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Feature-based molecular networking (FBMN) is a popular analysis approach for liquid chromatography-tandem mass spectrometry-based non-targeted metabolomics data. While processing liquid chromatography-tandem mass spectrometry data through FBMN is fairly streamlined, downstream data handling and statistical interrogation are often a key bottleneck. Especially users new to statistical analysis struggle to effectively handle and analyze complex data matrices. Here we provide a comprehensive guide for the statistical analysis of FBMN results, focusing on the downstream analysis of the FBMN output table. We explain the data structure and principles of data cleanup and normalization, as well as uni- and multivariate statistical analysis of FBMN results. We provide explanations and code in two scripting languages (R and Python) as well as the QIIME2 framework for all protocol steps, from data clean-up to statistical analysis. All code is shared in the form of Jupyter Notebooks ( https://github.com/Functional-Metabolomics-Lab/FBMN-STATS ). Additionally, the protocol is accompanied by a web application with a graphical user interface ( https://fbmn-statsguide.gnps2.org/ ) to lower the barrier of entry for new users and for educational purposes. Finally, we also show users how to integrate their statistical results into the molecular network using the Cytoscape visualization tool. Throughout the protocol, we use a previously published environmental metabolomics dataset for demonstration purposes. Together, the protocol, code and web application provide a complete guide and toolbox for FBMN data integration, cleanup and advanced statistical analysis, enabling new users to uncover molecular insights from their non-targeted metabolomics data. Our protocol is tailored for the seamless analysis of FBMN results from Global Natural Products Social Molecular Networking and can be easily adapted to other mass spectrometry feature detection, annotation and networking tools., (© 2024. Springer Nature Limited.)

Objectives: The pathophysiology of multiple sclerosis (MS) involves inflammatory neurodegeneration in the brainstem, cerebellum, and retina. The clinical relevance of oculomotor involvement in MS, however, remains uncertain., Methods: In this cross-sectional study, we evaluated heterophoria as a (sub)clinical tool in 54 MS patients and 55 age-matched healthy controls (HCs). We quantified heterophoria in prism diopters for distance and near range with orthoptic examination. Our primary outcome was high degrees of horizontal heterophoria (HDHH) defined as measurements beyond ±2 standard deviations from the mean prism diopter of heterophoria of our HCs., Results: More than one-third (37%, n=20/54) of MS patients but only 11% (n=6/55) of HCs were classified as HDHH [distance, MS=9% (n=5/54) versus HC=6% (n=3/55); near, MS=19% (n=10/54) versus HC=5% (n=3/55)]. Our MS patients presented more combined vertical and horizontal deviations at near range [MS 19% (n=10/54) versus for HC 7% (n=4/55)]. We observed the combination of HDHH both at distance and at near testing in 9% (n=5/54) of MS patients but not at all in HCs (n=0/55)., Discussion: Despite the high prevalence of heterophoria, HDHH may be an additional (sub)clinical tool of subclinical involvement in MS. Thus, orthoptic examination may be an additional tool to improve MS diagnostic procedures., Competing Interests: JG has received within the past 3 years a Research Fellowship from the German Research Foundation DFG, project number 438899010. SM received honoraria for lecturing and travel expenses for attending meetings from Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, Celgene, Diamed, Genzyme, MedDay Pharmaceuticals, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis, Chugai Pharma, QuintilesIMS, and Teva. His research funded by the German Ministry for Education and Research BMBF, German Research Foundation DFG, Else Kröner Fresenius Foundation, German Academic Exchange Service, Hertie Foundation, Interdisciplinary Center for Clinical Studies IZKF Muenster, German Foundation Neurology, and by Almirall, Amicus Therapeutics Germany, Biogen, Diamed, Fresenius Medical Care, Genzyme, Merck Serono, Novartis, ONO Pharma, Roche, and Teva. OA received, with approval of the Rector of Heinrich-Heine University, grants from the German Research Foundation DFG, the German Ministry for Education and Research BMBF as part of the German Competence Network Multiple Sclerosis KKNMS; for NEMOS NationNMO-PAT FKZ 01GI1602B, the Eugène Devic European Network EU-FP7, honoraria and travel/accommodation/meeting expenses from Alexion, Almirall, Biogen, Horizon, Janssen Cilag, Merck Serono, Novartis, Roche, and Sanofi-Genzyme. OA is founding member and co-coordinator of the German Neuromyelitis optica-Study Group NEMOS, www.nemos-net.de and member of the European Reference Network for Rare Eye Diseases ERN-EYE, co-funded by the Health Program of the European Union under the Framework Partnership Agreement No 739534 “ERN-EYE.” PA received, with approval of the Rector of Heinrich-Heine University and the CEO of University of Düsseldorf Hospital, personal fees, research grants, and/or non-financial support from Allergan, Biogen, Bristol Myers Squibb, Celgene, German Research Foundation DFG, EFRE-NRW, Janssen Cilag, Ipsen, Merck Serono, Merz Pharmaceuticals, Novartis, and Roche, and personal fees and non-financial support from Bayer Healthcare, Teva, and Sanofi-Aventis/Genzyme, outside the submitted work. The remaining 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 © 2024 Graf, Weise, Guthoff, Balloff, Gasis, Link, Küchlin, Lagrèze, Meuth, Aktas and Albrecht.)

The incidence of antibiotic-resistant bacterial infections is increasing, and development of new antibiotics has been deprioritised by the pharmaceutical industry. Interdisciplinary research approaches, based on the ecological principles of bacterial fitness, competition, and transmission, could open new avenues to combat antibiotic-resistant infections. Many facultative bacterial pathogens use human mucosal surfaces as their major reservoirs and induce infectious diseases to aid their lateral transmission to new host organisms under some pathological states of the microbiome and host. Beneficial bacterial commensals can outcompete specific pathogens, thereby lowering the capacity of the pathogens to spread and cause serious infections. Despite the clinical relevance, however, the understanding of commensal-pathogen interactions in their natural habitats remains poor. In this Personal View, we highlight directions to intensify research on the interactions between bacterial pathogens and commensals in the context of human microbiomes and host biology that can lead to the development of innovative and sustainable ways of preventing and treating infectious diseases., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes including osmolyte uptake, cell wall homeostasis, as well as antibiotic and heat resistance. This study investigates the role of the c-di-AMP receptor protein DarA in the osmotic stress response in Bacillus subtilis . Through a series of experiments, we demonstrate that DarA plays a central role in the cellular response to osmotic fluctuations. Our findings show that DarA becomes essential under extreme potassium limitation as well as upon salt stress, highlighting its significance in mediating osmotic stress adaptation. Suppressor screens with darA mutants reveal compensatory mechanisms involving the accumulation of osmoprotectants, particularly potassium and citrulline. Mutations affecting various metabolic pathways, including the citric acid cycle as well as glutamate and arginine biosynthesis, indicate a complex interplay between the osmotic stress response and metabolic regulation. In addition, the growth defects of the darA mutant during potassium starvation and salt stress in a strain lacking the high-affinity potassium uptake systems KimA and KtrAB can be rescued by increased affinity of the remaining potassium channel KtrCD or by increased expression of ktrD, thus resulting in increased potassium uptake. Finally, the darA mutant can respond to salt stress by the increased expression of MleN , which can export sodium ions.IMPORTANCEEnvironmental bacteria are exposed to rapidly changing osmotic conditions making an effective adaptation to these changes crucial for the survival of the cells. In Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in this adaptation by controlling (i) the influx of physiologically compatible organic osmolytes and (ii) the biosynthesis of such osmolytes. In several bacteria, cyclic di-adenosine monophosphate (c-di-AMP) can bind to a signal transduction protein, called DarA, in Bacillus subtilis . So far, no function for DarA has been discovered in any organism. We have identified osmotically challenging conditions that make DarA essential and have identified suppressor mutations that help the bacteria to adapt to those conditions. Our results indicate that DarA is a central component in the integration of osmotic stress with the synthesis of compatible amino acid osmolytes and with the homeostasis of potassium, the first response to osmotic stress., Competing Interests: The authors declare no conflict of interest.

High-grade endometrial stromal sarcoma is a rare and aggressive soft tissue tumor characterized by YWHAE::NUTM2A/B translocations, diagnosis at a median of 50-60 years, and a poor prognosis (overall survival 30%-40%). We describe a 16-year-old patient with high-grade endometrial stromal sarcoma and regional nodal and pulmonary metastases who is a long-term survivor after grossly complete tumor resection, intensive chemotherapy, and pelvic radiotherapy. We discovered a previously undescribed YWHAE::NUTM2E translocation in the tumor. Our patient's favorable outcome suggests that intensive multimodality therapy with curative intent is appropriate for young patients with high-grade endometrial stromal sarcoma and highlights the importance of fertility preservation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Significant improvements in treatments for children with cancer have resulted in a growing population of childhood cancer survivors who may face long-term adverse outcomes. Here, we aimed to diagnose high-dose methotrexate-induced brain injury on [ 18 F]FDG PET/MRI and correlate the results with cognitive impairment identified by neurocognitive testing in pediatric cancer survivors. Methods: In this prospective, single-center pilot study, 10 children and young adults with sarcoma ( n = 5), lymphoma ( n = 4), or leukemia ( n = 1) underwent dedicated brain [ 18 F]FDG PET/MRI and a 2-h expert neuropsychologic evaluation on the same day, including the Wechsler Abbreviated Scale of Intelligence, second edition, for intellectual functioning; Delis-Kaplan Executive Function System (DKEFS) for executive functioning; and Wide Range Assessment of Memory and Learning, second edition (WRAML), for verbal and visual memory. Using PMOD software, we measured the SUV mean , cortical thickness, mean cerebral blood flow (CBF mean ), and mean apparent diffusion coefficient of 3 different cortical regions (prefrontal cortex, cingulate gyrus, and hippocampus) that are routinely involved during the above-specified neurocognitive testing. Standardized scores of different measures were converted to z scores. Pairs of multivariable regression models (one for z scores < 0 and one for z scores > 0) were fitted for each brain region, imaging measure, and test score. Heteroscedasticity regression models were used to account for heterogeneity in variances between brain regions and to adjust for clustering within patients. Results: The regression analysis showed a significant correlation between the SUV mean of the prefrontal cortex and cingulum and DKEFS-sequential tracking (DKEFS-TM4) z scores ( P = 0.003 and P = 0.012, respectively). The SUV mean of the hippocampus did not correlate with DKEFS-TM4 z scores ( P = 0.111). The SUV mean for any evaluated brain regions did not correlate significantly with WRAML-visual memory (WRAML-VIS) z scores. CBF mean showed a positive correlation with SUV mean ( r = 0.56, P = 0.01). The CBF mean of the cingulum, hippocampus, and prefrontal cortex correlated significantly with DKEFS-TM4 (all P < 0.001). In addition, the hippocampal CBF mean correlated significantly with negative WRAML-VIS z scores ( P = 0.003). Conclusion: High-dose methotrexate-induced brain injury can manifest as a reduction in glucose metabolism and blood flow in specific brain areas, which can be detected with [ 18 F]FDG PET/MRI. The SUV mean and CBF mean of the prefrontal cortex and cingulum can serve as quantitative measures for detecting executive functioning problems. Hippocampal CBF mean could also be useful for monitoring memory problems., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO 2 . Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for Escherichia coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis ( pgi ), central-carbon regulation ( crp ), and RNA transcription ( rpoB ). The pgi mutation reduces the enzyme's activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module ( rpiB ) and the energy module ( fdoGH ), as well as an increased ratio of NADH/NAD + - the cycle's electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs., Competing Interests: RB, SG, EN, RM We declare the following provisional patent related to the manuscript, "An Engineered Autotrophic E. coli Strain for CO2 Conversion to Organic Materials."European Patent Office: EP4051695A1, EM, VP, Bd, GJ, DL, HY, NN, DE, HL No competing interests declared, (© 2023, Ben Nissan, Milshtein et al.)

In this issue of Cell Reports Methods, Rachwalski et al. describe a high-throughput method to screen genetic interactions in bacteria using a conjugative CRISPR interference (CRISPRi) plasmid. The method enables systematic studies of gene essentiality in diverse genomic and environmental contexts and is applicable to Escherichia coli as well as other bacteria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Anemia in cancer should be diagnosed and treated according to guideline recommendations. The implementation of ESMO and German guidelines and their effect on anemia correction was analyzed., Methods: This retrospective epidemiological study, representative for Germany, analyzed data on anemia management of cancer patients with anemia ≥ grade 2. The Guideline Adherence Score (GLAD) for diagnosis (GLAD-D) and therapy (GLAD-T) was defined as follows: 2 points for complete, 1 point for partial, 0 point for no adherence., Results: Data were analyzed for 1046 patients. Hb levels at diagnosis of anemia were 8-10 g/dL in 899 (85.9%) patients, 7-8 g/dL in 92 (8.7%), and < 7 g/dL (5.0%) in 52. Transferrin saturation was determined in 19% of patients. Four hundred fifty-six patients received RBC (43.6%), 198 (18.9%) iron replacement, 106 (10.1%) ESA, and 60 (5.7%) vitamin B12 replacement. 60.6% of patients receiving iron replacement were treated intravenously and 39.4% were treated orally. Two hundred eighty-eight (36.6%) of 785 patients receiving transfusions had no guideline-directed indication. GLAD-D was 2 in 310 patients (29.6%), 1 in 168 (16.1%), and 0 in 568 (54.3%). GLAD-T was 2 in 270 patients (25.8%), 1 in 320 patients (30.6%), and 0 in 456 patients (43.6%). Higher GLAD-D significantly correlated with higher GLAD-T (τB = 0.176, p < 0.001). GLAD-T 2 was significantly associated with greater Hb increase than GLAD-T 0/1 (p < 0.001) at 28 days (10.2 vs. 9.7 g/dL) and at 2 months (10.4 vs. 9.9 g/dL)., Conclusions: Anemia assessment is inadequate, transfusion rates too high, and iron and ESA therapy too infrequent., Trial Registration: ClinicalTrials.gov, NCT05190263, date: 2022-01-13., (© 2024. The Author(s).)