Biology subjects

Klatt, N.

Publications and source records attributed to Klatt, N..

3 recordsLinked to original sources

bioRxiv2026

Sample preparation for direct nanopore based protein identification via tryptic peptide recognition

Rapid, cost-efficient and miniaturized protein analysis is essential for point-of-care clinical diagnostics. Nanopores offer a promising alternative to mass spectrometry, offering high-resolution, single-molecule sensing with a minimal footprint. However, peptide-level protein identification remains challenging due to difficulties in assigning nanopore signals to peptide sequences and the incompatibility of conventional proteolysis workflows with nanopore sensing. Here we integrate a single-pot, solid-phase-enhanced sample preparation (SP3) workflow with nanopore sensing. The SP3 protocol decouples proteolysis from interfering components via magnetic bead cleanup, yielding a digest compatible with nanopore sensing. As a proof of concept, tryptic digests of myelin basic protein measured with an engineered aerolysin variant (AeL-R220S) generated a reproducible five-peak peptide fingerprint. Cross-validation against a synthetic peptide library and LC-MS/MS enabled reference-based assignment of blockade signatures to four MBP-derived peptides. This approach establishes SP3 as a sample-preparation workflow for nanopore sensing, paving the way for label-free identification and characterization of protein targets.

biochemistry↗

bioRxiv2026

Single-cell DNA cytometry with magnetic- and fluorescence-activated bead sorting

Many clinically important cell populations are defined by intracellular DNA or RNA signatures, but isolating and profiling these cells remains difficult. Probe-based in situ hybridization strategies that are compatible with cytometry have difficulty resolving low-abundance transcripts, integrated provirus, or single-copy genomic mutations. In contrast, droplet digital PCR achieves single-molecule sensitivity; however, subsequent cell isolation requires specialized microfluidic sorters that are slow and not widely available. Here, we introduce magniFIND-seq, a nucleic acid cytometry platform that combines the sensitivity of digital PCR with commercial magnetic-activated (MACS) and fluorescence-activated (FACS) sorting instruments. Single-cell genomes and transcriptomes are captured in agarose beads, target sequences are detected by digital PCR, and bead-bound amplicons are labeled with magnetic or fluorescent probes. Controlled evaporation shrinks beads from 55 to 20 m, enabling scalable magnetic separation, fast single-bead FACS sorting, and their combination for high-purity recovery of rare populations. Using magniFIND-seq, we demonstrate multiplexed single-copy detection and FACS-based isolation of simian immunodeficiency virus proviral pol and env targets. Separately, using BCR::ABL1 as a disease-defining target, we enrich chronic myeloid leukemia cells and recover single-cell transcriptomes that resolve tyrosine kinase inhibitor-resistance programs. magniFIND-seq extends the throughput and accessibility of nucleic acid cytometry by engineering compatibility with commercial instruments.

bioengineering↗

bioRxiv2022

The Defenders of the Alveolus Succumb in COVID-19 Pneumonia to SARS-CoV-2, Necroptosis, Pyroptosis and Panoptosis

The alveolar type II (ATII) pneumocyte has been called the defender of the alveolus because, amongst the cells many important roles, repair of lung injury is particularly critical. We investigated the extent to which SARS-CoV-2 infection incapacitates the ATII reparative response in fatal COVID-19 pneumonia, and describe massive infection and destruction of ATI and ATII cells. We show that both type I interferon-negative infected ATII and type I-interferon-positive uninfected ATII cells succumb to TNF-induced necroptosis, BTK-induced pyroptosis and a new PANoptotic hybrid form of inflammatory cell death that combines apoptosis, necroptosis and pyroptosis in the same cell. We locate pathway components of these cell death pathways in a PANoptosomal latticework that mediates emptying and disruption of ATII cells and destruction of cells in blood vessels associated with microthrombi. Early antiviral treatment combined with inhibitors of TNF and BTK could preserve ATII cell populations to restore lung function and reduce hyperinflammation from necroptosis, pyroptosis and panoptosis. Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/503050v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8bf28dorg.highwire.dtl.DTLVardef@1e1335eorg.highwire.dtl.DTLVardef@1f3a0e2org.highwire.dtl.DTLVardef@1c77c62_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIn fatal COVID-19 pneumonia, the initial destruction of Type II alveolar cells by SARS-CoV-2 infection is amplified by infection of the large numbers of spatially contiguous Type II cells supplied by the proliferative reparative response. C_LIO_LIInterferon-negative infected cells and interferon-positive uninfected cells succumb to inflammatory forms of cell death, TNF-induced necroptosis, BTK-induced pyroptosis, and PANoptosis. C_LIO_LIAll of the cell death pathway components, including a recently identified NINJ1 component, are localized in a PANoptosome latticework that empties in distinctive patterns to generate morphologically distinguishable cell remnants. C_LIO_LIEarly combination treatment with inhibitors of SARS-CoV-2 replication, TNF and BTK could reduce the losses of Type II cells and preserve a reparative response to regenerate functional alveoli. C_LI

immunology↗