Structural basis for T cell immunoglobulin and mucin protein 3 and Toxascaris leonina galectin complex.

T-cell immunoglobulin and mucin protein 3 (Tim-3), also known as Hepatitis A virus cellular receptor 2, has been discovered to have a negative regulatory effect on murine T-cell responses. Galectin-9 exhibits various biological effects, including cell aggregation, eosinophil chemoattraction, activation, and apoptosis, observed in murine thymocytes, T-cells, and human melanoma cells. Such approach demonstrated that Galectin-9 acts as a binding partner on Tim-3 and mediates the T-cell inhibitory effects. Tl-gal is a homologous protein to galectin-9, isolated from the adult stage of the canine gastrointestinal nematode parasite Toxascaris leonina. However, molecular mechanism between Tim-3 and galectin-9 is still remain unknown. Here, we describe the cryo-electron microscopy and X-ray structures and interactions of the Tim-3 and Tl-gal complex as well as their biochemical and biophysical characterization. In the structure, Ser46 residue of Tl-gal NCRD was bound to Asp25 residue of hTim-3. Compared to our previous study, the binding site of the complex is the same as the sugar binding site (the Ser46 residue) of Tl-gal. In addition, analysis of the complex structure revealed that the four Tl-gal molecules were in an open form packing and one mTim-3 peptide was bound to one Tl-gal molecule. These observations suggest that how Tl-gal binds hTim3 is essential to understanding the molecular mechanism for the Tim-3-galectin 9 interaction that regulates immune responses. This could potentially serve as a therapeutic target for inflammatory diseases.

Anisotropic Bimetallic Core-Satellite-Poly(aniline) Nanohybrids for Detection of Autoantibodies.

Bimetallic core-satellite nanoparticles are widely exploited in surface-enhanced Raman scattering (SERS)-based applications due to their enhanced optical properties compared to single-component metallic nanoparticles (MNPs). In addition, anisotropic hybrid nanostructures containing both MNPs and polymeric compartments constitute a new class of functional nanomaterials for photonic applications because they show different functionalities and physicochemical characteristics at two distinct compartments. Herein, synthesis of two kinds of anisotropic bimetallic core-satellite-poly(aniline) nanohybrids (ABCPNs) using small or polymeric ligand-coated gold nanospheres or gold nanorods as seeds is reported. The ABCPNs exhibit enhanced optical properties due to a local electromagnetic field generated in the narrow interparticle gap between core and satellite nanoparticles. Furthermore, a SERS-based quantitative analysis of autoantibodies against cyclic citrullinated peptide using the ABCPNs as SERS nanoprobes for a diagnosis of early rheumatoid arthritis is demonstrated, suggesting that these multifunctional nanostructures will be potential for advanced SERS-based biosensors.

Oppositely Charged, Stimuli-Responsive Anisotropic Nanoparticles for Colloidal Self-Assembly.

Anisotropic nanoparticles (ANPs) composed of distinct compartments are of interest as advanced materials because they offer unique physicochemical properties controlled by polymer composition, distribution of functional groups, and stimuli responsiveness of each compartment. Furthermore, colloidal self-assembly of ANPs via noncovalent interactions between compartments can create superstructures with additional functionality. In this study, ANPs with two compartments composed of oppositely charged and thermally responsive ternary copolymers were prepared using electrohydrodynamic cojetting. One compartment was composed of poly( N-isopropylacrylamide- co-stearyl acrylate- co-allylamine), which is positively charged in aqueous solution at pH 7, and the other compartment was composed of poly( N-isopropylacrylamide- co-stearyl acrylate- co-acrylic acid), which is negatively charged. The ANPs were stabilized in aqueous solution by physical cross-linking because of hydrophobic interactions between the 18-carbon alkyl chains of their stearyl acrylate moieties and self-assembled into supracolloidal nanostructures via electrostatic interactions. Colloidal self-assembly and thermal responsiveness were controlled by compartment charge density and solution ionic strength. The supracolloidal nanostructures exhibited both the intrinsic temperature-responsive properties of the ANPs and collective properties from self-assembly. These multifunctional, stimuli-responsive nanostructures will be useful in a variety of applications, including switchable displays, drug delivery carriers, and ion-sensitive gels.

PEGylated nanographene-mediated metallic nanoparticle clusters for surface enhanced Raman scattering-based biosensing.

Surface-enhanced Raman scattering (SERS) is an optical spectroscopy technique that can detect a variety of analytes with high sensitivity and selectivity without any labels. Controlled clustering of metallic nanoparticles to prepare a new class of SERS nanotags is crucial for the ultra-sensitive detection of specific biological and chemical moieties because increased plasmonic hotspot junctions produce a greatly enhanced SERS signal. We report herein that controlled clustering of Au nanoparticles (AuNPs) was mediated by PEGylated nano-sized graphene (PNG) and that the PNG-induced AuNP clusters (PNG-AuNPCs) were highly sensitive SERS nanotags with colloidal stability for SERS-based biosensing. The AuNPs labeled with 4-mercaptopyridine as a Raman reporter were surface-modified with 1-aminomethylpyrene for the introduction of hydrophobic moieties, and were non-covalently complexed with PNG via π-π stacking and van der Waals forces. It resulted in the formation of PNG-AuNPCs that increased SERS intensity with an enhancement factor of 1.34 × 1011. The PNG induced a high degree of AuNP clustering by enhancing the non-covalent interactions between them, resulting in increased hotspot junctions at highly localized plasmonic centers. Furthermore, to show that the PNG-AuNPCs would serve as stable, reproducible, and highly sensitive SERS nanotags for biosensing, we formed sandwich-type immunocomplexes composed of the PNG-AuNPCs, immunoglobulin G (IgG) as the antigen, and magnetic beads. We found a linear relationship between SERS intensity and IgG concentration, with a limit of detection lower than 31.0 fM for IgG detection. Thus, the PNG-AuNPCs could be useful as SERS nanotags for highly sensitive SERS-based biosensing applications.

The relationship between anemia and pulse pressure and hypertension: The Korea National Health and Nutrition Examination Survey 2010-2012.

The present study was conducted to assess the relationship between anemia and pulse pressure (PP) and hypertension (HTN). Data from 16,060 adults (aged ≥20 years) in the fifth Korean National Health and Nutrition Examination Survey (2010-2012) were analyzed. Several key findings were identified. First, after adjusting for related variables, the odds ratio (OR) of anemia (hemoglobin <13 and <12 g/dL, in men and women, respectively), using the normal PP group (PP ≤61 mmHg) as a reference, was significant for the high PP cohort (PP >61 mmHg; OR, 1.517; 95% confidence interval [CI], 1.270-1.812). Second, after adjusting for related variables (except body mass index [BMI] and waist measurement [WM]), the OR of anemia, with a normal blood pressure group as a reference, was significant for the HTN group (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg or use of HTN medications; OR, 0.835; 95% CI, 0.709-0.983). However, when further adjusted for BMI and WM, anemia was not associated with HTN (OR, 0.884; 95% CI, 0.750-1.042). In conclusion, anemia was positively associated with high PP, but was not associated with HTN.

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin.

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg61/Arg196 and Glu80/Glu215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response.

The relationship between chronic kidney function and homeostasis model assessment of insulin resistance and beta cell function in Korean adults with or without type 2 diabetes mellitus.

The present study was conducted to assess the relationship between chronic kidney disease (CKD) and the homeostasis model assessment of insulin resistance (HOMA-IR) and beta cell function (HOMA-B) in Korean adults with or without type 2 diabetes mellitus (T2DM). This study included 5,188 adults aged 20 or older using the 2015 Korea National Health and Nutrition Examination Survey (KNHANES) data, which represents national data in Korea. A covariance test adjusted for covariates was performed for HOMA-IR and HOMA-B in relation to CKD. The present study has several key findings. First, in T2DM, HOMA-IR (p = 0.035) was higher in the CKD group than in the non-CKD group after adjusting for the related variables but HOMA-B (p = 0.141) was not significant. Second, in non-T2DM, HOMA-IR (p = 0.163) and HOMA-B (p = 0.658) were not associated with CKD after adjusting for the related variables (except age). However, when further adjusted for age, HOMA-IR (p = 0.020) and HOMA-B (p = 0.006) were higher in the CKD group than in the non-CKD group. In conclusion, insulin resistance was positively associated CKD with in Korean adults with or without T2DM. Beta cell function was positively associated CKD with in Korean adults without T2DM but not in Korean adults with T2DM.

Arabidopsis AtERF71/HRE2 functions as transcriptional activator via cis-acting GCC box or DRE/CRT element and is involved in root development through regulation of root cell expansion.

KEY MESSAGE: AtERF71/HRE2 binds to GCC box or DRE/CRT as transcription activator and plays an important role in root development via root cell expansion regulation. AtERF71/HRE2 transcription factor, a member of the AP2/ERF family, plays a key role in the stress response. GCC box and DRE/CRT, both essential cis-acting elements, have been shown to be recognized by AP2/ERF family transcription factors. However, it remains unclear whether or not AtERF71/HRE2 directly interacts with GCC box and/or DRE/CRT. Here, we showed that AtERF71/HRE2 binds to GCC box and DRE/CRT by electrophoretic mobility shift assay (EMSA). Binding of AtERF71/HRE2 to GCC box and DRE/CRT was also detected by fluorescence measurement and surface plasmon resonance spectroscopy (BIAcore) experiments. Folding properties of AtERF71/HRE2 proteins were characterized by CD spectroscopy, and AtERF71/HRE2 showed thermal stability as evidenced by two endothermic peaks (T d) at 53 and 65 °C. In addition, AtERF71/HRE2 showed transcriptional activation activity via GCC box and DRE/CRT in Arabidopsis protoplasts. Interestingly, AtERF71/HRE2 OXs showed increased primary root length due to elevated root cell expansion. Our data indicate that AtERF71/HRE2 binds to both GCC box and DRE/CRT, transactivates expression of genes downstream via GCC box or DRE/CRT, and plays an important role in root development through regulation of root cell expansion.

A genome-wide association study of seed protein and oil content in soybean.

BACKGROUND: Association analysis is an alternative to conventional family-based methods to detect the location of gene(s) or quantitative trait loci (QTL) and provides relatively high resolution in terms of defining the genome position of a gene or QTL. Seed protein and oil concentration are quantitative traits which are determined by the interaction among many genes with small to moderate genetic effects and their interaction with the environment. In this study, a genome-wide association study (GWAS) was performed to identify quantitative trait loci (QTL) controlling seed protein and oil concentration in 298 soybean germplasm accessions exhibiting a wide range of seed protein and oil content. RESULTS: A total of 55,159 single nucleotide polymorphisms (SNPs) were genotyped using various methods including Illumina Infinium and GoldenGate assays and 31,954 markers with minor allele frequency >0.10 were used to estimate linkage disequilibrium (LD) in heterochromatic and euchromatic regions. In euchromatic regions, the mean LD (r2) rapidly declined to 0.2 within 360 Kbp, whereas the mean LD declined to 0.2 at 9,600 Kbp in heterochromatic regions. The GWAS results identified 40 SNPs in 17 different genomic regions significantly associated with seed protein. Of these, the five SNPs with the highest associations and seven adjacent SNPs were located in the 27.6-30.0 Mbp region of Gm20. A major seed protein QTL has been previously mapped to the same location and potential candidate genes have recently been identified in this region. The GWAS results also detected 25 SNPs in 13 different genomic regions associated with seed oil. Of these markers, seven SNPs had a significant association with both protein and oil. CONCLUSIONS: This research indicated that GWAS not only identified most of the previously reported QTL controlling seed protein and oil, but also resulted in narrower genomic regions than the regions reported as containing these QTL. The narrower GWAS-defined genome regions will allow more precise marker-assisted allele selection and will expedite positional cloning of the causal gene(s).

Structural characterization and interaction of periostin and bone morphogenetic protein for regulation of collagen cross-linking.

Periostin appears to be a unique extracellular protein secreted by fibroblasts that is upregulated following injury to the heart or changes in the environment. Periostin has the ability to associate with other critical extracellular matrix (ECM) regulators such as TGF-ß, tenascin, and fibronectin, and is a critical regulator of fibrosis that functions by altering the deposition and attachment of collagen. Periostin is known to be highly expressed in carcinoma cells, but not in normal breast tissues. The protein has a structural similarity to insect fasciclin-1 (Fas 1) and can be induced by transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP)-2. To investigate the molecular interaction of periostin and bone morphogenetic protein, we modeled these three-dimensional structures and their binding sites. We demonstrated direct interaction between periostin and BMP1/2 in vitro using several biochemical and biophysical assays. We found that the structures of the first, second, and fourth Fas1 domains in periostin are similar to that of the fourth Fas 1 domain of TGFBIp. However, the structure of the third Fas 1 domain in periostin is different from those of the first, second, and fourth Fas1 domains, while it is similar to the NMR structure of Fasciclin-like protein from Rhodobacter sphaeroides. These results will useful in further functional analysis of the interaction of periostin and bone morphogenetic protein.

A study on the formative usability testing for modular powered wheelchair.

The increasing prevalence of mobility impairments underscores the urgent need for accessible and affordable mobility aids. To overcome the mobility limitations of people with disabilities, there is an increasing need for the development of lightweight and portable powered wheelchairs that can be easily loaded. This study aimed to perform an early health technology assessment and a formative usability evaluation on a modular (detachable) powered wheelchair. It aimed to gauge device satisfaction among users, pinpoint areas for improvement, and detect any unforeseen errors to inform future development. Engaging 16 participants, including powered wheelchair users, healthcare professionals, and caregivers, the research evaluated the wheelchair's functionality in various scenarios, emphasizing safety, effectiveness, and convenience. Statistical analyses of task performance and satisfaction surveys highlighted that, while powered wheelchair users successfully completed tasks focusing on driving and power control, healthcare professionals and caregivers encountered difficulties with the wheelchair's assembly and disassembly. Despite general positivity, the surveys indicated mixed satisfaction levels regarding safety, validity, and convenience, with specific issues related to frame durability, seat comfort, and control mechanisms. These findings suggest that refining the wheelchair's design and addressing user concerns could significantly enhance satisfaction and mobility services. Future efforts will include a thorough review of an advanced prototype and further satisfaction assessments.

We believe that our study makes a significant contribution to the literature by addressing a critical gap in the understanding of user-centric design and usability testing for powered wheelchairs.By emphasizing the importance of early assessments and incorporating user feedback into the development process, our research offers practical insights for creating more accessible and user-friendly mobility solutions.This contribution is particularly relevant in the context of advancing assistive technology and improving the quality of life for individuals with disabilities.

Effects of a brief Qigong-based stress reduction program (BQSRP) in a distressed Korean population: a randomized trial.

BACKGROUND: Distressed individuals in Korea may benefit from the practice of mind-body exercises such as Qigong. However, the effectiveness of such techniques needs to be investigated. METHODS: Fifty participants who were eligible to this study were randomized into a group receiving a 4-week intervention of a brief Qigong-based stress reduction program (BQSRP) or a wait-list control group. Before and after the intervention period, saliva samples were collected and questionnaires were completed on perceived stress, anxiety, "Hwa-Byung" (anger syndrome), and quality of life. Salivary cortisol has emerged in mind-body therapy research as an easy-to-collect, relatively inexpensive, biologic marker of stress. Salivary corisol were collected to evaluate physiological effect of BQSRP. Between-group comparisons of change from baseline to study completion were analyzed by analysis of covariance for the Perceived Stress Scale and independent two sample t-tests for other measures. RESULTS: Compared with the control group, the BQSRP intervention group displayed significantly larger decreases in Perceived Stress Scale scores (p = 0.0006), State Anxiety scores (p = 0.0028), Trait Anxiety scores (p < 0.0001), personality subscale scores of the Hwa-Byung Scale (p = 0.0321), symptoms scores of the Hwa-Byung Scale (p = 0.0196), and a significantly larger increase in World Health Organization Quality of Life Abbreviated version scores (ps < .05). Salivary cortisol levels were not changed. CONCLUSIONS: The BQSRP appears to be effective in reducing stress perception, anxiety, anger, and improving quality of life (KCT0000056).

Stimuli-responsive plasmonic core-satellite hybrid nanostructures with tunable nanogaps.

Incorporating stimuli-responsive block copolymers to hierarchical metallic nanoparticles (MNPs) is of particular interest due to their tunable plasmonic properties responding to environmental stimuli. We herein report thermo-responsive plasmonic core-satellite hybrid nanostructures with tunable nanogaps as surface-enhanced Raman scattering (SERS) nanotags. Two different diblock copolymers with opposite charges, poly(acrylic acid-b-N-isopropylacrylamide) (p(AAc-b-NIPAM)) and poly(N,N-dimethylaminoethyl methacrylate-b-N-isopropylacrylamide) (p(DMAEMA-b-NIPAM)), were synthesized. The negatively charged p(AAc-b-NIPAM)s were bound to gold nanospheres (GNSs), while the positively charged p(DMAEMA-b-NIPAM)s were conjugated to gold nanorods (GNRs) via gold-sulfur bonds. When p(AAc-b-NIPAM)-GNSs and p(DMAEMA-b-NIPAM)-GNRs were electrostatically complexed, plasmonic hybrid nanostructures consisting of both GNS satellites and a GNR core were formed. Dynamic tuning of electromagnetic coupling of their nanogaps was achieved via a temperature-triggered conformational change of p(NIPAM) blocks. Furthermore, a sandwich-type immunoassay for the detection of immunoglobulin G was performed to demonstrate these core-satellites as potential SERS nanotags. Our results showed that these plasmonic core-satellites with stimuli-responsiveness are promising for SERS-based biosensing applications.

Functional improvement by body-powered 3D-printed prosthesis in patients with finger amputation: Two case reports.

RATIONALE: The most common upper limb amputations are finger amputations, resulting in functional limitations that lead to problems with activities of daily living or job loss. For many years, prosthetic options for finger amputations have been limited to passive prostheses. In many countries including South Korea, body-powered finger prostheses have rarely been prescribed due to high cost, lack of experience of physicians and prosthetists, low interest and no coverage by insurance benefits. We report 2 cases of work-related finger amputations in patients who received body-powered 3D-printed finger prostheses. PATIENT CONCERNS AND DIAGNOSIS: Patient 1 was a 25-year-old woman with second and third finger amputations at the proximal interphalangeal level. Patient 2 was a 26-year-old man who sustained a second finger amputation at proximal interphalangeal level. INTERVENTIONS: We created body-powered 3D-printed finger prostheses that mimicked distal interphalangeal joint motion through patient-driven metacarpophalangeal joint motion using a string connected to a wrist strap and a linkage system. The source code "Knick Finger" was downloaded from e-NABLE. OUTCOMES: After 1 month of prosthesis training, both patients were satisfied with the prostheses and showed improved performance in patient-derived goals of cooking (patient 1) and typing on a computer (patient 2). LESSONS: Over the past decade, significant advances have been made in 3D-printed prosthetics owing to their light weight, low cost, on-site fabrication, and easy customization. Although there are still several limitations in the general application of 3D-printed finger prostheses, our study suggests that for patients with finger amputations, body-powered 3D-printed finger prostheses have high potential as an additional prosthetic option to the existing passive cosmetic prostheses.

Directional self-assembly of anisotropic bimetal-poly(aniline) nanostructures for rheumatoid arthritis diagnosis in multiplexing.

Anisotropic organic-inorganic hybrid nanoparticles possessing different functionalities and physicochemical properties from each compartment have attracted significant interest for the development of advanced functional materials. Moreover, their self-assembled structures exhibit unique optical properties for photonics-based biosensing. We report herein the fabrication of anisotropic bimetal-polymer nanoparticles (ABPNs) via combination of oxidative polymerization and additional growth of metallic nanoparticles on Au seeds as well as their directional clustering mediated via noncovalent interactions. Polymerization of anilines for poly (aniline) shell was conducted by reducing silver nitrate onto the Au seed in the presence of a surfactant, giving rise to spatially distinct bimetallic Au core and Ag shell compartment and the poly (aniline) counter-one that comprise the ABPNs. Furthermore, ABPNs were directionally clustered in a controlled manner via hydrophobic interaction, when the bimetallic compartment was selectively modified. These nanoclusters showed highly enhanced optical properties owing to the increased electromagnetic fields while the poly (aniline) being used to offer antibody binding capacity. Taking advantages of those properties of the ABPN nanoclusters, surface-enhanced Raman scattering (SERS) intensity-based quantification of two different biomarkers: autoantibodies against cyclic citrullinated peptide and rheumatoid factor was demonstrated using ABPN nanoclusters as SERS nanoprobes. Conclusively, this work has great potential to satisfy a need for multiplexing in diagnosis of early stage of rheumatoid arthritis.

Janus bimetallic nanorod clusters-poly(aniline) nanocomposites with temperature-responsiveness for Raman scattering-based biosensing.

Herein, Janus bimetallic nanorod clusters-poly(aniline) nanocomposites (JRCPCs) with gold nanorod clusters (GNRCs) in side-by-side (SBS) or end-to-end (ETE) configuration are synthesized, and applied to surface-enhanced Raman scattering (SERS)-based biosensing of carcinoembryonic antigen (CEA). Taking advantage of their geometrical and chemical anisotropy, GNRCs in both SBS and ETE configurations are prepared by addition of negatively charged citrate anions and poly(acrylic acid)-block-poly(N-isopropylacrylamide) (PAAc-b-PNIPAM), respectively, to electrostatically interact with cationic cetyltrimethylammonium bromide surfactant on the side of the gold nanorods (GNRs). Subsequently, the JRCPCs are prepared by unidirectional growth of polyaniline and additional growth of Ag onto these GNRCs. JRCPCs with GNRCs in either the SBS or the ETE configuration show strong enhancement of electromagnetic field at both GNR aggregates and GNRC core-Ag shell gaps of bimetallic nanorod cluster components. In particular, because temperature-responsive PAAc-b-PNIPAM of JRCPCs is embedded at GNR junctions, interparticle gaps generated in GNRCs in ETE configuration are controlled via temperature-triggered hydration-dehydration of the PAAc-b-PNIPAM chains such that optical properties are largely changed. With distinct surface functionalities from JRCPCs, SERS-based quantitative analysis of CEA is achieved using JRCPCs as SERS nanoprobes. This work presents the great potential of advanced Janus nanocomposites for SERS-based biosensing applications.

High-throughput SNP discovery and assay development in common bean.

BACKGROUND: Next generation sequencing has significantly increased the speed at which single nucleotide polymorphisms (SNPs) can be discovered and subsequently used as molecular markers for research. Unfortunately, for species such as common bean (Phaseolus vulgaris L.) which do not have a whole genome sequence available, the use of next generation sequencing for SNP discovery is much more difficult and costly. To this end we developed a method which couples sequences obtained from the Roche 454-FLX system (454) with the Illumina Genome Analyzer (GA) for high-throughput SNP discovery. RESULTS: Using a multi-tier reduced representation library we discovered a total of 3,487 SNPs of which 2,795 contained sufficient flanking genomic sequence for SNP assay development. Using Sanger sequencing to determine the validation rate of these SNPs, we found that 86% are likely to be true SNPs. Furthermore, we designed a GoldenGate assay which contained 1,050 of the 3,487 predicted SNPs. A total of 827 of the 1,050 SNPs produced a working GoldenGate assay (79%). CONCLUSIONS: Through combining two next generation sequencing techniques we have developed a method that allows high-throughput SNP discovery in any diploid organism without the need of a whole genome sequence or the creation of normalized cDNA libraries. The need to only perform one 454 run and one GA sequencer run allows high-throughput SNP discovery with sufficient sequence for assay development to be performed in organisms, such as common bean, which have limited genomic resources.

Tunable Decoupling of Dual Drug Release of Oppositely Charged, Stimuli-Responsive Anisotropic Nanoparticles.

Multicompartmentalized nanostructures are of interest because they can provide unique physicochemical properties and multifunctionalities in each compartment. Furthermore, stimuli-responsive anisotropic nanostructures (ANPs) with distinct opposite charges would be useful for drug delivery systems because different drug release kinetics could be achieved from each compartment in response to both charge and stimuli. In this study, stimuli-responsive ANPs were formed via electrohydrodynamic cojetting of poly(N-isopropylacrylamide)-based copolymers with opposite charges. The positively charged compartment consisted of poly(N-isopropylacylamide-co-stearyl acrylate-co-allylamine) (poly(NIPAM-co-SA-co-AAm)) (i.e., PNSAAm) and poly(N-isopropylacylamide-co-stearyl acrylate-co-acrylic acid) (poly(NIPAM-co-SA-co-AAc)) (i.e., PNSAAc). The two distinct compartments of ANPs were physically cross-linked through hydrophobic interactions within the copolymers. Oppositely charged, small-molecule model drugs (fluorescein sodium salt and rhodamine 6G) were separately encapsulated within each compartment and released based on changes in noncovalent interactions and temperature. Furthermore, two different biomacromolecule drugs with opposite charges, bovine serum albumin and lysozyme (which were complexed with polysaccharides by hydrophobic ion pairing), were loaded within the ANPs. Electrostatic interactions between the encapsulated drugs and each ANP compartment controlled the rate of drug release from the ANPs. In addition, these ANPs showed a thermally induced actuation, leading to drug release at different rates due to the collapse of poly(NIPAM)-based copolymers under aqueous conditions. This work may be useful for decoupled drug release kinetics.

Purification of caudal-related homeodomain transcription factor and its binding characterization.

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in the Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatography. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2 binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

Thermally-Induced Actuations of Stimuli-Responsive, Bicompartmental Nanofibers for Decoupled Drug Release.

Stimuli-responsive anisotropic microstructures and nanostructures with different physicochemical properties in discrete compartments, have been developed as advanced materials for drug delivery systems, tissue engineering, regenerative medicine, and biosensing applications. Moreover, their stimuli-triggered actuations would be of great interest for the introduction of the functionality of drug delivery reservoirs and tissue engineering scaffolds. In this study, stimuli-responsive bicompartmental nanofibers (BCNFs), with completely different polymer compositions, were prepared through electrohydrodynamic co-jetting with side-by-side needle geometry. One compartment with thermo-responsiveness was composed of methacrylated poly(N-isopropylacrylamide-co-allylamine hydrochloride) (poly(NIPAM-co-AAh)), while the counter compartment was made of poly(ethylene glycol) dimethacrylates (PEGDMA). Both methacrylated poly(NIPAM-co-AAh) and PEGDMA in distinct compartments were chemically crosslinked in a solid phase by UV irradiation and swelled under aqueous conditions, because of the hydrophilicity of both poly(NIPAM-co-AAh) and PEGDMA. As the temperature increased, BCNFs maintained a clear interface between compartments and showed thermally-induced actuation at the nanoscale due to the collapsed poly(NIPAM-co-AAh) compartment under the PEGDMA compartment of identical dimensions. Different model drugs, bovine serum albumin, and dexamethasone phosphate were alternately loaded into each compartment and released at different rates depending on the temperature and molecular weight of the drugs. These BCNFs, as intelligent nanomaterials, have great potential as tissue engineering scaffolds and multi-modal drug delivery reservoirs with stimuli-triggered actuation and decoupled drug release.