210 Citations
The richness of our somatosensory experience is reflected in the functional diversity of somatic sensory neurons Single-cell RNA sequencing of sensory neurons has revealed a molecular basis for such diversity However sensory neuron diversity has yet to be captured at the level of the proteome Here we combined electrophysiology with deep visual proteomics to quantify over proteins from phenotypically-defined sensory neurons in mice and identified proteomic markers of sensory neuron subtypes Comparative analysis revealed both concordance and meaningful divergence between transcriptomes and proteomes We further show that up to proteins can be quantified from one-fourth of a single neuron demonstrating ... More
The richness of our somatosensory experience is reflected in the functional diversity of somatic sensory neurons. Single-cell RNA sequencing of sensory neurons has revealed a molecular basis for such diversity1,2,3. However, sensory neuron diversity has yet to be captured at the level of the proteome. Here, we combined electrophysiology with deep visual proteomics 4 to quantify over 6000 proteins from phenotypically-defined sensory neurons in mice and identified proteomic markers of sensory neuron subtypes. Comparative analysis revealed both concordance and meaningful divergence between transcriptomes and proteomes. We further show that up to 3000 proteins can be quantified from one-fourth of a single neuron, demonstrating subset-specific protein signatures. In culture, nociceptive neurons can be acutely sensitized to mechanical stimuli by nerve growth factor (NGF) which normally drives inflammatory pain in vivo5. Indeed, overnight exposure of peptidergic nociceptors to NGF and a protein kinase C (PKC) activator produced functional sensitization associated with proteome changes. Functional knockdown experiments identified the up-regulated B3GNT2 enzyme as a potential effector of nociceptor sensitization. In summary, we present a high-resolution proteomic resource linking molecular identity to function, enabling the discovery of mechanisms underlying somatic sensation and pain sensitization. Less
Virtual reality VR devices are increasingly being utilized within operating theaters and intensive care units where appropriate sanitation is vital to ensure that patients do not unnecessarily acquire hospital-associated infections The morphology of VR devices in conjunction with the variety of materials and internal components provides challenges to their repurposing This study aimed to evaluate the microorganisms remaining on VR headsets following sanitation by laboratory staff in a medical education anatomy teaching facility The external components and internal facial interface were swabbed and separately cultured on four AGAR plates Horse Blood Nutrient bile Esculin and Mannitol Salt Colonies were counted ... More
Virtual reality (VR) devices are increasingly being utilized within operating theaters and intensive care units where appropriate sanitation is vital to ensure that patients do not unnecessarily acquire hospital-associated infections. The morphology of VR devices in conjunction with the variety of materials and internal components provides challenges to their repurposing. This study aimed to evaluate the microorganisms remaining on VR headsets following sanitation by laboratory staff in a medical education anatomy teaching facility. The external components and internal facial interface were swabbed and separately cultured on four AGAR plates (Horse Blood, Nutrient, bile Esculin, and Mannitol Salt). Colonies were counted, sampled, pooled and subsequently processed for shotgun metagenomic sequencing. A higher number of colonies were present on surfaces closest to the eyes and facial interface compared to the external components. Metagenomic analysis identified 27 pathogenic bacteria including 4 “ESKAPE” pathogens (Enterobacter sp., Staphylococcus aureus, Klebsiella spp. and, Escherichia coli) and numerous organisms associated with ocular infections. A broad range of antimicrobial resistance genes were identified conveying resistance to Methicillin, Aminoglycosides, Macrolides, Tetracyclines, and Polymixins. Further research is required to ensure that current sanitization practices of VR head mounted displays are appropriate within high-risk hospital settings. Less
The PD- PD-L immune checkpoint is a pivotal target for cancer immunotherapy Monoclonal antibodies mAbs targeting the PD- PD-L interaction have achieved clinical success but face limitations including high production costs suboptimal tumor penetration and potential immunogenicity To address these challenges we present the DNA-linked Inhibitor Antibody Assay DIANA a robust high-throughput screening platform optimized for identifying and characterizing low-molecular-weight inhibitors of human PD-L DIANA integrates competitive binding with qPCR detection enabling single-well determination of dissociation constants Kd and rapid screening of thousands of compounds The assay was validated using three FDA-approved mAbs atezolizumab avelumab and durvalumab the PD-L -binding ... More
The PD-1/PD-L1 immune checkpoint is a pivotal target for cancer immunotherapy. Monoclonal antibodies (mAbs) targeting the PD-1/PD-L1 interaction have achieved clinical success but face limitations, including high production costs, suboptimal tumor penetration, and potential immunogenicity. To address these challenges, we present the DNA-linked Inhibitor Antibody Assay (DIANA)─a robust, high-throughput screening platform optimized for identifying and characterizing low-molecular-weight inhibitors of human PD-L1. DIANA integrates competitive binding with qPCR detection, enabling single-well determination of dissociation constants (Kd) and rapid screening of thousands of compounds. The assay was validated using three FDA-approved mAbs (atezolizumab, avelumab, and durvalumab), the PD-L1-binding macrocyclic peptide WL12, and the native PD-1 receptor, yielding Kd values consistent with the literature. DIANA demonstrated a broad dynamic range spanning more than 4 orders of magnitude, excellent robustness (Z′-factor = 0.94), and high tolerance to DMSO (up to 10%). We applied DIANA to screen two libraries: a 5,280-compound in-house library (pooled format) and a 1,298-compound commercial peptidomimetic library (individual format). While very weak initial hits were detected, none were confirmed in follow-up manual (non-HTS) experiments or in an orthogonal cell-based assay. Nonetheless, DIANA’s sensitivity, scalability, and minimal sample requirements establish it as a powerful tool for accelerating the discovery of next-generation PD-1/PD-L1 inhibitors and overcoming key limitations of conventional screening methods. Less
Antisense oligonucleotides ASOs are promising therapeutics but safety concerns such as liver toxicity and off-target OffT effects necessitate thorough evaluation during the compound selection process This study leverages time course global proteomics and transcriptomics to assess ASO-induced changes in vitro comparing liver toxic versus non-liver toxic ASOs The research confirms that ASOs perturb different cellular pathways at both RNA and protein levels effectively discriminating between liver toxic and non-liver toxic ASOs Contrary to expectations protein level reduction isn t delayed relative to ASO-induced RNA reduction highlighting the importance of understanding RNA and protein level relationships in specific model systems Furthermore ... More
Antisense oligonucleotides (ASOs) are promising therapeutics, but safety concerns such as liver toxicity and off-target (OffT) effects necessitate thorough evaluation during the compound selection process. This study leverages time course global proteomics and transcriptomics to assess ASO-induced changes in vitro, comparing liver toxic versus non-liver toxic ASOs. The research confirms that ASOs perturb different cellular pathways at both RNA and protein levels, effectively discriminating between liver toxic and non-liver toxic ASOs. Contrary to expectations, protein level reduction isn’t delayed relative to ASO-induced RNA reduction, highlighting the importance of understanding RNA and protein level relationships in specific model systems. Furthermore, many OffT effects observed at the RNA level do not directly translate to corresponding protein level changes. These findings suggest that current RNA-focused OffT assessment strategies capture predicted OffTs but could benefit from protein level studies that could potentially de-risk oligonucleotide drug (OND) candidates with seemingly problematic OffT profiles at the RNA level. The study underscores the value of global proteomics as a complement to RNAseq in ASO drug development, refining safety assessment and improving candidate selection. Less
The Smart-seq family of methods represents the gold standard for high-sensitivity full-length single-cell RNA sequencing Despite iterative improvements fundamental challenges remain the generation of non-specific PCR products that limit sensitivity the inability to capture precise Transcription End Sites TES and the insidious generation of phantom UMIs artificial molecular barcodes created during PCR that systematically inflate molecular counts Here we present ESPeR-seq a novel architecture that resolves these barriers To enable precise stranded TES capture we developed an Omega-dT primer that bypasses synthetic poly-T tracts restoring high-quality sequencing directly at transcript termini To eliminate both PCR background and phantom UMIs we ... More
The Smart-seq family of methods represents the gold standard for high-sensitivity, full-length single-cell RNA sequencing. Despite iterative improvements, fundamental challenges remain: the generation of non-specific PCR products that limit sensitivity, the inability to capture precise Transcription End Sites (TES), and the insidious generation of “phantom UMIs”—artificial molecular barcodes created during PCR that systematically inflate molecular counts. Here, we present ESPeR-seq, a novel architecture that resolves these barriers. To enable precise, stranded TES capture, we developed an “Omega-dT” primer that bypasses synthetic poly-T tracts, restoring high-quality sequencing directly at transcript termini. To eliminate both PCR background and phantom UMIs, we implemented a biochemical “multi-lock” mechanism utilizing uracil-containing TSOs and a uracil-intolerant DNA polymerase. We validate this approach using the logQ-slope, a novel metric that sensitively diagnoses UMI fidelity. Benchmarking reveals that while state-of-the-art methods still exhibit signs of UMI inflation, ESPeR-seq strictly prevents it. Furthermore, the strandedness and precise end-delineation provided by TSO and dT reads support robust de novo gene model reconstruction, enabling the discovery of novel multi-exon genes, unannotated 3’ UTR extensions, and candidate eRNAs across aggregated single-cell populations. Thus, ESPeR-seq establishes a robust framework for absolute quantitative accuracy and full-length isoform resolution. Less
Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems Here we present the ZenoTOF system a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system Zeno trap-enhanced MS MS electron-activated dissociation and scanning quadrupole data-independent acquisition ZT Scan DIA We show that ZT Scan DIA outperforms conventional variable-window DIA Zeno SWATH DIA in both identifications and quantitative reproducibility and demonstrate the platform s versatility across proteomics applications thousands of protein groups from bulk samples at up to samples per day single-cell proteomics yielding ... More
Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems. Here we present the ZenoTOF 8600 system, a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system, Zeno trap-enhanced MS/MS, electron-activated dissociation, and scanning quadrupole data-independent acquisition (ZT Scan DIA). We show that ZT Scan DIA outperforms conventional variable-window DIA (Zeno SWATH DIA) in both identifications and quantitative reproducibility, and demonstrate the platform’s versatility across proteomics applications: thousands of protein groups from bulk samples at up to 500 samples per day, single-cell proteomics yielding up to 4,700 proteins, accurate ratio recovery in mixed-species quantitative benchmarks, low-attomole targeted quantitation, and detection of disease-relevant phosphorylation in a Parkinson’s disease cellular model using complementary CID and EAD fragmentation. The instrument’s compact footprint makes it attractive for settings where both analytical breadth and operational robustness are required. Less
The chikungunya virus CHIKV outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies So far there are no specific drugs against CHIKV A CHIKV macrodomain is critical for virulence and counteracts the host immune response representing a promising antiviral drug target Here we describe small molecule inhibitors targeting the CHIKV macrodomain Compound MDOLL- was identified through a high-throughput screening using a fluorescence resonance energy transfer FRET -based assay and its inhibitory activity was validated through multiple orthogonal assays Compound has a dual thiobarbiturate-indole scaffold and exhibits an IC of M X-ray crystallography ... More
The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity was validated through multiple orthogonal assays. Compound 1 has a dual thiobarbiturate-indole scaffold and exhibits an IC50 of 8.9 µM. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Notably, the inhibitor shows high selectivity for the CHIKV macrodomain over a panel of human and viral ADP-ribosyl binding and hydrolyzing proteins. Structure-activity relationship studies and medicinal chemistry efforts provide a promising starting point for further hit optimization. Less
Imidazolium LipidBrick cationic lipid nanoparticles LNPs provide a pH-independent alternative to conventional ionizable systems for nucleic acid delivery Through a high-throughput screen of formulations spanning eight imidazolium cores three helper lipids and varying PEG densities we found that more than half of the library outperformed the clinical ionizable benchmark ALC- in multiple representative mammalian cell types Top-performing candidates showed robust cellular uptake efficient endosomal escape and strong transgene expression both in vitro and following intramuscular administration A lead formulation C LNP incorporating an imidazolium lipid core bearing a hydroxyethyl substituent with mol DOPE achieved comparable intramuscular luciferase expression and antibody ... More
Imidazolium LipidBrick® cationic lipid nanoparticles (LNPs) provide a pH-independent alternative to conventional ionizable systems for nucleic acid delivery. Through a high-throughput screen of 1,944 formulations spanning eight imidazolium cores, three helper lipids, and varying PEG densities, we found that more than half of the library outperformed the clinical ionizable benchmark ALC-0315 in multiple representative mammalian cell types. Top-performing candidates showed robust cellular uptake, efficient endosomal escape, and strong transgene expression both in vitro and following intramuscular administration. A lead formulation (C3 LNP), incorporating an imidazolium lipid core bearing a hydroxyethyl substituent, with 30 mol% DOPE, achieved comparable intramuscular luciferase expression and antibody titers to ALC-0315, while eliciting ∼ 3-fold stronger ovalbumin-specific IFN-γ+ T-cell responses and maintaining low cytotoxicity. Machine-learning analysis of the dataset further distilled transferable design rules to inform future formulation strategies. Collectively, these findings establish cationic LipidBrick® LNPs as a versatile platform for mRNA delivery, offering a generalizable framework for the high-throughput discovery of ionization-independent systems that effectively prime adaptive immune responses. Less
Tankyrases are poly-ADP-ribosyltransferases that orchestrate numerous biological processes involved in disease Their established regulatory roles particularly within the WNT -catenin pathway have driven notable drug discovery efforts aimed at inhibiting their catalytic activity Targeting tankyrases interaction with proteins through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions In this article we employed a pre-established FRET-based assay to screen the EU-OPENSCREEN libraries for identification of ARC inhibitors We discovered a series of pyrrolone-based compounds and we synthesized compound S ARCher- which binds selectively to ARC with a potency of M NMR ... More
Tankyrases are poly-ADP-ribosyltransferases that orchestrate numerous biological processes involved in disease. Their established regulatory roles, particularly within the WNT/β-catenin pathway, have driven notable drug discovery efforts aimed at inhibiting their catalytic activity. Targeting tankyrases’ interaction with proteins through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions. In this article, we employed a pre-established FRET-based assay to screen the EU-OPENSCREEN libraries for identification of ARC4 inhibitors. We discovered a series of pyrrolone-based compounds, and we synthesized compound S8 (ARCher-142), which binds selectively to ARC4 with a potency of 8 μM. NMR analysis and X-ray crystallography allowed us to identify the binding site and to rationalize the observed selectivity. Despite binding exclusively to ARC4, the inhibitor can attenuate the WNT/β-catenin signaling pathway in cells. Our work demonstrates that targeting single ARC domains is possible, offering an inhibition approach tailored to tankyrase ARC4. Less
Autosomal recessive congenital ichthyosis ARCI refers to a group of rare highly debilitating skin disorders that significantly impair patients quality of life and lack any effective treatment options Here we report clinically relevant in situ correction of the most common ARCI-causing mutation TGM c - A G a splice-site aberration in human disease models Targeted skin barrier modulation followed by topical application of the cytosine base editor eTd packaged into lipid nanoparticles yielded functional restoration of of wild-type transglutaminase activity in skin tissue Toxicity studies and comprehensive off-target analysis demonstrated an excellent safety profile even after repeated application without systemic ... More
Autosomal recessive congenital ichthyosis (ARCI) refers to a group of rare, highly debilitating skin disorders that significantly impair patients’ quality of life and lack any effective treatment options. Here, we report clinically relevant in situ correction of the most common ARCI-causing mutation, TGM1 c.877-2A>G, a splice-site aberration, in human disease models. Targeted skin barrier modulation followed by topical application of the cytosine base editor eTd packaged into lipid nanoparticles yielded functional restoration of ∼30% of wild-type transglutaminase 1 activity in skin tissue. Toxicity studies and comprehensive off-target analysis demonstrated an excellent safety profile even after repeated application, without systemic distribution of the lipid nanoparticles or the genetic cargo as determined via highly sensitive methods, including desorption electrospray ionization (DESI) metabolic imaging. This study presents comprehensive preclinical data on the feasibility of in situ gene correction of genodermatoses-causing mutations, showcasing its therapeutic potential and paving the way for curative next-generation treatments for severe genetic skin diseases. Less
Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet trophic interactions and metabolic fluxes but conventional isotope ratio mass spectrometry requires milligram-scale samples limiting its applicability to small or rare biological specimens Fourier Transform Isotopic Ratio Mass Spectrometry FT IsoR MS enables amino acid resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale Here we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics style sample preparation Using human HeLa cells cultured in C-glucose enriched and control media we show ... More
Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet, trophic interactions, and metabolic fluxes, but conventional isotope ratio mass spectrometry requires milligram-scale samples, limiting its applicability to small or rare biological specimens. Fourier Transform Isotopic Ratio Mass Spectrometry (FT IsoR MS) enables amino acid–resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale. Here, we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics–style sample preparation. Using human HeLa cells cultured in 13C-glucose–enriched and control media, we show that reliable relative δ13C measurements can be obtained from as few as 50 cells, corresponding to <10 ng of total protein, with a precision of approximately ±9‰. The observed amino acid–specific labeling patterns are metabolically coherent and consistent with bulk measurements, while smaller cell numbers (≤10 cells) do not yield statistically robust results. These findings establish the practical sensitivity threshold of FT IsoR MS at the low-nanogram level and demonstrate its suitability for isotope-resolved analyses of small cell populations, micro-organoids, and other low-input biological samples, thereby extending stable isotope analysis toward single-cell–scale applications. Less
Functional testing of cytotoxic lymphocytes is essential for research and quality control QC but most assays require freshly prepared target cells and extensive handling A ready-to-thaw no-wash flow cytometry based cytotoxicity assay was developed using pre-labeled K targets cryopreserved in STEM-CELLBANKER EX SCB as suitably sized aliquots SCB tolerability was evaluated in K NK- and primary natural killer NK cells post-cryopreservation label stability of CellTrace Violet CTV and carboxyfluorescein succinimidyl ester CFSE was assessed freezing and thawing conditions were optimized and wash versus no-wash workflows were compared using viability-based and absolute-count readouts across effector-to-target E T ratios with NK donors ... More
Functional testing of cytotoxic lymphocytes is essential for research and quality control (QC), but most assays require freshly prepared target cells and extensive handling. A ready-to-thaw, no-wash, flow cytometry–based cytotoxicity assay was developed using pre-labeled K562 targets cryopreserved in STEM-CELLBANKER® EX (SCB) as suitably sized aliquots. SCB tolerability was evaluated in K562, NK-92, and primary natural killer (NK) cells; post-cryopreservation label stability of CellTrace™ Violet (CTV) and carboxyfluorescein succinimidyl ester (CFSE) was assessed; freezing and thawing conditions were optimized; and wash versus no-wash workflows were compared using viability-based and absolute-count readouts, across effector-to-target (E:T) ratios with NK donors and NK-92 cells. Effector viability remained high at SCB concentrations up to 10%, and 5% SCB was selected for assay design. After cryopreservation, CTV labeling remained stable over the tested storage period, whereas CFSE showed substantial signal loss. Warm-medium thawing performed comparably to water-bath thawing, and the consolidated protocol (SCB plus fetal calf serum and thermal buffering) maintained high post-thaw target viability and recovery. In killing assays, lysis increased with increasing E:T ratios; omission of the post-thaw wash had minimal impact, and 5% SCB did not impair cytotoxic function. This ready-to-thaw workflow reduces hands-on time and sample manipulation, while improving standardization for reproducible results and enabling high-throughput functional testing and QC. Less
Liver-enriched antimicrobial peptide LEAP can act as a natural antagonist or inverse agonist of the growth hormone secretagogue receptor also known as the ghrelin receptor In our previous studies palm-LEAP a truncated palmitoylated analogue of LEAP proved highly stable and exerted an acute anorexigenic effect Ghrelin the endogenous agonist of the ghrelin receptor and the anorexigenic hormone leptin have opposing effects on appetite We hypothesized that leptin supplementation combined with long-term antagonism of the ghrelin receptor using palm-LEAP might have a beneficial and potentially additive effect on obesity-related metabolic stress in leptin-deficient ob ob mice influencing diabetes liver steatosis impaired ... More
Liver-enriched antimicrobial peptide 2 (LEAP2) can act as a natural antagonist or inverse agonist of the growth hormone secretagogue receptor, also known as the ghrelin receptor. In our previous studies, palm-LEAP2(1–14), a truncated palmitoylated analogue of LEAP2, proved highly stable and exerted an acute anorexigenic effect. Ghrelin, the endogenous agonist of the ghrelin receptor, and the anorexigenic hormone leptin have opposing effects on appetite. We hypothesized that leptin supplementation combined with long-term antagonism of the ghrelin receptor using palm-LEAP2(1–14) might have a beneficial and potentially additive effect on obesity-related metabolic stress in leptin-deficient ob/ob mice, influencing diabetes, liver steatosis, impaired locomotor activity, and hypothermia.
Here, we demonstrated a clear additive effect of combined leptin and palm-LEAP2(1–14) treatment, which increased locomotor activity, reduced plasma cholesterol, liver steatosis, and mRNA expression of the metabolic stress marker Fgf21, and upregulated hypothalamic Pomc gene expression – outcomes not achieved by either treatment alone. Additionally, leptin, palm-LEAP2(1–14), and both treatments combined each reduced cumulative food intake and circulating FGF21 levels. Only leptin reduced body weight, elevated rectal temperature, increased Ucp1 and Pgc1 mRNA expression in brown adipose tissue, and attenuated hyperinsulinemia, indicating that the weight-reducing and antidiabetic effects were attributable to leptin – not palm-LEAP2(1–14). Liver metabolic stress was further alleviated by decreases in plasma FGF21 and hepatic expression of the lipogenic enzymes.
Our study demonstrated the beneficial effects of palm-LEAP2(1–14) on obesity-induced metabolic stress, which were further attenuated when palm-LEAP2(1–14) was coadministered with leptin in ob/ob mice. Less
Here, we demonstrated a clear additive effect of combined leptin and palm-LEAP2(1–14) treatment, which increased locomotor activity, reduced plasma cholesterol, liver steatosis, and mRNA expression of the metabolic stress marker Fgf21, and upregulated hypothalamic Pomc gene expression – outcomes not achieved by either treatment alone. Additionally, leptin, palm-LEAP2(1–14), and both treatments combined each reduced cumulative food intake and circulating FGF21 levels. Only leptin reduced body weight, elevated rectal temperature, increased Ucp1 and Pgc1 mRNA expression in brown adipose tissue, and attenuated hyperinsulinemia, indicating that the weight-reducing and antidiabetic effects were attributable to leptin – not palm-LEAP2(1–14). Liver metabolic stress was further alleviated by decreases in plasma FGF21 and hepatic expression of the lipogenic enzymes.
Our study demonstrated the beneficial effects of palm-LEAP2(1–14) on obesity-induced metabolic stress, which were further attenuated when palm-LEAP2(1–14) was coadministered with leptin in ob/ob mice. Less
Quantitative proteomics relies on accurate selection of fragment ions for quantification yet most current algorithms apply simple strategies such as median intensity or single quality filters Modern data-independent acquisition DIA searches generate rich features such as fragment ion correlations retention time and many others that could be leveraged to assess fragment quality We introduce QuantSelect a novel strategy to select optimal fragments by systematically integrating these features via self-supervised deep learning QuantSelect uses a regularized weighted-variance loss on intensity traces normalized via our directLFQ algorithm This allows learning a fragment quality score without ground truth labels enabling on-the-fly training on ... More
Quantitative proteomics relies on accurate selection of fragment ions for quantification, yet most current algorithms apply simple strategies such as median intensity or single quality filters. Modern data-independent acquisition (DIA) searches generate rich features such as fragment ion correlations, retention time and many others that could be leveraged to assess fragment quality. We introduce QuantSelect, a novel strategy to select optimal fragments by systematically integrating these features via self-supervised deep learning. QuantSelect uses a regularized, weighted-variance loss on intensity traces normalized via our directLFQ algorithm. This allows learning a fragment quality score without ground truth labels, enabling on-the-fly training on label-free DIA datasets. Integrated within our alphaDIA pipeline, QuantSelect significantly improves quantitative accuracy and in some cases substantially corrects protein intensity estimation. Sensitivity in differential expression improved by 68% in a mixed-species benchmarking dataset and by 18% in single-cell data. QuantSelect provides a practical framework for data-driven fragment selection that improves accuracy, precision and downstream inference in DIA proteomics. Less
Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing chromatography and MS acquisition Here we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients and samples per day along with three common DIA acquisition schemes on a timsUltra AIP mass spectrometer We found that tissue type was as important as gradient length and sample amount in determining proteome coverage Moreover the benefit of increased tissue sampling was gradient- and dynamic range-dependent Analyzing mouse liver a high dynamic range tissue over tenfold more tissue sampling led to only ... More
Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing, chromatography, and MS acquisition. Here, we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients (20, 40, 80, and 120 samples per day), along with three common DIA acquisition schemes on a timsUltra AIP mass spectrometer. We found that tissue type was as important as gradient length and sample amount in determining proteome coverage. Moreover, the benefit of increased tissue sampling was gradient- and dynamic range-dependent. Analyzing mouse liver, a high dynamic range tissue, over tenfold more tissue sampling led to only ~30% gain in protein identification for short gradients (120 SPD and 80 SPD). However, even the lowest tested tissue amount (0.04 nL, 40,000 µm3) yielded 3,200 reproducibly quantified proteins for the 120 SPD method. Longer gradients (40 SPD and 20 SPD) instead significantly benefited from more tissue sampling, quantifying over 7,500 proteins from 0.5 nL of tonsil T-cell niches. Finally, we applied our workflow to a rare squamous cell carcinoma of the oral cavity, uncovering disease-associated pathways and region-specific protein level changes. Our study demonstrates that more than 100 high-quality spatial tissue proteomes can be prepared and acquired daily, laying a strong foundation for cohort-size spatial tissue proteomics in translational research. Less
Plasma and cerebrospinal fluid are complementary sources of biomarkers for neurodegenerative diseases The wide dynamic range of protein abundances particularly in plasma hampers detection of low-abundance proteins Depletion of high-abundance proteins and efficient enzymatic digestion can improve proteome coverage but must be carefully optimized for reproducibility throughput and cost-efficiency for use in large-scale clinical proteomic studies We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid CSF that integrates depletion of high-abundance proteins optimized digestion using Lys-C and trypsin and compatibility with both label-free and tandem mass tag TMTpro -based quantification We systematically evaluated protein depletion and enzyme ... More
Plasma and cerebrospinal fluid are complementary sources of biomarkers for
neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in
plasma, hampers detection of low-abundance proteins. Depletion of high-abundance proteins
and efficient enzymatic digestion can improve proteome coverage but must be carefully
optimized for reproducibility, throughput, and cost-efficiency for use in large-scale clinical
proteomic studies.
We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF)
that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and
trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based
quantification. We systematically evaluated protein depletion and enzyme digestion conditions,
and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and
the quantitation precision. Less
neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in
plasma, hampers detection of low-abundance proteins. Depletion of high-abundance proteins
and efficient enzymatic digestion can improve proteome coverage but must be carefully
optimized for reproducibility, throughput, and cost-efficiency for use in large-scale clinical
proteomic studies.
We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF)
that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and
trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based
quantification. We systematically evaluated protein depletion and enzyme digestion conditions,
and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and
the quantitation precision. Less
The Maternal-to-Zygotic transition MZT is a reprograming process encompassing zygotic genome activation ZGA and the clearance of maternally-provided mRNAs While some factors regulating MZT have been identified there are thousands of maternal RNAs whose function has not been ascribed yet Here we have performed a proof-of-principle CRISPR-RfxCas d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT Bckdk mRNA knockdown caused epiboly defects ZGA deregulation H K ac reduction and a partial impairment of miR- processing Phospho-proteomic analysis revealed that Phf Baf a a chromatin remodeling factor is ... More
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. Less
High-grade serous ovarian cancer HGSOC is a devastating disease that is frequently detected at an advanced and incurable stage Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential Here we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma STIC the HGSOC precursor lesion covering more than proteins from ultralow input archival tissue We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level revealing a similar level of phenotypic and molecular heterogeneity Using cell-type resolved tissue ... More
High-grade serous ovarian cancer (HGSOC) is a devastating disease that is frequently detected at an advanced and incurable stage. Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential. Here, we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma (STIC), the HGSOC precursor lesion, covering more than 10,000 proteins from ultralow input archival tissue. We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level, revealing a similar level of phenotypic and molecular heterogeneity. Using cell-type resolved tissue proteomics, we revealed strong cell-of-origin signatures preserved in STICs and invasive tumors and identified early dysregulated pathways of therapeutic relevance. These include proliferation and DNA damage repair signatures, as well as onco-metabolic changes, such as increased cholesterol biosynthesis. Finally, we uncovered substantial remodeling of the co-evolving tumor microenvironment, affecting approximately one-third of the stromal proteome, and derived a common signature associated with progressive immunosuppression and ECM restructuring. In summary, our study highlights the power of spatially resolved quantitative proteomics to dissect the molecular underpinnings of early carcinogenesis and provides a rich proteomic resource for future biomarker and drug target discovery. Less
Poly-ADP-ribosylation at sites of DNA damage catalyzed by PARP enzymes activates the DNA damage response chromatin remodeling and DNA repair The modification is reversed by two enzymes in humans PARG which efficiently hydrolyzes the poly-ADP-ribose chains and ARH which is the key enzyme for removing the last proximal mono-ADP-ribose from serine residues While inhibitor development has largely focused on PARPs and PARG no potent and selective inhibitors for ARH are currently available We optimized a FRET-based competition assay for ARH and carried out high-throughput screening of small-molecule inhibitors One hit compound with a potency of M was discovered and through ... More
Poly-ADP-ribosylation at sites of DNA damage, catalyzed by PARP enzymes, activates the DNA damage response, chromatin remodeling, and DNA repair. The modification is reversed by two enzymes in humans: PARG, which efficiently hydrolyzes the poly-ADP-ribose chains, and ARH3, which is the key enzyme for removing the last proximal mono-ADP-ribose from serine residues. While inhibitor development has largely focused on PARPs and PARG, no potent and selective inhibitors for ARH3 are currently available. We optimized a FRET-based competition assay for ARH3 and carried out high-throughput screening of small-molecule inhibitors. One hit compound, 1, with a potency of 22 μM was discovered, and through structure–activity relationship studies and synthesis, we improved its potency 10-fold to 2 μM (compound 27, MDOLL-0286). We demonstrate that the compound inhibits ARH3’s poly-ADP-ribose hydrolytic activity on cellular substrates. Intriguingly, it does not effectively inhibit the hydrolysis of mono-ADP-ribosylation from natural protein substrates. This is despite the fact that the cocrystal structure of compound 1 bound to ARH3 reveals its overlap with the enzyme’s ADP-ribose binding site, agreeing with the competition in the FRET assay. The first experimental ARH3 inhibitor complex provides a valuable starting point for developing more potent chemical probes to study DNA damage response mechanisms in the future. Less
Liquid dispensing technologies that enhance experimental efficiency and deliver high accuracy across multiple volume ranges are critically important in high-throughput experimentation HTE workflows for reaction exploration and optimization The Mantis liquid handler stands out as a compact highly modular system featuring a wide array of input options and near plug-and-play functionality with carousels all while employing positive displacement dispensing technology for precise liquid delivery that is largely independent of liquid properties However the system was originally designed to handle aqueous-based biological media at low volumes L dispense limiting its applications in nonaqueous chemistry Herein we report a hardware development effort ... More
Liquid dispensing technologies that enhance experimental efficiency and deliver high accuracy across multiple volume
ranges are critically important in high-throughput experimentation (HTE) workflows for reaction exploration and optimization. The
Mantis liquid handler stands out as a compact, highly modular system featuring a wide array of input options and near plug-and-play
functionality with carousels, all while employing positive displacement dispensing technology for precise liquid delivery that is largely
independent of liquid properties. However, the system was originally designed to handle aqueous-based biological media at low
volumes (1−5 μL/dispense), limiting its applications in nonaqueous chemistry. Herein, we report a hardware development effort
from a cross-industry collaboration aimed at enhancing Mantis’ capabilities to handle organic solvents and chemical reagents, even at
larger deliverable volumes (up to 50 μL/dispense). Various chemistry examples are provided to demonstrate the implementation of
newly developed chip designs, which allow the acquisition of accurate, reproducible, and robust data, enabling more efficient
workflows for new reaction discovery, reaction optimization, and data set production. Most importantly, by implementing the Mantis
liquid handler from the outset, an increase of 77% in HTE execution efficiency relative to a manual workflow incorporating
traditional liquid handling operations was achieved in an amide coupling demonstration. Less
ranges are critically important in high-throughput experimentation (HTE) workflows for reaction exploration and optimization. The
Mantis liquid handler stands out as a compact, highly modular system featuring a wide array of input options and near plug-and-play
functionality with carousels, all while employing positive displacement dispensing technology for precise liquid delivery that is largely
independent of liquid properties. However, the system was originally designed to handle aqueous-based biological media at low
volumes (1−5 μL/dispense), limiting its applications in nonaqueous chemistry. Herein, we report a hardware development effort
from a cross-industry collaboration aimed at enhancing Mantis’ capabilities to handle organic solvents and chemical reagents, even at
larger deliverable volumes (up to 50 μL/dispense). Various chemistry examples are provided to demonstrate the implementation of
newly developed chip designs, which allow the acquisition of accurate, reproducible, and robust data, enabling more efficient
workflows for new reaction discovery, reaction optimization, and data set production. Most importantly, by implementing the Mantis
liquid handler from the outset, an increase of 77% in HTE execution efficiency relative to a manual workflow incorporating
traditional liquid handling operations was achieved in an amide coupling demonstration. Less
Genetic studies have identified thousands of individual disease-associated non-coding alleles but identification of the causal alleles and their functions remain critical bottlenecks Even though CRISPR-Cas editing has enabled targeted modification of DNA inefficient editing leads to heterogeneous outcomes across individual cells limiting the ability to detect functional consequences of disease alleles To overcome these challenges we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits assays the transcriptome and measures cell surface protein expression We apply this approach to investigate the effects of gene disruption deletions in regulatory regions and non-coding single nucleotide polymorphisms We identify ... More
Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells. Less
Electroporation-mediated gene delivery is a cornerstone of synthetic biology offering several advantages over other methods higher efficiencies broader applicability and simpler sample preparation Yet electroporation protocols are often challenging to integrate into highly multiplexed workflows owing to limitations in their scalability and tunability These challenges ultimately increase the time and cost per transformation As a result rapidly screening genetic libraries exploring combinatorial designs or optimizing electroporation parameters requires extensive iterations consuming large quantities of expensive custom-made DNA and cell lines or primary cells To address these limitations we have developed a High-Throughput Microfluidic Electroporation HTME platform that includes a -well ... More
Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME’s core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform’s ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME’s potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck. 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
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
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
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
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
Background Selective and potent Toll-like receptor TLR agonists are currently under evaluation in preclinical models and clinical studies to understand how the innate immune system can be harnessed for therapeutic potential These molecules are designed to modulate innate and adaptive immune responses making them promising therapeutic candidates for treating diseases such as cancer or chronic viral infections Much is known about the expression and signaling of TLRs which varies based on cell type cellular localization and tissue distribution However the downstream effects of different TLR agonists on cellular populations and phenotypes are not well understood This study aimed to investigate ... More
Background: Selective and potent Toll-like receptor (TLR) agonists are currently under evaluation in preclinical models and clinical studies to understand how the innate immune system can be harnessed for therapeutic potential. These molecules are designed to modulate innate and adaptive immune responses, making them promising therapeutic candidates for treating diseases such as cancer or chronic viral infections. Much is known about the expression and signaling of TLRs which varies based on cell type, cellular localization, and tissue distribution. However, the downstream effects of different TLR agonists on cellular populations and phenotypes are not well understood. This study aimed to investigate the impact of TLR pathway stimulation on peripheral blood mononuclear cell (PBMC) cultures from people living with HIV (PLWH) and healthy donors.
Methods: The effects of TLR4, TLR7, TLR7/8, TLR8 and TLR9 agonists were evaluated on cytokine production, cell population frequencies, and morphological characteristics of PBMC cultures over time. Changes in the proportions of different cell populations in blood and morphological features were assessed using high-content imaging and analyzed using an AI-driven approach.
Results: TLR4 and TLR8 agonists promoted a compositional shift and accumulation of small round (lymphocyte-like) PBMCs, whereas TLR9 agonists led to an accumulation of large round (myeloid-like) PBMCs. A related increase was observed in markers of cell death, most prominently with TLR4 and TLR8 agonists. All TLR agonists were shown to promote some features associated with cellular migration. Furthermore, a comparison of TLR agonist responses in healthy and HIV-positive PBMCs revealed pronounced differences in cytokine/chemokine responses and morphological cellular features. Most notably, higher actin contraction and nuclear fragmentation was observed in response to TLR4, TLR7, TLR7/8 and TLR9 agonists for antiretroviral therapy (ART)-suppressed PLWH versus healthy PBMCs.
Conclusions: These data suggest that machine learning, combined with cell imaging and cytokine quantification, can be used to better understand the cytological and soluble immune responses following treatments with immunomodulatory agents in vitro. In addition, comparisons of these responses between disease states are possible with the appropriate patient samples. Less
Methods: The effects of TLR4, TLR7, TLR7/8, TLR8 and TLR9 agonists were evaluated on cytokine production, cell population frequencies, and morphological characteristics of PBMC cultures over time. Changes in the proportions of different cell populations in blood and morphological features were assessed using high-content imaging and analyzed using an AI-driven approach.
Results: TLR4 and TLR8 agonists promoted a compositional shift and accumulation of small round (lymphocyte-like) PBMCs, whereas TLR9 agonists led to an accumulation of large round (myeloid-like) PBMCs. A related increase was observed in markers of cell death, most prominently with TLR4 and TLR8 agonists. All TLR agonists were shown to promote some features associated with cellular migration. Furthermore, a comparison of TLR agonist responses in healthy and HIV-positive PBMCs revealed pronounced differences in cytokine/chemokine responses and morphological cellular features. Most notably, higher actin contraction and nuclear fragmentation was observed in response to TLR4, TLR7, TLR7/8 and TLR9 agonists for antiretroviral therapy (ART)-suppressed PLWH versus healthy PBMCs.
Conclusions: These data suggest that machine learning, combined with cell imaging and cytokine quantification, can be used to better understand the cytological and soluble immune responses following treatments with immunomodulatory agents in vitro. In addition, comparisons of these responses between disease states are possible with the appropriate patient samples. Less
Insulin is a key hormone in glucose homeostasis Its lack causes severe health complications and has to be compensated by regular administration of insulin Despite intense long-lasting research a more stable substitute has yet to be discovered to alleviate patients' issues Here we report the development of a novel assay for screening potential insulin analogues based on the recently published method DIANA Our assay meets the need for a fast non-radioactive method as a sensitive alternative to the commonly used radioactive immunoassay
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
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 down-regulation trend in pathways associated with ribosome functioning, CO2 fixation, and carbohydrate catabolism characteristic of the third transcriptional state. In addition, carry-over rRNA reads recapitulated the taxonomic identity of the target while revealing distinct bacterial communities, in co-culture with the eukaryote, each associated with distinct transcriptional states. This study underscores single-cell transcriptomics as a powerful tool for characterizing metabolic states in microeukaryotes without a reference genome, offering insights into unknown physiological states and individual-level interactions with different bacterial taxa. This approach holds broad applicability to describe the ecological roles of environmental microeukaryotes, culture-free and reference-free, surpassing alternative methods like metagenomics or metatranscriptomics. Less
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
ackground The Safety and Immunogenicity of COVID- Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases SUCCEED study was created to better understand COVID- vaccination in immune-mediated inflammatory disease IMID Knowing the frequency of COVID- breakthrough infections is important particularly in IMID Our objective was to assess these events in IMID Methods We prospectively studied IMID participants who had received three COVID- vaccine doses Individuals provided saliva samples monthly September to August These were evaluated by polymerase chain reaction PCR for SARS-CoV- We also assessed antibodies against SARS-CoV- anti-spike SmT receptor binding domain RBD and nucleocapsid NP based on dried blood spots Multivariable ... More
ackground: The Safety and Immunogenicity of COVID-19 Vaccines in Systemic Autoimmune-Mediated Inflammatory Diseases (SUCCEED) study was created to better understand COVID-19 vaccination in immune-mediated inflammatory disease (IMID). Knowing the frequency of COVID-19 breakthrough infections is important, particularly in IMID. Our objective was to assess these events in IMID. Methods: We prospectively studied IMID participants who had received ≥three COVID-19 vaccine doses. Individuals provided saliva samples monthly (September 2022 to August 2023). These were evaluated by polymerase chain reaction (PCR) for SARS-CoV-2. We also assessed antibodies against SARS-CoV-2 (anti-spike, SmT1, receptor binding domain, RBD, and nucleocapsid, NP) based on dried blood spots. Multivariable general estimating equation regression produced odd ratios (OR) for PCR SARS-CoV-2 positivity, related to demographics, immunosuppressives, and antibody levels. Results: Diagnoses included rheumatoid arthritis RA (N = 161, 44% of the total), systemic lupus, psoriatic arthritis, spondylarthritis, vasculitis, systemic sclerosis, and inflammatory bowel disease. Of the 366 participants, most were taking immunosuppressive medication. Of 1266 saliva samples, 56 (5.1%) were positive for SARS-CoV-2 on PCR. Higher anti-SmT1 antibodies were inversely associated with SARS-CoV-2 detection on PCR (adjusted OR 0.66, 95% confidence interval 0.45–0.97). Antibodies to SmT1, RBD, and NP were correlated and thus could not be included in a single model, but when anti-RBD was used in place of anti-SmT1, the results were similar. No other factor (including prior COVID-19 infection) was clearly associated with SARS-CoV-2 detection. Conclusions: This is the first study of SARS-CoV-2 in a large prospective cohort of triple (or more) vaccinated individuals with IMIDs. Anti-SmT1 antibodies appeared to be protective against later SARS-CoV-2 positivity, although recent past infection was not clearly related. This suggests the importance of maintaining robust vaccine-induced immunity through vaccination in IMID. Less
Recombinant adeno-associated virus rAAV has emerged as the vector of choice for in vivo gene delivery with numerous clinical trials underway for the treatment of various human diseases Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product The conventional single-channel droplet digital PCR D ddPCR method offers only partial information regarding the viral vector genome titer lacking insights into its integrity In our pursuit of further advancing rAAV analysis we have developed a novel D ddPCR assay with advanced D linkage ... More
Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow’s output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases. Less
Background Whole genome resequencing WGRS platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program Therefore there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs The objective was to develop a comprehensive panel of soybean cyst nematode SCN resistance TaqMan assays via selecting the causative genes and analyzing their associated alleles Methods The Soybean Allele Catalog ... More
Background
Whole genome resequencing (WGRS) platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program. Therefore, there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs. The objective was to develop a comprehensive panel of soybean cyst nematode (SCN) resistance TaqMan® assays via selecting the causative genes and analyzing their associated alleles.
Methods
The Soybean Allele Catalog was utilized to investigate WGRS-derived variants which are predicted to cause a change in the amino acid sequence of a gene product. This panel of TaqMan® assays reflects current knowledge about known SCN resistance-causing genes and their associated alleles: GmSNAP18-a and -b, GmSNAP11, GmSHMT08, GmSNAP15, GmNSFRAN07, and GmSNAP02-ins and -del. Developed assays were tested using elite breeding lines and segregating populations. TaqMan assays were compared to other currently available KASP and CAPS assays.
Conclusion
All assays showed excellent allele determination efficiencies. This SCN genotyping assay panel can be utilized as a simplified, accurate and reliable genotyping platform further equipping the updated soybean breeding toolbox. Less
Whole genome resequencing (WGRS) platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program. Therefore, there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs. The objective was to develop a comprehensive panel of soybean cyst nematode (SCN) resistance TaqMan® assays via selecting the causative genes and analyzing their associated alleles.
Methods
The Soybean Allele Catalog was utilized to investigate WGRS-derived variants which are predicted to cause a change in the amino acid sequence of a gene product. This panel of TaqMan® assays reflects current knowledge about known SCN resistance-causing genes and their associated alleles: GmSNAP18-a and -b, GmSNAP11, GmSHMT08, GmSNAP15, GmNSFRAN07, and GmSNAP02-ins and -del. Developed assays were tested using elite breeding lines and segregating populations. TaqMan assays were compared to other currently available KASP and CAPS assays.
Conclusion
All assays showed excellent allele determination efficiencies. This SCN genotyping assay panel can be utilized as a simplified, accurate and reliable genotyping platform further equipping the updated soybean breeding toolbox. Less
T cells are key players in adaptive immunity The specificity of T cells is determined by the sequences of the hypervariable T cell receptor TCR and chains Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling it does not provide chain pairings which are essential for determining T cell specificity In contrast single-cell TCR sequencing technologies produce paired chain data but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies Here we present TIRTL-seq Throughput-Intensive Rapid TCR Library sequencing a novel approach that generates ready-to-sequence TCR libraries from live cells in ... More
ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired αβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ~$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire. Less
We developed an automated high-throughput Smart-seq HT Smart-seq workflow that integrates best practices and an optimized protocol to enhance efficiency scalability and method reproducibility This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity In a rigorous comparison with the X platform using human primary CD T-cells HT Smart-seq demonstrated higher cell capture efficiency greater gene detection sensitivity and lower dropout rates Additionally when sufficiently scaled HT Smart-seq achieved a comparable resolution of cellular heterogeneity to X Notably through T-cell receptor TCR reconstruction HT Smart-seq identified a greater number of productive alpha and beta chain ... More
We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples. Less
Primordial follicle activation PFA is a pivotal event in female reproductive biology coordinating the transition from quiescent to growing follicles This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells GCs and oocytes with the progression of the PFA process Wntless Wls conditional knockout cKO mice served as a unique model suppressing the transition from pre-GCs to GCs and disrupting somatic cell-derived WNT signaling in the ovary Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice leading to a divergent trajectory while ... More
Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, coordinating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells (GCs) and oocytes with the progression of the PFA process. Wntless (Wls) conditional knockout (cKO) mice served as a unique model, suppressing the transition from pre-GCs to GCs, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified the molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, the loss of WNT signaling in (pre-)GCs impacted gene expression in oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and the microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis. Less
Background Pseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis CF and bronchiectasis associated with poorer outcomes Unlike CF bronchiectasis has been a neglected disease More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P aeruginosa infections to guide novel and improved treatments Methods We have performed genome sequencing of P aeruginosa isolates from patients attending clinics worldwide to analyse the genomic diversity between and within patient infections Results We observed high genetic diversity between infections with low incidence of ... More
Background Pseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis (CF) and bronchiectasis, associated with poorer outcomes. Unlike CF, bronchiectasis has been a neglected disease. More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P. aeruginosa infections to guide novel and improved treatments.
Methods We have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections.
Results We observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms.
Conclusions Our findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes.
Summary The largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection. Less
Methods We have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections.
Results We observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms.
Conclusions Our findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes.
Summary The largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection. Less
A considerable number of antibacterial agents are derived from bacterial metabolites Similarly numerous known compounds that impede bacterial virulence stem from bacterial metabolites Enteropathogenic Escherichia coli EPEC is a notable human pathogen causing intestinal infections particularly affecting infant mortality in developing regions These infections are characterized by microvilli effacement and intestinal epithelial lesions linked with aberrant actin polymerization This study aimed to identify potential antivirulence compounds for EPEC infections among bacterial metabolites harvested from marine actinobacteria Kocuria sp and Rhodococcus spp from the Arctic Sea by the application of virulence-based screening assays Moreover we demonstrate the suitability of these antivirulence ... More
A considerable number of antibacterial agents are derived from bacterial metabolites. Similarly, numerous known compounds that impede bacterial virulence stem from bacterial metabolites. Enteropathogenic Escherichia coli (EPEC) is a notable human pathogen causing intestinal infections, particularly affecting infant mortality in developing regions. These infections are characterized by microvilli effacement and intestinal epithelial lesions linked with aberrant actin polymerization. This study aimed to identify potential antivirulence compounds for EPEC infections among bacterial metabolites harvested from marine actinobacteria (Kocuria sp. and Rhodococcus spp.) from the Arctic Sea by the application of virulence-based screening assays. Moreover, we demonstrate the suitability of these antivirulence assays to screen actinobacteria extract fractions for the bioassay-guided identification of metabolites. We discovered a compound in the fifth fraction of a Kocuria strain that interferes with EPEC-induced actin polymerization without affecting growth. Furthermore, a growth-inhibiting compound was identified in the fifth fraction of a Rhodococcus strain. Our findings include the bioassay-guided identification, HPLC-MS-based dereplication, and isolation of a large phospholipid and a likely antimicrobial peptide, demonstrating the usefulness of this approach in screening for compounds capable of inhibiting EPEC virulence. Less
Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis Despite their implications for sexual and reproductive health sialidases from other vaginal bacteria have not been characterized Here we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin These sialidases are highly conserved across clades of Prevotella from different ... More
Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis. These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment. Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis. Despite their implications for sexual and reproductive health, sialidases from other vaginal bacteria have not been characterized. Here, we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin. These sialidases are highly conserved across clades of Prevotella from different geographies, hinting at their importance globally. Finally, we find that Prevotella sialidases, including mucin-degrading enzymes from Prevotella timonensis, are highly prevalent and abundant in human vaginal metagenomes and metatranscriptomes, Together, our results identify Prevotella as a critical source of sialidases in the vaginal microbiome, improving our understanding of this detrimental bacterial activity. Less
Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations or patches For pathogens these patches can form at infection sites persist during antibiotic treatment and develop into mature biofilms Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding as well as cell-cell signaling However it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution Here we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival CS during antibiotic treatment Modeling predicts that the seeding heterogeneity of ... More
Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations, or patches. For pathogens, these patches can form at infection sites, persist during antibiotic treatment, and develop into mature biofilms. Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding, as well as cell-cell signaling. However, it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution. Here, we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival (CS) during antibiotic treatment. Modeling predicts that the seeding heterogeneity of these bacteria is amplified by local CS and global resource competition, leading to patch formation. Increasing the dose of a non-eradicating antibiotic treatment increases the degree of patchiness. Experimentally, we first demonstrated the mechanism using engineered Escherichia coli and then demonstrated its applicability to a pathogen, Pseudomonas aeruginosa. We further showed that the formation of P. aeruginosa patches promoted the evolution of antibiotic resistance. Our work provides new insights into population dynamics and resistance evolution during surface-attached bacterial growth. Less
Background Neutrophils the most abundant white blood cells in humans play pivotal roles in innate immunity rapidly migrating to sites of infection and inflammation to phagocytose neutralize and eliminate invading pathogens Neutrophil extracellular trap NET formation is increasingly recognized as an essential rapid innate immune response but when dysregulated it contributes to pathogenesis of sepsis and immunothrombotic disease Objectives Current NETosis models are limited routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood To ... More
Background
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment. Less
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment. Less
Drug sensitivity testing of patient-derived tumor organoids PDTOs is a promising tool for personalizing cancer treatment Here we present a protocol for generation of and high-throughput drug testing with PDTOs We describe detailed steps for PDTO establishment from colorectal cancer tissues preparation of PDTOs for high-throughput drug testing and quantification of drug testing results using image analysis This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies along with exploring the activity of new agents for translational research
Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions ligating an adapter to create a circular molecule sequencing the circular molecule to obtain sequence reads generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample
Nowadays an efficient and robust virtual screening procedure is crucial in the drug discovery process especially when performed on large and chemically diverse databases Virtual screening methods like molecular docking and classic QSAR models are limited in their ability to handle vast numbers of compounds and to learn from scarce data respectively In this study we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures The developed protocol yielded times faster binding energy predictions than classical docking-based screening The proposed predictive model learns from docking results allowing ... More
Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase. Less
The activities of the phospholipase C gamma PLC and enzymes are essential for numerous cellular processes Unsurprisingly dysregulation of PLC or PLC activity is associated with multiple maladies including immune disorders cancers and neurodegenerative diseases Therefore the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases To aid in the discovery of PLC family enzyme modulators that could be developed into therapeutic agents we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C CF -coumarin Herein the ability of PLC and PLC to enzymatically process C CF -coumarin was confirmed ... More
The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes. Less
Corticospinal neurons CSNs synapse directly on spinal neurons a diverse assortment of cells with unique structural and functional properties necessary for body movements CSNs modulating forelimb behavior fractionate into caudal forelimb area CFA and rostral forelimb area RFA motor cortical populations Despite their prominence the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted Here we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback CFA and RFA CSNs target similar spinal neuron types ... More
Corticospinal neurons (CSNs) synapse directly on spinal neurons, a diverse assortment of cells with unique structural and functional properties necessary for body movements. CSNs modulating forelimb behavior fractionate into caudal forelimb area (CFA) and rostral forelimb area (RFA) motor cortical populations. Despite their prominence, the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted. Here, we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types, favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback. CFA and RFA CSNs target similar spinal neuron types, with notable exceptions that suggest that these populations differ in how they influence behavior. Finally, axon collaterals of CFA and RFA CSNs target similar brain regions yet receive highly divergent inputs. These results detail the rules of CSN connectivity throughout the brain and spinal cord for two regions critical for forelimb behavior. Less