Quiescence Modulates Stem Cell Maintenance and Regenerative Capacity in the Aging Brain.

The function of somatic stem cells declines with age. Understanding the molecular underpinnings of this decline is key to counteract age-related disease. Here, we report a dramatic drop in the neural stem cells (NSCs) number in the aging murine brain. We find that this smaller stem cell reservoir is protected from full depletion by an increase in quiescence that makes old NSCs more resistant to regenerate the injured brain. Once activated, however, young and old NSCs show similar proliferation and differentiation capacity. Single-cell transcriptomics of NSCs indicate that aging changes NSCs minimally. In the aging brain, niche-derived inflammatory signals and the Wnt antagonist sFRP5 induce quiescence. Indeed, intervention to neutralize them increases activation of old NSCs during homeostasis and following injury. Our study identifies quiescence as a key feature of old NSCs imposed by the niche and uncovers ways to activate NSCs to repair the aging brain.

Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues.

Regulatory T cells (Treg cells) perform two distinct functions: they maintain self-tolerance, and they support organ homeostasis by differentiating into specialized tissue Treg cells. We found that epigenetic modifications defined the molecular characteristics of tissue Treg cells. Tagmentation-based whole-genome bisulfite sequencing revealed more than 11,000 regions that were methylated differentially in pairwise comparisons of tissue Treg cell populations and lymphoid T cells. Similarities in the epigenetic landscape led to the identification of a common tissue Treg cell population that was present in many organs and was characterized by gain and loss of DNA methylation that included many gene sites associated with the TH2 subset of helper T cells, such as the gene encoding cytokine IL-33 receptor ST2, as well as the production of tissue-regenerative factors. Furthermore, the ST2-expressing population was dependent on the transcriptional regulator BATF and could be expanded by IL-33. Thus, tissue Treg cells integrate multiple waves of epigenetic reprogramming that define their tissue-restricted specialization.

Corrigendum: Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues.

This corrects the article DOI: 10.1038/ni.3799.

Precursors for Nonlymphoid-Tissue Treg Cells Reside in Secondary Lymphoid Organs and Are Programmed by the Transcription Factor BATF.

Specialized regulatory T (Treg) cells accumulate and perform homeostatic and regenerative functions in nonlymphoid tissues. Whether common precursors for nonlymphoid-tissue Treg cells exist and how they differentiate remain elusive. Using transcription factor nuclear factor, interleukin 3 regulated (Nfil3) reporter mice and single-cell RNA-sequencing (scRNA-seq), we identified two precursor stages of interleukin 33 (IL-33) receptor ST2-expressing nonlymphoid tissue Treg cells, which resided in the spleen and lymph nodes. Global chromatin profiling of nonlymphoid tissue Treg cells and the two precursor stages revealed a stepwise acquisition of chromatin accessibility and reprogramming toward the nonlymphoid-tissue Treg cell phenotype. Mechanistically, we identified and validated the transcription factor Batf as the driver of the molecular tissue program in the precursors. Understanding this tissue development program will help to harness regenerative properties of tissue Treg cells for therapy.

Resolving therapy resistance mechanisms in multiple myeloma by multiomics subclone analysis.

Intratumor heterogeneity as a clinical challenge becomes most evident after several treatment lines, when multidrug-resistant subclones accumulate. To address this challenge, the characterization of resistance mechanisms at the subclonal level is key to identify common vulnerabilities. In this study, we integrate whole-genome sequencing, single-cell (sc) transcriptomics (scRNA sequencing), and chromatin accessibility (scATAC sequencing) together with mitochondrial DNA mutations to define subclonal architecture and evolution for longitudinal samples from 15 patients with relapsed or refractory multiple myeloma. We assess transcriptomic and epigenomic changes to resolve the multifactorial nature of therapy resistance and relate it to the parallel occurrence of different mechanisms: (1) preexisting epigenetic profiles of subclones associated with survival advantages, (2) converging phenotypic adaptation of genetically distinct subclones, and (3) subclone-specific interactions of myeloma and bone marrow microenvironment cells. Our study showcases how an integrative multiomics analysis can be applied to track and characterize distinct multidrug-resistant subclones over time for the identification of molecular targets against them.

Distinct human circulating NKp30+FcεRIγ+CD8+ T cell population exhibiting high natural killer-like antitumor potential.

CD8+ T cells are considered prototypical cells of adaptive immunity. Here, we uncovered a distinct CD8+ T cell population expressing the activating natural killer (NK) receptor NKp30 in the peripheral blood of healthy individuals. We revealed that IL-15 could de novo induce NKp30 expression in a population of CD8+ T cells and drive their differentiation toward a broad innate transcriptional landscape. The adaptor FcεRIγ was concomitantly induced and was shown to be crucial to enable NKp30 cell-surface expression and function in CD8+ T cells. FcεRIγ de novo expression required promoter demethylation and was accompanied by acquisition of the signaling molecule Syk and the "innate" transcription factor PLZF. IL-15-induced NKp30+CD8+ T cells exhibited high NK-like antitumor activity in vitro and were able to synergize with T cell receptor signaling. Importantly, this population potently controlled tumor growth in a preclinical xenograft mouse model. Our study, while blurring the borders between innate and adaptive immunity, reveals a unique NKp30+FcεRIγ+CD8+ T cell population with high antitumor therapeutic potential.

Controlling the Covalent Reactivity of a Kinase Inhibitor with Light.

Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acrylamide electrophile. To increase the tissue selectivity of kinase inhibitors, it could be advantageous to control the reactivity of these electrophiles with light. Here, we introduce covalent inhibitors of the kinase JNK3 that function as photoswitchable affinity labels (PALs). Our lead compounds contain a diazocine photoswitch, are poor non-covalent inhibitors in the dark, and become effective covalent inhibitors after irradiation with visible light. Our proposed mode of action is supported by X-ray structures that explain why these compounds are unreactive in the dark and undergo proximity-based covalent attachment following exposure to light.

Influence of structure properties on protein-protein interactions-QSAR modeling of changes in diffusion coefficients.

Information about protein-protein interactions provides valuable knowledge about the phase behavior of protein solutions during the biopharmaceutical production process. Up to date it is possible to capture their overall impact by an experimentally determined potential of mean force. For the description of this potential, the second virial coefficient B22, the diffusion interaction parameter kD, the storage modulus G', or the diffusion coefficient D is applied. In silico methods do not only have the potential to predict these parameters, but also to provide deeper understanding of the molecular origin of the protein-protein interactions by correlating the data to the protein's three-dimensional structure. This methodology furthermore allows a lower sample consumption and less experimental effort. Of all in silico methods, QSAR modeling, which correlates the properties of the molecule's structure with the experimental behavior, seems to be particularly suitable for this purpose. To verify this, the study reported here dealt with the determination of a QSAR model for the diffusion coefficient of proteins. This model consisted of diffusion coefficients for six different model proteins at various pH values and NaCl concentrations. The generated QSAR model showed a good correlation between experimental and predicted data with a coefficient of determination R2 = 0.9 and a good predictability for an external test set with R2 = 0.91. The information about the properties affecting protein-protein interactions present in solution was in agreement with experiment and theory. Furthermore, the model was able to give a more detailed picture of the protein properties influencing the diffusion coefficient and the acting protein-protein interactions. Biotechnol. Bioeng. 2017;114: 821-831. © 2016 Wiley Periodicals, Inc.

Squeeze flow rheometry as a novel tool for the characterization of highly concentrated protein solutions.

This study aims at defining rheological parameters for the characterization of highly concentrated protein solutions. As a basis for comparing rheological behavior with protein solution characteristics the protein phase behavior of Lysozyme from chicken egg white with concentrations up to 225 mg/mL, changing pH values and additive concentrations was studied in a microbatch scale format. The prepared phase diagrams, scored after 40 days (t40) give insights into the kind and kinetics of the phase transitions that occur. Oscillatory frequency sweep measurements of samples with exactly the same conditions were conducted immediately after preparation (t0). The protein solutions behave viscoelastic and show a characteristic curve shape of the storage modulus (G') and the loss modulus (G″). The graphs provide information about the cross-linking degree of the respective sample. The measured rheological parameters were sensitive concerning solution composition, protein concentration and solution inner structure. The rheological moduli G' and G″ and especially the ratio of these parameters over a frequency range from 100 to 40000 rad/sec give information about the aggregation tendency of the protein under tested conditions. We succeeded to correlate protein phase behavior with the defined rheological key parameter ωCO. This point represents the frequency value of the intersection point from G' and G″. In our study Lysozyme expressed a ωCO threshold value of 20000 rad/sec as a lower limit for stable protein solutions. The predictability of lysozyme aggregation tendency and crystallization by means of squeeze flow rheometry is shown.

Detailed assessment of incontinence in boys with fragile-X-syndrome in a home setting.

UNLABELLED: Fragile-X-syndrome (FXS) is caused by a mutation on the X chromosome (Xq27.3). Males with a full mutation have typical dysmorphic signs, moderate intellectual disability and psychological disorders. Twenty-five to fifty percent are affected by incontinence. The aim of the study was to assess subtypes of incontinence and psychological problems in children with FXS in their home environments. Twenty-two boys with FXS (mean age 11.0 years) and 22 healthy controls (mean age 11.1 years) were examined with sonography, uroflowmetry, 48-h bladder diary, physical examination, IQ test, parental psychiatric interview and questionnaires regarding incontinence and psychological symptoms in a home setting. Boys with FXS had higher rates of incontinence than controls (59.1 vs. 4.8 %). The most common subtypes in FXS boys were primary non-monosymptomatic nocturnal enuresis, urge incontinence and nonretentive faecal incontinence. 90.9 % boys with FXS had a psychological comorbidity. Incontinence and behavioural symptoms were not associated. CONCLUSION: Boys with FXS have a higher risk for physical disabilities, psychological disorders and incontinence than healthy boys. Constipation is not a major problem in FXS. As effective treatment is available for children with ID, we recommend offering assessment and therapy to all children with FXS and incontinence. WHAT IS KNOWN: • Boys with fragile-X-syndrome (FXS) have higher rates of incontinence, psychological disorders and somatic conditions than typically developing boys. What is New: • Constipation is a rare condition in FXS in contrast to other genetic syndromes. • Although incontinence rates are higher, urological findings (uroflowmetry, sonography) are not more pathological per se in FXS.

Non-invasive high throughput approach for protein hydrophobicity determination based on surface tension.

The surface hydrophobicity of a protein is an important factor for its interactions in solution and thus the outcome of its production process. Yet most of the methods are not able to evaluate the influence of these hydrophobic interactions under natural conditions. In the present work we have established a high resolution stalagmometric method for surface tension determination on a liquid handling station, which can cope with accuracy as well as high throughput requirements. Surface tensions could be derived with a low sample consumption (800 µL) and a high reproducibility (<0.1‰ for water) within a reasonable time (3.5 min per sample). This method was used as a non-invasive HTP compatible approach to determine surface tensions of protein solutions dependent on protein content. The protein influence on the solutions' surface tension was correlated to the hydrophobicity of lysozyme, human lysozyme, BSA, and α-lactalbumin. Differences in proteins' hydrophobic character depending on pH and species could be resolved. Within this work we have developed a pH dependent hydrophobicity ranking, which was found to be in good agreement with literature. For the studied pH range of 3-9 lysozyme from chicken egg white was identified to be the most hydrophilic. α-lactalbumin at pH 3 exhibited the most pronounced hydrophobic character. The stalagmometric method occurred to outclass the widely used spectrophotometric method with bromophenol blue sodium salt as it gave reasonable results without restrictions on pH and protein species.

Astrocytes regulate myelin clearance through recruitment of microglia during cuprizone-induced demyelination.

Recent evidence suggests that astrocytes play an important role in regulating de- and remyelination in multiple sclerosis. The role of astrocytes is controversial, and both beneficial as well as detrimental effects are being discussed. We performed loss-of-function studies based on astrocyte depletion in a cuprizone-induced rodent model of demyelination. This led to strong astrogliosis accompanied by microgliosis and demyelination in C57BL/6 wild-type mice. Ablation of astrocytes in glial fibrillary acidic protein-thymidine kinase transgenic mice was associated with a failure of damaged myelin removal and a consecutive delay in remyelination. Despite oligodendrocyte death, myelin was still present, but ultrastructual investigations showed that the myelin structure was loosened and this damaged myelin did not protect axons. These alterations were associated with a decrease in microglial activation. Thus, our results show that astrocyte loss does not prevent myelin damage, but clearance of damaged myelin through recruitment of microglia is impaired. Further studies suggest that this process is regulated by the chemokine CXCL10. As a consequence of the delayed removal of myelin debris, remyelination and oligodendrocyte precursor cell proliferation were impaired. Experiments omitting the influence of myelin debris demonstrated an additional beneficial effect of astrocytes on oligodendrocyte regeneration during remyelination. In conclusion, these data demonstrate for the first time in vivo that astrocytes provide the signal environment that forms the basis for the recruitment of microglia to clear myelin debris, a process required for subsequent repair mechanisms. This is of great importance to understanding regenerative processes in demyelinating diseases such as multiple sclerosis.

Comparing parasitological vs serological determination of Schistosoma haematobium infection prevalence in preschool and primary school-aged children: implications for control programmes.

To combat schistosomiasis, the World Health Organization (WHO) recommends that infection levels are determined prior to designing and implementing control programmes, as the treatment regimens depend on the population infection prevalence. However, the sensitivity of the parasitological infection diagnostic method is less reliable when infection levels are low. The aim of this study was to compare levels of Schistosoma haematobium infection obtained by the parasitological method vs serological technique. Infection levels in preschool and primary school-aged children and their implications for control programmes were also investigated. Infection prevalence based on serology was significantly higher compared with that based on parasitology for both age groups. The difference between infection levels obtained using the two methods increased with age. Consequentially, in line with the WHO guidelines, the serological method suggested a more frequent treatment regimen for this population compared with that implied by the parasitological method. These findings highlighted the presence of infection in children aged ⩽5 years, further reiterating the need for their inclusion in control programmes. Furthermore, this study demonstrated the importance of using sensitive diagnostic methods as this has implications on the required intervention controls for the population.

Evolution of SARS-CoV-2 in the Rhine-Neckar/Heidelberg Region 01/2021 - 07/2023.

In January 2021, the monitoring of circulating variants of SARS-CoV-2 was initiated in Germany under the Corona Surveillance Act, which was discontinued after July 2023. This initiative aimed to enhance pandemic containment, as specific amino acid changes, particularly in the spike protein, were associated with increased transmission and reduced vaccine efficacy. Our group conducted whole genome sequencing using the ARTIC protocol (currently V4) on Illumina's NextSeq 500 platform (and, starting in May 2023, on the MiSeq DX platform) for SARS-CoV-2 positive specimen from patients at Heidelberg University Hospital, associated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. In total, we sequenced 26,795 SARS-CoV-2-positive samples between January 2021 and July 2023. Valid sequences, meeting the requirements for upload to the German electronic sequencing data hub (DESH) operated by the Robert Koch Institute (RKI), were determined for 24,852 samples, and the lineage/clade could be identified for 25,912 samples. The year 2021 witnessed significant dynamics in the circulating variants in the Rhine-Neckar/Heidelberg region, including A.27.RN, followed by the emergence of B.1.1.7 (Alpha), subsequently displaced by B.1.617.2 (Delta), and the initial occurrences of B.1.1.529 (Omicron). By January 2022, B.1.1.529 had superseded B.1.617.2, dominating with over 90%. The years 2022 and 2023 were then characterized by the dominance of B.1.1.529 and its sublineages, particularly BA.5 and BA.2, and more recently, the emergence of recombinant variants like XBB.1.5. Since the global dominance of B.1.617.2, the identified variant distribution in our local study, apart from a time delay in the spread of new variants, can be considered largely representative of the global distribution. om a time delay in the spread of new variants, can be considered largely representative of the global distribution.

Compartments in medulloblastoma with extensive nodularity are connected through differentiation along the granular precursor lineage.

Medulloblastomas with extensive nodularity are cerebellar tumors characterized by two distinct compartments and variable disease progression. The mechanisms governing the balance between proliferation and differentiation in MBEN remain poorly understood. Here, we employ a multi-modal single cell transcriptome analysis to dissect this process. In the internodular compartment, we identify proliferating cerebellar granular neuronal precursor-like malignant cells, along with stromal, vascular, and immune cells. In contrast, the nodular compartment comprises postmitotic, neuronally differentiated malignant cells. Both compartments are connected through an intermediate cell stage resembling actively migrating CGNPs. Notably, we also discover astrocytic-like malignant cells, found in proximity to migrating and differentiated cells at the transition zone between the two compartments. Our study sheds light on the spatial tissue organization and its link to the developmental trajectory, resulting in a more benign tumor phenotype. This integrative approach holds promise to explore intercompartmental interactions in other cancers with varying histology.

Resolving the spatial architecture of myeloma and its microenvironment at the single-cell level.

In multiple myeloma spatial differences in the subclonal architecture, molecular signatures and composition of the microenvironment remain poorly characterized. To address this shortcoming, we perform multi-region sequencing on paired random bone marrow and focal lesion samples from 17 newly diagnosed patients. Using single-cell RNA- and ATAC-seq we find a median of 6 tumor subclones per patient and unique subclones in focal lesions. Genetically identical subclones display different levels of spatial transcriptional plasticity, including nearly identical profiles and pronounced heterogeneity at different sites, which can include differential expression of immunotherapy targets, such as CD20 and CD38. Macrophages are significantly depleted in the microenvironment of focal lesions. We observe proportional changes in the T-cell repertoire but no site-specific expansion of T-cell clones in intramedullary lesions. In conclusion, our results demonstrate the relevance of considering spatial heterogeneity in multiple myeloma with potential implications for models of cell-cell interactions and disease progression.

Hydrophobic Modification of Biopolymer Aerogels by Cold Plasma Coating.

The aim of this work was to evaluate the potential of cold plasma polymerization as a simple, fast and versatile technique for deposition of protective hydrophobic and oleophobic polymer layers on hydrophilic biopolymer aerogels. Polymerization of different fluorinated monomers (octafluorocyclobutane C4F8 and perfluoro-acrylates PFAC-6 and PFAC-8) on aerogel monoliths derived from alginate, cellulose, whey protein isolate (WPI) and potato protein isolate (PPI) resulted in fast and significant surface hydrophobization after short process times of 5 min and led to superhydrophobic surfaces with static water contact angles up to 154° after application of poly-C4F8 coatings. Simultaneous introduction of hydro- and oleophobicity was possible by deposition of perfluoro-acrylates. While the porous structure of aerogels stayed intact during the process, polymerization inside the aerogels pores led to the generation of new porous moieties and resulted therefore in significant increase in the specific surface area. The magnitude of the effect depended on the individual process settings and on the overall porosity of the substrates. A maximization of specific surface area increase (+179 m2/g) was obtained by applying a pulsed wave mode in the C4F8-coating of alginate aerogels.

High Na+ Environments Impair Phagocyte Oxidase-Dependent Antibacterial Activity of Neutrophils.

Infection and inflammation can augment local Na+ abundance. These increases in local Na+ levels boost proinflammatory and antimicrobial macrophage activity and can favor polarization of T cells towards a proinflammatory Th17 phenotype. Although neutrophils play an important role in fighting intruding invaders, the impact of increased Na+ on the antimicrobial activity of neutrophils remains elusive. Here we show that, in neutrophils, increases in Na+ (high salt, HS) impair the ability of human and murine neutrophils to eliminate Escherichia coli and Staphylococcus aureus. High salt caused reduced spontaneous movement, degranulation and impaired production of reactive oxygen species (ROS) while leaving neutrophil viability unchanged. High salt enhanced the activity of the p38 mitogen-activated protein kinase (p38/MAPK) and increased the interleukin (IL)-8 release in a p38/MAPK-dependent manner. Whereas inhibition of p38/MAPK did not result in improved neutrophil defense, pharmacological blockade of the phagocyte oxidase (PHOX) or its genetic ablation mimicked the impaired antimicrobial activity detected under high salt conditions. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) overcame high salt-induced impairment in ROS production and restored antimicrobial activity of neutrophils. Hence, we conclude that high salt-impaired PHOX activity results in diminished antimicrobial activity. Our findings suggest that increases in local Na+ represent an ionic checkpoint that prevents excessive ROS production of neutrophils, which decreases their antimicrobial potential and could potentially curtail ROS-mediated tissue damage.

Subclone-specific microenvironmental impact and drug response in refractory multiple myeloma revealed by single-cell transcriptomics.

Virtually all patients with multiple myeloma become unresponsive to treatment over time. Relapsed/refractory multiple myeloma (RRMM) is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations and profound changes of the bone marrow microenvironment (BME). However, the molecular mechanisms that drive drug resistance remain elusive. Here, we analyze the heterogeneous tumor cell population and its complex interaction network with the BME of 20 RRMM patients by single cell RNA-sequencing before/after treatment. Subclones with chromosome 1q-gain express a specific transcriptomic signature and frequently expand during treatment. Furthermore, RRMM cells shape an immune suppressive BME by upregulation of inflammatory cytokines and close interaction with the myeloid compartment. It is characterized by the accumulation of PD1+ γδ T-cells and tumor-associated macrophages as well as the depletion of hematopoietic progenitors. Thus, our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming with implications for clinical decision-making.

All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia.

Ecotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML.