Repetitive Motifs Research Articles

Laccaria bicolor MiSSP8 is a small‐secreted protein decisive for the establishment of the ectomycorrhizal symbiosis

The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long-term and intimate relationship between the soil-borne fungi and the roots of trees. Mycorrhiza-induced Small-Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8-RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C-terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small-secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.

Characterization of Helicobacter pylori genotypes from Iranian patients with gastric clinical diseases: Predominance of vacA s1a and cagA EPIYA-ABC genotypes

ObjectivesHelicobacter pylori is a causative agent of peptic ulcers and associated with gastric cancer. The major virulence factors of this organism are vacA and cagA genes. The vacA comes in a mosaic structure and the cagA become activated through a repetitive motif including glutamine (E), proline (P), isoleucine (I), tyrosine (Y) and alanine (A), shortly be called EPIYA. The aim of this study was to characterize the genotypes and the antibiotics resistance pattern of H. pylori isolates in Iranian patients with chronic gastritis, non-ulcer dyspepsia (NUD) and peptic ulcer disease (PUD). Methods191 biopsy samples were obtained from patients referred to Afzalipour hospital in Kerman, during November 2017 to October 2018 and cultured to isolate H. pylori. Isolates were confirmed as H. pylori using PCR with the 16s rRNA specific primers. Antimicrobial susceptibility testing was performed using the modified disk diffusion method and genotyping was done by PCR using the specific primers for vacA, cagA and glmM. ResultsFrom 191 samples, 63 H. pylori isolates were obtained. These, isolated from patients with chronic active gastritis (73%, n = 46), PUD (9.5%, n = 6) and NUD (17.5%, n = 11). Resistance to metronidazole was 55.5%, while the resistance to clarithromycin was 37%. There was no resistance to furazolidone. The most prevalent virulence gene among our isolates was vacA with 89%, while the prevalence of cagA and glmM genes were 59% and 81% respectively. CagA-EPIYA-ABC was the most prevalent motif in chronic gastritis (83%), PUD (75%), and NUD (61%). CagA-EPIYA-ABCC was more prevalent in NUD cases (23%). ConclusionIn this study there was no statistical significant relation between the gastric diseases and the genotypes of H. pylori. This is the first report of H. pylori isolates genotyping in south east of Iran which indicating a diversity among these isolates.

Abstract LB-215: CMSI: A Bayesian model for estimating clonal micro-satellites instability from NGS data

Abstract Genomic micro-satellites are the genomic regions consisting of short and repetitive DNA motifs. Micro-satellite region usually exposes intrinsic polymorphism in terms of the numbers of repetitive motifs, which is often described as a probability distribution of the numbers of repeats. In cancer genomics, for any micro-satellite region, it is considered as a micro-satellite instability (MSI) event, if the probability distribution sampled from tumor tissue is significantly different from the distribution sampled from the corresponding normal tissue. Since recent studies have emphasized the importance of micro-satellite instability events in cancer diagnosis and treatments, a series of computational approaches have been developed to detect MSI events from the sequencing data. However, the existing methods suffer an accuracy loss when clonal micro-satellites exist, which are recently observed in some TCGA/ICGC samples. For a clonal micro-satellite, different sub-clones may carry different distributions, while the observed “distribution” from the sequencing data is actually a convolution of the sub-clonal ones. In this case, a sub-clonal distribution may present a true MSI event, but the convolutional one dilutes the data signal and misleads the detection algorithm to report a micro-satellite stability (MSS) event, which introduces type-I error. In addition, a comprehensive understanding of the micro-satellite distribution of each sub-clone is also quite informative for downstream analyses. Thus, to overcome the potential weakness of existing approaches and further improve the computational model, here, we proposed a probabilistic framework, named CMSI, to identify the MSI events under tumor heterogeneous structure. Similar to other approaches, CMSI works on the next generation sequencing data. The proposed framework follows the assumption that the probability density function of the numbers of repeats of a micro-satellite region usually follows a normal distribution. Then, when clonal micro-satellite exists, the convolution distribution observed from the sequencing data should obey a Gaussian mixture distribution. CMSI establishes a variational Bayesian mixture model for the Gaussian distribution calculated from the sequencing reads. This mixture model clusters the reads by the numbers of repeats they bring or infer, and further provide a probabilistic assignment to each read by maximizing the global posterior distribution. By solving this computational model by an EM algorithm, CMSI estimates the number of sub-clones, the proportion of each sub-clone and the parameters of each distribution. Finally, each sub-clonal distribution is examined by statistical test by weighting the clonal proportion, and CMSI outputs the MSI events of sub-clones. To verify the performance of the proposed framework, we conducted several experiments on both simulation datasets and real datasets, where CMSI effectively identified an acceptable percentage of the preset MSI events. Note: This abstract was not presented at the meeting. Citation Format: Yixuan Wang, Xuanping Zhang, Yi Huang, Tao Liu, Xiao Xiao, Jiayin Wang. CMSI: A Bayesian model for estimating clonal micro-satellites instability from NGS data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-215.

Toward Spider Glue: Long Read Scaffolding for Extreme Length and Repetitious Silk Family Genes AgSp1 and AgSp2 with Insights into Functional Adaptation.

An individual orb weaving spider can spin up to seven different types of silk, each with unique functions and material properties. The capture spiral silk of classic two-dimensional aerial orb webs is coated with an amorphous glue that functions to retain prey that get caught in a web. This unique modified silk is partially comprised of spidroins (spider fibroins) encoded by two members of the silk gene family. The glue differs from solid silk fibers as it is a viscoelastic, amorphic, wet material that is responsive to environmental conditions. Most spidroins are encoded by extremely large, highly repetitive genes that cannot be sequenced using short read technology alone, as the repetitive regions are longer than read length. We sequenced for the first time the complete genomic Aggregate Spidroin 1 (AgSp1) and Aggregate Spidroin 2 (AgSp2) glue genes of orb weaving spider Argiope trifasciata using error-prone long reads to scaffold for high accuracy short reads. The massive coding sequences are 42,270 bp (AgSp1) and 20,526 bp (AgSp2) in length, the largest silk genes currently described. The majority of the predicted amino acid sequence of AgSp1 consists of two similar but distinct motifs that are repeated ∼40 times each, while AgSp2 contains ∼48 repetitions of an AgSp1-similar motif, interspersed by regions high in glutamine. Comparisons of AgSp repetitive motifs from orb web and cobweb spiders show regions of strict conservation followed by striking diversification. Glues from these two spider families have evolved contrasting material properties in adhesion (stickiness), extensibility (stretchiness), and elasticity (the ability of the material to resume its native shape), which we link to mechanisms established for related silk genes in the same family. Full-length aggregate spidroin sequences from diverse species with differing material characteristics will provide insights for designing tunable bio-inspired adhesives for a variety of unique purposes.

Modulation of in vitro antimalarial responses by polymorphisms in Plasmodium falciparum ABC transporters (pfmdr1 and pfmdr5)

The emergence of resistance to artemisinin-based combination therapies (ACT) was described in Southeast Asia. In this context, the identification of molecular markers of ACT resistance partner drugs is urgently needed for monitoring the emergence and spread of resistance. Polymorphisms in transporter genes, especially of the ATP-binding cassette (ABC) superfamily, have been involved in anti-malarial drug resistance. In this study, the association between the mutations in the P. falciparum multidrug resistance 1 gene (pfmdr1, N86Y, Y184 F, S1034C, N1042D and D1246Y) or repetitive amino acid motifs in pfmdr5 and the ex vivo susceptibility to anti-malarial drugs was evaluated. Susceptibility to chloroquine, quinine, monodesethylamodiaquine, lumefantrine, piperaquine, pyronaridine, mefloquine and dihydroartemisinin was assessed in 67 Senegalese isolates. The shorter DNNN motif ranged from to 2 to 11 copy repeats, and the longer DHHNDHNNDNNN motif ranged from 0 to 2 in pfmdr5. The present study showed the association between repetitive amino acid motifs (DNNN-DHHNDDHNNDNNN) in pfmdr5 and in vitro susceptibility to 4-aminoquinoline-based antimalarial drugs. The parasites with 8 and more copy repeats of DNNN in pfmdr5 were significantly more susceptible to piperaquine. There was a significant association between parasites whose DHHNDHNNDNNN motif was absent and replaced by DHHNDNNN, DHHNDHNNDHNNDNNN or DHHNDHNNDHNNDHNNDNNN and increased susceptibility to chloroquine, monodesethylamodiaquine and pyronaridine. A significant association between both the wild-type allele N86 in pfmdr1 and the N86-184 F haplotype and reduced susceptibility to lumefantrine was confirmed. Further studies with a large number of samples are required to validate the association between these pfmdr5 alleles and the modulation of 4-aminoquinoline-based antimalarial drug susceptibility.

The bagworm genome reveals a unique fibroin gene that provides high tensile strength

Arthropod silk is known as a versatile tool, and its variability makes it an attractive biomaterial. Eumeta variegata is a bagworm moth (Lepidoptera, Psychidae) that uses silk throughout all life stages. Notably, the bagworm-specific uses of silk include larval development in a bag coated with silk and plant materials and the use of silk attachments to hang pupae. An understanding at the molecular level of bagworm silk, which enables such unique purposes, is an opportunity to expand the possibilities for artificial biomaterial design. However, very little is known about the bagworm fibroin gene and the mechanical properties of bagworm silk. Here, we report the bagworm genome, including a silk fibroin gene. The genome is approximately 700 Mbp in size, and the newly found fibroin gene has a unique repetitive motif. Furthermore, a mechanical property test demonstrates a phylogenetic relationship between the unique motif and tensile strength of bagworm silk.

Identification of Bombyx mori sericin 4 protein as a new biological adhesive.

Sericins are large proteins with molecular weights >70 kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280 kDa and 260 kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks.

A Preview of a Construction of a Crystal Lattice Based on Intermolecular Interactions

A general procedure of crystal packing reconstruction using a certain number of intermolecular interactions is introduced and demonstrated on the crystal structure of l-histidine·HCl·H2O. Geometric restrictions based on intermolecular interactions are used for formation of a molecular pair as a basic repetitive motif of the crystal packing. The geometric restrictions were applied gradually within a supervised procedure, narrowing the scope of possible arrangement of two adjacent molecules. Subsequently, a pair of histidine molecules was used for construction of a molecular chain. The chain formed contained translation information on histidine molecules in one dimension, which coincided with one of the cell parameters. Furthermore, the periodicity in the second and third dimensions can be accomplished by chain assembly into sheets (2D), and sheets can be arranged into the final 3D structure. For this purpose, the rest of the available intermolecular interactions could be used to control the mutual assembly of molecular chains and sheets. Complete molecular packing would enable derivation of standard crystallographic parameters that can be used for verification of the structural model obtained. However, the procedure described for construction of the whole 3D structure from molecular chains was not attempted, and is only briefly outlined here. The procedure described can be employed especially when standard crystallographic parameters are not available and traditional methods based on X-ray diffraction fail.

Turtles of the genera Geoemyda and Pangshura (Testudines: Geoemydidae) lack differentiated sex chromosomes: the end of a 40-year error cascade for Pangshura.

For a long time, turtles of the family Geoemydidae have been considered exceptional because representatives of this family were thought to possess a wide variety of sex determination systems. In the present study, we cytogenetically studied Geoemyda spengleri and G. japonica and re-examined the putative presence of sex chromosomes in Pangshura smithii. Karyotypes were examined by assessing the occurrence of constitutive heterochromatin, by comparative genome hybridization and in situ hybridization with repetitive motifs, which are often accumulated on differentiated sex chromosomes in reptiles. We found similar karyotypes, similar distributions of constitutive heterochromatin and a similar topology of tested repetitive motifs for all three species. We did not detect differentiated sex chromosomes in any of the species. For P. smithii, a ZZ/ZW sex determination system, with differentiated sex chromosomes, was described more than 40 years ago, but this finding has never been re-examined and was cited in all reviews of sex determination in reptiles. Here, we show that the identification of sex chromosomes in the original report was based on the erroneous pairing of chromosomes in the karyogram, causing over decades an error cascade regarding the inferences derived from the putative existence of female heterogamety in geoemydid turtles.

Adaptor functions of the Ca2+-binding protein ALG-2 in protein transport from the endoplasmic reticulum.

Apoptosis-linked gene 2 (ALG-2) is a Ca2+-binding protein with five repetitive EF-hand motifs, named penta-EF-hand (PEF) domain. It interacts with various target proteins and functions as a Ca2+-dependent adaptor in diverse cellular activities. In the cytoplasm, ALG-2 is predominantly localized to a specialized region of the endoplasmic reticulum (ER), called the ER exit site (ERES), through its interaction with Sec31A. Sec31A is an outer coat protein of coat protein complex II (COPII) and is recruited from the cytosol to the ERES to form COPII-coated transport vesicles. I will overview current knowledge of the physiological significance of ALG-2 in regulating ERES localization of Sec31A and the following adaptor functions of ALG-2, including bridging Sec31A and annexin A11 to stabilize Sec31A at the ERES, polymerizing the Trk-fused gene (TFG) product, and linking MAPK1-interacting and spindle stabilizing (MISS)-like (MISSL) and microtubule-associated protein 1B (MAP1B) to promote anterograde transport from the ER.

Microsatellite marker set for genetic diversity assessment of primitive Chitala chitala (Hamilton, 1822) derived through SMRT sequencing technology

In present study, single molecule-real time sequencing technology was used to obtain a validated set of microsatellite markers for application in population genetics of the primitive fish, Chitala chitala. Assembly of circular consensus sequencing reads resulted into 1164 sequences which contained 2005 repetitive motifs. A total of 100 sequences were used for primer designing and amplification yielded a set of 28 validated polymorphic markers. These loci were used to genotype n = 72 samples from three distant riverine populations of India, namely Son, Satluj and Brahmaputra, for determining intraspecific genetic variation. The microsatellite loci exhibited high level of polymorphism with PIC values ranging from 0.281 to 0.901. The genetic parameters revealed that mean heterozygosity ranged from 0.6802 to 0.6826 and the populations were found to be genetically diverse (Fst 0.03-0.06). This indicated the potential application of these microsatellite marker set that can used for stock characterization of C. chitala, in the wild. These newly developed loci were assayed for cross transferability in another notopterid fish, Notopterus notopterus.

Extraordinary Mechanical Properties of Composite Silk Through Hereditable Transgenic Silkworm Expressing Recombinant Major Ampullate Spidroin

Spider dragline silk is a remarkable material that shows excellent mechanical properties, diverse applications, biocompatibility and biodegradability. Transgenic silkworm technology was used to obtain four types of chimeric silkworm/spider (termed composite) silk fibres, including different lengths of recombinant Major ampullate Spidroin1 (re-MaSp1) or recombinant Major ampullate Spidroin2 (re-MaSp2) from the black widow spider, Latrodectus hesperus. The results showed that the overall mechanical properties of composite silk fibres improved as the re-MaSp1 chain length increased, and there were significant linear relationships between the mechanical properties and the re-MaSp1 chain length (p < 0.01). Additionally, a stronger tensile strength was observed for the composite silk fibres that included re-MaSp1, which only contained one type of repetitive motif, (GA)n/An, to provide tensile strength, compared with the silk fibres that includedre-MaSp2, which has the same protein chain length as re-MaSp1 but contains multiple types of repetitive motifs, GPGXX and (GA)n/An. Therefore, the results indicated that the nature of various repetitive motifs in the primary structure played an important role in imparting excellent mechanical properties to the protein-based silk fibres. A silk protein with a single type of repetitive motif and sufficiently long chains was determined to be an additional indispensable factor. Thus, this study forms a foundation for designing and optimizing the structure of re-silk protein using a heterologous expression system.

Development of microsatellite loci in zoonotic tapeworm Dibothriocephalus latus (Linnaeus, 1758), Lühe, 1899 (syn. Diphyllobothrium latum) using microsatellite library screening

The broad fish tapeworm Dibothriocephalus latus is a causative agent of human food-borne disease called diphyllobothriosis. Medical importance, scattered geographical distribution and unknown origin of D. latus in Europe and North America make this species to be an interesting model for population genetics. Microsatellite markers were originally designed by library screening using NGS approach and validated as tools for future studies on population genetics of D. latus. Out of 122 candidates selected after NGS analysis, 110 yielded PCR products of the expected size, and in 78 of them, a declared repetitive motif was confirmed by Sanger sequencing. After the fragment analysis, six loci were proved to be polymorphic and tested for observed (Ho) and expected (He) heterozygosity, and deviations from Hardy-Weinberg equilibrium (HWE). They promise future application in studies on genetic interrelationships, origin and migratory routes of this medically important emerging tapeworm.

A Workflow to Improve Variant Calling Accuracy in Molecular Barcoded Sequencing Reads.

Multiplexed molecular barcoded amplicon sequencing has been previously demonstrated to improve the sensitivity of low-frequency variant detection. Molecular barcoded reads can be used to identify and correct amplification biases introduced during library preparation and sequencing errors. We propose a generic workflow to improve variant calling accuracy that takes advantage of molecular barcoded sequencing reads by applying a base score correction method to duplicate or overlapping read pairs across the targeted panel. The workflow is able to reduce the false-positive rate of variant calls in homopolymer and repetitive regions where the sequencer commonly encounters phasing errors interfering with base calling. The analysis was focused on three specific regions within a custom QIAseq targeted DNA (Qiagen, USA) of 220 genes observed to have a high false-positive rate in a clinical validation study. Uncorrected and corrected datasets were compared at targeted regions to reference calls from NIST and exome data from a reference laboratory (Macrogen, USA). Base correction removed all false positives identified without the correction method and retained the true positive call in the dataset. We have shown that our workflow incorporating base correction of molecular barcoded sequencing data can be applied to germline sequencing to improve variant calling accuracy in genomic regions with certain repetitive sequence motifs.

A survey of recent unusual high-resolution DNA structures provoked by mismatches, repeats and ligand binding.

The structure of the DNA duplex is arguably one of the most important biological structures elucidated in modern history. DNA duplex structure is closely associated with essential biological functions such as DNA replication and RNA transcription. In addition to the classical A-, B- and Z-DNA conformations, DNA duplexes are capable of assuming a variety of alternative conformations depending on the sequence and environmental context. A considerable number of these unusual DNA duplex structures have been identified in the past decade, and some of them have been found to be closely associated with different biological functions and pathological conditions. In this manuscript, we review a selection of unusual DNA duplex structures, particularly those originating from base pair mismatch, repetitive sequence motifs and ligand-induced structures. Although the biological significance of these novel structures has not yet been established in most cases, the illustrated conformational versatility of DNA could have relevance for pharmaceutical or nanotechnology development. A perspective on the future directions of this field is also presented.

Mechanical elasticity of proline-rich and hydroxyproline-rich collagen-like triple-helices studied using steered molecular dynamics.

Identity of the amino acids in Gly-X-Y repetitive motives governs biomechanical features of collagen such as elasticity in the extracellular matrix. Proline and hydroxyproline are the most abundant residues at the X and Y sites of collagen repetitive motives, respectively. However, their effects on the elasticity of collagen have not been identified. Here, we investigated the molecular mechanics of five different proline-rich collagens with Gly-Pro-Y repetitive motives, four hydroxyproline-rich collagens with Gly-X-Hyp repetitive motives as well as a collagen with Gly-Pro-Hyp repetitive motif, focusing on molecular stiffness and elasticity. The proteins were virtually built and stiffness, Young's modulus, cross-sectional radius, intermolecular and protein-solvent hydrogen bonds of the collagens were investigated using steered molecular dynamic simulation. Results showed a higher stiffness and Young's moduli of the proline-rich collagens compared to the hydroxyproline-rich collagens. Young's modulus of the proline-rich collagens was negatively correlated with the cross-sectional radius. There was no significant difference between the proline-rich and the hydroxyproline-rich collagen from the point of view of intermolecular and protein-water hydrogen bonds. However, a decreased and an increased number of protein-water hydrogen bonds in response to stretching was found for the proline-rich and the hydroxyproline-rich collagens respectively. Interestingly, the collagen with Gly-Pro-Hyp repetitive motif showed an intermediate stiffness, Young's moduli and cross-sectional radius. The collagen also had a lower number of intermolecular and protein -water hydrogen bonds when compared to the proline-rich and hydroxproline-rich collagens. These results suggest that the presence of proline in the structure of collagen reduces elasticity and increases stiffness, whereas presence of hydroxyproline increases elasticity of the collagen. We conclude that any codon substitution in the collagen genes causing alteration of proline and/or hydroxyproline residues may result in drastic collagen deficiency.

CagY-Dependent Regulation of Type IV Secretion in Helicobacter pylori Is Associated with Alterations in Integrin Binding.

ABSTRACTStrains of Helicobacter pylori that cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4+ T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human α5β1 integrin, we tested the hypothesis that recombination in the CagY MRR regulates T4SS function by modulating binding to α5β1 integrin. Using a cell-free microfluidic assay, we found that H. pylori binding to α5β1 integrin under shear flow is dependent on the CagY MRR, but independent of the presence of the T4SS pili, which are only formed when H. pylori is in contact with host cells. Similarly, expression of CagY in the absence of other T4SS genes was necessary and sufficient for whole bacterial cell binding to α5β1 integrin. Bacteria with variant cagY alleles that reduced T4SS function showed comparable reduction in binding to α5β1 integrin, although CagY was still expressed on the bacterial surface. We speculate that cagY-dependent modulation of H. pylori T4SS function is mediated by alterations in binding to α5β1 integrin, which in turn regulates the host inflammatory response so as to maximize persistent infection.

Byssus Structure and Protein Composition in the Highly Invasive Fouling Mussel Limnoperna fortunei.

Biofouling mediated by byssus adhesion in invasive bivalves has become a global environmental problem in aquatic ecosystems, resulting in negative ecological and economic consequences. Previous studies suggested that mechanisms responsible for byssus adhesion largely vary among bivalves, but it is poorly understood in freshwater species. Understanding of byssus structure and protein composition is the prerequisite for revealing these mechanisms. Here, we used multiple methods, including scanning electron microscope, liquid chromatography–tandem mass spectrometry, transcriptome sequencing, real-time quantitative PCR, inductively coupled plasma mass spectrometry, to investigate structure, and protein composition of byssus in the highly invasive freshwater mussel Limnoperna fortunei. The results indicated that the structure characteristics of adhesive plaque, proximal and distal threads were conducive to byssus adhesion, contributing to the high biofouling capacity of this species. The 3,4-dihydroxyphenyl-α-alanine (Dopa) is a major post-transnationally modification in L. fortunei byssus. We identified 16 representative foot proteins with typical repetitive motifs and conserved domains by integrating transcriptomic and proteomic approaches. In these proteins, Lfbp-1, Lffp-2, and Lfbp-3 were specially located in foot tissue and highly expressed in the rapid byssus formation period, suggesting the involvement of these foot proteins in byssus production and adhesion. Multiple metal irons, including Ca2+, Mg2+, Zn2+, Al3+, and Fe3+, were abundant in both foot tissue and byssal thread. The heavy metals in these irons may be directly accumulated by L. fortunei from surrounding environments. Nevertheless, some metal ions (e.g., Ca2+) corresponded well with amino acid preferences of L. fortunei foot proteins, suggesting functional roles of these metal ions by interacting with foot proteins in byssus adhesion. Overall, this study provides structural and molecular bases of adhesive mechanisms of byssus in L. fortunei, and findings here are expected to develop strategies against biofouling by freshwater organisms.

DNA‐based molecular tools for monitoring cellular activity

Methods to determine levels of protein‐protein interactions are essential in analysis of cellular signaling activity. High‐resolution analysis of proteins or protein complexes requires either advanced microscopy, or molecular methods that combine distance dependence with signal amplification. By using DNA molecules as tools, we have over the years developed several molecular methods utilizing a variety of enzymes to modify and amplify the DNA molecules used in the methods. One such method is Proximity Ligation Assay (PLA) where pairs of antibodies equipped with DNA oligonucleotides (so‐called proximity probes) are used to target proteins. Proximal binding of such probes template the creation of a circular DNA molecule, which can be amplified using rolling circle amplification (RCA). The single‐stranded RCA product from a single recognition event will contain several hundreds of repetitive motifs that can be visualized by hybridization of fluorophore‐conjugated oligonucleotides.Information on levels of both free and interacting proteins is important to determine if the interactions monitored are a result of a post‐translational protein modification, e.g. phosphorylations, that changes the conformation of the proteins and subsequently results in a different dissociation constant (KD), or if the amount of interactions reflects mere expression levels of the proteins. New more complex measurements are needed in order to model signaling pathway activity and information flow in single cells. We have hence further developed PLA to provided information on levels of both free and interacting proteins.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting Suppressive Oligonucleotide to Lymph Nodes Inhibits Toll-like Receptor-9-Mediated Activation of Adaptive Immunity.

This paper aims to investigate the immunoinhibitory properties of a lymph nodes-targeting suppressive oligonucleotide (ODN) for the potential treatment of autoimmune diseases or chronic inflammation. Synthetic suppressive ODN engineered with an albumin-binding diacyl lipid at the 5'-terminal (lipo-ODN) was synthesized. In vitro and in vivo experiments were designed to compare the immune suppressive properties of lipo-ODN and unmodified ODN. Cellular uptake and distribution, inhibition of Toll-like receptor (TLR) activation, lymph nodes (LN) draining, and the suppression of antigen-specific immune responses in an ovalbumin protein model was investigated. Compared to unmodified ODN, lipid functionalized suppressive ODN demonstrated enhanced cellular uptake and TLR-9 specific immune suppression in TLR reporter cells. Additionally, injection of a low dose of lipid-modified suppressive ODN, but not the unconjugated ODN, accumulated in the draining LNs and exhibited potent inhibition of antigen-specific CD8+ T cell and B cell responses in vivo. Targeting suppressive ODN to antigen presenting cells (APCs) in the local LNs is an effective approach to amplify the immune modulation mediated by ODN containing repetitive TTAGGG motif. This approach might be broadly applicable to target molecular adjuvants to the key immune cells in the LNs draining from disease site, providing a simple strategy to improve the efficacy of many molecular immune modulators.