Weak and saturable protein-surfactant interactions in the denaturation of apo-α-lactalbumin by acidic and lactonic sophorolipid

Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However, knowledge of such interactions is limited. Here, we present a study of the interactions between the model protein apo-alpha-lactalbumin (apo-aLA) and the biosurfactant sophorolipid (SL) produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL) is sparingly soluble and has a lower critical micelle concentration (cmc) than the acidic form [non-acetylated acidic sophorolipid (acidSL)]. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL), with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that acidSL has weak and saturable interactions with apo-aLA at low concentrations; due to the relatively low cmc of acidSL (which means that the monomer concentration is limited to ca. 0-1 mM SL), it is only possible to observe interactions with monomeric acidSL at high apo-aLA concentrations. However, the denaturation kinetics of apo-aLA in the presence of acidSL are consistent with a collaboration between monomeric and micellar surfactant species, similar to RL and non-ionic or zwitterionic surfactants. Inclusion of diacetylated lactonic sophorolipid (lactSL) as mixed micelles with acidSL lowers the cmc and this effectively reduces the rate of unfolding, emphasizing that SL like other biosurfactants is a gentle anionic surfactant. Our data highlight the potential of these biosurfactants for future use in the detergent and pharmaceutical industry

Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3'DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5'fluorophore and a 3'quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition

The Healthy Start project: a randomized, controlled intervention to prevent overweight among normal weight, preschool children at high risk of future overweight

BACKGROUND: Research shows that obesity prevention has to start early. Targeting interventions towards subgroups of individuals who are predisposed, but yet normal weight, may prove more effective in preventing overweight than interventions towards unselected normal weight subsets. Finally, interventions focused on other factors than diet and activity are lacking. The objectives were to perform a randomized, controlled intervention aiming at preventing overweight in children aged 2–6 years, who are yet normal weight, but have high predisposition for future overweight, and to intervene not only by improving diet and physical activity, but also reduce stress and improve sleep quality and quantity. METHODS/DESIGN: Based on information from the Danish National Birth Registry and administrative birth forms, children were selected based on having either a high birth weight, a mother who was overweight prior to pregnancy, or a familial low socioeconomic status. Selected children (n = 5,902) were randomized into three groups; an intervention group, a shadow control group followed in registers exclusively, and a control group examined at the beginning and at the end of the intervention. Approximately 21% agreed to participate. Children who presented as overweight prior to the intervention were excluded from this study (n = 92). In the intervention group, 271 children were included, and in the control group 272 were included. Information obtained from the shadow control group is on-going, but it is estimated that 394 children will be included. The intervention took place over on average 1½ year between 2009 and 2011, and consisted of optional individual guidance in optimizing diet and physical activity habits, reducing chronic stress and stressful events and improving sleep quality and quantity. The intervention also included participation in cooking classes and play arrangements. Information on dietary intake, meal habits, physical activity, sleep habits, and overall stress level was obtained by 4–7 day questionnaire diaries and objective measurements. DISCUSSION: If the Healthy Start project is effective in preventing excessive weight gain, it will provide valuable information on new determinants of obesity which should be considered in future interventions, and on new strategies to prevent development of overweight and obesity at an early age. TRIAL REGISTRATION: ClinicalTrials.gov, ID NCT01583335

A Model for the Development of the Rhizobial and Arbuscular Mycorrhizal Symbioses in Legumes and Its Use to Understand the Roles of Ethylene in the Establishment of these two Symbioses

We propose a model depicting the development of nodulation and arbuscular mycorrhizae. Both processes are dissected into many steps, using Pisum sativum L. nodulation mutants as a guideline. For nodulation, we distinguish two main developmental programs, one epidermal and one cortical. Whereas Nod factors alone affect the cortical program, bacteria are required to trigger the epidermal events. We propose that the two programs of the rhizobial symbiosis evolved separately and that, over time, they came to function together. The distinction between these two programs does not exist for arbuscular mycorrhizae development despite events occurring in both root tissues. Mutations that affect both symbioses are restricted to the epidermal program. We propose here sites of action and potential roles for ethylene during the formation of the two symbioses with a specific hypothesis for nodule organogenesis. Assuming the epidermis does not make ethylene, the microsymbionts probably first encounter a regulatory level of ethylene at the epidermis–outermost cortical cell layer interface. Depending on the hormone concentrations there, infection will either progress or be blocked. In the former case, ethylene affects the cortex cytoskeleton, allowing reorganization that facilitates infection; in the latter case, ethylene acts on several enzymes that interfere with infection thread growth, causing it to abort. Throughout this review, the difficulty of generalizing the roles of ethylene is emphasized and numerous examples are given to demonstrate the diversity that exists in plants

A Set of Lotus japonicus Gifu × Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype ‘Gifu’ was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation

Registration of ‘NS presser CLP’ hard red spring wheat

‘NS Presser CLP’ (Reg. No. CV-1132, PI 679964) hard red spring wheat (Triticum aestivum L.) was developed by the Montana Agricultural Experiment Station and released in 2016 to the commercial partner Northern Seed LLC. NS Presser CLP is a two-gene Clearfield wheat intended for use with the selective imidazolinone herbicide imazamox (Beyond, BASF Corp.). NS Presser CLP was developed by a single backcross of alleles for resistance to the imidazolinone herbicide class into the recurrent parent ‘Vida’. Vida has been the most widely grown hard red spring wheat cultivar in Montana since 2010. Alleles for herbicide resistance at TaAHAS1D and TaAHAS1B were selected during the backcrossing process and line derivation using polymerase chain reaction markers developed by BASF. Three years of replicated yield trials at a total of 23 sites showed that NS Presser CLP has yield potential under dryland production similar to Vida. NS Presser CLP showed tolerance to a 2× rate of imidazolinone herbicide (87.6 g a.i. ha-1) applied at three testing sites in each of 2 yr. It provides a high-yielding Clearfield cultivar for dryland production in Montana and surrounding areas

Analysis of promoter activity of the early nodulin Enod40 in Lotus japonicus

Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-ben-zylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.Animal science

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5

Agrobacterium rhizogenes-Mediated Transformation of the Parasitic Plant Phtheirospermum japonicum

Background: Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. Methodology/Principal Findings: We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. Conclusions: We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocol