Detection of Cucumber green mottle mosaic virus in low-concentration virus-infected seeds by improved one-step pre-amplification RT-qPCR.

BACKGROUND: Seeds were an important medium for long-distance transmission of plant viruses. Therefore, appropriate, more sensitive methods for detecting low concentrations of virus-infected in seeds were crucial to ensure the quality of seed lots. In this study, we have developed a one-step pre-amplification reverse transcription quantitative PCR (RT-qPCR) assay based on the TaqMan technology to detect Cucumber green mottle mosaic virus (CGMMV) in zucchini seeds. RESULT: Seed powder samples with simulated CGMMV-infected at a low concentration were prepared (the mass ratio 1:900 and 1:1000), and their uniformity were verified using one-step pre-amplification RT-qPCR. We used one-step pre-amplification RT-qPCR to detect CGMMV in low-concentration virus-infected seeds and compared this method with universal RT-qPCR and double antibody sandwich-enzyme-linked immunosorbent (DAS-ELISA) assay, the main methods used for virus detection in seeds. The minimum limit of detection (LOD) of the improved one-step pre-amplification RT-qPCR assays for simulated CGMMV-infected seeds in large lots seeds samples were 0.1%. CONCLUSIONS: One-step pre-amplification RT-qPCR assays could reliably and stably detected a single CGMMV-infected seed in 1000 seeds and demonstrated a higher detection sensitivity than universal RT-qPCR (infected seeds versus healthy seeds 1:900) and DAS-ELISA assay (infected seeds versus healthy seeds 1:500). Our improved one-step pre-amplification RT-qPCR assay have proved to be very suitable for the analysis of large seed lots.

Factors associated with consecutive and non-consecutive crashes on freeways: A two-level logistic modeling approach.

A consecutive crash consists of a primary crash and one or more secondary crashes that occur subsequently in a short period of time within a certain distance. It often affects a relatively large area of road space and the traffic disruption created can be difficult for traffic managers to control and resolve. This study identifies the factors delineating a primary crash that results in secondary crashes within a minute from a regular crash that does not result in any secondary crashes. Random-effects, random-parameter and two-level binary logistic regression models are applied to data collected on 8779 crashes on the freeway network of the Guizhou Province, China in 2018, of which 299 are consecutive crashes. According to the AIC values, the two-level logistic model outperforms the other two models. Rear-end primary crashes have a significant random effect varying across road segments on the occurrence of consecutive crashes. Various crash types (rear-end, roll-over and side-swipe), tunnel crash and foggy weather are positively associated with the possibility to cause subsequent consecutive crashes, whereas single-vehicle crash, truck involvement and the time periods with poorer natural lighting are less likely to incur consecutive crashes. Recommendations are provided to minimize the possibility of the occurrence of consecutive crashes on a freeway.

Research on the synchronous detection of neuronal signals under a nerve stimulation system control.

Deep brain stimulation (DBS) provides a recognized research intervention for neurological disease currently. However, there is a lack of traditional electrical stimulator to observe neuronal firing activity synchronously. The aim of the present study was to realize concurrent detection of neuronal signals better under a nerve stimulation system control. Herein, we designed an integrated software, which could control not only neuro-stimulator but also detection instrument at the same time. Moreover, the actual stimulation signals applied to the experiment object could be collected back to data acquisition card and in consistent with the electrophysiological signals. As to basic performance of self-building stimulator, the accuracy of output square signal was verified to be greater than 99.05 % with the change of voltage amplitude. Practicably, combined with homemade microelectrode array (MEA) detecting device, medial forebrain bundle (MFB) DBS effects were observed significantly through the changes of electrophysiological signals in caudate putamen (CPu) of Sprague-Dawley (SD) rat, and the signal-to-noise ratio (SNR) was 5:1 after stimulation. Therefore, the comprehensive nerve stimulation system, which consists of neuro-stimulator and integrated software, could be widely used in the field of neuroscience research with high precision and synchronization.

[Clinical application of cone beam computed tomography combined with micro-ultrasound technique in treating three mesial canals in mandibular first molars].

Objective The aim of this study is to investigate the influence of cone beam computed tomography (CBCT) and micro-ultrasound technique for the treatment of three mesial canals in mandibular first molars. The three mesial canals according to Pomeranz's classification were characterized. Methods A total of 75 permanent mandibular first molars for root canal treatment were randomly selected from patients belonging to the age group of 14-60 years. After preparing the access cavity and locating the main canals, the middle mesial canal orifices in all teeth were determined with an endodontic explorer under direct vision (StageⅠ), under magnification with the aid of micro-ultrasound (Stage Ⅱ), and with the combined use of CBCT and micro-ultrasound to remove the dentin wall and calcifications (Stage Ⅲ). Results Middle mesial canals were detected in 4.0%, 18.7%, and 22.7% of the teeth in StagesⅠ-Ⅲ, respectively. Statistical analysis showed significant differences (P<0.05) between StagesⅠand Ⅱ with regard to middle mesial canal detection. The number of Stage Ⅲ was more than that of Stage Ⅱ. The difference between the two stages was no significant. Among the 17 middle mesial canals, "confluent", "fin" and "independent" anatomies were 52.9%, 35.3%, and 11.8%. Conclusion When used with adjunctive aids, including CBCT, micro-ultrasound facilitates dental clinicians in the location and treatment of middle mesial canals.

[Application of single-retainer all-ceramic resin-bonded fixed partial denture in replacing single anterior tooth].

Objective In this study, we aimed to evaluate the clinical effect of single-retainer all-ceramic resin-bonded fixed partial denture (RBFPD) on the single anterior tooth loss patients. Methods A total of 20 single-retainer all-ceramic RBFPD
were fabricated and evaluated in a two-year follow-up observation. The restorations were examined on the basis of the American Public Health Association (APHA) criteria. Results A total of 20 single-retainer all-ceramic RBFPD achieved class A evaluation after a six-month follow-up observation. One single-retainer all-ceramic RBFPD was classified as class B for secondary caries after a one-year follow-up observation. After a two-year follow-up observation, one single-retainer all-ceramic RBFPD was classified as class B because of secondary caries, and one single-retainer all-ceramic RBFPD was classified as class B because of fracture. Conclusion Single-retainer all-ceramic RBFPD is a promising and optional method in replacing single anterior tooth.

An implantable microelectrode array for dopamine and electrophysiological recordings in response to L-dopa therapy for Parkinson's disease.

Dual-mode multielectrode recordings have become routine in rodent neuroscience research. However, robust and reliable application of acute, multielectrode recording methods in brain especially for in vivo research remains a challenge. In patients with Parkinson's disease (PD), the efficacy of L-dopa therapy depends on its ability to restore Dopamine (DA) neurotransmission in the striatum. In this paper, We describe a low cost thin film 16 sites implantable microelectrode array (MEA) chip fabricated by standard lithography technology for in vivo test. In urethane anesthetized rats, the MEA probes were implanted acutely for simultaneous recording of local field potentials, spikes, and L-dopa therapy evoked dopamine overflow on the same spatiotemporal scale. We present a detailed protocol for array fabrication, then show that the device can record Spikes, LFPs and dopamine variation in real time. Across any given microelectrode, spike amplitudes ranged from 80 to 300 µν peak to peak, with a mean signal-tonoise ratio of better than 5:1. Calibration results showed the MEA probe had high sensitivity and good selectivity for DA. Comparison with existing methods allow single mode recording, our neural probes would be useful for examining specific spatiotemporal relationships between electrical and chemical signaling in the brain.

Human tissue kallikrein ameliorates cerebral vasospasm in a rabbit model of subarachnoid hemorrhage.

OBJECTIVES: Cerebral vasospasm (CVS) and early brain injury are major causes of morbidity and mortality following subarachnoid hemorrhage (SAH). We investigated the efficiency of human tissue kallikrein (HTK) to prevent CVS in a rabbit model of SAH. METHODS: Forty-eight Japanese white rabbits were randomly divided into four groups (n = 12 each): control (sham-operated), SAH, SAH + phosphate-buffered saline (PBS, vehicle), and SAH + HTK. Basilar artery (BA) diameters were measured by three-dimensional computed tomography angiography at three time points. Endothelin-1 (ET-1) and nitric oxide (NO) levels in the cerebrospinal fluid (CSF) were assayed 24 h before and 5 and 7 days after SAH. After the last measurement, the animals were killed, and endothelial cell apoptosis was assessed. Bax and Bcl-2 levels in the BA were measured by western blotting. RESULTS: HTK was found to significantly reduce CVS following SAH in rabbits. Inverse changes were observed in ET-1 and NO levels in the CSF collected from the SAH group. HTK increased levels of NO, which has a vasodilatory effect, but did not affect levels of ET-1, which has a vasoconstrictive effect. CTA revealed that HTK treatment significantly increased BA diameter. Moreover, HTK treatment reduced the number of apoptotic cells following SAH, presumably by increasing and decreasing Bcl-2 and Bax expression, respectively. CONCLUSION: HTK ameliorated CVS and inhibited apoptosis in the BA in a rabbit model of SAH.

Factors associated with intramedullary MRI abnormalities in patients with ossification of the posterior longitudinal ligament.

STUDY DESIGN: A retrospective clinical study of 113 patients with ossification of the posterior longitudinal ligament (OPLL), who underwent either anterior or posterior surgery between 2006 and 2009. OBJECTIVE: To evaluate the risk factors affecting the intramedullary spinal cord changes in signal intensity on magnetic resonance imaging (MRI) for the patients with OPLL. SUMMARY OF BACKGROUND DATA: The relationship between the intramedullary spinal cord changes in signal intensity on MRI and neurological deficits, as well as the surgical outcomes, has been described. To obtain better prognosis, early surgery should be conducted in patients with OPLL who have potential abilities to develop intramedullary spinal cord changes in signal intensity on MRI. Various factors may be affecting the development of intramedullary spinal cord changes in signal intensity on MRI. MATERIALS AND METHODS: The clinical and radiographic data of 113 patients with OPLL who underwent either anterior or posterior surgery between 2006 and 2009 were reviewed. Age, sex, complication, mean occupying ratio of OPLL (the greatest thickness of OPLL divided by the anteroposterior diameter of the bony spinal canal), duration of symptoms, type of OPLL, preoperative Japanese Orthopedic Association (JOA) score, and range of motion of the cervical spine were collected. Logistic regression analysis was used. RESULTS: Changes in the intramedullary signal intensity on MRI were observed in 33 of the 113 patients. Statistical results show that duration of symptoms, occupying ratio of OPLL, preoperative JOA score, kyphosis, and instability of the cervical spine are the relevant risk factors for intramedullary spinal cord changes in signal intensity on MRI, with regression coefficients of 2.437, 0.953, -1.952, 2.093, and 1.516, respectively. For patients with OPLL, the longer the duration of the symptoms, or the higher occupying ratio of OPLL, or the lower preoperative JOA score, the greater the likelihood of intramedullary spinal cord changes in signal intensity on MRI. CONCLUSIONS: As intramedullary spinal cord changes in signal intensity on MRI indicated severe damage to spinal cord and poor prognosis as we described before, early surgery is suggested for patients with OPLL who manifest one of the following factors: prolonged symptoms, high occupying ratio, low preoperative JOA score, kyphosis, or instability of the cervical spine. These factors are closely related to the intramedullary spinal cord changes in signal intensity on MRI.

Vesicle consisted of calcified core and intervening turbid fluid, a possible composition of calcification in intervertebral disc calcification in children.

Intervertebral disc calcification (IDC) is one of the uncommon diseases in children. Normally, it is a benign lesion which is self-limited and has an excellent prognosis under conservative treatments and symptomatic support. Surgical treatment is usually carried out only for patients with progressive neurological deterioration in order to prevent the spinal cord from being irreversible injured. After conservative treatments for months or years, the calcification reduces gradually or even disappears through imaging. Until now, the etiology remains unclear and the mechanism for resorption of IDC is still unknown. Surgery was performed on an IDC patient with progressive neurological deterioration, it was found that the high density calcification region on CT is actually not "hard" but more like an enlarged cell. In such a cell, a calcified nuclear was surrounded by limewater-like liquid inside a large membrane. This study aims to unveil the mechanism for the resorption of IDC. We hypothesize that the high density calcification on imaging is a vesicle consisted of calcified core and intervening turbid fluid. Furthermore, the increase or diminution of calcification is caused by the production or resorption of inflammatory fluid around the calcified core in lesion disc. This could explain the mechanism of IDC resorption in children.

Pulmonary edema and hemorrhage, possible causes of pulmonary infection and respiratory failure in the early stage of lower spinal cord injury.

Pulmonary infection and respiratory failure are frequently encountered in the early stage of acute spinal cord injury (SCI) and are thought of as the chief causes of death. Unfortunately, there is little knowledge concerned with the pathogenesis of pulmonary infection, respiratory failure and other pathological changes in the lung in the early stage of SCI. Pulmonary embolism, respiratory muscle dysfunction, poor expectoration caused by position, and decreased ability to cough up respiratory secretions were the main causes. These explanations may be beyond criticism in high-level paraplegia in SCI, but are unconvincing in lower SCI such as in low-thoracic cord injury where the phenomenon of pneumonia and respiratory dysfunction remains. There might be some more important factors that lead to pulmonary infection and respiratory failure in the early stage of SCI. In SCI rats, pulmonary edema and hemorrhage were occurred in the early stage of SCI while the other organs were almost normal. And the location of lung edema and hemorrhage were the same as that of pulmonary infection. The purpose of this paper is to propose pathological changes in the lung and possible causes for pulmonary infection and respiratory failure. We hypothesize that pulmonary edema and hemorrhage in the early stage of SCI might be the chief factor contributing to pulmonary infection and respiratory failure in lower SCI.

Global activation of CD8+ cytotoxic T lymphocytes correlates with an impairment in regulatory T cells in patients with generalized vitiligo.

Melanocyte-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a pivotal role in vitiligo-induced depigmentation. Yet, the mechanisms underlying the high frequency of generalized autoimmune disorders associated with generalized vitiligo (GV) are unknown. We hypothesized that an imbalance between activated CD8(+) CTLs and regulatory T cells (Tregs) exists in patients with GV . Assessment of the circulating CD8(+) CTLs and Tregs by flow cytometric analysis revealed an obvious expansion of CD8(+) CTLs and a concomitant decrease in Treg cells in GV patients. The percentages of skin infiltrating CD8(+) CTLs and Tregs were evaluated by immunohistochemistry and revealed dramatically increased numbers of both CD8(+) CTLs and Tregs in the perilesional skin of GV patients. However, peripheral Tregs were impaired in their ability to suppress the proliferation and cytolytic capacity of autologous CD8(+) T cells, suggesting that a functional failure of Tregs and the hyper-activation of CD8(+) CTLs may contribute to progressive GV. Our data indicate that reduced numbers and impaired function of natural Tregs fail to control the widespread activation of CD8(+) CTLs, which leads to the destruction of melanocytes and contributes to the elevated frequency of various associated autoimmune diseases. This knowledge furthers our understanding of the mechanisms of immune tolerance that are impaired in GV patients and may aid in the future development of effective immunotherapy for GV patients.

Segmental anterior decompression and fusion for multilevel ossification of the posterior longitudinal ligament.

The purpose of this study was to evaluate the outcome of segmental anterior decompression and fusion for multilevel ossification of the posterior longitudinal ligament. Data were collected from 23 patients with multilevel ossification of the posterior longitudinal ligament. Average operative time and blood loss were 121 minutes and 201.6 mL, respectively. The Nurick score significantly decreased from 2.7±0.9 preoperatively to 1.8±0.9 at last follow-up (P<.01). The preoperative Japanese Orthopaedic Association score was 8.2, which significantly increased to 13.8 points at last follow-up (P<.01), with an improvement rate of 64.5%. The operation also significantly increased cervical lordosis (P<.01) from 7.7° preoperatively to 13.3° postoperatively. The fusion rate was 95.7% at 6 months postoperatively, and 100% at 12 months postoperatively. The loss of cervical lordosis and height of fusion segments were 1.2° and 0.9 mm at last follow-up, respectively. No hardware complications occurred. Cerebrospinal fluid leakage occurred in 2 patients, and hematoma occurred in 1 patient who needed an emergency operation. Segmental anterior decompression and fusion was generally effective and safe in the treatment of multilevel ossification of the posterior longitudinal ligament if indications were well controlled.

Chromatographic separation of (-)-ephedrine and (+)-pseudoephedrine in the traditional Chinese medicinal preparation Jiketing granule.

(-)-Ephedrine (EP) and (+)-Pseudoephedrine (PEP) in Jiketing granule were separated by TLC, RP-HPLC, GC, HPLC-MS, and the chromatographic phenomenon was interpreted by the topological indices. The observed chromatographic separations of investigated components were compared. This comparison indicated that simply methods as C18 RP-HPLC and GC are the more effective techniques, and more suitable than others for this preparation, for the separation of two alkaloids.