Mechanism of metamifop resistance in Digitaria ciliaris var. chrysoblephara from Jiangsu, China.

Digitaria ciliaris var. chrysoblephara is one of the most competitive and problematic grass weeds in China. Metamifop is an aryloxyphenoxypropionate (APP) herbicide that inhibits the activity of acetyl-CoA carboxylase (ACCase) of sensitive weeds. Following the introduction of metamifop to China in 2010, it has been continuously used in rice paddy fields, thereby substantially increasing selective pressure for resistant D. ciliaris var. chrysoblephara variants. Here, populations of D. ciliaris var. chrysoblephara (JYX-8, JTX-98, and JTX-99) were observed to be highly resistant to metamifop, with resistance index (RI) values of 30.64, 14.38, and 23.19, respectively. Comparison of resistant and sensitive population ACCase gene sequences revealed that a single nucleotide substitution from TGG to TGC resulted in an amino acid substitution from tryptophan to cysteine at position 2,027 in the JYX-8 population. No corresponding substitution was observed for JTX-98 and JTX-99 populations. The ACCase cDNA of D. ciliaris var. chrysoblephara was successfully obtained by PCR and RACE methods, representing the first amplification of full length ACCase cDNA from Digitaria spp. Investigation of the relative expressions of ACCase gene revealed the lack of significant differences between sensitive and resistant populations before and after herbicide treatments. ACCase activities in resistant populations were less inhibited than in sensitive populations and recovered to the same or even higher levels compared to untreated plants. Whole-plant bioassays were also conducted to assess resistance to other ACCase inhibitors, acetolactate synthase (ALS) inhibitors, auxin mimic herbicide, and protoporphyrinogen oxidase (PPO) inhibitor. Cross-resistance and some multi-resistance were observed in the metamifop-resistant populations. This study is the first to focus on the herbicide resistance of D. ciliaris var. chrysoblephara. These results provide evidence for a target-site resistance mechanism in metamifop-resistant D. ciliaris var. chrysoblephara, while providing a better understanding of cross- and multi-resistance characteristics of resistant populations that will help in the management of herbicide-resistant D. ciliaris var. chrysoblephara.

Experimental study on co-culture of DiI-labeled rat bone marrow mesenchymal stem cells and neonatal rat cardiomyocytes to induce differentiation into cardiomyocyte-like cells.

BACKGROUND: Myocardial infarction is a serious clinical disease with high mortality and poor prognosis. Cardiomyocytes (CMs) have limited regeneration abilities after ischemic injury. Their growth and differentiation can be enhanced by contact co-culture with stem cells. OBJECTIVE: The aim was to study the contact co-culture of Dil-labeled bone marrow mesenchymal stem cells (BMSCs) and CMs for inducing differentiation of CMs from stem cells for treating myocardial infarction. METHODS: After contact co-culture, the differentiation of BMSCs into CMs was analyzed qualitatively by detecting myocardial markers (cardiac troponin T and α-smooth muscle actin) using immunofluorescence and quantitatively using flow cytometry. To examine the mechanism, possible gap junctions between BMSCs and CMs were analyzed by detecting gap junction protein connexin 43 (C×43) expression in BMSCs using immunofluorescence. The functionality of gap junctions was analyzed using dye transfer experiments. RESULTS: The results revealed that BMSCs in contact with CMs exhibited myocardial markers and a significant increase in differentiation rate (P < 0.05); they also proved the existence and function of gap junctions between BMSCs and CMs. CONCLUSIONS: It was shown that contact co-culture can induce Dil-labeled BMSCs to differentiate into CM-like cells and examined the principle of gap junction-mediated signaling pathways involved in inducing stem cells to differentiate into cardiomyocytes.

Preparation of myocardial patches from DiI-labeled rat bone marrow mesenchymal stem cells and neonatal rat cardiomyocytes contact co-cultured on polycaprolactone film.

To provide better treatment of myocardial infarction, DiI-labeled bone marrow mesenchymal stem cells (BMSCs) were contact co-cultured with cardiomyocytes (CMs) on polycaprolactone (PCL) film to prepare myocardial patches. BMSCs from Sprague Dawley rats were isolated, cultured, and characterized for expression of surface markers by flow cytometry. CMs were isolated from suckling rats. After BMSCs were cultured for three generations, they were labeled with DiI dye. DiI-labeled BMSCs were co-cultured with CMs on PCL film in the experimental group, while CMs were replaced with the same amount of unlabeled BMSCs in the control group. After 24 h, cell growth was observed by light microscopy and cells were fixed for scanning electron microscopy (SEM). After 7 d of co-culture, cells were stained for immunofluorescence detection of myocardial markers cardiac troponin T (cTnT) andα-actin. Differentiation of BMSCs on PCL was observed by fluorescence microscopy. The efficiency of BMSC differentiation into CMs was analyzed by flow cytometry on the first and seventh days of co-culture. CMs were stained with calcein alone and contact co-cultured with DiI-labeled BMSCs on PCL film to observe intercellular dye transfer. Finally, cells were stained for immunofluorescence detection of connexin 43 (Cx43) expression and to observe the relationship between gap junctions and contact co-culture. BMSCs were identified by flow cytometry as strongly positive for CD90 and CD44H, and negative for CD11b/c and CD45. After co-culture for 24 h, cells were observed to have attached to PCL by light microscopy. Upon appropriate excitation, DiI-labeled BMSCs exhibited red fluorescence, while unlabeled CMs did not. SEM revealed a large number of cells on the PCL membrane and their cell state appeared normal. On the seventh day, some DiI-labeled BMSCs expressed cTnT andα-actin. Flow cytometry showed that the rate of stem cell differentiation in the experimental group was significantly higher than the control group on the seventh day (20.12% > 3.49%,P< 0.05). From the second day of co-culture, immunofluorescence staining for Cx43 revealed green fluorescent puncta in some BMSCs; from the third day of co-culture, a portion of BMSCs exhibited green fluorescence in dye transfer tests. Contact co-culture of DiI-labeled BMSCs and CMs on PCL film generated primary myocardial patches. The mechanism by which contact co-culture promoted differentiation of the myocardial patch may be related to gap junctions and gap junction-mediated intercellular signaling pathways.

[Effects of different barnyardgrass species on grain yield of rice and their physiological characteristics under alternate wetting and drying irrigation].

In order to investigate the influence of different barnyardgrass species on rice yield and physiological characteristics of rice, two rice cultivars, Liangyoupeijiu (an indica hybrid cultivar) and Nanjing 9108 (a japonica cultivar) , were employed to co-culture with four barnyardgrass species during the period from transplanting to maturity under alternate wetting and moderate drying ir- rigation condition. The treatments were separately designed as follow: weed free ( control) , rice with Echinochloa crusgalli var. mitis (T1), rice with E. crusgalli (T2), rice with E. crusgali var. zelayensis (T3) and rice with E. colonum (T4). The results showed that T1, T2, T3 and T4 treatments reduced the Liangyoupeijiu yield by 13.8%, 10.6%, 23.8% and 0.5%, but the corresponding yield loss of Nanjing 9108 could reach up to 45.5%, 36.9%, 60.7% and 15.1%, respectively. The results above showed that T1, T2 and T3 treatments all significantly reduced grain yield, and T4 treatment only reduced grain yield for Nanjing 9108 but not for Liangyoupeijiu. All treatments elevated malondialehyde contents of rice leaf, but the activities of peroxidase, catalase, superoxide dimutase, dry matter accumulation in maturity stage, root oxidation activities and contents of indole-3-acetic acid as well as zeatin + zeatin riboside in roots during rice grain filling stage were all decreased. The influence degree of four barnyardgrass against physiological indices of rice had the order of T3 > T1 >T2 > T4. It showed that the reductions in enzyme activities of antioxidant system, root oxidation activities, contents of indole-3-acetic acid, zeatin + zeatin riboside during grain filling stage and accumulation of dry matter in maturity as well as increase in contents of malondialehyde of rice during grain filling stage might be important reasons for grain yield reduction when grew with barnyardgrass.

[Influence of weeds in Echinochloa on growth and yield of rice].

In order to investigate effects of different barnyardgrass species on growth and yiled of rice, two rice cultivars, Xinliangyou 6 hao (an indica hybrid cultivar) and Nanjing 46 (a japonica cultivar), were co-cultured with four barnyardgrass species grown at a density of six plants · m(-2) from 10 days after transplanting to maturity. The treatments were designed as follow: weed free (control), rice with Echinochloa crusgalli var. mitis (T1), rice with E. crusgalli (T2), rice with E. crusgali var. zelayensis (T3), and rice with E. colonum (T4). The result showed that barnyardgrass-induced reductions in grain yield of rice were obviously different among the four treatments. T1, T2 and T3 treatments reduced the grain yield of indicia cultivar by 19.2%, 10.8% and 21.9%, and the respective reductions in japonica cultivar were 39.7%, 25.3% and 47.3%, re- spectively. However, no significant difference was detected for T4 treatment. During rice co-culture with barnyardgrass, T1, T2 and T3 significantly reduced rice dry matter accumulation at maturity, flag leaf photosynthetic rate, root oxidation activity and the activity of adenosine triphosphate enzyme (ATPsse) in rice grains at the filling stage, and the magnitude of decrease was in the order of T3 > T1 > T2, while no significant difference was observed between T4 and CK. In addition, all treatments had no effects on the final tiller number and plant height of rice. It indicated that the negative effect of barnyardgrass on rice growth and yield differed among the four species of barnyardgrass, in the order of T3 > T1 > T2 > T4. Barnyardgrass reduced the flag leaf photosynthetic rate of rice, both root oxidation activity and ATPsse activity in grains, which resulted in the reduction in final productivity of rice when co-cultured with barnyardgrass.