Biology subjects

Breuer, L.

Publications and source records attributed to Breuer, L..

9 recordsLinked to original sources

bioRxiv2026

Land-use influence on benthic algal assemblage structure and biomass in Afrotropical headwater streams

Benthic algae integrate catchment-and reach-scale pressures in streams. We assessed land-use effects on benthic algal assemblage structure, chlorophyll-a, ash-free dry mass (AFDM), and autotrophic index across 24 headwater streams in the Sondu-Miriu River basin, Kenya, and explored wet-dry patterns in two monitoring streams. We sampled across four land-use categories: natural forest (NF), tea and tree plantations (TTP), smallholder agriculture (SHA), and smallholder tea (SHT). Synoptic sampling occurred in August 2025, and one NF stream and one SHA stream were sampled five times between November 2024 and May 2025. Shannon and Simpson diversity were highest in NF streams and lowest in SHT streams. Chlorophyll-a and AFDM were highest in SHA streams; chlorophyll-a was lowest in SHT, while AFDM and autotrophic index were lowest in NF. Relative abundance patterns showed that SHA streams were dominated by Lyngbya and Stigeoclonium, whereas SHT streams were dominated mainly by Stigeoclonium; NF streams supported persistent diatom-rich assemblages. Non-metric multidimensional scaling showed land-use separation of benthic algal assemblages, while redundancy analysis indicated weak, non-significant evidence of environmental associations in the synoptic dataset. Composition and biomass metrics tracked land-use gradients and wet-dry patterns, supporting periphyton-based bioassessment for monitoring disturbance in Afrotropical headwater streams.

ecology↗

bioRxiv2026

A calcium imaging pipeline to detect and quantify compound-specific effects in human and mouse astrocytes and astrocyte-neuron cocultures

Astrocytes are crucial mediators of diverse aspects of brain function, including energy metabolism and synapse formation and maturation. Calcium is the primary information carrier in astrocytes, reporting cellular health and activity, and can be measured using fluorescent indicators. However, this readout is not yet widely used to screen and evaluate disease models and drug candidates. Here, we adapted a simple automated calcium imaging pipeline with key output parameters that characterize changes in astrocytic calcium signaling. We compared calcium responses in mouse astrocyte monocultures and astrocyte-neuron cocultures using GFAP-driven membrane-targeted GCaMP6f, with human astrocytes differentiated from two different induced pluripotent stem-cell lines using the calcium dye Cal520-AM. Event-based analysis reported similarities and differences in mean fluorescence, amplitude, frequency, duration, and area of calcium responses. We benchmarked the pipeline using the purinergic receptor agonist ATP to increase astrocyte activity, and the ER calcium pump blocker CPA to decrease activity across all culture models. Glutamatergic and serotonergic receptor function was tested with glutamate and lysergic acid diethylamide (LSD). LSD decreased activity in mouse cocultured astrocytes, but increased activity in human astrocytes. Furthermore, the addition of human recombinant Tau oligomers, an in vitro model of Alzheimers disease pathology, decreased activity in both mouse and human astrocytes. This pipeline can be used to quickly and easily characterize effects of astrocyte-targeting compounds, effects of non-astrocyte-targeting compounds on astrocyte activity, and rescue of disease models that affect astrocyte function, in mouse and human astrocytes and astrocyte-neuron cocultures.

neuroscience↗

bioRxiv2026

A human iPS cell line for ready-to-use human iAstrocytes that support human neurons

Human iPSC-derived neuronal networks are increasingly being employed in basic and applied research to enhance translation. Astrocytes are essential for neuronal network function, but are often not included, or replaced with mouse astrocytes, which compromises translation. Current protocols produce hiPSC-derived astrocytes by stepwise differentiation using small molecules and cytokines, or by forward programming by inducing transcription factors introduced by lentiviral transduction. Here we created a stable, inducible hiPSC line capable of producing iAstrocytes by introducing the transcription factors NFIB and SOX9 into the AAVS1 locus of the BIHi005-A hiPSC line. iAstrocytes induced from this line upregulated astrocytic genes over four weeks in culture, expressed GFAP and S100B and exhibited spontaneous calcium waves and responses to ATP and CPA. In co-cultures, iAstrocytes supported the growth and function of mature iNeuron networks. Pre- and post-synaptic markers and synchronous neuronal activity measured by high-density multi-electrode array recordings and neuronal calcium imaging, appeared by four weeks. The use of iAstrocytes will help to standardize the use of human astrocytes to support human neural networks and enhance translation.

neuroscience↗

bioRxiv2026

A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

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(R) 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.

immunology↗

bioRxiv2025

Cryopreservation of siRNA-treated cells is feasible

Cryopreservation is a routine step in the manufacturing process of adoptive cell therapies, providing critical logistic flexibility. RNAi-based therapies are increasingly being explored as enhancers or modulators of adoptive cell therapies. However, the impact of cryopreservation on cells treated with RNAi-based therapies has not been investigated before. In this study, we addressed this knowledge gap by examining silencing efficacy in siRNA-treated cells that undergo cryopreservation. Our findings demonstrate that silencing in cryopreserved cells is comparable to that in cells maintained continuously in culture. Moreover, we found that the duration of siRNA exposure plays a significant role in cells that later undergo cryopreservation, with extended exposure improving silencing efficiency. However, this effect diminishes at higher siRNA concentrations. Additionally, we showed that siRNA treatment is feasible at low temperatures (2-8{degrees}C), and siRNA-treated cells can be cryopreserved for extended periods (at least one month) without loss of efficacy. Furthermore, we demonstrated the feasibility of cryopreserving siRNA-treated primary cells, including those resembling leukapheresis material. Our work establishes the feasibility of integrating siRNA treatments into current manufacturing processes for adoptive cell therapies.

bioengineering↗

bioRxiv2025

Formation of the β-sheet at the extracellular opening of the intimin β-barrel domain is necessary for stability and efficient passenger secretion

Attaching and effacing pathogens, such as enteropathogenic and enterohaemorrhagic E. coli, rely on an adhesin, intimin, for attachment to enterocytes and downstream effects leading to actin pedestal formation. Intimin belongs to the inverse autotransporter family (type 5e secretion systems), which secrete the extracellular adhesive domain or passenger via a hairpin intermediate. While many of the next steps in secretion are now understood, how the hairpin initially forms is not known. We sought to investigate this by making point mutations at several positions in the {beta}-barrel domain of intimin, as this domain forms at least part of the secretion pore. We made the mutations in a wild-type background and in a stalled intermediate caught in the hairpin conformation, which allowed us to uncouple passenger secretion from hairpin formation. Surprisingly, most of the point mutations did not have an appreciable effect on hairpin formation or passenger secretion, and larger changes such as replacing the entire linker region with a flexible glycine-serine stretch showed only a modest reduction in passenger secretion. By contrast, mutations affecting a small {beta}-sheet at the extracellular face of the intimin {beta}-barrel between two extracellular loops and the C-terminus of the linker had a more pronounced effect on secretion, and abolishing this {beta}-sheet prevented hairpin formation and led to complete loss of the wild-type protein. Our results show that the intimin {beta}-barrel is remarkably tolerant to changes and that the {beta}-sheet on the extracellular side of this domain plays a central role in passenger secretion and protein stability.

microbiology↗

bioRxiv2025

Tissue-specific effects of dietary protein on cellular senescence are mediated by branched-chain amino acids

Dietary protein is a key regulator of healthy aging in both mice and humans. In mice, reducing dietary levels of the branched-chain amino acids (BCAAs) recapitulates many of the benefits of a low protein diet; BCAA-restricted diets extend lifespan, reduce frailty, and improve metabolic health, while BCAA supplementation shortens lifespan, promotes obesity, and impairs glycemic control. Recently, high protein diets have been shown to promote cellular senescence, a hallmark of aging implicated in many age-related diseases, in the liver of mice. Here, we test the hypothesis that the effects of high protein diets on metabolic health and on cell senescence are mediated by BCAAs. We find that reducing dietary levels of BCAAs protects male and female mice from the negative metabolic consequences of both normal and high protein diets. Further, we identify tissue-specific effects of BCAAs on cellular senescence, with restriction of all three BCAAs - but not individual BCAAs - protecting from hepatic cellular senescence while potentiating cell senescence in white adipose tissue. We find that the effects of BCAAs on hepatic cellular senescence are cell-autonomous, with lower levels of BCAAs protecting cultured cells from antimycin-A induced senescence. Our results demonstrate a direct effect of a specific dietary component on a hallmark of aging and suggest that cellular senescence may be highly susceptible to dietary interventions.

physiology↗

bioRxiv2023

Towards substitution of invasive telemetry: An integrated home cage concept for unobstrusive monitoring of objective physiological parameters in rodents

This study presents a novel concept for a smart cage designed to monitor the physiological parameters of mice and rats in animal-based experiments. The system focuses on monitoring key clinical parameters, including heart rate, respiratory rate, body temperature, activity, and circadian rhythm. To create the smart home cage system, an in-depth analysis of the requirements was performed, including camera positioning, imaging system types, resolution, frame rates, external illumination, video acquisition, data storage, and synchronization. Two different camera perspectives were considered, and specific camera models, including two near-infrared and two thermal cameras, were selected to meet the requirements. During the first testing phase, the system demonstrated the potential of extracting vital parameters such as respiratory and heart rate. This technology has the potential to reduce the need for implantable sensors while providing reliable and accurate physiological data, leading to refinement and improvement in laboratory animal care.

bioengineering↗

bioRxiv2022

Blind spots on western blots: a meta-research study highlighting opportunities to improve figures and methods reporting

Western blotting is a standard laboratory method used to detect proteins and assess their expression levels. Unfortunately, poor western blot image display practices and a lack of detailed methods reporting can limit a readers ability to evaluate or reproduce western blot results. While several groups have studied the prevalence of image manipulation or provided recommendations for improving western blotting, data on the prevalence of common publication practices are scarce. We systematically examined 551 articles published in the top 25% of journals in neurosciences (n=151) and cell biology (n=400) that contained western blot images, focusing on practices that may omit important information. Our data show that most published western blots are cropped and blot source data are not made available to readers in the supplement. Publishing blots with visible molecular weight markers is rare, and many blots additionally lack molecular weight labels. Western blot methods sections often lack information on the amount of protein loaded on the gel, blocking steps and antibody labeling protocol. Important antibody identifiers like source, catalog number or RRID were omitted frequently for primary antibodies, and regularly for secondary antibodies. We present detailed descriptions and visual examples to help scientists, peer reviewers and editors to publish more informative western blot figures and methods. Additional resources include a toolbox to help scientists produce more reproducible western blot data, teaching slides in English and Spanish and an antibody reporting template.

cell biology↗