A Micromanipulation-Actuated Large-Scale Screening to Identify Optimized Microphysiological Model Parameters in Skeletal Muscle Regeneration.

Hydrogel-based 3D cell cultures are extensively utilized to create biomimetic cellular microstructures. However, there is still lack of effective method for both evaluation of the complex interaction of cells with hydrogel and the functionality of the resulting micro-structures. This limitation impedes the further application of these microstructures as microphysiological models (microPMs) for the screening of potential culture condition combinations to enhance the skeletal muscle regeneration. This paper introduces a two-probe micromanipulation method for the large-scale assessment of viscoelasticity and contractile force (CF) of skeletal muscle microPMs, which are produced in high-throughput via microfluidic spinning and 96-well culture. The collected data demonstrate that viscoelasticity parameters (E* and tanδ) and CF both measured in a solution environment are indicative of the formation of cellular structures without hydrogel residue and the subsequent generation of myotubes, respectively. This study have developed screening criterias that integrate E*, tanδ, and CF to examine the effects of multifactorial interactions on muscle fiber repair under hypoxic conditions and within bioprinted bipennate muscle structures. This approach has improved the quality of hypoxic threshold evaluation and aligned cell growth in 3D. The proposed method is useful in exploring the role of different factors in muscle tissue regeneration with limited resources.

Laboratory automation and high-throughput biology.

BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, Annona et al present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, the authors compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.

Fusion/fission protein family identification in Archaea.

The majority of newly discovered archaeal lineages remain without a cultivated representative, but scarce experimental data from the cultivated organisms show that they harbor distinct functional repertoires. To unveil the ecological as well as evolutionary impact of Archaea from metagenomics, new computational methods need to be developed, followed by in-depth analysis. Among them is the genome-wide protein fusion screening performed here. Natural fusions and fissions of genes not only contribute to microbial evolution but also complicate the correct identification and functional annotation of sequences. The products of these processes can be defined as fusion (or composite) proteins, the ones consisting of two or more domains originally encoded by different genes and split proteins, and the ones originating from the separation of a gene in two (fission). Fusion identifications are required for proper phylogenetic reconstructions and metabolic pathway completeness assessments, while mappings between fused and unfused proteins can fill some of the existing gaps in metabolic models. In the archaeal genome-wide screening, more than 1,900 fusion/fission protein clusters were identified, belonging to both newly sequenced and well-studied lineages. These protein families are mainly associated with different types of metabolism, genetic, and cellular processes. Moreover, 162 of the identified fusion/fission protein families are archaeal specific, having no identified fused homolog within the bacterial domain. Our approach was validated by the identification of experimentally characterized fusion/fission cases. However, around 25% of the identified fusion/fission families lack functional annotations for both composite and split states, showing the need for experimental characterization in Archaea.IMPORTANCEGenome-wide fusion screening has never been performed in Archaea on a broad taxonomic scale. The overlay of multiple computational techniques allows the detection of a fine-grained set of predicted fusion/fission families, instead of rough estimations based on conserved domain annotations only. The exhaustive mapping of fused proteins to bacterial organisms allows us to capture fusion/fission families that are specific to archaeal biology, as well as to identify links between bacterial and archaeal lineages based on cooccurrence of taxonomically restricted proteins and their sequence features. Furthermore, the identification of poorly characterized lineage-specific fusion proteins opens up possibilities for future experimental and computational investigations. This approach enhances our understanding of Archaea in general and provides potential candidates for in-depth studies in the future.

Optimization of inoculum production of Stemphylium botryosum for large-scale resistance screening of lentils.

BACKGROUND: Stemphylium blight incited by Stemphylium botryosum poses a significant threat to lentil crops worldwide, inducing severe defoliation and causing substantial yield losses in susceptible varieties under favorable conditions. While some moderate levels of resistance have been identified within lentil germplasm, a low number of resistant cultivars are available to farmers. Adding to the common constraints of resistance breeding, a notable challenge is generating a sufficient number of spores for large-scale screenings, which are essential for pinpointing additional sources of resistance for integration into breeding programs. Therefore, there is a pressing need to improve existing screening methods and tailor them for large-scale material selection. In pursuit of this objective, a protocol for the efficient production of fungal material has been adapted. RESULTS: Optimization of fungal material production was successfully achieved by comparing the use of fungal mycelia and spores. Spore production was found to be optimal when produced on solid V8-PDA(hi) medium, while liquid Richard's medium was identified as superior for mycelium yield. Furthermore, a refined screening method was developed by evaluating the resistance of six lentil accessions to stemphylium blight. This assessment included the use of either fungal mycelia (at densities ranging from 1 to 5 g L- 1) or spores (with densities ranging from 5 × 104 to 2 × 105 conidia mL- 1) under three different relative humidity levels (from 50 to 100%). Both humidity levels and inoculum dose significantly influenced the final disease rating (DR) and the relative Area Under the Disease Progress Curve (rAUDPC). Differences among genotypes in final symptom severity (DR) became more pronounced after inoculation with inoculum densities of 5 g L- 1 of mycelium or of 105 and 2 × 105 conidia mL- 1 of spore under 100% relative humidity. Given the challenges associated with the large-scale production of S. botryosum spores, inoculations with 5 g L- 1 of mycelium is highly recommended as a practical alternative for conducting mass-scale screenings. CONCLUSIONS: The findings from this study underscore the critical importance of maintaining high level of humidity during inoculation and disease progression development for accurately assessing resistance to stemphylium blight. The optimization of mycelial production for suspension inoculation emerges as a more reliable and efficient approach for conducting large-scale screening to assess germplasm resistance against stemphylium blight in lentil crops.

Base editor-mediated large-scale screening of functional mutations in bacteria for industrial phenotypes.

Base editing, the targeted introduction of point mutations into cellular DNA, holds promise for improving genome-scale functional genome screening to single-nucleotide resolution. Current efforts in prokaryotes, however, remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library, which falls far short of fully releasing the potential of base editors. Here, we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli. We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E. coli, and screened for adaptive mutations under isobutanol and furfural selective conditions. Guided by the screening results, we successfully identified several known and novel functional mutations. Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.

Reaping the benefits of liquid handlers for high-throughput gene expression profiling in a marine model invertebrate.

BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, we present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, we compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.

Connectivity Analysis of Adsorption Sites in Metal-Organic Frameworks for Facilitated Water Adsorption.

Metal-organic frameworks (MOFs) have recently drawn considerable attention as promising adsorbents to harvest atmospheric water. To achieve an efficient harvesting process, seeking MOFs that demonstrate sharp condensation behavior is the key. Given that the clustering of water molecules in MOFs should be driven by not only MOF-water interactions but also water-water interactions, the spatial arrangement of water adsorption sites in a MOF is therefore crucial. Specifically, this study demonstrates the critical role of continuous adsorption channels (CACs) in MOFs. Such CACs will enable water molecules to stay in proximity and in a continuous manner, thus promoting the formation of hydrogen bonds and, consequently, the clustering of water molecules. We have developed an automatic algorithm to detect CACs based on the energy grid of host-guest interactions and applied the algorithm to more than 2000 diverse structures. The results show that more than 80% of the studied MOFs displaying water condensation at 298 K and 20% relative humidity predicted by Monte Carlo simulations indeed have CACs. The developments herein are anticipated to largely facilitate the future discovery of optimal adsorbents for water harvesting or water-adsorption-related applications in general. A Python-based code for detecting CACs in porous materials is also provided along with this article to employ this approach.

Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations.

Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

Large-scale screening of yeast strains that can utilize proline.

Proline contributes to the taste and flavor of foods. The yeast Saccharomyces cerevisiae poorly assimilates proline during fermentation processes, resulting in the accumulation of proline in fermentative products. We performed here a screening of in total 1138 yeasts to obtain strains that better utilize proline. Our results suggest that proline utilization occurs in the genera of Zygoascus, Galactomyces, and Magnusiomyces.

Efficacy of a Deep Learning System for Screening Myopic Maculopathy Based on Color Fundus Photographs.

INTRODUCTION: The maculopathy in highly myopic eyes is complex. Its clinical diagnosis is a huge workload and subjective. To simply and quickly classify pathologic myopia (PM), a deep learning algorithm was developed and assessed to screen myopic maculopathy lesions based on color fundus photographs. METHODS: This study included 10,347 ocular fundus photographs from 7606 participants. Of these photographs, 8210 were used for training and validation, and 2137 for external testing. A deep learning algorithm was trained, validated, and externally tested to screen myopic maculopathy which was classified into four categories: normal or mild tessellated fundus, severe tessellated fundus, early-stage PM, and advanced-stage PM. The area under the precision-recall curve, the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, accuracy, and Cohen's kappa were calculated and compared with those of retina specialists. RESULTS: In the validation data set, the model detected normal or mild tessellated fundus, severe tessellated fundus, early-stage PM, and advanced-stage PM with AUCs of 0.98, 0.95, 0.99, and 1.00, respectively; while in the external-testing data set of 2137 photographs, the model had AUCs of 0.99, 0.96, 0.98, and 1.00, respectively. CONCLUSIONS: We developed a deep learning model for detection and classification of myopic maculopathy based on fundus photographs. Our model achieved high sensitivities, specificities, and reliable Cohen's kappa, compared with those of attending ophthalmologists.

Explainable artificial intelligence for precision medicine in acute myeloid leukemia.

Artificial intelligence (AI) can unveil novel personalized treatments based on drug screening and whole-exome sequencing experiments (WES). However, the concept of "black box" in AI limits the potential of this approach to be translated into the clinical practice. In contrast, explainable AI (XAI) focuses on making AI results understandable to humans. Here, we present a novel XAI method -called multi-dimensional module optimization (MOM)- that associates drug screening with genetic events, while guaranteeing that predictions are interpretable and robust. We applied MOM to an acute myeloid leukemia (AML) cohort of 319 ex-vivo tumor samples with 122 screened drugs and WES. MOM returned a therapeutic strategy based on the FLT3, CBFß-MYH11, and NRAS status, which predicted AML patient response to Quizartinib, Trametinib, Selumetinib, and Crizotinib. We successfully validated the results in three different large-scale screening experiments. We believe that XAI will help healthcare providers and drug regulators better understand AI medical decisions.

Detection of Carbohydrate Antigen 50 Based on a Novel Miniaturized Chemiluminescence Analyzer Enables Large-Scale Cancer Early Screening in Grassroots Community.

Early screening of cancer can effectively prolong survival time and reduce cancer mortality. However, the existing health-monitoring devices can only be carried out in professional laboratories, so large-scale early cancer screening in resource-limited settings is hardly achieved. To embrace the challenge, we developed a novel chemiluminescence immunoassay (CLIA) analyzer that does not require a professional operation. Then, it was applied to detect carbohydrate antigen 50 (CA50), a non-organ-specific tumor marker for screening various cancers. As a result, the analyzer exhibited excellent performance that the total assay time was only 15 min, and the detection limit reached 0.057 U ml-1. A coefficient of variance (CV) less than 15% was well-controlled for both intra- and inter-assay precision, and the linear range was 0-500 U ml-1. More importantly, this analyzer can continuously detect 60 samples per hour without any professional paramedic. Finally, this analyzer has been applied to evaluate clinical samples and the detected results showed a good correlation with the clinical test results (correlation coefficient, 0.9958). These characteristics exactly meet large-scale and high-throughput early screening of cancer. Thus, this miniaturized analyzer for CA50 detection is promising to achieve early large-scale screening of cancer in the resource-limited grassroots community.

Monitoring Cell Death Via Ion Leakage and PAM Fluorometry.

Cell death in plants plays a major role during development as well as in response to certain biotic and abiotic stresses. For example, plant cell death can be triggered in a tightly regulated way during the hypersensitive response (HR) in defense against pathogens or be elicited by pathogenic toxin deployment. Monitoring cell death and its impact on plant health can aid in the quantification of plant disease symptoms and help to identify the underlying molecular pathways. Here, we describe our current protocol for monitoring plant cell death via ion leakage and Pulse-Amplitude-Modulation (PAM) fluorometry. We further provide a detailed protocol for the sample preparation, the measurement, and the data evaluation and discuss the complementary nature of ion leakage and PAM fluorometry as well as the potential of PAM fluorometry for high-throughput screenings.

A Large-Scale Screening Responding Sporadic Epidemic of COVID-19 in China by an Integrated Health-Care System.

Introduction: In the post-pandemic period of COVID-19, the majority of cities in China try to balance the normalization of epidemic prevention and social-economic development. However, the appearance of asymptomatic infected patients poses threats to public health, which might be infectious without clinical symptoms and only be detected by testing approaches. Methods: Along with the appearance of one symptomatic case, a regional large-scale screening program was carried out in Shenzhen City charged by a regionally integrated healthcare system. After describing the screening program, a retrospective cross-sectional study for the screening outcome and efficacy was conducted. Discussion: According to the screening results, the asymptomatic case was infectious and their close contacts should be quarantined cautiously as the close contacts of symptomatic cases. Besides, after integrating medical resources in Luohu district of Shenzhen, the medical capability of Luohu district improved greatly which could be demonstrated in inspection and organization abilities in this screening program. Conclusion: The large-scale screening contributed to preventing epidemic transmission. In the post-pandemic period, regular surveillance of asymptomatic cases and rapid response capability for emergent screening program are both crucial for the prevention and control of COVID-19 epidemic. The integrated healthcare system coordinating regional medical institutions and optimizing regional medical recourse has advantages to address public health emergencies.

Real-world Evaluation of a Sample Pooling Strategy for Large-Scale Rapid COVID-19 Testing.

BACKGROUND: The worldwide outbreak of COVID-19 has become a public health crisis of unprecedented proportions. The fast spread of emerging variants increases the needs of rapid diagnostic and screening testing. Sample pooling efficiently expands the testing capacity under limited resources. OBJECTIVES: We evaluated the performance of sample pooling on the Point-of-Care (POC) Liat® and cobas® 6800 systems and provided real-world experiences for implementing these systems in large-scale screenings. METHODS: Positive nasopharyngeal (NP) specimens with Ct values < 25, 25∼30 or > 30 were tested individually and in pools to optimize the POC Liat® and cobas® 6800 systems, which were then implemented in community screenings. RESULTS: The 5-sample pooling strategy did not affect the positive detection rates on Liat® or cobas® 6800 in samples with Ct values <25 or 25∼30. However, in samples with low viral loads (Ct values >30), five-sample pooling has a higher positive detection rate on POC Liat® (20/20; 100%), compared to cobas® 6800 (9/20; 45%). Five-sample pooled on POC Liat® and two-sample pooled on cobas® 6800 appear to be appropriate for SARS-CoV-2 detection. By implementing the pooling strategies in two large-scale community screenings, 7,606 NP specimens was tested within 36 h; the average turn-around time was 4.8 h for cobas® 6800 and 1.3 h for POC Liat®. Eight positive specimens (0.11%; 8/7,606) were identified, with Ct values ranging from 18.85 to 37.68. CONCLUSION: The performance of sample pooling on POC Liat® was demonstrated to be an effective, accurate, and economical approach for large-scale community screenings for COVID-19.

The challenge of targets and drug discovery using large-scale screening approaches in onco-hematology.

Target identification and drug discovery roads have been widely improved over the past decades in onco-hematology. In this review, we summarize recent improvements in the use of physio-pathologically relevant models and innovative screening approaches to accelerate efficient drug development. Using acute myeloid leukemia as an example, we also discuss the main encountered pitfalls and propose alternative roads to improve the drug discovery journey in onco-hematology.

Compounds from the Medicines for Malaria Venture Box Inhibit In Vitro Growth of Babesia divergens, a Blood-Borne Parasite of Veterinary and Zoonotic Importance.

Babesiosis is an infectious disease with an empty drug pipeline. A search inside chemical libraries for novel potent antibabesial candidates may help fill such an empty drug pipeline. A total of 400 compounds (200 drug-like and 200 probe-like) from the Malaria Box were evaluated in the current study against the in vitro growth of Babesia divergens (B. divergens), a parasite of veterinary and zoonotic importance. Novel and more effective anti-B. divergens drugs than the traditionally used ones were identified. Seven compounds (four drug-like and three probe-like) revealed a highly inhibitory effect against the in vitro growth of B. divergens, with IC50s ≤ 10 nanomolar. Among these hits, MMV006913 exhibited an IC50 value of 1 nM IC50 and the highest selectivity index of 32,000. The atom pair fingerprint (APfp) analysis revealed that MMV006913 and MMV019124 showed maximum structural similarity (MSS) with atovaquone and diminazene aceturate (DA), and with DA and imidocarb dipropionate (ID), respectively. MMV665807 and MMV665850 showed MMS with each other and with ID. Of note, a high concentration (0.75 IC50) of MMV006913 caused additive inhibition of B. divergens growth when combined with DA at 0.75 or 0.50 IC50. The Medicines for Malaria Venture box is a treasure trove of anti-B. divergens candidates according to the obtained results.

Selective screening for lysosomal storage disorders in a large cohort of minorities of African descent shows high prevalence rates and novel variants.

Population studies point to regional and ethnicity-specific differences in genetic predisposition for some lysosomal storage disorders (LSDs). The aim of the study was to determine the prevalence of the three treatable forms of lysosomal storage disorders (Gaucher disease [GD], Pompe disease [PD], and Fabry disease [FD]) in a cohort of mostly urban-dwelling individuals of African ancestry, a previously unknown genetic landscape for LSDs. Large-scale selective multistep biochemical and genetic screening was performed in patients seeking healthcare for various health concerns. Fluorimetric enzyme assays for GD, PD, and FD were performed on dried blood spots. Targeted gene sequencing was performed on samples that showed significantly lower enzyme activities (<10% of control mean) after two tiers of enzymatic screening. A total of 5287 unique samples representing a cross section of patients who visited Howard University Hospital and College of Medicine from 2015 to 2017 were included in the study. Study samples were obtained from a population where ~90% reported as African-American, ~5% Hispanic, and <5% Caucasian or other. Regarding GD, three subjects had either homozygous or heterozygous mutations in the GBA gene. As to PD, eight subjects were either homozygous or compound heterozygous for GAA mutations, including three novel mutations: (a) c.472 A > G; p.T158A, (b) c.503G > T; p.R168L, (c) c.1985del. Regarding FD, two subjects had pathogenic GLA mutations, and four had single nucleotide polymorphisms in the 5'UTR, previously implicated in modulating gene expression. The findings highlight a higher incidence of abnormal enzyme levels and pathogenic mutations in the target population reflecting ancestry-based specific genotype and phenotype variations.

General population screening for atrial fibrillation with an automated rhythm-detection blood pressure device.

BACKGROUND: Screening strategies to diagnose previously undetected atrial fibrillation (AF), especially silent AF (SAF), in at-risk populations may help reduce the number of strokes. We prospectively assessed the incidence rate of AF, including SAF, using an automated AF-detection capable sphygmomanometer in the General Practitioner (GP) setting. METHODS: This was a population-based prospective study of unselected general population of ≥65 years without prior AF. Participating GPs were requested, in the period February 2018-April 2019, to record all AF diagnoses including those derived from the AF-detection capable sphygmomanometer and confirmed by 12­lead ECG or ECG Holter in asymptomatic patients. RESULTS: Overall, 14,987 patients assisted by 76 GPs accumulated 16,838 patient-years of follow up. The incidence rate of AF was 2.25% patient-years (95%CI 2.03-2.48). AF was more frequently detected in male, older, overweight, and patients with prior stroke, congestive heart failure, and chronic kidney disease. One in four patients had device-detected SAF (0.56% patient-years, 95%CI 0.46-0.69). Age, overweight, and the number of annual visits, were independent predictors of both SAF and AF. In addition, congestive heart failure, mitral valve disease were independent predictors of AF. Due to the interaction between blood pressure and age the risk of AF increased exponentially after 75 years of age in patients with higher systolic blood pressure values. CONCLUSION: We found a higher than previously reported incidence rate of AF possibly by capturing SAF. Our simple protocol might be feasible in large-scale screening for AF and SAF in routine GP care.

A novel HER2-targeting antibody 5G9 identified by large-scale trastuzumab-based screening exhibits potent synergistic antitumor activity.

BACKGROUND: Pertuzumab is currently used in combination with trastuzumab as the first-line treatment for HER2-positive metastatic breast cancer. However, pertuzumab was originally developed independently from trastuzumab and was later incidentally found to have synergistic efficacy when combined with trastuzumab, it remains to be seen whether a more potent synergistic efficacy partner exists for trastuzumab. METHODS: A trastuzumab-based functional assay was used to screen anti-HER2 antibodies harboring trastuzumab-synergistic antitumor activity. The lead candidate 5G9, in combination with trastuzumab, was further characterized for its bioactivities in cell proliferation, cell apoptosis, antigen-antibody endocytosis and HER2-mediated cell signaling pathway blocking. Finally, animal models were used to evaluate the in vivo synergistic antitumor efficacy of 5G9 in combination with trastuzumab. FINDINGS: Compared to pertuzumab, 5G9 demonstrated more potent synergistic cell growth inhibitory activity when combined with trastuzumab (85% vs. 55%, P<0.001). In addition, 5G9 exhibited a higher internalization rate than pertuzumab (20% vs. 9%, P<0.05), and was able to further synergize with trastuzumab to promote antigen-antibody endocytosis. The internalization rate of the combination of 5G9 and trastuzumab was higher than that of pertuzumab and trastuzumab (35% vs. 14%, P<0.001). In vivo animal studies demonstrated that 5G9 in combination with trastuzumab showed more potent synergistic antitumor efficacy than the combination of pertuzumab and trastuzumab. INTERPRETATION: 5G9, together with trastuzumab, may provide a potential opportunity for more efficacious treatment of HER2-positive cancers. FUNDING: National Natural Science Foundation of China; State Key Laboratory of Analytical Chemistry for Life Science.