1209 Citations
The Olink Explore platform enables high-throughput protein biomarker discovery through Proximity Extension Assay PEA technology combined with Next Generation Sequencing NGS on Illumina instruments This approach allows for the simultaneous measurement of thousands of human plasma proteins with minimal sample volumes The Explore library offers approximately protein assays while the smaller Explore -plex panels cater to more targeted studies The platform excels in detecting low-abundance proteins such as cytokines and chemokines and is particularly effective for challenging sample types like cerebrospinal fluid CSF where protein content is typically low In this chapter we emphasize critical dry-lab considerations including CSF handling ... More
The Olink® Explore platform enables high-throughput protein biomarker discovery through Proximity Extension Assay (PEA) technology combined with Next Generation Sequencing (NGS) on Illumina instruments. This approach allows for the simultaneous measurement of thousands of human plasma proteins with minimal sample volumes. The Explore 3072 library offers approximately 3000 protein assays, while the smaller Explore 384-plex panels cater to more targeted studies. The platform excels in detecting low-abundance proteins, such as cytokines and chemokines, and is particularly effective for challenging sample types like cerebrospinal fluid (CSF), where protein content is typically low. In this chapter, we emphasize critical dry-lab considerations, including CSF handling, study design, sample size determination, instrumentation requirements, and post-experiment data management. Proper planning and execution of these factors are essential for optimizing performance and ensuring reliable outcomes when using Olink®'s platform. Less
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
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
Introduction The MHC-class-I-related molecule MR presents small metabolites of microbial and self-origin to T cells bearing semi-invariant or variant T cell receptors One such T cell receptor MC G was previously shown to confer broad MR -restricted reactivity to tumor cells but not normal cells sparking interest in the development of non-MHC-restricted immunotherapy approaches Methods Results Here we provide cellular biophysical and crystallographic evidence that the MC G TCR does not have pan-cancer specificity but is restricted to a rare allomorph of MR bearing the R H mutation Discussion Our results underscore the importance of in-depth characterization of MR -reactive ... More
Introduction: The MHC-class-I-related molecule MR1 presents small metabolites of microbial and self-origin to T cells bearing semi-invariant or variant T cell receptors. One such T cell receptor, MC.7.G5, was previously shown to confer broad MR1-restricted reactivity to tumor cells but not normal cells, sparking interest in the development of non-MHC-restricted immunotherapy approaches.
Methods/Results: Here we provide cellular, biophysical, and crystallographic evidence that the MC.7.G5 TCR does not have pan-cancer specificity but is restricted to a rare allomorph of MR1, bearing the R9H mutation.
Discussion: Our results underscore the importance of in-depth characterization of MR1-reactive TCRs against targets expressing the full repertoire of MR1 allomorphs. Less
Methods/Results: Here we provide cellular, biophysical, and crystallographic evidence that the MC.7.G5 TCR does not have pan-cancer specificity but is restricted to a rare allomorph of MR1, bearing the R9H mutation.
Discussion: Our results underscore the importance of in-depth characterization of MR1-reactive TCRs against targets expressing the full repertoire of MR1 allomorphs. Less
Human lens fiber membrane intrinsic protein MP is the second most abundant membrane protein of the human eye lens Despite decades of effort its structure and function remained elusive Here we determined the MicroED structure of full-length human MP in lipidic-cubic phase to a resolution of MP forms tetramers each of which contain transmembrane -helices that are packed against one another forming a helical bundle Both the N- and C-termini of MP are cytoplasmic We found that each MP tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion These interactions were mediated by the extracellular loops of ... More
Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C-termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency. Less
Background Selective and potent Toll-like receptor TLR agonists are currently under evaluation in preclinical models and clinical studies to understand how the innate immune system can be harnessed for therapeutic potential These molecules are designed to modulate innate and adaptive immune responses making them promising therapeutic candidates for treating diseases such as cancer or chronic viral infections Much is known about the expression and signaling of TLRs which varies based on cell type cellular localization and tissue distribution However the downstream effects of different TLR agonists on cellular populations and phenotypes are not well understood This study aimed to investigate ... More
Background: Selective and potent Toll-like receptor (TLR) agonists are currently under evaluation in preclinical models and clinical studies to understand how the innate immune system can be harnessed for therapeutic potential. These molecules are designed to modulate innate and adaptive immune responses, making them promising therapeutic candidates for treating diseases such as cancer or chronic viral infections. Much is known about the expression and signaling of TLRs which varies based on cell type, cellular localization, and tissue distribution. However, the downstream effects of different TLR agonists on cellular populations and phenotypes are not well understood. This study aimed to investigate the impact of TLR pathway stimulation on peripheral blood mononuclear cell (PBMC) cultures from people living with HIV (PLWH) and healthy donors.
Methods: The effects of TLR4, TLR7, TLR7/8, TLR8 and TLR9 agonists were evaluated on cytokine production, cell population frequencies, and morphological characteristics of PBMC cultures over time. Changes in the proportions of different cell populations in blood and morphological features were assessed using high-content imaging and analyzed using an AI-driven approach.
Results: TLR4 and TLR8 agonists promoted a compositional shift and accumulation of small round (lymphocyte-like) PBMCs, whereas TLR9 agonists led to an accumulation of large round (myeloid-like) PBMCs. A related increase was observed in markers of cell death, most prominently with TLR4 and TLR8 agonists. All TLR agonists were shown to promote some features associated with cellular migration. Furthermore, a comparison of TLR agonist responses in healthy and HIV-positive PBMCs revealed pronounced differences in cytokine/chemokine responses and morphological cellular features. Most notably, higher actin contraction and nuclear fragmentation was observed in response to TLR4, TLR7, TLR7/8 and TLR9 agonists for antiretroviral therapy (ART)-suppressed PLWH versus healthy PBMCs.
Conclusions: These data suggest that machine learning, combined with cell imaging and cytokine quantification, can be used to better understand the cytological and soluble immune responses following treatments with immunomodulatory agents in vitro. In addition, comparisons of these responses between disease states are possible with the appropriate patient samples. Less
Methods: The effects of TLR4, TLR7, TLR7/8, TLR8 and TLR9 agonists were evaluated on cytokine production, cell population frequencies, and morphological characteristics of PBMC cultures over time. Changes in the proportions of different cell populations in blood and morphological features were assessed using high-content imaging and analyzed using an AI-driven approach.
Results: TLR4 and TLR8 agonists promoted a compositional shift and accumulation of small round (lymphocyte-like) PBMCs, whereas TLR9 agonists led to an accumulation of large round (myeloid-like) PBMCs. A related increase was observed in markers of cell death, most prominently with TLR4 and TLR8 agonists. All TLR agonists were shown to promote some features associated with cellular migration. Furthermore, a comparison of TLR agonist responses in healthy and HIV-positive PBMCs revealed pronounced differences in cytokine/chemokine responses and morphological cellular features. Most notably, higher actin contraction and nuclear fragmentation was observed in response to TLR4, TLR7, TLR7/8 and TLR9 agonists for antiretroviral therapy (ART)-suppressed PLWH versus healthy PBMCs.
Conclusions: These data suggest that machine learning, combined with cell imaging and cytokine quantification, can be used to better understand the cytological and soluble immune responses following treatments with immunomodulatory agents in vitro. In addition, comparisons of these responses between disease states are possible with the appropriate patient samples. Less
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
While contemporary short-read single cell RNA-sequencing allows to decipher tissue composition discrimination between transcript isoforms remains challenging Here we propose single cell long-read isoform sequencing scLIS-seq and highlight its performance on Jurkat and HEK T cells in direct comparison to Smart-seq xpress SS X scLIS-seq demonstrates sensitive gene and transcript detection with high correlation compared to SS X and detects at least isoforms of over genes while of the reads supported novel isoforms Direct comparison of the scLIS-seq isoforms to SS X-reconstructed isoforms demonstrated scLIS-seq s superiority Overall scLIS-seq provides a powerful scRNA-seq strategy enabling long-read transcriptome analysis and isoform ... More
While contemporary short-read single cell RNA-sequencing allows to decipher tissue composition, discrimination between transcript isoforms remains challenging. Here, we propose single cell long-read isoform sequencing (scLIS-seq), and highlight its performance on Jurkat and HEK293T cells in direct comparison to Smart-seq3xpress (SS3X). scLIS-seq demonstrates sensitive gene and transcript detection with high correlation compared to SS3X and detects at least 10 isoforms of over 2600 genes, while 17.1–21.6% of the reads supported novel isoforms. Direct comparison of the scLIS-seq isoforms to SS3X-reconstructed isoforms demonstrated scLIS-seq’s superiority. Overall, scLIS-seq provides a powerful scRNA-seq strategy, enabling long-read transcriptome analysis and isoform detection. Less
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
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
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
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
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
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
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
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 downregulation 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
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
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
Abstract Background Chronic inflammation and oxidative stress are central to the pathophysiology of Type Diabetes Mellitus T DM contributing to the progression of metabolic dysfunction and related complications Objective The aim of this study was to explore the therapeutic potential of combining hypoxia-preconditioned mesenchymal stem cell SH-MSC with alkaline water in a T DM rat model Methods T DM was induced in Wistar rats through a high-fat diet HFD followed by streptozotocin STZ administration A total of healthy male Wistar rats were randomly assigned to five groups healthy control T DM T DM Metformin T DM SH-MSC and T DM ... More
Abstract
Background:
Chronic inflammation and oxidative stress are central to the pathophysiology of Type 2 Diabetes Mellitus (T2DM), contributing to the progression of metabolic dysfunction and related complications.
Objective:
The aim of this study was to explore the therapeutic potential of combining hypoxia-preconditioned mesenchymal stem cell SH-MSC with alkaline water in a T2DM rat model.
Methods:
T2DM was induced in Wistar rats through a high-fat diet (HFD) followed by streptozotocin (STZ) administration. A total of 30 healthy male Wistar rats were randomly assigned to five groups: healthy control, T2DM, T2DM + Metformin, T2DM + SH-MSC, and T2DM + SH-MSC + alkaline water.
Results:
The combination of SH-MSC and alkaline water significantly reduced malondialdehyde (MDA) levels, a key indicator of lipid peroxidation, and suppressed the expression of p65 mRNA, a crucial component of the NF-κB signaling pathway. Notably, the most pronounced reduction in p65 mRNA expression was observed in the group receiving both SH-MSC and alkaline water, suggesting a synergistic effect in mitigating oxidative stress and inflammation.
Conclusion:
These findings highlight the potential of SH-MSC and alkaline water as a novel therapeutic strategy for alleviating T2DM. Less
Background:
Chronic inflammation and oxidative stress are central to the pathophysiology of Type 2 Diabetes Mellitus (T2DM), contributing to the progression of metabolic dysfunction and related complications.
Objective:
The aim of this study was to explore the therapeutic potential of combining hypoxia-preconditioned mesenchymal stem cell SH-MSC with alkaline water in a T2DM rat model.
Methods:
T2DM was induced in Wistar rats through a high-fat diet (HFD) followed by streptozotocin (STZ) administration. A total of 30 healthy male Wistar rats were randomly assigned to five groups: healthy control, T2DM, T2DM + Metformin, T2DM + SH-MSC, and T2DM + SH-MSC + alkaline water.
Results:
The combination of SH-MSC and alkaline water significantly reduced malondialdehyde (MDA) levels, a key indicator of lipid peroxidation, and suppressed the expression of p65 mRNA, a crucial component of the NF-κB signaling pathway. Notably, the most pronounced reduction in p65 mRNA expression was observed in the group receiving both SH-MSC and alkaline water, suggesting a synergistic effect in mitigating oxidative stress and inflammation.
Conclusion:
These findings highlight the potential of SH-MSC and alkaline water as a novel therapeutic strategy for alleviating T2DM. Less
Non-alcoholic fatty liver disease NAFLD - characterized by excess accumulation of fat in the liver - now affects one third of the world s population As NAFLD progresses extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis a major determinant of disease severity and mortality To identify transcriptional regulators of fibrosis we computationally inferred the activity of transcription factors TFs relevant to fibrosis by profiling the matched transcriptomes and epigenomes of human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD CRISPR-based genetic knockout of the top TFs identified ZNF as ... More
Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis. Less
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
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
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
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
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
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
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
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
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
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
Hydrogen sulfide H S is an endogenous gasotransmitter with cardioprotective and antiviral effects In this work new cysteine-selective nucleoside-H S-donor hybrid molecules were prepared by conjugating nucleoside biomolecules with a thiol-activatable dithioacetyl group -Dithioacetate derivatives were synthesized from the canonical nucleosides uridine adenosine cytidine guanosine and thymidine and the putative -thio metabolites were also produced from uridine and adenosine According to our measurements made with an H S-specific sensor nucleoside dithioacetates are moderately fast H S donors the guanosine derivative showed the fastest kinetics and the adenosine derivative the slowest The antioxidant activity of -thionucleosides is significantly higher than that ... More
Hydrogen sulfide (H2S) is an endogenous gasotransmitter with cardioprotective and antiviral effects. In this work, new cysteine-selective nucleoside-H2S-donor hybrid molecules were prepared by conjugating nucleoside biomolecules with a thiol-activatable dithioacetyl group. 5′-Dithioacetate derivatives were synthesized from the canonical nucleosides (uridine, adenosine, cytidine, guanosine and thymidine), and the putative 5′-thio metabolites were also produced from uridine and adenosine. According to our measurements made with an H2S-specific sensor, nucleoside dithioacetates are moderately fast H2S donors, the guanosine derivative showed the fastest kinetics and the adenosine derivative the slowest. The antioxidant activity of 5′-thionucleosides is significantly higher than that of trolox, but lower than that of ascorbic acid, while intact dithioacetates have no remarkable antioxidant effect. In human Calu cells, the guanosine derivative showed a moderate anti-SARS-CoV-2 effect which was also confirmed by virus yield reduction assay. Dithioacetyl-adenosine and its metabolite showed similar acute cardiac effects as adenosine, however, it is noteworthy that both 5′-thio modified adenosines increased left ventricular ejection fraction or stroke volume, which was not observed with native adenosine. Less
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
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
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
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
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
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
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
Transcription factors of the bHLH-PAS family play vital roles in animal development physiology and disease Two members of the family require binding of low-molecular weight ligands for their activity the vertebrate aryl hydrocarbon receptor AHR and the insect juvenile hormone receptor JHR In the fly Drosophila melanogaster the paralogous proteins GCE and MET constitute the ligand-binding component of JHR complexes Whilst GCE MET and AHR are phylogenetically heterologous their mode of action is similar JHR is targeted by several synthetic agonists that serve as insecticides disrupting the insect endocrine system AHR is an important regulator of human endocrine homeostasis and ... More
Transcription factors of the bHLH-PAS family play vital roles in animal development, physiology, and disease. Two members of the family require binding of low-molecular weight ligands for their activity: the vertebrate aryl hydrocarbon receptor (AHR) and the insect juvenile hormone receptor (JHR). In the fly Drosophila melanogaster, the paralogous proteins GCE and MET constitute the ligand-binding component of JHR complexes. Whilst GCE/MET and AHR are phylogenetically heterologous, their mode of action is similar. JHR is targeted by several synthetic agonists that serve as insecticides disrupting the insect endocrine system. AHR is an important regulator of human endocrine homeostasis and it responds to environmental pollutants and endocrine disruptors. Whether AHR signaling is affected by compounds that can activate JHR has not been reported. To address this question, we screened a chemical library of 50,000 compounds to identify 93 novel JHR agonists in a reporter system based on Drosophila cells. Of these compounds, 26% modulated AHR signaling in an analogous reporter assay in a human cell line, indicating a significant overlap in the agonist repertoires of the two receptors. To explore the structural features of agonist-dependent activation of JHR and AHR, we compared the ligand-binding cavities and their interactions with selective and common ligands of AHR and GCE. Molecular dynamics modeling revealed ligand-specific as well as conserved side chains within the respective cavities. Significance of predicted interactions was supported through site-directed mutagenesis. The results have indicated that synthetic insect juvenile hormone agonists might interfere with AHR signaling in human cells. Less
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
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
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
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
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
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
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
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
Background Chronic inflammation is central to the pathophysiology of Type Diabetes Mellitus T DM contributing to the progression of metabolic dysfunction characterized by hyperglycaemia and insulin resistance This study aims to investigate the therapeutic potential of the hypoxic MSCs secretome SH-MSCs in reducing inflammation of a T DM rat model Methods T DM was induced in Wistar rats through a high-fat diet HFD followed by streptozotocin STZ administration A total of healthy male Wistar rats were randomly assigned to five groups healthy control T DM T DM metformin T DM SH-MSCs Results SH-MSCs significantly reduced IL- mRNA expression a key ... More
Background: Chronic inflammation is central to the pathophysiology of Type 2 Diabetes Mellitus (T2DM), contributing to the progression of metabolic dysfunction characterized by hyperglycaemia and insulin resistance. This study aims to investigate the therapeutic potential of the hypoxic MSCs secretome (SH-MSCs) in reducing inflammation of a T2DM rat model. Methods: T2DM was induced in Wistar rats through a high-fat diet (HFD) followed by streptozotocin (STZ) administration. A total of 24 healthy male Wistar rats were randomly assigned to five groups: healthy control, T2DM, T2DM + metformin, T2DM + SH-MSCs. Results: SH-MSCs significantly reduced IL-18 mRNA expression, a key indicator of proinflammation, and suppressed the expression of AP-1 mRNA, a crucial proinflammatory transcription factor. Conclusion: These findings highlight the therapeutic potential of SH-MSCs as an alternative approach to alleviate inflammation in T2DM. Less
The transcription factor BCL A is a critical regulator of the switch from fetal hemoglobin HbF to adult hemoglobin HbA during development BCL A binds at a cognate recognition site TGACCA in the -globin gene promoter and represses its expression DNA-binding is mediated by a triple zinc finger domain designated ZnF Here we report comprehensive investigation of ZnF leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA We delve into the dynamics and mode of interaction with DNA Moreover we discovered that the last zinc finger of BCL A ZnF plays a ... More
The transcription factor BCL11A is a critical regulator of the switch from fetal hemoglobin (HbF: α 2 γ 2 ) to adult hemoglobin (HbA: α 2 β 2 ) during development. BCL11A binds at a cognate recognition site (TGACCA) in the γ-globin gene promoter and represses its expression. DNA-binding is mediated by a triple zinc finger domain, designated ZnF456. Here, we report comprehensive investigation of ZnF456, leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA. We delve into the dynamics and mode of interaction with DNA. Moreover, we discovered that the last zinc finger of BCL11A (ZnF6) plays a special role in DNA binding and γ-globin gene repression. Our findings help account for some rare γ-globin gene promoter mutations that perturb BCL11A binding and lead to increased HbF in adults (hereditary persistence of fetal hemoglobin). Comprehending the DNA binding mechanism of BCL11A opens avenues for the strategic, structure-based design of novel therapeutics targeting sickle cell disease and β-thalassemia. Less
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
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
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
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
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
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
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
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
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
T cells are key players in adaptive immunity The specificity of T cells is determined by the sequences of the hypervariable T cell receptor TCR and chains Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling it does not provide chain pairings which are essential for determining T cell specificity In contrast single-cell TCR sequencing technologies produce paired chain data but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies Here we present TIRTL-seq Throughput-Intensive Rapid TCR Library sequencing a novel approach that generates ready-to-sequence TCR libraries from live cells in ... More
ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired αβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ~$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire. Less
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
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
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
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
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
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
In this study we integrate Ro s target evaluation SpectraView and DL-driven virtual screening HydraScreen tools alongside Strateos' robotic cloud labs high-throughput screening platform to accelerate target and hit identification Using SpectraView to select IRAK as the target we prospectively validate HydraScreen a structure-based deep learning model We demonstrate that HydraScreen could identify up to of all IRAK hits in the top of the ranked compounds simultaneously identifying the three most potent nanomolar scaffolds present in the library The three nanomolar scaffolds identified in our project are novel for IRAK and lend themselves for future development HydraScreen outperforms traditional virtual ... More
In this study, we integrate Ro5’s target evaluation SpectraView and DL-driven virtual screening HydraScreen tools alongside Strateos' robotic cloud labs high-throughput screening platform to accelerate target and hit identification. Using SpectraView to select IRAK1 as the target, we prospectively validate HydraScreen, a structure-based deep learning model. We demonstrate that HydraScreen could identify up to 23.8% of all IRAK1 hits in the top 1% of the ranked compounds, simultaneously identifying the three most potent (nanomolar) scaffolds present in the library. The three nanomolar scaffolds identified in our project are novel for IRAK1 and lend themselves for future development. HydraScreen outperforms traditional virtual screening methods in an unbiased prospective evaluation and offers advanced features such as ligand pose confidence scoring. Thus, SpectraView and HydraScreen are innovative tools which can aid and expedite early stages of drug discovery. Less
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
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
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
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
Primordial follicle activation PFA is a pivotal event in female reproductive biology coordinating the transition from quiescent to growing follicles This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells GCs and oocytes with the progression of the PFA process Wntless Wls conditional knockout cKO mice served as a unique model suppressing the transition from pre-GCs to GCs and disrupting somatic cell-derived WNT signaling in the ovary Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice leading to a divergent trajectory while ... More
Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, coordinating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells (GCs) and oocytes with the progression of the PFA process. Wntless (Wls) conditional knockout (cKO) mice served as a unique model, suppressing the transition from pre-GCs to GCs, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified the molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, the loss of WNT signaling in (pre-)GCs impacted gene expression in oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and the microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis. Less
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
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
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
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
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
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
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
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
A group of three deep learning tools referred to collectively as CHiMP Crystal Hits in My Plate were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source DLS synchrotron UK The first tool a classification network assigns images into categories relating to experimental outcomes The other two tools are networks that perform both object detection and instance segmentation resulting in masks of individual crystals in the first case and masks of crystallisation droplets in addition to crystals in the second case allowing positions and sizes of these entities to be recorded Creation of these tools ... More
A group of three deep learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate) were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case, and masks of crystallisation droplets in addition to crystals in the second case, allowing positions and sizes of these entities to be recorded. Creation of these tools used transfer learning, where weights from a pre-trained deep learning network were used as a starting point and re-purposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS where they absolve the need for any user input both for monitoring crystallisation experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug discovery screening platform, also at DLS, to allow automatic targeting of acoustic compound dispensing into crystallisation droplets. Less
-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
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
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
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
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
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