Corneal stromal structure replicating humanized hydrogel patch for sutureless repair of deep anterior-corneal defect.

A critical shortage of donor corneas exists worldwide. Hydrogel patches with a biological architecture and functions that simulate those of native corneas have garnered considerable attention. This study introduces a stromal structure replicating corneal patch (SRCP) composed of a decellularized cornea-templated nanotubular skeleton, recombinant human collagen, and methacrylated gelatin, exhibiting a similar ultrastructure and transmittance (above 80 %) to natural cornea. The SRCP is superior to the conventional recombinant human collagen patch in terms of biomechanical properties and resistance to enzymatic degradation. Additionally, SRCP promotes corneal epithelial and stromal cell migration while preventing the trans-differentiation of stromal cells into myofibroblasts. When applied to an ocular surface (37 °C), SRCP releases methacrylated gelatin, which robustly binds SRCP to the corneal stroma after activation by 405 nm light. Compared to gelatin-based photocurable hydrogel, the SRCP better supports the restoration of normal corneal curvature and withstands deformation under an elevated intraocular pressure (100 mmHg). In an in vivo deep anterior-corneal defect model, SRCP facilitated epithelial healing and vision recovery within 2 weeks, maintained graft structural stability, and inhibited stromal scarring at 4 weeks post-operation. The ideal performance of the SRCP makes it a promising humanized corneal equivalent for sutureless clinical applications.

Diosmetin-7-O-ß-D-glucopyranoside from Pogostemonis Herba alleviated SARS-CoV-2-induced pneumonia by reshaping macrophage polarization and limiting viral replication.

ETHNOPHARMACOLOGICAL RELEVANCE: Viral pneumonia is the leading cause of death after SARS-CoV-2 infection. Despite effective at early stage, long-term treatment with glucocorticoids can lead to a variety of adverse effects and limited benefits. The Chinese traditional herb Pogostemonis Herba is the aerial part of Pogostemon Cablin (Blanco) Benth., which has potent antiviral, antibacterial, anti-inflammatory, and anticancer effects. It was used widely for treating various throat and respiratory diseases, including COVID-19, viral infection, cough, allergic asthma, acute lung injury and lung cancer. AIM OF THE STUDY: To investigate the antiviral and anti-inflammatory effects of chemical compounds from Pogostemonis Herba in SARS-CoV-2-infected hACE2-overexpressing mouse macrophage RAW264.7 cells and hACE2 transgenic mice. MATERIALS AND METHODS: The hACE2-overexpressing RAW264.7 cells were exposed with SARS-CoV-2. The cell viability was detected by CCK8 assay and cell apoptotic rate was by flow cytometric assay. The expressions of macrophage M1 phenotype markers (TNF-α and IL-6) and M2 markers (IL-10 and Arg-1) as well as the viral loads were detected by qPCR. The mice were inoculated intranasally with SARS-CoV-2 omicron variant to induce viral pneumonia. The levels of macrophages, neutrophils, and T cells in the lung tissues of infected mice were analyzed by full spectrum flow cytometry. The expressions of key proteins were detected by Western blot assay. RESULTS: Diosmetin-7-O-ß-D-glucopyranoside (DG) presented the strongest anti-SARS-CoV-2 activity. Intervention with DG at the concentrations of 0.625-2.5 µM not only reduced the viral replication, cell apoptosis, and the productions of inflammatory cytokines (IL-6 and TNF-α) in SARS-CoV-2-infected RAW264.7 cells, but also reversed macrophage polarity from M1 to M2 phenotype. Furthermore, treatment with DG (25-100 mg/kg) alleviated acute lung injury, and reduced macrophage infiltration in SARS-COV-2-infected mice. Mechanistically, DG inhibited SARS-COV-2 gene expression and HK3 translation via targeting YTHDF1, resulting in the inactivation of glycolysis-mediated NF-κB pathway. CONCLUSIONS: DG exerted the potent antiviral and anti-inflammatory activities. It reduced pneumonia in SARS-COV-2-infected mice via inhibiting the viral replication and accelerating M2 macrophage polarization via targeting YTHDF1, indicating its potential for COVID-19 treatment.

Replication marketplaces would help science to become more self-correcting.

Independent replications are very rare in the behavioural and social sciences. This is problematic because they can help to detect 'false positives' in published research and, in turn, contribute to scientific self-correction. The lack of replication studies is, among other factors, due to a rather passive editorial approach concerning replications by many journals, which does not encourage and may sometimes even actively discourage submission of replications. In this Perspective article, we advocate for a more proactive editorial approach concerning replications and suggest introducing journal-based replication marketplaces as a new publication track. We argue that such replication marketplaces could solve the long-standing problem of lacking independent replications. To establish these marketplaces, a designated part of a journal's editorial board identifies the most relevant new findings reported within the journal's pages and publicly offers them for replication. This public offering could be combined with small grants for authors to support these replications. Authors then compete for the first accepted registered report to conduct the related replications and can thus be sure that their replication will be published independent of the later findings. Replication marketplaces would not only increase the prevalence of independent replications but also help science to become more self-correcting.

NMR assignment of the conserved bacterial DNA replication protein DnaA domain IV.

Chromosomal replication is a ubiquitous and essential cellular process. In bacteria, the master replication initiator DnaA plays a key role in promoting an open complex at the origin (oriC) and recruiting helicase in a tightly regulated process. The C-terminal domain IV specifically recognises consensus sequences of double-stranded DNA in oriC, termed DnaA-boxes, thereby facilitating the initial engagement of DnaA to oriC. Here, we report the 13Cß and backbone 1H, 15N, and 13C chemical shift assignments of soluble DnaA domain IV from Bacillus subtilis at pH 7.6 and 298 K.

Association study reveals a susceptibility locus with male pattern baldness in the Han Chinese population.

Introduction: Male pattern baldness (MPB), also known as androgenetic alopecia, represents the most prevalent form of progressive hair loss in humans. It is characterized by a distinctive pattern of hair loss progression from the scalp; however, its underlying mechanism remains elusive and is influenced by hereditary, immune, and environmental factors. Genome-wide association studies (GWASs) have uncovered numerous risk genes/loci among European individuals with MPB. However, the validation of these susceptibility genes/loci within Han Chinese men remains largely unexplored. The aim of this study was to investigate whether the 71 susceptibility loci identified in a recent GWAS among European men also confer risk for MPB in Chinese men. Methods: Forty-seven single nucleotide polymorphisms (SNPs) previously reported in GWASs of MPB were selected and genotyped in independent individuals comprising 499 Han Chinese cases and 1,489 controls using the Sequenom MassArray system. After stringent quality control measures, 25 SNPs were subjected to statistical analyses. Cochran-Armitage trend test was used to evaluate the association between SNPs and disease susceptibility. To address multiple tests, Bonferroni correction was conducted, setting the threshold for statistical significance at a p-value <2 × 10-3 (0.05/25). Results: The rs13405699 SNP located at 2q31.1 exhibited a significant association with MPB in Han Chinese men (p = 4.84 × 10-5, OR = 1.37, 95% CI: 1.18-1.59). Moreover, the difference in rs13405699 genotype distribution between MPB cases and controls was statistically significant (p = 7.00 × 10-5). Genotype-based association analysis suggested that the recessive model provided the best fit for the rs13405699 polymorphism. Conclusion: This study represents the first confirmation of the association between the rs13405699 SNP at 2q31.1 and MPB within the Han Chinese population, thereby enhancing our understanding of the genetic underpinnings of MPB.

Proapoptotic activity of JNK-sensitive BH3-only proteins underpins ovarian cancer response to replication checkpoint inhibitors.

Recent studies indicate that replication checkpoint modulators (RCMs) such as inhibitors of CHK1, ATR, and WEE1 have promising monotherapy activity in solid tumors, including platinum-resistant high grade serous ovarian cancer (HGSOC). However, clinical response rates are generally below 30%. While RCM-induced DNA damage has been extensively examined in preclinical and clinical studies, the link between replication checkpoint interruption and tumor shrinkage remains incompletely understood. Here we utilized HGSOC cell lines and patient-derived xenografts (PDXs) to study events leading from RCM treatment to ovarian cancer cell death. These studies show that RCMs increase CDC25A levels and CDK2 signaling in vitro, leading to dysregulated cell cycle progression and increased replication stress in HGSOC cell lines independent of homologous recombination status. These events lead to sequential activation of JNK and multiple BH3-only proteins, including BCL2L11/BIM, BBC3/PUMA and the BMF, all of which are required to fully initiate RCM-induced apoptosis. Activation of the same signaling pathway occurs in HGSOC PDXs that are resistant to poly(ADP-ribose) polymerase inhibitors but respond to RCMs ex vivo with a decrease in cell number in 3-dimensional culture and in vivo with xenograft shrinkage or a significantly diminished growth rate. These findings identify key cell death-initiating events that link replication checkpoint inhibition to antitumor response in ovarian cancer.

Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells.

Human genomes are susceptible to damage by a variety of endogenous and exogenous agents. If not repaired, the resulting DNA lesions can potentially lead to mutations, genome instability, and cell death. While existing in vitro experiments allow for characterizing replication outcomes from the use of purified translesion synthesis (TLS) DNA polymerases, such studies often lack the sophistication and dynamic nature of cellular contexts. Here, we present a strand-specific PCR-based Competitive Replication and Adduct Bypass (ssPCR-CRAB) assay designed to investigate quantitatively the impact of DNA lesions on replication efficiency and fidelity in mammalian cells. Combined with genetic manipulation, this approach facilitates the revelation of diverse functions of TLS polymerases in replication across DNA lesions.

How bacteria initiate DNA replication comes into focus.

The ability to initiate DNA replication is a critical step in the proliferation of all organisms. In bacteria, this process is mediated by an ATP-dependent replication initiator protein, DnaA, which recognizes and melts replication origin (oriC) elements. Despite decades of biochemical and structural work, a mechanistic understanding of how DnaA recognizes and unwinds oriC has remained enigmatic. A recent study by Pelliciari et al. provides important new structural insights into how DnaA from Bacillus subtilis recognizes and processes its cognate oriC, showing how DnaA uses sequence features encoded in the origin to engage melted DNA. Comparison of the DnaA-oriC structure with archaeal/eukaryl replication origin complexes based on Orc-family proteins reveals a high degree of similarity in origin engagement by initiators from di domains of life, despite fundamental differences in origin melting mechanisms. These findings provide valuable insights into bacterial replication initiation and highlight the intriguing evolutionary history of this fundamental biological process.

Incorporating replication in higher education: Supervisors' perspectives and institutional pressures.

Background: Even though replication research has gained traction within academia over the recent years, it is not often well-received as a stand-alone thesis topic by supervisors and university administrators.Methods: In this qualitative investigation, we delve into the perspectives of academic supervisors on the feasibility of replication as a thesis topic within the field of applied linguistics (AL). Drawing on Institutional Theory, administrative pressures facing supervisors on what to be considered permissible for a thesis were also explored. By conducting semi-structured e-mail interviews with a global cohort of AL supervisors and a thematic analysis of their responses, a nuanced landscape was brought to light.Results: Supervisors outlined numerous benefits associated with replication including fostering academic advancement as well as providing opportunities for reevaluating prior research. Nonetheless, they also pointed to several obstacles along the way, such as concerns over originality, constraints on time and resources, and the necessity for mentorship. Moreover, supervisors emphasized their pivotal role as decision-makers in accepting or rejecting replication for a thesis project, while acknowledging the partial influence of institutional pressures.Conclusions: Lastly, some implications and recommendations on allocating more resources to replication research are provided.

Infant-derived human nasal organoids exhibit relatively increased susceptibility, epithelial responses, and cytotoxicity during RSV infection.

BACKGROUND: Respiratory syncytial virus (RSV) causes significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe compared to healthy adults is not fully understood. METHODS: We used ex-vivo human nasal organoid platforms from infants and adults to investigate the underlying mechanism of this disease disparity at the initial site of RSV replication, the nasal epithelium. RESULTS: Infant-derived human nasal organoid-air liquid interface (HNO-ALIs) lines were more susceptible to early RSV replication. Moreover, infant-derived HNO-ALIs elicited a statistically significant greater overall cytokine response, enhanced mucous production, and greater cellular damage compared to their adult counterparts. Furthermore, the adult cytokine response was associated with a superior regulatory cytokine response, which could explain less cellular damage than in infant lines. CONCLUSIONS: Our data highlights substantial differences in how infant and adult upper respiratory tract epithelium responds to RSV infection at the cellular level. These differences in epithelial cellular response can lead to impaired mucociliary clearance, a more dysregulated innate immune response predisposing infants to more severe RSV infection compared to adults.

Life should be redefined: Any molecule with the ability to self-replicate should be considered life.

Understanding the nature of life and its propensity for reproduction has long been a question that humans aspire to answer. Reproduction, a defining characteristic of life, fundamentally involves the replication of genetic material, be it DNA or RNA. The driving force behind this replication process has always intrigued scientists. In recent years, theories involving selfish genes, the RNA world, and entropic forces have been proposed by some scholars. These theories seem to suggest that life, as we know it, exists solely in Earth's environment and is based on a single type of genetic material, either DNA or RNA. However, if we broaden our definition of life to include any replicable molecules, we might be able to transcend traditional thought. This could potentially enhance our understanding of the impetus behind DNA replication and provide deeper insights into the essence of life.

Closing the Dissemination Gap: Accessible Toolkits for the Rapid Replication of Evidence-Informed Interventions to Improve Health Outcomes Among People with HIV.

Despite advances in HIV care and treatment in the U.S., disparities in outcomes along the HIV care continuum persist. The widespread replication of effective and sustainable interventions that prioritize the engagement of underserved populations has been identified as a promising path to ending the HIV epidemic in the U.S. Intervention dissemination products, however, rarely provide the comprehensive and accessible information needed to replicate interventions within community settings. To bridge the divide between research and community-based implementation, the Using Evidence-informed Interventions to Improve Health Outcomes among People Living with HIV (E2i) initiative-grounded in the HIV/AIDS Bureau Implementation Science Framework-created a suite of tools to promote the rapid replication of interventions focused on transgender women, Black men who have sex with men, behavioral health integration, and identifying and addressing trauma. The resulting dissemination products are detailed and digestible multimedia toolkits that follow adult learning theory principles and align with the Template for Intervention Description and Replication criteria for adapting non-pharmacological interventions. Each E2i toolkit consists of five components: implementation guides, narrative videos of site implementation, best practice demonstration videos, interactive learning modules, and recruitment posters and brochures. Over 2 years (2022-2024), the E2i toolkit webpages amassed 7703 unique users and 17,666 pageviews. These toolkits can serve as a blueprint for designing comprehensive and accessible dissemination products for replication of HIV interventions in care settings. Dissemination products that bridge the gap between intervention research and replication in community settings are a crucial missing tool for ending the HIV epidemic.

Phosphorylation of Ser711 residue in the hypervariable region of zoonotic genotype 3 hepatitis E virus is important for virus replication.

Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both in vitro and in vivo. We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.

DeOri 10.0: An Updated Database of Experimentally Identified Eukaryotic Replication Origins.

DNA replication is a complex and crucial biological process in eukaryotes. To facilitate the study of eukaryotic replication events, we present a database of eukaryotic DNA replication origins (DeOri), which collects scattered data and integrates extensive sequencing data on eukaryotic DNA replication origins. With continuous updates of DeOri, the number of datasets in the new release increased from 10 to 151 and the number of sequences increased from 16,145 to 9,742,396. Besides nucleotide sequences and bed files, corresponding annotation files, such as coding sequences (CDS), mRNA, and other biological elements within replication origins, are also provided. The experimental techniques used for each dataset, as well as other statistical data, are also presented on web page. Differences in experimental methods, cell lines, and sequencing technologies have resulted in distinct replication origins, making it challenging to differentiate between cell-specific and non-specific replication. We combined multiple replication origins at the species level, scored them, and screened them. The screened regions were considered as species-conservative origins. They are integrated and presented as reference replication origins (rORIs), including Homo sapiens, Gallus gallus, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans. Additionally, we analyzed the distribution of relevant genomic elements associated with replication origins at the genome level, such as CpG island (CGI), transcription start site (TSS), and G-quadruplex (G4). These analysis results allow users to select the desired data based on it. DeOri is available at http://tubic.tju.edu.cn/deori/.

Effect of mutations in GDNV motif of viral hemorrhagic septicemia virus (VHSV) L protein on polymerase activity, viral growth, and in vivo virulence.

Most Mononegavirales viruses have a GDNQ motif within the L protein, whereas Novirhabdovirus species feature a GDNV motif. This study examined the function of the GDNV motif within the L protein of viral hemorrhagic septicemia virus (VHSV) by modifying its amino acid composition. Substituting the aspartic acid (D) with valine (V) completely abolished polymerase activity in a minigenome assay. Replacing GDNV with GDNQ showed no significant difference in luciferase activity. Further characterization using reverse genetically engineered recombinant viruses revealed that rVHSV-LGDNQ exhibited an accelerated replication rate and higher virus titer in EPC cells than rVHSV-wild. Olive flounder infected with rVHSV-LGDNQ experienced higher early-stage mortality but lower overall mortality than those infected with rVHSV-wild. These findings suggest that while the GDNQ motif may positively influence VHSV replication speed, it may not confer an overall advantage for the ultimate viral pathogenicity.

AAV2 can replicate its DNA by a rolling hairpin or rolling circle mechanism, depending on the helper virus.

Adeno-associated virus type 2 (AAV2) is a small, non-pathogenic, helper virus-dependent parvovirus with a single-stranded (ss) DNA genome of approximately 4.7 kb. AAV2 DNA replication requires the presence of a helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1) and is generally assumed to occur as a strand-displacement rolling hairpin (RHR) mechanism initiated at the AAV2 3' inverted terminal repeat (ITR). We have recently shown that AAV2 replication supported by HSV-1 leads to the formation of double-stranded head-to-tail concatemers, which provides evidence for a rolling circle replication (RCR) mechanism. We have revisited AAV2 DNA replication and specifically compared the formation of AAV2 replication intermediates in the presence of either HSV-1 or AdV5 as the helper virus. The results confirmed that the AAV2 DNA replication mechanism is helper virus-dependent and follows a strand-displacement RHR mechanism when AdV5 is the helper virus and primarily an RCR mechanism when HSV-1 is the helper virus. We also demonstrate that recombination plays a negligible role in AAV2 genome replication. Interestingly, the formation of high-molecular-weight AAV2 DNA concatemers in the presence of HSV-1 as the helper virus was dependent on an intact HSV-1 DNA polymerase. IMPORTANCE: AAV is a small helper virus-dependent, non-pathogenic parvovirus. The AAV genome replication mechanism was extensively studied in the presence of AdV as the helper virus and described to proceed using RHR. Surprisingly, HSV-1 co-infection facilitates RCR of the AAV2 DNA. We directly compared AdV5 and HSV-1 supported AAV2 DNA replication and showed that AAV2 can adapt its replication mechanism to the helper virus. A detailed understanding of the AAV replication mechanism expands our knowledge of virus biology and can contribute to increase gene therapy vector production.

Identification and characterization of a novel small viral peptide (VSP59) encoded by Bombyx mori cypovirus (BmCPV) that negatively regulates viral replication.

Bombyx mori cypovirus (BmCPV), a member of the Reoviridae family, is a well-established research model for double-stranded RNA (dsRNA) viruses with segmented genomes. Despite its small genome size, the coding potential of BmCPV remains largely unexplored. In this study, we identified a novel small open reading frame within the S10 dsRNA genome, encoding a small viral peptide (VSP59) with 59 amino acid residues. Functional characterization revealed that VSP59 acts as a negative regulator of viral replication. VSP59 predominantly localizes to the cytoplasm, where it interacts with prohibitin 2 (PHB2), an inner membrane mitophagy receptor. This interaction targets mitochondria and triggers caspase 3-dependent apoptosis. Transient expression of vsp59 in BmN cells suppressed viral replication, an effect that was reversed by silencing PHB2 expression. Moreover, recombinant BmCPV with a mutated vsp59 exhibited reduced replication. Our findings demonstrate that VSP59 interacts with PHB2 on mitochondria, inducing apoptosis and thereby diminishing viral replication. This study expands our understanding of the genetic information encoded by the BmCPV genome and highlights the role of novel small peptides in host-virus interactions. IMPORTANCE: A novel small open reading frame (sORF) from the viral genome was identified and characterized. The sORF could encode a small viral peptide (VSP59) that targeted mitochondria and induced prohibitin 2-related apoptosis, further attenuating Bombyx mori cypovirus replication.

X-ray crystal structure of proliferating cell nuclear antigen 1 from Aeropyrum pernix.

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. In this study, the crystal structure of ApePCNA1, one of three PCNAs from the thermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Šresolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols.

Enhancing quantitative imaging to study DNA damage response: A guide to automated liquid handling and imaging.

Laboratory automation and quantitative high-content imaging are pivotal in advancing diverse scientific fields. These innovative techniques alleviate the burden of manual labour, facilitating large-scale experiments characterized by exceptional reproducibility. Nonetheless, the seamless integration of such systems continues to pose a constant challenge in many laboratories. Here, we present a meticulously designed workflow that automates the immunofluorescence staining process, coupled with quantitative high-content imaging to study DNA damage signalling as an example. This is achieved by using an automatic liquid handling system for sample preparation. Additionally, we also offer practical recommendations aimed at ensuring the reproducibility and scalability of experimental outcomes. We illustrate the high level of efficiency and reproducibility achieved through the implementation of the liquid handling system but also addresses the associated challenges. Furthermore, we extend the discussion into critical aspects such as microscope selection, optimal objective choices, and considerations for high-content image acquisition. Our study streamlines the image analysis process, offering valuable recommendations for efficient computing resources and the integration of cutting-edge deep learning techniques. Emphasizing the paramount importance of robust data management systems aligned with the FAIR data principles, we provide practical insights into suitable storage options and effective data visualization techniques. Together, our work serves as a comprehensive guide for life science laboratories seeking to elevate their high-content quantitative imaging capabilities through the seamless integration of advanced laboratory automation.