Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.     

Characteristics of Nasal-Associated Lymphoid Tissue (NALT) and Nasal Absorption Capacity in Chicken

As the main mucosal immune inductive site of nasal cavity, nasal-associated lymphoid tissue (NALT) plays an important role in both antigen recognition and immune activation after intranasal immunization. However, the efficiency of intranasal vaccines is commonly restricted by the insufficient intake of antigen by the nasal mucosa, resulting from the nasal mucosal barrier and the nasal mucociliary clearance. The distribution of NALT and the characteristic of nasal cavity have already been described in humans and many laboratory rodents, while data about poultry are scarce. For this purpose, histological sections of the chicken nasal cavities were used to examine the anatomical structure and histological characteristics of nasal cavity. Besides, the absorptive capacity of chicken nasal mucosa was also studied using the materials with different particle size. Results showed that the NALT of chicken was located on the bottom of nasal septum and both sides of choanal cleft, which mainly consisted of second lymphoid follicle. A large number of lymphocytes were distributed under the mucosal epithelium of inferior nasal meatus. In addition, there were also diffuse lymphoid tissues located under the epithelium of the concha nasalis media and the walls of nasal cavity. The results of absorption experiment showed that the chicken nasal mucosa was capable to absorb trypan blue, OVA, and fluorescent latex particles. Inactivated avian influenza virus (IAIV) could be taken up by chicken nasal mucosa except for the stratified squamous epithelium sites located on the forepart of nasal cavity. The intake of IAIV by NALT was greater than that of the nasal mucosa covering on non-lymphoid tissue, which could be further enhanced after intranasal inoculation combined with sodium cholate or CpG DNA. The study on NALT and nasal absorptive capacity will be benefit for further understanding of immune mechanisms after nasal vaccination and development of nasal vaccines for poultry.     

Using Bosentan to Treat Paraquat Poisoning-Induced Acute Lung Injury in Rats

Background

Paraquat poisoning is well known for causing multiple organ function failure (MODS) and high mortality. Acute lung injury and advanced pulmonary fibrosis are the most serious complications. Bosentan is a dual endothelin receptor antagonist. It plays an important role in treating PF. There is no related literature on the use of bosentan therapy for paraquat poisoning.

Objective

To study the use of bosentan to treat acute lung injury and pulmonary fibrosis as induced by paraquat.

Method

A total of 120 adult Wister male rats were randomly assigned to three groups: the paraquat poisoning group (rats were intragastrically administered with paraquat at 50 mg/kg body weight once at the beginning); the bosentan therapy group (rats were administered bosentan at 100 mg/kg body weight by intragastric administration half an hour after paraquat was administered, then the same dose was administered once a day); and a control group (rats were administered intragastric physiological saline). On the 3rd, 7th, 14th, and 21st days following paraquat exposure, rats were sacrificed, and samples of lung tissue and venous blood were collected. The levels of transforming growth factor-β1 (TGF-β1), endothelin-1 (ET-1), and hydroxyproline (HYP) in the plasma and lung homogenate were determined. Optical and electronic microscopes were used to examine pathological changes.

Result

The TGF-β1, ET-1, and HYP of the paraquat poisoning group were significantly higher than in the control group, and they were significantly lower in the 21st day therapy group than in the paraquat poisoning group on the same day. Under the optical and electronic microscopes, lung tissue damage was observed to be more severe but was then reduced after bosentan was administered.

Conclusion

Bosentan can reduce inflammation factor release. It has a therapeutic effect on acute lung injury as induced by paraquat.     

Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins

Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.     

IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer

Homeobox D10 (HoxD10) plays important roles in the differentiation of embryonic cells and progression of breast cancer. Our previous report revealed that insulin-like growth factor binding protein-3 (IGFBP3) was regulated by HoxD10 in gastric cancer cells; however, the functional roles and underlying mechanisms of IGFBP3 in gastric cancer remain unclear. Here, we found that the expression of IGFBP3 were upregulated after ectopic expression of HoxD10 in gastric cancer cells. Chromatin immunoprecipitation assay showed that HoxD10 bound to three potential regions of IGFBP3 promoter. Exogenous HoxD10 significantly enhanced the activity of luciferase reporter containing these binding regions in gastric cancer cells. Further data showed that all of these binding sites had Hox binding element “TTAT”. Immunohistochemical staining results revealed that IGFBP3 expression was significantly downregulated in 86 gastric adenocarcinomas tissues relative to their adjacent non-cancerous tissues (p<0.001). Moreover, IGFBP3 expression was significantly lower in gastric tumor with lymph node metastasis compared with that without lymph node metastasis (p=0.045). Patients with high expression level of IGFBP3 showed favorable 5 year overall survival (p=0.011). Knockdown of IGFBP3 accelerated gastric cancer cell migration and invasion and induced the expression of invasive factors including MMP14, uPA and uPAR. Thus, our data suggest that HoxD10-targeted gene IGFBP3 may suppress gastric cancer cell invasion and favors the survival of gastric cancer patients.     

Intratumoral Hepatic Stellate Cells as a Poor Prognostic Marker and a New Treatment Target?for?Hepatocellular Carcinoma

Hepatic stellate cells (HSCs), a specialized stromal cytotype in the liver, have been demonstrated to actively contribute to hepatocellular carcinoma (HCC) development. However, the previous studies were performed using HSC cell lines, and the prognostic value of intratumoral HSCs (tHSCs) was unclear. Here we isolated tHSCs from fresh human HCC tissues, and analyzed the abilities of tHSCs to promote HCC progression by using in vitro assays for cell viability, migration and invasion as well as epithelial-mesenchymal transition (EMT) phenotype. 252 HCC patients who underwent hepatectomy were enrolled for analysis of tHSCs and E-cadherin expression in tumor tissues, and 55 HCC patients for analysis of tHSCs in tumor tissues and circulating tumor cells (CTCs) in blood. Prognostic factors were then identified. The results showed that coculture of tHSCs with HCC cells had a stronger effect on HCC cell viability, migration and invasion, accompanied with the acquisition of epithelial-mesenchymal transition (EMT) phenotype. In vivo cotransplantation of HCC cells with tHSCs into nude mice more efficiently promoted tumor formation and growth. Icaritin, a known apoptosis inducer of HSCs, was demonstrated to effectively inhibit tHSC proliferation in vitro and tHSC-induced HCC-promoting effects in vivo. Clinical evidence indicated that tHSCs were rich in 45% of the HCC specimens, tHSC-rich subtypes were negatively correlated either with E-cadherin expression in tumor tissues (r = -0.256, p < 0.001) or with preoperative CTCs in blood (r = -0.287, p = 0.033), and were significantly correlated with tumor size (p = 0.027), TNM staging (p = 0.018), and vascular invasion (p = 0.008). Overall and recurrence-free survival rates of tHSC-rich patients were significantly worse than those for tHSC-poor patients. Multivariate analysis revealed tHSC-rich as an independent factor for overall and recurrence-free survival. In conclusion, tHSCs provide a promising prognostic biomarker and a new treatment target for HCC.     

Directed Forgetting of Negative Self-Referential Information Is Difficult: An fMRI Study

A large body of evidence suggested that both emotion and self-referential processing can enhance memory. However, it remains unclear how these two factors influence directed forgetting. This study speculates that directed forgetting of negative self-referential memory is more difficult than forgetting of other-referential memory. To verify this speculation, we combined the directed forgetting paradigm with the self-reference task. The behavioral result suggested that although both self-referential and other-referential information can be directly forgotten, less self-referential information can be forgotten than other-referential information. At the neural level, the forget instruction strongly activated the frontal cortex, suggesting that directed forgetting is not memory decay but an active process. In addition, compared with the negative other-referential information, forgetting of the negative self-referential information were associated with a more widespread activation, including the orbital frontal gyrus (BA47), the inferior frontal gyrus (BA45, BA44), and the middle frontal gyrus. Our results suggest that forgetting of the self-referential information seems to be a more demanding and difficult process.     

The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.     

13-Methyltetradecanoic Acid Exhibits Anti-Tumor Activity on T-Cell Lymphomas In Vitro and In Vivo by Down-Regulating p-AKT and Activating Caspase-3

13-Methyltetradecanoic acid (13-MTD), a saturated branched-chain fatty acid purified from soy fermentation products, induces apoptosis in human cancer cells. We investigated the inhibitory effects and mechanism of action of 13-MTD on T-cell non-Hodgkin’s lymphoma (T-NHL) cell lines both in vitro and in vivo. Growth inhibition in response to 13-MTD was evaluated by the cell counting kit-8 (CCK-8) assay in three T-NHL cell lines (Jurkat, Hut78, EL4 cells). Flow cytometry analyses were used to monitor the cell cycle and apoptosis. Proteins involved in 13-MTD-induced apoptosis were examined in Jurkat cells by western blotting. We found that 13-MTD inhibited proliferation and induced the apoptosis of T-NHL cell lines. 13-MTD treatment also induced a concentration-dependent arrest of Jurkat cells in the G₁-phase. During 13-MTD-induced apoptosis in Jurkat cells, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP, a caspase enzymolysis product) were detected after incubation for 2 h, and increased after extending the incubation time. However, there was no change in the expression of Bcl-2 or c-myc proteins. The appearance of apoptotic Jurkat cells was accompanied by the inhibition of AKT and nuclear factor-kappa B (NF-κB) phosphorylation. In addition, 13-MTD could also effectively inhibit the growth of T-NHL tumors in vivo in a xenograft model. The tumor inhibition rate in the experimental group was 40%. These data indicate that 13-MTD inhibits proliferation and induces apoptosis through the down-regulation of AKT phosphorylation followed by caspase activation, which may provide a new approach for treating T-cell lymphomas.     

Monitoring Hip and Elbow Dysplasia Achieved Modest Genetic Improvement of 74 Dog Breeds over 40 Years in USA

Hip (HD) and Elbow Dysplasia (ED) are two common complex developmental disorders of dogs. In order to decrease their prevalence and severity, the Orthopedic Foundation for Animals (OFA) has a voluntary registry of canine hip and elbow conformation certified by boarded radiologists. However, the voluntarily reports have been severely biased against exposing dogs with problems, especially at beginning period. Fluctuated by additional influential factors such as age, the published raw scores barely showed trends of improvement. In this study, we used multiple-trait mixed model to simultaneously adjust these factors and incorporate pedigree to derive Estimated Breeding Values (EBV). A total of 1,264,422 dogs from 74 breeds were evaluated for EBVs from 760,455 hip scores and 135,409 elbow scores. These EBVs have substantially recovered the reporting bias and the other influences. Clear and steady trends of genetic improvement were observed over the 40 years since 1970. The total genetic improvements were 16.4% and 1.1% of the phenotypic standard deviation for HD and ED, respectively. The incidences of dysplasia were 0.83% and 2.08%, and the heritabilities were estimated as 0.22 and 0.17 for hip and elbow scores, respectively. The genetic correlation between them was 0.12. We conclude that EBV is more effective than reporting raw phenotype. The weak genetic correlation suggested that selection based on hip scores would also slightly improve elbow scores but it is necessary to allocate effort toward improvement of elbow scores alone.