Rounds Of Selection Research Articles

Genetic improvement of Egyptian cotton (Gossypium barbadense L.) for high yield and fiber quality properties under semi arid conditions

Between 2016 and 2018, the Agriculture Research Center's Sakha Agriculture Research Station conducted two rounds of pedigree selection on a segregating population of cotton (Gossypium barbadense L.) using the F2, F3, and F4 generations resulting from crossing Giza 94 and Suvin. In 2016, the top 5% of plants from the F2 population were selected based on specific criteria. The superior families from the F3 generation were then selected to produce the F4 families in 2017, which were grown in the 2018 summer season in single plant progeny rows and bulk experiments with a randomized complete block design of three replications. Over time, most traits showed increased mean values in the population, with the F2 generation having higher Genotypic Coefficient of Variance (GCV) and Phenotypic Coefficient of Variance (PCV) values compared to the succeeding generations for the studied traits. The magnitude of GCV and PCV in the F3 and F4 generations was similar, indicating that genotype had played a greater role than the environment. Moreover, the mean values of heritability in the broad sense increased from generation to generation. Selection criteria I2, I4, and I5 were effective in improving most of the yield and its component traits, while selection criterion I1 was efficient in improving earliness traits. Most of the yield and its component traits showed a positive and significant correlation with each other, highlighting their importance in cotton yield. This suggests that selecting to improveone or more of these traits would improve the others. Families number 9, 13, 19, 20, and 21 were the best genotypes for relevant yield characters, surpassing the better parent, check variety, and giving the best values for most characters. Therefore, the breeder could continue to use these families in further generations as breeding genotypes to develop varieties with high yields and its components.

Abstract 6025: Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display

Abstract Radioligand therapeutics (RLT) aim to selectively deliver radioisotopes to cancer tissues to eradicate tumor cells while limiting the damage to surrounding tissues. They are composed of a tumor-targeting ligand and a radioactive payload. The therapeutic potential of RLT strongly depends on their biostability and residence time in tumor and healthy tissue after injection. Macrocyclic peptides are effective tumor-targeting ligands because their size and molecular characteristics confer optimal pharmacologic properties that can be fine-tuned during the development process. mRNA display is a powerful platform for macrocyclic peptide drug discovery that identifies high affinity peptide ligands through in vitro selection. Here, we describe the selection and initial characterization of macrocyclic peptides discovered by mRNA display that bind to the extracellular domain of a tumor target. Enrichment of the library for peptides that bound to the recombinant target protein was observed after four rounds of selection with no further enrichment in round five. The library of round 5 was sequenced by Illumina sequencing and the top 1,000 sequences (highest counts) were used to perform cluster analysis. We tested the top sequences of 43 different clusters in in vitro translation binding assays to validate the selection output and identify sequences for hit confirmation. Most of the sequences tested showed clear binding to the target protein and only a few peptides showed binding to a closely related protein from the same protein family. No non-specific binding of the discovered peptides to an irrelevant protein was observed showing the high specificity of the generated library. From this confirmatory selection data, we chose a subset of peptides for chemical synthesis. Binding affinity measurements were determined by surface plasmon resonance (SPR) and cellular binding of selected candidates to target-positive and -negative tumor cells was analyzed by flow cytometry. From this hit confirmation data, in vitro internalization and in vitro anti-tumor efficacy of radiolabeled peptide candidates for RLT are assessed. Our data highlights the potential of mRNA display platforms for fast and efficient discovery of highly specific macrocyclic peptides with optimal binding properties suitable for RLT. Citation Format: Anika Jaekel, Kathryn Sherry, Prachi Desai, David Fox, Tang Guo-Qing, Manuel Sturzbecher-Hoehne, Dennis Mewis, Kaori Noridomi, Liam Rayman, Ping Ye. Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6025.

RNA-catalyzed evolution of catalytic RNA

An RNA polymerase ribozyme that was obtained by directed evolution can propagate a functional RNA through repeated rounds of replication and selection, thereby enabling Darwinian evolution. Earlier versions of the polymerase did not have sufficient copying fidelity to propagate functional information, but a new variant with improved fidelity can replicate the hammerhead ribozyme through reciprocal synthesis of both the hammerhead and its complement, with the products then being selected for RNA-cleavage activity. Two evolutionary lineages were carried out in parallel, using either the prior low-fidelity or the newer high-fidelity polymerase. The former lineage quickly lost hammerhead functionality as the population diverged toward random sequences, whereas the latter evolved new hammerhead variants with improved fitness compared to the starting RNA. The increase in fitness was attributable to specific mutations that improved the replicability of the hammerhead, counterbalanced by a small decrease in hammerhead activity. Deep sequencing analysis was used to follow the course of evolution, revealing the emergence of a succession of variants that progressively diverged from the starting hammerhead as fitness increased. This study demonstrates the critical importance of replication fidelity for maintaining heritable information in an RNA-based evolving system, such as is thought to have existed during the early history of life on Earth. Attempts to recreate RNA-based life in the laboratory must achieve further improvements in replication fidelity to enable the fully autonomous Darwinian evolution of RNA enzymes as complex as the polymerase itself.

“Pharmacotrophy”: a playful tournament for game- and team-based learning in pharmacology education - assessing its impact on students’ performance

BackgroundAt the Faculty of Pharmacy of Paris, we conducted a pharmacology tournament in 2021 and 2022, named “Pharmacotrophy”, to offer a game-, team- and competitive-based learning innovation based on fun and challenge. This article aims to (1) provide a detailed overview of the organisation of “Pharmacotrophy,” (2) present and compare feedback from both students and teachers, and (3) assess the impact of student participation on their exam marks.Methods“Pharmacotrophy” took place in 2021 and 2022 over a two-week period at the beginning of the exam revision phase. It involved a combination of remote matches using the online quiz creation tool Kahoot!® and in-person matches. Teams, consisting of three students from the 4th or 5th year, participated in several selection rounds leading up to the final match. The questions covered various topics from the pharmacology curriculum. Using an anonymous online survey, we collected the feedback from students and teacher regarding the organisation of the tournament and the interest and difficulty of the different type of questions. We retrospectively compared the exam marks of 4th year students who took part in “Pharmacotrophy” (n2021 = 19 and n2022 = 20) with those of the rest of the 4th year (n2021 = 315–320 and n2022 = 279–281), both in the year before “Pharmacotrophy” and just after the tournament.ResultsStudents highlighted the educational benefits of team-based and game-based learning. This novel approach positively and constructively motivated students to review pharmacology. Additionally, students appreciated the establishment of a trust-based relationship with their teachers. All students had a similar pharmacology level based on their exam results in the year before “Pharmacotrophy.” After the tournament, participants had marks 20.1% higher in pharmacology questions compared to non-participants (p = 0.02), while they had comparable overall levels, as evidenced by their final grade averages and marks in non-pharmacology questions. Moreover, participants who advanced further in the competition achieved higher marks in pharmacology questions compared to those who were eliminated early in the tournament.ConclusionThe implementation of “Pharmacotrophy” provided students with an enjoyable way to review pharmacology coursework and revived the interest in pharmacology for some. Specifically, participating in “Pharmacotrophy” led to an increase in pharmacology marks for students who were not among the top performers in the class or did not excel in pharmacology in the previous year. This study quantified the pedagogical value of this innovative curriculum in terms of knowledge acquisition.

Light‐Up Sensing Citrate Using a Capture‐Selected DNA Aptamer

AbstractCitrate is a key metabolite and nutrient in humans. Its level is associated with many diseases from tumor growth to bone diseases. Detection of citrate has relied on its high negative charge, metal chelating properties and as an enzyme substrate. In this work, the capture‐selection method is used to isolate DNA aptamers for citrate. After 18 rounds of selection, a highly converged library is obtained and the first two sequences reached 99.6% of the library. Using the most abundant sequence named CA1, thioflavin T fluorescence spectroscopy and isothermal titration calorimetry show dissociation constants of 7.4 and 4.4 µm citrate, respectively. CA1 does not require sodium for binding but requires 1.0 mm magnesium. Among the tested carboxylate molecules, only citrate can bind to the aptamer. A light‐up fluorescence strand displacement biosensor is developed and it can detect citrate in simulated urine with a detection limit of 1.1 µm. This short 42‐nucleotide aptamer can be readily adapted to other types of sensing mechanisms for the detection of citrate.

Inference of annealed protein fitness landscapes with AnnealDCA.

The design of proteins with specific tasks is a major challenge in molecular biology with important diagnostic and therapeutic applications. High-throughput screening methods have been developed to systematically evaluate protein activity, but only a small fraction of possible protein variants can be tested using these techniques. Computational models that explore the sequence space in-silico to identify the fittest molecules for a given function are needed to overcome this limitation. In this article, we propose AnnealDCA, a machine-learning framework to learn the protein fitness landscape from sequencing data derived from a broad range of experiments that use selection and sequencing to quantify protein activity. We demonstrate the effectiveness of our method by applying it to antibody Rep-Seq data of immunized mice and screening experiments, assessing the quality of the fitness landscape reconstructions. Our method can be applied to several experimental cases where a population of protein variants undergoes various rounds of selection and sequencing, without relying on the computation of variants enrichment ratios, and thus can be used even in cases of disjoint sequence samples.

Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

New approach to acquired drug resistance and toxicity in cancer chemotherapy

Acquired drug resistance is a major cause of failure of cancer chemotherapy. Rapidly dividing cancer cells quickly evolve DNA mutations to bypass any single drug targeted metabolic pathway. Several drugs targeting different cancer biomarkers, can be used in combination, to target multiple biomarkers simultaneously, to reduce the tumor’s ability to provide sufficient mutations to evade the simultaneous attack. We hypothesize a new cancer treatment paradigm to overwhelm the ability of the cancer to rapidly evolve sufficient drug resistant pathways to survive. Simultaneous administration of a large array of hundreds of different drugs targeting cancer specific proteins at many sites on these proteins will overwhelm the ability of the cancer cells to survive by simultaneously developing pathways around the effects of all these drugs.We propose that hundreds of small molecule oligonucleotide aptamers be used to selectively bind the surface proteins of cancer cells and hinder their function. Therapeutic radionuclides added to the selected array of aptamers will enhance the cancer treatment. Unlike single drugs which must target a unique focus on a cancer specific protein, radiolabeled aptamers can target anywhere on a cancer specific protein to exert their effects.Toxicity of cancer chemotherapy, another major cause of cancer chemotherapy failure, is caused by binding of chemotherapeutic drugs to components present in normal cells. To reduce therapeutic toxicity, aptamers which bind normal cell proteins can be subtracted from the array of aptamers binding cancer cell proteins. Libraries of hundreds of million inexpensive commercially obtained random aptamers which bind proteins extracted from human cancer tissue, can be selected in vitro. Although the cancer proteins will not retain all of their in vivo configurations, the use of a large array of aptamers will assure that many aptamers will bind many of the in vitro sites that retain their configuration in vivo. Subtraction of aptamers binding to normal cell proteins to reduce toxicity will also remove aptamers binding normal proteins in the cancer cells. The final selected array of hundreds of aptamers will represent aptamers binding only the cancer specific molecules.As proof of principle of our hypothesis, proteins extracted from tumors that develop after implanation of cancer cells in xenogenic mice can be used as targets for multiple rounds of aptamer selection, reimplanation and reselection. To reduce toxicity, aptamers binding to proteins extracted from a collection of many different normal mouse tissues can be subtracted from the array of selected aptamers.

P058 The identification of three linear epitopes on infliximab all map to complementarity-determining regions

Abstract Background Infliximab (IFX) is a mainstay of treatment for inflammatory bowel disease (IBD). Its use is limited by the development of anti-drug antibodies (ADA), which can cause loss of response and adverse drug reactions. The antigenic epitopes have not been clearly defined, and large antigenic areas suggested. The aim of this study was to identify clinically relevant distinct epitopes in IBD patients treated with IFX with ADA and different clinical outcomes. Methods Sera were pooled from 8 ADA positive IBD patients (ADA >10 AU/mL; IDKmonitor® Infliximab total ADA) treated with CT -P13 (Remsima® Celltrion Inc., Korea) exhibiting: loss of response, response with therapeutic drug levels, response with subtherapeutic drug levels and adverse drug reactions. A novel whole gene fragment library (GFL) was synthesised using pools of overlapping synthetic oligonucleotides covering the IFX heavy chain and light chain (Twist Bioscience, California, United States). This was cloned into a phage display vector to generate a library >106 clones. Three rounds of selection of the GFL were performed using pooled sera. DNA from the selection outputs were analysed by next generation sequencing (NGS) (Illumina, San Diego, United States). Outputs from one round of selection using individual sera were also analysed by NGS. Results ADA ranged 17->600 AU/mL. 23,383 gene sequences analysed from the GFL showed complete coverage of the IFX gene. After three rounds of selection, DNA sequencing showed the enrichment of three peptides – all overlapping the IFX complementary-determining regions (CDR)- Figure 1. The three peptides enriched: heavy chain peptide 1 included heavy chain CDR 1, light chain peptide 2 overlap light chain CDR 1 and light chain peptide 3 overlap light chain CDR 2. NGS assessment of the outputs from round 1-3 using pooled ADA positive sera confirmed enrichment of the three peptides. NGS analysis of the selections on individual sera showed differences in enrichment profile of the three peptides. Conclusion Using ADA positive sera from IBD patients treated with IFX with differing clinical outcomes, three linear epitopes were identified from a novel whole IFX-GFL. All epitopes overlapped with CDRs which is consistent with being potential sites for neutralising ADA. NGS has suggested that individual ADA positive sera have differing recognition profiles for the selected epitopes. Identifying distinct epitopes may lead to the identification of a biomarker for loss of response to infliximab.

Blocking pathogenic Leptospira invasion with aptamer molecules targeting outer membrane LipL32 protein

This study aimed to develop aptamers targeting LipL32, a most abundant lipoprotein in pathogenic Leptospira, to hinder bacterial invasion. The objectives were to identify high-affinity aptamers through SELEX and evaluate their specificity and inhibitory effects. SELEX was employed to generate LipL32 aptamers (L32APs) over 15 rounds of selection. L32APs' binding affinity and specificity for pathogenic Leptospira were assessed. Their ability to inhibit LipL32-ECM interaction and Leptospira invasion was investigated. Animal studies were conducted to evaluate the impact of L32AP treatment on survival rates, Leptospira colonization, and kidney damage. Three L32APs with strong binding affinity were identified. They selectively detected pathogenic Leptospira, sparing non-pathogenic strains. L32APs inhibited LipL32-ECM interaction and Leptospira invasion. In animal studies, L32AP administration significantly improved survival rates, reduced Leptospira colonies, and mitigated kidney damage compared to infection alone. This pioneering research developed functional aptamers targeting pathogenic Leptospira. The identified L32APs exhibited high affinity, pathogen selectivity, and inhibition of invasion and ECM interaction. L32AP treatment showed promising results, enhancing survival rates and reducing Leptospira colonization and kidney damage. These findings demonstrate the potential of aptamers to impede pathogenic Leptospira invasion and aid in recovery from Leptospira-induced kidney injury (190 words).

Highly sensitive and specific graphene oxide-based FRET aptasensor for quantitative detection of human soluble growth stimulating gene protein 2

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1–100 μg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established.

Development of a lambda Red based system for gene deletion in Chlamydia.

The ability to efficiently target loci in the Chlamydia trachomatis genome for deletion remains a desirable goal in the field and new strategies need to be developed and refined. Here we describe the development and application of a lambda red recombineering system for Chlamydia. Using a non-replicative plasmid encoding key lambda Red components and targeting sequences, we demonstrate the efficient deletion of numerous gene targets in the model chlamydial strains C. trachomatis L2/434 and C. muridarum. For initial development of the system, we targeted the incA gene of C. trachomatis for deletion. Deletion mutants containing a chloramphenicol resistance marker were recovered within 3 rounds of selection, or 2 rounds of passaging, in McCoy cells and the resulting clones (CTΔincA) were verified by PCR-based genotyping and whole genome sequencing. Phenotypic assessment of host cells infected with CTΔincA was performed by immunofluorescence microscopy and confirmed the lack of IncA expression and the uniform presence of nonfusogenic vacuoles (inclusions) across CTΔincA-infected monolayers. To explore the utility of this system, we deleted 5 additional candidate virulence factors in C. trachomatis and C. muridarum, including deletions of single and multiple genes. We expect lambda Red recombineering to offer a powerful new strategy for making gene deletion and/or replacement mutants in Chlamydia.

Guide to the preparation of speech reports for implanted children: opinion of specialists.

To develop a guide for the preparation of speech-language reports of implanted children to be shared among speech-language pathologists of cochlear implant (CI) services and rehabilitation professionals. The Delphi method was used to select the relevant and fundamental items that should be included in the two versions proposed for the guide: Guide 1 - Speech-language reports provided by the CI services to rehabilitators, and Guide 2 - Speech-language reports provided by the rehabilitators to CI services. Twenty-one speech therapists specialized and with experience in cochlear implants and auditory rehabilitation participated in the discussion and judgment of the items during the selection rounds. Consensus was considered when the item reached agreement equal to or greater than 80% among participants, being selected to compose the two guides. After the two rounds, 21 items from Guide 1 reached consensus among therapists, that is, more than 80% of them agreed that these items should be present in the report sent by the CI service. For Guide 2, 22 items analyzed by speech therapists working in CI services in the postoperative sector were selected in the second round. Based on the analysis of the two rounds, the "Guide for the preparation of speech-language pathology reports: intersection between CI service and rehabilitators" was developed. This material can be applied in the follow-up of implanted children, standardizing the information shared about the electronic device, evaluation results, monitoring of results and therapeutic process of this population.

Guia para a elaboração de relatórios fonoaudiológicos de crianças implantadas: opinião de especialistas

ABSTRACT Purpose To develop a guide for the preparation of speech-language reports of implanted children to be shared among speech-language pathologists of cochlear implant (CI) services and rehabilitation professionals. Methods The Delphi method was used to select the relevant and fundamental items that should be included in the two versions proposed for the guide: Guide 1 - Speech-language reports provided by the CI services to rehabilitators, and Guide 2 - Speech-language reports provided by the rehabilitators to CI services. Twenty-one speech therapists specialized and with experience in cochlear implants and auditory rehabilitation participated in the discussion and judgment of the items during the selection rounds. Consensus was considered when the item reached agreement equal to or greater than 80% among participants, being selected to compose the two guides. Results After the two rounds, 21 items from Guide 1 reached consensus among therapists, that is, more than 80% of them agreed that these items should be present in the report sent by the CI service. For Guide 2, 22 items analyzed by speech therapists working in CI services in the postoperative sector were selected in the second round. Conclusion Based on the analysis of the two rounds, the “Guide for the preparation of speech-language pathology reports: intersection between CI service and rehabilitators” was developed. This material can be applied in the follow-up of implanted children, standardizing the information shared about the electronic device, evaluation results, monitoring of results and therapeutic process of this population.

Evolving membrane-associated accessory protein variants for improved adeno-associated virus production

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1×106 MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

Prototype Guided Pseudo Labeling and Perturbation-based Active Learning for domain adaptive semantic segmentation

This work aims at active domain adaptation to transfer knowledge from a fully-labeled source domain to an entirely unlabeled target domain. During the active learning period, some pixels in the target domain are selected and annotated as active labels through several selection rounds. Such active labels can improve the target domain model performance greatly. However, existing approaches solely rely on pseudo labels, highly-confident classifier predictions on target images, to train the initial target domain model, resulting in a sub-optimal solution for model training. This initial model will be used for active label selection. Meanwhile, previous methods use entropy-based measurement to select pixels for annotation, which fails to detect high-confidence errors in earlier selection rounds due to the absence of target information.To address these issues, we propose a prototype-guided pseudo-label generating approach that leverages the relationships between source prototypes and target features. It generates target pseudo labels based on diverse source prototypes, thereby alleviating the issue of classifier predictions. Furthermore, perturbation-based uncertainty measurement, calculating the discrepancy between the target image and the augmented one, is introduced to find the areas with unstable predictions. Extensive experiments demonstrate that our approach outperforms state-of-the-art active domain adaptation methods on two benchmarks, GTAV → Cityscapes, and SYNTHIA → Cityscapes. Comparable performance is also achieved when compared to fully-supervised methods.

Development of alpha-cypermethrin resistance and its effect on biological parameters of yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culicidae).

Alpha-cypermethrin interacts with the sodium channel and causes nerve blockage in insects. It is used to manage Aedes aegypti (Linnaeus) (Diptera: Culicidae), a primary vector of dengue worldwide. It not only affects both target and non-target organisms, but overuse of this insecticide increases the chances of resistance development in insect pests. In this study, resistance development, biological parameters, and stability of alpha-cypermethrin resistance were studied in a laboratory-selected strain of Ae. aegypti. The alpha-cypermethrin selected strain (Alpha Sel) developed an 11.86-fold resistance level after 12 rounds of alpha-cypermethrin selection compared to the unselected strain (Unsel). In biological parameters, Alpha Sel and Cross1 (Unsel ♂ and Alpha Sel♀) had shorter larval durations compared to Unsel and Cross2 (Unsel ♀ and Alpha Sel ♂) populations. The pupal duration of Alpha Sel and both crosses was shorter than that in the Unsel strain. The relative fitness of Alpha Sel, Cross1, and Cross2 was significantly less than that of the Unsel strain. These results indicate that alpha-cypermethrin resistance comes with fitness costs. Moreover, the frequency of alpha-cypermethrin resistance decreased when the Alpha Sel population was reared without further selection pressure for four generations. So, resistance was unstable and reversed when insecticide pressure ceased. We concluded that the judicious and rotational use of different insecticides with different modes of action and the adoption of other IPM-recommended practices would suppress resistance development for more extended periods in Ae. aegypti.

Selection of DNA aptamers for the aptamer-assisted magnetic capture of the purified xylanase from Aspergillus niger

Xylanases are a group of enzymes that catalyze the hydrolysis of xylan. Xylanases have wide industrial applications, and they can produced by various organisms. In this study, we aimed to develop aptamers for the capture of xylanase produced by a wild-type Aspergillus niger strain. Xylanase was produced by Aspergillus niger in a 5-liter stirred-tank bioreactor and then purified by column chromatography. Magnetic bead-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was performed to select DNA aptamers specific to the purified xylanase. After nine rounds of selection, next-generation sequencing (NGS) analysis was performed. Four aptamers, namely AXYL-1, AXYL-2, AXYL-3, and AXYL-4, were identified for further characterization. The binding properties of the selected aptamers were characterized by fluorescence quenching (FQ) analysis and an enzyme-linked aptamer assay (ELAA). The Kd values were found to be in the low μM range. Then, each aptamer was immobilized on streptavidin-coated magnetic particles, and the recovery ratio of xylanase was determined. Although AXYL-1 wasn't effective, AXYL-2, AXYL-3, and AXYL-4 were proven to capture the xylanase. The maximum recovery rate of xylanase was found to be approximately 54 %.

Phage display selection of a Pb(II) specific peptide and its application as a biorecognition unit for colorimetric detection of Pb(II) ions.

Phage display technology employs a library of engineered filamentous M13 viruses infecting only bacteria. In this study, the genuine phage display selection technique was applied to identify a Pb(II) specific peptide. After three rounds of positive selection against Pb(II) coated agarose-based beads and the consecutive negative screenings against interfering metal ions (Al(III), Co(II), Fe(III), Ni(II), and Zn(II)), a final phage library with enhanced Pb(II) binding was obtained. Enzyme Linked Immunosorbent Assay (ELISA) analyses confirmed the selective Pb(II) binding of the enriched viruses. Twenty monoclonal phage plaques were randomly selected, single stranded DNAs (ssDNAs) were isolated and sequenced. Sequencing results revealed four different peptide sequences. Pb9 peptide (KASPYIT) showing the most specific Pb(II) binding was selected for detection studies. Pb9 was synthetically synthesized with additional three cysteine (3xCys) units at C-terminal. Twenty nanometers AuNPs were functionalized with Pb9-3xCys peptides through Au-thiol (Au-S) interaction. A colorimetric Pb(II) detection system was validated using the engineered peptide-AuNP complex at a calculated LOD of around 11nM (3σ/k, n=6) for the case study. The detection system was Pb(II) selective over various metal ions (Ag(II), Al(III), Au(III), Cd(II), Co(II), Cr (III), Cu(II), Fe(III), Hg(II), Mg(II), Mn(II), Ni(II), and Zn(II)). Such metal ion specific peptides can be further studied to develop simple, user friendly and cost-effective tools to design alternative detection and bioremediation systems for a circular economy.

Selection and application of aptamers for p-hydroxybenzyl hydrogen sulfite after Gastrodia elata Bl. fumigated with sulfur

Gastrodia elata Bl. is a widely used traditional Chinese medicine known for its medicinal properties. However, during the drying process, G. elata is often fumigated with sulfur to prevent corrosion and improve its appearance. Sulfur-fumigation can result in a reduction in the effective components of the herb and can also be hazardous to human health due to the remaining sulfur dioxide. Sulfur-fumigation of G. elata poses a significant challenge to both end-users and researchers. The detection of p-hydroxybenzyl hydrogen sulfite (p-HS) is a useful tool in determining whether G. elata has been fumigated with sulfur. Unfortunately, the current method for detecting p-HS is costly and requires sophisticated instruments. Therefore, there is a need to develop a more cost-effective and user-friendly method for the detection of p-HS. This study utilized the Capture-SELEX technique to screen high-affinity aptamers for p-HS, which were subsequently characterized by isothermal titration calorimetry (ITC). An aptamer sequence (seq 6) with a high affinity of Kd = 26.5 μM was obtained following 8 rounds of selection against p-HS. With the aptamer serving as the recognition element and gold nanoparticles as the colorimetric indicator, a simple and efficient colorimetric sensor was developed for the specific detection of p-HS. This detection method exhibited a limit of detection of 1 μg/ml, while the p-HS recoveries demonstrated a range of between 88.5 % and 105 % for samples of G. elata obtained in the market. In summary, the aptamer exhibited a high affinity for p-HS, and the sensor developed through the use of a colloidal gold detector based on nucleic acid aptamer can be utilized for rapid detection of sulfur-fumigated G. elata. With these findings, this research paper provides valuable scientific insights and highlights significant potential for future studies in this area.