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Current screens to assess tumour drug resistance require a large amount of material, normally not available from patients. Here the authors report CombiSeq, a scalable microfluidic workflow to screen hundreds of drug combinations in picoliter-size droplets using transcriptome changes as a readout.

Abstract Background Deregulated transcription is a major driver of diseases such as cancer. Bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are chromatin readers essential for maintaining proper gene transcription by specifically binding acetylated lysine residues. Targeted displacement of BET proteins from chromatin, using BET inhibitors (I-BETs), is a promising therapy, especially for acute myeloid leukemia (AML), and evaluation of resistance mechanisms is necessary to optimize the clinical efficacy of these drugs. Results To uncover mechanisms of intrinsic I-BET resistance, we quantified chromatin binding and displacement for BRD2, BRD3 and BRD4 after dose response treatment with I-BET151, in sensitive and resistant in vitro models of leukemia, and mapped BET proteins/I-BET interactions genome wide using antibody- and compound-affinity capture methods followed by deep sequencing. The genome-wide map of BET proteins sensitivity to I-BET revealed a bimodal pattern of binding flanking transcription start sites (TSSs), in which drug-mediated displacement from chromatin primarily affects BRD4 downstream of the TSS and prolongs the pausing of RNA Pol II. Correlation of BRD4 binding and drug-mediated displacement at RNA Pol II pause sites with gene expression revealed a differential behavior of sensitive and resistant tumor cells to I-BET and identified a BRD4 signature at promoters of sensitive coding and non-coding genes. Conclusions We provide evidence that I-BET-induced shift of Pol II pausing at promoters via displacement of BRD4 is a determinant of intrinsic I-BET sensitivity. This finding may guide pharmacological treatment to enhance the clinical utility of such targeted therapies in AML and potentially other BET proteins-driven diseases.

The formation and development of glacial lakes in mountainous regions is one of the consequences of glacier recession. Such lakes may drain partially or completely when the stability of their dams is disturbed or as a consequence of impacts. We present a case study from the Central Asian mountain range of Tien Shan – a north-oriented tributary of the Adygine Valley, where the retreat of a polythermal glacier surrounded by permafrost has resulted in the formation of several generations of lakes. The aim of this study was to analyse the past development of different types of glacial lakes influenced by the same glacier, to project the site's future development, and to evaluate the outburst susceptibility of individual lakes with an outlook for expected future change. We addressed the problem using a combination of methods, namely bathymetric, geodetic and geophysical on-site surveys, satellite images and digital elevation model analysis, and modelling of glacier development. Based on this case of the glacial lakes being of varied age and type, we demonstrated the significance of glacier ice in lake development. Lake 3, which is in contact with the glacier terminus, has changed rapidly over the last decade, expanding both in area and depth and increasing its volume by more than 13 times (7800 to 106 000 m3). The hydrological connections and routing of glacier meltwater have proved to be an important factor as well, since most lakes in the region are drained by subsurface channels. As the site is at the boundary between continuous and discontinuous permafrost, the subsurface water flow is strongly governed by the distribution of non-frozen zones above, within, or beneath the perennially frozen ground. In the evaluation of lake outburst susceptibility, we have highlighted the importance of field data, which can provide crucial information on lake stability. In our case, an understanding of the hydrological system at the site, and its regime, helped to categorise Lake 2 as having low outburst susceptibility, while Lake 1 and Lake 3 were labelled as lakes with medium outburst susceptibility. Further development of the site will be driven mainly by rising air temperatures and increasingly negative glacier mass balance. All three climate model scenarios predicted a significant glacier areal decrease by 2050, specifically leaving 73.2 % (A1B), 62.3 % (A2), and 55.6 % (B1) of the extent of the glacier in 2012. The glacier retreat will be accompanied by changes in glacier runoff, with the first peak expected around 2020, and the formation of additional lakes.

The aim was to develop an embedded system for educational purposes with functions of emulation of some peripherals which are intended for automotive electronic stability control unit (ESC ECU). Emulators of two key ECU peripherals were developed: one for four wheel speed sensors (both two and three current levels types) and second for electronic parking break (EPB) switch based on LIN bus. As real-time processors the Arduino Micro platform was chosen. The up level information system architecture is based on the web server (Raspberry Pi 3 platform) and web browser client and programming was done with JavaScript language for the client (AngularJS framework) and also for server (Node.js).

In the Czech Republic, there is still a predominance of obsolete apple orchards that were established more than 15 years ago and that are not profitable under contemporary conditions. Typical features of these orchards are low or medium tree densities, freely growing semi-standard trees or hedgerows on semi-dwarf, or sometimes also on vigorous, rootstocks. The farmers are not always in a position to completely renovate them, and therefore they are interested in their topworking. The present paper studied the effectiveness of this measure under the current economic and market conditions of this country, using 3 types of orchards with different spacings and rootstocks and 5 groups of tree densities. Four cultivars were chosen as examples of different starting statuses for the modelling of subsequent development in three time horizons and for the prediction of profitability of this treatment. The profitability is based on an increase in farmer prices for cultivars that are presently recommended for replacement of the older ones according to the recent development of these prices on the fresh market. In the case of topworking for Spartan cv., an economic return of the measure can be expected at the earliest after 8 years of running the treated orchard. The greatest increase in profit can be achieved in orchards on the rootstock M 9. Nevertheless, with the decline of tree numbers in the orchard, or with the increase in tree losses, the general economic effectiveness of topworking notably falls. In the case of trees on the rootstock MM 106, this measure can have an economic effect only if at least 80% of the trees is in a good health state and it is presumed that the orchard will be used for another 10 years at least. A list of recommended cultivars to be used for replacement of the old ones is given.

Background: The posttranslational modification of cellular macromolecules by glycosylation is considered to contribute to disease pathogenesis in autoimmune and inflammatory conditions. In a subgroup of patients with common variable immunodeficiency (CVID), the occurrence of such complications is associated with an expansion of naïve-like CD21 low B cells during a chronic type 1 immune activation. The glycosylation pattern of B cells in CVID patients has not been addressed to date., Objective: The objective of this study was to examine the surface glycome of B cells in patients with CVID and associated immune dysregulation., Methods: We performed surface lectin staining on B cells from peripheral blood and tonsils, both ex vivo and after in vitro stimulation. Additionally, we examined the expression of glycosylation-related genes by RNAseq in naïve-like CD21 low B cells ex vivo , as well as in naïve CD21 pos B cells from healthy controls after in vitro stimulation., Results: Unlike CD21 pos B cells, naïve-like CD21 low B cells from CVID patients and CD21 low B cells from healthy controls exhibited a unique glycosylation pattern with high levels of α2,6 sialic acids and fucose. This hypersialylation and hyperfucosylation were particularly induced by activation with anti-IgM and interferon-γ (IFN-γ). Transcriptome analysis suggested that naïve-like CD21 low B cells possess a comprehensively reorganised glycosylation machinery, with anti-IgM/IFN-γ having the potential to initiate these changes in vitro ., Conclusion: CD21 low B cells are hypersialylated and hyperfucosylated. This may implicate altered lectin-ligand interactions on the cell surface potentially affecting the CD21 low B-cell function. These glycome changes appear to be driven by the prominent type I immune response in complicated CVID patients. A better understanding of how altered glycosylation influences immune cell function could lead to new therapeutic strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Felixberger, Andrieux, Maul-Pavicic, Goldacker, Harder, Gutenberger, Landry, Benes, Jakob, Boerries, Nitschke, Voll, Warnatz and Keller.)

Pancreatic ductal adenocarcinoma (PDAC) often arises from preexisting cystic lesions such as intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN). This study investigated the molecular heterogeneity and mutational landscape of MCN in relation to PDAC, highlighting the significance of KRAS mutations in tumor progression. Utilizing targeted next-generation sequencing on low-grade MCN and invasive PDAC samples, we identified a substantial overlap in mutational profiles, particularly mutations in KRAS, TP53, and FBXW7. Specifically, 69.2% of MCN exhibited somatic mutations, with KRAS mutations being a predominant oncogenic driver. The characterization of mutant versus wildtype KRAS variant allele frequencies (VAF) indicated higher mutation levels in PDAC compared to MCN, suggesting an evolutionary trajectory toward malignancy. Further histological analysis of 12 additional MCN cases revealed significant intratumor heterogeneity, with variant KRAS mutation distributions correlating with distinct cellular morphologies and dysplastic features. Additionally, we explored the potential of liquid biopsies, demonstrating a concordance rate of 71.4% for KRAS mutation detection in circulating tumor DNA (ctDNA) relative to tissue biopsies across cohorts. Our findings underscore the relevance of evaluating KRAS mutations-herein referred to as VAF per microdissected region-as they relate to histopathological markers of dysplasia, contributing to improved stratification of pancreatic lesions and facilitating personalized treatment strategies. In conclusion, this comprehensive analysis of MCN highlights the importance of KRAS as a crucial biomarker for both malignant progression and therapeutic decision-making in pancreatic pathology. Ultimately, our study suggests that characterizing the mutational landscape and histological features of MCN can enhance early detection and intervention strategies for at-risk patients. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

The histological grade is crucial for therapeutic management, and its reliable preoperative detection can significantly influence treatment approach. Lacking established risk factors, this study identifies preoperative predictors of high-grade skull base meningiomas and discusses the implications of non-invasive detection. A multicentric study was conducted on 552 patients with skull base meningiomas who underwent primary surgical resection between 2014 and 2019. Data were gathered from clinical, surgical and pathology records and radiological diagnostics. The predictive factors of higher WHO grade were analysed in univariate analysis and multivariate stepwise selection logistic regression analysis. Histological analysis revealed 511 grade 1 (92.6%) and 41 grade 2 (7.4%) meningiomas. A prognostic model predicting the probability of WHO grade 2 skull base meningioma (AUC 0.79; SE 0.04; 95% Wald Confidence Limits (0.71; 0.86)) based on meningioma diameter, presence of an arachnoid plane and cranial nerve palsy was built. Accurate preoperative detection of WHO grade in skull base meningiomas is essential for effective treatment planning. Our logistic regression model, based on diameter, cranial nerve palsy, and arachnoid plane, is tailored for detecting WHO grade 2 skull base meningiomas, even in outpatient settings., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval: All procedures performed in studies involving human participants were in accordancewith the ethical standards of the Ethical Committee of the University Hospital in Ostrava(reference number 530/2018) and with the 1964 Helsinki Declaration and its lateramendments or comparable ethical standards Consent to participate: General informed consent to the use of the anonymised data for research purposes wasobtained from all individual participants included in the study Consent for publication: The authors affirm that human research participant provided informed consent for publication of the image in Fig. 2., (© 2025. The Author(s).)

To identify the differences between aged and young human hematopoiesis, we performed a direct comparison of aged and young human hematopoietic stem and progenitor cells (HSPCs). Alterations in transcriptome profiles upon aging between humans and mice were then compared. Human specimens consist of CD34+ cells from bone marrow, and mouse specimens of hematopoietic stem cells (HSCs; Lin- Kit+ Sca1+ CD150+). Single-cell transcriptomic studies, functional clustering, and developmental trajectory analyses were performed. A significant increase in multipotent progenitor 2A (MPP2A) cluster is found in the early HSC trajectory in old human subjects. This cluster is enriched in senescence signatures (increased telomere attrition, DNA damage, activation of P53 pathway). In mouse models, the accumulation of an analogous subset was confirmed in the aged LT-HSC population. Elimination of this subset has been shown to rejuvenate hematopoiesis in mice. A significant activation of the P53-P21WAF1/CIP1 pathway was found in the MPP2A population in humans. In contrast, the senescent HSCs in mice are characterized by activation of the p16Ink4a pathway. Aging in the human HSC compartment is mainly caused by the clonal evolution and accumulation of a senescent cell cluster. A population with a similar senescence signature in the aged LT-HSCs was confirmed in the murine aging model. Clearance of this senescent population with senotherapy in humans is feasible and potentially beneficial.

Here, we present Link-Seq, a highly efficient droplet microfluidic method for combined sequencing of antibody-encoding genes and the transcriptome of individual B cells at large scale. The method is based on 3' barcoding of the transcriptome and subsequent single-molecule PCR in droplets, which freely shift the barcode along specific gene regions, such as the antibody heavy- and light-chain genes. Using the immune repertoire of COVID-19 patients and healthy donors as a model system, we obtain up to 91.7% correctly paired immunoglobulin heavy and light chains. Furthermore, we map the V(D)J usage and obtain sensitivities comparable with the current gold-standard 10× Genomics commercial systems while offering full flexibility in experimental setup and significant cost savings. A further unique feature of Link-Seq is the possibility of barcoding multiple target genes in a site-specific manner. Based on the open character of the platform and its conceptual advantages, we expect Link-Seq to become a versatile tool for single-cell analysis, especially for applications requiring additional processing steps that cannot be implemented on commercially available platforms., (© The Author(s) 2025. Published by Oxford University Press on behalf of National Academy of Sciences.)

Background: Regeneration is the replacement of lost or damaged tissue with a functional copy. In axolotls and zebrafish, regeneration involves stem cells produced by de-differentiation. These cells form a growth zone which expresses developmental patterning genes at its apex. This system resembles an embryonic developmental field where cells undergo pattern formation. Some lizards, including geckos, can regenerate their tails, but it is unclear whether they show a "development-like" regeneration pathway., Results: Using the tokay gecko (Gekko gecko) model species, we examined seven stages of tail regeneration, and three stages of embryonic tail bud development, using transcriptomics, single-cell sequencing, and in situ hybridization. We find no apical growth zone in the regenerating tail. The transcriptomes of the regenerating vs. embryonic tails are quite different with respect to developmental patterning genes. Posterior HOXC genes were activated in a temporally collinear sequence in the regenerating tail. The major precursor populations were stromal cells (regenerating tail) vs. pluripotent stem cells (embryonic tail). Segmented skeletal muscles were regenerated with no expression of classical segmentation genes, but with the early activation of satellite cell markers., Conclusions: Our study suggests that tail regeneration in the tokay gecko-unlike tail development-might rely on the activation of resident stem cells, guided by pre-existing positional information., Competing Interests: Declarations. Ethics approval and consent to participate: The tokay gecko (Gekko gecko) is listed as “least concern” in the International Union for Conservation of Nature Red List ( https://www.iucnredlist.org/species/195309/2378260 ). All animals were collected in Yogyakarta, Indonesia, under a license issued by the Ministry of Environment and Forestry, the Republic of Indonesia (permit number SK.83/KSDAE/SET/KSA.2/5/2021 signed on 7 May 2021) and a recommendation letter issued by The Indonesian Institute of Sciences (Lembaga Ilmu Pengetahuan Indonesia/LIPI; Recommendation Number: B-2158 /IV/KS.01.04/3/2021 signed on 19 March 2021). The transportation of samples from Indonesia to the Netherlands was conducted under CITES permit numbers 05717/IV/SATS-LN/2021 and 09160/IV/SATS-LN/2022 issued by the Ministry of Environment and Forestry, the Republic of Indonesia. All experimental and surgical procedures needed for sample collection were done at the Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia, with the approval from The Ethical Committee of the Integrated Laboratory for Research and Testing (Laboratorium Penelitian dan Pengujian Terpadu/LPPT) Universitas Gadjah Mada (Ethical Clearance number: Ref. 00014/04/LPPT/IV/2021 signed on 30 April 2021). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Objectives: Corallium rubrum, the precious red coral, is an octocoral endemic to the western Mediterranean Sea. Like most octocorals, it produces tiny, calcified structures called sclerites. Uniquely, it also produces a completely calcified axial skeleton that is a bright red color. This combination of color and hardness has made the red coral prized for centuries, leading to extensive fishing and trade for use in jewelry. Understanding how it produces this red skeleton is thus a central question in economics, culture, and biology. To gain insights into this process, we sequenced the C. rubrum genome., Data Description: Our C. rubrum genome assembly is 655 megabases (Mb) in size, distributed across 2910 scaffolds with a very low level of unknown nucleotides (0.95%). We used a pipeline based on the MaSuRCA hybrid assembler, combining long PacBio reads and short Illumina reads, followed by several steps to improve the assembly, including scaffolding, merging, and polishing. This represents the third published genome of an octocoral and the first within the order Scleralcyonacea., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Excessive deposition of fibrillar collagen in the interstitial extracellular matrix (ECM) of human lung tissue causes fibrosis, which can ultimately lead to organ failure. Despite our understanding of the molecular mechanisms underlying the disease, no cure for pulmonary fibrosis has yet been found. We screened a drug library and found that dextromethorphan (DXM), a cough expectorant, reduced the amount of excess fibrillar collagen deposited in the ECM in cultured primary human lung fibroblasts, a bleomycin mouse model, and a cultured human precision-cut lung slice model of lung fibrosis. The reduced extracellular fibrillar collagen upon DXM treatment was due to reversible trafficking inhibition of collagen type I (COL1) in the endoplasmic reticulum (ER) in TANGO1- and HSP47-positive structures. Mass spectrometric analysis showed that DXM promoted hyperhydroxylation of proline and lysine residues on various collagens (COL1, COL3, COL4, COL5, COL7, and COL12) and latent transforming growth factor-β-binding protein (LTBP1 and LTBP2) peptides, coinciding with their secretion block. Additionally, proteome profiling of DXM-treated cells showed increased thermal stability of prolyl-hydroxylases P3H2, P3H3, P3H4, P4HA1, and P4HA2, suggesting a change in their activity. Transcriptome analysis of profibrotic stimulated primary human lung fibroblasts and human ex vivo lung slices after DXM treatment showed activation of an antifibrotic program through regulation of multiple pathways, including the MMP-ADAMTS axis, WNT signaling, and fibroblast-to-myofibroblast differentiation. Together, these data obtained from in vitro, in vivo, and ex vivo models of lung fibrogenesis show that DXM has the potential to limit fibrosis through inhibition of COL1 membrane trafficking in the ER.

Purpose: Endoscopically assisted sagittal strip craniotomy with subsequent cranial orthosis is a frequently used surgical approach for non-syndromic sagittal synostosis. Originally, this technique involved a wide sagittal strip craniectomy with bilateral wedge osteotomies. More recent studies suggest omitting wedge osteotomies, achieving similar outcomes. The controversy surrounding wedge osteotomies and our efforts to refine our technique led us to create models and evaluate the mechanical impact of wedge osteotomies., Methods: We conducted a 3D-print study involving preoperative CT scans of non-syndromic scaphocephaly patients undergoing minimally invasive-assisted remodelation (MEAR) surgery. The sagittal strip collected during surgery underwent thickness measurement, along with a 3-point bending test. These results were used to determine printing parameters for accurately replicating the skull model. Model testing simulated gravitational forces during the postoperative course and assessed lateral expansion under various wedge osteotomy conditions., Results: The median sagittal strip thickness was 2.00 mm (range 1.35-3.46 mm) and significantly positively correlated (p = 0.037) with the median force (21.05 N) of the 3-point bending test. Model testing involving 40 models demonstrated that biparietal wedge osteotomies significantly reduced the force required for lateral bone shift, with a trend up to 5-cm-long cuts (p = 0.007). Additional cuts beyond this length or adding the occipital cut did not provide further significant advantage (p = 0.1643; p = 9.6381)., Conclusion: Biparietal wedge osteotomies reduce the force needed for lateral expansion, provide circumstances for accelerated head shape correction, and potentially reduce the duration of cranial orthosis therapy., Competing Interests: Declarations. Ethics approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Ethics Committee of the University Hospital Motol and 2nd Faculty of Medicine, Charles University in Prague (No. EK- 362/24). Competing interests: This project was supported by the Charles University Grant Agency (GAUK No. 32122)., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Allergen-specific immunotherapy (AIT) induces immune tolerance, showing the highest success rate (>95%) for insect venom while a much lower chance for pollen allergy. However, the molecular switches leading to successful durable tolerance restoration remain elusive. The primary outcome of this observational study is the comprehensive immunological cellular characterization during the AIT initiation phase, whereas the secondary outcomes are the serological and Th2-cell-type-specific transcriptomic analyses. Here we apply a multilayer-omics approach to reveal dynamic peripheral immune landscapes during the AIT-initiation phase in venom allergy patients (VAP) versus pollen-allergic and healthy controls. Already at baseline, VAP exhibit altered abundances of several cell types, including classical monocytes (cMono), CD4 + hybrid type 1-type 17 cells (Th1-Th17 or Th1/17) and CD8 + counterparts (Tc1-Tc17 or Tc1/17). At 8-24 h following AIT launch in VAP, we identify a uniform AIT-elicited pulse of late-transitional/IL-10-producing B cells, IL-6 signaling within Th2 cells and non-inflammatory serum-IL-6 levels. Sequential induction of activation and survival protein markers also immediately occur. A disequilibrium between serum IL-6 and cMono in VAP baseline is restored at day seven following AIT launch. Our longitudinal analysis discovers molecular switches during initiation-phase insect-venom AIT that secure long-term outcomes. Trial number: NCT02931955., Competing Interests: Competing interests: Pending patent application on the protection of predictive biomarkers for AIT efficacy (patent applicant: Luxembourg Institute of Health; inventors: F.Q.H. and M.O.; EP Patent Application No. 23192753.4 entitled “EARLY RESPONSE BIOMARKERS FOR ALLERGEN IMMUNOTHERAPY”). The remaining authors of this work declare no competing interests., (© 2024. The Author(s).)

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii (laboratory culture reference and a single individual assembly) and of the closely related P. massiliensis and P. megalops (single individual assembly) to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ~1.47 Gbp for P. dumerilii , of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 105 miRNA loci. We further explore the high genetic variation (~3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.

Human development relies on the correct replication, maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages, and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells, we identify that several patterning signals-including WNT, BMP, and FGF-converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers, but re-emerges in neural progenitors. In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development., (© 2024. The Author(s).)

In January 2021, Germany commenced surveillance of SARS-CoV-2 variants under the Corona Surveillance Act, which ceased in July 2023. The objective was to bolster pandemic control, as specific alterations in amino acids, particularly within the spike protein, were linked to heightened transmission and decreased vaccine effectiveness. Consequently, our team conducted whole genome sequencing using the commercially accessible ARTIC protocol on Illumina's NextSeq500 platform and MiSeq for SARS-CoV-2 positive samples obtained from patients at Heidelberg University Hospital, affiliated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. Throughout the pandemic, we refined the existing ARTIC V4 protocol as well as our bioinformatics pipeline, the details of which are outlined in this report. This report reflects the protocol for the MiSeq analysis, the protocol for the NextSeq500 can be found in our previous publication., (© 2024 The Authors.)

Within the thymus, thymic epithelial cells (TECs) create dedicated microenvironments for T cell development and selection. Considering that TECs are sensitive to distinct pathophysiological conditions, uncovering the molecular elements that coordinate their thymopoietic role has important fundamental and clinical implications. Particularly, medullary thymic epithelial cells (mTECs) play a crucial role in central tolerance. Our previous studies, along with others, suggest that mTECs depend on molecular factors linked to genome-protecting pathways, but the precise mechanisms underlying their function remain unknown. These observations led us to examine the role of Foxo3, as it is expressed in TECs and involved in DNA damage response. Our findings show that mice with TEC-specific deletion of Foxo3 (Foxo3 cKO ) displayed a disrupted mTEC compartment, with a more profound impact on the numbers of CCL21 + and thymic tuft mTEC lo subsets. At the molecular level, Foxo3 controls distinct functional modules in the transcriptome of cTECs and mTECs under normal conditions, which includes the regulation of ribosomal biogenesis and DNA damage response, respectively. These changes in the TEC compartment resulted in a reduced total thymocyte cellularity and specific changes in regulatory T cell and iNKT cell development in the Foxo3 cKO thymus. Lastly, the thymic defects observed in adulthood correlated with mild signs of altered peripheral immunotolerance in aged Foxo3 cKO mice. Moreover, the deficiency in Foxo3 moderately aggravated the autoimmune predisposition observed in Aire-deficient mice. Our findings highlight the importance of Foxo3 in preserving the homeostasis of TECs and in supporting their role in T cell development and tolerance., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest None declared., (Copyright © 2023. Published by Elsevier B.V.)

This study aims at identifying molecular biomarkers differentiating responders and non-responders to treatment with Tumor Necrosis Factor inhibitors (TNFi) among patients with axial spondyloarthritis (axSpA). Whole blood mRNA and plasma proteins were measured in a cohort of biologic-naïve axSpA patients ( n = 35), pre and post (14 weeks) TNFi treatment with adalimumab. Differential expression analysis was used to identify the most enriched pathways and in predictive models to distinguish responses to TNFi. A treatment-associated signature suggests a reduction in inflammatory activity. We found transcripts and proteins robustly differentially expressed between baseline and week 14 in responders. C-reactive protein (CRP) and Haptoglobin (HP) proteins showed strong and early decrease in the plasma of axSpA patients, while a cluster of apolipoproteins (APOD, APOA2, APOA1) showed increased expression at week 14. Responders to TNFi treatment present higher levels of markers of innate immunity at baseline, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP, gender, and AFF3 , the top differentially expressed gene at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC = 0.97). In conclusion, innate and adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axSpA patients. A model including clinical and gene expression variables should also be considered.

In this study, we provide a comprehensive clinical and molecular biological characterization of radiation-induced gliomas (RIG), including a risk assessment for developing gliomas. A cohort of 12 patients who developed RIG 9.5 years (3-31 years) after previous cranial radiotherapy for brain tumors or T-cell acute lymphoblastic leukemia was established. The derived risk of RIG development based on our consecutive cohort of 371 irradiated patients was 1.6% at 10 years and 3.02% at 15 years. Patients with RIG glioma had a dismal prognosis with a median survival of 7.3 months. We described radiology features that might indicate the suspicion of RIG rather than the primary tumor recurrence. Typical molecular features identified by molecular biology examination included the absence of Histon3 mutation, methylation profile of pedHGG-RTK1 and the presence of recurrent PDGFRA amplification and CDKN2A/B deletion. Of the two long-term surviving patients, one had gliomatosis cerebri, and the other had pleomorphic xanthoastrocytoma with BRAF V600E mutation. In summary, our experience highlights the need for tissue diagnostics to allow detailed molecular biological characterization of the tumor, differentiation of the secondary tumor from the recurrence of the primary disease and potentially finding a therapeutic target., (© 2024. The Author(s).)

Bidirectional interactions between the immune system and the gut microbiota are key contributors to various physiological functions. Immune-associated diseases such as cancer and autoimmunity, and efficacy of immunomodulatory therapies, have been linked to microbiome variation. Although COVID-19 infection has been shown to cause microbial dysbiosis, it remains understudied whether the inflammatory response associated with vaccination also impacts the microbiota. Here, we investigate the temporal impact of COVID-19 vaccination on the gut microbiome in healthy and immuno-compromised individuals; the latter included patients with primary immunodeficiency and cancer patients on immunomodulating therapies. We find that the gut microbiome remained remarkably stable post-vaccination irrespective of diverse immune status, vaccine response, and microbial composition spanned by the cohort. The stability is evident at all evaluated levels including diversity, phylum, species, and functional capacity. Our results indicate the resilience of the gut microbiome to host immune changes triggered by COVID-19 vaccination and suggest minimal, if any, impact on microbiome-mediated processes. These findings encourage vaccine acceptance, particularly when contrasted with the significant microbiome shifts observed during COVID-19 infection., (© 2024 Boston et al.)

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Background: Immunotherapeutic targets in multiple myeloma (MM) have variable expression height and are partly expressed in subfractions of patients only. With increasing numbers of available compounds, strategies for appropriate choice of targets (combinations) are warranted. Simultaneously, risk assessment is advisable as patient's life expectancy varies between months and decades., Methods: We first assess feasibility of RNA-sequencing in a multicenter trial (GMMG-MM5, n=604 patients). Next, we use a clinical routine cohort of untreated symptomatic myeloma patients undergoing autologous stem cell transplantation (n=535, median follow-up (FU) 64 months) to perform RNA-sequencing, gene expression profiling (GEP), and iFISH by ten-probe panel on CD138-purified malignant plasma cells. We subsequently compare target expression to plasma cell precursors, MGUS (n=59), asymptomatic (n=142) and relapsed (n=69) myeloma patients, myeloma cell lines (n=26), and between longitudinal samples (MM vs. relapsed MM). Data are validated using the independent MMRF CoMMpass-cohort (n=767, FU 31 months)., Results: RNA-sequencing is feasible in 90.8% of patients (GMMG-MM5). Actionable immune-oncological targets (n=19) can be divided in those expressed in all normal and >99% of MM-patients (CD38, SLAMF7, BCMA, GPRC5D, FCRH5, TACI, CD74, CD44, CD37, CD79B), those with expression loss in subfractions of MM-patients (BAFF-R [81.3%], CD19 [57.9%], CD20 [82.8%], CD22 [28.4%]), aberrantly expressed in MM (NY-ESO1/2 [12%], MUC1 [12.7%], CD30 [4.9%], mutated BRAF V600E/K [2.1%]), and resistance-conveying target-mutations e.g., against part but not all BCMA-directed treatments. Risk is assessable regarding proliferation, translated GEP- (UAMS70-, SKY92-, RS-score) and de novo (LfM-HRS) defined risk scores. LfM-HRS delineates three groups of 40%, 38%, and 22% of patients with 5-year and 12-year survival rates of 84% (49%), 67% (18%), and 32% (0%). R-ISS and RNA-sequencing identify partially overlapping patient populations, with R-ISS missing, e.g., 30% (22/72) of highly proliferative myeloma., Conclusion: RNA-sequencing based assessment of risk and targets for first choice treatment is possible in clinical routine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Emde-Rajaratnam, Beck, Benes, Salwender, Bertsch, Scheid, Hänel, Weisel, Hielscher, Raab, Goldschmidt, Jauch, Maes, De Bruyne, Menu, De Veirman, Moreaux, Vanderkerken, Seckinger and Hose.)

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning., (© 2023. The Author(s).)

Despite the importance of functional outcome, only a few scoring systems exist to predict neurologic outcome in meningioma surgery. Therefore, our study aims to identify preoperative risk factors and develop the receiver operating characteristics (ROC) models estimating the risk of a new postoperative neurologic deficit and a decrease in Karnofsky performance status (KPS). A multicentric study was conducted in a cohort of 552 consecutive patients with skull base meningiomas who underwent surgical resection from 2014 to 2019. Data were gathered from clinical, surgical, and pathology records as well as radiological diagnostics. The preoperative predictive factors of functional outcome (neurologic deficit, decrease in KPS) were analyzed in univariate and multivariate stepwise selection analyses. Permanent neurologic deficits were present in 73 (13.2%) patients and a postoperative decrease in KPS in 84 (15.2%). Surgery-related mortality was 1.3%. A ROC model was developed to estimate the probability of a new neurologic deficit (area 0.74; SE 0.0284; 95% Wald confidence limits (0.69; 0.80)) based on meningioma location and diameter. Consequently, a ROC model was developed to predict the probability of a postoperative decrease in KPS (area 0.80; SE 0.0289; 95% Wald confidence limits (0.74; 0.85)) based on the patient's age, meningioma location, diameter, presence of hyperostosis, and dural tail. To ensure an evidence-based therapeutic approach, treatment should be founded on known risk factors, scoring systems, and predictive models. We propose ROC models predicting the functional outcome of skull base meningioma resection based on the age of the patient, meningioma size, and location and the presence of hyperostosis and dural tail., (© 2023. The Author(s).)

Inter-patient variability and the similarity of healthy and leukemic stem cells (LSCs) have impeded the characterization of LSCs in acute myeloid leukemia (AML) and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. Although residual healthy and preleukemic cells dominated the dormant stem cell compartment, active LSCs resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors constituted a highly aberrant, disease-defining compartment: their gene expression and differentiation state affected both the chemotherapy response and leukemia's ability to differentiate into transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers misregulated specifically in leukemic cells. Taken together, CloneTracer reveals a differentiation landscape that mimics its healthy counterpart and may determine biology and therapy response in AML., Competing Interests: Declaration of interests The Department of Medicine V (Director C.M.-T.) receives research funding from multiple pharmaceutical and biotech companies especially for clinical trials but also for translational research., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pterostilbene (PTS) is a drug candidate with low water solubility and poor bioavailability. On the other hand, drug:cyclodextrins complexes frequently provide bulk powders with low drug concentrations, which is crucial for obtention solid or semi-solid pharmaceutical dosage forms. In order to determine the optimal conditions for enhancing the solubility of PTS:BCD (β-cyclodextrin) complex, a Box-Behnken design was performed. Although the optimal conditions have been applied, low complexation efficiency (0.127) and the bulk powder remained. A PTS:BCD:HPMC (HPMC, hydroxypropyl methylcellulose) ternary system was developed to overcome this limitation, comparing two media, water and a mixture of ethanol-water. When ethanol was used as a co-solvent, the PTS:BCD:HPMC ternary system (freeze-dried) contained 116.65 ± 1.40 mg/g of PTS. This value was 3.4-fold higher than the PTS content observed when the same ternary system was obtained in aqueous media (34.8 mg/g) and 2.8-fold higher than the PTS content observed for PTS:BCD complex (freeze-dried) obtained using ethanol as a co-solvent. Dissolution tests revealed that after 120 min, in a buffer with a pH value of 1.2, only 43% of PTS dissolved. In contrast, 80% and 90% of PTS were dissolved from the PTS:BCD complex and PTS:BCD:HPMC ternary system, respectively. Moreover, the dissolution was fast in a buffer with a pH value of 6.8. PTS:BCD complex reached the maximum PTS dissolution at 75 min and PTS:BCD:HPMC at 45 min. In summary, the results of this study demonstrated, for the first time, that low-bulk powders with a high content of PTS can be obtained from PTS:BCD:HPMC ternary systems using ethanol as a co-solvent. This new finding offers a valuable alternative for producing solid or semi-solid formulations containing highly soluble PTS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Purpose: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute., Methods: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest., Results: VA development was connected to the lack of intestinal (IgA + ) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme + CD8 + T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA + ) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples., Conclusions: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination., (© 2022. The Author(s).)

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the flippase (FLP) and cyclization recombination (Cre) enzymes has proved particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes, and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes., Competing Interests: Conflicts of interest: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Background: The utility for liquid biopsy of tumor-associated circulating single-nucleotide variants, as opposed to mutations, of the mitochondrial (mt) and nuclear genomes in neuroblastoma (NB) is unknown., Procedure: Variants of the mt and nuclear genomes from tumor, blood cells, and consecutive plasma samples of five patients with metastatic NB that relapsed or progressed were analyzed. Targeted parallel sequencing results of the mt genome, and of the coding region of 139 nuclear genes and 22 miRNAs implicated in NB, were correlated with clinical imaging and laboratory data., Results: All tumors harbored multiple somatic mt and nuclear single nucleotide variants with low allelic frequency, most of them not detected in the circulation. In one patient a tumor-associated mt somatic variant was detected in the plasma before and during progressive disease. In a second patient a circulating nuclear tumor-associated DNA variant heralded clinical relapse. In all patients somatic mt and nuclear variants not evident in the tumor biopsy at time of diagnosis were found circulating at varying timepoints. This suggests either tumor heterogeneity, evolution of tumor variants or a confounding contribution of normal tissues to somatic variants in patient plasma. The number and allelic frequency of the circulating variants did not reflect the clinical course of the tumors. Mutational signatures of mt and nuclear somatic variants differed. They varied between patients and were detected in the circulation without mirroring the patients' course., Conclusions: In this limited cohort of NB patients clinically informative tumor-associated mt and nuclear circulating variants were detected by targeted parallel sequencing in a minority of patients., (© 2022 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon-responsive genes in primary mouse ( Mus musculus ) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis., Competing Interests: AC, DP, EK, NP, HC, RG, AB, KW, TV, CM, IS, PP, SC, FM, JF, Sd, VB, JA, SW, MS, AG, JN, MS, AM, AN, LM No competing interests declared, (© 2022, Chora, Pedroso et al.)

The purpose of the Maleth Program, also known as Project Maleth, is Malta's first space program to evaluate human skin tissue microbiome changes in type 2 diabetes mellitus (T2DM) patients afflicted with diabetic foot ulcers (DFU). This was carried out in both ground-based models and spaceflight. The first mission (Maleth I) under this program was carried out to uncover the effects of spaceflight, microgravity and radiation on human skin tissue microbiome samples from six T2DM patients recruited into the study. Each patient human skin tissue sample was split in three, with one section processed immediately for genomic profiling by 16S typing and the rest were processed for longer term ground-control and spaceflight experiments. Ground-control and spaceflight human skin tissue samples were also processed for genomic profiling upon mission re-entry and completion. Maleth I's overall objective was achieved, as human skin tissue samples with their microbiomes travelled to space and back yielding positive results by both standard microbiology techniques and genetic typing using 16S rRNA amplicon sequencing. Preliminary findings of this mission are discussed in light of its innovative approach at DFU microbiome research, and the clinical implications that may emerge from this and other future similar studies., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Background and Purpose: DSA (digital subtraction angiography) is the gold standard for measuring carotid artery stenosis (CS). Yet, the correlation between DSA and stenosis is not well documented., Material and Methods: We compared CS as measured by DSA to carotid artery specimens obtained from carotid endarterectomy surgery. Patients were divided into three groups according to NASCET criteria (North American Symptomatic Carotid Endarterectomy Trial): stenosis of 30-49% (mild), stenosis of 50-69% (moderate), and stenosis of 70-99% (severe)., Results: This prospective cohort study involved 644 patients. The mean stenosis in the mild stenosis group (n = 128 patients) was 54% ECST (European Carotid Surgery Trial), 40% NASCET, and 72% ESs (endarterectomy specimens). The mean absolute difference between ECST and NASCET was 14%. The mean stenosis in the moderate stenosis group (n = 347 patients) was 66% ECST, 60% NASCET, and 77% ES. The mean absolute difference between ECST and NASCET was 6%. The mean stenosis in the severe group (n = 169 patients) was 80% ECST, 76% NASCET, and 79% ES. No significant correlation coefficients were found between DSA and ES methods. In the mild group, the CC was 0.16 (ESCT) and 0.13 (NASCET); in the moderate group, the CC was 0.05 (ESCT) and 0.01 (NASCET); and in the severe group, the CC was 0.23 (ESCT) and 0.10 (NASCET). For all groups combined, CC was 0.22 for the ECST and 0.20 for the NASCET method., Conclusion: The relationship between DSA and ES methods to measure CS is almost random. This lack of a relationship between the DSA and ES techniques questions the validity of current DSA-based guidelines., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Nanoscale zero-valent iron (nZVI) has increasingly been applied to remediate aquifers polluted by organochlorines or heavy metals. As a result, bacteria in the vicinity of remediate action can be stressed by surplus iron released from nZVI. However, the understanding of the iron stress defense pathways during this process is currently incomplete. Therefore, we aimed to elucidate the physiological and transcriptomic response of the bacterium, Pseudomonas putida NCTC 10936, to 100 mg/L of nZVI and 44.5 µg/L of dissolved iron obtained from nZVI suspension. Cell viability was neither affected by nZVI nor dissolved iron, although the dissolved iron caused stress that altered the cell physiology and caused the generation of smaller cells, whereas cells were elongated in the presence of nZVI. Transcriptomic analysis confirmed the observed stronger physiological effect caused by dissolved iron (in total 3839 differentially expressed genes [DEGs]) than by nZVI (945 DEGs). Dissolved iron (but not nZVI) activated genes involved in oxidative stress-related pathways, antioxidant activity, carbohydrate and energy metabolism, but downregulated genes associated with flagellar assembly proteins and two-component systems involved in sensing external stimuli. As a result, bacteria very effectively faced oxidative insults and cell viability was not affected., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)