One-step Selection Research Articles

Terminal Alkyne-Modified DNA Aptamers with Enhanced Protein Binding Affinities.

Nucleic acid-based receptors, known as aptamers, are relatively fast to discover and manufacture but lack the diverse functional groups of protein receptors (e.g., antibodies). The binding properties of DNA aptamers can be enhanced by attaching abiotic functional groups; for example, aromatic groups such as naphthalene slow dissociation from proteins. Although the terminal alkyne is a π-electron-rich functional group that has been used in small molecule drugs to enhance binding to proteins through noncovalent interactions, it remains unexplored for enhancing DNA aptamer binding affinity. Here, we demonstrate the utility of the terminal alkyne for improving the binding of DNA to proteins. We prepared a library of 256 terminal-alkyne-bearing variants of HD22, a DNA aptamer that binds the protein thrombin with nanomolar affinity. After a one-step thrombin-binding selection, a high-affinity aptamer containing two alkynes was discovered, exhibiting 3.2-fold tighter thrombin binding than the corresponding unmodified sequence. The tighter binding was attributable to a slower rate of dissociation from thrombin (5.2-fold slower than HD22). Molecular dynamics simulations with enhanced sampling by Replica Exchange with Solute Tempering (REST2) suggest that the π-electron-rich alkyne interacts with an asparagine side chain N-H group on thrombin, forming a noncovalent interaction that stabilizes the aptamer-protein interface. Overall, this work represents the first case of terminal alkynes enhancing the binding properties of an aptamer and underscores the utility of the terminal alkyne as an atom economical π-electron-rich functional group to enhance binding affinity with minimal steric perturbation.

CDSM: C ascaded D eep S emantic M atching on Textual Graphs Leveraging Ad-hoc Neighbor Selection

Deep semantic matching aims at discriminating the relationship between documents based on deep neural networks. In recent years, it becomes increasingly popular to organize documents with a graph structure, then leverage both the intrinsic document features and the extrinsic neighbor features to derive discrimination. Most of the existing works mainly care about how to utilize the presented neighbors, whereas limited effort is made to filter appropriate neighbors. We argue that the neighbor features could be highly noisy and partially useful. Thus, a lack of effective neighbor selection will not only incur a great deal of unnecessary computation cost but also restrict the matching accuracy severely. In this work, we propose a novel framework, C ascaded D eep S emantic M atching ( CDSM ), for accurate and efficient semantic matching on textual graphs. CDSM is highlighted for its two-stage workflow. In the first stage, a lightweight CNN-based ad-hod neighbor selector is deployed to filter useful neighbors for the matching task with a small computation cost. We design both one-step and multi-step selection methods. In the second stage, a high-capacity graph-based matching network is employed to compute fine-grained relevance scores based on the well-selected neighbors. It is worth noting that CDSM is a generic framework which accommodates most of the mainstream graph-based semantic matching networks. The major challenge is how the selector can learn to discriminate the neighbors’ usefulness which has no explicit labels. To cope with this problem, we design a weak-supervision strategy for optimization, where we train the graph-based matching network at first and then the ad-hoc neighbor selector is learned on top of the annotations from the matching network. We conduct extensive experiments with three large-scale datasets, showing that CDSM notably improves the semantic matching accuracy and efficiency thanks to the selection of high-quality neighbors. The source code is released at https://github.com/jingjyyao/CDSM.

P-084 Novel Single-Step Microfluidic Chamber Outperforms Density Gradient Centrifugation in Post-Selection Motility And DNA Integrity

Abstract Study question Does sperm selection by a novel single-step microfluidic chamber offer effective selection of motile sperm without DNA damage compared with Density Gradient Centrifugation (DGC)? Summary answer The novel single-step microfluidic chamber is a good alternative to conventional DGC, increasing DNA integrity and progressive sperm motility in recovered sperm populations. What is known already High levels of sperm DNA fragmentation (SDF) can have negative impacts on reproductive outcomes such as clinical pregnancy and miscarriage rates. Microfluidic sperm selection technologies have been developed to select sperm with lower level of SDF and higher levels of motility to improve Assisted Reproductive technology (ART) outcomes. While these methods have achieved variable success, very few have proven clinically relevant and fail to perform selection in a robust and simple methodology that can be adopted in clinics. Study design, size, duration A prospective cohort study including 21 donated semen samples processed in 2021. Each sample was split to perform DGC and microfluidic sperm selections side-by-side. Laboratory outcomes were evaluated using semen parameters pre and post processing and included concentration, motility, and DNA fragmentation index (DFI). Participants/materials, setting, methods The study was performed using samples donated at the Department of Obstetrics and Gynaecology, Monash University, Melbourne, Australia. DGC (10 minutes at 300g and 5 minutes wash at 300g) and microfluidic sperm selection (one-step sperm selection;15-minute incubation for selection) were performed with Sydney IVF 80/40 gradients and gamete buffers, respectively. DFI was assessed by chromatin sperm dispersion assay (SCD). Results were analysed by Friedman’s test and differences were considered significant when p-value < 0.05. Main results and the role of chance For the unprocessed raw semen, the starting DFI values varied from 3.0 to 23.3% with an average of 11.38% ± 5.51 and motility varied between 25.3% and 86.7% with an average of 56.42% ± 18.57. The device consistently outperformed DGC in all 21 samples for DFI (0.94% ± 1.12 vs 4.87% ±3.69, P = 0.0012) and in 20 of the 21 samples for motility post-processing DGC (90.88% ± 6.8 vs 73.58% ± 14.97, P = 0.0061). The average concentrations of the raw, DGC, and microfluidic sperm samples were 94.5, 74.12, and 3.55 million sperm/mL, respectively. Thus, both DGC and the microfluidic device showed improvements in SDF, motility, and concentration over the raw sample. However, the microfluidic device significantly outperformed DGC in terms of percentage SDF and motility, indicating its potential for clinical implementation. Limitations, reasons for caution This cohort study did not include samples from infertile patients. Furthermore, the effect of each sperm selection methods on reproductive outcomes was not a part of the study. A larger number of samples across a range of clinically infertile samples is required to fully characterise the microfluidic devices efficacy. Wider implications of the findings The microfluidic device we report here has the potential to become a new tool to improve the efficacy and consistency of sperm selections. When compared to DGC it offers improved sperm motility and DNA integrity from a platform that is simple to use and less skill-based than conventional methods. Trial registration number not applicable

Monoclonal Antibody Discovery Based on Precise Selection of Single Transgenic Hybridomas with an On-Cell-Surface and Antigen-Specific Anchor.

Hybridoma technology is widely used for monoclonal antibody (mAb) discovery, whereas the generation and identification of single hybridomas by the limiting dilution method (LDM) are tedious, inefficient, and time- and cost-consuming, especially for hapten molecules. Here, we describe a single transgenic hybridoma selection method (STHSM) that employs a transgenic Sp2/0 with an artificial and stable on-cell-surface anchor. The anchor was designed by combining the truncated variant transmembrane domain of EGFR with a biotin acceptor peptide AVI-tag, which was stably integrated into the genome of Sp2/0 via a piggyBac transposon. To ensure the subsequent precise selection of the hybridoma, the number of on-cell-surface anchors of the transfected Sp2/0 for fusion with immunized splenocytes was further normalized by flow cytometry at the single cell level. Then the single antigen-specific transgenic hybridomas were precisely identified and automatically selected using a CellenONE platform based on the fluorescence assay of the on-cell-surface anchor with the corresponding secreted antigen-specific mAb. The STHSM produced 579 single chloramphenicol (CAP)-specific transgenic hybridomas with a positive rate of 62.7% in 10 plates within 2 h by one-step selection, while only 12 single CAP-specific hybridomas with a positive rate of 6.3% in 40 plates required at least 32 days using the LDM with multiple subcloning steps. The best affinity of mAbs from the STHSM was more than 2-fold higher than that of those from the LDM, and this was mainly due to the preaffinity selection based on the on-cell-surface anchors and more interactions between the mAb and CAP. Then the mAbs from the STHSM and LDM were used to develop an immunoassay for CAP in spiked and natural biological samples. The method displayed satisfactory sensitivity, accuracy, and precision, demonstrating that the STHSM we developed is a versatile, practical, and efficient method for mAb discovery.

Design of Multipartite Transcription Factors for Multiplexed Logic Genome Integration Control in Mammalian Cells.

Synthetic biology relies on rapid and efficient methods to stably integrate DNA payloads encoding for synthetic biological systems into the genome of living cells. The size of designed biological systems increases with their complexity, and novel methods are needed that enable efficient and simultaneous integration of multiple payloads into single cells. By assembling natural and synthetic protein–protein dimerization domains, we have engineered a set of multipartite transcription factors for driving heterologous target gene expression. With the distribution of single parts of multipartite transcription factors on piggyback transposon-based donor plasmids, we have created a logic genome integration control (LOGIC) system that allows for efficient one-step selection of stable mammalian cell lines with up to three plasmids. LOGIC significantly enhances the efficiency of multiplexed payload integration in mammalian cells compared to traditional cotransfection and may advance cell line engineering in synthetic biology and biotechnology.

A model selection approach to jointly testing for structural breaks and cointegration with application to the Eurocurrency interest rates market

All tests involving both structural breaks and cointegration are parametric. As a complement to the classical hypothesis testing for empirical researchers, we suggest the use of a one-step model selection approach to simultaneously specifying lag orders, cointegration ranks, and structural breaks. The performances of the four popular information criteria along with a LM-based parametric test are shown in extensive simulation studies. Applying the approach to study linkages in the Eurocurrency interest rates market, we find that six major short-term rates were subject to a structural break and the cointegration rank also changed following the break.

Pre-Emptive Donor-Derived Innate Immune Cell Add-Back after Haploidentical Hematopoietic Stem Cell Transplantation to Reduce High Rate of Infection, Gvhd, and Non-Relapse Mortality

<h3>Introduction</h3> Post-transplant cyclophosphamide has allowed alloHSCT using haploidentical donors. PTCy is very effective at reducing GVHD-causing T cells, but it has the unwanted effect of eliminating Natural Killer cells. We have developed a new reduced-intensity haploHSCT transplant conditioning regimen based on an a regimen developed for fully-matched donors with lymphoma: bendamustine 130 mg/m2 IV daily, and fludarabine 30 mg/m2 IV daily, for 3 days, +/- rituximab. We made additional modifications designed to improve NK immune recovery: 1.) no mycophenolate, 2.) no routine G-CSF, 3.) early tacrolimus taper starting on day +60, 4.) pre-emptive NK-enriched donor lymphocyte infusion on Day +8. <h3>Objectives</h3> Conduct a prospective clinical trial to evaluate the safety of bendamustine-fludarabine conditioning, PTCy, NK cell add-back, and short course tacrolimus for patients with myeloma or lymphoma undergoing haploHSCT. Assess clinical outcomes over the first year after transplantation. Efficacy Endpoints: Death or engraftment failure at D+30, death or severe chronic extensive GVHD at D+100. Test rate of survival, engraftment, and GVHD. Measure reconstitution of adaptive and innate lymphocyte subsets. Measure NK KIR repertoire recovery and assess for ‘missing self'. <h3>Methods</h3> This is a Phase I, single center, single cohort, open-label, proof-of-concept clinical trial to evaluate the safety of the BFRHaplo regimen (Figure 1) and preemptive CD56-selected DLI following PTCy for adults with myeloma or lymphoma. <h3>Results</h3> We have safety data on six patients (Table 1). Recovery of neutrophils and platelets is prompt. No primary or secondary graft failures have occurred. No patients have developed Grade III-IV or steroid-refractory acute GVHD. No organ toxicities, and no alkylator-alkylator toxicity from bendamustine – PTCy, have been observed. No instances of either veno-occlusive disease or transplant-related thrombotic microangiopathy, and no deaths, serious infections, or re-hospitalizations. One patient relapsed and is alive without GVHD receiving salvage therapy. We used single-step CD56 selection to prepare CD56-enriched donor lymphocyte infusions (Table 2). The one-step CD56 selection method has the attractive property of preserving CD3+/CD56+ cells, including NK/T and gdT. The target cell dose (> 1 million CD56+ cells/kg and < 1 million T cells/kg) was achieved for all patients. <h3>Conclusion</h3> The trial will establish whether BFRHaplo, and the infusion of cytotoxic NK and some T cells (< 1million/kg) without mycophenolate, is safe. Completion of this Phase I trial will lead to double-blind, placebo- controlled studies to assess the efficacy of CD56-selected immune cell add-back after PTCy in reducing the high rate of infection, GVHD, and mortality following haploidentical stem cell transplantation.

Functional proteomic atlas of HIV infection in primary human CD4+ T cells.

Viruses manipulate host cells to enhance their replication, and the identification of cellular factors targeted by viruses has led to key insights into both viral pathogenesis and cell biology. In this study, we develop an HIV reporter virus (HIV-AFMACS) displaying a streptavidin-binding affinity tag at the surface of infected cells, allowing facile one-step selection with streptavidin-conjugated magnetic beads. We use this system to obtain pure populations of HIV-infected primary human CD4+ T cells for detailed proteomic analysis, and quantitate approximately 9000 proteins across multiple donors on a dynamic background of T cell activation. Amongst 650 HIV-dependent changes (q < 0.05), we describe novel Vif-dependent targets FMR1 and DPH7, and 192 proteins not identified and/or regulated in T cell lines, such as ARID5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of host factors subverted by the virus in its natural target cell.

Ideal-Filter Capillary Electrophoresis (IFCE) Facilitates the One-Step Selection of Aptamers.

Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one-step selection of aptamers. We introduce partitioning by ideal-filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE-based partitioning reaches 109 , which is ten million times higher than that of typical solid-phase partitioning methods. One step of IFCE-based partitioning is sufficient for the selection of a high-affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.

Identification of CDC25-P1306L, a novel mutant allele of CDC25, conferring tolerance to multiple stresses associated with food production on Saccharomyces cerevisiae

Mutations conferring tolerance to diverse stresses (i.e. multistress tolerance) on budding yeast Saccharomyces cerevisiae are useful for industrial yeast strains such as baker’s and wine yeast. However, little is known about the mutations conferring multistress tolerance. Previously, we developed a simple method for isolating multistress-tolerant semidominant mutants of S. cerevisiae by one-step selection under lethal hydrogen peroxide stress, which we named the LCH method. In this study, we applied a combination of genetics and next-generation sequencing (NGS) technology to identify the causal mutation for multistress tolerance of the mutant isolated using the LCH method. The haploid mutant strain was crossed with the wild-type strain and the resulting diploids were sporulated. The 20 haploid progeny strains showing multistress tolerance were mixed and subjected to DNA extraction for NGS. CDC25-P1306L, a novel mutant allele of CDC25 encoding a Ras guanine nucleotide exchange factor, was detected 86 times; however, a wild-type CDC25 allele was not detected in the NGS data from the mixture of the multistress-tolerant progeny strains, suggesting that all of the progeny strains showing multistress tolerance have a CDC25-P1306L allele instead of a wild-type CDC25 allele. Substitution of CDC25 in the wild-type strain with CDC25-P1306L rendered the strain tolerant to ethanol, heat shock, freeze-thaw, chronological aging and high concentrations of glucose. These results indicate that CDC25-P1306L is a multistress-tolerant mutation and is promising for breeding multistress-tolerant S. cerevisiae strains for food production.

Cost-efficient community-driven breeding programs for local pig breeds in Northwest Vietnam

In villages of Northwest Vietnam, a community-based breeding program is being implemented that serves distinct supply chains for pork from local pig breeds. The present study evaluated different selection scenarios for an effective breeding program under the specific framework conditions of smallholder pig production in the uplands of Northwest Vietnam. One- and two-step selection scenarios were considered for two nucleus populations of the local Ban and Mong Cai breeds. Two scenarios additionally considered the use of marker information on the estrogen receptor gene (ESR) in the Ban breed. The breeding planning calculations were performed with the software ZPLAN which is based on the gene flow and selection index methodology. In a deterministic approach, ZPLAN calculates genetic gains for breeding objective traits as well as the breeding profit sow−1 in a defined period. Selected input parameters were varied to identify optimum levels to ensure the profitability of the breeding program. The most promising option with regard to economic viability and acceptable genetic gains is two-step selection. However, the population size in the breeding program has to be increased. One-step selection can also lead to acceptable genetic gains and economic success, but again only by increasing the population size parallel to a reduction in the nucleus size as well as the productive lifetime of nucleus sows. Using marker information on the ESR gene for selection in the Ban breed in the first selection step improves the genetic trend in number of piglets born alive (NBA). The use of marker information in the breeding program, however, seems questionable due to high costs, particularly because conventional index selection is supposed to also increase the frequency of the preferred ESR B allele in the Ban breed.

Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

Compressive Sensing-Inspired Dual-Sparse SLFNN for Hyperspectral Imagery Classification

In this letter we explore the sparse sensing and learning mechanism of the human visual system, to propose a dual-sparse single-hidden-layer feedforward neural network (SLFNN) for the hyperspectral imagery classification. Firstly a large SLFNN is randomly initialized and trained by an extreme learning algorithm, and then the input and hidden neurons are simultaneously reduced by imposing a sparse constraint on the weights of the network. Then a saliency map is derived via the recent developed compressive sensing theory, and a joint sparse optimization approach is proposed to realize a one-step rapid selection of significant neurons. The reduction of input neurons can realize an automatic band-subset-selection of hyperspectral bands to remove the redundancy of hyperspectral vectors, and the reduction of hidden neurons can avoid the high computational cost at runtime and potential overfitting. Some experiments are taken on AVIRIS imagery data to investigate the performance of the proposed method, and the results show that it can achieve accurate and rapid classification.

Comprehensive Identification of Mutations Responsible for Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA)-to-VISA Conversion in Laboratory-Generated VISA Strains Derived from hVISA Clinical Strain Mu3

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10(-6) or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.

Production of Monoclonal Antibodies

This unit describes the production of monoclonal antibodies beginning with immunization, cell fusion, and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one-step selection and cloning of hybridomas utilizing a semi-solid methylcellulose-based medium (ClonaCell-HY from StemCell Technologies).

A New Simple Method for Isolating Multistress-Tolerant Semidominant Mutants of &lt;i&gt;Saccharomyces cerevisiae&lt;/i&gt; by One-Step Selection under Lethal Hydrogen Peroxide Stress Condition

Tolerance of microorganisms to diverse stresses (i.e., multistress tolerance) is a very useful property with industrial applications. We have developed a simple method for isolating multistress-tolerant semidominant mutants of the budding yeast Saccharomyces cerevisiae by one-step selection under lethal hydrogen peroxide (H(2)O(2)) stress condition, which we named the lethal concentration of H(2)O(2) (LCH) method. This method involves simply isolating colonies after plating of mutagenized S. cerevisiae cells, which are cultivated overnight in liquid media, on agar plates containing a lethal concentration of H(2)O(2) for the wild-type strain. Phenotypic and genetic analyses of the ten strains isolated by this method revealed that two strains exhibiting stress tolerance to H(2)O(2), ethanol, heat shock, salt, organic solvent, freeze-thaw, chronological aging, and high concentrations of glucose possess semidominant and distinct single-gene mutations designated as MLT1-1 (multistress tolerance) and MLT2-1, which are responsible for multistress tolerance. From these results, we expect this method to confer multistress tolerance on industrial yeasts.

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

BackgroundNucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.Principal FindingsHerein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.ConclusionWe have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

Antibiotic susceptibility of coagulase-negative staphylococci (CoNS): emergence of teicoplanin-non-susceptible CoNS strains with inducible resistance to vancomycin

Coagulase-negative staphylococci (CoNS) have become increasingly recognized as important agents of nosocomial infection. One of the characteristics of CoNS is their resistance to multiple antimicrobial agents commonly used for the treatment of staphylococcal infections. CoNS strains (n = 745) isolated from a university teaching hospital in China between 2004 and 2009 were tested for antibiotic resistance. The antibiotics were placed into three categories based on resistance levels of the CoNS strains to these antibiotics: high resistance (resistance rate >70 %), including penicillin G, oxacillin and erythromycin; medium resistance (resistance rate between 30 and 70 %), including tetracycline, clindamycin, ciprofloxacin, trimethoprim/sulfamethoxazole and chloramphenicol; and low resistance (resistance rate <30 %), including rifampicin, ceftizoxime and gentamicin. We also found that the prevalence of strains non-susceptible to teicoplanin increased from 4.5 to 6.7 % between 2008 and 2009. A one-step vancomycin agar selection experiment and subsequent population analysis revealed potentially vancomycin-resistant subpopulations that have been selected from the teicoplanin-non-susceptible strains. Vigilant surveillance of nosocomial isolates of CoNS is needed to determine their resistance to glycopeptides.

B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies

The recent development of screening strategies based on the generation and display of large libraries of antibody fragments has allowed considerable advances for the in vitro isolation of monoclonal antibodies (mAbs). We previously developed a technology referred to as the ‘ADLib (Autonomously Diversifying Library) system’, which allows the rapid screening and isolation in vitro of antigen-specific monoclonal antibodies (mAbs) from libraries of immunoglobulin M (IgM) displayed by the chicken B-cell line DT40. Here, we report a novel application of the ADLib system to the production of chimeric human mAbs. We have designed gene knock-in constructs to generate DT40 strains that coexpress chimeric human IgG and chicken IgM via B-cell-specific RNA alternative splicing. We demonstrate that the application of the ADLib system to these strains allows the one-step selection of antigen-specific human chimeric IgG. In addition, the production of chimeric IgG can be selectively increased when we modulate RNA processing by overexpressing the polyadenylation factor CstF-64. This method provides a new way to efficiently design mAbs suitable for a wide range of purposes including antibody therapy.

SELECCIÓN MULTICRITERIO DE CONTRATISTAS DE OBRAS. ENFOQUE BASADO EN REDES NEURONALES

This paper presents two cases of applying neural networks to extract knowledge for, subsequently, using it to support multicriteria contractor selection, in traditional design-bid-build projects with one-step selection processes. Different qualitative and quantitative selection criteria are taken into account, up to 22 and 9, respectively. The ? rst case includes a high number of input variables, making up a complex system related to complex and medium or large-sized projects. The second case is related to small projects in a medium-sized municipality. One advantage of these systems is that they can serve to ‘homogenize’ speci? c decision making in medium and large organizations. The paper also analyzes other pros of this approach, as well as the main problems.