Sequences Of Candidates Research Articles

Does human homology reduce the potential immunogenicity of non-antibody scaffolds?

Biologics developers are moving beyond antibodies for delivery of a wide range of therapeutic interventions. These non-antibody modalities are often based on 'natural' protein scaffolds that are modified to deliver bioactive sequences. Both human-derived and non-human-sourced scaffold proteins have been developed. New types of "non-antibody" scaffolds are still being discovered, as they offer attractive alternatives to monoclonals due to their smaller size, improved stability, and ease of synthesis. They are believed to have low immunogenic potential. However, while several human-sourced protein scaffolds have not been immunogenic in clinical studies, this may not predict their overall performance in other therapeutic applications. A preliminary evaluation of their potential for immunogenicity is warranted. Immunogenicity risk potential has been clearly linked to the presence of T "helper" epitopes in the sequence of biologic therapeutics. In addition, tolerogenic epitopes are present in some human proteins and may decrease their immunogenic potential. While the detailed sequences of many non-antibody scaffold therapeutic candidates remain unpublished, their backbone sequences are available for review and analysis. We assessed 12 example non-antibody scaffold backbone sequences using our epitope-mapping tools (EpiMatrix) for this perspective. Based on EpiMatrix scoring, their HLA DRB1-restricted T cell epitope content appears to be lower than the average protein, and sequences that may act as tolerogenic epitopes are present in selected human-derived scaffolds. Assessing the potential immunogenicity of scaffold proteins regarding self and non-self T cell epitopes may be of use for drug developers and clinicians, as these exciting new non-antibody molecules begin to emerge from the preclinical pipeline into clinical use.

Mechanisms of Antimicrobial Peptides from Bagasse against Human Pathogenic Bacteria

Nonedible agricultural wastes (agricultural wastes, agro-industrial wastes, and fishery wastes) were chosen as potential sources of antimicrobial peptides and evaluated for antibacterial efficiency against human pathogens. Specifically, protein hydrolysates were first obtained by hydrolysis with pepsin. Filtrated peptides smaller than 3 kDa were then purified by C18 reversed-phase chromatography, cation exchange chromatography, and off-gel fractionation. NanoLC-MS/MS was used to investigate the amino acid sequences of active peptide candidates. Five candidate peptides were finally chosen for chemical synthesis and evaluation of growth inhibition against human pathogenic bacteria. Two synthetic peptides from bagasse, NLWSNEINQDMAEF (Asn-Leu-Trp-Ser-Asn-Glu-Ile-Asn-Gln-Asp-Met-Ala-Glu-Phe) and VSNCL (Val-Ser-Asn-Cys-Leu), showed the most potent antibacterial activity against three pathogens: Pseudomonas aeruginosa, Bacillus subtilis, and Burkholderia cepacia. The antibacterial mechanisms of these peptides were then examined using shotgun proteomics, which revealed their effects to involve both intracellular-active and membrane-active mechanisms. Further investigation and modification of peptides are needed to increase the efficiency of these peptides against human pathogens.

Molecular analysis of gene from ginger and Zingiber zerumbet encoding resistance to bacterial wilt

The bottleneck in ginger cultivation is bacterial wilt which causes crop damage of more than 70%. Since conventional cross-breeding in ginger is difficult, genetic engineering has allowed new ginger variety resistance to bacterial wilt development. Thus, a homologous sequence of resistance genes (RRS1-R) could be designed and constructed, then transformed to generate new ginger variety tolerance to R. solanacearum. The more resistant genotypes (red ginger and wild ginger/shampoo ginger) were subjected to gene isolation in this research. The red and wild ginger species were first inoculated with a suspension of R. solanacearum before gene isolation. Then, the generated primer was used to isolate homologous sequences of RRS1-R gene candidates from both species. Cloning and sequence results showed that induced red and wild ginger tissues to R. solanacearum were expressed on both species. However, the RRS1-R homologous gene was not detected. Furthermore, the full-length DNA gene cloned from the red and wild ginger species was homologous to the Kafirin gene group. These results indicated that different genes might have been involved in encoding resistance to bacterial wilt in ginger.

Effect of charged mutation on aggregation of a pentapeptide: Insights from molecular dynamics simulations.

Aggregation of therapeutic monoclonal antibodies (mAbs) can negatively affect their chemistry, manufacturing, and control attributes and lead to undesirable immune responses in patients. Therefore, optimization of lead mAb drug candidates during discovery stages to mitigate aggregation is increasingly becoming an integral part of their developability assessments. The disruption of short sequence motifs called aggregation prone regions (APRs) found in amino acid sequences of mAb candidates can potentially mitigate their aggregation. In this work, we have performed molecular dynamics simulations to study the aggregation of an APR (VLVIY) found in λ light chains of human antibodies and its single point mutant KLVIY. Eighteen different multicopy peptide simulation systems of "VLVIY" and "KLVIY" were constructed by varying their concentrations, temperatures, termini capping, and flanking gate-keeper regions. Within 20 ns of the simulation, peptide "VLVIY" formed an aggregate of 100 peptides at ~0.1M concentration with a 60% reduction in solvent accessible surface area (SASA). Furthermore, analysis of the SASA change, peptide cluster distribution, and water residence time demonstrated how Val ➔ Lys mutation resists aggregation and improves solubility. Presence of Lys slows down aggregation kinetics via charge-charge repulsions and by raising the kinetic barrier to formation of large oligomers. However, the effect of the Val ➔ Lys mutation is dependent on sequence and structural contexts around the APR. This mutation also alters the solvation shell around the peptide by favoring solute-solvent interactions, thereby increasing its solubility. This work has provided a detailed mechanistic explanation of how APR disruption can mitigate aggregation in biotherapeutics and improve their developability.

Peptide barcodes in dogs affected by mitral valve disease with and without pulmonary hypertension using MALDI-TOF MS and LC-MS/MS.

Mitral valve disease (MVD) is an important and most frequently acquired heart disease found in dogs. MVD is classified into different stages according to its severity. There is a challenge in differentiation between asymptomatic and symptomatic stages of the MVD. Moreover, pulmonary hypertension (PH) is a common complication in dogs affected by MVD. In clinical practice, there are also some limitations to identify PH. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that can characterize specific patterns of peptide mass called peptide barcodes from various samples. Besides, in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS), potential peptide sequences associated with specific conditions could be identified. The present study aimed to use MALDI-TOF coupled with LC-MS/MS to characterize specific peptide barcodes and potential peptide candidates in serum samples from healthy dogs, dogs with MVD stage B (MVD B, asymptomatic stage), MVD stage C (MVD C, symptomatic stage), MVD stage B with PH (MVD B PH), and MVD stage C with PH (MVD C PH). Discrete clusters of the 5 sample groups were identified by 3D plot analysis. Peptide barcodes also revealed differences in peptide patterns among the 5 groups. Six amino acid sequences of peptide candidates at 1,225.60, 1,363.85, 1,688.71, 1789.52, 2020.21, and 2156.42 Da were identified as part of the proteins CLCN1, CLUL1, EDNRA, PTEN, SLC39A7, and CLN6, respectively. The network interactions between these discovered proteins and common cardiovascular drugs were also investigated. These results demonstrate that MALDI-TOF MS has promise as an optional technique for diagnosing dogs affected by asymptomatic and symptomatic stages of MVD with and without PH. Further studies are required to identify peptide barcodes in dogs with other diseases to create peptide barcode databases in veterinary medicine before using this method as a novel diagnostic tool in the future.

Chemical Modification of the Yeast Cell Surface Allows the Switch Between Display and Soluble Secretion of Full-Length Antibodies.

The classical yeast surface display (YSD)-based antibody hit discovery relies on the enrichment of candidates from large antibody fragment libraries by fluorescence-activated cell sorting (FACS), followed by sequencing of the remaining diversity. The sequences of hit candidates are then transferred into plasmids, which allow their expression as full-length IgG in mammalian cells. After production and downstream processing, antibodies are further qualified in biochemical and functional assays. This multistep process has been proven to be successful in academia and has also found its way to the biopharmaceutical industry. However, during the recent decade, several groups tried to simplify this process by enabling either switchable or simultaneous secretion and display of antibodies or antibody fragments by yeast cells. Therefore, functional testing of candidates can be accomplished immediately during the screening process, while eliminating the need for tedious sequence reformatting and production host change. In this protocol, we give guidance for the establishment of a full-length antibody display system on yeast cells, which permits a simple switch to soluble antibody secretion.

What Do Teacher Candıdates Know About the Limits of the Sequences?

candidates. Research is a case study in which qualitative methods are adopted. The first phase of the study wasconducted with a total of 45 teacher candidates taking the course of Analysis III. At this stage, the "Limit KnowledgeTest in Sequences", which was developed by the researchers to investigate the concept knowledge of the sequenceconcept, was used as a data collection tool. At this stage, "Limit Knowledge Test in Sequences", which wasdeveloped by researchers to investigate the limit concept knowledge in sequences of the teacher candidates, included2 open-ended problems were used as data collection tool. In the second phase of the research, individual interviewswere made with 8 teacher candidates selected from the sample to conduct in-depth research. Content analysis wasused to analyze the obtained data. As a result of the analysis of the data, significant shortcomings were found in theknowledge of the concept of the limit topic in the sequences of teacher candidates. It is seen that the candidates donot know the importance of the concept of "accumulation point", which is indispensable to convergence.

Reconstruction of insect hormone pathways in an aquatic firefly, Sclerotia aquatilis (Coleoptera: Lampyridae), using RNA-seq.

Insect hormones: ecdysteroids and juvenile hormones have crucial functions during the regulation of different developmental pathways in insects. Insect metamorphosis is one of the primary pathways regulated by these hormones. The insect hormone biosynthetic pathway is conserved among arthropods, including insects, with some variations in the form of hormones used among each group of insects. In this study, the candidate genes involved in the insect hormone pathways and their functional roles were assessed in an aquatic firefly, Sclerotia aquatilis using a high-throughput RNA sequencing technique. Illumina next-generation sequencing (NGS) was used to generate transcriptome data for the different developmental stages (i.e., larva, pupa, and adult) of S. aquatilis. A total of 82,022 unigenes were generated across all different developmental stages. Functional annotation was performed for each gene, based on multiple biological databases, generating 46,230 unigenes. These unigenes were subsequently mapped using KEGG pathways. Accordingly, 221 protein-encoding genes involved in the insect hormone pathways were identified, including, JHAMT, CYP15A1, JHE, and Halloween family genes. Twenty potential gene candidates associated with the biosynthetic and degradation pathways for insect hormones were subjected to real-time PCR, reverse transcriptase PCR (RT-PCR) and sequencing analyses. The real-time PCR results showed similar expression patterns as those observed for transcriptome expression profiles for most of the examined genes. RT-PCR and Sanger sequencing confirmed the expressed coding sequences of these gene candidates. This study is the first to examine firefly insect hormone pathways, facilitating a better understanding of firefly growth and development.

EGAR, A Food Protein-Derived Tetrapeptide, Reduces Seizure Activity in Pentylenetetrazole-Induced Epilepsy Models Through α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionate Receptors.

A primary pathogeny of epilepsy is excessive activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs). To find potential molecules to inhibit AMPARs, high-throughput screening was performed in a library of tetrapeptides in silico. Computational results suggest that some tetrapeptides bind stably to the AMPAR. We aligned these sequences of tetrapeptide candidates with those from in vitro digestion of the trout skin protein. Among salmon-derived products, Glu-Gly-Ala-Arg (EGAR) showed a high biological affinity toward AMPAR when tested in silico. Accordingly, natural EGAR was hypothesized to have anticonvulsant activity, and in vitro experiments showed that EGAR selectively inhibited AMPAR-mediated synaptic transmission without affecting the electrophysiological properties of hippocampal pyramidal neurons. In addition, EGAR reduced neuronal spiking in an in vitro seizure model. Moreover, the ability of EGAR to reduce seizures was evaluated in a rodent epilepsy model. Briefer and less severe seizures versus controls were shown after mice were treated with EGAR. In conclusion, the promising experimental results suggest that EGAR inhibitor against AMPARs may be a target for antiepilepsy pharmaceuticals. Epilepsy is a common brain disorder characterized by the occurrence of recurring, unprovoked seizures. Twenty to 30% of persons with epilepsy do not achieve adequate seizure control with any drug. Here we provide a possibility in which a natural and edible tetrapeptide, EGAR, can act as an antiepileptic agent. We have combined computation with in vitro experiments to show how EGAR modulates epilepsy. We also used an animal model of epilepsy to prove that EGAR can inhibit seizures in vivo. This study suggests EGAR as a potential pharmaceutical for the treatment of epilepsy.

Assessment of promoters and a selectable marker for development of strawberry intragenic vectors

Conventional breeding of the cultivated strawberry (Fragaria × ananassa) is hindered by a complicated octoploid genome and a paucity of molecular markers. Genetic improvement may be accelerated by direct gene-transfer techniques that could address single-gene deficiencies in otherwise commercially valuable selections. ‘Cisgenic’ or “intragenic” approaches face fewer regulatory hurdles, yet more characterized promoters and selectable markers are needed for the development of such technologies in strawberry. This report tests a number of putative promoters and a potential selectable marker for use in cisgenic vector development. Based on RNAseq data, the 5′-proximal noncoding regions from highly expressed transcripts were tested for activity. These putative promoter sequences were fused to eGFP and GUS reporter genes and transiently expressed in both Nicotiana benthamiana leaves and F. × ananassa strawberry fruits. The promoter sequences of FanAPA1-related and FanUBCE2 present constitutive promoter candidates that are active in all tissues tested. A potential selectable marker from strawberry sequence, the coding region of the strawberry 5-enolpyruvylshikimate 3 phosphatase synthase (EPSPS) gene, was cloned and mutagenized, and then stably transformed into Arabidopsis plants. Overexpression of the non-mutagenized form confers resistance to glyphosate, showing potential utility as a selectable marker. The results of these trials define new tools for the development of intragenic vectors in strawberry.

Identification and characterization of peptide fragments for the direct and site-specific immobilization of functional proteins onto the surface of silicon nitride

In this study, we successfully identified peptide fragments that have a strong affinity toward the surface of a silicon nitride (SiN) substrate. An E. coli soluble protein, which was preferentially adsorbed onto the surface of a SiN substrate was isolated by 2D electrophoresis, and it was identified as “elongation factor Tu (ELN)” via the peptide MS fingerprinting method. A recombinant ELN that was originally cloned and produced, also maintained its adsorptive ability to a SiN substrate, by comparison with BSA that was used as a control protein. The peptide fragments derived from the recombinant ELN were prepared via 3 types of proteases with different recognition properties (trypsin, chymotrypsin and V8 protease). The peptide mixture was applied to the surface of a SiN substrate, and then, the SiN-binding peptide candidates were isolated and identified. The amino acid sequences of the peptide candidates were genetically fused with the C-terminal region of glutathione S-transferase as a model protein, and the adsorption properties of mutant-type GSTs on the surface of a SiN substrate were directly monitored using a reflectometric interference spectroscopy (RIfS) sensor system. Consequently, among the 8 candidates identified, the genetic fusion of TP14, V821 and CT22 peptides resulted in a significant enhancement of GST adsorption to the surface of the SiN substrate, while the adsorption of a wild-type GST was hardly detectable by RIfS sensor. These peptide fragments were located at the C-terminal region in the aminoacid sequence of recombinant ELN. Interestingly, the sequence with the shortest and strongest SiN-binding peptide, TP14 (GYRPQFYFR), was also found in that of V821 (GGRHTPFFKGYRPQFYFRTTDVTGTIE). The TP14 peptide might be the smallest unit of SiN-binding peptide, and a clarification of the amino acid contribution in TP14 peptide will be the next subject. Three-fold higher enzymatic activities were detected from the SiN substrate immobilized with GST-TP14 and GST-V821 due to a higher density of enzyme through the SiN-binding peptides.Thus, the SiN-binding peptides identified in this study will be considerably useful for the immobilization of target proteins with high density and biological activity onto the surfaces of SiN substrates, and these will be applicable to the task of coating proteins onto the surface of SiN-based RIfS sensors and semiconductors.

Melody Extraction From Polyphonic Music Signals Using Pitch Contour Characteristics

We present a novel system for the automatic extraction of the main melody from polyphonic music recordings. Our approach is based on the creation and characterization of pitch contours, time continuous sequences of pitch candidates grouped using auditory streaming cues. We define a set of contour characteristics and show that by studying their distributions we can devise rules to distinguish between melodic and non-melodic contours. This leads to the development of new voicing detection, octave error minimization and melody selection techniques. A comparative evaluation of the proposed approach shows that it outperforms current state-of-the-art melody extraction systems in terms of overall accuracy. Further evaluation of the algorithm is provided in the form of a qualitative error analysis and the study of the effect of key parameters and algorithmic components on system performance. Finally, we conduct a glass ceiling analysis to study the current limitations of the method, and possible directions for future work are proposed.

Building interacting partner predictors using co‐varying residue pairs between histidine kinase and response regulator pairs of 48 bacterial two‐component systems

The two-component system (TCS) is a signal transduction system that involves a histidine kinase (HK) and a response regulator (RR). Although up to hundreds of TCSs may operate in parallel in a bacterial cell, the high-fidelity of a TCS signaling is well maintained, minimizing irrelevant crosstalk between TCSs. When a HK gene and a RR gene in a given TCS system exist in neighboring positions, it is almost certain that their protein products (i.e., HK and RR) are interacting partners. However, large bacterial genomes often have multiple HK genes and/or cognate RR genes that are not neighboring positions. In many partially assembled genomes, some HK genes and RR genes belong to different contigs. In these cases, it is not clear which HK(s) and RR(s) interact. By combining information-theoretic and graph-theoretic approaches, we developed a computational method identifying co-evolving residue pairs between HKs and cognate RRs and predicting the interacting HK:RR pairs for each TCS. In addition, we built a TCSppWWW webserver (http://compath.org/platcom/tcs) that takes query sequences of pairing candidates and predicts their HK:RR pairing using precomputed models. The current release of TCSppWWW provides predictors for 48 TCSs using over 20,000 protein sequences from about 900 bacterial genomes. Three different types of predictors using Random Forest, RBF Network, and Naïve Bayes are provided. Once a set of HK and RR candidate sequences are submitted, TCSppWWW aligns query sequences to the precomputed multiple sequence alignment of HK:RR pairs, extracts co-evolving column positions, then returns prediction results with prediction margin and additional information.