Development Of New Molecular Tools Research Articles

Activatable organic probes for in situ imaging of biomolecules.

Biomolecules are fundamental for various chemical and biological processes of living organisms. High-resolution in situ imaging of the dynamics and local distribution of biomolecules may facilitate better interpretation of diverse complex cell events in the biomedicine field. In different advanced imaging tools, fluorescence imaging-based activatable organic probes can be noninvasively and effortlessly internalized into cells and can be easily modified, which is essential for the in situ imaging of targets in living organisms. We here briefly summarize the existing general design strategies of activatable organic probes for retaining the fluorescence signal inside cells. We particularly describe the bioapplication of these probes for the in situ bioimaging. This review is expected to promote the development of new molecular tools for extending the application of these in situ imaging strategies to other biomolecules.

Enhancement of SARS-CoV-2 receptor-binding domain activity by two microbial defensins.

Peptide binders are of great interest to both basic and biomedical research due to their unique properties in manipulating protein functions in a precise spatial and temporal manner. The receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein is a ligand that captures human angiotensin-converting enzyme 2 (ACE2) to initiate infection. The development of binders of RBDs has value either as antiviral leads or as versatile tools to study the functional properties of RBDs dependent on their binding positions on the RBDs. In this study, we report two microbe-derived antibacterial defensins with RBD-binding activity. These two naturally occurring binders bind wild-type RBD (WT RBD) and RBDs from various variants with moderate-to-high affinity (7.6-1,450 nM) and act as activators that enhance the ACE2-binding activity of RBDs. Using a computational approach, we mapped an allosteric pathway in WT RBD that connects its ACE2-binding sites to other distal regions. The latter is targeted by the defensins, in which a cation-π interaction could trigger the peptide-elicited allostery in RBDs. The discovery of the two positive allosteric peptides of SARS-CoV-2 RBD will promote the development of new molecular tools for investigating the biochemical mechanisms of RBD allostery.

G-Quadruplex Binding of an NIR Emitting Osmium Polypyridyl Probe Revealed by Solution NMR and Time-Resolved Infrared Studies.

G-quadruplexes are emerging targets in cancer research and understanding how diagnostic probes bind to DNA G-quadruplexes in solution is critical to the development of new molecular tools. In this study the binding of an enantiopure NIR emitting [Os(TAP)2 (dppz)]2+ complex to different G-quadruplex structures formed by human telomer (hTel) and cMYC sequences in solution is reported. The combination of NMR and time-resolved infrared spectroscopic techniques reveals the sensitivity of the emission response to subtle changes in the binding environment of the complex. Similar behaviour is also observed for the related complex [Os(TAP)2 (dppp2)]2+ upon quadruplex binding.

Seizing the moment: The opportunity and relevance of the California Conservation Genomics Project to state and federal conservation policy.

Conservation science and environmental regulation are sibling constructs of the latter half of the 20th century, part of a more general awakening to humanity's effect on the natural world in the wake of 2 world wars. Efforts to understand the evolution of biodiversity using the models of population genetics and the data derived from DNA sequencing, paired with legal and political mandates to protect biodiversity through novel laws, regulations, and conventions arose concurrently. The extremely rapid rate of development of new molecular tools to document and compare genetic identities, and the global goal of prioritizing species and habitats for protection are separate enterprises that have benefited from each other, ultimately leading to improved outcomes for each. In this article, we explore how the California Conservation Genomics Project has, and should, contribute to ongoing and future conservation implementation, and how it serves as a model for other geopolitical regions and taxon-oriented conservation efforts. One of our primary conclusions is that conservation genomics can now be applied, at scale, to inform decision-makers and identify regions and their contained species that are most resilient, and most in need of conservation interventions.

Comparative Transcriptome Analysis of Stage-Specific Changes in Gene Expression during Larval Development in Monochamus alternatus Hope

Monochamus alternatus Hope (Coleoptera: Cerambycidae) is an important trunk borer of pine trees and a major vector of pine wilt disease. Although chemicals are widely used in forest pest control, new strategies based on insect biology are offering promising approaches to manage the disease. Although there have been important research advances in this respect, there has not yet been a deep sequence analysis of M. alternatus describing the transcriptome, and no information is available about the gene function of this insect vector. We used next generation sequencing technology to provide a full transcriptome from the four larval instars of M. alternatus and successfully built an M. alternatus transcriptome database. In total, 67,456 unigenes were obtained with trinity software, information for 11,858 classified unigenes was obtained with the Clusters of Orthologous Groups (COGs) database, and 13,007 unigenes matched predicted pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, genes related to lignocellulose, and putative Bt receptors and genes related to digestion are described. Additionally, the differential gene expression of these genes in different larval stages was analyzed. This study provides valuable information to underpin the development of new molecular tools for M. alternatus control strategies.

Identification of DNA Methylation and Transcriptomic Profiles Associated With Fruit Mealiness in Prunus persica (L.) Batsch.

Peach (Prunus persica) fruits have a fast ripening process and a shelf-life of days, presenting a challenge for long-distance consuming markets. To prolong shelf-life, peach fruits are stored at low temperatures (0 to 7 °C) for at least two weeks, which can lead to the development of mealiness, a physiological disorder that reduces fruit quality and decreases consumer acceptance. Several studies have been made to understand this disorder, however, the molecular mechanisms underlying mealiness are not fully understood. Epigenetic factors, such as DNA methylation, modulate gene expression according to the genetic background and environmental conditions. In this sense, the aim of this work was to identify differentially methylated regions (DMRs) that could affect gene expression in contrasting individuals for mealiness. Peach flesh was studied at harvest time (E1 stage) and after cold storage (E3 stage) for 30 days. The distribution of DNA methylations within the eight chromosomes of P. persica showed higher methylation levels in pericentromeric regions and most differences between mealy and normal fruits were at Chr1, Chr4, and Chr8. Notably, differences in Chr4 co-localized with previous QTLs associated with mealiness. Additionally, the number of DMRs was higher in CHH cytosines of normal and mealy fruits at E3; however, most DMRs were attributed to mealy fruits from E1, increasing at E3. From RNA-Seq data, we observed that differentially expressed genes (DEGs) between normal and mealy fruits were associated with ethylene signaling, cell wall modification, lipid metabolism, oxidative stress and iron homeostasis. When integrating the annotation of DMRs and DEGs, we identified a CYP450 82A and an UDP-ARABINOSE 4 EPIMERASE 1 gene that were downregulated and hypermethylated in mealy fruits, coinciding with the co-localization of a transposable element (TE). Altogether, this study indicates that genetic differences between tolerant and susceptible individuals is predominantly affecting epigenetic regulation over gene expression, which could contribute to a metabolic alteration from earlier stages of development, resulting in mealiness at later stages. Finally, this epigenetic mark should be further studied for the development of new molecular tools in support of breeding programs.

The Expanding Spectrum of Mutations in Hereditary Angioedema

The evolution in the knowledge of rare genetic diseases such as hereditary angioedema (HAE) has increased at a parallel pace with the development of new molecular tools. The deficiency of C1 inhibitor (C1-INH) has been recognized as the main cause of HAE (HAE-C1-INH) since the 1960s, but the discovery of the wide spectrum of mutations affecting the C1-INH gene (SERPING1) was possible only from the late 1980s, when Sanger sequencing became available and more accessible worldwide. Nevertheless, the involvement of other genes in HAE was discovered only in 2006 with the description of mutations in the F12 gene in patients with HAE and normal C1-INH. In the last 3 years, advanced next-generation sequencing techniques allowed the identification of mutations in 5 new genes being associated with HAE and normal C1-INH: ANGPT1 (angiopoietin-1), PLG (plasminogen), KNG1 (kininogen), MYOF (myoferlin), and HS3ST6 (heparan sulfate-glucosamine 3-O-sulfotransferase 6). The knowledge provided by the new era of genomic studies was pivotal in the discovery of mutations in new genes responsible for this complex pathogenesis. Genomics advances promise a better understanding of unknown mechanisms leading to HAE, the establishment of new molecular targets for novel therapeutic agents, and personalized treatment.

Simple Regulation of the Self-Assembling Ability by Multimerization of Elastin-Derived Peptide (FPGVG) n Using Nitrilotriacetic Acid as a Building Block.

Elastin comprises hydrophobic repetitive sequences, such as Val-Pro-Gly-Val-Gly, which are thought to be important for the temperature-dependent reversible self-association (coacervation). Elastin and elastin-like peptides (ELPs), owing to their characteristics, are expected to be applied as base materials for the development of new molecular tools, such as drug-delivery system carrier and metal-scavenging agents. Recently, several studies have been reported on the dendritic or branching ELP analogues. Although the topological difference of the branched ELPs compared to their linear counterparts may lead to useful properties in biomaterials, the available information regarding the effect of branching on molecular architecture and thermoresponsive behavior of ELPs is scarce. To obtain further insight into the thermoresponsive behavior of branched ELPs, novel ELPs, such as nitrilotriacetic acid (NTA)–(FPGVG)n conjugates, that is, (NTA)-Fn analogues possessing 1–3 (FPGVG)n (n = 3, 5) molecule(s), were synthesized and investigated for their coacervation ability. Turbidity measurement of the synthesized peptide analogues revealed that (NTA)-Fn analogues showed strong coacervation ability with various strengths. The transition temperature of NTA-Fn analogues exponentially decreased with increasing number of residues. In the circular dichroism measurements, trimerization did not alter the secondary structure of each peptide chain of the NTA-Fn analogue. In addition, it was also revealed that the NTA-Fn analogue possesses one peptide chain that could be utilized as metal-scavenging agents. The study findings indicated that multimerization of short ELPs via NTA is a useful and powerful strategy to obtain thermoresponsive molecules.

Experience in the Use of Optogenetic Approaches to Studies of Brain Functions and Prosthetization of Degenerative Retinas

Optogenetics – a method allowing light to be used to control neuron activity via expression of light-activated proteins within cells – is a powerful tool in neurophysiological research. Optogenetics has made significant progress in studies of brain functions in the last decade. Progress in optogenetics depends decisively on the development of new molecular tools, i.e., light-activated proteins. The most widely used molecule for excitation of cells in optogenetics is the natural light-activated cation channelrhodopsin 2 (ChR2). Studies in 2015 identified the natural light-activated chloride channel GtACR2, which in optogenetic experiments can efficiently suppress neuron activity. We identified the unique properties of this channel and showed that not only do GtACR2-expressing neurons respond to the light signal with strong inhibition, but also that action potentials can be generated, evidently in the axon terminals of the neuron due to changes in the chloride reversion potential is this cell compartment. Our studies used optogenetic methods to investigate the cellular mechanisms of learning and memory. Using channelrhodopsin 2 expression in networks of presynaptic neurons, we employed light stimulation to study the properties of synaptic connections and their plasticity in whole populations of neurons in a single experiment. One potential direction in the clinical use of optogenetics is its use for prosthetization of degenerative retinas. One version of this approach consists of creating an ON/OFF receptor field of retinal ganglion cells by targeted expression of the excitatory light-activated protein in the central part of a ganglion cell and an inhibitory protein in the periphery. Within the framework of this approach, we created a two-layer construct carrying genes for excitatory and inhibitory opsins whose expression was able to lead to restoration of ON/OFF interactions typical of ganglion neurons.

Tumour metabolism and its unique properties in prostate adenocarcinoma.

Anabolic metabolism mediated by aberrant growth factor signalling fuels tumour growth and progression. The first biochemical descriptions of the altered metabolic nature of solid tumours were reported by Otto Warburg almost a century ago. Now, the study of tumour metabolism is being redefined by the development of new molecular tools, tumour modelling systems and precise instrumentation together with important advances in genetics, cell biology and spectroscopy. In contrast to Warburg's original hypothesis, accumulating evidence demonstrates a critical role for mitochondrial metabolism and substantial variation in the way in which different tumours metabolize nutrients to generate biomass. Furthermore, computational and experimental approaches suggest a dominant influence of the tissue-of-origin in shaping the metabolic reprogramming that enables tumour growth. For example, the unique metabolic properties of prostate adenocarcinoma are likely to stem from the distinct metabolism of the prostatic epithelium from which it emerges. Normal prostatic epithelium employs comparatively glycolytic metabolism to sustain physiological citrate secretion, whereas prostate adenocarcinoma consumes citrate to power oxidative phosphorylation and fuel lipogenesis, enabling tumour progression through metabolic reprogramming. Current data suggest that the distinct metabolic aberrations in prostate adenocarcinoma are driven by the androgen receptor, providing opportunities for functional metabolic imaging and novel therapeutic interventions that will be complementary to existing diagnostic and treatment options.

An integrative view of senescence in nature

Abstract Senescence—the decline in age‐specific contribution to fitness with increasing age—has been widely investigated in evolutionary ecology. A tremendous amount of detailed empirical analyses have now revealed the widespread occurrence of demographic senescence (i.e. both actuarial and reproductive senescence) and have started to identify factors (e.g. environmental conditions) that modulate its timing and intensity, both within and across species. In this special feature, we have built on this flourishing work to highlight several axes of research that would benefit from more integrative and multidisciplinary approaches. Several contributions compiled in this special feature emphasize that our understanding of senescence remains taxonomically limited, mostly focused on birds and mammals, and is therefore not representative of the biological diversity displayed across the tree of life. In line with this observation, the influence of some peculiar lifestyles (e.g. involving sociality or modularity) on the evolution of senescence is yet to be deciphered. Understanding of the diversity of senescence patterns across and within species and among traits will necessitate the establishment of new metrics as a golden standard to fully account for age‐specific changes recorded in individuals’ performance. This is illustrated with the specific case of actuarial senescence. This special feature also highlights that the diversity of biological samples collected from wild plants and animals, along with accurate demographic data, is expanding. The fast development of new molecular tools now offers a unique opportunity to launch research programmes at the interface of physiology, health and ageing in non‐model organisms. We argue that while these different research axes constitute key avenues of investigations for the coming years, they are only the tip of the iceberg. To appreciate the full complexity of the senescence process in nature, from its evolutionary causes to its demographic consequences, we also need a better understanding of the role played by both environmental conditions and gene–environment interactions, of constraints, and of senescence, an improved assessment of the influence of individual heterogeneity, and the consideration of transgenerational effects when quantifying the fitness consequences of senescence.

Опыт применения оптогенетических подходов к исследованию функций мозга и протезированию дегенеративной сетчатки

Optogenetics, which allows control the activity of neurons by means of light after expression of light-activated proteins in them, is a powerful tool in neuroscience that determined significant progress in the study of brain functions in the last decade. The progress of optogenetics crucially depends on the development of new molecular tools - light-activated proteins. In 2015, the natural light-activated chlorine channel GtACR2 was identified. This channel is capable of suppressing neuron activity with high efficiency in optogenetic experiments. We discovered the unique properties of this channel, showing that in GtACR2-expressing neurons, the light stimulus, in addition to strong inhibition, can also induce the generation of action potentials, which occurs, apparently, in axon terminals of the neuron due to the altered chlorine reversal potential in these cell compartments . In our work, we use optogenetic methods to study the cellular mechanisms of learning and memory. Using the expression of channelrhodopsin2 in the network of presynaptic neurons, we used light stimulation to study the properties of synaptic connections and their plasticity in a whole population of neurons in a single experiment. One of the promising areas of clinical application of optogenetics is its use for prosthetic of degenerative retina. A variant of this approach is to recreate the ON / OFF receptive field of ganglion neurons by targeted expression of the excitatory light-activated protein in the central part of the ganglion cell, and the inhibitory opsins in the peripheral. In the framework of this approach, we created a bicistronic construct that carries the genes of the excitatory and inhibitory opsins, the expression of which was able to lead to the restoration of ON / OFF interactions typical for ganglion cells.

Plasmodium genomics: an approach for learning about and ending human malaria.

Malaria causes high levels of morbidity and mortality in human beings worldwide. According to the World Health Organization (WHO), about half a million people die of this disease each year. Malaria is caused by six species of parasites belonging to the Plasmodium genus: P. falciparum, P. knowlesi, P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri. Currently, malaria is being kept under control with varying levels of elimination success in different countries. The development of new molecular tools as well as the use of next-generation sequencing (NGS) technologies and novel bioinformatic approaches has improved our knowledge of malarial epidemiology, diagnosis, treatment, vaccine development, and surveillance strategies. In this work, the genetics and genomics of human malarias have been analyzed. Since the first P. falciparum genome was sequenced in 2002, various population-level genetic and genomic surveys, together with transcriptomic and proteomic studies, have shown the importance of molecular approaches in supporting malaria elimination.

Development of New Molecular Tools for Antibody Detection and Purification

Primary antibody detection is crucial for biomarker detection in research and diagnosis. Molecular tools for antibody detection and purification currently available consist of secondary antibodies as well as bacterial proteins such as Proteins A and G. Disadvantages of the current methods include the fact that secondary antibody production is expensive and may require the use of animals. In the case of Proteins A and G, they either do not recognize all IgG isotypes or require acidic elution conditions for antibody purification, which may lead to antibody denaturation. We have produced chimeric proteins, using an IgG binding protein recently discovered (TRIM21) linked to detection proteins such as streptavidin, and alkaline phosphatase. TRIM21 is a cytosolic protein that binds all IgG isotypes from many species. Bacterial expression, purification and refolding of TRIM21‐streptavidin as well as TRIM21‐alkaline phosphatase were successful. TRIM21‐streptavidin chimeric protein was incubated with biotinylated HRP or alkaline phosphatase for immunoassays. ELISA and western blotting experiments demonstrated that the chimeric proteins efficiently detect monoclonal and polyclonal antibodies from a wide range of species, including human, mouse, rat, dog, horse and bovine. Binding experiments are currently underway to determine the binding constants for TRIM21 chimeric proteins and IgG from different species. Future experiments will be performed using imobilized TRIM21 for antibody purification.Support or Funding InformationThis work was supported by the “ State University of Santa Catarina” (UDESC), “Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina” (FAPESC) and CNPQ.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chloride conducting light activated channel GtACR2 can produce both cessation of firing and generation of action potentials in cortical neurons in response to light

Optogenetics is a powerful technique in neuroscience that provided a great success in studying the brain functions during the last decade. Progress of optogenetics crucially depends on development of new molecular tools. Light-activated cation-conducting channelrhodopsin2 was widely used for excitation of cells since the emergence of optogenetics. In 2015 a family of natural light activated chloride channels GtACR was identified which appeared to be a very promising tool for using in optogenetics experiments as a cell silencer. Here we examined properties of GtACR2 channel expressed in the rat layer 2/3 pyramidal neurons by means of in utero electroporation. We have found that despite strong inhibition the light stimulation of GtACR2-positive neurons can surprisingly lead to generation of action potentials, presumably initiated in the axonal terminals. Thus, when using the GtACR2 in optogenetics experiments, its ability to induce action potentials should be taken into account. Our results also open an interesting possibility of using the GtACR2 both as cell silencer and cell activator in the same experiment varying the pattern of light stimulation.

Switching on a transient endogenous ROS production in mammalian cells and tissues

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide.We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.

Versatile roles of brassinosteroid in plants in the context of its homoeostasis, signaling and crosstalks.

Brassinosteroids (BRs) are a class of steroidal plant hormones that play diverse roles in plant growth and developmental processes. Recently, the easy availability of biological resources, and development of new molecular tools and approaches have provided the required impetus for deeper understanding of the processes involved in BRs biosynthesis, transport, signaling and degradation pathways. From recent studies it is also evident that BRs interact with other phytohormones such as auxin, cytokinin, ethylene, gibberellin, jasmonic acid, abscisic acid, salicylic acid and polyamine in regulating wide range of physiological and developmental processes in plants. The inputs from these studies are now being linked to the versatile roles of BRs. The present review highlights the conceptual development with regard to BR homeostasis, signaling and its crosstalk with other phytohormones. This information will assist in developing predictive models to modulate various useful traits in plants and address current challenges in agriculture.

Modern molecular approaches for analyzing microbial diversity from mushroom compost ecosystem.

Biosphere is a store house of various microorganisms that may be employed to isolate and exploit microbes for environmental, pharmaceutical, agricultural and industrial applications. There is restricted data regarding the structure and dynamics of microbial communities in several ecosystems because only a little fraction of microbial diversity is accessible by culture methods. Owing to limitations of traditional enrichment methods and pure culture techniques, microbiological studies have offered a narrow portal for investigating microbial flora. The bacterial community represented by the morphological and nutritional criteria failed to provide a natural taxonomic order according to the evolutionary relationship. Genetic diversity among the isolates recovered from mushroom compost has not been widely studied. To understand genetic diversity and community composition of the mushroom compost microflora, different approaches are now followed by taxonomists, to characterize and identify isolates up to species level. Molecular microbial ecology is an emerging discipline of biology under molecular approach which can provide complex community profiles along with useful phylogenetic information. The genomic era has resulted in the development of new molecular tools and techniques for study of culturable microbial diversity including the DNA base ratio (mole% G + C), DNA–DNA hybridization, DNA microarray and reverse sample genome probing. In addition, non-culturable diversity of mushroom compost ecosystem can be characterized by employing various molecular tools which would be discussed in the present review.

The ITM2B (BRI2) gene is a target of BCL6 repression: Implications for lymphomas and neurodegenerative diseases

The human BCL6 gene encodes a transcriptional repressor that is crucial for germinal center B cell development and T follicular helper cell differentiation. It is involved in the pathogenesis of certain human lymphomas. In an effort to identify targets of BCL6 repression, we used a previously described cell system in which BCL6 repressive effects are inhibited, followed by subtractive hybridization, and identified the integral membrane 2B gene (ITM2B, formerly BRI2) as a potential target. Here we show that BCL6 can bind to its preferential consensus binding site within the first intron of ITM2B and represses its transcription. Knockdown of endogenous BCL6 in a human B cell lymphoma line increases ITM2B expression. Further, there is an inverse relationship between the expression levels of BCL6 and ITM2B proteins in 16 human B- and T-cell lymphomas studied by immunohistochemistry. Both the BCL6 and ITM2B proteins are expressed ubiquitously. Similar to some other targets of BCL6, a short form of the ITM2B protein generated by alternative splicing induces apoptosis in hematopoietic cell lines. Molecular alterations in the ITM2B gene are associated with two neurodegenerative diseases, Familial British and Familial Danish dementia. ITM2B dysfunction also may be relevant for the development of Alzheimer's disease. Our data confirm ITM2B as a target of BCL6 repression in lymphoma. A further understanding of the genes that function as regulators of the ITM2B protein may provide insights for the development of new molecular tools not only for targeted lymphoma therapy but also for the treatment of these dementias.

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: Carbohydrate–protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate–protein interactions. We investigated whether carbohydrate–lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp–GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.