1194 Citations
Fungal cell walls composed of polysaccharides and proteins play critical roles in adaptation cell division and protection against environmental stress Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases GHs and transferases GTs In Saccharomyces cerevisiae and other ascomycetes enzymes of the Dfg subfamily which belong as GTs to the GH family cleave an linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall s polyglucans In contrast the functions of other fungal GH subfamilies are not understood We characterized CtGH from the sordariomycete Chaetomium thermophilum a member of the Fungi ... More
Fungal cell walls, composed of polysaccharides and proteins, play critical roles in adaptation, cell division, and protection against environmental stress. Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases (GHs) and transferases (GTs). In Saccharomyces cerevisiae and other ascomycetes, enzymes of the Dfg5 subfamily, which belong as GTs to the GH76 family, cleave an α1,4 linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall’s polyglucans. In contrast, the functions of other fungal GH76 subfamilies are not understood. We characterized CtGH76 from the sordariomycete Chaetomium thermophilum, a member of the Fungi/Bacteria-mixed GH76 subfamily, revealing conserved structural features and functional divergence within the GH76 family. Notably, our structural characterization by X-ray crystallography combined with glycan fragment screening indicated that CtGH76 can recognize GPI-anchors like members of the Dfg5 subfamily but shows a broader promiscuity toward other glycans with central α1,6-mannobiose motifs due to the presence of an elongated glycan binding canyon. These findings provide new insights into GH76 enzyme diversity and fungal cell wall maturation. Less
Malaria poses an enormous threat to human health With ever-increasing resistance to currently deployed antimalarials new targets and starting point compounds with novel mechanisms of action need to be identified Here we explore the antimalarial activity of the Streptomyces sp natural product -O-sulfamoyl- -chloroadenosine dealanylascamycin DACM and compare it with the synthetic adenosine monophosphate AMP mimic -O-sulfamoyladenosine AMS These nucleoside sulfamates exhibit potent inhibition of P falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin Exposure of P falciparum to DACM leads to inhibition of protein translation driven by eIF phosphorylation We show that DACM ... More
Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5ʹ-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition. Less
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions ... More
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death. Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects. Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown. We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy, and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF. Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions in five types of intermolecular high-affinity sites, leading to a decrease in negative surface charge and reversible oligomerization of the protein, thereby masking the target recognition sites responsible for its neurotrophic and antiangiogenic activities and collagen binding. Notably, PEDF secretion is enhanced in both glaucoma and retinal cell models in response to zinc stress; however, zinc binding negatively affects axogenic, differentiative and prosurvival functions of PEDF by suppressing its ability to activate receptor PEDF-R/PNPLA2. We suggest that glaucomatous neurodegeneration is associated with direct inhibition of PEDF signaling by extracellular zinc, making their complex a promising target for neuroprotective therapy. Less
Glycyl tRNA synthetases GlyRSs are prospective drug targets for combating Mycobacterium tuberculosis Mtb and cancer in humans These synthetases are of the -subtype with the ortholog in humans being dual targeted to the cytosol and mitochondria Whereas the human enzyme has been structurally characterized previously in several liganded states no structures of MtbGlyRS have thus far been reported Here we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS MtrGlyRS which progressed through all phases of the structural genomics pipeline for the purpose of facilitating structure-based drug discovery MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in ... More
Glycyl tRNA synthetases (GlyRSs) are prospective drug targets for combating Mycobacterium tuberculosis (Mtb) and cancer in humans. These synthetases are of the α2-subtype, with the ortholog in humans being dual targeted to the cytosol and mitochondria. Whereas the human enzyme has been structurally characterized previously in several liganded states, no structures of MtbGlyRS have thus far been reported. Here, we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS (MtrGlyRS), which progressed through all phases of the structural genomics pipeline, for the purpose of facilitating structure-based drug discovery. MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in Escherichia coli. Crystal structures are described for complexes of the two enzymes with adenosine monophosphate (AMP) and glycyl-sulfamoyl-adenylate (glycyl-AMS) at resolutions of 1.65/2.90 and 2.25/1.95 Å, respectively, and for MtrGlyRS in its apo state at 2.85 Å. Despite crystallizing in the dimeric state characteristic of many class II synthetases, the two enzymes elute predominantly as monomers during size exclusion chromatography. Strikingly, significant portions of the dimer interface and active site are unstructured in the MtrGlyRS apoenzyme crystal. AMP orders two tRNA recognition loops and a section of the insertion domain, and glycyl-AMS further stabilizes the structure, including the closure of a lid motif. Both the active and anticodon binding sites display structural differences with the human GlyRS and thus the collection of crystal structures should be useful for guiding drug development efforts targeting the various characterized structural states. Less
Millions of platelet units are needed each year to manage thrombocytopenia and other conditions linked to excessive bleeding These life-saving treatments still depend entirely on donated platelets despite the numerous shortcomings associated with them such as limited shelf life supply shortages unpredictable functionality potential for infection as well as immune-incompatibility issues These challenges could be overcome with universal donor platelets generated from human induced pluripotent stem cell hiPSC -derived megakaryocytes MKs We recently developed expandable hiPSC-derived megakaryocytic cell lines imMKCLs as a potentially unlimited source for platelet production imMKCL-derived platelets are functional and have already been tested in patients In ... More
Millions of platelet units are needed each year to manage thrombocytopenia and other conditions linked to excessive bleeding. These life-saving treatments still depend entirely on donated platelets, despite the numerous shortcomings associated with them, such as limited shelf life, supply shortages, unpredictable functionality, potential for infection, as well as immune-incompatibility issues. These challenges could be overcome with universal donor platelets generated from human induced pluripotent stem cell (hiPSC)-derived megakaryocytes (MKs). We recently developed expandable hiPSC-derived megakaryocytic cell lines (imMKCLs) as a potentially unlimited source for platelet production. imMKCL-derived platelets are functional and have already been tested in patients. In this study, we demonstrate through single-cell time-course imaging that imMKCL maturation is heterogeneous and asynchronous, with only a few imMKCLs generating platelets at any given time under static culture conditions. Using a chemical screen, we identify microtubule (MT) destabilizing agents, including vincristine (VCR), as promising hits, with a larger proportion of VCR-exposed imMKCLs developing proplatelet extensions and more platelets being produced per imMKCL. VCR use reduces the MT content of imMKCLs and results in the production of platelets with a diminished peripheral MT ring structure. Nevertheless, these platelets are functional, as evidenced by their normal response to agonists, their ability to attach to and spread on fibrinogen-coated surfaces, and their capacity to restore hemostasis in vivo. Interestingly, we also observed a negative correlation between the MT content of imMKCLs and platelet yields when we compared imMKCLs differentiated under static conditions (MThigh, low yield) to our turbulence-optimized VerMES™ bioreactor (MTlow, high yield). Taken together, our findings highlight the importance of MT dynamics in megakaryocyte biology, provide a possible explanation for the still poorly understood link between vinca alkaloid in vivo use and thrombocytosis, and bring us closer to realizing the clinical potential of affordable, off-the-shelf hiPSC-derived platelets. Less
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space Here we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor NCS- protein-protein interactions PPIs This study represents the first implementation of a complete design-make-test-analyze cycle leading to the identification of micromolar affinity compounds with the potential to modulate NCS- interactions with key targets including the G-protein chaperone Ric- A and the dopamine D and cannabinoid CB receptors Through X-ray crystallographic fragment screening CFS diverse interaction patterns within the NCS- hydrophobic crevice were revealed Algorithmically guided fragment evolution and ... More
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space. Here, we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor 1 (NCS-1) protein-protein interactions (PPIs). This study represents the first implementation of a complete design-make-test-analyze cycle leading to the identification of micromolar affinity compounds with the potential to modulate NCS-1 interactions with key targets, including the G-protein chaperone Ric-8A and the dopamine D2 and cannabinoid CB1 receptors. Through X-ray crystallographic fragment screening (CFS), diverse interaction patterns within the NCS-1 hydrophobic crevice were revealed. Algorithmically guided fragment evolution and automated synthesis enabled the rapid generation of over 400 derivatives, with biophysical validation using LC-MS and waveRAPID technology. Structural analyses highlighted key pharmacophores, with selected compounds exhibiting favorable drug-like properties and potential blood-brain barrier penetration, making them promising candidates for neurodegenerative and neurodevelopmental disorders. Our results demonstrate the feasibility of accelerated hit-to-lead development at synchrotrons, demonstrating a robust, scalable platform for PPI-targeting drug discovery. The generated chemically diverse scaffolds provide a strong foundation for future therapeutic optimization. Less
Room-temperature RT X-ray diffraction experiments enable us to investigate protein dynamics efficiently probe fragment binding and perform time-resolved crystallography experiments The Versatile Macromolecular Crystallography in-situ VMXi beamline at Diamond Light Source DLS in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals improving crystal integrity for fragile crystals While many X-ray sources are now equipped to grow crystals on site for in-situ experiments to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ... More
Room-temperature (RT) X-ray diffraction experiments enable us to investigate protein dynamics, efficiently probe fragment binding, and perform time-resolved crystallography experiments. The Versatile Macromolecular Crystallography in-situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals, improving crystal integrity for fragile crystals. While many X-ray sources are now equipped to grow crystals on site for in-situ experiments, to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ambient temperature for RT data collection, while the equivalent methodology for cryo-cooled crystals is well established. Here we examine the impact of shipping on crystals grown on MiTeGen In Situ-1 plates at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI) in Buffalo, NY, United States transatlantic to DLS in Didcot, United Kingdom. We utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Box Thermal Shipper (Blue Box), which can maintain temperature for up to 168 hours, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping might compromise data quality through mechanical stress or temperature fluctuations. Instead, we found that room-temperature data collected at VMXi showed no significant differences for crystals set up at UB-HWI and shipped relative to crystals set up on site in the UK. High-resolution structures were successfully determined for all proteins in the study, demonstrating that long-distance shipment of crystals at non-cryogenic temperatures is feasible without compromising diffraction quality. This study provides a proof-of-concept workflow for expanding access to room-temperature crystallography worldwide, enabling more researchers to leverage cutting-edge techniques without needing to grow crystals on site. Less
ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD to an acceptor molecule While ADP-ribosylation is well-established as a post-translational modification of proteins rifamycin antibiotics are its only known small-molecule targets ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis whose Arr enzyme transfers the ADP-ribose moiety to the -hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation ... More
ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay, which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 µM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 µM. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds. Less
In this work we present the high-resolution structure of human aconitate decarboxylase hACOD in its true apo form active site empty as well in complex with the inhibitor citraconate These two new structures show the architecture of the active site and the structure-activity relationships of citraconate inhibition Careful analysis of the structures indicates probable dynamics required for substrate inhibitor binding and catalysis These observations were further explored using molecular dynamic simulations which show a clear open-close mechanism of hACOD between the A and A loops the lid- and helical-domain respectively As part of the biochemical characterization of the protein we ... More
In this work, we present the high-resolution structure of human aconitate decarboxylase 1 (hACOD1) in its true apo form (active site empty) as well in complex with the inhibitor citraconate. These two new structures show the architecture of the active site and the structure-activity relationships of citraconate inhibition. Careful analysis of the structures indicates probable dynamics required for substrate/inhibitor binding and catalysis. These observations were further explored using molecular dynamic simulations, which show a clear open-close mechanism of hACOD1 between the A1 and A2 loops, the lid- and helical-domain respectively. As part of the biochemical characterization of the protein, we also developed an alternative kinetic assay which measures the rate of catalysis of hACOD1 by direct observation of the conversion of cis-aconitate to itaconate by NMR spectroscopy. The work herein offers a foundation for structure- and dynamic-driven design of novel hACOD1 inhibitors as novel chemotherapeutics. Less
Microbial production of target molecules has advanced significantly in recent years driven by innovations in enzyme engineering DNA synthesis and genomic editing However to access the massive potential of microbial production a vast parametric space remains to be investigated to optimize these biobased processes for a robust bioeconomy Here we review the current state of the art some key challenges and possible solutions We see a critical role of automation high-throughput technologies self-driving and cloud labs and data management to enable Artificial Intelligence Machine Learning and mechanistic models to overcome the design space challenges and accelerate the development of novel ... More
Microbial production of target molecules has advanced significantly in recent years driven by innovations in enzyme engineering, DNA synthesis, and genomic editing. However, to access the massive potential of microbial production, a vast parametric space remains to be investigated to optimize these biobased processes for a robust bioeconomy. Here, we review the current state of the art, some key challenges and possible solutions. We see a critical role of automation, high-throughput technologies, self-driving and cloud labs, and data management to enable Artificial Intelligence/Machine Learning and mechanistic models to overcome the design space challenges and accelerate the development of novel bio-based solutions. Accurate models will expedite the development and scale-up of engineered microbes for a range of final products from many starting materials. Less
Flavin-binding proteins flavoproteins are widespread in nature revealing versatile oxidation-reduction reactions and photochemistry Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics as well as fluorescent markers and photogenerators of reactive oxygen species Despite extensiev efforts all currently used LOV-derived proteins have similar absorption spectra with maxima around - and - nm Here we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein LSSFbFP which can be obtainedin vivo and in vitro with absorption maxima at - and - nm Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ... More
Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments. Less
West Nile virus NS B-NS innactive fusion protease was crystallized using vapor diffusion in Morpheus screen conditions at pH Hexagonal rod-shaped crystals grew to m in length after days at C The crystals belonged to space group P and diffracted to resolution at Diamond Light Source beamline I The structure has been deposited as PDB ID CO In this version we added the Addgene id of the plasmid used for the protein expresssion and purification
Crystal-based structural methods including X-ray crystallography are frequently utilized for the determination of high-resolution structures of biomolecules All crystal-based diffraction methods first require the preparation of biomolecular crystals and careful sample preparation for crystallization experiments can increase the frequency of success In this article strategies to optimize factors that can impact crystallization are presented from which buffers and reducing agents are most favorable to which crystallization techniques could be used
Physical activity improves health yet the molecular mechanisms remain partially understood This study presents a high-resolution time-resolved atlas profiling proteins across plasma saliva and urine from healthy adults post-acute exercise Exercise regulated over proteins revealing distinct fluid-specific temporal dynamics By integrating fluid-specific exercise signatures with tissue and disease atlases we delineated the contribution of tissues and associations to various diseases Network analysis across body fluids elucidated coordinated remodeling in the extracellular matrix and immune activation orchestrating exercise-induced networks Many exercise-responsive plasma proteins were robust across age sex and exercise modalities indicating a conserved systemic signature Integration with genetic data established ... More
Physical activity improves health, yet the molecular mechanisms remain partially understood.
This study presents a high-resolution, time-resolved atlas profiling 10,127 proteins across
plasma, saliva, and urine from healthy adults post-acute exercise. Exercise regulated over
3,000 proteins, revealing distinct, fluid-specific temporal dynamics. By integrating fluid-specific
exercise signatures with tissue and disease atlases, we delineated the contribution of tissues
and associations to various diseases. Network analysis across body fluids elucidated
coordinated remodeling in the extracellular matrix and immune activation orchestrating
exercise-induced networks. Many exercise-responsive plasma proteins were robust across
age, sex, and exercise modalities, indicating a conserved systemic signature. Integration with
genetic data established exercise-regulated proteins as modulators of metabolic traits and
identified over 200 targeted by approved drugs, highlighting their impact on disease-relevant
pathways. This comprehensive atlas, available as an open-access resource
https://cbmr.ku.dk/research/research-groups/deshmukh-group/shiny-apps/, advances our
molecular insight into exercise adaptations and enables exerkine discovery, biomarker
development, and pharmacological exercise-mimetic strategies. Less
This study presents a high-resolution, time-resolved atlas profiling 10,127 proteins across
plasma, saliva, and urine from healthy adults post-acute exercise. Exercise regulated over
3,000 proteins, revealing distinct, fluid-specific temporal dynamics. By integrating fluid-specific
exercise signatures with tissue and disease atlases, we delineated the contribution of tissues
and associations to various diseases. Network analysis across body fluids elucidated
coordinated remodeling in the extracellular matrix and immune activation orchestrating
exercise-induced networks. Many exercise-responsive plasma proteins were robust across
age, sex, and exercise modalities, indicating a conserved systemic signature. Integration with
genetic data established exercise-regulated proteins as modulators of metabolic traits and
identified over 200 targeted by approved drugs, highlighting their impact on disease-relevant
pathways. This comprehensive atlas, available as an open-access resource
https://cbmr.ku.dk/research/research-groups/deshmukh-group/shiny-apps/, advances our
molecular insight into exercise adaptations and enables exerkine discovery, biomarker
development, and pharmacological exercise-mimetic strategies. Less
Mobile phones contaminated with pathogenic microorganisms have the potential to act as trojan horses The microbial signatures present on their surfaces most probably vary across different geographical regions As a result mobile phones belonging to international conference attendees may serve as a model for global microbial dissemination posing potential risks to public health and biosecurity This study aimed to profile the microbes present on mobile phones belonging to delegates attending an international scientific conference through use of metagenomic shotgun DNA sequencing Results A total of microbial hits were accumulated across mobile phones inclusive of bacteria viruses fungi and protozoa Of ... More
Mobile phones, contaminated with pathogenic microorganisms, have
the potential to act as “trojan horses”. The microbial signatures present on their surfaces most probably vary across different geographical regions. As a result, mobile phones belonging to
international conference attendees may serve as a model for global microbial dissemination,
posing potential risks to public health and biosecurity. This study aimed to profile the microbes present on mobile phones belonging to delegates
attending an international scientific conference through use of metagenomic shotgun DNA
sequencing. Results: A total of 2204 microbial hits were accumulated across 20 mobile phones inclusive of
882 bacteria, 1229 viruses, 88 fungi and 5 protozoa. Of particular concern was the identification of 65 distinct antibiotic resistance genes and 86 virulence genes. Plant, animal and human
pathogens, including ESKAPE and HACEK bacteria were found on mobile phones Less
the potential to act as “trojan horses”. The microbial signatures present on their surfaces most probably vary across different geographical regions. As a result, mobile phones belonging to
international conference attendees may serve as a model for global microbial dissemination,
posing potential risks to public health and biosecurity. This study aimed to profile the microbes present on mobile phones belonging to delegates
attending an international scientific conference through use of metagenomic shotgun DNA
sequencing. Results: A total of 2204 microbial hits were accumulated across 20 mobile phones inclusive of
882 bacteria, 1229 viruses, 88 fungi and 5 protozoa. Of particular concern was the identification of 65 distinct antibiotic resistance genes and 86 virulence genes. Plant, animal and human
pathogens, including ESKAPE and HACEK bacteria were found on mobile phones Less
In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae the essential extracellular cell-division-associated peptidoglycan PG hydrolase PcsB interacts directly with the cytoplasmic-membrane-bound complex between FtsE and FtsX PcsB contains a cysteine hishdine-dependent amidohydrolase pephdase CHAP domain responsible for PG hydrolysis as well as a coiled-coil domain required for interaction with FtsEX ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane which ultimately regulates the PG hydrolase on the outside of the cell In this work we show using in vitro and in vivo approaches that the CHAP domain of PcsB predominately functions as ... More
In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae, the essential extracellular cell-division-associated peptidoglycan (PG) hydrolase PcsB interacts directly with the cytoplasmic-membrane-bound complex between FtsE and FtsX (1–3). PcsB contains a cysteine, hishdine-dependent amidohydrolase/pephdase (CHAP) domain responsible for PG hydrolysis, as well as a coiled-coil domain required for interaction with FtsEX (1,4). ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane, which ultimately regulates the PG hydrolase on the outside of the cell (5). In this work we show using in vitro and in vivo approaches, that the CHAP domain of PcsB predominately functions as an iso-D-Glutaminyl-Lysyl D,L-endopeptidase, with particular substrate specificity for Lys-containing, amidated PG, cleaving between the second and third amino acids of the peptidoglycan stem peptide. The catalytic activity of PcsB is regulated and activated by conformation changes of the coiled-coil region of PcsB and in part by a short helical region immediately adjacent to the CHAP domain to guard against PcsB hydrolytic activation outside of its cell division specific functional requirement. This work supports a model for the overall biological activity of the FtsEX-PcsB complex, in which ATP hydrolysis by FtsE in the cytoplasm, drives conformational changes in FtsX and PcsB resulting in the liberation of the hydrolytic CHAP domain of PcsB from its regulatory helix to allow PG stem peptide cleavage that splits the septal disk and marks a region of the peptidoglycan sacculus for subsequent cell division remodelling. Less
Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life However chemical copying with the canonical nucleotides A U G and C strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U because of the stronger G C vs A U base pair and the weaker stacking interactions of U Recent advances in prebiotic chemistry suggest that the -thiopyrimidines were precursors to the canonical pyrimidines raising the possibility that they may have played an important early role in RNA copying chemistry Furthermore -thiouridine ... More
Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life. However, chemical copying with the canonical nucleotides (A, U, G, and C) strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U, because of the stronger G:C vs. A:U base pair, and the weaker stacking interactions of U. Recent advances in prebiotic chemistry suggest that the 2-thiopyrimidines were precursors to the canonical pyrimidines, raising the possibility that they may have played an important early role in RNA copying chemistry. Furthermore, 2-thiouridine (s2U) and inosine (I) form by deamination of 2-thiocytidine (s2C) and A respectively. We used thermodynamic and crystallographic analyses to compare the I:s2C and A:s2U base pairs. We find that the I:s2C base pair is isomorphic and isoenergetic with the A:s2U base pair. The I:s2C base pair is weaker than a canonical G:C base pair, while the A:s2U base pair is stronger than the canonical A:U base pair, so that a genetic alphabet consisting of s2U, s2C, I and A generates RNA duplexes with uniform base pairing energies. Consistent with these results, kinetic analysis of nonenzymatic template-directed primer extension reactions reveals that s2C and s2U substrates bind similarly to I and A in the template, and vice versa. Our work supports the plausibility of a potentially primordial genetic alphabet consisting of s2U, s2C, I and A, and offers a potential solution to the long-standing problem of biased nucleotide incorporation during nonenzymatic template copying. Less
Collagen prolyl -hydroxylase C-P H catalyzes the -hydroxylation of Y-prolines of the XYG-repeat of procollagen C-P Hs are tetrameric enzymes The -subunit provides the N-terminal dimerization domain the middle peptide-substrate binding PSB domain and the C-terminal catalytic CAT domain There are three isoforms of the -subunit complexed with a -subunit that is protein disulfide isomerase forming C-P H I-III The PSB domain of the -subunit binds proline-rich peptides but its function with respect to the prolyl hydroxylation mechanism is unknown An extended mode of binding of proline-rich peptides PPII polyproline type-II conformation to the PSB-I domain has previously been reported ... More
Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the 4-hydroxylation of Y-prolines of the XYG-repeat of procollagen. C-P4Hs are tetrameric α2β2 enzymes. The α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate–binding (PSB) domain, and the C-terminal catalytic (CAT) domain. There are three isoforms of the α-subunit, complexed with a β-subunit that is protein disulfide isomerase, forming C-P4H I-III. The PSB domain of the α-subunit binds proline-rich peptides, but its function with respect to the prolyl hydroxylation mechanism is unknown. An extended mode of binding of proline-rich peptides (PPII, polyproline type-II, conformation) to the PSB-I domain has previously been reported for the PPG-PPG-PPG and P9 peptides. Crystal structures now show that peptides with the motif PxGP (PPG-PRG-PPG, PPG-PAG-PPG) (where x, at Y-position 5, is not a proline) bind to the PSB-I domain differently, more deeply, in the peptide-binding groove. The latter mode of binding has previously been reported for structures of the PSB-II domain complexed with these PxGP-peptides. In addition, it is shown here by crystallographic binding studies that the POG-PAG-POG peptide (with 4-hydroxyprolines at Y-positions 2 and 8) also adopts the PxGP mode of binding to PSB-I as well as to PSB-II. Calorimetric binding studies show that the affinities of these peptides are lower for PSB-I than for PSB-II, with, respectively, KD values of about 70 μM for PSB-I and 20 μM for PSB-II. The importance of these results for understanding the reaction mechanism of C-P4H, in particular concerning the function of the PSB domain, is discussed. Less
Immunomodulatory imide drugs IMiDs degrade specific C H zinc finger degrons in transcription factors making them effective against certain cancers SALL a cancer driver contains seven C H zinc fingers in four clusters including an IMiD degron in zinc finger cluster two ZFC Surprisingly IMiDs do not inhibit growth of SALL expressing cancer cells To overcome this limit we focused on a non-IMiD degron SALL zinc finger cluster four ZFC By combining AlphaFold and the ZFC -DNA crystal structure we identified a potential ZFC drug pocket Utilizing an in silico docking algorithm and cell viability assays we screened chemical libraries ... More
Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. Less
The COVID- pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness Other than Severe Acute Respiratory Syndrome Coronavirus SARS-CoV- there are several human coronaviruses that are of pandemic concern these include SARS-CoV and Middle Eastern Respiratory Syndrome MERS-CoV MERS-CoV is a zoonotic virus that was first discovered in The disease has spread rapidly with large outbreaks as recent as and Currently there is no therapeutic intervention for MERS-CoV with of reported cases resulting in human death Like-wise to SARS-CoV- MERS-CoV produces a main protease Mpro which is essential for viral replication and therefore an ... More
The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness. Other than Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several human coronaviruses that are of pandemic concern, these include SARS-CoV and Middle Eastern Respiratory Syndrome (MERS-CoV). MERS-CoV is a zoonotic virus that was first discovered in 2012. The disease has spread rapidly with large outbreaks as recent as 2015 and 2018. Currently there is no therapeutic intervention for MERS-CoV with 35% of reported cases resulting in human death. Like-wise to SARS-CoV-2, MERS-CoV produces a main protease (Mpro) which is essential for viral replication and therefore an attractive target to inhibit the virus. In this new version we added the protein purification protocol and Addgene id, together with the solvent test and compound soaking conditions. Less
Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein Here we report engineering of soluble analogues of bacteriorhodopsin NeuroBRs which bind retinal and photocycle under illumination We also report the crystallographic structure of NeuroBR A determined at anisotropic resolution reaching that reveals a conserved chromophore binding pocket and tertiary structure Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins
The Fe II - and -oxoglutarate -OG -dependent dioxygenase FOGDD enzyme superfamily catalyzes the oxidation of RNA DNA and peptide substrates to install chemical modifications that regulate the diverse functions of these biomolecules For decades it has been appreciated that many FOGDDs require ascorbate Vitamin C as a cofactor for efficient catalysis but ascorbate requirements across different FOGDD enzymes its effects on the catalysis of different substrates and how it engages the FOGDD active site remain poorly understood Here we use RNA demethylases FTO and AlkBH as model FOGDD enzymes and show that their ascorbate requirements for efficient demethylation reactions ... More
The Fe(II)- and 2-oxoglutarate(2-OG)-dependent dioxygenase (FOGDD) enzyme superfamily catalyzes the oxidation of RNA, DNA, and peptide substrates to install chemical modifications that regulate the diverse functions of these biomolecules. For decades, it has been appreciated that many FOGDDs require ascorbate (Vitamin C) as a cofactor for efficient catalysis, but ascorbate requirements across different FOGDD enzymes, its effects on the catalysis of different substrates, and how it engages the FOGDD active site, remain poorly understood. Here, we use RNA demethylases FTO and AlkBH5 as model FOGDD enzymes and show that their ascorbate requirements for efficient demethylation reactions are dramatically different. Furthermore, FTO was found to have strikingly different ascorbate requirements for its own demethylation reactions with different methylated RNA substrates. Our enzymology experiments suggest FTO and AlkBH5, and likely FOGDDs generally, can have widely different ascorbate dependencies based on the balance between an enzyme’s intrinsic ability to decarboxylate co-substrate 2-oxoglutarate and the kinetics of its substrate oxidation reaction. Finally, we determined a crystal structure of FTO in complex with ascorbate, which for the first time shows how ascorbate selectively engages the FOGDD active site. Together, our biochemical and structural data show that ascorbate can selectively tune the RNA demethylation reactions of FTO and AlkBH5, and that more globally, differences in ascorbate concentrations in different environments or diseases may regulate the activity and specificity of diverse FOGDD-catalyzed oxidation reactions in the cell. Less
Solid-state nuclear magnetic resonance ssNMR is a powerful technique for studying membrane protein structure and dynamics Ideally measurements are performed with the protein in a lipid bilayer However homogenous reconstitution of functional protein into intact bilayers at sufficiently high concentrations is often difficult to achieve In this work we investigate the suitability of the lipid cubic phase LCP which incorporates a lipid bilayer as an alternative medium for ssNMR of integral membrane peptides and proteins The cubic mesophase has long been used to generate membrane protein crystals for use in X-ray crystallographic structure determination by the so-called in meso method ... More
Solid-state nuclear magnetic resonance (ssNMR) is a powerful technique for studying membrane protein structure and dynamics. Ideally, measurements are performed with the protein in a lipid bilayer. However, homogenous reconstitution of functional protein into intact bilayers at sufficiently high concentrations is often difficult to achieve. In this work, we investigate the suitability of the lipid cubic phase (LCP), which incorporates a lipid bilayer, as an alternative medium for ssNMR of integral membrane peptides and proteins. The cubic mesophase has long been used to generate membrane protein crystals for use in X-ray crystallographic structure determination by the so-called in meso method and for protein functional and biophysical characterization. Preparing and handling protein-laden LCP is straightforward. LCP may therefore provide a valuable alternative to native membranes and other membrane mimetics for ssNMR. We tested this idea by conducting standard magic-angle spinning ssNMR experiments on LCP into which gramicidin, a ∼4-kDa transmembrane peptide, or bacterial lipoprotein signal peptidase II (LspA), a ∼20-kDa integral membrane enzyme, had been reconstituted. We report one- and two-dimensional ssNMR spectra for both gramicidin and LspA and the parameters for optimizing spectral quality. The high protein-carrying capacity of the cubic phase facilitated 13C ssNMR at natural abundance. Lowering temperature and raising magic-angle spinning frequency enabled significant improvements in spectral quality. One-dimensional 13C and 15N spectra were collected for LspA. Two-dimensional ssNMR experiments provided information on LspA dynamics and its interaction with the water and lipid components of the cubic phase. Solution NMR measurements carried out in parallel yielded information on the effect of the antibiotic, globomycin, on LspA structure and dynamics. Less
The Elettra synchrotron radiation facility located in Trieste Italy is a third-generation storage ring operating in top-up mode at both and GeV The facility currently hosts one beamline fully dedicated to macromolecular crystallography XRD XRD is based on a superconducting wiggler and it has been open to users since On-site and remote access for data collection as well as monitoring tools and automatic data analysis pipelines are available to its users In addition since Elettra has operated a general-purpose diffraction beamline XRD offering the macromolecular community a wide spectrum extending to long wavelengths for phasing and ion identification Ancillary facilities ... More
The Elettra synchrotron radiation facility, located in Trieste, Italy, is a third-generation storage ring, operating in top-up mode at both 2.0 and 2.4 GeV. The facility currently hosts one beamline fully dedicated to macromolecular crystallography, XRD2. XRD2 is based on a superconducting wiggler, and it has been open to users since 2018. On-site and remote access for data collection, as well as monitoring tools and automatic data analysis pipelines are available to its users. In addition, since 1994 Elettra has operated a general-purpose diffraction beamline, XRD1, offering the macromolecular community a wide spectrum extending to long wavelengths for phasing and ion identification. Ancillary facilities support the beamlines, providing sample preparation and a high-throughput crystallization platform for the user community. A new CryoEM facility is being established on campus and jointly operated by the Consiglio Nazionale della Ricerche – Istituto Officina dei Materiali (CNR–IOM) and Elettra, providing further opportunities to the Elettra user community. This review outlines the current capabilities and anticipated developments for macromolecular crystallography at Elettra to accompany the upcoming upgrade to Elettra 2.0, featuring a six-bend enhanced achromat lattice. The new source is expected to deliver a high-brilliance beam, enabling the macromolecular crystallography community to better address the emerging and future scientific challenges. Less
Bacteria face a constant existential threat in the form of infection by viruses along with other forms of mobile genetic elements such as bacteriophage and transposable elements To survive bacteria and other prokaryotes have evolved various immune systems to evade these would-be invaders One such immune system is the CRISPR-Cas system an adaptive immune system able to record the genetic signature of invading viruses in order to recognize and destroy them should they be encountered again in the future In this thesis I present data that sheds light on the mechanism of one particular subtype of CRISPR-Cas systems the type ... More
Bacteria face a constant existential threat in the form of infection by viruses along with other forms of mobile genetic elements, such as bacteriophage and transposable elements. To survive, bacteria and other prokaryotes have evolved various immune systems to evade these would-be invaders. One such immune system is the CRISPR-Cas system, an adaptive immune system able to record the genetic signature of invading viruses in order to recognize and destroy them should they be encountered again in the future. In this thesis I present data that sheds light on the mechanism of one particular subtype of CRISPR-Cas systems: the type IV-A1 system from Pseudomonas aeruginosa. I also report on some of the newly identified tools used by viruses and plasmids to evade this system, called anti-CRISPRs.
The type IV-A1 system is unique in that unlike most CRISPR-Cas systems, it doesn’t appear to destroy or degrade the genome of invading viruses. Instead, it relies on an additional helicase protein called CasDinG to repress the expression of any genes near its target. I report data which explains the genetic signatures necessary to activate type IV-A CRISPR system, and I also explore the significance of a particular domain of the CasDinG helicase.
This thesis also identifies the first-ever reported anti-CRISPRs against the type IV-A system, along with hypothesized mechanisms by which they repress immunity. Less
The type IV-A1 system is unique in that unlike most CRISPR-Cas systems, it doesn’t appear to destroy or degrade the genome of invading viruses. Instead, it relies on an additional helicase protein called CasDinG to repress the expression of any genes near its target. I report data which explains the genetic signatures necessary to activate type IV-A CRISPR system, and I also explore the significance of a particular domain of the CasDinG helicase.
This thesis also identifies the first-ever reported anti-CRISPRs against the type IV-A system, along with hypothesized mechanisms by which they repress immunity. Less
DNA replication is tightly regulated to ensure genomic stability and prevent several diseases including cancers Eukaryotes and archaea partly achieve this regulation by strictly controlling the activation of hexameric minichromosome maintenance MCM helicase rings that unwind DNA during its replication In eukaryotes MCM activation critically relies on the sequential recruitment of the essential factors Cdc and a tetrameric GINS complex at the onset of the S-phase to generate a larger CMG complex We present the crystal structure of the tetrameric GINS complex from the archaeal organism Saccharolobus solfataricus Sso to reveal a core structure that is highly similar to the ... More
DNA replication is tightly regulated to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea partly achieve this regulation by strictly controlling the activation of hexameric minichromosome maintenance (MCM) helicase rings that unwind DNA during its replication. In eukaryotes, MCM activation critically relies on the sequential recruitment of the essential factors Cdc45 and a tetrameric GINS complex at the onset of the S-phase to generate a larger CMG complex. We present the crystal structure of the tetrameric GINS complex from the archaeal organism Saccharolobus solfataricus (Sso) to reveal a core structure that is highly similar to the previously determined GINS core structures of other eukaryotes and archaea. Using molecular modeling, we illustrate that a subdomain of SsoGINS would need to move to accommodate known interactions of the archaeal GINS complex and to generate a SsoCMG complex analogous to that of eukaryotes. Less
T cell receptors TCRs recognize specific peptides presented by human leukocyte antigens HLAs on the surface of antigen-presenting cells and are involved in fighting pathogens and cancer surveillance Canonical docking orientation of TCRs to their target peptide-HLAs pHLAs is essential for T cell activation with reverse binding TCRs lacking functionality TCR binding geometry and molecular interaction footprint with pHLAs are typically obtained by determining the crystal structure Here we describe the use of a cross-linking tandem mass spectrometry XL-MS MS method to decipher the binding orientation of several TCRs to their target pHLAs Cross-linking sites were localized to specific residues ... More
T cell receptors (TCRs) recognize specific peptides presented by human leukocyte antigens (HLAs) on the surface of antigen-presenting cells and are involved in fighting pathogens and cancer surveillance. Canonical docking orientation of TCRs to their target peptide-HLAs (pHLAs) is essential for T cell activation, with reverse binding TCRs lacking functionality. TCR binding geometry and molecular interaction footprint with pHLAs are typically obtained by determining the crystal structure. Here, we describe the use of a cross-linking tandem mass spectrometry (XL-MS/MS) method to decipher the binding orientation of several TCRs to their target pHLAs. Cross-linking sites were localized to specific residues and their molecular interactions showed differentiation between TCRs binding in canonical or reverse orientations. Structural prediction and crystal structure determination of two TCR-pHLA complexes validated these findings. The XL-MS/MS method described herein offers a faster and simpler approach for elucidating TCR-pHLA binding orientation and interactions. Less
Since the Macromolecular Crystallography MX group at Helmholtz-Zentrum Berlin HZB has been operating three MX beamlines at the BESSY II storage ring in Berlin These beamlines were established to support the emerging structural genomics initiatives founded in Germany Europe and overseas around the turn of the century Over the past two decades these beamlines have been continuously developed to enable state-of-the-art diffraction experiments and to provide supporting facilities such as a sample preparation laboratory a spectroscopy laboratory a Biosafety Level laboratory and all necessary computing resources for the MX and chemical crystallography user community Currently more than independent research groups ... More
Since 2003, the Macromolecular Crystallography (MX) group at Helmholtz-Zentrum Berlin (HZB) has been operating three MX beamlines at the BESSY II storage ring in Berlin. These beamlines were established to support the emerging structural genomics initiatives founded in Germany, Europe, and overseas around the turn of the century. Over the past two decades, these beamlines have been continuously developed to enable state-of-the-art diffraction experiments and to provide supporting facilities such as a sample preparation laboratory, a spectroscopy laboratory, a Biosafety Level 1 laboratory and all necessary computing resources for the MX and chemical crystallography user community. Currently, more than 100 independent research groups from the greater Berlin area, Germany, and Europe utilize these beamlines. Over time, more than 4500 Protein Data Bank depositions have been accrued based on data collected at the beamlines. This paper presents historical aspects of the beamlines, their current status including their research output, and future directions. Less
Personalised cell therapies utilising T cell receptors TCRs show tremendous clinical promise though TCR synthesis and validation techniques lag far behind current TCR repertoire sequencing capacity To address this gap we developed makeTCR a modular TCR cloning system that enables rapid single-step fidelity assembly of human or murine TCR sequences into diverse expression vectors We provide pre-cloned modules for and TCRs as well as many native and engineered constant regions We show how implementing cell-free manufacturing both facilitates the propagation of precloned modules and allows testable TCR material to be synthesised in hours enabling patient-derived TCRs to be prototyped prior ... More
Personalised cell therapies utilising T cell receptors (TCRs) show tremendous clinical promise, though TCR synthesis and validation techniques lag far behind current TCR repertoire sequencing capacity. To address this gap we developed makeTCR: a modular TCR cloning system that enables rapid, single-step, 100% fidelity assembly of human or murine TCR sequences into diverse expression vectors. We provide pre-cloned modules for αβ and γδ TCRs, as well as many native and engineered constant regions. We show how implementing cell-free manufacturing both facilitates the propagation of precloned modules, and allows testable TCR material to be synthesised in 24 hours, enabling patient-derived TCRs to be prototyped prior to use in personalised cell therapies. makeTCR scales to making thousands of TCRs, at high fidelity and at substantially reduced cost. makeTCR is facilitated by a free, open-source, extensible, graphical platform to simplify, standardise, and accelerate TCR functionality testing for personalised medicine and beyond. Less
Picornaviridae coxsackievirus A is the causative agent of paediatric hand-foot-and-mouth disease and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak The A protease of the virus is responsible for self-cleavage from the poly protein allowing for correct folding and assembly of capsid proteins in the final stages of viral replication Inhibition deranges capsid folding and assembly preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity This protocol was used to grow coxsackievirus A crystals PDB POA that were used in high-throughput crystallographic ... More
Picornaviridae coxsackievirus A16 is the causative agent of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of the virus is responsible for self-cleavage from the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. Inhibition deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. This protocol was used to grow coxsackievirus A16 crystals (PDB 8POA) that were used in high-throughput crystallographic fragment screening, and follow up compounds on the target. In this new version we added: the group deposition code; details about the fragment screen and solvent tolerance; also the protein production protocol. Less
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics A current cause for concern is the emerging pathogen Enterovirus D EV-D which primarily spreads through respiratory routes While it mostly causes mild to severe respiratory illness in severe cases it can lead to acute flaccid myelitis The C protease of EV-D is a potential target for antiviral drug development due to its essential role in the viral life cycle and high sequence conservation This protocol was used to grow EV-D C crystals that were subjected to high-throughput fragment screening crystallography PDB group deposition G ... More
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics. A current cause for concern is the emerging pathogen Enterovirus D68 (EV-D68), which primarily spreads through respiratory routes. While it mostly causes mild to severe respiratory illness, in severe cases it can lead to acute flaccid myelitis. The 3C protease of EV-D68 is a potential target for antiviral drug development due to its essential role in the viral life cycle and high sequence conservation. This protocol was used to grow EV-D68 3C crystals that were subjected to high-throughput fragment screening crystallography (PDB group deposition G_10002271). In this new version, we have added the protocols for protein expression and purification, soaking conditions, and fragment screening information, as well as the affiliation with the ASAP Discovery Consortium. Less
Zika virus ZIKV NS protease with its NS B cofactor is essential for the cleavage of Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target We optimized a robust crystal system of co-expressed NS protease with its NS B cofactor The crystals appeared within hours and diffracted to in average The NS B-NS structure is in closed conformation and has been deposited to PDB PDB code PN In this version we added the addgene id and the protein production protocol
Bacterial proton pumps proteorhodopsins PRs are a major group of light-driven membrane proteins found in marine bacteria They are functionally and structurally distinct from archaeal and eukaryotic proton pumps To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level high-resolution structures of PRs functional states are needed In this work we have determined atomic-resolution structures of MAR a PR from marine actinobacteria in various functional states notably the challenging late O intermediate state These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward ... More
Bacterial proton pumps, proteorhodopsins (PRs), are a major group of light-driven membrane proteins found in marine bacteria. They are functionally and structurally distinct from archaeal and eukaryotic proton pumps. To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level, high-resolution structures of PRs’ functional states are needed. In this work, we have determined atomic-resolution structures of MAR, a PR from marine actinobacteria, in various functional states, notably the challenging late O intermediate state. These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward proton pump xenorhodopsin allow for deducing key universal elements for light-driven proton pumping. First, long hydrogen-bonded chains characterize proton pathways. Second, short hydrogen bonds allow proton storage and inhibit their backflow. Last, the retinal Schiff base is the active proton donor and acceptor to and from hydrogen-bonded chains. Less
Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies bsAbs targeting CD or CD is emerging as a promising therapeutic strategy Conditional tumor-specific recruitment can offer a necessary layer of control and specificity We developed pH-selective CD xVISTA bsAbs to act specifically within the acidic tumor microenvironment TME aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome CRS risk CD agonism by CD xVISTA bsAbs relies on pH-selective engagement of VISTA a protein robustly expressed on myeloid cells highly prevalent in most solid tumors This modality avoids engagement of tumor-associated antigens TAAs with the ... More
Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 or CD137 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a necessary layer of control and specificity. We developed pH-selective CD28xVISTA bsAbs to act specifically within the acidic tumor microenvironment (TME), aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. CD28 agonism by CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells highly prevalent in most solid tumors. This modality avoids engagement of tumor-associated antigens (TAAs) with the potential to provide highly tumor specific activity with minimal on-target/off-tumor side effects.
We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. Less
We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. Less
NEMO is an essential component in the activation of the canonical NF B pathway and exerts its function by recruiting the I B kinases IKK to the IKK complex Inhibition of the NEMO IKKs interaction is an attractive therapeutic paradigm for diseases related to NF B mis-regulation but a difficult endeavor because of the extensive protein-protein interface Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO programmed to render this difficult protein domain amenable to NMR and X-ray characterization while preserving the biological function ZipNEMO binds IKK with nanomolar affinity is amenable ... More
NEMO is an essential component in the activation of the canonical NFκ B pathway and exerts its function by recruiting the I κ B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NFκ B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO, programmed to render this difficult protein domain amenable to NMR and X-ray characterization, while preserving the biological function. ZipNEMO binds IKK β with nanomolar affinity, is amenable to heteronuclear NMR techniques and structure determination by X-ray crystallography. We show that NMR spectra of zipNEMO allow to detect inhibitor binding in solution and resonance assignment. The X-ray structure of zipNEMO highlights a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors. Less
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
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
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
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