Discrimination of Maize Haploid Seeds from Hybrid Seeds Using Vis Spectroscopy and Support Vector Machine Method.

Doubled haploid (DH) lines are routinely applied in the hybrid maize breeding programs of many institutes and companies for their advantages of complete homozygosity and short breeding cycle length. A key issue in this approach is an efficient screening system to identify haploid kernels from the hybrid kernels crossed with the inducer. At present, haploid kernel selection is carried out manually using the"red-crown" kernel trait (the haploid kernel has a non-pigmented embryo and pigmented endosperm) controlled by the R1-nj gene. Manual selection is time-consuming and unreliable. Furthermore, the color of the kernel embryo is concealed by the pericarp. Here, we establish a novel approach for identifying maize haploid kernels based on visible (Vis) spectroscopy and support vector machine (SVM) pattern recognition technology. The diffuse transmittance spectra of individual kernels (141 haploid kernels and 141 hybrid kernels from 9 genotypes) were collected using a portable UV-Vis spectrometer and integrating sphere. The raw spectral data were preprocessed using smoothing and vector normalization methods. The desired feature wavelengths were selected based on the results of the Kolmogorov-Smirnov test. The wavelengths with p values above 0. 05 were eliminated because the distributions of absorbance data in these wavelengths show no significant difference between haploid and hybrid kernels. Principal component analysis was then performed to reduce the number of variables. The SVM model was evaluated by 9-fold cross-validation. In each round, samples of one genotype were used as the testing set, while those of other genotypes were used as the training set. The mean rate of correct discrimination was 92.06%. This result demonstrates the feasibility of using Vis spectroscopy to identify haploid maize kernels. The method would help develop a rapid and accurate automated screening-system for haploid kernels.

Activation of cGMP-PKG signaling pathway contributes to neuronal hyperexcitability and hyperalgesia after in vivo prolonged compression or in vitro acute dissociation of dorsal root ganglion in rats.

Injury or inflammation affecting sensory neurons in the dorsal root ganglia (DRG) causes hyperexcitability of DRG neurons that can lead to spinal central sensitization and neuropathic pain. Recent studies have indicated that, following chronic compression of DRG (CCD) or acute dissociation of DRG (ADD) treatment, both hyperexcitability of neurons in intact DRG and behaviorally expressed hyperalgesia are maintained by activity in cGMP-PKG signaling pathway. Here, we provide evidence supporting the idea that CCD or ADD treatment activates cGMP-PKA signaling pathway in the DRG neurons. The results showed that CCD or ADD results in increase of levels of cGMP concentration and expression of PKG-I mRNA, as well as PKG-I protein in DRG. CCD or ADD treated-DRG neurons become hyperexcitable and exhibit increased responsiveness to the activators of cGMP-PKG pathway, 8-Br-cGMP and Sp-cGMP. Hyperexcitability of the injured neurons is inhibited by cGMP-PKG pathway inhibitors, ODQ and Rp-8-pCPT-cGMPS. In vivo delivery of Rp-8-pCPT-cGMPS into the compressed ganglion within the intervertebral foramen suppresses CCD-induced thermal hyperalgesia. These findings indicate that the in vivo CCD or in vitro ADD treatment can activate the cGMP-PKG signaling pathway, and that continuing activation of cGMP-PKG pathway is required to maintain DRG neuronal hyperexcitability and/or hyperalgesia after these two dissimilar forms of injury-related stress.

EphB receptor signaling in mouse spinal cord contributes to physical dependence on morphine.

Cellular and molecular mechanisms underlying opioid tolerance and dependence remain elusive. We investigated roles of EphB receptor tyrosine kinases--which play important roles in synaptic connection and plasticity during development and in the matured nervous system--in development and maintenance of physical dependence on morphine in the mouse spinal cord (SC). Spinal administration of an EphB receptor blocking reagent EphB2-Fc prevents and/or suppresses behavioral responses to morphine withdrawal and associated induction of c-Fos and depletion of calcitonin gene-related peptide. Western blotting and immunohistochemical fluorescence staining demonstrates that EphB1 receptor protein is significantly up-regulated in the spinal dorsal horn following escalating morphine treatment. Chronic morphine exposure and withdrawal significantly increased phosphorylation of N-methyl-D-aspartate receptor subunit NR2B as well as the activated forms of extracellular signal-regulated kinase and the cAMP response element binding protein in SC. The increased levels of phosphorylation of these molecules, however, are significantly inhibited by the EphB receptor blocker. These findings indicate that EphB receptor signaling, probably by interacting with NR2B in SC, contributes to the development of opioid physical dependence and withdrawal effects. This novel role for EphB receptor signaling suggests that these molecules may be useful therapeutic targets for preventing, minimizing, or reversing the development of opiate dependence.

An in vivo mouse model of long-term potentiation at synapses between primary afferent C-fibers and spinal dorsal horn neurons: essential role of EphB1 receptor.

BACKGROUND: Long-term potentiation (LTP), a much studied cellular model of synaptic plasticity, has not been demonstrated at synapses between primary afferent C-fibers and spinal dorsal horn (DH) neurons in mice in vivo. EphrinB-EphB receptor signaling plays important roles in synaptic connection and plasticity in the nervous system, but its role in spinal synaptic plasticity remains unclear. RESULTS: This study characterizes properties of LTP at synapses of C-fibers onto neurons in the superficial DH following high-frequency stimulation (HFS) of a peripheral nerve at an intensity that activates C-fibers and examines associated activation of Ca2+/calmodulin-activated protein kinase II (p-CaMKII), extracellular signal-regulated kinase (p-ERK) and the cyclic AMP response element binding protein (p-CREB) and expression of c-Fos, and it investigates further roles for the EphB1 receptor in LTP. HFS induced LTP within 5 min and lasts for 3-8 h during the period of recording and resulted in upregulation of p-CaMKII, p-ERK and p-CREB protein levels in the spinal cord and expression of c-Fos in DH. Intrathecal pretreatment of MK-801 or EphB2-Fc prevented LTP and significantly reduced upregulation of p-CaMKII, p-ERK, p-CREB and c-Fos. Further, targeted mutation of EphB1 receptor prevented induction of LTP and associated increases in phosphorylation of CaMKII, ERK, and CREB. CONCLUSION: This study provides an in vivo mouse model of LTP at synapses of C-fibers onto the superficial DH neurons that will be valuable for studying the DH neuron excitability and their synaptic plasticity and hyperalgesia. It further takes advantage of examining functional implications of a specific gene targeted mice and demonstrates that the EphB1 receptor is essential for development of LTP.

Chronic compression or acute dissociation of dorsal root ganglion induces cAMP-dependent neuronal hyperexcitability through activation of PAR2.

Chronic compression (CCD) or dissociation of dorsal root ganglion (DRG) can induce cyclic adenosine monophosphate (cAMP)-dependent DRG neuronal hyperexcitability and behaviorally expressed hyperalgesia. Here, we report that protease-activated receptor 2 (PAR2) activation after CCD or dissociation mediates the increase of cAMP activity and protein kinase A (PKA) and cAMP-dependent hyperexcitability and hyperalgesia in rats. CCD and dissociation, as well as trypsin (a PAR2 activator) treatment, increased level of cAMP concentration, mRNA, and protein expression for PKA subunits PKA-RII and PKA-c and protein expression of PAR2, in addition to producing neuronal hyperexcitability and, in CCD rats, thermal hyperalgesia. The increased expression of PAR2 was colocalized with PKA-c subunit. A PAR2 antagonistic peptide applied before and/or during the treatment, prevented or largely diminished the increased activity of cAMP and PKA, neuronal hyperexcitability, and thermal hyperalgesia. However, posttreatment with the PAR2 antagonistic peptide failed to alter either hyperexcitability or hyperalgesia. In contrast, an adenylyl cyclase inhibitor, SQ22536, administrated after dissociation or CCD, successfully suppressed hyperexcitability and hyperalgesia, in vitro and/or in vivo. Trypsin-induced increase of the intracellular calcium [Ca(2+)](i) was prevented in CCD or dissociation DRG neurons. These alterations were further confirmed by knockdown of PAR2 with siRNA. In addition, trypsin and PAR2 agonistic peptide-induced increase of cAMP was prevented by inhibition of PKC, but not Gαs. These findings suggest that PAR2 activation is critical to induction of nerve injury-induced neuronal hyperexcitability and cAMP-PKA activation. Inhibiting PAR2 activation may be a potential target for preventing/suppressing development of neuropathic pain.

Topical application of compound Ibuprofen suppresses pain by inhibiting sensory neuron hyperexcitability and neuroinflammation in a rat model of intervertebral foramen inflammation.

UNLABELLED: There is lack of evidence that topical application of an anti-inflammatory reagent could reduce pain due to intervertebral foramen (IVF) inflammation (IVFI). We investigated analgesic effects and underlying mechanisms of topical application of a compound ibuprofen cream (CIC) onto the surface of back skin covering the inflamed L(5) IVF in a rat model. Repetitive CIC treatment (~.54 g each treatment daily for 5 consecutive days) significantly reduces severity and duration of IVFI-induced thermal hyperalgesia and mechanical allodynia by 80 to 100% and 50 to 66%, respectively. Electrophysiological studies and Western blot analysis demonstrated that CIC treatment significantly inhibited hyperexcitability of the inflamed dorsal root ganglion (DRG) neurons and upregulation of Nav1.7 and Nav1.8 protein, respectively. Pathological manifestations of the inflamed DRG were also markedly improved following CIC treatment. Further, in the inflamed DRGs, phosphorylation and expression of transcription factor NF-κB and pro-inflammatory enzyme cyclooxygenase-2 (COX-2) were significantly increased, while a cytokine IL-1ß level was increased. IVFI-induced upregulation of these molecules was significantly inhibited by CIC treatment. This study provides evidence that an anti-inflammatory reagent can be used topically to suppress pain due to IVFI and/or DRG inflammation through inhibition of sensory neuron hyperexcitability and the immune and inflammatory responses. PERSPECTIVE: This study suggests a convenient and safe clinical intervention for treating pain due to intervertebral foramen inflammation and similar syndromes.

Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy.

EphrinB-EphB receptor signaling plays diverse roles during development, but recently has been implicated in synaptic plasticity in the matured nervous system and in pain processes. The present study investigated the correlation between expression of ephrinB and EphB receptor proteins and chronic constriction injury (CCI) of the sciatic nerve and dorsal rhizotomy (DR) in dorsal root ganglion (DRG) and spinal cord (SC); and interaction of CCI and DR on expression of these signals. Adult, male Sprague-Dawley rats were employed and thermal sensitivity was determined in the sham operated CCI and DR rats. Western blot and immunobiochemistry analysis and immunofluorescence staining techniques were used to detect the expression and location of the ephrinB-EphB receptor proteins in DRG and SC. The results showed that expression of ephrinB1 and EphB1 receptor proteins was significantly upregulated in DRG and SC in a time-dependent manner corresponding to the development of thermal hyperalgesia after CCI. The increased expression is predominately located in the medium- and small-sized DRG neurons, the superficial layers of spinal dorsal horn (DH) neurons, and the IB4 positive nociceptive terminals. DR increases ephrinB1 in DRG, not SC and EphB receptor in SC, not DRG. DR suppressed CCI-induced upregulation of ephrinB1 in SC and EphB1 receptor in DRG and SC. These findings indicate that ephrinB-EphB receptor activation and redistribution in DRG and DH neurons after nerve injury could contribute to neuropathic pain. This study may also provide a new mechanism underlying DR-induced analgesia in clinic.

Role of TRAIL and IFN-gamma in CD4+ T cell-dependent tumor rejection in the anterior chamber of the eye.

Although the anterior chamber of the eye expresses immune privilege, some ocular tumors succumb to immune rejection. Previous studies demonstrated that adenovirus-induced tumors, adenovirus type 5 early region 1 (Ad5E1), underwent immune rejection following transplantation into the anterior chamber of syngeneic mice. Intraocular tumor rejection required CD4(+) T cells, but did not require the following: 1) CD8(+) T cells, 2) B cells, 3) TNF, 4) perforin, 5) Fas ligand, or 6) NK cells. This study demonstrates that CD4(+) T cell-dependent tumor rejection does not occur in IFN-gamma-deficient mice. Ad5E1 tumor cells expressed DR5 receptor for TRAIL and were susceptible to TRAIL-induced apoptosis. Although IFN-gamma did not directly induce apoptosis of the tumor cells, it rendered them 3-fold more susceptible to TRAIL-induced apoptosis. Both CD4(+) T cells and corneal endothelial cells expressed TRAIL and induced apoptosis of Ad5E1 tumor cells. The results suggest that Ad5E1 tumor rejection occurs via TRAIL-induced apoptosis as follows: 1) tumor cells express TRAIL-R2 and are susceptible to TRAIL-induced apoptosis, 2) IFN-gamma enhances TRAIL expression on CD4(+) T cells and ocular cells, 3) IFN-gamma enhances tumor cell susceptibility to TRAIL-induced apoptosis, 4) apoptotic tumor cells are found in the eyes of rejector mice, but not in the eyes of IFN-gamma knockout mice that fail to reject intraocular tumors, 5) CD4(+) T cells and corneal endothelial cells express TRAIL and induce apoptosis of tumor cells, and 6) apoptosis induced by either CD4(+) T cells or corneal cells can be blocked with anti-TRAIL Ab.