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Agroecological transition requires that innovative and diversified cropping systems be developed. Conducting system experiments is an approach well-suited to the analysis of performance of cropping systems when subjected to soil, weather and biotic stresses. Conducting system experiments nevertheless gives rise to methodological challenges. Using the Syppre network of experiments, consisting of five sites in France, we present an original case study that provides valuable methodological and agronomic lessons on system experiments. The innovative cropping systems tested there are based on crop diversification (including oilseeds and protein crops), as well as flexible tillage, technical innovations and optimized crop management. From a methodological standpoint, we show that (i) mixed models are adapted to a range of experimental questions and constraints; (ii) multifactorial analysis enables the characterization of relationships between performance indicators; (iii) a multisite experimental network is an efficient approach not only for answering agronomic questions, but also for addressing methodological issues. From an agronomic standpoint, we showed that reconciling multiple indicators of performance is still challenging. Overall, innovative and diversified systems improved the performance of input utilization and environmental impacts, but with lower productivity and profitability. Introducing legume crops is a promising strategy because this contributes significantly to reductions in mineral N fertilizer use, energy consumption and greenhouse gas emissions, without major trade-offs against other performance indicators. Finally, we showed that the nature of the production situation had a major influence on the performance profile. This led us to be cautious in making overall analyses especially with regard to general conclusions.

Many scientific studies often assumed that the most reliable methods for assessing sperm motility are those that give the highest values, and this leads to misinterpretation of the results. This study aims to propose an objective method to validate sperm motility reliability. Bovine and porcine semen samples were split into two equal fractions. Fraction A was kept alive with a motile population considered at maximum proportion, while fraction B was killed with 0% motile population. A range of motile/non motile sperm was performed by mixing both fractions. The Motility Ratio method, comparing measured and theoretical motility, was validated using LEJA slide and IVOS II and applied to other slides. All slides showed strong Concordance Correlation Coefficient between measured and theoretical motility. However, with IVOS II, LEJA slide showed the lowest bias (< 1) while MAKLER or coverslip showed higher bias (> 2 and > 7 respectively) between measured and theoretical motility. This study shows that the best sperm motility is not always the true motility and highlights the importance of implementing a gold standard methodology for motility reliability such as The Motility Ratio method., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background and Aims: Breast cancer is the most common malignancy observed in patients with inflammatory bowel diseases (IBD). The aim of our study was to evaluate incident cancer rate (recurrence or new-onset cancer) in a cohort of IBD patients with a history of breast cancer according to the subsequent IBD treatment provided., Methods: A multicenter retrospective study included consecutive IBD patients with prior breast cancer. The inclusion date corresponded to the diagnosis of index malignancy. Follow-up lasted from cancer diagnosis until the occurrence of incident cancer., Results: Among 207 patients included (median disease duration: 13 years [IQR 6 - 21]), first line treatment (median interval of 28 months [IQR 7 - 64]) was a conventional immunosuppressant in 19.3 % of patients, anti-TNF in 19.8 %, vedolizumab in 7.2 % and ustekinumab in 1.9 %. After a median follow-up of 71 months [IQR, 34 - 148], 42 (20%) incident cancers were observed (34 breast cancer recurrences). Adjusted incidence rates per 1000 person-years were 10.2 (95%CI 6.0- 16.4) for the untreated arm and 28.9 (95%CI 11.6-59.6) for exposed patients (p= 0.0519). There was no significant difference between treated patients and controls regarding incident-cancer free survival rates (p=0.4796). In multivariable analysis, factors associated with incident cancer were stage T4d (p=0.036), triple negative tumor (p=0.016) and follow-up of less than 71 months (p=0.005)., Conclusion: We did not find a statistically significant increase in incident breast cancer related to IBD treatment beyond the already known poor prognostic factors of breast cancer., (Copyright © 2024. Published by Elsevier Inc.)

Viruses compete with each other for limited cellular resources, and some deliver defence mechanisms that protect the host from competing genetic parasites 1 . The phage antirestriction induced system (PARIS) is a defence system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB) 2 . However, the mechanism by which AriA and AriB function in phage defence is unknown. Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-electron microscopy to determine the structure of this complex, thereby explaining how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving host lysine transfer RNA. Phage T5 subverts PARIS immunity through expression of a lysine transfer RNA variant that is not cleaved by PARIS, thereby restoring viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids 3 ., (© 2024. The Author(s).)

Prokaryotic CRISPR-Cas immune systems detect and cleave foreign nucleic acids. In type III CRISPR-Cas systems, the Cas10 subunit of the activated recognition complex synthesizes cyclic oligoadenylates (cOAs), second messengers that activate downstream ancillary effector proteins. Once the viral attack has been weathered, elimination of extant cOA is essential to limit the antiviral response and to allow cellular recovery. Various families of ring nucleases have been identified, specializing in the degradation of cOAs either as standalone enzymes or as domains of effector proteins. Here we describe the ring nuclease activity inherent in the SAVED domain of the cA4-activated CRISPR Lon protease CalpL. We characterize the kinetics of cA4 cleavage and identify key catalytic residues. We demonstrate that cA4-induced oligomerization of CalpL is essential not only for activation of the protease, but is also required for nuclease activity. Further, the nuclease activity of CalpL poses a limitation to the protease reaction, indicating a mechanism for regulation of the CalpL/T/S signaling cascade. This work is the first demonstration of a catalytic SAVED domain and gives new insights into the dynamics of transcriptional adaption in CRISPR defense systems., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

All vertebrate genomes encode for three large histone H2A variants that have an additional metabolite-binding globular macrodomain module, macroH2A. MacroH2A variants impact heterochromatin organization and transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how macroH2A is incorporated into chromatin and the identity of any chaperones required for histone deposition remain elusive. Here, we develop a split-GFP-based assay for chromatin incorporation and use it to conduct a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We show that the histone chaperone ANP32B is a regulator of macroH2A deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetrasomes. In cells, depletion of ANP32B strongly affects global macroH2A chromatin incorporation, revealing ANP32B as a macroH2A histone chaperone., Competing Interests: Declaration of interests A.G.L. is a founder, CSO, and managing director of Eisbach Bio GmbH, a biotechnology company in oncology and virology., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Patients with inflammatory bowel disease (IBD) may have a modified immune response to SARS-CoV-2. The objectives were to evaluate the prevalence of COVID-19 in patients treated with infliximab or vedolizumab, to analyze the factors associated with the infection, the impact of treatments and trough levels., Methods: Patients with IBD treated with intravenous biologics in 14 French centers were included between March and June 2020 and followed-up for 6 months. Blood samples were collected for serologies and trough levels. The analysis of factors associated with COVID-19 was conducted in a matched 1:1 case-control sub-study with positive patients., Results: In total, 1026 patients were included (74.9% infliximab). Over the follow-up period, 420 patients reported the occurrence of COVID-19 symptoms; 342 had been tested of whom 18 were positive. At the end of follow-up, 38 patients had a positive serology. Considering both nasal tests and serologies together, 46 patients (4.5%) had been infected. The risk of COVID-19 was related neither to the use of treatments (whatever the trough levels) nor to disease activity. Infections were more frequent when using public transport or living in flats in urban areas., Conclusions: The prevalence rate of COVID-19 in this IBD population treated with intravenous infliximab or vedolizumab was the same as the one in the French population before the start of the vaccination campaign. The risk was increased by urban living and was not influenced by disease activity or biologics. Sanitary barrier measures remain the best way to protect against SARS-CoV-2 in patients with IBD in biological therapy., Competing Interests: SN declares counseling, boards, transports or fees from Abbvie, Biogen, HAC-pharma, Janssen, MSD, Novartis, Pfizer, Takeda, Tillots, BMS, Amgen, Galapagos and Fresenius. Prof. Bouguen received lecture fees from Abbvie, Ferring, MSD, Takeda and Pfizer and consultant fees from Takeda, Janssen. MA reports consulting or lecture fees from AbbVie, Amgen, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Ferring, Genentech, Gilead, IQVIA, Janssen, Novartis, Pfizer, Roche, Takeda, and Tillots; and grant support from Innate Pharma, Janssen, Takeda, and Genentech/Roche. MS has received lecture or consulting fees from Abbvie, Ferring, Amgen, Celltrion, Janssen, Ferring, Takeda and Tillotts. LC received board and lecture fees from Abbvie, Janssen, Pfizer, Takeda, Amgen. CR has received payment for lectures from Abbvie, Biogen and Galapagos. PB has received payment for lectures from Abbvie, Pfizer, Tillotts, Fresenius Kabi and Gilead. DL declares counseling, boards, transports or fees from Abbvie, Biogaran, Biogen, Ferring, Galapagos, Janssen, Lilly, MSD, Novartis, Pfizer, Prometheus, Roche, Takeda. LP-B has served as a consultant for Abbvie, Alimentiv, Alma Bio Therapeutics, Amgen, Applied Molecular Transport, Arena, Biogen, BMS, Celltrion, CONNECT Biopharm, Cytoki Pharma, Enthera, Ferring, Fresenius Kabi, Galapagos, Genentech, Gilead, Gossamer Bio, GSK, HAC-Pharma, IAG Image Analysis, Index Pharmaceuticals, Inotrem, Janssen, Lilly, Medac, Mopac, Morphic, MSD, Norgine, Novartis, OM Pharma, ONO Pharma, OSE Immunotherapeutics, Pandion Therapeutics, Par’Immune, Pfizer, Prometheus, Protagonist, Roche, Sandoz, Takeda, Theravance, Thermo Fisher, Tigenix, Tillots, Viatris, Vifor, Ysopia, Abivax; has received payment for lectures from Galapagos, AbbVie, Janssen, Genentech, Ferring, Tillots, Celltrion, Takeda, Pfizer, Sandoz, Biogen, MSD, Amgen, Vifor, Arena, Lilly, Gilead, Viatris, Medac, Sanofi; reports grant support from Takeda, Fresenius Kabi, Celltrion; has received meeting support fees from Galapagos, AbbVie, Janssen, Genentech, Ferring, Tillots, Celltrion, Takeda, Pfizer, Gossamer Bio, Sandoz, MSD, Amgen, Lilly, Gilead, Thermo Fisher, Medac, CONNECT Biopharm. AM has received payment for lectures from Galapagos. CT-P has reveived lecture fees from Ferring, Janssen, Mayoly spindler, Norgine, Abbvie, MSD, and Takeda. AB declares lecture or consulting fees from Abbvie, Amgen, Celltrion, Ferring, Fresenius Kabi, Galapagos, Gilead, Janssen, MSD, OSE Immunotherapeutics, Pfizer, Roche, Takeda, and Tillotts. CLB has served as a consultant for Abbvie, Janssen and Gilead; has received payment for lectures from Abbvie, Amgen, Celltrion, Ferring, Fresenius Kabi, Galapagos, Janssen, Lilly, MSD, Pfizer, and Takeda; reports grant support from Abbvie and Takeda; has received meeting support fees from Abbvie, Ferring, Fresenius Kabi, Galapagos, Janssen, Lilly, Pfizer, Sandoz, and Takeda. 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., (Copyright © 2023 Lelong, Josien, Coste-Burel, Rimbert, Bressollette-Bodin, Nancey, Bouguen, Allez, Serrero, Caillo, Rouillon, Blanc, Laharie, Olivier, Peyrin-Biroulet, Dib, De Maissin, Montuclard, Trang-Poisson, Vavasseur, Gallot, Berthome, Braudeau, Chevreuil, Bourreille and Le Berre.)

Purpose: Hyper activation of the JAK-STAT signaling underlies the pathophysiology of many human immune-mediated diseases. Herein, the study of 2 adult patients with SOCS1 haploinsufficiency illustrates the severe and pleomorphic consequences of its impaired regulation in the intestinal tract., Methods: Two unrelated adult patients presented with gastrointestinal manifestations, one with Crohn's disease-like ileo-colic inflammation refractory to anti-TNF and the other with lymphocytic leiomyositis causing severe chronic intestinal pseudo-occlusion. Next-generation sequencing was used to identify the underlying monogenic defect. One patient received anti-IL-12/IL-23 treatment while the other received the JAK1 inhibitor, ruxolitinib. Peripheral blood, intestinal tissues, and serum samples were analyzed before-and-after JAK1 inhibitor therapy using mass cytometry, histology, transcriptomic, and Olink assay., Results: Novel germline loss-of-function variants in SOCS1 were identified in both patients. The patient with Crohn-like disease achieved clinical remission with anti-IL-12/IL-23 treatment. In the second patient with lymphocytic leiomyositis, ruxolitinib induced rapid resolution of the obstructive symptoms, significant decrease of the CD8+ T lymphocyte muscular infiltrate, and normalization of serum and intestinal cytokines. Decreased frequencies of circulating Treg cells, MAIT cells, and NK cells, with altered CD56 bright :CD16 lo :CD16 hi NK subtype ratios were not modified by ruxolitinib., Conclusion: SOCS1 haploinsufficiency can result in a broad spectrum of intestinal manifestations and need to be considered as differential diagnosis in cases of severe treatment-refractory enteropathies, including the rare condition of lymphocytic leiomyositis. This provides the rationale for genetic screening and considering JAK inhibitors in such cases., (© 2023. The Author(s).)

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes 1,2 . The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA 3-5 . Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates 6,7 , a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family 7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA 4 ), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite diagnostic advances in microbiology, the etiology of neutropenic fever remains elusive in most cases. In this study, we evaluated the utility of a metagenomic shotgun sequencing based assay for detection of bacteria and viruses in blood samples of patients with febrile neutropenia. We prospectively enrolled 20 acute leukemia patients and obtained blood from these patients at three time points: 1) anytime from onset of neutropenia until before development of neutropenic fever, 2) within 24 hours of onset of neutropenic fever, 3) 5-7 days after onset of neutropenic fever. Blood samples underwent sample preparation, sequencing and analysis using the iDTECT® Dx Blood v1® platform (PathoQuest, Paris, France). Clinically relevant viruses or bacteria were detected in three cases each by metagenomic shotgun sequencing and blood cultures, albeit with no concordance between the two. Further optimization of sample preparation methods and sequencing platforms is needed before widespread adoption of this technology into clinical practice., Competing Interests: M.M., C.R., S.C. and P.B. are employed by PathoQuest and own shares of PathoQuest. A.F. is a former employee of PathoQuest. M.R.S. reports receiving funds from TYRX Inc. and Medtronic for prior research unrelated to this study administered according to a sponsored research agreement between Mayo Clinic and study sponsor that prospectively defined the scope of the research effort and corresponding budget; and honoraria/consulting fees from Medtronic Inc. and Aziyo Biologics, Inc. Research Grant: Medtronic (significant - $40K), Honoraria: Medtronic (significant $20K), and Aziyo Biologics (modest $5K). M.M.P. serves on the Advisory Board for Kura Oncology. R.P reports grants from ContraFect, TenNor Therapeutics Limited, and BioFire. R.P. is or has been a consultant to Curetis, Specific Technologies, Next Gen Diagnostics, PathoQuest, Selux Diagnostics, 1928 Diagnostics, PhAST, Torus Biosystems, Day Zero Diagnostics, Mammoth Biosciences, CARB-X, and Qvella; monies are paid to Mayo Clinic. R.P. is also a consultant to Netflix. In addition, R.P. has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. R.P. receives travel reimbursement from ASM and IDSA and an editor’s stipend from IDSA, and honoraria from the NBME, Up-to-Date and the Infectious Diseases Board Review Course. All other authors report no potential conflicts of interest. this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Study Question: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)?, Summary Answer: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response., What Is Known Already: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin., Study Design, Size, Duration: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2)., Participants/materials, Setting, Methods: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9)., Main Results and the Role of Chance: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3β-hydroxysteroid dehydrogenase (3βHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3βHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9)., Large Scale Data: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048., Limitations, Reasons for Caution: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation., Wider Implications of the Findings: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS., Study Funding/competing Interest(s): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY » funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Within the field of DNA nanotechnology, numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging applications. These structures typically suffer from a low tolerance against non-optimal environmental conditions including elevated temperatures. Here, we apply a chemical ligation method to covalently seal the nicks between adjacent 5' phosphorylated and 3' amine-modified strands within the DNA nanostructures. Using a cost-effective enzymatic strand modification procedure, we are able to batch-modify all DNA strands even of large DNA objects, such as origami nanostructures. The covalent strand linkage increases the temperature stability of the structures by ∼10 K. Generally, our method also allows a 'surgical' introduction of covalent strand linkages at preselected positions. It can also be used to map the strand ligation into chains throughout the whole nanostructure and identify assembly defects. We expect that our method can be applied to a large variety of DNA nanostructures, in particular when full control over the introduced covalent linkages and the absence of side adducts and DNA damages are required.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the C α -helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 μs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

Reducing the variability in nuclear transfer outcome requires a better understanding of its cellular and epigenetic determinants, in order to ensure safer fish regeneration from cryobanked somatic material. In this work, clones from goldfish were obtained using cryopreserved fin cells as donor and non-enucleated oocytes as recipients. We showed that the high variability of clones survival was not correlated to spawn quality. Clones were then characterized for their first cleavages pattern in relation to their developmental fate up to hatching. The first cell cycle duration was increased in clones with abnormal first cleavage, and symmetric first two cleavages increased clone probability to reach later on 24 h- and hatching-stages. At 24 h-stage, 24% of the clones were diploids and from donor genetic origin only. However, ploidy and genetic origin did not determine clones morphological quality. DNA methylation reprogramming in the promoter region of pou2, nanog, and notail marker genes was highly variable, but clones with the nicest morphologies displayed the best DNA methylation reprogramming. To conclude, non-enucleated oocytes did allow authentic clones production. The first two cell cycles were a critical determinant of the clone ability to reach hatching-stage, and DNA methylation reprogramming significantly influenced clones morphological quality.

Introduction: Paternal low-protein diet can alter sperm methylation status, fetal growth and program offspring ill-health, however its impact on the placenta remains poorly defined. Here we examine the influence paternal low-protein diet has on fetal and placental development and the additional impact of supplementary methyl-donors on fetoplacental physiology., Methods: Male C57BL/6J mice were fed a control normal protein diet (NPD; 18% protein), a low-protein diet (LPD; 9% protein) or LPD with methyl-donor supplementation (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12) for a minimum of 8 weeks. Males were mated with 8-11 week old female C57BL/6J mice and fetal and placental tissue collected on embryonic day 17.5., Results: Paternal LPD was associated with increased fetal weights compared to NPD and MD-LPD with 22% fetuses being above the 90th centile for fetal weight. However, LPD and MD-LPD placental weights were reduced when compared to NPD. Placentas from LPD fathers demonstrated a reduced junctional zone area and reduced free-fatty acid content. MD-LPD placentas did not mirror these finding, demonstrating an increased chorion area, a reduction in junctional-specific glycogen staining and reduced placental Dnmt3bexpression, none of which were apparent in either NPD or LPD placentas., Discussion: A sub-optimal paternal diet can influence fetal growth and placental development, and dietary methyl-donor supplementation alters placental morphology and gene expression differentially to that observed with LPD alone. Understanding how paternal diet and micro-nutrient supplementation influence placental development is crucial for determining connections between paternal well-being and future offspring health., 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 © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The link between male diet and sperm quality has received significant investigation. However, the impact diet and dietary supplements have on the testicular environment has been examined to a lesser extent. Here, we establish the impact of a sub-optimal low protein diet (LPD) on testicular morphology, apoptosis and serum fatty acid profiles. Furthermore, we define whether supplementing a LPD with specific methyl donors abrogates any detrimental effects of the LPD. Male C57BL6 mice were fed either a control normal protein diet (NPD; 18% protein; n = 8), an isocaloric LPD (LPD; 9% protein; n = 8) or an LPD supplemented with methyl donors (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12; n = 8) for a minimum of 7 weeks. Analysis of male serum fatty acid profiles by gas chromatography revealed elevated levels of saturated fatty acids and lower levels of mono- and polyunsaturated fatty acids in MD-LPD males when compared to NPD and/or LPD males. Testes of LPD males displayed larger seminiferous tubule cross section area when compared to NPD and MD-LPD males, while MD-LPD tubules displayed a larger luminal area. Furthermore, TUNNEL staining revealed LPD males possessed a reduced number of tubules positive for apoptosis, while gene expression analysis showed MD-LPD testes displayed decreased expression of the pro-apoptotic genes Bax, Csap1 and Fas when compared to NPD males. Finally, testes from MD-LPD males displayed a reduced telomere length but increased telomerase activity. These data reveal the significance of sub-optimal nutrition for paternal metabolic and reproductive physiology.

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA 4 ), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an 'off-switch' for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA 4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict., Competing Interests: JA, SG, CR, SG, CC, MW No competing interests declared, (© 2020, Athukoralage et al.)

Nuclear transfer consists in injecting a somatic nucleus carrying valuable genetic information into a recipient oocyte to sire a diploid offspring which bears the genome of interest. It requires that the oocyte (maternal) DNA is removed. In fish, because enucleation is difficult to achieve, non-enucleated oocytes are often used and disappearance of the maternal DNA was reported in some clones. The present work explores which cellular events explain spontaneous erasure of maternal DNA, as mastering this phenomenon would circumvent the painstaking procedure of fish oocyte enucleation. The fate of the somatic and maternal DNA during oocyte activation and first cell cycle was studied using DNA labeling and immunofluorescence in goldfish clones. Maternal DNA was always found as an intact metaphase within the oocyte, and polar body extrusion was minimally affected after oocyte activation. During the first cell cycle, only 40% of the clones displayed symmetric cleavage, and these symmetric clones contributed to 80% of those surviving at hatching. Maternal DNA was often fragmented and located under the cleavage furrow. The somatic DNA was organized either into a normal mitotic spindle or abnormal multinuclear spindle. Scenarios matching the DNA behavior and the embryo fate are proposed.

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA 4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA 4 -activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA 4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems., (© 2019 Elsevier Inc. All rights reserved.)

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids 1-3 . Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA 4,5 . Cyclic oligoadenylate, in turn, binds to and activates ribonucleases and other factors-via a CRISPR-associated Rossman-fold domain-and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the 'off-switch' that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This 'ring nuclease' is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight to the CRISPR system.

We report the nearly complete 1 H, 15 N and 13 C resonance assignment and the solution structure of the external DII domain of the yeast Rvb2 protein, a member of the AAA+ATPase superfamily.

The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence., Competing Interests: CR, JA, SG, SG, MW No competing interests declared, (© 2018, Rouillon et al.)

In type I CRISPR-Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade-crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade-crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade-crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA.

The CRISPR-Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3' ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3' ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR-Cas elements and host proteins across CRISPR types.

Background: Despite the lack of scientific studies on biofield therapies, they are widely acclaimed by patients. The mechanisms of action are not explained by current allopathic medical approaches. Warts are common and contagious viral lesions that may be refractory to standard dermatologic treatments such as cryotherapy, laser therapy, and keratolytic ointments. Biofield therapies are efficient in various pathologies. Their ability to treat warts has never been demonstrated in a scientific study with a robust methodology. Patients with refractory warts often place their trust in these alternative therapies because of the poor results obtained from traditional medicine. We propose a prospective, randomized, single-blind, assessor-blind trial to evaluate the efficacy of treatment of warts by biofield therapy., Methods/design: Subjects with warts on their feet or hands will be randomized into two groups: real biofield therapy versus sham therapy. The diagnosis will be made at the time of inclusion, and follow-up will take place in week 3. Comparison of pictures of the warts at baseline and after 3 weeks will be used as the primary outcome measure. The hypothesis is that the extent of the disappearance of the original wart in the group treated by real biofield therapy will be 70% and that it will be 30% in the group treated by sham therapy. Using 90% power and an alpha risk of 5%, 31 subjects are required in each group for a two-tailed proportion comparison test., Discussion: To our knowledge, this is the first study to evaluate the efficacy of biofield therapy on warts. Therefore, the aim of this study is to extend knowledge of biofield therapy to another area of medicine such as dermatology and to propose complementary or alternative practices to improve patient well-being. The main strength of the study is that it is a randomized, single-blind, assessor-blind, placebo-controlled study., Trial Registration: ClinicalTrials.gov identifier: NCT02773719 . Registered on 22 April 2016.

Ribonucleoprotein (RNP) complexes from CRISPR-Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR-Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule. Specifically, we demonstrate how magnetic tweezers are applied to measure the local DNA unwinding at the target in real time. To this end we introduce the technique and the measurement principle. By studying modifications of the consensus target sequence, we show how different sequence elements contribute to the target recognition mechanism. From these data, a unified target recognition mechanism can be concluded for the RNPs Cascade and Cas9 from types I and II CRISPR-Cas systems. R-loop formation is hereby initiated on the target at an upstream element, called protospacer adjacent motif (PAM), from which the R-loop structure zips directionally toward the PAM-distal end of the target. At mismatch positions, the R-loop propagation stalls and further propagation competes with collapse of the structure. Upon full R-loop zipping conformational changes within the RNPs trigger degradation of the DNA target. This represents a shared labor mechanism in which zipping between nucleic acid strands is the actual target recognition mechanism while sensing of the R-loop arrival at the PAM-distal end just verifies the success of the full zipping., (© 2017 Elsevier Inc. All rights reserved.)

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I-III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Site-directed spin labeling and pulsed electron-electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets., (Copyright © 2012 Elsevier Inc. All rights reserved.)