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Staňurová et al. 2025 | Preprint | Link
Insulin is a key hormone in glucose homeostasis Its lack causes severe health complications and has to be compensated by regular administration of insulin Despite intense long-lasting research a more stable substitute has yet to be discovered to alleviate patients' issues Here we report the development of a novel assay for screening potential insulin analogues based on the recently published method DIANA Our assay meets the need for a fast non-radioactive method as a sensitive alternative to the commonly used radioactive immunoassay |Related Solutions: MANTIS
Escós et al. 2025 | Preprint | Link
Cells of the myeloid lineage particularly monocytes and macrophages play a key role in HIV infection by contributing to viral replication immune response and maintaining immune balance during suppressive therapy We hypothesized that metabolic reprogramming and altered chemokine signaling in people living with HIV PWH on long-term antiretroviral therapy ART affect monocyte transport and polarization due to ongoing inflammation Therefore the present study aimed to identify the mechanism of impaired monocyte macrophage function in PWH on well-treated ART that can lead to clinical intervention strategies to improve health Single-cell RNA sequencing immune-phenotyping and metabolic modeling identified altered expression of chemokine ... More |Related Solutions: MANTIS
Cells of the myeloid lineage, particularly monocytes and macrophages, play a key role in HIV infection by contributing to viral replication, immune response, and maintaining immune balance during suppressive therapy. We hypothesized that metabolic reprogramming and altered chemokine signaling in people living with HIV (PWH) on long-term antiretroviral therapy (ART) affect monocyte transport and polarization due to ongoing inflammation. Therefore, the present study aimed to identify the mechanism of impaired monocyte/macrophage function in PWH on well-treated ART that can lead to clinical intervention strategies to improve health. Single-cell RNA sequencing, immune-phenotyping, and metabolic modeling identified altered expression of chemokine and metabolite receptors and altered metabolic flux in PWH monocytes that decreased monocyte migration. The plasma secretome revealed a nonclassical inflammatory microenvironment in PWH. Integrative multi-omics and single-cell proteomics of differentiated monocyte-derived macrophages (MDMs) detected metabolic reprogramming orchestrated by α-ketoglutarate (AKG) that affected macrophage function and HIV infection. Increased levels of AKG in plasma were shown to occur in PWH under ART. Therefore, when differentiating MDM with serum from PWH or AKG, macrophage function was found polarized towards an M2-like state. AKG alone was shown to increase CCR5 levels and increase HIV-1 infection in MDM. Here, we utilize systems biology-driven identification and ex vivo assays to show impaired macrophage polarization, due to metabolic training, can leads to a low-grade nonclassical inflammatory environment in well-treated PWH. Less |Related Solutions: MANTIS
Makhmut et al. 2025 | Preprint | Link
High-grade serous ovarian cancer HGSOC is a devastating disease that is frequently detected at an advanced and incurable stage Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential Here we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma STIC the HGSOC precursor lesion covering more than proteins from ultralow input archival tissue We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level revealing a similar level of phenotypic and molecular heterogeneity Using cell-type resolved tissue ... More |Related Solutions: MANTIS
High-grade serous ovarian cancer (HGSOC) is a devastating disease that is frequently detected at an advanced and incurable stage. Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential. Here, we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma (STIC), the HGSOC precursor lesion, covering more than 10,000 proteins from ultralow input archival tissue. We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level, revealing a similar level of phenotypic and molecular heterogeneity. Using cell-type resolved tissue proteomics, we revealed strong cell-of-origin signatures preserved in STICs and invasive tumors and identified early dysregulated pathways of therapeutic relevance. These include proliferation and DNA damage repair signatures, as well as onco-metabolic changes, such as increased cholesterol biosynthesis. Finally, we uncovered substantial remodeling of the co-evolving tumor microenvironment, affecting approximately one-third of the stromal proteome, and derived a common signature associated with progressive immunosuppression and ECM restructuring. In summary, our study highlights the power of spatially resolved quantitative proteomics to dissect the molecular underpinnings of early carcinogenesis and provides a rich proteomic resource for future biomarker and drug target discovery. Less |Related Solutions: MANTIS
Douglas et al. 2025 | ACS Catalysis | Link
High-throughput experimentation HTE is a critical tool in modern pharmaceutical discovery and development The ability to perform multiple parallel experiments in miniaturized plate-based formats has revolutionized how chemical reactions are optimized HTE has been especially enabling for catalytic reactions where the complexity of factors influencing the outcome makes the HTE approach especially suitable We detail AstraZeneca s -year journey with HTE from early beginnings to a global community of HTE specialists that are critical to the delivery of our complex portfolio with reduced environmental impact With an emphasis on catalytic reactions we provide relevant case study examples from across discovery ... More |Related Solutions: FAST
High-throughput experimentation (HTE) is a critical tool in modern pharmaceutical discovery and development. The ability to perform multiple parallel experiments in miniaturized plate-based formats has revolutionized how chemical reactions are optimized. HTE has been especially enabling for catalytic reactions, where the complexity of factors influencing the outcome makes the HTE approach especially suitable. We detail AstraZeneca’s 20-year journey with HTE, from early beginnings to a global community of HTE specialists that are critical to the delivery of our complex portfolio with reduced environmental impact. With an emphasis on catalytic reactions, we provide relevant case study examples from across discovery and development, discuss current technology, data science and workflows, and provide insights into where we see future advances in HTE. Less |Related Solutions: FAST
Møller et al. 2025 | Synthetic and Systems Biotechnology | Link
The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds Advances in molecular engineering tools such as CRISPR have provided some access to the metabolic potential of Streptomyces but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols In this paper we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp using the affordable and open-sourced Opentrons OT- robotics platform To increase the accessibility of the workflow we provide an open-source protocol-creator ActinoMation ActinoMation is a literate programming environment using Python in Jupyter Notebook We validated ... More |Related Solutions: MANTIS
The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds. Advances in molecular engineering tools, such as CRISPR, have provided some access to the metabolic potential of Streptomyces, but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols. In this paper, we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp. using the affordable and open-sourced Opentrons (OT-2) robotics platform. To increase the accessibility of the workflow we provide an open-source protocol-creator, ActinoMation. ActinoMation is a literate programming environment using Python in Jupyter Notebook. We validated the method by transforming Streptomyces coelicolor (M1152 and M1146), S. albidoflavus (J1047), and S. venezuelae (DSM40230) with the plasmids pSETGUS and pIJ12551. We demonstrate conjugation efficiencies of 3.33*10-3/0.33% for M1152 with pSETGUS and pIJ12551; 2.96*10-3/0.29%for M1146 with pSETGUS and pIJ12551; 1.21*10-5/0.0012% for J1047 with pSETGUS and 4.70*10-4/0.047% with pIJ12551, and 4.97*10-2/4.97% for DSM40230 with pSETGUS and 6.13*10-2 /6.13% with pIJ12551 with a false positive rate between 8.33% and 54.54%. Automation of the conjugation workflow facilitates a streamlined workflow on a larger scale without any evident loss of conjugation efficiency. Less |Related Solutions: MANTIS
Jeevannavar et al. 2025 | The ISME Journal | Link
Single-cell transcriptomics is a key tool for unravelling metabolism and tissue diversity in model organisms Its potential for elucidating the ecological roles of microeukaryotes especially non-model ones remains largely unexplored This study employed the Smart-seq protocol on Ochromonas triangulata a microeukaryote lacking a reference genome showcasing how transcriptional states align with two distinct growth phases a fast-growing phase and a slow-growing phase Besides the two expected expression clusters each corresponding to either growth phase a third transcriptional state was identified across both growth phases Metabolic mapping revealed a boost of photosynthetic activity in the fast growth over the slow growth ... More |Related Solutions: MANTIS
Single-cell transcriptomics is a key tool for unravelling metabolism and tissue diversity in model organisms. Its potential for elucidating the ecological roles of microeukaryotes, especially non-model ones, remains largely unexplored. This study employed the Smart-seq2 protocol on Ochromonas triangulata, a microeukaryote lacking a reference genome, showcasing how transcriptional states align with two distinct growth phases: a fast-growing phase and a slow-growing phase. Besides the two expected expression clusters, each corresponding to either growth phase, a third transcriptional state was identified across both growth phases. Metabolic mapping revealed a boost of photosynthetic activity in the fast growth over the slow growth stage, as well as down-regulation trend in pathways associated with ribosome functioning, CO2 fixation, and carbohydrate catabolism characteristic of the third transcriptional state. In addition, carry-over rRNA reads recapitulated the taxonomic identity of the target while revealing distinct bacterial communities, in co-culture with the eukaryote, each associated with distinct transcriptional states. This study underscores single-cell transcriptomics as a powerful tool for characterizing metabolic states in microeukaryotes without a reference genome, offering insights into unknown physiological states and individual-level interactions with different bacterial taxa. This approach holds broad applicability to describe the ecological roles of environmental microeukaryotes, culture-free and reference-free, surpassing alternative methods like metagenomics or metatranscriptomics. Less |Related Solutions: MANTIS
Kanappe et al. 2025 | Bioconjugate Chemistry | Link
Nucleic acid nanoparticles NANPs fabricated by using the DNA origami method have broad utility in materials science and bioengineering Their site-specific heterovalent functionalization with secondary molecules such as proteins or fluorophores is a unique feature of this technology that drives its utility Currently however there are few chemistries that enable fast efficient covalent functionalization of NANPs with a broad conjugate scope and heterovalency To address this need we introduce synthetic methods to access inverse electron-demand Diels Alder chemistry on NANPs We demonstrate a broad conjugate scope characterize application-relevant kinetics and integrate this new chemistry with strain-promoted azide alkyne cycloaddition chemistry ... More |Related Solutions: FLO
Nucleic acid nanoparticles (NANPs) fabricated by using the DNA origami method have broad utility in materials science and bioengineering. Their site-specific, heterovalent functionalization with secondary molecules such as proteins or fluorophores is a unique feature of this technology that drives its utility. Currently, however, there are few chemistries that enable fast, efficient covalent functionalization of NANPs with a broad conjugate scope and heterovalency. To address this need, we introduce synthetic methods to access inverse electron-demand Diels–Alder chemistry on NANPs. We demonstrate a broad conjugate scope, characterize application-relevant kinetics, and integrate this new chemistry with strain-promoted azide–alkyne cycloaddition chemistry to enable heterovalent click reactions on NANPs. We applied these chemistries to formulate a prototypical chemical countermeasure against chemical nerve agents. We envision this additional chemistry finding broad utility in the synthetic toolkit accessible to the nucleic acid nanotechnology community. Less |Related Solutions: FLO
Alaviuhkola et al. 2025 | Preprint | Link
ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD to an acceptor molecule While ADP-ribosylation is well-established as a post-translational modification of proteins rifamycin antibiotics are its only known small-molecule targets ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis whose Arr enzyme transfers the ADP-ribose moiety to the -hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation ... More |Related Solutions: MANTIS
ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay, which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 µM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 µM. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds. Less |Related Solutions: MANTIS
Richardson et al. 2025 | Nature Microbiology | Link
The local arrangement of microbes can profoundly impact community assembly function and stability However our understanding of the spatial organization of the human gut microbiome at the micron scale is limited Here we describe a high-throughput and streamlined method called Split-And-pool Metagenomic Plot-sampling sequencing SAMPL-seq to capture spatial co-localization in a complex microbial consortium The method obtains microbial composition of micron-scale subcommunities through split-and-pool barcoding SAMPL-seq analysis of the healthy human gut microbiome identified bacterial taxa pairs that consistently co-occurred both over time and across multiple individuals These co-localized microbes organize into spatially distinct groups or spatial hubs dominated by ... More |Related Solutions: MANTIS
The local arrangement of microbes can profoundly impact community assembly, function and stability. However, our understanding of the spatial organization of the human gut microbiome at the micron scale is limited. Here we describe a high-throughput and streamlined method called Split-And-pool Metagenomic Plot-sampling sequencing (SAMPL-seq) to capture spatial co-localization in a complex microbial consortium. The method obtains microbial composition of micron-scale subcommunities through split-and-pool barcoding. SAMPL-seq analysis of the healthy human gut microbiome identified bacterial taxa pairs that consistently co-occurred both over time and across multiple individuals. These co-localized microbes organize into spatially distinct groups or ‘spatial hubs’ dominated by Bacteroidaceae, Ruminococcaceae and Lachnospiraceae families. Using inulin as a dietary perturbation, we observed reversible spatial rearrangement of the gut microbiome where specific taxa form new local partnerships. Spatial metagenomics using SAMPL-seq can unlock insights into microbiomes at the micron scale. Less |Related Solutions: MANTIS
Ling et al. 2025 | ACS publications | Link
Fatty acid esters of hydroxy fatty acids FAHFAs are a newly discovered lipid class known for their potential anti-inflammatory and insulin-sensitizing properties A sustainable and efficient synthesis route is essential to realize the potential of FAHFAs and enable cost-effective large-scale production Enzymatic synthesis favored for its scalability and environmental impact is the preferred approach Candida Moesziomyces antarctica lipase A CalA previously known for its thermostability and limited ability to catalyze FAHFA esterification was investigated along with its orthologues for their ability to produce a variety of FAHFAs We developed a systematic workflow to identify uncharacterized enzymes for FAHFA synthesis from ... More |Related Solutions: NT8
Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered lipid class known for their potential anti-inflammatory and insulin-sensitizing properties. A sustainable and efficient synthesis route is essential to realize the potential of FAHFAs and enable cost-effective, large-scale production. Enzymatic synthesis, favored for its scalability and environmental impact, is the preferred approach. Candida (Moesziomyces) antarctica lipase A (CalA), previously known for its thermostability and limited ability to catalyze FAHFA esterification, was investigated along with its orthologues for their ability to produce a variety of FAHFAs. We developed a systematic workflow to identify uncharacterized enzymes for FAHFA synthesis from natural sources, using an automation-compatible method, leading to the discovery of several novel lipases capable of synthesizing diverse FAHFAs. Among these lipases, two newly discovered enzymes, CL20 and CL23, demonstrated superior performance in FAHFA biosynthesis, achieving faster and higher yields than the benchmark enzyme, CalA. Our work advances methodologies and processes critical for industrial FAHFA production and provides a foundation for sustainable commercial-scale synthesis via synthetic enzymology. Less |Related Solutions: NT8
Mpontshane et al. 2025 | Preprint | Link
Candida albicans is a critical priority fungal pathogen causing invasive fungal infections with high mortality rates in immunocompromised patients The increasing fungal infection rate and resistance of fungal pathogens to existing antifungal treatments have emphasized the need for the development of novel antifungal medicine The ergosterol biosynthesis pathway has been a successful target for antifungal compounds but many enzymatic steps remain unexplored C-sterol methyltransferase C-SMT catalyzes a critical fungal-specific step in ergosterol biosynthesis When C-SMT is disrupted fungal pathogens are sensitized to temperature various inhibitors and antifungals and a loss of virulence can be observed In this study five C-SMT ... More |Related Solutions: FORMULATOR
Candida albicans is a critical priority fungal pathogen causing invasive fungal infections with high mortality rates in immunocompromised patients. The increasing fungal infection rate and resistance of fungal pathogens to existing antifungal treatments have emphasized the need for the development of novel antifungal medicine. The ergosterol biosynthesis pathway has been a successful target for antifungal compounds, but many enzymatic steps remain unexplored. 24C-sterol methyltransferase (24C-SMT) catalyzes a critical fungal-specific step in ergosterol biosynthesis. When 24C-SMT is disrupted, fungal pathogens are sensitized to temperature, various inhibitors, and antifungals, and a loss of virulence can be observed. In this study, five 24C-SMT variants with different lengths of N-termini were heterologously produced in Escherichia coli and three were purified to near-homogeneity with immobilized metal-affinity and size-exclusion chromatography. N-terminally truncated C. albicans 24C-SMT was utilized for crystallization trials due to its increased stability and higher purity compared to the full-length protein. 24C-SMT crystals were obtained in the presence of Sadenosyl-homocysteine, but diffracted to low resolution. Therefore, we established a starting point for 24C-SMT crystallization by providing an optimized protocol for heterologous 24C-SMT production, purification and initial crystallization conditions, which could be used for further downstream crystallographic studies. Less |Related Solutions: FORMULATOR
Stamos et al. 2025 | Preprint | Link
Crystallisation and stereochemical stability are pivotal factors in pharmaceutical development particularly for complex beyond Rule of bRo compounds In this study we explore the intricate interplay between atropisomerism and crystallisation using two model bRo compounds namely ACBI and BI both violating three of four Lipinski s rules One of the tool compounds exhibits Class atropisomeric behaviour and the other devoid of it A diverse array of crystallisation methods including solution-phase crystallisation cocrystallisation and salt formation was applied revealing the critical role of atropisomerism induced stereochemistry in polymorphism and nucleation outcomes In-silico torsion profile calculations and NMR studies were employed to ... More |Related Solutions: ROCKIMAGER
Crystallisation and stereochemical stability are pivotal factors in pharmaceutical development, particularly for complex beyond Rule of 5 (bRo5) compounds. In this study, we explore the intricate interplay between atropisomerism and crystallisation using two model bRo5 compounds, namely ACBI1 and BI201335, both violating three of four Lipinski’s rules. One of the tool compounds exhibits Class 2 atropisomeric behaviour and the other devoid of it. A diverse array of crystallisation methods—including solution-phase crystallisation, cocrystallisation, and salt formation—was applied, revealing the critical role of atropisomerism induced stereochemistry in polymorphism and nucleation outcomes. In-silico torsion profile calculations and NMR studies were employed to elucidate the rotational energy barriers and confirm the presence or absence of atropisomerism. This comprehensive analysis highlights the significance of understanding stereochemical phenomena like atropisomerism in designing and developing bRo5 compounds. By integrating advanced analytical techniques and crystallisation strategies, this work provides novel insights into tailoring pharmaceutical properties for nextgeneration therapeutics. Less |Related Solutions: ROCKIMAGER
Tan et al. 2025 | Vaccines | Link
ackground The Safety and Immunogenicity of COVID- Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases SUCCEED study was created to better understand COVID- vaccination in immune-mediated inflammatory disease IMID Knowing the frequency of COVID- breakthrough infections is important particularly in IMID Our objective was to assess these events in IMID Methods We prospectively studied IMID participants who had received three COVID- vaccine doses Individuals provided saliva samples monthly September to August These were evaluated by polymerase chain reaction PCR for SARS-CoV- We also assessed antibodies against SARS-CoV- anti-spike SmT receptor binding domain RBD and nucleocapsid NP based on dried blood spots Multivariable ... More |Related Solutions: MANTIS
ackground: The Safety and Immunogenicity of COVID-19 Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases (SUCCEED) study was created to better understand COVID-19 vaccination in immune-mediated inflammatory disease (IMID). Knowing the frequency of COVID-19 breakthrough infections is important, particularly in IMID. Our objective was to assess these events in IMID. Methods: We prospectively studied IMID participants who had received ≥three COVID-19 vaccine doses. Individuals provided saliva samples monthly (September 2022 to August 2023). These were evaluated by polymerase chain reaction (PCR) for SARS-CoV-2. We also assessed antibodies against SARS-CoV-2 (anti-spike, SmT1, receptor binding domain, RBD, and nucleocapsid, NP) based on dried blood spots. Multivariable general estimating equation regression produced odd ratios (OR) for PCR SARS-CoV-2 positivity, related to demographics, immunosuppressives, and antibody levels. Results: Diagnoses included rheumatoid arthritis RA (N = 161, 44% of the total), systemic lupus, psoriatic arthritis, spondylarthritis, vasculitis, systemic sclerosis, and inflammatory bowel disease. Of the 366 participants, most were taking immunosuppressive medication. Of 1266 saliva samples, 56 (5.1%) were positive for SARS-CoV-2 on PCR. Higher anti-SmT1 antibodies were inversely associated with SARS-CoV-2 detection on PCR (adjusted OR 0.66, 95% confidence interval 0.45–0.97). Antibodies to SmT1, RBD, and NP were correlated and thus could not be included in a single model, but when anti-RBD was used in place of anti-SmT1, the results were similar. No other factor (including prior COVID-19 infection) was clearly associated with SARS-CoV-2 detection. Conclusions: This is the first study of SARS-CoV-2 in a large prospective cohort of triple (or more) vaccinated individuals with IMIDs. Anti-SmT1 antibodies appeared to be protective against later SARS-CoV-2 positivity, although recent past infection was not clearly related. This suggests the importance of maintaining robust vaccine-induced immunity through vaccination in IMID. Less |Related Solutions: MANTIS
al. et al. 2025 | Pharmaceutical Research | Link
Background High concentration protein formulation HCPF development needs to balance protein stability attributes such as conformational colloidal stability chemical stability and solution properties such as viscosity and osmolality Methodology A three-phase design is established in this work In Phase conformational and colloidal stability are measured by -well-based high-throughput HT biophysical screening while viscosity reduction screening is performed with HT viscosity screening Collectively the biophysical and viscosity screening data are leveraged to design the phase of short-term stability study executed using -well plates under thermal and freeze thaw stresses In phase samples are analyzed by stability-indicating assays and processed with pair-wise ... More |Related Solutions: µPULSE
Background High concentration protein formulation (HCPF) development needs to balance protein stability attributes such
as conformational/colloidal stability, chemical stability, and solution properties such as viscosity and osmolality.
Methodology A three-phase design is established in this work. In Phase 1, conformational and colloidal stability are measured by 384-well-based high-throughput (HT) biophysical screening while viscosity reduction screening is performed with HT viscosity screening. Collectively, the biophysical and viscosity screening data are leveraged to design the phase 2 of short-term stability study, executed using 96-well plates under thermal and freeze/thaw stresses. In phase 2, samples are analyzed by stability-indicating assays and processed with pair-wise Student’s t-test analyses to choose the final formulations. In phase 3, the final formulations are then confirmed through a one-month accelerated stability in glass vials.
Results Using a model antibody A (mAb-A), the initial HT screening successfully established the 384-well based platform.
A lead formulation was chosen from the second round based on statistical analyses and subsequently tested against the commercial
formulation of mAb-A as a control. Compared to the control, the lead formulation reduced the viscosity of mAb-A by 30% and decreased subvisible particles after thermal stress by 80%.
Conclusions HT biophysical screening in 384-well plates was demonstrated to effectively guide the rational design of a high-throughput stability screening study using 96-well plates. This platform enables the identification of a high concentration formulation within seven weeks within the first two phases of study that strategically balance stability with solution
properties, thus achieving a rapid development of HCPF. Less |Related Solutions: µPULSE
Wright et al. 2025 | Preprint | Link
The rise of drug-resistant fungal pathogens including Candida auris highlights the urgent need for novel antifungal therapies We developed a cost-effective platform combining microbial extract prefractionation with rapid MS MS-bioinformatics-based dereplication to efficiently prioritize new antifungal scaffolds Screening C auris and C albicans revealed novel lipopeptaibiotics coniotins from Coniochaeta hoffmannii WAC which were undetectable in crude extracts Coniotins exhibited potent activity against critical fungal pathogens on the WHO Fungal Priority Pathogens List including C albicans C neoformans multidrug-resistant C auris and Aspergillus fumigatus with high selectivity and low resistance potential Coniotin A targets -glucan compromising fungal cell wall integrity remodelling ... More |Related Solutions: TEMPEST
The rise of drug-resistant fungal pathogens, including Candida auris, highlights the urgent need for novel antifungal therapies. We developed a cost-effective platform combining microbial extract prefractionation with rapid MS/MS-bioinformatics-based dereplication to efficiently prioritize new antifungal scaffolds. Screening C. auris and C. albicans revealed novel lipopeptaibiotics, coniotins, from Coniochaeta hoffmannii WAC11161, which were undetectable in crude extracts. Coniotins exhibited potent activity against critical fungal pathogens on the WHO Fungal Priority Pathogens List, including C. albicans, C. neoformans, multidrug-resistant C. auris, and Aspergillus fumigatus, with high selectivity and low resistance potential. Coniotin A targets β-glucan, compromising fungal cell wall integrity, remodelling, and sensitizing C. auris to caspofungin. Identification of a PKS-NRPS biosynthetic gene cluster further enables the discovery of related clusters encoding potential novel lipopeptaibiotics. This study demonstrates the power of natural product prefractionation in uncovering bioactive scaffolds and introduces coniotins as promising candidates for combating multidrug-resistant fungal pathogens. Less |Related Solutions: TEMPEST
Duong et al. 2025 | Scientific Reports | Link
Recombinant adeno-associated virus rAAV has emerged as the vector of choice for in vivo gene delivery with numerous clinical trials underway for the treatment of various human diseases Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product The conventional single-channel droplet digital PCR D ddPCR method offers only partial information regarding the viral vector genome titer lacking insights into its integrity In our pursuit of further advancing rAAV analysis we have developed a novel D ddPCR assay with advanced D linkage ... More |Related Solutions: MANTIS
Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow’s output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases. Less |Related Solutions: MANTIS
Böhm et al. 2025 | Preprint | Link
The C carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms In maize and sorghum the evolution of C -NADP-malic enzyme C -NADP-ME involved gene duplication and neofunctionalization leading to the emergence of two plastidic isoforms C -NADP-ME and nonC -NADP-ME each with distinct kinetic and structural features While C -NADP-ME functions primarily as a tetramer nonC -NADP-ME exists in an equilibrium between dimeric and tetrameric forms favoring the dimer in solution This study shows which evolutionary changes in amino acid sequences influence the structure and function ... More |Related Solutions: ROCKIMAGER
The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions. Less |Related Solutions: ROCKIMAGER
Krysan et al. 2025 | Preprint | Link
Acetyl CoA synthetases ACS have emerged as drug targets for the treatment of cancer metabolic diseases as well as fungal and parasitic infections Although a variety of small molecule ACS inhibitors have been discovered the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition Through a chemical genetic-based synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C neoformans Acs relative to human ACSS as well as other fungal ACSs Xray crystallographic characterization of the ... More |Related Solutions: NT8
Acetyl CoA synthetases (ACS) have emerged as drug targets for the treatment of cancer, metabolic diseases as well as fungal and parasitic infections. Although a variety of small molecule ACS inhibitors have been discovered, the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition. Through a chemical genetic-based, synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans, we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C. neoformans Acs1 relative to human ACSS2 as well as other fungal ACSs. Xray crystallographic characterization of the isoxazole-CnAcs1 complex revealed that the isoxazole functions as an acetyl CoA mimic and occupies both the acetyl- and CoA-binding sites of CnAcs1. Consistent with this novel mode of inhibition, the isoxazoles display uncompetitive inhibition kinetics that are similar to antimalarial ACS inhibitors also proposed to target the CoA binding site. Consequently, these data provide structural and mechanistic insights into the remarkable selectivity of Acetyl CoA pocket-targeting ACS inhibitors. In addition, these data provide strong proof-of-principle that targeting fungal and parasitic ACSs for the development of novel anti-infectives can be achieved with high selectivity and, thereby, low host toxicity. Less |Related Solutions: NT8
Kaundal et al. 2024 | Science Advances | Link
Many viral proteins form biomolecular condensates via liquid-liquid phase separation LLPS to support viral replication and evade host antiviral responses and thus they are potential targets for designing antivirals In the case of nonenveloped positive-sense RNA viruses forming such condensates for viral replication is unclear and less understood Human noroviruses HuNoVs are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide Here we show that the RNA-dependent RNA polymerase RdRp of pandemic GII HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication ... More |Related Solutions: ROCKIMAGER
Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA. Less |Related Solutions: ROCKIMAGER
Dunnett et al. 2024 | Preprint | Link
The human heterogeneous nuclear ribonucleoprotein hnRNP A is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells As a multifunctional protein with a key role in RNA metabolism deregulation of its functions has been linked to neurodegenerative diseases tumour aggressiveness and chemoresistance which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities Here using a combination of Molecular Dynamics MD simulations and graph neural network pockets predictions we showed that hnRNPA N-terminal RNA binding domain UP contains several cryptic pockets capable of binding small molecules To identify chemical entities for ... More |Related Solutions: ROCKIMAGER
The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pockets predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits which extensively samples UP1 functional regions involved in RNA recognition and binding, as well as mapping hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provides rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1 mediated splicing regulation. Less |Related Solutions: ROCKIMAGER
McFadden et al. 2024 | Cell | Link
Crimean-Congo hemorrhagic fever virus CCHFV is a tickborne virus that can cause severe disease in humans with case fatality rates of Although structures of CCHFV glycoproteins GP and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies the structure of glycoprotein Gn and its interactions with GP and Gc have remained elusive Here we use structure-guided protein engineering to produce a stabilized GP -Gn-Gc heterotrimeric glycoprotein complex GP -GnH-DS-Gc A cryo-electron microscopy cryo-EM structure of this complex provides the molecular basis for GP s association on the viral surface reveals the structure of Gn ... More |Related Solutions: NT8
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne virus that can cause severe disease in humans with case fatality rates of 10%–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we use structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-electron microscopy (cryo-EM) structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. Less |Related Solutions: NT8
Barekatain et al. 2024 | Science Signaling | Link
GPR is an orphan G protein coupled receptor with high constitutive activity found in D -type dopamine receptor expressing medium spiny neurons of the striatopallidal pathway which is aberrantly hyperactivated in Parkinson s disease Here we solved crystal structures of GPR without the addition of a ligand a pseudo-apo state and in complex with two inverse agonists including CVN which improved motor symptoms in patients with Parkinson s disease in clinical trials In addition we obtained a cryo electron microscopy structure of the signaling complex between GPR and its cognate Gs heterotrimer The pseudo-apo structure revealed a strong density in ... More |Related Solutions: NT8
GPR6 is an orphan G protein–coupled receptor with high constitutive activity found in D2-type dopamine receptor–expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson’s disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson’s disease in clinical trials. In addition, we obtained a cryo–electron microscopy structure of the signaling complex between GPR6 and its cognate Gs heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling. Less |Related Solutions: NT8
Usovsky et al. 2024 | Molecular Biology Reports | Link
Background Whole genome resequencing WGRS platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program Therefore there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs The objective was to develop a comprehensive panel of soybean cyst nematode SCN resistance TaqMan assays via selecting the causative genes and analyzing their associated alleles Methods The Soybean Allele Catalog ... More |Related Solutions: MANTIS
Background
Whole genome resequencing (WGRS) platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program. Therefore, there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs. The objective was to develop a comprehensive panel of soybean cyst nematode (SCN) resistance TaqMan® assays via selecting the causative genes and analyzing their associated alleles.

Methods
The Soybean Allele Catalog was utilized to investigate WGRS-derived variants which are predicted to cause a change in the amino acid sequence of a gene product. This panel of TaqMan® assays reflects current knowledge about known SCN resistance-causing genes and their associated alleles: GmSNAP18-a and -b, GmSNAP11, GmSHMT08, GmSNAP15, GmNSFRAN07, and GmSNAP02-ins and -del. Developed assays were tested using elite breeding lines and segregating populations. TaqMan assays were compared to other currently available KASP and CAPS assays.

Conclusion
All assays showed excellent allele determination efficiencies. This SCN genotyping assay panel can be utilized as a simplified, accurate and reliable genotyping platform further equipping the updated soybean breeding toolbox. Less |Related Solutions: MANTIS
Kornilov et al. 2024 | International Journal of Biological Macromolecules | Link
Heliorhodopsins HeRs constitute a novel and distinct group of microbial rhodopsins characterized by the inverted position of C- and N- termini relative to conventional Type I rhodopsins The production of HeRs for structural and functional investigations has proven challenging as evidenced by the structural elucidation of only two proteins and the functional characterization of a few others to date Notably no eukaryotic HeRs have been reported thus far In this study we report the first expression of three eukaryotic HeRs in the LEXSY expression system from marine and freshwater algae and a free-living marine unicellular eukaryote We spectroscopically characterized these ... More |Related Solutions: NT8
Heliorhodopsins (HeRs) constitute a novel and distinct group of microbial rhodopsins, characterized by the inverted position of C- and N- termini relative to conventional Type I rhodopsins. The production of HeRs for structural and functional investigations has proven challenging, as evidenced by the structural elucidation of only two proteins and the functional characterization of a few others to date. Notably, no eukaryotic HeRs have been reported thus far. In this study, we report the first expression of three eukaryotic HeRs in the LEXSY expression system: from marine and freshwater algae and a free-living marine unicellular eukaryote. We spectroscopically characterized these HeRs, demonstrating that they were expressed in the functional states. Finally, we report their successful crystallization, thus paving the way for their further structural and functional studies Less |Related Solutions: NT8
Naz et al. 2024 | International Journal of Molecular Sciences | Link
Phosphopentomutases catalyze the isomerization of ribose -phosphate and ribose -phosphate Thermococcus kodakarensis a hyperthermophilic archaeon harbors a novel enzyme PPMTk that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity Instead PPMTk catalyzes the interconversion of ribose -phosphate and ribose -phosphate Here we report biophysical analysis crystallization and three-dimensional structure determination of PPMTk by X-ray diffraction at resolution The solved structure revealed a novel catalytic motif unique to PPMTk which makes this enzyme distinct from the homologous counterparts We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose To the best of ... More |Related Solutions: ROCKIMAGER
Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPMTk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPMTk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPMTk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPMTk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea. Less |Related Solutions: ROCKIMAGER
Schneberger et al. 2024 | Communications Biology | Link
The tripartite ATP-independent periplasmic TRAP transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid aiding their colonization of human hosts This process depends on SiaP a substrate-binding protein SBP that captures and delivers sialic acid to the transporter We identified nanobodies that bind specifically to the SiaP proteins from H influenzae HiSiaP and V cholerae VcSiaP Two nanobodies inhibited sialic acid binding Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism preventing ligand binding and releasing pre-bound sialic acid A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and ... More |Related Solutions: ROCKIMAGER
The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules. Less |Related Solutions: ROCKIMAGER
Murugesan et al. 2024 | Nature Communications | Link
The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases Chronic Hepatitis B virus HBV infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope Env protein hepatitis B surface antigen HBsAg Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide Env - identified through bioinformatic predictions and verified by biochemical and cellular assays Using a soluble affinity-enhanced T cell receptor TCR a b -anti-CD bispecific molecule to probe HLA-E presentation of the Env - peptides we demonstrate that only the most stable ... More |Related Solutions: ROCKIMAGER
The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env371-379 peptides, we demonstrate that only the most stable Env371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env371-379 L6I-specific CD8+ T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules. Less |Related Solutions: ROCKIMAGER
Chuang et al. 2024 | BMC Genomics | Link
We developed an automated high-throughput Smart-seq HT Smart-seq workflow that integrates best practices and an optimized protocol to enhance efficiency scalability and method reproducibility This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity In a rigorous comparison with the X platform using human primary CD T-cells HT Smart-seq demonstrated higher cell capture efficiency greater gene detection sensitivity and lower dropout rates Additionally when sufficiently scaled HT Smart-seq achieved a comparable resolution of cellular heterogeneity to X Notably through T-cell receptor TCR reconstruction HT Smart-seq identified a greater number of productive alpha and beta chain ... More |Related Solutions: MANTIS
We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples. Less |Related Solutions: MANTIS
Nikolaev et al. 2024 | Preprint | Link
Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein Here we report engineering of soluble analogues of bacteriorhodopsin NeuroBRs which bind retinal and photocycle under illumination We also report the crystallographic structure of NeuroBR A determined at anisotropic resolution reaching that reveals a conserved chromophore binding pocket and tertiary structure Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins |Related Solutions: NT8
Karuppiah et al. 2024 | Scientific Reports | Link
Specificity of a T cell receptor TCR is determined by the combination of its interactions to the peptide and human leukocyte antigen HLA TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele Some peptides are presented on multiple HLA alleles and by engineering TCRs for specific recognition of more than one allele there is potential to expand the targetable patient population Here as a proof of concept we studied two TCRs S and S binding to the PRAME peptide antigen ELFSYLIEK presented by HLA alleles HLA-A and HLA-A By structure-guided affinity ... More |Related Solutions: ROCKIMAGER
Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations. Less |Related Solutions: ROCKIMAGER
Bildziukevich et al. 2024 | RSC Medical Chemistry | Link
A series of amides of selected plant triterpenoids moronic acid and morolic acid with the tripeptides MAG and GAM was designed and synthesized Two required tripeptides and were synthesized by a step-wise chain elongation of the ethyl esters of either glycine or L-methionine at their N-terminus using Boc-protected amino acids in each step The tripeptides and were used for the synthesis of the derivatives of moronic acid and morolic acid to get a series of amide derivatives of the less frequently studied triterpenoids and The target compounds and their intermediates were subjected to an investigation of their antimicrobial antiviral and ... More |Related Solutions: TEMPEST
A series of amides of selected plant triterpenoids, moronic acid and morolic acid, with the tripeptides MAG and GAM, was designed and synthesized. Two required tripeptides 5 and 10 were synthesized by a step-wise chain elongation of the ethyl esters of either glycine or L-methionine at their N-terminus using Boc-protected amino acids in each step. The tripeptides 5 and 10 were used for the synthesis of 13–23, the derivatives of moronic acid (11) and morolic acid (12), to get a series of amide derivatives of the less frequently studied triterpenoids 11 and 12. The target compounds, and their intermediates, were subjected to an investigation of their antimicrobial, antiviral and cytotoxic activity. Selectivity of the pharmacological effects was found. Generally, the target compounds inhibited only the G+ microorganisms. Compound 16 inhibited Staphylococcus aureus (I = 99.6%; c = 62.5 μM) and Enterococcus faecalis (I = 85%; c = 250 μM). Several compounds showed moderate antiviral effects, both anti-HIV-1, 19 (EC50 = 57.0 ± 4.1 μM, CC50 > 100 μM), 20 (EC50 = 17.8 ± 2.1 μM, CC50 = 41.0 ± 5.2 μM) and 23 (EC50 = 12.6 ± 0.82 μM, CC50 = 38.0 ± 4.2 μM), and anti-HSV-1, 22 (EC50 = 27.7 ± 3.5 μM, CC50 > 100 μM) and 23 (EC50 = 30.9 ± 3.3 μM, CC50 > 100 μM). The target compounds showed no cytotoxicity in cancer cells, however, several of their intermediates were cytotoxic. Compound 21 showed cytotoxicity in HeLa (IC50 = 7.9 ± 2.1 μM), G-361 (IC50 = 8.0 ± 0.6 μM) and MCF7 (IC50 = 8.6 ± 0.2 μM) cancer cell lines, while being non-toxic in normal fibroblasts (BJ; IC50 > 50 μM). Less |Related Solutions: TEMPEST
Zhang et al. 2024 | Nature Conferences | Link
Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns which then triggers an immune response Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages Although different immunity proteins can recognize different phage-encoded triggers individual bacterial immunity proteins have been found to sense only a single trigger during infection suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands Here we demonstrate that the antiphage defence protein CapRelSJ in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using ... More |Related Solutions: NT8
Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1,2,3,4,5,6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7,8,9,10,11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers. Less |Related Solutions: NT8
Jezewski et al. 2024 | Journal of Biological Chemistry | Link
Leishmania a protozoan parasite is responsible for significant morbidity and mortality worldwide manifesting as cutaneous mucocutaneous and visceral leishmaniasis These diseases pose a substantial burden especially in impoverished regions with limited access to effective medical treatments Current therapies are toxic have low efficacy and face growing resistance Understanding the metabolic pathways of Leishmania particularly those differing from its host can unveil potential therapeutic targets In this study we investigated the acetyl-CoA synthetase ACS enzyme from Leishmania infantum LiAcs which unlike many organisms also exhibits acetoacetyl-CoA synthetase KBC activity This dual functionality is unique among ANL superfamily enzymes and crucial for ... More |Related Solutions: NT8
Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease. Less |Related Solutions: NT8
Scharffenberger et al. 2024 | Cell reports | Link
Respiratory syncytial virus RSV causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age Vaccines based on the viral fusion protein are approved for adults over but infant protection relies on passive immunity via antibody transfer or maternal vaccination An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need Antibodies arising from the VH - VL - gene pairing can neutralize RSV without the need for affinity maturation making them attractive to target through vaccination Here we develop an anti-idiotypic monoclonal antibody ai-mAb immunogen that is specific for unmutated VH - ... More |Related Solutions: NT8
Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies. Less |Related Solutions: NT8
Rodarte et al. 2024 | ACS publications | Link
The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues F FAs in both street mixtures and counterfeit pills To expand current treatment options drug-targeting monoclonal antibodies mAbs offer a viable therapeutic for both pre- and postexposure clinical scenarios This study reports the isolation in vitro characterization and in vivo efficacy of two murine mAb families targeting fentanyl carfentanil or both Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil crystal structures of mAbs in complex with fentanyl or carfentanil were ... More |Related Solutions: NT8
The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest. Less |Related Solutions: NT8
Sotiropoulou et al. 2024 | Structural Biology | Link
-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharo lyticus Bgl has been denoted as having an attractive catalytic profile for various industrial applications Bgl catalyses the final step of in the decomposition of cellulose an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere With the aim of enhancing the thermostability of Bgl for a broad spectrum of biotechnological processes it has been subjected to structural studies Crystal structures of Bgl and its complex with glucose were determined at and resolution respectively Bgl ... More |Related Solutions: ROCKIMAGER
β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharo­lyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles. Less |Related Solutions: ROCKIMAGER
Ishii et al. 2024 | The FEBS journal | Link
Human - exonuclease PLD a member of the phospholipase D family of enzymes has been validated as a therapeutic target for treating Alzheimer's disease Here we have determined the crystal structure of the luminal domain of the enzyme at resolution revealing a bilobal structure with a catalytic site located between the lobes We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues previously shown to be key for phospholipase activity are not conserved or are absent This led us to test whether the enzyme ... More |Related Solutions: NT8
Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease. Less |Related Solutions: NT8
Cao et al. 2024 | Thesis/ Dessertation | Link
This thesis focusses on developing novel data driven oral drug formulation analysis methods by employing technologies such as Fourier transform analysis and generative adversarial learning Data driven measurements have been addressing challenges in advanced manufacturing and analysis for pharmaceutical development for the last two decade Data science combined with analytical chemistry holds the future to solving key problems in the next wave of industrial research and development Data acquisition is expensive in the realm of pharmaceutical development and how to leverage the capability of data science to extract information in data deprived circumstances is a key aspect for improving such ... More |Related Solutions: FRAP
This thesis focusses on developing novel data driven oral drug formulation analysis methods by employing technologies such as Fourier transform analysis and generative adversarial learning. Data driven measurements have been addressing challenges in advanced manufacturing and analysis for pharmaceutical development for the last two decade. Data science combined with analytical chemistry holds the future to solving key problems in the next wave of industrial research and development. Data acquisition is expensive in the realm of pharmaceutical development, and how to leverage the capability of data science to extract information in data deprived circumstances is a key aspect for improving such data driven measurements. Among multiple measurement techniques, chemical imaging is an informative tool for analyzing oral drug formulations. However, chemical imaging can often fall into data deprived situations, where data could be limited from the time-consuming sample preparation or related chemical synthesis. An integrated imaging approach, which folds data science techniques into chemical measurements, could lead to a future of informative and cost-effective data driven measurements. In this thesis, the development of data driven chemical imaging techniques for the analysis of oral drug formulations via Fourier transformation and generative adversarial learning are elaborated. Chapter 1 begins with a brief introduction of current techniques commonly implemented within the pharmaceutical industry, their limitations, and how the limitations are being addressed. Chapter 2 discusses how Fourier transform fluorescence recovery after photobleaching (FT-FRAP) technique can be used for monitoring the phase separated drug-polymer aggregation. Chapter 3 follows the innovation presented in Chapter 1 and illustrates how analysis can be improved by incorporating diffractive optical elements in the patterned illumination. While previous chapters discuss dynamic analysis aspects of drug product formulation, Chapter 4 elaborates on the innovation in composition analysis of oral drug products via use of novel generative adversarial learning methods for linear analyses. Less |Related Solutions: FRAP
Chretien et al. 2024 | Thesis/ Dessertation | Link
Photosensory receptors essential molecular entities across all domains of life enable organisms to detect and respond to light stimuli underpinning their critical involvement in regulating biological processes such as phototropism circadian rhythms photomorphogenesis and photosynthesis Among the myriad types of photosensory receptors blue light sensing proteins such as Blue Light Using Flavin BLUF photoreceptors distinguish themselves through their ability to utilize blue light for signalling Characterized by the conserved structure of their sensor domain BLUF photoreceptors are found in a wide array of organisms from bacteria and algae to plants and certain fungi Known for their capacity to bind flavin ... More |Related Solutions: NT8
Photosensory receptors, essential molecular entities across all domains of life, enable organisms to
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools Less |Related Solutions: NT8
Yu et al. 2024 | Preprint | Link
Human T-cell Leukemia Virus type HTLV- is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology especially when compared to the closely related virus HIV A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host Importantly the first in class anti-capsid drug approved lenacapavir can provide long-acting pre-exposure prophylaxis Such a property would provide a means to prevent the transmission of HTLV- but its capsid has not previously ... More |Related Solutions: NT8
Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development. Less |Related Solutions: NT8
Pinto et al. 2024 | Thesis/ Dessertation | Link
Neurodegenerative diseases NDDs characterized by progressive neuronal death and misfolded protein aggregation pose significant clinical social and personal challenges Parkinson's Disease PD the second most common neurological disorder is notably associated with the aggregation of alpha-synuclein aSyn Despite its prominence the transition of monomeric aSyn to aggregates remains inadequately understood Recent studies suggest that Liquid-Liquid Phase Separation LLPS and disease related metal ions involve the transition in the molecular pathogenesis of PD LLPS involves the separation of biomolecules into distinct phases without a membrane potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits I aim to investigate ... More |Related Solutions: ROCKIMAGER
Neurodegenerative diseases (NDDs), characterized by progressive neuronal death and misfolded protein aggregation, pose significant clinical, social, and personal challenges. Parkinson's Disease (PD), the second most common neurological disorder, is notably associated with the aggregation of alpha-synuclein (aSyn). Despite its prominence, the transition of monomeric aSyn to aggregates remains inadequately understood. Recent studies suggest that Liquid-Liquid Phase Separation (LLPS) and disease related metal ions involve the transition in the molecular pathogenesis of PD. LLPS involves the separation of biomolecules into distinct phases without a membrane, potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits. I aim to investigate LLPS of aSyn and macroscopic dynamics of its formed droplets over time, and examine how PD related metal ions, affect the dynamic process of LLPS and modulate its toxicity to neuroblastoma cells. These metal ions, prevalent in the brain and specifically interacting with aSyn are presumably modulating LLPS, toxicity and aggregation of aSyn, making it crucial to understand their roles in the molecular pathogenesis of PD I expressed α-synuclein (aSyn) proteins in E. coli and studied the biophysical properties and toxicities of aSyn-metal ion coacervates using various techniques, including a protein crystallization robotic dispenser and confocal microscopy. In the presence of metal ions such as CuCl₂, MnCl₂, ZnCl₂, and FeCl₃, the number of droplets significantly decreased. I found that CuCl₂ ions immobilize aSyn condensates and increase their toxicity. In contrast, MnCl₂, ZnCl₂, and FeCl₃ help maintain a longer metastable state of the condensates, reducing their toxicity. This project highlights the crucial role of metal ions in modulating aSyn phase behavior, condensate toxicity and their potential involvement in the progression of PD. Less |Related Solutions: ROCKIMAGER
Agrawal et al. 2024 | Journal of Virology | Link
VRC -class broadly neutralizing antibodies bnAbs have been isolated from people with HIV- but they have not yet been elicited by vaccination They are extensively somatically mutated and sometimes accumulate CDRL deletions Such indels may allow VRC -class antibodies to accommodate the glycans expressed on a conserved N N-linked glycosylation site in loop D of the gp subunit These glycans constitute a major obstacle in the development of VRC -class antibodies as unmutated antibody forms are unable to accommodate them Although immunizations of knock-in mice expressing human VRC -class B-cell receptors BCRs with specifically designed Env-derived immunogens lead to the ... More |Related Solutions: NT8
VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. Less |Related Solutions: NT8
Skeens et al. 2024 | Preprint | Link
Dual-specificity mitogen-activated protein kinase MAPK phosphatases MKPs directly dephosphorylate and inactivate the MAPKs Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored Here we sought to define the molecular mechanism of MKP regulation through an allosteric site within its catalytic domain We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y is required to maintain the structural integrity of the allosteric pocket Along with molecular dynamics simulations these data provide insight into how changes in ... More |Related Solutions: NT8
Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK Less |Related Solutions: NT8
Kovar et al. 2024 | SLAS Discovery | Link
The SARS-CoV- main protease Mpro is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates A library of fluorogenic substrates of various lengths sequences and dye quencher positions was prepared and tested against full length SARS-CoV- Mpro enzyme for optimal activity The addition of buffers containing strongly hydrated kosmotropic anion salts such as citrate from the Hofmeister series significantly ... More |Related Solutions: TEMPEST
The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration. Less |Related Solutions: TEMPEST
al. et al. 2024 | International Journal of Cell and Biomedical Science | Link
Background Psoriasis is a chronic immune-mediated skin disease that also has systemic manifestations Case In this report we discuss our findings about a -years old psoriasis suffering male patient with a Psoriasis Area Severity Index PASI score of treated with Wharton s Jelly Mesenchymal Stem Cells-derived Secretome S-MSCs Remarkably complete regression was recorded within a treatment period of a week only Result The patient demonstrated a decrease in PASI from to after infusion and followed by intramuscular injections of S-MSCs Bioactive factors secreted by MSCs cytokines and growth factors are very likely to be the principal molecules which play a ... More |Related Solutions: µPULSE
Background: Psoriasis is a chronic, immune-mediated skin disease that also has systemic manifestations. Case: In this report, we discuss our findings about a 47-years old psoriasis suffering male patient with a Psoriasis Area Severity Index (PASI) score of 10.8, treated with Wharton’s Jelly Mesenchymal Stem Cells-derived Secretome (S-MSCs). Remarkably, complete regression was recorded within a treatment period of a week only. Result: The patient demonstrated a decrease in PASI, from 10.8 to 3.2 after 1 infusion and followed by 4 intramuscular injections of S-MSCs. Bioactive factors secreted by MSCs, cytokines and growth factors, are very likely to be the principal molecules which play a vital role in inflammatory modulation and skin tissue regeneration. No serious adverse events were noted for the patient as a result of secretome infusion and intramuscular injection. Conclusion: This report demonstrates safety and promises to be an effective strategy using S-MSCs treatment for managing the psoriatic issue and, thus, may offer as an alternative approach to overcome the limitations of the cell-based therapy. Less |Related Solutions: µPULSE
Paul et al. 2024 | Preprint | Link
Regulation of Ras GTPases by GTPase activating proteins GAP is essential for their normal signaling Nine of the ten GAPs for Ras contain a C domain immediately proximal to their canonical GAP domain and in RasGAP p GAP p RasGAP RASA mutation of this domain is associated with vascular malformations in humans Here we show that the C domain of RasGAP is required for full catalytic activity towards Ras Analysis of the RasGAP C -GAP crystal structure AlphaFold models and sequence conservation reveal direct C domain interaction with the Ras allosteric lobe This is achieved by an evolutionarily conserved surface ... More |Related Solutions: NT8
Regulation of Ras GTPases by GTPase activating proteins (GAP) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity towards Ras. Analysis of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of Ras GTPase activating proteins. Less |Related Solutions: NT8
Saberi et al. 2024 | The FEBS journal | Link
Bacillus circulans xylanase BcX from the glycoside hydrolase family degrades xylan through a retaining double-displacement mechanism The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large active site cleft with six subsites allowing the binding of six xylose units Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways In the crystal structure of the catalytically inactive variant BcX E Q the substrate xylotriose is observed in the active site as well as bound to the known secondary binding site and a third site on the protein surface Nuclear ... More |Related Solutions: NT8
Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site. Less |Related Solutions: NT8
Lee et al. 2024 | Preprint | Link
A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action SARS-CoV- nsp -mac is a viral macrodomain with ADP-ribosylhydrolase activity which counteracts host immune response Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV- compared to approved therapeutics which target viral replication directly Here we report a fragment-based lead generation campaign guided by computational approaches We discover tool compounds which inhibit nsp -mac activity at low nanomolar concentrations and with responsive structure-activity relationships high selectivity and drug-like properties Using our inhibitors we show that inhibition ... More |Related Solutions: ROCKIMAGER
A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunitydriven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development. Less |Related Solutions: ROCKIMAGER
Baumgarten et al. 2024 | Chemistry Europe | Link
The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa which is on the WHO list of antibiotic resistant priority pathogens In this study the isomerase PhzF a key bacterial enzyme of the pyocyanin biosynthetic pathway was investigated as a pathoblocker target The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it thus preventing the rapid development of resistance Based on crystal structures of PhzF derivatives of the inhibitor hydroxyanthranilic acid were designed Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF ... More |Related Solutions: ROCKIMAGER
The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant “priority pathogens”. In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3–hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3–hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors. Less |Related Solutions: ROCKIMAGER
Aschenbrenner et al. 2024 | Protocols part of Springer nature | Link
The COVID- pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus SARS-CoV- This protocol details an optimized crystallization method for the SARS-CoV- nsp macrodomain a potential drug target Using sitting drop vapor diffusion with seeding we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening The method yields crystals that diffract to an average resolution of enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments All structures solved during the development of tool compounds for the ... More |Related Solutions: ROCKIMAGER
The COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion with seeding, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.5 Å, enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283). Less |Related Solutions: ROCKIMAGER