Recording of elapsed time and temporal information about biological events using Cas9

1. Download : Download high-res image (204KB)
2. Download : Download full-size image

     

Bone marrow-derived mesenchymal stem cells improve post-ischemia neurological function in rats via the PI3K/AKT/GSK-3β/CRMP-2 pathway

Background: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. However, the underlying protective mechanism remains undetermined. Here, we tested the hypothesis that transplantation of BMSCs via intravenous injection can alleviate neurological functional deficits through activating PI3K/AKT signaling pathway after cerebral ischemia in rats.

Methods: A cerebral ischemic rat model was established by the 2 h middle cerebral artery occlusion (MCAO). Twenty-four hours later, BMSCs (1?×?10⁶ in 1 ml PBS) from SD rats were injected into the tail vein. Neurological function was evaluated by modified neurological severity score (mNSS) and modified adhesive removal test before and on d1, d3, d7, d10 and d14 after MCAO. Protein expressions of AKT, GSK-3β, CRMP-2 and GAP-43 were detected by Western-bolt. NF-200 was detected by immunofluorescence.

Results: BMSCs transplantation did not only significantly improve the mNSS score and the adhesive-removal somatosensory test after MCAO, but also increase the density of NF-200 and the expression of p-AKT, pGSK-3β and GAP-43, while decrease the expression of pCRMP-2. Meanwhile, these effects can be suppressed by LY294002, a specific inhibitor of PI3K/AKT.

Conclusion: These data suggest that transplantation of BMSCs could promote axon growth and neurological deficit recovery after MCAO, which was associated with activation of PI3K/AKT /GSK-3β/CRMP-2 signaling pathway.

     

Focus on Weed Control: The Impact of Herbicide-Resistant Rice Technology on Phenotypic Diversity and Population Structure of United States Weedy Rice

The use of herbicide-resistant (HR) Clearfield rice (Oryza sativa) to control weedy rice has increased in the past 12 years to constitute about 60% of rice acreage in Arkansas, where most U.S. rice is grown. To assess the impact of HR cultivated rice on the herbicide resistance and population structure of weedy rice, weedy samples were collected from commercial fields with a history of Clearfield rice. Panicles from each weedy type were harvested and tested for resistance to imazethapyr. The majority of plants sampled had at least 20% resistant offspring. These resistant weeds were 97 to 199 cm tall and initiated flowering from 78 to 128 d, generally later than recorded for accessions collected prior to the widespread use of Clearfield rice (i.e. historical accessions). Whereas the majority (70%) of historical accessions had straw-colored hulls, only 30% of contemporary HR weedy rice had straw-colored hulls. Analysis of genotyping-by-sequencing data showed that HR weeds were not genetically structured according to hull color, whereas historical weedy rice was separated into straw-hull and black-hull populations. A significant portion of the local rice crop genome was introgressed into HR weedy rice, which was rare in historical weedy accessions. Admixture analyses showed that HR weeds tend to possess crop haplotypes in the portion of chromosome 2 containing the ACETOLACTATE SYNTHASE gene, which confers herbicide resistance to Clearfield rice. Thus, U.S. HR weedy rice is a distinct population relative to historical weedy rice and shows modifications in morphology and phenology that are relevant to weed management.Weedy rice (Oryza sativa), a conspecific weed of cultivated rice, is a global threat to rice production (Delouche et al., 2007). Classified as the same species as cultivated rice, it is highly competitive (Diarra et al., 1985; Pantone and Baker, 1991; Burgos et al., 2006), difficult to control without damaging cultivated rice, and can cause almost total crop failure (Diarra et al., 1985). The competition of cultivated rice with weedy rice can lead to yield losses from less than 5% to 100% (Kwon et al., 1991; Watanabe et al., 2000; Chen et al., 2004; Ottis et al., 2005; Shivrain et al., 2009b). Besides being difficult to control, weedy rice persists in rice fields because of key weedy traits, including variable emergence (Shivrain et al., 2009b), high degree of seed shattering (Eleftherohorinos, et al., 2002; Thurber et al., 2010), high diversity in seed dormancy (Do Lago, 1982; Noldin, 1995; Vidotto and Ferrero, 2000; Burgos et al., 2011; Tseng et al., 2013), and its seed longevity in soil (Goss and Brown, 1939). Weedy rice is a problem mainly in regions with large farm sizes where direct-seeded rice culture is practiced (Delouche et al., 2007). It is not a major problem in transplanted rice culture, where roguing weeds is possible and hand labor is available. The severity of the problem has increased in recent decades because of the significant shift to direct seeding from transplanting (Pandey and Velasco, 2002; Rao et al., 2007; Chauhan et al., 2013), which is driven by water scarcity (Kummu et al., 2010; Turral et al., 2011), increasing labor costs, and migration of labor to urban areas (Grimm et al., 2008).The herbicide-resistant (HR) Clearfield rice technology (Croughan, 2003) provides an option to control weedy rice in rice using imidazolinone herbicides, in particular, imazethapyr. Imidazolinones belong to group 2 herbicides, also known as ACETOLACTATE SYNTHASE (ALS) inhibitors. Examples of herbicides in this group are imazamox, imazapic, imazaquin, and imazethapyr. Developed through mutagenesis of the ALS locus (Croughan, 1998), Clearfield rice was first commercialized in 2002 in the southern U.S. rice belt (Tan et al., 2005). Low levels of natural hybridization are known to occur between the crop and weedy rice. Gene flow generally ranges from 0.003% to 0.25% (Noldin et al., 2002; Song et al., 2003; Messeguer et al., 2004; Gealy, 2005; Shivrain et al., 2007, 2008). After the adoption of Clearfield technology, resistant weedy outcrosses were soon detected in commercial fields (Fig. 1), generally after two cropping seasons of Clearfield rice, where escaped weedy rice was able to produce seed (Zhang et al., 2006; Burgos et al., 2007, 2008). Similar observations have been reported outside the United States, in other regions adopting the technology (Gressel and Valverde, 2009; Busconi et al., 2012).Open in a separate windowFigure 1.Suspected herbicide-resistant weedy rice in a rice field previously planted with Clearfield rice along the Mississippi River Delta in Arkansas. More than 10 morphotypes of weedy rice were observed in this field, with different maturity periods. In the foreground is a typical weedy rice with pale green leaves; the rice cultivar has dark green leaves. The inset shows a weedy morphotype that matured earlier than cultivated rice.Despite this complication, the adoption of Clearfield rice technology is increasing, albeit at a slower pace than that of glyphosate-resistant crops. After a decade of commercialization, 57% of the rice area in Arkansas was planted with Clearfield rice cultivars in 2013 (J. Hardke, personal communication). Clearfield technology has been very successful at controlling weedy rice, and polls among rice growers suggest that farmers have kept the problem of HR weeds in check by following the recommended stewardship practices (Burgos et al., 2008). The most notable of these are (1) implementation of herbicide programs that incorporate all possible modes of action available for rice production; (2) ensuring maximum efficacy of the herbicides used; (3) preventing seed production from escaped weedy rice, remnant weedy rice after crop harvest, or volunteer rice and weedy rice in the next crop cycle; (4) rotating Clearfield rice with other crops to break the weedy rice cycle; and (5) practicing zero tillage to avoid burying HR weedy rice seed (Burgos et al., 2008).Clearfield rice has gained a foothold in Asia, where rice cultivation originated (Londo and Schaal, 2007; Zong et al., 2007). Clearfield rice received government support for commercialization in Malaysia in 2010 (Azmi et al., 2012) because of the severity of the weedy rice problem there. Dramatic increases in rice yields (from 3.5 to 7 metric tons ha⁻¹) were reported in Malaysia where Clearfield rice was planted (Sudianto et al., 2013). However, the risk of gene flow and evolution of resistant weedy rice populations is high in the tropics, where up to three rice crops are planted each year, and freezing temperatures, which would reduce the density of volunteer plants, do not occur.In the United States, where Clearfield technology originated and has been used for the longest time, the interaction between HR cultivated rice and weedy rice is not yet fully understood. Two main populations of weedy rice are known to occur in the southern United States and can be found in the same cultivated rice fields. These populations are genetically differentiated, are largely distinct at the phenotypic level, and have separate evolutionary origins (Reagon et al., 2010). One group tends to have straw-colored hulls and is referred to as the SH population; a second group tends to have black-colored hulls and awns and is referred to as the BHA population (Reagon et al., 2010). Genomic evidence suggests that both groups descended from cultivated ancestors but not from the tropical japonica subgroup varieties that are grown commercially in the United States. Instead, the SH group evolved from indica, a subgroup of rice commonly grown in the lowland tropics, and the BHA group descended from aus, a related cultivated subgroup typically grown in Bangladesh and the West Bengal region (Reagon et al., 2010). Weed-weed and weed-crop hybrids are also known to occur, but prior to Clearfield commercialization, these hybrids had occurred at low frequency (Reagon et al., 2010; Gealy et al., 2012). With the advent and increased adoption of Clearfield cultivars, the impact on U.S. weedy rice population structure and the prevalence of the SH and BHA groups are unknown.Efforts to predict the possible consequences of HR or genetically modified rice on weedy rice have been a subject of discussion for many years. Both weedy rice and cultivated rice are primarily self-fertilizing, but, as mentioned above, low levels of gene flow are known to occur. Additional environmental and intrinsic genetic factors can act as prezygotic and postzygotic mating barriers between cultivated and weedy rice and influence the possibility and levels of gene flow between these groups (Craig et al., 2014; Thurber et al., 2014). However, once gene flow occurs between cultivated and weedy rice, and if the resulting hybrids are favored by selection, the resulting morphological, genetic, and physiological changes in weedy rice populations can alter the way that weedy rice evolves and competes. For example, herbicide-resistant weed outcrosses in an experimental field have been observed to be morphologically diverse (Shivrain et al., 2006), with some individuals carrying major weedy traits and well adapted to rice agriculture. Such weedy plants could be more problematic than their normal weedy counterparts. Thus, introgression of crop genes into weedy populations has the potential to change the population dynamic, genetic structure, and morphological profile of weedy plants. This, in turn, must alter our crop management practices. To increase our understanding of the impact of HR rice on the evolution of weedy rice, in this article we aim to (1) assess the frequency of herbicide resistance in weedy rice in southern U.S. rice fields with a history of Clearfield use; (2) characterize the weedy attributes of resistant populations; and (3) determine the genetic origins of herbicide-resistant weeds in U.S. fields.     

Cloning and analysis of a type II polyketide synthase gene cluster from Streptomyces toxytricini NRRL 15,443

A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), beta-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.     

Electrophysiological modelling of pulmonary artery smooth muscle cells in the rabbits--special consideration to the generation of hypoxic pulmonary vasoconstriction

In vascular smooth muscle cells, it has been suggested that membrane potential is an important component that initiates contraction. We developed a mathematical model to elucidate the quantitative contributions of major ion currents [a voltage-gated L-type Ca²⁺ current (I_CaL), a voltage-sensitive K⁺ current (I_KV), a Ca²⁺-activated K⁺ current (I_KCa) and a nonselective cation current (I_NSC)] to membrane potential. In order to typify the diverse nature of pulmonary artery smooth muscle cells (PASMCs), we introduced parameters that are not fixed (variable parameters). The population of cells with different parameters was constructed and the cells that have the electrophysiological properties of PASMCs were selected. The contributions of each membrane current were investigated by sensitivity analysis and modification of the current parameters. Consequently, I_KV and I_NSC were found to be the most important currents that affect the membrane potential. The occurrence of depolarisation in hypoxic pulmonary vasoconstriction (HPV) was also examined. In hypoxia, I_KV and I_KCa were reduced, but the consequent depolarisation in simulation was not enough to initiate contractions. If we add an increase of I_NSC (2.5-fold), the calculated membrane potential was enough to induce contraction. From the results, we conclude that the balance of various ion channel activities determines the resting membrane potential of PASMCs and our model was successful in explaining the depolarisation in HPV. Therefore, this model can be a powerful tool to investigate the various electrical properties of PASMCs in both normal and pathological conditions.     

Effects of BmK AS on Nav1.2 expressed in <Emphasis Type="Italic">Xenopus laevis</Emphasis> oocytes

In the present study, the pharmacological effects of BmK AS, a β-like scorpion toxin on rNav1.2 α-subunit expressed in Xenopus laevis oocytes were investigated using a two-electrode voltage-clamp recording. It was found that the voltage dependence of rNav1.2 inactivation was significantly shifted towards positive membrane potential by 500 nM BmK AS, whereas the activation curves of rNav1.2 were unruffled at the same dosage. The inactivation curves of both slow and fast inactivation currents were positively moved about 12.8 and 9.7 mV, respectively. In addition, the persistent currents of rNav1.2 were invariable. The effects of BmK AS on the rNav1.2 inactivation were opposite to the previous results found in the peripheral sensory neurons. The results suggested that Nav1.2 might be the target of BmK AS in the central nervous system, and BmK AS might have an excitatory effect on the central neuron through enhancing Nav1.2.     

Development of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: implications for bone regeneration

We made a three-dimensional (3-D) nanofibrous fibroin scaffold (NFS) with high porosity (94%) and examined its feasibility in bone regeneration. Under scanning electron microscopy, MC3T3-E1 preosteoblasts on the scaffold showed more spread on the first day after seeding compared with a 2-D scaffold. MTT assay showed significantly increased proliferation in 3-D NFS compared with 2-D NFS 7 days after seeding (P < 0.05). Western immunoblotting for activated paxillin, FAK, AKT, C-Src, and ERK1/2 antibodies showed signals from the extracellular matrix were significantly increased in 3-D NFS. Newly developed 3-D electrospun NFS may be a good candidate for use in bone regeneration. Ki and Park equally contributed in this paper.     

Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.)

We previously identified a salt and drought stress-responsive TFIIIA-type zinc finger protein gene ZFP252 from rice. Here we report the functional analysis of ZFP252 using gain- and loss-of-function strategies. We found that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes and enhanced rice tolerance to salt and drought stresses, as compared with ZFP252 antisense and non-transgenic plants. Our findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced tolerance to salt and drought stresses.     

Neoparamoeba perurans is a cosmopolitan aetiological agent of amoebic gill disease

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry.