Biology subjects

Schmid, J.

Publications and source records attributed to Schmid, J..

15 recordsLinked to original sources

bioRxiv2026

NMR assignment and functional characterization of biotechnologically modified xanthan

Xanthan gum is one of the most important industrial microbial polysaccharides, yet comprehensive molecular-level structural characterization remains limited because of its high molecular weight, conformational rigidity, and severe spectral overlap in nuclear magnetic resonance spectroscopy. Here, we present a near-complete 1H/13C NMR chemical shift assignment of the xanthan repeating unit by combining multidimensional NMR spectroscopy with targeted glycosyltransferase knockout strains and controlled enzymatic depolymerization. Comparative analysis of wild-type xanthan and{Delta} gumI and{Delta} gumK variants enabled the unambiguous assignment of {beta}-mannose and glucuronic acid resonances, and, by extension, of the remaining sugars, supporting future structural studies of engineered xanthan variants. In addition, we investigated how side-chain composition affects xanthan properties by analyzing genetically modified variants lacking specific acetyl, pyruvyl, or sugar residues. Temperature-dependent 1H NMR determined order-disorder transition temperatures and revealed that removing pyruvate or negatively charged side-chain components increased thermal stability, whereas losing the terminal {beta}-mannose destabilized the ordered conformation. Together, these results establish a robust NMR assignment for xanthan and provide new insights into how individual side-chain substituents influence the structural stability of this polysaccharide, facilitating the rational characterization of engineered xanthan variants and structurally closely related polysaccharides.

biochemistry↗

bioRxiv2026

Cyst-type epithelial heterogeneity shapes therapeutic responsiveness in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) exhibits substantial interpatient variability in disease course and therapeutic response, but the cellular basis for this variability remains poorly understood. Here, we combine single-nucleus RNA sequencing of human cyst epithelia with machine learning-based histological analysis of >1,800 cysts to resolve three epithelial cyst types--proximal tubule-like, collecting duct-like, and mixed. These cyst types display distinct injury states, metabolic programs, and stromal microenvironments, including a mixed-cyst niche enriched for CCL2-associated inflammatory signaling. Expression of key therapeutic targets was highly cell-type specific with CFTR enriched in proximal-like epithelia, whereas AVPR2 expression was confined to AQP2-positive collecting duct-like cells. Cyst-type composition varied widely across patients and in an orthologous mouse model (Pkd1RC/RC) in which the burden of AQP2-positive cysts correlated with responsiveness to tolvaptan. These findings identify cyst-type heterogeneity as a major determinant of molecular pathway activation and predictability of therapeutic response in ADPKD.

molecular biology↗

bioRxiv2025

Bioprospecting c-di-GMP activated exopolysaccharides in bacteria

The genetic and physiological diversity of bacteria are critical resources to discover new exopolysaccharides (EPS) as raw materials with biotechnological applications. However, uncovering new EPS is limited by their lack of production in laboratory cultures, as EPS are often cryptic and their biosynthesis only proceed upon unknown environmental cues. The dinucleotide cyclic di-GMP (c-di-GMP) has emerged as a universal second messenger in bacteria and common activator of many EPS. Here, a genetic modification to elevate intracellular c-di-GMP levels and a carbohydrate fingerprinting analysis, were combined for a High-Throughput Screening (HTS) of 330 bacterial strains in search of c-di-GMP activated EPS. Nearly 10% of strains were revealed as promising candidates to overproduce novel EPS composites, in a c-di-GMP dependent manner. In these conditions, Sphingomonas sp. C10 massively produced a EPS with an unusual monosaccharide composition, compared to known biotechnologically relevant sphingans.

microbiology↗

bioRxiv2025

Memory CD4 T cells orchestrate neoadjuvant-responsive niches in colorectal cancer liver metastases

Colorectal cancer frequently progresses to liver metastases (CRLM), a stage with limited treatment options and poor prognosis. Neoadjuvant chemotherapy is used to control tumor growth and enable resection, yet many patients fail to respond, and the mechanisms underlying this variability remain unclear. To identify determinants of treatment response, we profiled T cell states and their spatial organization in CRLM. We found that spatial arrangement and polarization of CD4 memory T cell networks determine treatment outcome. In responders, Th1-like CD4 memory T cells organized with effector-memory CD8 T cells and antigen-presenting cells (APCs) into therapy-responsive immune niches (TRINs) that support CD4-mediated APC licensing and local immune engagement. Non-responders lacked such immune architecture, exhibiting myeloid-rich regions dominated by circulating-like CD4 memory and regulatory T cells. CD4-driven TRINs thus emerge as key determinants of chemotherapy efficacy and provide a rationale for developing biomarkers and strategies that enhance CD4-APC-CD8 crosstalk within organized immune niches. Statement of significanceTh1-polarized CD4 memory T cells form therapy-responsive immune niches (TRINs) that orchestrate CD4, CD8, and APC function in colorectal cancer liver metastases, a clinically challenging and immunologically cold tumor type. TRINs define chemotherapy response and provide a mechanistic foundation for biomarker development and immunotherapy strategies designed to restore anti-tumor immunity.

immunology↗

bioRxiv2025

Organoid-based modeling of platinum resistance identifies KRT17 as both a response mediator and biomarker for targeted therapy in ovarian cancer

Variable platinum responses drive high mortality in high-grade serous ovarian cancer (HGSOC), but to date, there is no molecular approach to define resistance levels for clinical decision-making. Here, we developed the organoid drug resistance assay (ODR-test) with patient-derived organoids from our ovarian cancer biobank and found that all HGSOC patients develop molecular resistance under exposure to carboplatin, although with varying clinical implications. Sustained phenotypic reprogramming and cellular plasticity under carboplatin pressure emerged as a conserved mechanism irrespective of the basal resistance level. Transcriptional and proteomic analyses revealed changes in cell adhesion and differentiation in post-platinum lines as adaptive responses that drive the increase in resistance. We identified Keratin 17 (KRT17) as a mediator of developing platinum resistance and validated its function by CRISPR/Cas9 and overexpression. Additionally, we found that KRT17 expression status (K-score) is a significant negative prognostic histopathological biomarker in a large cohort (N=384) of advanced HGSOC patients. In organoids, increased KRT17 levels enhanced sensitivity to PI3K/Akt inhibitors Alpelisib and Afuresertib, highlighting the potential of KRT17 as a stratification biomarker for targeted therapies.

cancer biology↗

bioRxiv2023

InSituCor: a toolkit for discovering non-trivial spatial correlations in spatial transcriptomics

Spatial transcriptomics presents the best kind of problem: how to find the many biological insights hidden within complex datasets. Spatially correlated genes can reveal high-interest phenomena like cell-cell interactions and latent variables. We introduce InSituCor, a toolkit for discovering modules of spatially correlated genes. A major contribution is that InSituCor returns only correlations not explainable by obvious factors like the cell type landscape; this spares precious analyst effort for non-trivial findings. InSituCor supports both unbiased discovery of whole-dataset correlations and knowledge-driven exploration of genes of interest. As a special case, it evaluates ligand-receptor pairs for spatial co-regulation.

bioinformatics↗

bioRxiv2023

Rapid redirection of auxin fluxes during root gravitropism by translocation of NGR proteins driving polarization of PIN-activating kinases

Root gravitropic bending represents a fundamental aspect of terrestrial plant physiology. Gravity is perceived by sedimentation of starch-rich plastids (statoliths) to the bottom of the central root cap cells. Following gravity perception, intercellular auxin transport is redirected downwards leading to an asymmetric auxin accumulation at the lower root side causing inhibition of cell expansion, ultimately resulting in downwards bending. How gravity-induced statoliths repositioning is translated into asymmetric auxin distribution remains unclear despite PIN auxin efflux carriers and the Negative Gravitropic Response of roots (NGR) proteins polarize along statolith sedimentation, thus providing a plausible mechanism for auxin flow redirection. In this study, using a functional NGR1-GFP construct, we visualized the NGR1 localization on the statolith surface and plasma membrane (PM) domains in close proximity to the statoliths, correlating with their movements. We determined that NGR1 binding to these PM domains is indispensable for NGR1 functionality and relies on cysteine acylation and adjacent polybasic regions as well as on lipid and sterol PM composition. Detailed timing of the early events following graviperception suggested that both NGR1 repolarization and initial auxin asymmetry precede the visible PIN3 polarization. This discrepancy motivated us to unveil a rapid, NGR-dependent translocation of PIN-activating AGCVIII kinase D6PK towards lower PMs of gravity-perceiving cells, thus providing an attractive model for rapid redirection of auxin fluxes following gravistimulation.

plant biology↗

bioRxiv2022

Adaptation to spindle assembly checkpoint inhibition through the selection of specific aneuploidies

Both the presence of an abnormal complement of chromosomes (aneuploidy) and an increased frequency of chromosome missegregation (chromosomal instability) are hallmarks of cancer. Analyses of cancer genome data have identified certain aneuploidy patterns in tumors; however, the bases behind their selection are largely unexplored. By establishing time-resolved long-term adaptation protocols, we found that human cells adapt to persistent spindle assembly checkpoint (SAC) inhibition by acquiring specific chromosome arm gains and losses. Independently adapted populations converge on complex karyotypes, which over time are refined to contain ever smaller chromosomal changes. Of note, the frequencies of chromosome arm gains in adapted cells correlate very well with those detected in cancers, suggesting that our cellular adaptation approach recapitulates selective traits that dictate pan-cancer aneuploidy patterns. We further engineered specific aneuploidies to determine the genetic basis behind the observed karyotype patterns. These experiments demonstrated that the adapted and engineered aneuploid cell lines limit CIN by extending mitotic duration. Heterozygous deletions of key SAC and APC/C genes recapitulated the rescue phenotypes of the monosomic chromosomes. We conclude that aneuploidy-induced gene dosage imbalances of individual mitotic regulators are sufficient for altering mitotic timing to reduce CIN.

cell biology↗

bioRxiv2022

Arabidopsis PFA-DSP-type phosphohydrolases target specific inositol pyrophosphate messengers

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologs display phosphohydrolase activity with a high specificity for the 5-{beta}-phosphate of inositol pyrophosphates. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin-sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-{beta}-phosphate specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

plant biology↗

bioRxiv2022

Insights in the complex DegU, DegS, Spo0A regulation system of Paenibacillus polymyxa by CRISPR-Cas9-based targeted point mutations

Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated: the single mutants DegU Q218*, DegS L99F, Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild type and the engineered strains revealed differences in swarming behavior, genetic competence, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed significant increase in regard to the genetic competence as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences of the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights in the complex regulatory system of P. polymyxa DSM 365. ImportanceTo date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate three regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations are introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, with the model bacterium Bacillus subtilis in regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance in comparison to the wild type strain. Overall, our study provides novel insights which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.

molecular biology↗

bioRxiv2021

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

The use of molecular tools based on the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems has rapidly advanced genetic engineering. These molecular biological tools have been applied for different genetic engineering purposes in multiple organisms, including the quite rarely explored Paenibacillus polymyxa. However, only limited studies on large cluster deletion and multiplex genome editing have been described for this highly interesting and versatile bacterium. Here, we demonstrate the utilization of a Cas9-based system to realize targeted deletions of four biosynthetic gene clusters in the range of 12 kb to 41 kb by the use of a single targeting sgRNA. Furthermore, we also harness the system for multiplex editing of genes and large genomic regions. Multiplex deletion was achieved with more than 80 % efficiency, while simultaneous integration at two distantly located sites was obtained with 58 % efficiency. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

synthetic biology↗

bioRxiv2021

Control of Arabidopsis shoot stem cell homeostasis by two antagonistic CLE peptide signalling pathways

Stem cell homeostasis in plant shoot meristems requires tight coordiantion between stem cell proliferation and cell differentiation. In Arabidopsis, stem cells express the secreted dodecapeptide CLAVATA3 (CLV3), which signals through the leucine-rich repeat (LRR)-receptor kinase CLAVATA1 (CLV1) and related CLV1-family members to downregulate expression of the homeodomain transcription factor WUSCHEL (WUS). WUS protein moves from cells below the stem cell domain to the meristem tip and promotes stem cell identity, together with CLV3 expression, generating a negative feedback loop. How stem cell activity in the meristem centre is coordinated with organ initiation and cell differentiation at the periphery is unknown. We show here that the CLE40 gene, encoding a secreted peptide closely related to CLV3, is expressed in the SAM in differentiating cells in a pattern complementary to that of CLV3. CLE40 promotes WUS expression via BAM1, a CLV1-family receptor, and CLE40 expression is in turn repressed in a WUS-dependent manner. Together, CLE40-BAM1-WUS establish a second negative feedback loop. We propose that stem cell homeostasis is achieved through two intertwined pathways that adjust WUS activity and incorporate information on the size of the stem cell domain, via CLV3-CLV1, and on cell differentiation via CLE40-BAM1.

plant biology↗

bioRxiv2020

A novel prokaryotic CRISPR-Cas12a based tool for programmable transcriptional activation and repression

Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9 based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable programmable activation of gene expression. Proof of principle of transcriptional regulation was demonstrated based on fluorescence reporter assays using the alternative host organism Paenibacillus polymyxa as well as Escherichia coli. Single target and multiplex CRISPR interference targeting the exopolysaccharide biosynthesis of P. polymyxa was shown to emulate polymer compositions of gene knock-outs. Simultaneous expression of 11 gRNAs targeting multiple lactate dehydrogenases and a butanediol dehydrogenase resulted in decreased lactate formation, as well as an increased butanediol production in microaerobic fermentation processes. Even though Cas12a is more restricted in terms of its genomic target sequences compared to Cas9, its ability to efficiently process its own guide RNAs in vivo makes it a promising tool to orchestrate sophisticated genetic reprogramming of bacterial cells or to screen for engineering targets in the genome. The developed tool will accelerate metabolic engineering efforts in the alternative host organism P. polymyxa and might be also applied for other bacterial cell factories.

synthetic biology↗

bioRxiv2020

Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7.

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T cell differentiation remains inefficient. We generated mice expressing human interleukin (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

immunology↗

bioRxiv2017

Facilitation between plants shapes pollination networks

SignificanceAlthough it is known that plant-plant and plant-pollinator interactions can strongly influence biodiversity and its effects on ecosystem functioning, the details of how competition and facili-tation among plants scale up to mutualistic interactions with pollinators and thus affect pollina-tion networks are poorly understood. We introduce a simple experimental system in which we control local plant interactions, measure pollinator responses and characterize plant-pollinator networks. We find that facilitation among plants produces synergistic and antagonistic effects on the pollinator community affecting the architecture and robustness of plant-pollinator net-works. Our results provide evidence for bottom-up non-additive effects of plant interactions on pollination networks and have implications for the way we study and manage ecosystems.

ecology↗