Scalable CRISPR Screens in Zebrafish Using MIC-Drop.

CRISPR-Cas9 is a powerful tool to interrogate gene function in a targeted and systematic manner. Although the technology has been scaled up for large-scale genetic screens in cell culture, similar scale screens in vivo have been extremely challenging due to the cost, labor, and time required to generate and keep track of thousands of mutant animals. We reported the development of Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform that makes large-scale reverse genetic screens possible in zebrafish. In this chapter, we provide a detailed protocol to conduct large-scale genetic screens using this novel platform.

A NIR ratiometric fluorescent probe for the rapid detection of hydrogen sulfide in living cells and zebrafish.

Hydrogen sulfide (H2S) acts as a gas transporter and cell protector and plays a role in a number of disorders and signaling processes. Given that the half-life of H2S in biological systems is between seconds and minutes, the development of rapid and accurate technologies for reliable monitoring H2S levels and dynamics in organisms is critical. However, it is still difficult to design innovative near-infrared fluorescent probes that can quickly and accurately detect H2S. Here, we constructed a novel NIR ratiometric fluorescent probe based on the "aldehyde group auxiliary strategy", Cy-H2S, for the quantitative detection and precise imaging of H2S in living cells and zebrafish. Cy-H2S responded quickly (150 s) and was highly sensitive (0.179 µM) to H2S donor. Cy-H2S was further successfully employed to track endogenous H2S fluctuation in HCT116 cells and zebrafish and evaluated the release efficiency of the H2S prodrug in a NIR ratiometric imaging way. Cy-H2S has the potential to be used as a reliable indication of H2S levels in living cells and zebrafish, as well as an innovative and practical instrument for furthering the physiological research of H2S, which will encourage the creation of advanced NIR ratiometric probes for a variety of biological applications.

Teleconsulta de enfermagem: desenvolvimento de plataforma para atendimento de casos de Covid-19 / Nursing teleconsultation: development of a platform for Covid-19 case management / Telecosulta en enfermería: desarrollo de una plataforma para el manejo de casos de Covid-19

Objetivo: Desenvolver uma plataforma virtual de Teleconsulta para atendimento a casos suspeitos de Síndromes Gripais e infecção por COVID-19. Metodologia: Trata-se de um estudo de natureza aplicada, com desenvolvimento de produção tecnológica e inovadora, prospectivo, ecológico, descritivo, de série temporal. A população do estudo foi formada por qualquer pessoa sintomática para Síndromes Gripais por COVID-19, suspeitos ou confirmados, de qualquer local do Brasil. Este estudo foi realizado em duas etapas, a saber: Etapa I: Desenvolvimento da Aplicação para Plataforma de Teleconsulta. Etapa II: atendimento por meio de Teleconsulta de Casos suspeitos de COVID-19 e Sindromes Gripais. A metodologia utilizada para o desenvolvimento da aplicação proposta foi a modelagem por prototipação evolucionária. Resultados: Foram realizados 209 atendimentos na Plataforma de Teleconsulta, sendo 151 (70%) do sexo feminino e 65 (30%) do sexo masculino, com prevalência de idade variando de 20 a 29 anos (41%). Quanto ao risco de infecção por COVID-19, 42 (20%) tinham alto risco, 75 (36%) médio risco e 92 (44%) baixo risco. Os sintomas mais prevalentes foram: secreção nasal ou espirros (53%), dores no corpo (49%), dor de cabeça (47%), dor de garganta (46%), tosse seca (35%), Febre (31%), falta de ar (25%) e diarreia (23%). Inicialmente o teleatendimento foi composto por teletriagem com classificação de risco com base na sintomatologia dos pacientes que foram codificados com pontuações conforme a gravidade do sintoma para formas graves de COVID-19. A classificação de risco categorizou os pacientes em risco baixo (1 a 9 pontos), risco médio (10 a 19 pontos) e risco alto (20 a 36 pontos). Em seguida, a teleconsulta foi agendada conforme disponibilidade do paciente por meio do método SBAR para comunicação efetiva e ao término do atendimento um plano de cuidados com Sistematização da Assistência de Enfermagem ­ SAE era encaminhado ao paciente por meio de WhatsApp ou e-mail. Conclusão: A plataforma de teleconsulta possibilitou a triagem dos pacientes, reduziu as visitas desnecessárias às unidades de emergência, permitiu a avaliação e monitoramento dos casos, bem como o acompanhamento de pacientes ambulatoriais que não necessitam de avaliação presencial.

Objective: To develop a virtual Teleconsultation platform for care of suspected cases of influenza syndromes and infection by COVID-19. Methodology: This is a study of applied nature, with development of technological and innovative production, prospective, ecological, descriptive, time series. The study population was made up of any person symptomatic for COVID-19 influenza syndromes, suspected or confirmed, from any location in Brazil. This study was conducted in two stages, namely: Stage I: Development of the Application for Teleconsultation Platform. Stage II: care through Teleconsultation of suspected cases of COVID-19 and influenza syndromes. The methodology used to develop the proposed application was evolutionary prototyping modeling. Results: There were 209 consultations in the Teleconsultation Platform, 151 (70%) were female and 65 (30%) were male, with prevalence of age ranging from 20 to 29 years (41%). As for the risk of infection by COVID-19, 42 (20%) had high risk, 75 (36%) medium risk and 92 (44%) low risk. The most prevalent symptoms were: nasal discharge or sneezing (53%), body aches (49%), headache (47%), sore throat (46%), dry cough (35%), fever (31%), shortness of breath (25%), and diarrhea (23%). Initially, the telecare was composed of teletry with risk classification based on the symptomatology of the patients who were coded with scores according to symptom severity for severe forms of COVID-19. The risk classification categorized patients into low risk (1 to 9 points), medium risk (10 to 19 points), and high risk (20 to 36 points). Then, the teleconsultation was scheduled according to the patient's availability through the SBAR method for effective communication and at the end of the service a care plan with Nursing Assistance Systematization - SAE was forwarded to the patient through WhatsApp or e-mail. Conclusion: Teleconsultation platform enabled patient triage, reduced unnecessary visits to emergency units, allowed the evaluation and monitoring of cases, as well as the follow- up of outpatients who do not need face-to-face evaluation.

Objetivo: Desarrollar una plataforma de Teleconsulta virtual para atender casos sospechosos de síndromes gripales e infección por COVID-19. Metodología: Se trata de un estudio aplicado, con desarrollo de producción tecnológica e innovadora, prospectivo, ecológico, descriptivo, con serie de tiempo. La población de estudio estuvo formada por cualquier persona sintomática de síndromes gripales por COVID-19, sospechada o confirmada, de cualquier localidad de Brasil. Este estudio se realizó en dos etapas, a saber: Etapa I: Desarrollo de Aplicaciones para la Plataforma de Teleconsulta. Etapa II: atención mediante teleconsulta de casos sospechosos de COVID-19 y síndromes gripales. La metodología utilizada para el desarrollo de la aplicación propuesta fue el modelado por prototipo evolutivo. Resultados: Se realizaron 209 consultas en la Plataforma de Teleconsulta, 151 (70%) del sexo femenino y 65 (30%) del masculino, con prevalencia de edades entre 20 a 29 años (41%). En cuanto al riesgo de infección por COVID-19, 42 (20%) fueron de alto riesgo, 75 (36%) de riesgo medio y 92 (44%) de bajo riesgo. Los síntomas más prevalentes fueron: secreción nasal o estornudos (53%), dolor de cuerpo (49%), dolor de cabeza (47%), dolor de garganta (46%), tos seca (35%), fiebre (31%), falta de aliento (25%) y diarrea (23%). Inicialmente, la teleasistencia consistía en teleselección con clasificación de riesgo en función de la sintomatología de los pacientes a los que se codificaba con puntuaciones según la gravedad del síntoma para formas graves de COVID-19. La clasificación de riesgo clasificó a los pacientes en riesgo bajo (1 a 9 puntos), riesgo medio (10 a 19 puntos) y riesgo alto (20 a 36 puntos). Luego, se programó la teleconsulta de acuerdo a la disponibilidad del paciente a través del método SBAR para una comunicación efectiva y al final de la atención se remitió al paciente un plan de cuidados con Sistematización de Atención de Enfermería - SAE vía WhatsApp o correo electrónico. Conclusión: La plataforma de teleconsulta posibilitó el triaje de pacientes, redujo las visitas innecesarias a las unidades de emergencia, permitió la evaluación y seguimiento de casos, así como el seguimiento de pacientes ambulatorios que no requieren evaluación presencial.

Construction of Human Immune and Naive scFv Phage Display Libraries.

Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such high-quality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications.

Antibody Selection via Phage Display in Microtiter Plates.

The most common and robust in vitro technology to generate monoclonal human antibodies is phage display. This technology is a widely used and powerful key technology for recombinant antibody selection. Phage display-derived antibodies are used as research tools, in diagnostic assays, and by 2022, 14 phage display-derived therapeutic antibodies were approved. In this review, we describe a fast high-throughput antibody (scFv) selection procedure in 96-well microtiter plates. The given detailed protocol allows the antibody selection ("panning"), screening, and identification of monoclonal antibodies in less than 2 weeks. Furthermore, we describe an on-rate panning approach for the selection of monoclonal antibodies with fast on-rates.

High-Throughput IgG Reformatting and Expression Using Hybrid Secretion Signals and InTag Positive Selection Technology.

We have previously published protocols for high-throughput IgG reformatting and expression, that enable rapid reformatting of phage-displayed antibody Fab fragments into a single dual expression vector for full IgG expression in Expi293F cells (Chen et al. Nucleic Acids Res 42:e26, 2014; Chen et al. Methods in Molecular Biology, vol 1701, 2018). However, when working with phage clones from a naïve library containing highly diverse N-terminal sequences, where the 5' PCR primers bind, the PCR step can become cumbersome. To overcome this limitation, we have investigated and found that the C-terminal 7 amino acid residues of the human antibody VH1 secretion signal can be replaced with those from ompA or pelB bacterial signals to form hybrid signal sequences that can drive strong IgG expression in Expi293F cells. The use of such hybrid signals allows any Fab fragment in the library to be amplified and cloned into the IgG expression vector using only a single 5' PCR primer targeting the bacterial secretion signal of the light or heavy chain, thus dramatically simplifying the IgG reformatting workflow.

Deep Mining of Complex Antibody Phage Pools.

An important, and rapidly growing class of drugs are antibodies which can be discovered through phage display technology. In this technique, antibodies are typically first enriched through consecutive rounds of selection on a target antigen with amplification in bacteria between each selection round. Thereafter, a subset of random individual clones is analyzed for binding in a screening procedure. This results in discovery of the most abundant antibodies in the pool. However, there are multiple factors affecting the enrichment of antibodies during the selection resulting in a very complex output pool of antibodies. A few antibodies are present in many copies and others only in a few copies, where the most abundant antibodies are not necessarily the functionally best ones. In order to utilize the full potential of the output from a phage display selection, and enable discovery of low abundant, potentially functionally important clones, deep mining technologies are needed. In this chapter, two methods for deep mining of an antibody pool are described, protein depletion and antibody blocking. The methods can be applied both when the target is a single antigen and on complex antigen mixtures such as whole cells and tissues.

Antibody Selection in Solution Using Magnetic Beads.

Antibody phage display is a valuable in vitro technology to generate recombinant, sequence-defined antibodies for research, diagnostics, and therapy. Up to now (autumn 2022), 14 FDA/EMA-approved therapeutic antibodies were developed using phage display, including the world best-selling antibody adalimumab. Additionally, recombinant, sequence-defined antibodies have significant advantages over their polyclonal counterparts.For a successful in vitro antibody generation by phage display, a suitable panning strategy is highly important. We present in this book chapter the panning in solution and its advantages over panning with immobilized antigens and give detailed protocols for the panning and screening procedure.

Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering.

Next-generation DNA sequencing (NGS) technologies have made it possible to interrogate antibody repertoires to unprecedented depths, typically via sequencing of cDNAs encoding immunoglobulin variable domains. In the absence of heavy-light chain pairing, the variable domains of heavy chain-only antibodies (HCAbs), referred to as single-domain antibodies (sdAbs), are uniquely amenable to NGS analyses. In this chapter, we provide simple and rapid protocols for producing and sequencing multiplexed immunoglobulin variable domain (VHH, VH, or VL) amplicons derived from a variety of sources using the Illumina MiSeq platform. Generation of such amplicon libraries is relatively inexpensive, requiring no specialized equipment and only a limited set of PCR primers. We also present several applications of NGS to sdAb discovery and engineering, including: (1) evaluation of phage-displayed sdAb library sequence diversity and monitoring of panning experiments; (2) identification of sdAbs of predetermined epitope specificity following competitive elution of phage-displayed sdAb libraries; (3) direct selection of B cells expressing antigen-specific, membrane-bound HCAb using antigen-coupled magnetic beads and identification of antigen-specific sdAbs, and (4) affinity maturation of lead sdAbs using tandem phage display selection and NGS. These methods can easily be adapted to other types of proteins and libraries and expand the utility of in vitro display technology.

The ChIP-Hub Resource: Toward plantEncode.

Recent advances in sequencing technologies lead to the generation of an enormous amount of regulome and epigenome data in a variety of plant species. However, a comprehensive standardized resource is so far not available. In this chapter, we present ChIP-Hub, an integrative platform that has been developed based on the ENCODE standards by collecting and reanalyzing regulatory genomic datasets from 41 plant species. The ChIP-hub website is introduced in this chapter, including information on detailed steps of searching, data download, and online analyses, which facilitates users to explore ChIP-Hub. We also provide a cross-species comparison of chromatin accessibility information that gives a thorough view of evolutionary regulatory networks in plants.

Untargeted Proteomics and Metabolomics Analysis of Plant Organ Development.

Our understanding of major developmental transitions in plants and animals has been transformed by the emergence of omics technologies. The majority of leaf growth research has been conducted at the transcriptional level. Although historically understudied, alterations at the protein and metabolite levels have begun to gain traction in recent years. Here, we present a protocol for metabolite and protein extraction followed by untargeted metabolomics and proteomics analysis of the growing leaves.

[Gene therapy: Where are we? Where are we going?] / Terapia génica: ¿dónde estamos?, ¿a dónde vamos?

Gene therapy has achieved significant advancements in the treatment of genetic diseases, especially in rare and monogenic diseases. Gene therapies have been developed and approved to treat diseases such as spinal muscular atrophy, offering hope to patients and demonstrating the effectiveness of this therapy. Currently, numerous clinical trials are being conducted to evaluate the safety and efficacy of gene therapy in various diseases, particularly in the field of pediatric neurology. These studies are generating encouraging data and contributing to the knowledge on how to improve gene therapy techniques. Despite the advancements, gene therapy faces significant challenges. It is a costly and technically complex therapy, limiting its accessibility. Additionally, aspects such as efficient gene delivery, immune response to vectors, and duration of therapeutic response require improvements and are actively being investigated. Regarding the future of gene therapy, advances in gene editing technology, such as CRISPR-Cas9, are expected to allow for greater precision and efficiency in gene modification. Research on gene therapy vectors is expected to enhance the delivery capacity and safety of treatments. New generations of viral and non-viral vectors are being developed that could overcome current limitations and enable more efficient and precise administration of therapeutic genes.

La terapia génica ha logrado avances significativos en el tratamiento de enfermedades genéticas, especialmente en enfermedades raras y monogénicas. Se han desarrollado y aprobado terapias génicas para tratar enfermedades como la atrofia muscular espinal, brindando esperanza a los pacientes y demostrando la eficacia de esta terapia. Actualmente, se están realizando numerosos ensayos clínicos para evaluar la seguridad y eficacia de la terapia génica en diversas enfermedades, particularmente en el campo de la neurología pediátrica. Estos estudios están generando datos alentadores y contribuyen al conocimiento sobre cómo mejorar las técnicas de terapia génica. A pesar de los avances, la terapia génica enfrenta desafíos importantes. Es una terapia costosa y técnicamente compleja, lo que limita su accesibilidad. Además, aspectos como la entrega eficiente de genes, la respuesta inmunológica a los vectores y la duración de la respuesta terapéutica requieren mejoras. se está investigando activamente. En cuanto al futuro de la terapia génica, se espera que los avances en tecnología de edición génica, como CRISPR-Cas9, permitan una mayor precisión y eficiencia en la modificación de genes. Se espera que la investigación en vectores de terapia génica mejore la capacidad de entrega y la seguridad de los tratamientos. Se están desarrollando nuevas generaciones de vectores virales y no virales que podrían superar las limitaciones actuales y permitir una administración más eficiente y precisa de genes terapéuticos.

Phase-appropriate Application of Process Analytical Technology for Early Pharmaceutical Development of Oral Solid Dosage Forms-the Case Study of Uniformity Screening of Dosage Units and Blends.

Process analytical technology (PAT) in late-stage drug product development is typically used for real-time process monitoring, in-process control, and real-time release testing. In early research and development (R&D), PAT usage is limited as the manufacturing scale is relatively small with frequent changes and only a few batches are produced on an annual basis. However, process understanding is critical at early R&D in order to identify process and formulation boundaries, so PAT applications could be particularly useful in early-stage R&D. For oral solid dosage form, conventional HPLC-based content uniformity (CU) methods with sampling of 3 tablets per stratified sampling location in early R&D are typically not sufficient to identify these manufacturing process boundaries and temporal profile. Here, we report a screening CU method based on a multivariate model using transmission Raman spectroscopy (TRS) data on a phase-appropriate calibration set of only 16 tablets. This initial model was used for multiple pre-GMP development batches to provide critical information about blend uniformity and content uniformity (CU). In this work, the precision of the TRS method was evaluated; multiple spectral preprocessing approaches were compared regarding their effects on measurement precision as well as their ability to mitigate the photo bleaching effects during precision experiments. Overall, the TRS-based CU method was much faster than a traditional HPLC-based method allowing a much larger number of tablets to be screened. This larger number of analyzed tablets enabled the processes boundaries and temporal changes in CU to be identified while providing proper statistical assurance on product quality.

Control of erucic acid biosynthesis in Camelina (Camelina sativa) by antisense technology.

Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistance to abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elangase 1 (FAE1) gene encoded the enzyme ß-ketoacyl COA synthase (KCS), which is a key factor in the biosynthesis of erucic acid. In this study, isolation and cloning of the FAE1 from the Camelina sativa were performed to prepare an antisense structure.  The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning FAE1 into the Bluescriprt II SK+ vector and sequencing, these fragments were used for the preparation of antisense structure in the pBI121 plant expression vector. The approved structure was transferred to the camelina plant via the Agrobacterium-mediated method. Also, the conditions of tissue culture and gene transfer were optimized. Moreover, the erucic acid content of the immature seeds of T0 transgenic plants was analyzed with gas chromatography (GC). Results showed significant changes in erucic acid levels of two control plants (0.88%), while two lines of the RFAE1 transgenic plants showed a decrease of approximately 0% in erucic acid level. It can be concluded that the antisense structure can be effective in reducing erucic acid.

CRISPR-based engineering of RNA viruses.

CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.

Leveraging Data Science and Novel Technologies to Develop and Implement Precision Medicine Strategies in Critical Care.

Precision medicine aims to identify treatments that are most likely to result in favorable outcomes for subgroups of patients with similar clinical and biological characteristics. The gaps for the development and implementation of precision medicine strategies in the critical care setting are many, but the advent of data science and multi-omics approaches, combined with the rich data ecosystem in the intensive care unit, offer unprecedented opportunities to realize the promise of precision critical care. In this article, the authors review the data-driven and technology-based approaches being leveraged to discover and implement precision medicine strategies in the critical care setting.

Three-input logic gate based on DNA strand displacement reaction.

In this paper, three kinds of three-input logic gates are designed based on DNA strand displacement reaction, which are three-input OR logic gate, three-input AND logic gate, and three-input MAJORITY logic gate. The logic gates designed in this paper takes different DNA strands as input and fluorescence signals as output. The biochemical experimental results verify my designs. The results show that DNA strand displacement technology has important application value in DNA computing, especially in the construction of DNA molecular logic gates.

Climate change adaptation and the back of the invisible hand.

A good deal of contemporary work in cultural evolutionary theory focuses on the adaptive significance of culture. In this paper, we make the case that scientifically accurate and politically feasible responses to the climate crisis require a complex understanding of human cultural practices of niche construction that moves beyond the adaptive significance of culture. We develop this thesis in two related ways. First, we argue that cumulative cultural practices of niche construction can generate stable equilibria and runaway selection processes that result in long-term existential risks within and across cultural groups. We dub this the back of the invisible hand. Second, we argue that the ability of cultural groups to innovate technological solutions to environmental problems is highly constrained in ways that are exacerbated by sustained intergroup conflict, inequality and by inherently unpredictable cascades in climate change and human migration patterns. After developing these theoretical points about human cultural practices of niche construction in detail, we conclude our discussion with some tentative practical suggestions about the way that cultural evolutionary history can more fruitfully be used in efforts to remit the climate crisis and contribute to sustainable practices of human climate change adaptation. This article is part of the theme issue 'Climate change adaptation needs a science of culture'.