Biology subjects

Kiem, A.

Publications and source records attributed to Kiem, A..

4 recordsLinked to original sources

bioRxiv2026

Single-Cell Translation and Apoptosis Profiling to Define Human CD34⁺ Cell Response to Specific Factors

Global mRNA translation is a defining functional property of hematopoietic stem cells (HSCs) and is increasingly recognized as a critical axis of dysregulation in myelodysplastic syndromes (MDS) and other clonal hematopoietic disorders. Yet the quantitative measurement of protein synthesis at single-cell resolution across phenotypically defined HSPC subpopulations, in parallel with apoptotic state, is technically challenging. Here we describe and validate a single-tube flow cytometry protocol that simultaneously quantifies global protein synthesis by O-propargyl-puromycin (OP-Puro) incorporation and intracellular cleaved Caspase-3 with cell immunophenotyping across the canonical CD34+ HSPC hierarchy in cryopreserved human cord blood (CB) CD34+ cells. The protocol enables quantitative assessment of key dynamic cell processes in defined subsets of primary hematopoietic cells on a standard flow cytometer. We apply this assay to a four-condition factor-omission analysis of the canonical SR1 + UM729 + dmPGE2 ex vivo expansion cocktail across three independent CB donors. The analysis assigns each compound a distinct functional profile: UM729 constrains protein synthesis and supports apoptotic priming across the hierarchy; SR1 maintains a pro-survival state without modulating translation; and dmPGE2 promotes HSC cycling and progressive exit from the primitive state, with minimal direct effect on the translation or apoptotic axes measured here. This analysis resolves three mechanistically distinct small-molecule signatures using a protocol directly transferable to clinical biobank specimens. We propose it as a functional-state analytic platform that may be useful for patient-derived CD34+ cells from MDS and other myeloid neoplasms in which translational dysregulation is a recognized pathological feature.

cell biology↗

bioRxiv2026

Microbiome depletion rejuvenates the aging brain

Aging is associated with cognitive decline and increased vulnerability to neurodegeneration driven by an array of molecular and cellular changes like impaired vascular integrity, demyelination, reduced neurogenesis, and chronic inflammation. Recent studies implicate the gut microbiome as a modulator of brain aging, but the underlying mechanisms remain elusive. Here, we show that depleting the gut microbiome by administering antibiotics to aged mice induces widespread molecular and structural rejuvenation in the brain. Our transcriptomic analyses by single-nucleus RNA sequencing revealed pronounced transcriptional shifts across multiple brain cell types. We confirmed that antibiotic treatment improves vascular density, promotes myelination, enhances neurogenesis, and reduces microglial reactivity. Functionally, microbiome-depleted mice showed improved hippocampal memory performance. Analyses of brain and plasma cytokine levels showed a decrease in several pro-inflammatory factors post-treatment and identified candidate factors, including the chemokine eotaxin-1. Inhibiting eotaxin-1 alone can reverse several aspects of brain aging. Our findings demonstrate that age-associated microbial inflammation contributes to brain aging and that its attenuation can restore youthful features at the molecular, cellular, and functional levels. Targeting the gut microbiome or its circulating mediators may therefore represent a non-invasive approach to promote brain health and cognitive resilience in aging.

neuroscience↗

bioRxiv2025

Differentiation stage-specific use of cap-independent and cap-dependent translation initiation in hematopoiesis

Cell stress can increase the use of m7G-cap-independent, IRES-mediated translation initiation relative to cap-dependent translation (IRES/Cap). Reporters that quantify IRES/Cap have demonstrated differential activity across cultured cell types and stress conditions. By generating an IRES/Cap reporter mouse, we were able to systematically evaluate IRES/Cap across distinct tissues and cell types during physiological stresses and lineage commitment. Caloric stress invoked the expected boost in IRES/Cap translation regardless of differentiation state, but unexpectedly IRES/Cap progressively increased during hematopoietic and epithelial (hair follicle) differentiation under normal, homeostatic conditions. This was independent of total protein output or cell cycle. Even within cells of a given differentiation state, cells with lower relative-IRES utilization had markedly higher multipotent capability in vivo. The RNA processing protein PTBP1 is a mediator of this translation initiation preference. Therefore, low IRES/Cap is a signature of high stemness and suggests modulation of translation initiation participates in cell differentiation state.

developmental biology↗

bioRxiv2024

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

bioengineering↗