mRNA-1273 vaccine-induced antibodies maintain Fc effector functions across SARS-CoV-2 variants of concern.

SARS-CoV-2 mRNA vaccines confer robust protection against COVID-19, but the emergence of variants has generated concerns regarding the protective efficacy of the currently approved vaccines, which lose neutralizing potency against some variants. Emerging data suggest that antibody functions beyond neutralization may contribute to protection from the disease, but little is known about SARS-CoV-2 antibody effector functions. Here, we profiled the binding and functional capacity of convalescent antibodies and Moderna mRNA-1273 COVID-19 vaccine-induced antibodies across SARS-CoV-2 variants of concern (VOCs). Although the neutralizing responses to VOCs decreased in both groups, the Fc-mediated responses were distinct. In convalescent individuals, although antibodies exhibited robust binding to VOCs, they showed compromised interactions with Fc-receptors. Conversely, vaccine-induced antibodies also bound robustly to VOCs but continued to interact with Fc-receptors and mediate antibody effector functions. These data point to a resilience in the mRNA-vaccine-induced humoral immune response that may continue to offer protection from SARS-CoV-2 VOCs independent of neutralization.

Unveiling contact-mediated cellular crosstalk.

Cell-cell interactions orchestrate complex functions in multicellular organisms, forming a regulatory network for diverse biological processes. Their disruption leads to disease states. Recent advancements - including single-cell sequencing and spatial transcriptomics, coupled with powerful bioengineering and molecular tools - have revolutionized our understanding of how cells respond to each other. Notably, spatial transcriptomics allows us to analyze gene expression changes based on cell proximity, offering a unique window into the impact of cell-cell contact. Additionally, computational approaches are being developed to decipher how cell contact governs the symphony of cellular responses. This review explores these cutting-edge approaches, providing valuable insights into deciphering the intricate cellular changes influenced by cell-cell communication.

More than one million barriers fragment Europe's rivers.

Rivers support some of Earth's richest biodiversity1 and provide essential ecosystem services to society2, but they are often fragmented by barriers to free flow3. In Europe, attempts to quantify river connectivity have been hampered by the absence of a harmonized barrier database. Here we show that there are at least 1.2 million instream barriers in 36 European countries (with a mean density of 0.74 barriers per kilometre), 68 per cent of which are structures less than two metres in height that are often overlooked. Standardized walkover surveys along 2,715 kilometres of stream length for 147 rivers indicate that existing records underestimate barrier numbers by about 61 per cent. The highest barrier densities occur in the heavily modified rivers of central Europe and the lowest barrier densities occur in the most remote, sparsely populated alpine areas. Across Europe, the main predictors of barrier density are agricultural pressure, density of river-road crossings, extent of surface water and elevation. Relatively unfragmented rivers are still found in the Balkans, the Baltic states and parts of Scandinavia and southern Europe, but these require urgent protection from proposed dam developments. Our findings could inform the implementation of the EU Biodiversity Strategy, which aims to reconnect 25,000 kilometres of Europe's rivers by 2030, but achieving this will require a paradigm shift in river restoration that recognizes the widespread impacts caused by small barriers.

Identification of mammalian transcription factors that bind to inaccessible chromatin.

Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion, which includes TFs that can open the chromatin, their co-factors and TFs with similar motifs. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF, USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.

CellNeighborEX: deciphering neighbor-dependent gene expression from spatial transcriptomics data.

Cells have evolved their communication methods to sense their microenvironments and send biological signals. In addition to communication using ligands and receptors, cells use diverse channels including gap junctions to communicate with their immediate neighbors. Current approaches, however, cannot effectively capture the influence of various microenvironments. Here, we propose a novel approach to investigate cell neighbor-dependent gene expression (CellNeighborEX) in spatial transcriptomics (ST) data. To categorize cells based on their microenvironment, CellNeighborEX uses direct cell location or the mixture of transcriptome from multiple cells depending on ST technologies. For each cell type, CellNeighborEX identifies diverse gene sets associated with partnering cell types, providing further insight. We found that cells express different genes depending on their neighboring cell types in various tissues including mouse embryos, brain, and liver cancer. Those genes are associated with critical biological processes such as development or metastases. We further validated that gene expression is induced by neighboring partners via spatial visualization. The neighbor-dependent gene expression suggests new potential genes involved in cell-cell interactions beyond what ligand-receptor co-expression can discover.

Charge transport in electrospinning of polyelectrolyte solutions.

This study elucidates the electrical charge transport during electrospinning of weak polyelectrolyte (poly(acrylic acid) (PAA)) solutions by employing current emission measurements. With pH variation, the PAA ionization degree could be controlled from uncharged at low pH to weakly charged at intermediate solution pH. Electrospinning neutral poly(vinylpyrrolidone) (PVP) as a reference polymer solution confirmed established current-flow rate scaling relationships as shown by De La Mora and Loscertales (1994), I ∼ (γKQ)ν, independent of the applied electric field polarity, where ν = 0.5, K is the conductivity, γ is the surface tension, Q is the flow rate, and I is the current. Similarly, the uncharged PAA did not display any polarity dependence, yet ν ≈ 0.8. Negatively charged PAA, however, showed a marked deviation in the current-flow rate behavior, which was affected by the applied electric field polarity. In the case of negative polarity, ν = 0.99, whereas for a positive polarity ν = 0.68. Similarly, the voltage required for stable cone-jet electrospinning of charged PAA was significantly higher in the negative polarity configuration for all tested flow rates (300-1600 µL h-1). As opposed to merely surface charges typically considered when electrospinning leaky dielectric fluids, as suggested by Melcher and Taylor (1969), our results suggest that the measured current is also affected by volumetric charges from charged PAA in the bulk of the jet. The proposed additional charge transport might affect the orientational order within PE-based nanofibers and their diameter.

Latest Trends in Lipase-Catalyzed Synthesis of Ester Carbohydrate Surfactants: From Key Parameters to Opportunities and Future Development.

Carbohydrate-based surfactants are amphiphilic compounds containing hydrophilic moieties linked to hydrophobic aglycones. More specifically, carbohydrate esters are biosourced and biocompatible surfactants derived from inexpensive renewable raw materials (sugars and fatty acids). Their unique properties allow them to be used in various areas, such as the cosmetic, food, and medicine industries. These multi-applications have created a worldwide market for biobased surfactants and consequently expectations for their production. Biobased surfactants can be obtained from various processes, such as chemical synthesis or microorganism culture and surfactant purification. In accordance with the need for more sustainable and greener processes, the synthesis of these molecules by enzymatic pathways is an opportunity. This work presents a state-of-the-art lipase action mode, with a focus on the active sites of these proteins, and then on four essential parameters for optimizing the reaction: type of lipase, reaction medium, temperature, and ratio of substrates. Finally, this review discusses the latest trends and recent developments, showing the unlimited potential for optimization of such enzymatic syntheses.

Thinking through critical posthumanism: Nursing as political and affirmative becoming.

As a rejection and continuous reframing of theoretical humanism, critical posthumanism questions and imagines the human condition in the current context, aligning it with nonhuman and more than human entities, past and future. While this philosophical approach has been referenced in many academic disciplines since the 1990s, it has been gradually garnering interest among nursing scholars, leading to questions such as what it means to be human and what it means to be a nurse in the here and now. As a deeply ethical and political project, posthumanism, which we associate with poststructuralist concepts of power and resistance, questions the formation of posthuman subjects who more accurately reflect complex times, characterized by capitalistic commodification of life-human and nonhuman. In this article, we aim to explore how the ontological and epistemological underpinnings of critical posthumanism, specifically through Rosi Braidotti's works, can be useful to understand a posthuman subjectivity that favors affirmative actions aimed at actualizing our world in becoming. Through examples in nursing practice, education, and research, we will explore not only how critical posthumanism allows us to frame transformations in the current situation that we are embedded in as nurses and more generally as beings but also how these examples allow us to move beyond critique to the actualization of affirmative actions that correspond to the creation of new worlds.

Subjectivity through the lens of Guattari: A key concept for nursing.

Félix Guattari, a French philosopher and psychotherapist often recognized for his collaboration with Gilles Deleuze, also published important work of his own. The way he conceptualizes subjectivity and schizoanalysis (later developed into institutional analysis) can incite us to interpret our social contexts differently and to help frame an emancipatory path in nursing. At La Borde, a psychiatric clinic, subjectivity was seen as the real power that lies within the institutions; invisible and flowing through all levels of the hierarchal structure-like waves-each of them unique but still part of the same ocean. Even with its elusive character, this concept can be wielded through psychotherapeutic techniques of analysis which aim to reduce hierarchies, encourage collaborations, decentralize levers of power and promote initiatives that arise from the base. These concepts deserve further exploration when it comes to modern institutional issues like the ones present in Quebec's (Canada) healthcare system. Therefore, this article borrows theorizations elaborated through psychotherapy and applies them to the hospital institution which is seen as an organized, stable structure (the molar line), while paying attention to fluid, changing processes and the multiplicity of desires for transformation (the molecular line), to promote nursing movements that escape and abolish these structures, creating new possibilities and new forms of thinking (the line of flight).

Vesalius: high-resolution in silico anatomization of spatial transcriptomic data using image analysis.

Characterization of tissue architecture promises to deliver insights into development, cell communication, and disease. In silico spatial domain retrieval methods have been developed for spatial transcriptomics (ST) data assuming transcriptional similarity of neighboring barcodes. However, domain retrieval approaches with this assumption cannot work in complex tissues composed of multiple cell types. This task becomes especially challenging in cellular resolution ST methods. We developed Vesalius to decipher tissue anatomy from ST data by applying image processing technology. Vesalius uniquely detected territories composed of multiple cell types and successfully recovered tissue structures in high-resolution ST data including in mouse brain, embryo, liver, and colon. Utilizing this tissue architecture, Vesalius identified tissue morphology-specific gene expression and regional specific gene expression changes for astrocytes, interneuron, oligodendrocytes, and entorhinal cells in the mouse brain.

Electrically Mediated Static Contact Angle and Hysteresis of Polyelectrolyte Solutions.

Manipulating a droplet by electrowetting-on-dielectric (EWOD) is vital in various fields ranging from industrial applications to life sciences. As of now, EWOD research has focused primarily on aqueous electrolytes and ionic liquids. This paper investigates the electrowetting behavior of weak polyelectrolyte solutions containing poly(acrylic acid) (PAA). The study reveals distinct wetting behavior of weakly and fully charged PAA droplets controlled by their solution pH. Under an applied electric field, strongly ionized PAA wets more effectively than weakly charged PAA. The electrowetting hysteresis of fully ionized PAA droplets was also higher than that of weakly charged droplets. The reason may be the suppression of retraction flow near the contact line. In this thin region, the electric field aligns the stretched polymer chains perpendicular to the dielectric surface, thus affecting the bulk rheological properties. The results reveal how charge-connectivity and polyelectrolyte conformation under an external electric field can control the electrowetting gain and the hysteresis. This previously unexplored electrowetting mechanism of polyelectrolyte solutions might help order and manipulate biological polyelectrolytes, such as deoxyribonucleic acid (DNA), polypeptides, and glycosaminoglycans.

Application of Doehlert Experimental Design for Optimization of a New-Based Hydrophilic Interaction Solid-Phase Extraction of Phenolic Acids from Olive Oils.

In this work, a rapid, precise, and cost-valuable method has been established to quantify phenolic compounds in olive oil using new-based hydrophilic interaction solid-phase extraction (SPE). Boehlert's experimental design applied the determination of the optimal operating conditions. An investigation into the effects of the methanol composition (50-100%), the volume of eluent (1-12 mL), and pH (1-3) on the extraction of phenols acids and total phenols from Tunisian olive oils was performed. The results showed that the extraction conditions had a significant effect on the extraction efficiency. The experiment showed that the greatest conditions for the SPE of phenolic acids were the methanol composition at 90.3%, pH at 2.9, and volume at 7.5 mL, respectively. The optimal conditions were applied to different types of olive oils, and it could be concluded that larger concentrations of polyphenols were found in extra virgin olive oil (89.15-218), whereas the lowest levels of these compounds (66.8 and 5.1) were found in cold-pressed crude olive oil and olive pomace oil, respectively.

Optimization of Alkali Treatment for Production of Fermentable Sugars and Phenolic Compounds from Potato Peel Waste Using Topographical Characterization and FTIR Spectroscopy.

Potato peel waste (PPW) was utilized as a bio-template for the production of valuable compounds such as reducing sugars (RS), total sugar (TS) and total phenolic compounds (TPC). Two methods of alkali treatments, i.e., chemical (NaOH) and thermochemical (NaOH assisted with autoclaving) processes, were employed for the deconstruction of PPW. Response surface methodology (RSM) was used to study the effects of alkali concentration (0.6-1.0 w/v), substrate concentration (5-15 g) and time (4-8 h) on the extraction of RS, TS and TP from PPW. The application of alkali plus steam treatment in Box-Behnken design (BBD) with three levels yielded the optimum releases of RS, TS and TP as 7.163, 28.971 and 4.064 mg/mL, respectively, corresponding to 10% substrate loading, in 0.6% NaOH for 8 h. However, the alkali treatment reported optimum extractions of RS, TS and TP as 4.061, 17.432 and 2.993 mg/mL, respectively. The thermochemical pretreatment was proven a beneficial process as it led to higher productions of TP. FTIR and SEM were used to analyze the deterioration levels of the substrate. The present work was used to explore the sustainable management of PPW, which is a highly neglected substrate bioresource but is excessively dumped in open environment, raising environmental concerns. The cost-effective methods for the breakdown of PPW starch into fermentable sugars might be utilized to extract valuable compounds.

Physicochemical Properties, Antioxidant Markers, and Meat Quality as Affected by Heat Stress: A Review.

Heat stress is one of the most stressful events in livestock life, negatively impacting animal health, productivity, and product quality. Moreover, the negative impact of heat stress on animal product quality has recently attracted increasing public awareness and concern. The purpose of this review is to discuss the effects of heat stress on the quality and the physicochemical component of meat in ruminants, pigs, rabbits, and poultry. Based on PRISMA guidelines, research articles were identified, screened, and summarized based on inclusion criteria for heat stress on meat safety and quality. Data were obtained from the Web of Science. Many studies reported the increased incidences of heat stress on animal welfare and meat quality. Although heat stress impacts can be variable depending on the severity and duration, the exposure of animals to heat stress (HS) can affect meat quality. Recent studies have shown that HS not only causes physiological and metabolic disturbances in living animals but also alters the rate and extent of glycolysis in postmortem muscles, resulting in changes in pH values that affect carcasses and meat. It has been shown to have a plausible effect on quality and antioxidant activity. Acute heat stress just before slaughter stimulates muscle glycogenolysis and can result in pale, tender, and exudative (PSE) meat characterized by low water-holding capacity (WHC). The enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) act by scavenging both intracellular and extracellular superoxide radicals and preventing the lipid peroxidation of the plasma membrane. Therefore, understanding and controlling environmental conditions is crucial to successful animal production and product safety. The objective of this review was to investigate the effects of HS on meat quality and antioxidant status.

A pioneer morphological and genetic study of the intertidal fauna of the Gerlache Strait (Antarctic Peninsula).

The underexplored intertidal ecosystems of Antarctica are facing rapid changes in important environmental factors. Associated with temperature increase, reduction in coastal ice will soon expose new ice-free areas that will be colonized by local or distant biota. To enable detection of future changes in faunal composition, a biodiversity baseline is urgently required. Here, we evaluated intertidal faunal diversity at 13 locations around the Gerlache Strait (western Antarctic Peninsula), using a combination of a quadrat approach, morphological identification and genetic characterization. Our data highlight a community structure comprising four generally distributed and highly abundant species (the flatworm Obrimoposthia wandeli, the bivalve Kidderia subquadrata, and the gastropods Laevilitorina umbilicata and Laevilitorina caliginosa) as well as 79 rarer and less widely encountered species. The most abundant species thrive in the intertidal zone due to their ability to either survive overwinter in situ or to rapidly colonize this zone when conditions allow. In addition, we confirmed the presence of multiple trophic levels at nearly all locations, suggesting that complex inter-specific interactions occur within these communities. Diversity indices contrasted between sampling locations (from 3 to 32 species) and multivariate approaches identified three main groups. This confirms the importance of environmental heterogeneity in shaping diversity patterns within the investigated area. Finally, we provide the first genetic and photographic baseline of the Antarctic intertidal fauna (106 sequences, 137 macrophotographs), as well as preliminary insights on the biogeography of several species. Taken together, these results provide a timely catalyst to assess the diversity and to inform studies of the potential resilience of these intertidal communities.

What can anarchism do for nursing?

The notion of mutual aid, which Peter Kropotkin introduced in the 19th century, goes against the logic of competition as a natural condition, and instead shows how mutual aid is a more important factor to consider for the survival and flourishing of a group. The best cooperation strategies allow organisms to adapt to different types of changes in their environment-and we have witnessed a lot of these changes since the start of the COVID-19 pandemic. This propensity towards cooperation is not a foreign concept, despite how it seems to be overshadowed by individualism in Western societies. These reflections then lead us to believe it is possible to apply the anarchist philosophical principle of mutual aid to our social organizations, rather than giving priority, again and again, to competition and professional hierarchies, especially in healthcare systems, and particularly in hospitals were the majority of nurses work. For us, anarchist philosophical precepts, including but not limited to mutual aid, can be the key to a more adequate functioning of healthcare institutions. Anarchism can help to imagine the first steps needed to take to gradually move away from ideologies that encourage competition, professional hierarchies, and illegitimate authority. In this paper, we will first explore some anarchist philosophical precepts before turning to mutual aid as it is currently conceptualised, then highlight several concrete ways it is visible in nursing, as well as ways it can be applied in hospitals, and healthcare systems.

Mattering: Per/forming nursing philosophy in the Chthulucene.

This paper presents an overview of the process of entanglement at the 25th International Philosophy of Nursing Conference (IPNC) at University of California at Irvine held on August 18, 2022. Representing collective work from the US, Canada, UK and Germany, our panel entitled 'What can critical posthuman philosophies do for nursing?' examined critical posthumanism and its operations and potential in nursing. Critical posthumanism offers an antifascist, feminist, material, affective, and ecologically entangled approach to nursing and healthcare. Rather than focusing on the arguments of each of the three distinct but interrelated panel presentation pieces, this paper instead focuses on process and performance (per/formance) and performativity as relational, connected and situated, with connections to nursing philosophy. Building upon critical feminist and new materialist philosophies, we describe intra-activity and performativity as ways to dehierarchise knowledge making practices within traditional academic conference spaces. Creating critical cartographies of thinking and being are actions of possibility for building more just and equitable futures for nursing, nurses, and those they accompany-including all humans, nonhumans, and more than human matter.

Effects of vertical violence on the work of nurses in hospital settings: An exploratory study / Effets de la violence verticale sur le travail d'infirmières soignantes exerçant en milieux hospitaliers : une étude exploratoire

Introduction: More than 39.7% of nurses report being victims of psychological harassment in the workplace. In 60% of cases, the abuse is vertical, involving a person in a position of authority. Context: Few studies have examined this phenomenon without conflating it with other forms of workplace violence. Objective: The purpose of this study was to shed light on cases of vertical violence experienced by nurses working in hospitals. Method: Data was collected by means of semi-structured interviews with six nurses working in hospitals in the province of Quebec (Canada). Descriptive phenomenology was used to analyze the collected data. Results: The overall effects of the vertical violence experienced by nurses in hospital settings resulted in less diligent and individualized nursing care. Discussion: It is recommended that organizational policies against vertical violence put in place in hospital be enforced in a rigorous and transparent manner. Further investigation is needed to identify the organizational factors that contribute to vertical violence in hospital settings.

Introduction: Plus de 39,7 % des infirmières se déclarent victimes de harcèlement psychologique dans leur milieu de travail. Dans 60 % des cas, il est question de violence verticale impliquant une personne en supériorité hiérarchique. Contexte: Peu d'études abordent ce phénomène sans le confondre avec d'autres formes de violence en milieu de travail. Objectifs: Cette étude visait à décrire l'effet de la violence verticale vécue par des infirmières soignantes exerçant en milieux hospitaliers. Méthode: Une collecte des données par entrevues semi-dirigées a été réalisée auprès de six infirmières de milieux hospitaliers de la province de Québec (Canada). La phénoménologie descriptive a été utilisée dans le cadre de la présente étude. Résultats: La représentation globale des effets de la violence verticale vécue par des infirmières soignantes en milieux hospitaliers se traduit par un détournement des initiatives infirmières qui renvoient à des soins diligents et personnalisés. Discussion: Il est recommandé que les politiques organisationnelles contre la violence verticale, mises en place dans les centres hospitaliers, soient appliquées de façon rigoureuse et transparente. D'autres études seraient appropriées afin de préciser les facteurs organisationnels favorisant la violence verticale en milieux hospitaliers.

Disentangling cause and consequence: genetic dissection of the DANGEROUS MIX2 risk locus, and activation of the DM2h NLR in autoimmunity.

Nucleotide-binding domain-leucine-rich repeat-type immune receptors (NLRs) protect plants against pathogenic microbes through intracellular detection of effector proteins. However, this comes at a cost, as NLRs can also induce detrimental autoimmunity in genetic interactions with foreign alleles. This may occur when independently evolved genomes are combined in inter- or intraspecific crosses, or when foreign alleles are introduced by mutagenesis or transgenesis. Most autoimmunity-inducing NLRs are encoded within highly variable NLR gene clusters with no known immune functions, which were termed autoimmune risk loci. Whether risk NLRs differ from sensor NLRs operating in natural pathogen resistance and how risk NLRs are activated in autoimmunity is unknown. Here, we analyzed the DANGEROUS MIX2 risk locus, a major autoimmunity hotspot in Arabidopsis thaliana. By gene editing and heterologous expression, we show that a single gene, DM2h, is necessary and sufficient for autoimmune induction in three independent cases of autoimmunity in accession Landsberg erecta. We focus on autoimmunity provoked by an EDS1-yellow fluorescent protein (YFP)NLS fusion protein to characterize DM2h functionally and determine features of EDS1-YFPNLS activating the immune receptor. Our data suggest that risk NLRs function in a manner reminiscent of sensor NLRs, while autoimmunity-inducing properties of EDS1-YFPNLS in this context are unrelated to the protein's functions as an immune regulator. We propose that autoimmunity, at least in some cases, may be caused by spurious, stochastic interactions of foreign alleles with coincidentally matching risk NLRs.

Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants.

Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2 , respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1 , we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.