Biology subjects

Morgenstern, M.

Publications and source records attributed to Morgenstern, M..

9 recordsLinked to original sources

bioRxiv2026

Persistent Staphylococcus aureus in human musculoskeletal tissues despite targeted antibiotic therapy

Staphylococcus aureus is a leading cause of musculoskeletal infection with frequent recurrence despite surgery and antibiotic therapy. Its persistence in human tissues under antibiotic pressure remains unclear. We performed longitudinal in situ analyses of matched tissue samples before and during therapy from patients undergoing repeated surgery using advanced imaging and tissue-based metagenomics. S. aureus remained detectable in all samples--including culture-negative specimens--and whole-genome sequencing confirmed persistence of the infecting strain. Antibiotic initiation reduced bacterial burden by 1.0Slog10 bacteria/mm3 but residual loads stabilized thereafter. Quantitative imaging identified a culture detection threshold ([~]103bacteria/mm3), indicating that culture negativity reflects diagnostic sensitivity limits rather than true bacterial clearance. Persistence under antibiotic therapy was associated with high baseline burden and intracellular sequestration, whereas long-term persistence was characterized by predominantly extracellular bacteria capable of driving relapse. These findings reveal sustained tissue persistence despite apparent microbiological clearance and guide precision strategies to target resilient bacterial reservoirs.

microbiology↗

bioRxiv2026

Rapid Peptide Mapping of Monoclonal Antibodies with Direct Infusion Mass Spectrometry

Peptide mapping is a critical tool for characterizing biotherapeutic proteins and is essential for the development of monoclonal antibody drugs. Here we describe a new direct infusion technology that streamlines peptide mapping data collection and analysis, accelerating the method by up to 100-fold. This method, which we term RaPiD-mAb-MS, combines high-throughput plate-based sample preparation with direct infusion mass spectrometry analysis. RaPiD-mAb-MS allows analysis of 96 samples within [~] 1.5 to 2 hours, routinely achieves >95% sequence coverage, and has been successfully applied to 28 unique antibodies and over 2,000 samples. Here we demonstrate that RaPiD-mAb-MS detects and quantifies oxidation, deamidation, isomerization, glycosylation, and sequence variants with results comparable to conventional LC-MS based methods in a fraction of the time. Further, by eliminating chromatography, data analysis is greatly streamlined and simplified. By allowing for the collection of [~] 1,000 peptide maps per day, RaPiD-mAb-MS is positioned to accelerate all phases of antibody-based drug discovery & development and sets the stage for collection of massive datasets that would allow artificial intelligent prediction of optimal antibody variants and formulations.

biochemistry↗

bioRxiv2026

Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

Nitric oxide (NO*) is an important signaling molecule in many biological processes, including immune response. During response to classical activation stimuli lipopolysaccharide (LPS) and interferon-{gamma} (IFN{gamma}), macrophages generate NO* via inducible nitric oxide synthase (iNOS). To comprehensively define the effects of NO*, we applied a multi-omic strategy integrating proteomics and transcriptomics to profile murine macrophages across conditions with or without LPS/IFN{gamma}-activation, with or without iNOS expression or exogenous NO* donor treatment. The results revealed NO* has broad, yet selected and controlled, regulatory effects, playing a key role in coordinating the systematic remodeling during macrophage classical activation. Among the proteins that are most suppressed in a NO*-dependent manner, electron transport chain (ETC) is the most enriched. NO* drives complex-specific remodeling of ETC, causing selected downregulation of complex I, II, and IV, through a different combination of transcriptional and post-transcriptional mechanisms for each complex. Functionally, we found NO* is required, but not sufficient, for the strong suppression of cellular respiration upon macrophage activation. Among the most consistently upregulated proteins are many enzymes involved in redox defense. AKR1A1 was identified as a top hit. We found Akr1a1 induction requires both NO* and LPS/IFN{gamma} stimulation. The S-nitroso-CoA reductase activity of AKR1A1 mitigates NO*-driven inhibition of pyruvate dehydrogenase complex by limiting the inhibitory modifications targeting its lipoyl cofactor. Knocking out Akr1a1 causes accelerated remodeling of TCA cycle, dysregulated immunoregulatory metabolite level, and altered functional gene expression and cytokine production at later stage of immune response. Thus, the NO*-dependent upregulation of AKR1A1 forms a negative regulatory loop to fine-tune NO*-mediated metabolic and functional remodeling during immune response. Together, this work provided a systems-level map of NO*-dependent regulation, revealed the crosstalk between NO* and immune signaling, and demonstrated mechanisms providing redox adaptation and precise control of NO*s effects.

immunology↗

bioRxiv2025

Non-Optimal Codon Usage Shapes Cell Cycle Regulators: Codon Optimization Studies on CDK1 and NUF2

The expression and functions of genes are largely dependent on genome integrity and stability. Codon usage plays a significant role in maintaining stability and functions of genes. Perturbation in codon sequence can lead to functional and structural dysfunction of a gene. In our study, we performed the codon usage analyses of cell cycle-dependent genes. Various codon usage parameters analyzed using nucleotide compositions like RSCU, ENC, and GC analyses showed that cell cycle dependent genes follow non-optimal codon usage. Genes preferred AT-rich ending over GC-rich ending, thereby suggesting preference for non-optimal codons. Neutrality and parity plot showed that the codon preference is a result of mutation selection pressure. To study the codon usage implications, we optimized the codons of cell cycle dependent genes (CDK1 and NUF2) to study their effects on cell cycle and apoptosis in vitro. We observed that codon optimization alters the cell cycle length in cell cycle-dependent genes impacting cell fate and survival. Our studies revealed that codon usage preferences directly affect the stability of both mRNA and proteins. Specifically, genes and proteins with non-optimal codons exhibited reduced stability compared to their optimized counterparts, suggesting critical implications for cell cycle regulation and apoptosis.

molecular biology↗

bioRxiv2025

Microgeography of staphyloccoci in human tissue explains antibiotic failure

Summary paragraphBacterial infections remain a major health threat, yet pathogen biology in human tissues is poorly understood. Using AI-guided imaging, we mapped [~]15,500 Staphylococcus aureus cells in biopsies from 33 patients undergoing surgery for musculoskeletal infections. Despite substantial interindividual variability, consistent patterns emerged. Most bacteria resided within non-classical monocytes/macrophages, challenging models of primarily extracellular pathogenesis. Both intra- and extracellular bacteria were predominantly isolated single cells or doublets with low rRNA content, suggesting limited replication. Complementary proteomics implicated inflammation-associated hypoxia and host glucose-to-lactate metabolism as growth constraints. Preoperative antibiotic therapy failed to clear bacteria across microenvironments and cluster sizes, challenging assumptions that antibiotic tolerance is confined to intracellular niches or biofilms and underscoring the clinical need for debridement. In vitro models replicating diverse tissues conditions impaired antibiotic activity, indicating multifactorial resilience. Together, these findings redefine S. aureus infection biology in musculoskeletal infections and establish a framework for mechanism-based prevention and therapy.

microbiology↗

bioRxiv2025

SLX4IP acts in parallel to FANCM to limit BLM-dependent replication stress at ALT telomeres

Alternative Lengthening of Telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that enables cancer cells to gain unlimited replicative capacity. ALT relies on recombination-mediated telomere elongation and is promoted by telomeric replication stress. However, ALT requires strict regulation, as excessive replication stress or recombination are cytotoxic. Central to ALT is the RecQ helicase BLM, which regulates telomeric replication stress and promotes telomere recombination and DNA synthesis. Despite its key role in the ALT pathway, BLM must be tightly regulated to prevent deleterious outcomes. Here, we identify SLX4IP as a key suppressor of BLM-driven replication stress at ALT telomeres. Loss of SLX4IP in ALT-positive cells leads to BLM-dependent telomeric replication stress and impaired replication fork progression. Mechanistically, SLX4IP limits the unwinding of unligated Okazaki fragments by BLM on the lagging strand during telomere replication. This reduces the formation of toxic 5' DNA flaps and prevents hyperactivation of ATR signalling and deleterious recombination levels. We also uncover a synthetic lethal interaction between SLX4IP and FANCM, an ATPase/translocase that is a known regulator of BLM at telomeric replication forks in ALT cells. We demonstrate that SLX4IP and FANCM act in parallel to restrain BLM activity, thereby maintaining the balance of replication stress and recombination that is necessary for productive ALT. These findings reveal a vulnerability in ALT-positive cancers lacking SLX4IP and establish SLX4IP as a potential biomarker for therapeutic strategies targeting FANCM. HIGHLIGHTSO_LISLX4IP depletion activates the replication stress response at ALT telomeres C_LIO_LISLX4IP acts in parallel to FANCM to limit replication stress at ALT telomeres C_LIO_LIThe synthetic lethal interaction between SLX4IP and FANCM is dependent on BLM C_LIO_LISLX4IP depletion causes BLM-dependent lagging-strand replication stress C_LI

cell biology↗

bioRxiv2025

Adeno-Associated Virus 2 (AAV2) - induced RPA exhaustion generates cellular DNA damage and restricts viral gene expression

Parvoviruses are single-stranded DNA viruses that have been modified to serve as vehicles for therapeutic transgene delivery in the form of recombinant Adeno-Associated Virus (rAAV2) vectors or rodent parvovirus-derived oncolytic agents. Infection with viruses of the Parvoviridae family induces a cellular DNA Damage Response (DDR) signal that supports virus replication. However, it remains unknown whether rAAV2 vectors or non-replicative AAV2 genomes induce cellular DDR signals, which might be deleterious to the cell. To determine the impact of AAV2/rAAV2 genomes on the integrity of the host chromosome, we have pulsed AAV2/rAAV2 infected cells with BrdU analogs followed by single-molecule imaging of the cellular replisomes and proteomic analysis of the host replication forks. We discovered that non-replicative AAV2/rAAV2 genomes are sufficient to induce replication stress on the host genome, leading to DDR signals in a dose-dependent manner. Moreover, infection with replication-competent AAV2 leads to enrichment of replication stress proteins, DNA repair factors and RNA processing machinery on cellular replication forks. However, neither the AAV2 Inverted Terminal Repeats (ITRs) that are retained in rAAV2s nor empty capsids are sufficient to induce host-cell replication stress. Strikingly, incoming AAV2 genomes associate with the single-stranded DNA binding protein RPA in host cells in a dose-dependent manner, progressively shortening cellular replication forks. These elevated levels of AAV2-induced cellular replication stress eventually leads to accumulation of DDR signals in the nucleus. Chemical inhibition of RPA activity and RNAi-mediated knockdown leads to de-repression of the AAV2 genome, increasing Rep 68/78 gene expression. Ectopic expression of RPA rescues AAV2-induced replication stress. Taken together, our findings suggest that depletion of cellular stores of RPA molecules by competing AAV2 genomes restrict viral gene expression and cause cellular DNA damage. AUTHOR SUMMARYAdeno-Associated Viruses 2 (AAV2) are modified to design therapeutic gene therapy vectors, but how they interact with the guardians of host DNA remains unknown. In this work, we show that AAV2 genomes compete with the host cell for the single-stranded DNA binding protein RPA, rendering the host vulnerable to replication stress leading to both suppression of the viral gene expression and induction of cellular DNA breaks. These findings provide insights into how gene therapies delivered at high doses could have genotoxic effects, underscoring the importance of engineering AAV2-based gene therapy platforms that express efficiently at lower doses.

microbiology↗

bioRxiv2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of elongation factor Tu or slowing translation elongation with chloramphenicol suppress the effects of G4 stabilization. In contrast, reducing expression of certain translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses demonstrate that ribosome assembly factors and other proteins involved in translation are less abundant in G4-stabilizing conditions. Our integrated systems approach allowed us to propose a model for how RNA G4s can present barriers to E. coli growth and that reducing the rate of translation can compensate for G4-related stress.

biochemistry↗

bioRxiv2023

A Far-Red Fluorescent Probe to Visualize Staphylococcus aureus in Patient Samples

Staphylococcus aureus (S. aureus) is the leading bacterial cause of death in high-income countries and can cause invasive infections at various body sites. These infections are associated with prolonged hospital stays, a large economic burden, considerable treatment failure, and mortality rates. So far, there is only limited knowledge about the specific locations where S. aureus resides in the human body during various infections. Hence, the visualization of S. aureus holds significant importance in microbiological research. Herein, we report the development and validation of a far-red-fluorescent probe to detect S. aureus in human biopsies from deep-seated infections. This probe displays strong fluorescence and low background in human tissues, outperforming current tools for S. aureus detection. Several applications are demonstrated, including fixed- and live-cell imaging, flow cytometry, and super-resolution bacterial imaging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/556223v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1bcf4e4org.highwire.dtl.DTLVardef@71bf45org.highwire.dtl.DTLVardef@1c468fdorg.highwire.dtl.DTLVardef@1c8baf3_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗