CHARACTERIZATION OF GENIC MICROSATELLITE MARKERS (EST-SSRS) IN THE ENDANGERED OAK SPECIES QUERCUS GEORGIANA M.A.CURTIS

Quercus is important ecologically and economically because it provides food and habitat for wildlife, wood and paper products for humans. Oaks are endangered due to various factors like shifting climates, habitat loss, drought, pathogens and genetic swamping. Quercus georgiana (M.A. Curtis) is an endangered and restricted oak species which is remaining only in the southeastern part of the US. Efforts are required to conserve this endangered species from extinction. Conservation of this species can be done through these methods: ex-situ conservation (arboretum and botanical garden) and in-situ conservation strategies which protect the species in its natural habitat. For this conservation strategy, it is important to ensure that sample collections capture as much of the gene pool as possible so that the biodiversity is maintained. A variety of molecular markers are available for Quercus. These markers which are highly polymorphic, co-dominant and multiallelic loci will be useful in the study of population genetics of Q. georgiana. Genetic variations in both, among and within the populations, have to be considered if sampling and conservation strategies are developed for this rare and endangered species. These analyses are important in the future for sample collection trips so that the conservation goal is obtained

Taxonomic similarity does not predict necessary sample size for ex situ conservation: A comparison among five genera

Effectively conserving biodiversity with limited resources requires scientifically informed and efficient strategies. Guidance is particularly needed on how many living plants are necessary to conserve a threshold level of genetic diversity in ex situ collections. We investigated this question for 11 taxa across five genera. In this first study analysing and optimizing ex situ genetic diversity across multiple genera, we found that the percentage of extant genetic diversity currently conserved varies among taxa from 40% to 95%. Most taxa are well below genetic conservation targets. Resampling datasets showed that ideal collection sizes vary widely even within a genus: one taxon typically required at least 50% more individuals than another (though Quercus was an exception). Still, across taxa, the minimum collection size to achieve genetic conservation goals is within one order of magnitude. Current collections are also suboptimal: they could remain the same size yet capture twice the genetic diversity with an improved sampling design. We term this deficiency the ‘genetic conservation gap’. Lastly, we show that minimum collection sizes are influenced by collection priorities regarding the genetic diversity target. In summary, current collections are insufficient (not reaching targets) and suboptimal (not efficiently designed), and we show how improvements can be made

Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands †

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((β-D-galactopyranosyl)-(1→4)-β-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3′ of each of the two sugars, resulting in a 3′-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl β-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3′ position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series

Comparing genetic diversity in three threatened oaks

Genetic diversity is a critical resource for species’ survival during times of environmental change. Conserving and sustainably managing genetic diversity requires understanding the distribution and amount of genetic diversity (in situ and ex situ) across multiple species. This paper focuses on three emblematic and IUCN Red List threatened oaks (Quercus, Fagaceae), a highly speciose tree genus that contains numerous rare species and poses challenges for ex situ conservation. We compare the genetic diversity of three rare oak species-Quercus georgiana, Q. oglethorpensis, and Q. boyntonii-to common oaks; investigate the correlation of range size, population size, and the abiotic environment with genetic diversity within and among populations in situ; and test how well genetic diversity preserved in botanic gardens correlates with geographic range size. Our main findings are: (1) these three rare species generally have lower genetic diversity than more abundant oaks; (2) in some cases, small population size and geographic range correlate with genetic diversity and differentiation; and (3) genetic diversity currently protected in botanic gardens is inadequately predicted by geographic range size and number of samples preserved, suggesting non-random sampling of populations for conservation collections. Our results highlight that most populations of these three rare oaks have managed to avoid severe genetic erosion, but their small size will likely necessitate genetic management going forward

DETECTION AND MUTATIONAL ANALYSIS OF A HUMAN PROTEIN ASSOCIATED WITH CANCER AND CARDIOVASCULAR DISEASES

Biomarkers are disease specific proteins that are overexpressed under pathological conditions. These proteins save lives because they play key roles in early diagnosis of diseases. Many of these biomarkers are glycosylated proteins (glycoproteins) and their receptors (lectins). Current biomarker detection techniques depend on antibodies and/or tagged reagents. Thus, there is a constant need to develop fast and simple detection techniques that do not require antibodies and labeled reagents. We recently developed such a simple method for (glyco)protein purification/identification and found that this method could also be used for biomarker detection. Our method was able to detect biomarker proteins in blood plasma and in other complex mixtures. One of those detected proteins was human Galectin-3 (Gal-3), a protein that serves as a biomarker for multiple diseases such as cancer, cardiovascular diseases, and diabetes. Gal-3 is a multifunctional protein (lectin), which is overexpressed in a wide variety of cellular and pathological events. However, the molecular basis of its multifunctional property and the reasons behind its occurrence in multiple diseases are poorly understood. Our research has revealed that the binding site of Gal-3 shows considerable plasticity to accommodate a completely unexpected group of ligands, namely proteoglycans and their constituents glycosaminoglycans (GAGs). Our findings explain, at least in part, the multifunctional ability of Gal-3 and provide a reason why this protein is involved in so many pathological events. To understand the plasticity of the binding site of Gal-3, several mutants were generated and characterized. I will present the biomarker detection technique and will discuss the binding site plasticity of Gal-3 as revealed by mutational analysis

Detection and purification of lectins and glycoproteins by a non-column chromatographic technique

Proteins including glycoproteins and lectins play important roles in many biological processes. Therefore, they are vigorously studied in academic, clinical and industrial research. Such research activities often need these proteins in their purest forms. Thus, protein purification constitutes an important step in many scientific projects. Conventional protein purification techniques include affinity chromatography, ion-exchange chromatography and size-exclusion chromatography, and electrophoresis. These techniques have their own limitations. Conventional approaches are generally tedious, multi-step, expensive and time consuming. They generally require elaborate infrastructure and larger starting crude materials. In addition, these techniques sometimes encounter non-specific binding. In order to overcome some of the limitations associated with these conventional methods, our lab developed a protein detection/purification method named “Capture and Release” (CaRe). In this method, a target capturing agent (TCA) captures a specific target (lectin or glycoprotein) in the crude solution and form insoluble complex. The complex is spun down while the other unwanted proteins are washed off. Captured target is released from the TCA by the addition of competitive monovalent ligand, separated by membrane filtration and visualized by gel electrophoresis. We were successful in purifying recombinant human Galectin-3 by CaRe. This method was able to purify glycoproteins as well. CaRe was validated by purifying known lectins and glycoproteins. Compare to conventional techniques, our method is relatively fast, simple, precise and less expensive and it can detect/purify lectins and glycoproteins even from a small volume (~1 ml) of starting material. Thus CaRe can serve as a valuable tool to discover unknown proteins and glycoproteins

Revealing the Identity of Human Galectin-3 as a Glycosaminoglycan-Binding Protein

Human galectin-3 (Gal-3) is a β-galactoside-binding lectin. This multitasking protein preferentially interacts with N-acetyllactosamine moieties on glycoconjugates. Specific hydroxyl groups (4-OH, 6-OH of galactose, and 3-OH of glucose/N-acetylglucosamine) of lactose/LacNAc are essential for their binding to Gal-3. Through hemagglutination inhibition, microcalorimetry, and spectroscopy, we have shown that despite being a lectin, Gal-3 possesses the characteristics of a glycosaminoglycan (GAG)-binding protein (GAGBP). Gal-3 interacts with sulfated GAGs [heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC)] and chondroitin sulfate proteoglycans (CSPGs). Heparin, CSA, and CSC showed micromolar affinity for Gal-3, while the affinity of CSPGs for Gal-3 was much higher (nanomolar). Interestingly, CSA, CSC, and a bovine CSPG, not heparin and CSB, were multivalent ligands for Gal-3, and they formed reversible noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs to Gal-3 was completely inhibited when Gal-3 was preincubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was also reversed by β-lactose. These findings strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans. The GAG- and CSPG-binding properties of Gal-3 make the lectin a potential competitor/collaborator of other GAGBPs such as growth factors, cytokines, morphogens, and extracellular matrix proteins

Mechanism of Mucin Recognition by Lectins: A Thermodynamic Study

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, heat of binding (enthalpy) and indirectly the entropy, and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limitedly in applications to lectin–glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high-affinity lectin–mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions

The resilience of cancer-specific protein galectin-3: Implications in biological functions

Structural degradation is detrimental for protein activity and functions. We recently found that the tumor-associated protein galectin-3 (Gal-3) is apparently an exception to this rule. Gal-3 plays important roles in cancer but its functions are not fully understood. We have found that the affinity purified Gal-3, when stored with lactose at 4°C for more than six weeks, undergoes complete degradation even in the absence of collagenases. Full-length Gal-3 after full degradation produced a single protein band of 17 kDa. This molecular weight is similar to that of the CRD (carbohydrate recognition domain) portion of Gal-3. Conventionally, the CRD is separated from intact Gal-3 by enzymatic digestion using collagenases. However, in the present case, Gal-3 apparently self-degraded to produce the CRD without the catalytic action of collagenases. Our data indicate that degradation of Gal-3 significantly alters the protein but the CRD remains intact and functionally active. Like the full-length Gal-3, the CRDs interacted with thyroglobulin, chondroitin sulfate A and C. However, the CRDs could not cross-link these multivalent ligands as revealed by our spectroscopic analysis. Our current observations suggest that Gal-3 retains it ligand recognition properties even when it is substantially degraded in a challenging cellular environment. Thus the degraded Gal-3 can still carry out some of the functions of intact Gal-3. Although the CRDs retain the binding property of its intact precursor (Gal-3), they lose the ability to cross-link cellular receptors. As a result, the CRDs generated through degradation may influence cell signaling by competing with intact Gal-3 for the same receptors in cellular environment

Rapid Detection and Purification of Galectin-3 by the Capture and Release (CaRe) Method

Specific interactions between lectins and glycoproteins determine the outcomes of numerous biological processes. To elucidate the roles of lectins and glycoproteins in those processes, it is essential to detect these proteins in biological samples and purify them to homogeneity. Conventional protein detection and purification techniques are multi-step, time-intensive, and expensive. They often require rigorous trial and error experimentations and fairly larger volumes of crude extracts. To minimize some of these challenges, we recently formulated a new method named Capture and Release (CaRe). This method is rapid, facile, precise, and inexpensive, and it works even when the sample volume is smaller. We developed this method to detect and purify recombinant human Galectin-3 and subsequently validated this method by purifying several other lectins. Besides lectins, CaRe is capable of detecting/purifying glycoproteins. In this method, targets (lectins and glycoproteins) are captured by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. CaRe can potentially be used as a tool to discover new lectins and glycoconjugates and elucidate their functions