) neurons depolarizes passive neighbors that share the same ganglion. We asked whether age or prior nerve injury affect this 'cross-depolarization' signal. Intracellular recordings made from excised DRGs in vitro revealed that the prevalence and duration of cross-depolarization were no greater in adult than in young rats, and that its amplitude was significantly smaller in adults. The amplitude of cross-depolarization was not affected by nerve injury. The decrease in membrane input resistance (R(in)) observed during cross-depolarization was less than that expected from equivalent depolarization alone. This affirms prior evidence that the neural process underlying cross-depolarization causes a net increase in R(in).

Digital processing for improvement of ultrasonic abdominal images.

Digital processing that increases resolution by spatial deconvolution and histogram-based amplitude mapping has been used to improve ultrasonic abdominal image quality. The processing was applied to pulse-echo ultrasound data obtained from clinical imaging instrumentation modified to permit digital recording of signals in either RF or video forms for subsequent off-line analysis. Spatial deconvolution was accomplished both along the axis and across the width of the ultrasonic beam. Axial deconvolution was carried out on RF data with a point spread function derived from the echo of a wire target. Lateral deconvolution was performed on the video envelope placed in a matrix by an inverse filter with parameters that adjust themselves to the spatial frequency content of the image being processed. Resultant image amplitudes were mapped into a hyperbolic distribution to increase image contrast for improved demonstration of low amplitudes. The combination of processing produced resolution improvements to show boundaries more sharply and contrast changes to demonstrate more detail in the images.

The effects of subcortical sensory and motor lesions on conditioned tactile placing in cats with pyramid section.

Cats after unilateral bulbar pyramid section showed permanent loss of tactile placing in the contralateral limbs. However, the cats could be trained to place the forelimb following light touch in a conditioning situation with food as a reward. This conditioned response survived subsequent unilateral red nucleus and cerebellar interposed nucleus lesions. Dorsal quadrant spinal cord lesion at the cervical level produced initial loss of the conditioned tactile placing but is recovered without retraining. Since those lesions interrupted subcortical afferent and efferent pathways essential to reflex tactile placing, these results indicate that reflex and conditioned responses involve different pathways and mechanisms. Training recruits or activates additional pathways for conditioned responses rather than strengthening those subserving the reflex responses.

The effects of cerebral sensory cortical ablation on instrumental conditioned reflex in cats with cerebellar lesions.

Cats were trained to reach with their fore limbs for food placed in horizontally mounted cylinders of various diameters. The chain instrumental reflex (entering cylinder, reaching for, grasping and bringing food toward the mouth) survived cerebellar paravermal cortical ablation or interposite-dentate nuclear lesion, with some exaggerated flexor or extensor responses respectively. Movements improved with time. Cats were then subjected to ablation of cerebral sensory cortex, SI and SII. SI or compined SI and SII lesion resulted in an initial period of decompensation of cerebellar symptoms. Sensory loss was also noted for 30-40 days after SI lesion and throughout the observation period up to 53 days after combined SI and SII lesion. The conditioned responses were soon accomplished at preoperative level. Furthermore, visual occlusion did not change the performance in conditioning situation. The result indicates that the compensation of cerebellar symptoms does not depend on an intact cerebral sensory cortex. It also suggests that functional accomplishment can be obtained through training despite neurological deficits following certain brain damage.

Cerebral influence on postural effects of cerebellar vermal zonal lesions or eighth nerve section in monkeys.

In monkeys, cerebellar vermal cortical or fastigial nuclear lesion resulted in no significant postural asymmetry. Combined decerebration (but not bulbar pyramid section) and unilateral vermal cortical or fastigial nuclear lesion gave marked ipsilateral hyperextension and contralateral hyperflexion of limbs. Unilateral eighth nerve section resulted in only ipsilateral head tilt but combined unilateral eighth nerve section and decerebration or bilateral or contralateral cerebral cortical areas 4 and 6 lesion gave also ipsilateral flexion and contralateral extension of limbs. Cervical deafferentation or postbrachial spinal cord transection did not alter these results. This study indicates a powerful cerebral influence on postural effects of cerebellar vermal zonal lesion or eighth nerve section in monkeys. Possible mechanisms mediating these effects in monkeys as compared to cats were discussed.

Instrumental conditioned responses in cats with bulbar pyramid and red nucleus lesions.

Adult cats were trained to insert a forelimb into horizontally mounted transparent cylinders and then either depress a barrier or pull a sliding tray against resistance in order to receive food reward. Unilateral lesions of both bulbar pyramid and red nucleus resulted in severe neurological deficits. However, the instrumental tasks were soon accomplished at preoperative levels of rapidity and strength, although movements of the digits and wrist were reduced and adjustive movements occurred chiefly at more proximal joints. These results support our hypothesis that conditioned and unconditioned behaviors are mediated through different mechanisms.

The role of the medial lemniscal and spinocervicothalamic pathways on tactile reactions in cats.

The role of dorsal column (DC)-medial lemniscus and spinocervicothalamic systems on tactile reactions, placing and tactile localization (turning to localize a point touched on the body) was studied in cats with spinal and bulbar lesions. The cats with vision occluded were trained preoperately on horizontal bars for tactile localization. The bulbar lesions of cervicothalamic tract (CTT), which anatomically overlaps with gracile nuclear efferent projection, resulted in permanent loss of tactile placing and localization in contralateral hindlimb. Section of dorsal half of lateral funiculus at C3, which interrupts afferents to lateral cervical nucleus but also involves descending motor pathways, gave loss of tactile placing without impairment of tactile localization. However, combined lesions of both systems abolished tactile placing and localization permanently. Results suggest that tactile placing and localization persists after CTT lesion alone; temporarily loss after DC section but permanently abolished after destruction of both systems. It. is also noted that cats after lesions of both systems without involving motor pathways could walk on bars without vision. The deficits following combined lesions are suggested to be primarily sensory in nature.

Effects of exercise on reflexes in paraplegic monkeys.

Eight monkeys, after complete spinal cord transection at the midthoracic level, were placed in a supine position and an exercise program was given to one hindlimb but not the other. Within 3 days, the exercised limb showed more muscle tone and more active cutaneous and proprioceptive reflexes. The difference increased progressively throughout the observation period of 11 to 36 days. These results suggest an important role of afferent influence on the recovery of spinal shock. The exercise program probably protects the neuromuscular apparatus from deterioration and thus maximize the recovery.

The effects of cerebellar lesions upon skilled movements and instrumental conditioned reflexes.

Eight cats were trained in reaching for food with their forelegs in cylinders 19-21.6 cm long of various diameters (3.9-10.1 cm). When the pre-operative training was completed, in six cats the interposite and dentate intracerebellar nuclei were destroyed, whereas in two cats the paravermal cortex was ablated. Interposite animals displayed big oscillatory movements around the entrance to the cylinder and overshooting in reaching for food with occasional forced grasping. Paravermal cats displayed overflexion leading to missing the entrance of the cylinder and wild batting movements observed in the large cylinder. The physiological mechanism of these disorders is discussed.

Effects of nicotine on spontaneous and amphetamine-induced motor behaviors: the differences between nicotine tolerant and nontolerant rats.

This study was designed to investigate the effect of nicotine on spontaneous and amphetamine (AMP)-induced motor activity in rats with or without tolerance to nicotine. Tolerance were induced by treating the rats with nicotine (0.3 mg/kg, s.c.) 2 hr before receiving challenge doses. Motor activity including locomotion and stereotypy was monitored automatically by videocamera every 15 min for 90 min. The results indicated that: (1) Nicotine increased spontaneous locomotion at 0.15 or 0.3 mg/kg (s.c.) in naive rats and at 0.6 mg/kg in tolerant rats. Nicotine also slightly affected AMP-induced locomotion at 0.15, 0.3 or 0.6 mg/kg in both naive and tolerant rats, and (2) Nicotine increased spontaneous stereotypy at 0.3 or 0.6 mg/kg in naive rats only and showed no effect on AMP-induced stereotypy in either naive or tolerant rats. Comparing the results of spontaneous motor activity between naive and tolerant rats, it revealed behavioral desensitization in locomotion at low doses (0.15 or 0.3 mg/kg) and hyperlocomotion at higher dose (0.6 mg/kg), and revealed desensitization in stereotypy at 0.3 or 0.6 mg/kg. Moreover, nicotine had temporary (at 0-15 min interval) attenuating effect on AMP-induced locomotion in naive rats but showed a potentiating effect on AMP-induced locomotion in tolerant rats. The present results indicated that acute tolerance modified the action of nicotine in both spontaneous and AMP-induced locomotion, while stereotypy was changed only in the spontaneous one but not in the AMP-induced one. In other words, acute tolerance modified the effect of nicotine on locomotion-related dopaminergic system, and it affected the stereotypy-related dopaminergic system only in the spontaneous one but not in the AMP-induced one.