Obtaining transgenic plants is a common method for analyzing gene function. Unfortunately, stable genetic transformation is difficult to achieve, especially for plants（e.g., soybean）, which are ...recalcitrant to genetic transformation. Transient expression systems, such as Arabidopsis protoplast, Nicotiana leaves, and onion bulb leaves are widely used for gene functional studies. A simple method for obtaining transgenic soybean callus tissues was reported recently. We extend this system with simplified culture conditions to gene functional studies, including promoter analysis, expression and subcellular localization of the target protein, and protein-protein interaction. We also evaluate the plasticity of this system with soybean varieties, different vector constructs, and various Agrobacterium strains. The results indicated that the callus transformation system is efficient and adaptable for gene functional investigation in soybean genotype-, vector-, and Agrobacterium strain-independent modes. We demonstrated an easy set-up and practical homologous strategy for soybean gene functional studies.
Background
Interstitial cells of Cajal associated with the submuscular plexus (ICC‐SMP) generate omnipresent slow‐wave activity in the colon and are associated with prominent motor patterns. Our aim ...was to investigate colon motor dysfunction in W/Wv mice in which the ICC are reportedly reduced.
Methods
Whole organ colon motility was studied using spatio‐temporal mapping; immunohistochemical staining was carried out for c‐Kit and Ano1; calcium imaging was applied to ICC‐SMP.
Key Results
Discrepancies between Ano1 and c‐Kit staining were found in both wild‐type and W/Wv colon. ICC‐SMP were reduced to ~50% in the W/Wv mouse colon according to c‐Kit immunohistochemistry, but Ano1 staining indicated a normal network of ICC‐SMP. The latter was consistent with rhythmic calcium transients occurring at the submucosal border of the colon in W/Wv mice, similar to the rhythmic transients in wild‐type ICC‐SMP. Furthermore, the motor pattern associated with ICC‐SMP pacemaking, the so‐called ‘ripples’ were normal in the W/Wv colon.
Conclusions & Inferences
c‐Kit is not a reliable marker for quantifying ICC‐SMP in the mouse colon. Ano1 staining revealed a normal network of ICC‐SMP consistent with the presence of a normal ‘ripples’ motor pattern. We detected a class of Ano1 positive c‐Kit negative cells that do not depend on Kit expression for maintenance, a feature shared with ICC progenitors.
c‐Kit is not a reliable marker for quantifying ICC‐SMP in the mouse colon. Ano1 staining revealed a normal network of ICC‐SMP consistent with the presence of a normal ‘ripples’ motor pattern. We detected a class of Ano1 positive c‐Kit negative cells that do not depend on Kit expression for maintenance, a feature shared with ICC progenitors.
The objective of this prospective study of the risks of treatment failure in patients with drug-susceptible pulmonary tuberculosis （PTB） was to provide reference data to help develop a disease ...control strategy. Participants were recruited in eight provinces of China from October 2008 to December 2010. A total of 1447 patients with drug-susceptible PTB and older than 15 years of age were enrolled.
Jin‐Nao‐Ning mixture (JNNm) has been clinically used to treat insomnia in China. However, the major constituents of JNNm were not revealed. In this study, an ultra‐high‐performance liquid ...chromatography‐tandem mass spectrometry was established and validated for simultaneous quantitation of 19 representative constituents of JNNm. The method had good specificity and adequate sensitivity. The calibration curves of the constituents displayed good linearity (r > 0.990) in appropriate ranges. The intra‐ and inter‐day precision at three quality control (QC) levels ranged from 0.68% to 5.17% and 0.42% to 5.37%, respectively. The recovery ranged from 91.76% to 110.06%, with a precision of less than 5.07%. In addition, the method had good repeatability (relative standard deviation RSD < 4.93%). The analytes were all stable for 24 h (RSD < 7.98%) at room temperature. The results of the quantitative analysis showed that salvianolic acid B (495.7–606.3 μg/mL) is the most abundant constituent, followed by betaine (411.3–468.3 μg/mL), salvianic acid A (10.7–34.9 μg/mL), lobetyolin (11.6–14.6 μg/mL), spinosin (4.0–7.6 μg/mL), schisandrol A (1.5–4.1 μg/mL), and schisandrol B (1.2–1.5 μg/mL). Concentrations of other constituents were below 1 μg/mL. This study would be helpful for the QC of JNNm and the revelation of its effective constituents.
Soil-borne pathogen Phytophthora sojae is an oomycete that causes devastating damage to soybean yield. To mine original resistant genes in soybean is an effective and environmentally-friend approach ...controlling the disease. In this study, soybean proteins were extracted from the first trifoliolates infected by predominant P. sojae race 1 and analyzed by twodimensional gel electrophoresis. Nineteen differently-expressed protein spots were detected, and 10 of them were further applied for Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry Assay. One protein containing a dirigent (DIR) domain was identified and belonged to the DIR-b/d family. Therefore, it was named as GmDRR1 (Glycine max Disease Resistance Response 1). Then, GmDRR1 gene was pathologically confirmed to be involved in the resistant to P. sojae in soybean. GmDRR1-GFP (green fluorescent protein) fusion proteins localized in the cell membrane. qRTPCR results showed GmDRR1 gene expressed differently in P. sojae resistant- and susceptible-soybean cultivars. By the promoter analysis, we found a haplotype H8 was existing in most resistant soybean varieties, while a haplotype H77 was existing in most susceptible soybean varieties. The H77 haplotype had seven SNPs (C to A, G to C, C to A, T to A, T to C, T to C, and T to A) and two single nucleotide insertions. The results supported that the expression difference of GmDRR1 genes between P. sojae resistant- and susceptible-soybean cultivars might depend on the GmDRR1 promoter SNPs. The results suggested that GmDRR1 was a dirigent protein involved in soybean resistant to P. sojae and paved a novel way for investigation of the molecular regulatory mechanism of the defense response to P. sojae in soybean.
We assessed the role of diabetes mellitus（DM） on treatment effects in drug‐susceptible initial pulmonary tuberculosis（PTB） patients. A prospective study was conducted in eight provinces of China from ...October 2008 to December 2010. We enrolled 1,313 confirmed drug‐susceptible initial PTB patients, and all subjects received the treatment regimen（2 H3 R3 E3 Z3/4 H3 R3） as recommended by the national guidelines. Of the 1,313 PTB patients, 157（11.9%） had DM; these patients had more sputum smear‐positive rates at the end of the second month adjusted odds ratios（aO R） 2.829, 95% confidence intervals（CI） 1.783‐4.490, and higher treatment failure（aO R 2.120, 95% CI 1.565‐3.477） and death rates（aO R 1.536, 95% CI 1.011‐2.628）. DM was a contributing factor for culture‐positive rates at the end of the second month and treatment failure and death of PTB patients, thus playing an unfavorable role in treatment effects of PTB.
The ability of T cells that have been genetically engineered to express T-cell receptors (TCRs) directed against tumor antigens to mediate tumor regression has been demonstrated in several clinical ...trials. These TCRs have primarily targeted HLA-A*0201-restricted TCRs, as approximately 50% of whites, who represent the predominant population of patients who develop melanomas, expresses this HLA class I allele. These therapies could be extended to additional patients through the use of TCRs that target epitopes that are presented by additional class I alleles that are prevalent in this population such as HLA-C*07 and HLA-A*01, which are expressed by approximately 50% and 30% of the patient population respectively. Therefore, 2 TCRs that recognize an epitope of MAGE-A12 in the context of HLA-C*07 and 2 TCRs that recognize an epitope of MAGE-A3 in the context of HLA-A*01 were isolated from tumor-reactive T-cell clones and cloned in a recombinant retroviral expression vector. Comparative studies indicated that one of the 2 MAGE-A3-reactive TCRs and one of the 2 MAGE-A12-reactive TCRs were superior to the additional TCRs in conferring transduced peripheral blood mononuclear cells with the capacity to recognize a broad array of antigen and MHC-positive target cells. These results provide support for the use of these TCRs in cancer adoptive immunotherapy trials.
Causes of neuropathic pain following nerve injury remain unclear, limiting the development of mechanism-based therapeutic approaches. Animal models have provided some directions, but little is known ...about the specific sensory neurons that undergo changes in such a way as to induce and maintain activation of sensory pain pathways. Our previous studies implicated changes in the Aβ, normally non-nociceptive neurons in activating spinal nociceptive neurons in a cuff-induced animal model of neuropathic pain and the present study was directed specifically at determining any change in excitability of these neurons. Thus, the present study aimed at recording intracellularly from Aβ-fiber dorsal root ganglion (DRG) neurons and determining excitability of the peripheral receptive field, of the cell body and of the dorsal roots.
A peripheral neuropathy was induced in Sprague Dawley rats by inserting two thin polyethylene cuffs around the right sciatic nerve. All animals were confirmed to exhibit tactile hypersensitivity to von Frey filaments three weeks later, before the acute electrophysiological experiments. Under stable intracellular recording conditions neurons were classified functionally on the basis of their response to natural activation of their peripheral receptive field. In addition, conduction velocity of the dorsal roots, configuration of the action potential and rate of adaptation to stimulation were also criteria for classification. Excitability was measured as the threshold to activation of the peripheral receptive field, the response to intracellular injection of depolarizing current into the soma and the response to electrical stimulation of the dorsal roots.
In control animals mechanical thresholds of all neurons were within normal ranges. Aβ DRG neurons in neuropathic rats demonstrated a mean mechanical threshold to receptive field stimulation that were significantly lower than in control rats, a prolonged discharge following this stimulation, a decreased activation threshold and a greater response to depolarizing current injection into the soma, as well as a longer refractory interval and delayed response to paired pulse electrical stimulation of the dorsal roots.
The present study has demonstrated changes in functionally classified Aβ low threshold and high threshold DRG neurons in a nerve intact animal model of peripheral neuropathy that demonstrates nociceptive responses to normally innocuous cutaneous stimuli, much the same as is observed in humans with neuropathic pain. We demonstrate further that the peripheral receptive fields of these neurons are more excitable, as are the somata. However, the dorsal roots exhibit a decrease in excitability. Thus, if these neurons participate in neuropathic pain this differential change in excitability may have implications in the peripheral drive that induces central sensitization, at least in animal models of peripheral neuropathic pain, and Aβ sensory neurons may thus contribute to allodynia and spontaneous pain following peripheral nerve injury in humans.
In past decades, the development of microelectronics has moved along with constant speed of scaling to maximize transistor density as driven by the need for electrical and functional performance. For ...further development, the propagation velocity of electromagnetic waves becomes increasingly important due to their unyielding constraints on interconnect delay. To minimize it, it was forced to the introduction of the Cu/low-k dielectric interconnects to very large scale integrated circuits (VLSI) where k denotes the dielectric constant. In addition, reliable barrier structures, which are the thinnest part among the device parts to maximize space availability for the actual Cu IWs, are required to prevent penetration of different materials. In light of the above statements, this review will focus recent patents and some studies on Cu interconnects including Cu interconnect wires, low-k dielectrics and related barrier materials as well manufacturing techniques in VLSI, which are one of the most essential concerns in microelectronic industry and decides the further development of VLSI. In addition, possible future development in this field is considered.
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