Single Experiment Research Articles

Development of a Multiplex Real-Time Quantitative PCR Assay for Detecting Vaginal Microbiota in Chinese Women - China, 2021-2022.

The Nugent score, limited by subjectivity and personnel requirements, lacks accuracy. Establishing a precise and simple molecular test is therefore essential for detecting vaginal microbiota compositions and evaluating vaginal health. We evaluated the vaginal health of Chinese women using quantitative polymerase chain reaction (qPCR) to target Lactobacillus crispatus (L. crispatus), L. iners, Gardnerella vaginalis (G. vaginalis), Atopobium vaginae (A. vaginae), and Megasphaera phylotype1. bacterial vaginosis (BV)-related bacteria shared a fluorescent channel. Using 16S rDNA sequencing as a reference standard, we evaluated and validated the diagnostic accuracy of the qPCR assay. Both qPCR and 16S rDNA sequencing demonstrated 90.5% concordance in segregating vaginal community state type (CST), as visualized through heatmaps and PCoA. Spearman's correlation analysis revealed strong correlations between the two methods in calculating the RA of L. crispatus (CST I), L. iners (CST III), and BV-related bacteria (CST IV), with coefficients of 0.865, 0.837, and 0.827, respectively. Receiver operating characteristic analysis showed that qPCR had significant diagnostic accuracy for CST I, CST III, and CST IV (molecular BV), with area under the curve values of 0.967, 0.815, and 0.950, respectively, indicating strong predictive power. Vaginal health can be evaluated using a single qPCR amplification experiment, making the multiplex qPCR assay a highly accurate tool for this purpose.

Quantifying surface tension and viscosity in biomolecular condensates by FRAP-ID

Many proteins with intrinsically disordered regions undergo liquid-liquid phase separation under specific conditions in vitro and in vivo. These complex biopolymers form a metastable phase with distinct mechanical properties defining the timescale of their biological functions. However, determining these properties is nontrivial, even in vitro, and often requires multiple techniques. Here we report the measurement of both viscosity and surface tension of biomolecular condensates via correlative fluorescence microscopy and atomic force microscopy (AFM) in a single experiment (fluorescence recovery after probe-induced dewetting, FRAP-ID). Upon surface tension evaluation via regular AFM-force spectroscopy, controlled AFM indentations induce dry spots in fluorescent condensates on a glass coverslip. The subsequent rewetting exhibits a contact line velocity that is used to quantify the condensed-phase viscosity. Therefore, in contrast with fluorescence recovery after photobleaching (FRAP), where molecular diffusion is observed, in FRAP-ID fluorescence recovery is obtained through fluid rewetting and the subsequent morphological relaxation. We show that the latter can be used to cross-validate viscosity values determined during the rewetting regime. Making use of fluid mechanics, FRAP-ID is a valuable tool to evaluate the mechanical properties that govern the dynamics of biomolecular condensates and determine how these properties impact the temporal aspects of condensate functionality.

Extraction efficiency of loganic acid and gentiopicroside from Gentianae radix et rhizoma by deep eutectic solvent-based ultrasound-assisted extraction

This study describes the extraction efficiency of loganic acid and gentiopicroside from Gentianae radix et rhizoma using the deep eutectic solvent-based ultrasound-assisted extraction (DES-UAE) method. A comparison was made against 12 deep eutectic solvents (DESs) as alternative extraction solutions. The extraction efficiency of DES-4 (prepared from choline chloride and lactic acid) exceeded that of other DESs. A single factor experiment and response surface methodology (RSM) were adopted, and the optimal processing parameters of DES-UAE were: the water content of DES-4 was 19.9 %, solid/liquid was 29.3 mg/ml, extraction temperature was 50.8 °C and extraction time was 26.8 min. Under these conditions, the extraction efficiency of loganic acid and gentiopicroside was 7.6986 mg/100 g. The results of scanning electron microscopy (SEM) indicated that the unextracted powder exhibited a granular shape with rough surface and complete structure. Moreover, the ultrasonic extraction of Gentianae radix et rhizoma powder with DES-4 as extraction solution caused significant damage to the surface structure of the powder, creating numerous fine pores on the surface. This suggested that DES-4 had higher permeability and erosive force, which effectively destroyed the cell wall.

Using neural networks to obtain NMR spectra of both small and macromolecules from blood samples in a single experiment

Metabolomics plays a crucial role in understanding metabolic processes within biological systems. Using specific pulse sequences, NMR-based metabolomics detects small and macromolecular metabolites that are altered in blood samples. Here we proposed a method called spectral editing neural network, which can effectively edit and separate the spectral signals of small and macromolecules in 1H NMR spectra of serum and plasma based on the linewidth of the peaks. We applied the model to process the 1H NMR spectra of plasma and serum. The extracted small and macromolecular spectra were then compared with experimentally obtained relaxation-edited and diffusion-edited spectra. Correlation analysis demonstrated the quantitative capability of the model in the extracted small molecule signals from 1H NMR spectra. The principal component analysis showed that the spectra extracted by the model and those obtained by NMR spectral editing methods reveal similar group information, demonstrating the effectiveness of the model in signal extraction.

Simultaneous Multiscale 4DEnVar With Scale Dependent Localization (SDL) in Hurricane Analysis and Forecast System (HAFS)

AbstractThis study presents the implementation and evaluation of scale‐dependent localization (SDL) in the hurricane analysis and forecast system 4D Ensemble Variational (4DEnVar) Data Assimilation (DA) system. The SDL capability is compared with the traditional Single‐Scale Localization (SSL) method to assess its benefits and necessity for hurricane prediction. The experiments focus on Hurricane Laura (2020) and involve single observation experiments as well as real observation DA cycling experiments. The results indicate that the SDL experiment, which incorporates the Fast Almost Gaussian Filtering approach for scale decomposition, consistently outperforms the corresponding SSL configurations in almost all aspects. Further diagnostics show that due to its multiscale nature, the SDL approach demonstrates better track prediction over small‐scale SSL due to improved environmental analysis and better analyzed vortex position and structure, and superior intensity prediction during the rapid intensification over both the large‐scale SSL and the small‐scale SSL owing to enhanced inner‐core thermodynamic analysis.

Mechanism and threshold of environmental stressors on seagrass in high-turbidity estuary: case of Zostera japonica in Yellow River Estuary, China

Zostera japonica (Z. japonica), the most widely distributed seagrass species in temperate estuaries, has experienced a dramatic decline of nearly 75% over the past decade. While previous research has investigated the adaptation of seagrass individuals and populations to single stress factors, the molecular mechanisms underlying the interaction of multiple stressors remain poorly understood. This study conducted laboratory experiments to examine the response of Z. japonica at different life stages to environmental pressures, specifically salinity and turbidity, as indicated by changes in free amino acids (FAAs). The results demonstrate that Z. japonica exhibits stronger adaptability to high salinity environments but displays weaker adaptability to freshwater conditions. Through single stress experiments, the salinity and turbidity thresholds for FAA homeostatic disturbance in Z. japonica were determined at seedling, juvenile, and mature stages. As Z. japonica matures, its metabolic pathways expand and diversify, allowing the regulation of key FAAs to enhance stress resistance. Turbidity stress exerts a more pronounced negative impact on the cellular homeostasis of Z. japonica compared to salinity stress, and when turbidity levels exceed 150 NTU, they significantly intensify the negative effects of salinity stress on the seagrass. Furthermore, under strong salinity-turbidity interactions, the concentration of key FAAs generally decreases by 20-30%, indicating inhibition of growth and development in Z. japonica. These findings have important implications for the conservation of intertidal seagrass beds and estuarine ecosystems in the face of multiple human activities and environmental stressors. The study provides valuable insights into the molecular mechanisms underlying Z. japonica’s adaptations to salinity and turbidity stress, contributing to the development of targeted strategies to mitigate the impacts of environmental pressures on seagrass populations and promote the resilience of these critical marine ecosystems.

Revisiting the Effect of the Resistance to Gas Accumulation in Constant Volume Systems on the Membrane Time Lag.

The time-lag method is commonly used to determine membrane permeability, diffusivity and solubility in a single gas permeation experiment in a constant volume system. An unwritten assumption on which this method relies is that there is no resistance to gas accumulation in the downstream receiver of the system. However, this is not the case, even with the specially designed receiver used in this study when, in addition to tubing, the receiver utilizes an additional accumulation tank. The resistance to gas accumulation originates from a finite diffusivity (Knudsen diffusion) of gases in tubing, which are magnified by "resistance-free" accumulation tank(s). As a result of the resistance to gas accumulation, the time lag of the membrane is underestimated, which leads to an overestimation of gas diffusivity in the membrane. The experimentally predicted resistances in different configurations of the receiver, expressed by the difference in the time lag at two different receiver locations, were several times greater than the theoretically predicted values. A high molecular PPO membrane was used to demonstrate this effect. The time lags measured at different locations differed by as much as 30%. The diffusivity of nitrogen in a PPO of 4.04 × 10-12 m2/s determined at the optimum configuration of the receiver is at least 50% lower than the literature-reported values.

Cell-intrinsic platinum response and associated genetic and gene expression signatures in ovarian cancer cell lines and isogenic models.

Ovarian cancers are still largely treated with platinum-based chemotherapy as the standard of care, yet few biomarkers of clinical response have had an impact on clinical decision making as of yet. Two particular challenges faced in mechanistically deciphering platinum responsiveness in ovarian cancer have been the suitability of cell line models for ovarian cancer subtypes and the availability of information on comparatively how sensitive ovarian cancer cell lines are to platinum. We performed one of the most comprehensive profiles to date on 36 ovarian cancer cell lines across over seven subtypes and integrated drug response and multiomic data to improve on our understanding of the best cell line models for platinum responsiveness in ovarian cancer. RNA-seq analysis of the 36 cell lines in a single batch experiment largely conforms with the currently accepted subtyping of ovarian cancers, further supporting other studies that have reclassified cell lines and demonstrate that commonly used cell lines are poor models of high-grade serous ovarian carcinoma. We performed drug dose response assays in the 32 of these cell lines for cisplatin and carboplatin, providing a quantitative database of IC 50 s for these drugs. Our results demonstrate that cell lines largely fall either well above or below the equivalent dose of the clinical maximally achievable dose (C max ) of each compound, allowing designation of cell lines as sensitive or resistant. We performed differential expression analysis for high-grade serous ovarian carcinoma cell lines to identify gene expression correlating with platinum-response. Further, we generated two platinum-resistant derivatives each for OVCAR3 and OVCAR4, as well as leveraged clinically-resistant PEO1/PEO4/PEO6 and PEA1/PEA2 isogenic models to perform differential expression analysis for seven total isogenic pairs of platinum resistant cell lines. While gene expression changes overall were heterogeneous and vast, common themes were innate immunity/STAT activation, epithelial to mesenchymal transition and stemness, and platinum influx/efflux regulators. In addition to gene expression analyses, we performed copy number signature analysis and orthogonal measures of homologous recombination deficiency (HRD) scar scores and copy number burden, which is the first report to our knowledge applying field-standard copy number signatures to ovarian cancer cell lines. We also examined markers and functional readouts of stemness that revealed that cell lines are poor models for examination of stemness contributions to platinum resistance, likely pointing to the fact that this is a transient state. Overall this study serves as a resource to determine the best cell lines to utilize for ovarian cancer research on certain subtypes and platinum response studies, as well as sparks new hypotheses for future study in ovarian cancer.

Thymine DNA glycosylase combines sliding, hopping, and nucleosome interactions to efficiently search for 5-formylcytosine.

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylctyosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we studied TDG in the presence and absence of 5-formylctyosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. We probed TDG active site variants and truncated N-terminus revealing how these variants alter the lesion search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG was found to either bypass, colocalize with, or encounter but not bypass the nucleosome. However, truncating the N-terminus reduced the number of interactions with the nucleosome. Our findings provide unprecedented mechanistic insights into how TDG searches for DNA lesions in chromatin.

Real-time observation of coherent spin wave handedness

Magnonics, a crucial domain in information science and technology, utilizes spin waves in magnets as efficient information carriers. While antiferromagnets have been suggested for versatile magnonic platform because of the coexistence of right- and left-handed spin waves, their energetic degeneracy poses challenges for observation through spectral measurements, limiting their applicability. Recent observations of distinct spin wave handedness within the gigahertz regime have reported but, are yet to be demonstrated in terahertz (THz) frequencies of antiferromagnetic spin waves. Most of all, the coherence of spin waves is a key aspect of quantum information. Here, employing THz time-domain spectroscopy—a direct, precise, and easy probe for monitoring coherent spin wave dynamics—we discern chiral antiferromagnetic spin waves of opposite phase windings in the time domain, noting their handedness reversal across the angular momentum compensation temperature in ferrimagnets. We establish a principle for directly measuring the handedness of coherent antiferromagnetic spin waves in ferrimagnets with net magnetic moment M ≠ 0 but angular momentum L = 0. Our multidimensional access in the time and spectral domain enables the accurate determination of critical temperature and the dynamic observation of coherent chiral spin waves simultaneously in a single experiment, with potential applications in exploring other quantum chiral entities.

Scalable assessment of genome editing off-targets associated with genetic variants.

Genome editing with RNA-guided DNA binding factors carries risk of off-target editing at homologous sequences. Genetic variants may introduce sequence changes that increase homology to a genome editing target, thereby increasing risk of off-target editing. Conventional methods to verify candidate off-targets rely on access to cells with genomic DNA carrying these sequences. However, for candidate off-targets associated with genetic variants, appropriate cells for experimental verification may not be available. Here we develop a method, Assessment By Stand-in Off-target LentiViral Ensemble with sequencing (ABSOLVE-seq), to integrate a set of candidate off-target sequences along with unique molecular identifiers (UMIs) in genomes of primary cells followed by clinically relevant gene editor delivery. Gene editing of dozens of candidate off-target sequences may be evaluated in a single experiment with high sensitivity, precision, and power. We provide an open-source pipeline to analyze sequencing data. This approach enables experimental assessment of the influence of human genetic diversity on specificity evaluation during gene editing therapy development.

Research on Library Resource Management Based on Modern Information Technology and Reconfigurable Mobile Information System

Library resource library management based on modern information technology and reconfigurable mobile information systems have attracted much attention from researchers of academia. This paper introduces the LSTM model into the field of Chinese library resource management, and tries to propose a classification system with practical application value. The main research work:The Chinese library resource is designed LSTM model, and three experiments are designed on this basis: the coarse-grained book single classification experiment, different measures on the classification results; the fine-grained book single classification experiment, to compare The advantages and disadvantages of direct classification and layer-by-layer classification; coarse-grained book multi-classification experiments to explore the feasibility of multi-classification. After analyzing the experimental results, the model has achieved a good classification effect, and overcomes some deficiencies in the library resource library predecessors, and has a strong practical application value.

Light and Guest Responsive Behavior in a Porous Coordination Network Enabled by Reversible [2+2] Photocycloaddition.

Stimuli-responsive physisorbents that undergo reversible structural transformations induced by external stimuli (e.g. light, guests, or heat) offer the promise of utility in gas storage and separation. Whereas reports on guest or light-responsive sorbents have increased in recent years, we are unaware of reports on sorbents that exhibit both light and guest-induced structural transformations. Herein, we report that the square lattice, sql, topology coordination network Zn(fba)(bis) ⋅ 2DMF (sql-5,6-Zn-α, 5=trans-4,4'-bis(1-imidazolyl)stilbene=bis, 6=2,2-bis(4-carboxyphenyl)hexafluoropropane=H2fba) underwent single-crystal-to-single-crystal transformation (SCSC) upon activation, affording nonporous sql-5,6-Zn-β. Parallel alignment at 3.23 Å of olefinic moieties on adjacent bis ligands in sql-5,6-Zn-α enabled SCSC [2+2] photocycloaddition upon exposure to UV light (365 nm) or sunlight. sql-5,6-Zn-α thereby transformed to mot-5,6-Zn-α, which was subsequently activated to the narrow pore phase mot-5,6-Zn-β. sql-5,6-Zn-β and mot-5,6-Zn-β both exhibited S-shaped adsorption isotherms characteristic of guest-induced structural changes when exposed to CO2 at 195 K (type-F-IV and type F-I, respectively). Cycling experiments conducted upon sql-5,6-Zn-β reduced particle size after cycle 1 and induced transformation into a rare example of a shape memory coordination network, sql-5,6-Zn-γ. Insight into this smorgasbord of SCSC phase changes was gained from in situ PXRD, single crystal XRD and 1H NMR spectroscopy experiments.

Asymmetries in the Southern Ocean contribution to global heat and carbon uptake

The Southern Ocean provides dominant contributions to global ocean heat and carbon uptake, which is widely interpreted as resulting from its unique upwelling and circulation. Here we show a large asymmetry in these contributions, with the Southern Ocean accounting for 83 ± 33% of global heat uptake versus 43 ± 3% of global ocean carbon uptake over the historical period in state-of-the-art climate models. Using single radiative forcing experiments, we demonstrate that this historical asymmetry is due to suppressed heat uptake by northern oceans from enhanced aerosol forcing. In future projections, such as SSP2-4.5 where greenhouse gases increasingly dominate radiative forcing, the Southern Ocean contributions to global heat and carbon uptake become more comparable, 52 ± 5% and 47 ± 4%, respectively. Hence, the past is not a reliable indicator of the future, with the northern oceans becoming important for heat uptake while the Southern Ocean remains important for both heat and carbon uptake.

Design, synthesis and antiviral activity of indole derivatives containing quinoline moiety.

A series of indole derivatives containing quinoline structures were designed and synthesized. The synthesized compounds were characterized by NMR and HRMS. And W14 was performed by single crystal X-ray diffraction experiments. The antiviral activity studies showed that some of the target compounds possessed significant activity against tobacco mosaic virus (TMV). In particular, W20 had significant activity. The results of in vivo anti-TMV activity assay showed that W20 possessed the best curative and protective activities with EC50 values of 84.4 and 65.7 μg/mL, which were better than ningnanmycin (NNM) 205.1 and 162.0 μg/mL, respectively. The results of Microscale thermophoresis (MST) showed that W20 had a strong binding affinity for the tobacco mosaic virus coat protein (TMV-CP) with a dissociation constant (Kd) of 0.00519 μmol/L, which was superior to that of NNM (1. 65320 μmol/L). The molecular docking studies were in accordance with the experimental results. In addition, the determination of malondialdehyde (MDA) content in tobacco leaves showed that W20 improved the disease resistance of tobacco. Overall, this study shows that indole derivatives containing quinoline can be used as new antiviral agents for plant viruses for further research.

Scour variations around pier groups at a sharp bend with triad vanes

This study applied triad submerged vanes upstream of piers and varied their submergence ratios in order to explore and reduce scouring around piers in a bend. The most important findings are presented as follows: Triad vanes decreased the scour hole volume by a minimum of 22 % in the triad transverse pier and by a maximum of 53 % in the single pier experiment. The highest maximum scour hole reduction was achieved in the longitudinal triad piers with triad vanes for about 35 % in comparison with the case of piers without any vanes. The pier test with three vanes of 50 % submergence ratio resulted in the lowest hole volume, scour area, maximum scour depth and maximum sedimentary ridge of all the tests.

Light and Guest Responsive Behavior in a Porous Coordination Network Enabled by Reversible [2+2] Photocycloaddition

AbstractStimuli‐responsive physisorbents that undergo reversible structural transformations induced by external stimuli (e.g. light, guests, or heat) offer the promise of utility in gas storage and separation. Whereas reports on guest or light‐responsive sorbents have increased in recent years, we are unaware of reports on sorbents that exhibit both light and guest‐induced structural transformations. Herein, we report that the square lattice, sql, topology coordination network Zn(fba)(bis) ⋅ 2DMF (sql‐5,6‐Zn‐α, 5=trans‐4,4′‐bis(1‐imidazolyl)stilbene=bis, 6=2,2‐bis(4‐carboxyphenyl)hexafluoropropane=H2fba) underwent single‐crystal‐to‐single‐crystal transformation (SCSC) upon activation, affording nonporous sql‐5,6‐Zn‐β. Parallel alignment at 3.23 Å of olefinic moieties on adjacent bis ligands in sql‐5,6‐Zn‐α enabled SCSC [2+2] photocycloaddition upon exposure to UV light (365 nm) or sunlight. sql‐5,6‐Zn‐α thereby transformed to mot‐5,6‐Zn‐α, which was subsequently activated to the narrow pore phase mot‐5,6‐Zn‐β. sql‐5,6‐Zn‐β and mot‐5,6‐Zn‐β both exhibited S‐shaped adsorption isotherms characteristic of guest‐induced structural changes when exposed to CO2 at 195 K (type‐F–IV and type F–I, respectively). Cycling experiments conducted upon sql‐5,6‐Zn‐β reduced particle size after cycle 1 and induced transformation into a rare example of a shape memory coordination network, sql‐5,6‐Zn‐γ. Insight into this smorgasbord of SCSC phase changes was gained from in situ PXRD, single crystal XRD and 1H NMR spectroscopy experiments.

Development of a novel variable-curvature soft gripper used for orientating broccoli in the trimming line

Orientating the broccoli plays an important part in the trimming line, which helps reduce the redundant stalk length and provides accurate throughput data for sale. However, this work is still manual with high labor intensity and low work efficiency, and current grippers show limitations in replacing manual operations, which need the ability to self-adaptively grasp broccoli without damage and guide them upright. This study presented a novel pneumatic variable-curvature soft gripper (VCSG) for orientating broccoli, which mainly comprised three soft variable-curvature fiber-reinforced bending actuators (VCFRBAs). The VCFRBAs can generate additional lifting forces to help guide the broccoli upright, thus orientating the broccoli. A design concept was built for the VCFRBA, and an analytical model and finite element method (FEM) were proposed to evaluate the bending deformation of VCFRBA, achieving a good match with a root mean squared error of below 5.8°. Step response tests of VCFRBA showed a response time of 0.35 s, hysteresis tests demonstrated a hysteresis error of less than 10 %, and durability tests in 5,000 cycles showed two error bands of both less than 5 %, indicating a fast response, good repeatability, and good durability, respectively. Single actuator force experiments demonstrated the VCFRBA can generate a clamping force of 1.13 N with an additional lifting force of 1.38 N when grasping under the pressure of 0.2 MPa. Furthermore, a maximum pull-off force of 35.06 N was measured for the VCSG at this pressure. Results of grasping experiments showed a 100 % success rate without damage when the VCSG grasped broccoli positioned upright, and broccoli can still be securely and self-adaptively grasped and guided upright when grasping them inclinedly. Further orientating tests suggested that the VCSG had a good performance with an 85 % success rate. These results demonstrate that the proposed VCSG has a good ability to orientate the broccoli self-adaptively without complex operations. This study showcases a novel VCSG, providing a unique solution to orientating broccoli in the trimming line.

Tandem "One-Shot" Measurement of Residual Chemical Shift Anisotropy and Residual Dipolar Coupling using Biphasic Supramolecular Peptide Liquid Crystals.

Both solitary and tandem applications of residual chemical shift anisotropy (RCSA) and residual dipolar coupling (RDC) show great potential for the structural and configurational determination of organic molecules. A critical component of both RDC and RCSA methodologies is the alignment medium, whose availability is limited, especially for RCSA measurement. Moreover, reported RDC and RCSA acquisitions mainly rely on two experiments conducted under two different conditions, which are relatively time-consuming and easily cause experimental errors. Herein, a biphasic supramolecular lyotropic liquid crystalline (LLC) system was developed through the self-assembly of C21H43-CONH-V4K3-CONH2, which could act as an alignment medium for not only RDC but also RCSA extraction in DMSO-d6. Notably, the RCSA extraction was easily achieved via one-shot measurement from a single one-dimensional 13C NMR experiment, with no need for special instruments, devices, and correction. Relying on the biphasic LLC medium, meanwhile, RDC data were simply extracted from a single F1-coupled HSQC experiment, different from the standard protocol that requires two spectral acquisitions corresponding to the isotropic and anisotropic conditions. Collectively, the biphasic LLC medium is applicable for tandem RCSA and RDC measurements in one single sample, advancing the stereochemical elucidation of molecules of interest.

Allosteric nanobodies to study the interactions between SOS1 and RAS

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.