Biology subjects

Kaminski, T. S.

Publications and source records attributed to Kaminski, T. S..

12 recordsLinked to original sources

bioRxiv2026

Precision single-cell profiling of Circulating Tumour Cells: novel markers and data-driven characterization by CTCeek

Circulating tumour cells (CTCs) represent a minimally invasive method for monitoring cancer evolution in patients. CTCs are nowadays commonly isolated using antibodies against EPCAM protein. A key limitation regards the extent of EPCAM-negative CTCs, such as those that undergo EMT or whose tumour of origin is EPCAM-low or negative. We studied 3,302 RNA single-cell transcriptomes reported as CTCs in public repositories. Using copy number variation and cell type-specific markers, we discriminated bona fide CTCs from contaminating blood cells, often mislabelled as CTCs. The integration of bona fide CTCs and PBMCs, from multiple datasets, allowed us to identify novel markers, such as CLDN4, CLDN7, EFNA1 and TACSTD2 for epithelial CTCs, KCNK15 and LY6K for epithelial B CTCs, and ITGB4 for both epithelial B and mesenchymal CTCs. We revealed PODXL, AXL, CAV1, and TGM2 as markers of mesenchymal CTCs, which might be undetectable using anti-EPCAM antibodies, and TM4SF1 as universal marker, expressed in all CTC subclasses. Additionally, we found platelets to be physically associated with the epithelial A, but not with the epithelial B or the mesenchymal subtypes. Finally, we developed and implemented CTCeek, the first web-based and public reference tool that automatically annotates bona fide CTCs from scRNA-sequencing profiles.

cancer biology↗

bioRxiv2025

Droplet microfluidic PicoSorter for high throughput and active selection of cellulolytic microorganisms.

Classical enrichment methods for microorganisms rely on growth in selective media, but such practices are relatively expensive, low-throughput, and may result in a biased representation of taxa. Alternatively, microorganisms can be cultivated in thousands of picoliter droplets of equal volume, with the most efficient strains selected through quantitative assays at unprecedented ultrahigh throughput. Here, we present a novel high-throughput microfluidic technology for the characterization of cellulolytic microbial communities at the single-cell level. Individual microbial cells are encapsulated in picoliter droplets for clonal cultivation, after which a colorimetric assay using Congo red is applied to identify positive droplets containing cellulose-degrading strains. These positive droplets are then actively sorted at high throughput using absorbance-activated droplet sorting. The critical component of the enrichment assay we propose is the PicoSorter module, which expands the range of available droplet-based enrichment methods. The platform introduces a new design that enables buffer picoinjection required for the assay, followed by droplet sorting on a millisecond timescale. Highlights- Development of a novel device (PicoSorter) capable of performing multiple microfluidic operations simultaneously. - Implementation of a colorimetric droplet-based assay for the detection of cellulose. - The proposed absorbance-based HTS enables active and efficient enrichment of cellulolytic bacteria.

microbiology↗

bioRxiv2025

Induced Droplet Ovalisation (IDO): Image-based microfluidic method for high-throughput and label-free characterization of microbial proteolytic strains from wastewater sludge.

Traditional bacterial isolation methods are costly, labour-intensive, and often fail to detect rare or slow-growing taxa. In contrast, droplet-based microfluidic assays have emerged as powerful alternatives. Here, we describe a novel, label-free microfluidic assay for screening microbial proteolytic activity based on oil-induced droplet ovalization. This method outperformed two traditional isolation techniques in terms of performance, time, and overall cost-efficiency. We present Induced Droplet Ovalisation (IDO): a droplet-based protocol that combines single-cell encapsulation with automated image analysis. Using a custom-made microfluidic device and bespoke image-processing scripts, our system efficiently detects microbial proteolytic activity by monitoring droplet deformability. This approach significantly reduces the time and resources required to study proteolytic consortia, offering an automated, easy-to-implement, and label-free alternative to conventional screening. Our device achieves high-throughput screening (HTS) at 0.2 kHz while significantly reducing the cultivation medium volume used compared to traditional methods. Furthermore, in-droplet microbial recovery is enhanced by two orders of magnitude, enabling the enumeration of significantly more colonies compared to traditional screenings. Beyond microbiological applications, this versatile platform serves as a powerful tool for studying hydrogel degradation and polymerization dynamics, extending its applicability across research and industry.

microbiology↗

bioRxiv2024

Ancestral aryl sulfatases in the alkaline phosphatase family allow for greater increases in catalytic efficiency than extant variants

Characterizing the dynamics and functional shifts during protein evolution is essential, both for understanding protein evolution and for rationalizing efficient strategies for e.g. enzymes with desired and effective functions. Most proteins organize in families, sets of divergent sequences which share a common ancestor and have a similar structural fold. We study aryl sulfatases (ASs), a subfamily of the large and evolutionary old alkaline phosphatase superfamily. In this manuscript we present how ultrahigh-throughput droplet micro-fluidics can be used for studying aryl sulfatases and their computationally reconstructed putative common ancestors. We compare the evolvability and robustness of three ancestors and four extant ASs which all exhibit catalytic promiscuity towards a range of substrate classes. Fourteen libraries with varying mutation rates were expressed in single-cell microdroplets. In general, higher mutation rates resulted in wider distribution of active variants but fewer improved variants overall. However, the impact of mutation rate differed between enzymes, with some benefiting from higher and others from lower mutation rate, underscoring the need to test diverse mutagenesis regimes.

evolutionary biology↗

bioRxiv2024

Passive droplet microfluidic platform for high-throughput screening of microbial proteolytic activity.

Traditional bacterial isolation methods are often costly, have limited throughput, and may not accurately reflect the true microbial community composition. Consequently, identifying rare or slow-growing taxa becomes challenging. Over the last decade, a new approach has been proposed to replace traditional flasks or multi-well plates with ultrahigh-throughput droplet microfluidic screening assays. In this study, we present a novel passive droplet-based method designed for isolating proteolytic microorganisms, which are crucial in various biotechnology industries. Following the encapsulation of single cells in gelatin microgel compartments and their subsequent clonal cultivation, microcultures are passively sorted at high throughput based on the deformability of droplets. Our novel chip design offers a 50-fold improvement in throughput compared to previously developed deformability-based droplet sorter. This method expands an array of droplet-based microbial enrichment assays and significantly reduces the time and resources required to isolate proteolytic bacteria strains.

bioengineering↗

bioRxiv2024

Microdroplet screening rapidly profiles a biocatalyst to enable its AI-assisted engineering

Engineering enzymes for increased efficiency is key to enabling sustainable, green biocatalytic production processes in the chemical and pharmaceutical industries. This challenge can be tackled from two angles: by directed evolution, based on labour-intensive experimental testing of enzyme variant libraries, or by computational methods, where data-dependent algorithms relating sequence and function are used to predict biocatalyst improvements. Here, we combine both approaches into a two-week, low-cost workflow, in which ultra-high throughput screening of a library of imine reductases (IREDs) in microfluidic devices provides not only selected hits, but also long-read sequence data linked to fitness scores of >17 thousand enzyme variants. We demonstrate the engineering of an IRED for chiral amine synthesis by mapping its local fitness landscape in one go, ready to be used for interpretation and extrapolation by protein engineers with the help of machine learning (ML). We calculate position-dependent mutability and combinability scores of mutations and comprehensively illuminate a complex interplay of mutations driven by synergistic, often positively epistatic effects. When interpreted by easy-to-use regression and tree-based ML algorithms designed for random whole-gene mutagenesis data, 3-fold improved hits initially obtained from experimental screening are extrapolated further to give another order of magnitude improvement (23-fold in kcat) after testing only a handful of designed mutants. Predictions succeed in >80% of cases. The catalytic features discovered in one IRED are shown to be portable and confer activity on IREDs with [~]50% homology. Our campaigns yield biocatalytically efficient IREDs and are paradigmatic for future enzyme engineering efforts that rely on large sequence-function maps, profiling how a biocatalyst responds to mutation. In the age of predictive biology, these maps will chart the way to improved function by exploiting the synergy of rapid experimental screening combined with ML evaluation and extrapolation.

synthetic biology↗

bioRxiv2023

Ultrahigh throughput evolution of tryptophan synthase in droplets via an aptamer-biosensor

Tryptophan synthase catalyzes the synthesis of a wide array of non-canonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (>107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic read-outs which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a biosensor to quantitatively report on tryptophan production in droplets. The utility of the biosensor was validated by identifying 5-fold improved tryptophan synthases from [~]100,000 protein variants. More generally this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

biochemistry↗

bioRxiv2023

spinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these unique properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

developmental biology↗

bioRxiv2023

Microfluidics-enabled fluorescence-activated cell sorting of single pathogen-specific antibody secreting cells for the rapid discovery of monoclonal antibodies

Monoclonal antibodies are increasingly used to prevent and treat viral infections, playing a pivotal role in pandemic response efforts. Antibody secreting cells (ASCs, plasma cells and plasmablasts) are an excellent source of high-affinity antibodies with therapeutic potential. Current methodologies to study antigen-specific ASCs either have low throughput, require expensive and labour-intensive screening or are technically demanding and therefore not accessible to the wider research community. Here, we present a straightforward technology for the rapid discovery of monoclonal antibodies from ASCs: we combine microfluidic encapsulation of single cells into an antibody capture hydrogel with antigen bait sorting by conventional flow cytometry. With our technology, we screened millions of mouse and human ASCs and obtained anti-SARS-CoV-2 monoclonal antibodies with high affinity (pM) and neutralising capacity (<100 ng/mL) in two weeks with a high hit rate (>85%). By facilitating access into the underexplored ASC compartment, we enable fast and efficient antibody discovery as well as immunological studies into the generation of protective antibodies.

immunology↗

bioRxiv2022

Ultrahigh-throughput Absorbance Activated Droplet Sorting (UHT-AADS) for enzyme screening at kilohertz frequencies

Droplet microfluidics is a valuable method to beat the odds in high throughput screening campaigns such as directed evolution, where valuable hits are infrequent and large library sizes are required. Absorbance-based sorting expands the landscape of range of enzyme families that can be subjected to droplet screening by expanding possible assays beyond fluorescence detection. However, absorbance activated droplet sorting (AADS) is currently [~]10-fold slower than typical fluorescence activated droplet sorting (FADS), meaning that, in comparison, a larger portion of sequence space is inaccessible due to throughput constraints. Here we improve AADS to reach kHz sorting speeds in an order of magnitude increase over previous designs, with close-to-ideal sorting accuracy. This is achieved by a combination of (i) the use of refractive index matching oil that improves signal quality by removal of side scattering (increasing the sensitivity of absorbance measurements); (ii) a sorting algorithm capable of reaching 4 kHz with an Arduino Due; and (iii) a chip design that transmits product detection better into sorting decisions without false positives, namely a single-layered inlet to space droplets further apart and injections of bias oil providing a fluidic barrier preventing droplets from entering the incorrect sorting channel. The updated ultrahigh-throughput absorbance activated droplet sorter (UHT-AADS) increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/507731v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1c81dc3org.highwire.dtl.DTLVardef@119fe88org.highwire.dtl.DTLVardef@195c06borg.highwire.dtl.DTLVardef@8bce56_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Ultrahigh-throughput directed evolution of a metal-free α/β-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase

The recent massive release of new, man-made substances into the environment requires bioremediation, but a very limited number of enzymes evolved in response are available. When environments have not encountered the potentially hazardous materials in their evolutionary history, existing enzymes have to be repurposed. The recruitment of accidental, typically low-level promiscuous activities provides a head start that, after gene duplication, can adapt and provide a selectable advantage. This evolutionary scenario raises the question whether it is possible to adaptively improve the low-level activity of enzymes recruited from non- (or only recently) contaminated environments quickly to the level of evolved bioremediators. Here we address the evolution of phosphotriesterases (enzymes for hydrolysis of organophosphate pesticides or chemical warfare agents) in such a scenario: In a previous functional metagenomics screening we had identified a promiscuous phosphotriesterase activity of the /{beta}-hydrolase P91, with an unexpected Cys-His-Asp catalytic triad as the active site motif. We now probe evolvability of P91 using ultrahigh-throughput screening in microfluidic droplets, and test for the first time whether the unique catalytic motif of a cysteine-containing triad can adapt to achieve rates that rival existing phosphotriesterases. These mechanistically distinct enzymes achieve their high rates based on catalysis involving a metal-ion cofactor. A focussed, combinatorial library of P91 (> 105 members) was screened on-chip in microfluidic droplets by quantification of the reaction product, fluorescein. Within only two rounds of evolution P91s phosphotriesterase activity was increased {approx} 400-fold to a kcat/KM of {approx} 106 M-1s-1, matching the catalytic efficiencies of naturally evolved metal-dependent phosphotriesterases. In contrast to its homologue acetylcholinesterase that suffers suicide inhibition, P91 shows fast de-phosphorylation rates and is rate-limited by the formation of the covalent adduct rather than by its hydrolysis. Our analysis highlights how the combination of focussed, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in Nature, resulting in alternative reagents with a novel catalytic machinery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/480337v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@5e6f7aorg.highwire.dtl.DTLVardef@1e8cd72org.highwire.dtl.DTLVardef@10809c9org.highwire.dtl.DTLVardef@ba6fc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development

In recent years, single-cell transcriptome sequencing has revolutionized biology, allowing for the unbiased characterization of cellular subpopulations. However, most methods amplify the termini of polyadenylated transcripts capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts. Additionally, most workflows do not sequence the full transcript hindering the analysis of alternative splicing. We therefore developed VASA-seq to detect the total transcriptome in single cells. VASA-seq is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to over 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. The dynamics of the total single-cell transcriptome result in the discovery of novel cell type markers many based on non-coding RNA, an in vivo cell cycle analysis and an improved RNA velocity characterization. Moreover, it provides the first comprehensive analysis of alternative splicing during mammalian development.

molecular biology↗