Short Amino Acid Sequences Research Articles

Absent from DNA and protein: genomic characterization of nullomers and nullpeptides across functional categories and evolution

Nullomers and nullpeptides are short DNA or amino acid sequences that are absent from a genome or proteome, respectively. One potential cause for their absence could be their having a detrimental impact on an organism.ResultsHere, we identify all possible nullomers and nullpeptides in the genomes and proteomes of thirty eukaryotes and demonstrate that a significant proportion of these sequences are under negative selection. We also identify nullomers that are unique to specific functional categories: coding sequences, exons, introns, 5′UTR, 3′UTR, promoters, and show that coding sequence and promoter nullomers are most likely to be selected against. By analyzing all protein sequences across the tree of life, we further identify 36,081 peptides up to six amino acids in length that do not exist in any known organism, termed primes. We next characterize all possible single base pair mutations that can lead to the appearance of a nullomer in the human genome, observing a significantly higher number of mutations than expected by chance for specific nullomer sequences in transposable elements, likely due to their suppression. We also annotate nullomers that appear due to naturally occurring variants and show that a subset of them can be used to distinguish between different human populations. Analysis of nullomers and nullpeptides across vertebrate evolution shows they can also be used as phylogenetic classifiers.ConclusionsWe provide a catalog of nullomers and nullpeptides in distinct functional categories, develop methods to systematically study them, and highlight the use of variability in these sequences in other analyses

The intervening domain is required for DNA-binding and functional identity of plant MADS transcription factors

The MADS transcription factors (TF) are an ancient eukaryotic protein family. In plants, the family is divided into two main lineages. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the MADS domain (M domain) called the Intervening domain (I domain) that was previously defined only in type II lineage MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a conserved fold with the I domain acting to stabilise the M domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters genome-wide DNA-binding specificity and dimerisation specificity. Introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant resulted in strong complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts as an integral part of the DNA-binding domain and significantly contributes to the functional identity of the MADS TF.

Evolution of hes gene family in vertebrates: the hes5 cluster genes have specifically increased in frogs

Backgroundhes genes are chordate homologs of Drosophila genes, hairy and enhancer of split, which encode a basic helix-loop-helix (bHLH) transcriptional repressor with a WRPW motif. Various developmental functions of hes genes, including early embryogenesis and neurogenesis, have been elucidated in vertebrates. However, their orthologous relationships remain unclear partly because of less conservation of relatively short amino acid sequences, the fact that the genome was not analyzed as it is today, and species-specific genome duplication. This results in complicated gene names in vertebrates, which are not consistent in orthologs. We previously revealed that Xenopus frogs have two clusters of hes5, named “the hes5.1 cluster” and “the hes5.3 cluster”, but the origin and the conservation have not yet been revealed.ResultsHere, we elucidated the orthologous and paralogous relationships of all hes genes of human, mouse, chicken, gecko, zebrafish, medaka, coelacanth, spotted gar, elephant shark and three species of frogs, Xenopus tropicalis (X. tropicalis), X. laevis, Nanorana parkeri, by phylogenetic and synteny analyses. Any duplicated hes5 were not found in mammals, whereas hes5 clusters in teleost were conserved although not as many genes as the three frog species. In addition, hes5 cluster-like structure was found in the elephant shark genome, but not found in cyclostomata.ConclusionThese data suggest that the hes5 cluster existed in the gnathostome ancestor but became a single gene in mammals. The number of hes5 cluster genes were specifically large in frogs.

Dissection of phospholipases A2 reveals multifaceted peptides targeting cancer cells, Leishmania and bacteria

Cationic peptides bio-inspired by natural toxins have been recognized as an efficient strategy for the treatment of different health problems. Due to the specific interaction with substrates from biological membranes, snake venom phospholipases (PLA2s) represent valuable scaffolds for the research and development of short peptides targeting parasites, bacteria, and cancer cells. Considering this, we evaluated the in vitro therapeutic potential of three biomimetic peptides (pCergo, pBmTxJ and pBmje) based on three different amino acid sequences from Asp49 PLA2s. First, short amino acid sequences (12–17 in length) derived from these membranolytic toxins were selected using a combination of bioinformatics tools, including AntiCP, AMPA, PepDraw, ToxinPred, and HemoPI. The peptide, from each polypeptide sequence, with the greatest average antimicrobial index, no toxicity, and no hemolysis predicted was synthesized, purified, and characterized. According to in vitro assays performed, pBmje showed moderate cytotoxicity specifically against MCF–7 (breast cancer cells) with an EC50 of 464.85 µM, whereas pBmTxJ showed an antimicrobial effect against Staphylococcus aureus (ATCC 25923) with an MIC of 37.5 µM, and pCergo against E. coli (ATCC 25922) with an MIC of 75 µM. In addition, pCergo showed antileishmanial activity with an EC50 of 93.69 µM and 110.40 µM against promastigotes of Leishmania braziliensis and L. amazonensis, respectively. Altogether, these results confirmed the versatility of PLA2-derived synthetic peptides, highlighting the relevance of the use of these membrane-interacting toxins as specific archetypes for drug design focused on public health problems.

The Peptide Functionalized Inorganic Nanoparticles for Cancer-Related Bioanalytical and Biomedical Applications.

In order to improve their bioapplications, inorganic nanoparticles (NPs) are usually functionalized with specific biomolecules. Peptides with short amino acid sequences have attracted great attention in the NP functionalization since they are easy to be synthesized on a large scale by the automatic synthesizer and can integrate various functionalities including specific biorecognition and therapeutic function into one sequence. Conjugation of peptides with NPs can generate novel theranostic/drug delivery nanosystems with active tumor targeting ability and efficient nanosensing platforms for sensitive detection of various analytes, such as heavy metallic ions and biomarkers. Massive studies demonstrate that applications of the peptide–NP bioconjugates can help to achieve the precise diagnosis and therapy of diseases. In particular, the peptide–NP bioconjugates show tremendous potential for development of effective anti-tumor nanomedicines. This review provides an overview of the effects of properties of peptide functionalized NPs on precise diagnostics and therapy of cancers through summarizing the recent publications on the applications of peptide–NP bioconjugates for biomarkers (antigens and enzymes) and carcinogens (e.g., heavy metallic ions) detection, drug delivery, and imaging-guided therapy. The current challenges and future prospects of the subject are also discussed.

Structural and Mechanismic Studies of Lactophoricin Analog, Novel Antibacterial Peptide.

Naturally derived antibacterial peptides exhibit excellent pharmacological action without the risk of resistance, suggesting a potential role as biologicals. Lactophoricin-I (LPcin-I), found in the proteose peptone component-3 (PP3; lactophorin) of bovine milk, is known to exhibit antibiotic activity against Gram-positive and Gram-negative bacteria. Accordingly, we derived a new antibacterial peptide and investigated its structure–function relationship. This study was initiated by designing antibacterial peptide analogs with better antibacterial activity, less cytotoxicity, and shorter amino acid sequences based on LPcin-I. The structural properties of antibacterial peptide analogs were investigated via spectroscopic analysis, and the antibacterial activity was confirmed by measurement of the minimal inhibitory concentration (MIC). The structure and mechanism of the antibacterial peptide analog in the cell membrane were also studied via solution-state nuclear magnetic resonance (NMR) and solid-state NMR spectroscopy. Through 15N one-dimensional and two-dimensional NMR experiments and 31P NMR experiments, we suggest the 3D morphology and antibacterial mechanism in the phospholipid bilayer of the LPcin analog. This study is expected to establish a system for the development of novel antibacterial peptides and to establish a theoretical basis for research into antibiotic substitutes.

Structural stability of antimicrobial peptides rich in tryptophan, proline and arginine: a computational study

The host defense peptides or antimicrobial peptides (AMPs) often contain short sequence of amino acids, either positive or negatively charged and express broad-spectrum antibacterial, antiviral and antifungal activity. Many researchers had reported that tryptophan, arginine and proline rich AMPs have a promising source of next-generation antibiotics. Nowadays, AMPs are used as a possible therapeutic source for future antibiotics. In the present study, the amino acid sequences of 2924 AMPs belonging to various sources rich in Tryptophan, Proline and Arginine was chosen for investigation. The AMPs were further categorized according to their source, structure and antimicrobial activities. The AMPs with tryptophan, arginine, proline residues in abundance with maximum sequence length of 20 amino acids alone was obtained. Homology modeling was performed with PEP-FOLD and the modeled structures were evaluated using RAMPAGE to identify the structural information. Further, the stability of peptide in aqueous condition was probed using molecular dynamics simulations. Communicated by Ramaswamy H. Sarma

Antibiofilm Activity of Natural Products: Promising Strategies for Combating Microbial Biofilms

Abstract Microbial biofilm is defined as an association of bacterial and fungal cells, fixed to surfaces, biotic or abiotic, included in a complex extracellular matrix of polymeric substances (EPS). It is estimated that approximately 65% of microbial infections are caused by biofilm-forming microorganisms, representing a serious public health problem. In addition, the integrity of machinery and equipment in industries can be compromised by the presence of biofilms, causing significant economic losses. Therefore, there is an urgent need to discover new drugs that are effective against biofilms, which are non-toxic. Natural products have been extensively studied due to their enormous antimicrobial efficacy, like garlic, chitosan derivatives, various peptide spices, products derived from plants and essential oils. Thus, the purpose of this review is to describe the most promising anti-biofilm compounds derived from natural products for bacteria and fungi. One way of treating infections is to use medicinal plants. They have several compounds that have antimicrobial properties and raise your interest in medicine for the treatment of infections. Essential oils are secondary metabolites produced by plants; they act by damaging the extracellular matrix and eliminate adherence factors, contributing to the failure in the construction of the biofilm. Other strategy is the Cationic antimicrobial peptides (AMPs). They are short linear amino acid sequences, which display antimicrobial activity against a wide range of bacterial species. It is a promising novel antimicrobials since they have shown bactericidal effects against multiresistant bacteria. Bacteria and fundi are also capable of producing substances such as peptides and other compounds, which can act as antimicrobials, inhibiting the growth of other microorganisms. This review describes the most promising anti-biofilm compounds, derived from natural products that act against bacteria and fungi. However, the treatment with these compounds must bestudied in detail, since biofilms increase microbial resistance and have strategies to decrease the penetration of antimicrobial substances through their polysaccharide matrix.

Nanovaccine based on self-assembling nonstructural protein 1 boosts antibody responses to Zika virus

Self-assembling proteins may be generated after the addition of short specific amino acid sequences at both the N- and C-terminal ends. To date, this approach has not been evaluated regarding the impact of self-assembled proteins on the induction of immune responses. In the present study, we report the application of this experimental approach to the immunogenicity of protein antigens by measuring the antibody responses in mice immunized with nanoparticles made with a recombinant form of Zika virus nonstructural protein 1 (∆NS1). The results clearly indicated that ∆NS1-derived nanoparticles (NP-∆NS1) are assembled into a 3-dimensional structure with a high degree of multimerization. While ∆NS1 proved to be a weak immunogen, immunization with NP-∆NS1 enhanced subunit vaccines' immunogenicity with improved longevity in vaccinated mice. Thus, immunization with self-assembled antigens (nanovaccines) represents a new and promising strategy to enhance NS1-specific antibodies' induction based on purified recombinant proteins.

Predicting and clustering plant CLE genes with a new method developed specifically for short amino acid sequences

BackgroundThe CLV3/ESR-RELATED (CLE) gene family encodes small secreted peptides (SSPs) and plays vital roles in plant growth and development by promoting cell-to-cell communication. The prediction and classification of CLE genes is challenging because of their low sequence similarity.ResultsWe developed a machine learning-aided method for predicting CLE genes by using a CLE motif-specific residual score matrix and a novel clustering method based on the Euclidean distance of 12 amino acid residues from the CLE motif in a site-weight dependent manner. In total, 2156 CLE candidates—including 627 novel candidates—were predicted from 69 plant species. The results from our CLE motif-based clustering are consistent with previous reports using the entire pre-propeptide. Characterization of CLE candidates provided systematic statistics on protein lengths, signal peptides, relative motif positions, amino acid compositions of different parts of the CLE precursor proteins, and decisive factors of CLE prediction. The approach taken here provides information on the evolution of the CLE gene family and provides evidence that the CLE and IDA/IDL genes share a common ancestor.ConclusionsOur new approach is applicable to SSPs or other proteins with short conserved domains and hence, provides a useful tool for gene prediction, classification and evolutionary analysis.

Extremely Amyloidogenic Single-Chain Analogues of Insulin's H-Fragment: Structural Adaptability of an Amyloid Stretch.

Relatively short amino acid sequences often play a pivotal role in triggering protein aggregation leading to the formation of amyloid fibrils. In the case of insulin, various regions of A- and B-chains have been implicated as the most relevant to the protein’s amyloidogenicity. Here, we focus on the highly amyloidogenic H-fragment of insulin comprising the disulfide-bonded N-terminal parts of both chains. Analysis of the aggregation behavior of single-chain peptide derivatives of the H-fragment suggests that the A-chain’s part initiates the aggregation process while the disulfide-tethered B-chain reluctantly adapts to amyloid structure. Merging of both A- and B-parts into single-chain continuous peptides (A–B and B–A) results in extreme amyloidogenicity exceeding that of the double-chain H-fragment as reflected by almost instantaneous de novo fibrillization. Amyloid fibrils of A–B and B–A present distinct morphological and infrared traits and do not cross-seed insulin. Our study suggests that the N-terminal part of insulin’s A-chain containing the intact Cys6–Cys11 intrachain disulfide bond may constitute insulin’s major amyloid stretch which, through its bent conformation, enforces a parallel in-register alignment of β-strands. Comparison of the self-association behavior of H, A–B, and B–A peptides suggests that A-chain’s N-terminal amyloid stretch is very versatile and adaptive to various structural contexts.

Abstract 4879: Identification of degron motifs and mutations by analyzing a deep neural network

Abstract Degrons, short amino acid sequences on protein substrates, play a critical role in the ubiquitin-proteasome system (UPS), which regulates over 80 percent of protein degradation in cells. When recognized by E3 ubiquitin ligases, degrons signal the destruction of its substrate protein, determining the regulatory specificity of the UPS. While a few degrons have been reported to be affected by mutations driving cancer growth, the effect of specific mutations on degrons remains largely unidentified. In this study, we analyze a deep neural network model that predicts the likelihood of an input protein sequence to contain a degron. To characterize the important C-terminal degron motifs learned by the model, we ran computational methods including sequence ranking and selection, enrichment analyses, and sequence logo visualization. This revealed the discovery of 101 significant degron motifs across lengths 2, 3, and 4. While previous studies suggested that di-amino acid motifs might be the primary source of c-end degron, the new motifs suggest that additional complexity might exist with a longer extended degron. Using these putative motifs as targets for mutations, we performed in silico mutagenesis to identify high-impact amino acid mutations that cause degron loss or gain. Our discovery of degron motif-mutation pairs could inform the development of targeted small-molecule drugs for cancer treatments and identification mutational biomarkers used for early detection of cancers. Citation Format: Cynthia Chen, Collin Tokheim, Shirley Liu. Identification of degron motifs and mutations by analyzing a deep neural network [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4879.

The structure of a novel membrane-associated 6-phosphogluconate dehydrogenase from Gluconacetobacterdiazotrophicus (Gd6PGD) reveals a subfamily of short-chain 6PGDs.

The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacterdiazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333AA) 6PGDs, compared to the long-chain subfamily (480AA; e.g., Ovisaries, Homosapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.

Newly synthesized peptide, Ara-27, exhibits significant improvement in cell-penetrating ability compared to conventional peptides.

Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 μM Ara-27 in both HDF and hDPSC. In contrast, 2 μM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 μM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.

Does Early Childhood Vaccination Protect Against COVID-19?

The coronavirus disease 2019 (COVID-19) is an on-going pandemic caused by the SARS-coronavirus-2 (SARS-CoV-2) which targets the respiratory system of humans. The published data show that children, unlike adults, are less susceptible to contracting the disease. This article aims at understanding why children constitute a minor group among hospitalized COVID-19 patients. Here, we hypothesize that the measles, mumps, and rubella (MMR) vaccine could provide a broad neutralizing antibody against numbers of diseases, including COVID-19. Our hypothesis is based on the 30 amino acid sequence homology between the SARS-CoV-2 Spike (S) glycoprotein (PDB: 6VSB) of both the measles virus fusion (F1) glycoprotein (PDB: 5YXW_B) and the rubella virus envelope (E1) glycoprotein (PDB: 4ADG_A). Computational analysis of the homologous region detected the sequence as antigenic epitopes in both measles and rubella. Therefore, we believe that humoral immunity, created through the MMR vaccination, provides children with advantageous protection against COVID-19 as well, however, an experimental analysis is required.

P1609HLA EPITOPE MATCHING IN KIDNEY TRANSPLANTATION AND PRACTICAL APPLICATION

Abstract Background and Aims HLA compatibility between donor and recipient is an important factor that influences graft outcomes after kidney transplantation. Increasing number of class I (HLA-AB) and class II (HLA-DR) mismatches are associated with an increased risk of acute rejection and graft loss. Recent developments have increased our understanding of the structural basis of HLA antigenicity and may allow accurate evaluation of the immunogenic potential of broad antigen HLA mismatches. The immunogenicity of HLA antigens is determined by continuous and discontinuous short sequences of amino acids that form the antibody-accessible regions within each HLA allele known as epitopes. HLAMatchmaker, a computer algorithm that calculates the number of epitope mismatches between donors and recipients by considering each HLA allele as a combination of distinct epitopes known as triplets (continuous amino acid sequences) or eplets (closely located contiguous amino acid sequences). Method A total number of 55 adult patients with LDKT were included in the study. The inclusion criteria: first transplantation of one organ - kidney, use of living donor related or unrelated, emotionally related (spouses) donor. Data concerning donor - sex, age of the donor, type of donation (related or unrelated donor), and data concerning the recipients: sex, age, hemodialysis vintage, underlying disease, type of immunosuppressive therapy, were analyzed. The number of eplet HLA mismatches for each recipient and donor pair at HLA class I (HLA-AB) and class II (HLA-DR) loci was calculated using HLAMatchmaker (Version 1.2) Results In our study we have included 55 patients. 44 patients have related and 11 have unrelated donors. From the group of related donors 31 patients were haploidentycal, with 3 HLA miss match, in the group of unrelated donors-recipients 5 of them have 6 miss match. According HLA MM triplets Class 1 + 2 &amp;lt; or&amp;gt; 20 we have devided 2 groups, but no statistical difference on the graft function was observed in the follow up period. Three acute rejections were reported in the group of patients with HLA miss match triplets Class 1 + 2&amp;gt; 20. Conclusion An increasing number of epitope HLA mismatches is associated with an increased risk of acute rejection. Triplet HLA mismatches may provide better risk stratification for acute rejection and graft survival among those who are otherwise classified as low immunological risk at the broad antigen level.

Identification and Expression Profiling of Toll-Like Receptors of Brown Trout (Salmo trutta) during Proliferative Kidney Disease.

Proliferative kidney disease is an emerging disease among salmonids in Europe and North America caused by the myxozoan parasite Tetracapsuloides bryosalmonae. The decline of endemic brown trout (Salmo trutta) in the Alpine streams of Europe is fostered by T. bryosalmonae infection. Toll-like receptors (TLRs) are a family of pattern recognition receptors that acts as sentinels of the immune system against the invading pathogens. However, little is known about the TLRs’ response in salmonids against the myxozoan infection. In the present study, we identified and evaluated TLR1, TLR19, and TLR13-like genes of brown trout using data-mining and phylogenetic analysis. The expression pattern of TLRs was examined in the posterior kidney of brown trout infected with T. bryosalmonae at various time points. Typical Toll/interleukin-1 receptor protein domain was found in all tested TLRs. However, TLR13-like chr2 had a short amino acid sequence with no LRR domain. Phylogenetic analysis illustrated that TLR orthologs are conserved across vertebrates. Similarly, a conserved synteny gene block arrangement was observed in the case of TLR1 and TLR19 across fish species. Interestingly, all tested TLRs showed their maximal relative expression from 6 to 10 weeks post-exposure to the parasite. Our results suggest that these TLRs may play an important role in the innate defense mechanism of brown trout against the invading T. bryosalmonae.

A Moso bamboo gene VQ28 confers salt tolerance to transgenic Arabidopsis plants.

Overexpression ofPeVQ28in Arabidopsis regulated the expression of salt/ABA-responsive genes and indicated thatPeVQ28may affect the ABA synthesis induced by stress in plants by regulating salt tolerance. Plant-specific VQ proteins, which contain a conserved short FxxhVQxhTG amino acid sequence motif, play an important role in abiotic stress responses, but their functions have not been previously studied in Moso bamboo (Phyllostachys edulis). In this study, real-time quantitative PCR analysis indicated that expression of PeVQ28 was induced by salt and abscisic acid stresses. A subcellular localization experiment showed that PeVQ28 was localized in the nuclei of tobacco leaf cells. Yeast two-hybrid and bimolecular fluorescence complementation analyses indicated that PeVQ28 and WRKY83 interactions occurred in the nucleus. The PeVQ28-overexpressing Arabidopsis lines showed increased resistance to salt stress and enhanced sensitivity to ABA. Compared with wild-type plants under salt stress, PeVQ28-transgenic plants had lower malondialdehyde and higher proline contents, which might enhance stress tolerance. Overexpression of PeVQ28 in Arabidopsis enhanced expression of salt- and ABA-responsive genes. These results suggest that PeVQ28 functions in the positive regulation of salt tolerance mediated by an ABA-dependent signaling pathway.

Coordination crosslinking of helical substituted oligoamide nanorods with Cu(II)

ABSTRACT Substituted oligoamides are short sequences of unnatural amino acids. Oligoamides made entirely of β3 amino acids yield helical monomers that, if N-acylated, assemble into nanorod structures via a supramolecular assembly motif. In this work, coordination crosslinking was used to create complex nanomaterials from oligoamides WKLWEL (KE) and WELWEL (EE) (the letters denote the analogous α-amino acids). Upon Cu(II) addition, atomic force microscopy and small angle neutron scattering revealed morphologic changes specific to KE but absent in EE. Vibration spectroscopy measurements revealed that Cu(II) can coordinate to the amine moieties of the side chains, without direct effect on the backbone amides. While coordination in excess solvent lead to regular nanostructures, fast drying of the sample yielded oligoamide templated crystallization of CuCl2. The metal coordination crosslinking of supramolecular assemblies as reported here is the first realization of a metallosupramolecular framework structure.

93 Efficient introduction of green fluorescent protein-9R, a protein with cell-penetrating peptides, into oocytes using intracytoplasmic sperm injection

The introduction of functional molecules such as oligonucleotides, nucleic acids, peptides, and antibodies into gametes has been beneficial not only for research purposes but is also expected to provide a treatment modality for failure of assisted reproductive technology (ART). The use of cell-penetrating peptides (CPPs) has been established as a method to introduce proteins and nucleic acids, which cannot normally pass through the cell membrane, into cells. Cell-penetrating peptides are short sequences of amino acids that facilitate the penetration of conjugated cargoes across mammalian cell membranes. To date, reports on the introduction of proteins via CPP into germ cells have been limited, and the method has not achieved success in preimplantation embryos. Simple polyarginine peptides have been known to induce higher cell penetration rates among CPPs. In this study, we examined whether green fluorescent protein-conjugated nine arginines (GFP-9R) can be effectively introduced into mouse oocytes and spermatozoa. Oocytes were collected from oviducts of 8- to 10-week-old female ICR mice after administration of equine chorionic gonadotropin and human chorionic gonadotropin. Spermatozoa were collected from epididymides of 8- to 10-week-old male mice. Some oocytes were used to perform IVF and subsequently cultured. These oocytes, spermatozoa, and embryos were cultured with GFP-9R or GFP for 30min to 1h and were then fixed with 4% paraformaldehyde and observed with a confocal laser microscope to check the introduction of GFP-9R into intact germ cells. In addition, we also attempted to introduce GFP-9R into oocytes by performing IVF or intracytoplasmic sperm injection (ICSI) using GFP-9R-introduced spermatozoa. In the case of ICSI, the tails of sperm were removed by sonication. After performing IVF or ICSI, embryos were cultured and observed with a fluorescence microscope. As a result, the GFP fluorescence signal was not detected in the ooplasm incubated with GFP-9R but was detected in the perivitelline space, the zona pellucida, and the collapsed first polar body. No GFP fluorescence signal was detected in the oocytes incubated with GFP. On the other hand, GFP fluorescence was detected in some spermatozoa that were incubated with GFP-9R (GFP-9R 43.3% (26 out of 60) but not with GFP alone 0% (0 out of 67)). However, when IVF was performed using these spermatozoa, the fluorescence signal was not detected in spermatozoa attached to the zona pellucida, but a strong signal was detected in dead spermatozoa, which are not involved in fertilization. Furthermore, we found that efficiency of introduction of GFP-9R into sperm heads increased after sonication (GFP-9R 93.1% (27 of 29) vs. GFP 0% (0 of 23)). Finally, we performed ICSI with GFP-9R-introduced sperm heads which were sonicated. These ICSI embryos developed to blastocysts normally (GFP-9R 46.7% (7 of 15) vs. non-GFP-9R 50.0% (8 of 16)). In this study, we demonstrated efficient CPP protein delivery of sonicated sperm heads into oocytes via ICSI in mice, even though the CPPs did not enter effectively into intact germ cells directly.