Arylsulfatases and neuraminidases modulate engagement of CCR5 by chemokines by removing key electrostatic interactions.

The chemokine receptor CCR5 is known to exist in cell surface subpopulations that differ in their capacity to engage ligands. One proposed explanation for this phenomenon is the presence of CCR5 species with different levels of post-translational modifications (PTMs). Tyrosine sulfation and O-glycan sialylation are PTMs that add negative charges to the extracellular domain of CCR5 and make strong contributions to chemokine binding but it is not known whether cellular mechanisms to control their levels exist. In this study we used a combination of sulfation-sensitive and sulfation-insensitive CCR5 ligands to show that the rate of turnover of CCR5 tyrosine sulfation is more rapid than the rate of turnover of the receptor itself. This suggests that the steady state level of CCR5 sulfation is maintained through the combination of tyrosine protein sulfotransferase (TPST), the trans-Golgi network (TGN)-resident 'source enzyme, and a 'sink' activity that removes tyrosine sulfation from CCR5. By measuring the effects on ligand binding of knockdown and overexpression experiments, we provided evidence that non-lysosomal cellular arylsulfatases, particularly ARSG, ARSI and ARSJ, are CCR5 sulfation 'sink' enzymes. We also used targeted knockdown and sialylation-sensitive and insensitive chemokines to identify the sialidase NEU3 as a candidate 'sink' enzyme for CCR5 O-glycan sialylation. This study provides the first experimental evidence of activity of sulfatase and sialidase 'sink' enzymes on CCR5, providing a potential mechanism for cells to control steady-state levels of these PTMs and thereby exert dynamic control over receptor-ligand interactions at the cell surface and during receptor desensitization.

Chitosan Nano/Microformulations for Antimicrobial Protection of Leather with a Potential Impact in Tanning Industry.

Tanned leather can be attacked by microorganisms. To ensure resistance to bacteria on leather surfaces, protection solutions need to be developed, addressing both environmental issues and economic viability. In this work, chitosan nano/microparticles (CNP) and chitosan/silver nano/microstructures (CSNP), containing silver nanoparticles around 17 nm size, were incorporated into leather, obtained from the industrial process. Low loads of chitosan-based nano/microformulations, 0.1% mass ratio, resulted in total bacteria reduction (100%) after 2 h towards Gram-positive Staphylococcus aureus, both with CNP and CSNP coatings. Otherwise, comparable tests with the Gram-negative bacteria, Klebsiella pneumoniae, Escherichia coli, showed no significant improvement under the coating acidic conditions. The antimicrobial activity was evaluated by standard test methods: (1) inhibition halo and (2) dynamic contact conditions. The developed protection of leather either with CNP or CSNP is much higher than the one obtained with a simple chitosan solution.

Involving society in science: Reflections on meaningful and impactful stakeholder engagement in fundamental research.

Open Science calls for transparent science and involvement of various stakeholders. Here are examples of and advice for meaningful stakeholder engagement.

Corticosteroids in adult respiratory distress syndrome - an inconvenient truth?

OBJECTIVES: Recent studies have demonstrated mortality benefits from corticosteroid use in COVID-19 patients requiring respiratory support. However, clinical practice may warrant the use of corticosteroids outside the context of a clinical trial. Such data are rarely, if ever, reported. We explored the use of corticosteroids for adult respiratory distress syndrome (ARDS) indications in patients with non-COVID ARDS. METHODS: We retrospectively studied patients with moderate-to-severe ARDS, admitted to our intensive care unit (ICU) between January 2018 and March 2020. KEY FINDINGS: Of the 91 patients with ARDS identified, 80% were treated with a corticosteroid during their ICU admission. Of these, 73 (82%) had corticosteroids administered for reasons other than ARDS. CONCLUSIONS: Corticosteroid use for non-ARDS indications is commonplace in ARDS patients in our ICU. The use of corticosteroids outside a randomisation process in randomised clinical trials may be more common than appreciated and needs to be routinely reported.

Is it just an obstructive pyelonephritis?

Pyelocaliceal obstruction is a diagnostic challenge, and it is important to identify the obstruction cause. Some patients present extra-renal compressive masses that need further imagiologic investigation and a biopsy, to establish the diagnosis.

Thinking "ethical" when designing an international, cross-disciplinary biomedical research consortium.

This commentary outlines challenges with identifying and implementing ethical, legal and societal considerations when initiating large-scale scientific programs and suggests best practices to ensure responsible research.

LifeTime and improving European healthcare through cell-based interceptive medicine.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

Author Correction: In-cell identification and measurement of RNA-protein interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SPEN integrates transcriptional and epigenetic control of X-inactivation.

Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN1-3, the loss of which has been associated with deficient XCI at multiple loci2-6. Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.

In-cell identification and measurement of RNA-protein interactions.

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.

X chromosome inactivation: new players in the initiation of gene silencing.

X chromosome inactivation (XCI) is a dosage compensation process that was adopted by female mammals to balance gene dosage between XX females and XY males. XCI starts with the upregulation of the non-coding RNA Xist, after which most X-linked genes are silenced and acquire a repressive chromatin state. Even though the chromatin marks of the inactive X have been fairly well described, the mechanisms responsible for the initiation of XCI remain largely unknown. In this review, we discuss recent developments that revealed unexpected factors playing a role in XCI and that might be of crucial importance to understand the mechanisms responsible for the very first steps of this chromosome-wide gene-silencing event.

Alterations of Nuclear Architecture and Epigenetic Signatures during African Swine Fever Virus Infection.

Viral interactions with host nucleus have been thoroughly studied, clarifying molecular mechanisms and providing new antiviral targets. Considering that African swine fever virus (ASFV) intranuclear phase of infection is poorly understood, viral interplay with subnuclear domains and chromatin architecture were addressed. Nuclear speckles, Cajal bodies, and promyelocytic leukaemia nuclear bodies (PML-NBs) were evaluated by immunofluorescence microscopy and Western blot. Further, efficient PML protein knockdown by shRNA lentiviral transduction was used to determine PML-NBs relevance during infection. Nuclear distribution of different histone H3 methylation marks at lysine's 9, 27 and 36, heterochromatin protein 1 isoforms (HP1α, HPß and HPγ) and several histone deacetylases (HDACs) were also evaluated to assess chromatin status of the host. Our results reveal morphological disruption of all studied subnuclear domains and severe reduction of viral progeny in PML-knockdown cells. ASFV promotes H3K9me3 and HP1ß foci formation from early infection, followed by HP1α and HDAC2 nuclear enrichment, suggesting heterochromatinization of host genome. Finally, closeness between DNA damage response factors, disrupted PML-NBs, and virus-induced heterochromatic regions were identified. In sum, our results demonstrate that ASFV orchestrates spatio-temporal nuclear rearrangements, changing subnuclear domains, relocating Ataxia Telangiectasia Mutated Rad-3 related (ATR)-related factors and promoting heterochromatinization, probably controlling transcription, repressing host gene expression, and favouring viral replication.

Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity.

Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation. The H3K9me1 is converted in the nucleus to H3K9me3 by the Suv39h enzymes to reinforce heterochromatin. Simultaneous depletion of Prdm3 and Prdm16 abrogates H3K9me1 methylation, prevents Suv39h-dependent H3K9me3 trimethylation, and derepresses major satellite transcription. Most strikingly, DNA-FISH and electron microscopy reveal that combined impairment of Prdm3 and Prdm16 results in disintegration of heterochromatic foci and disruption of the nuclear lamina. Our data identify Prdm3 and Prdm16 as H3K9me1 methyltransferases and expose a functional framework in which anchoring to the nuclear periphery helps maintain the integrity of mammalian heterochromatin.

Conjugation of human topoisomerase 2 alpha with small ubiquitin-like modifiers 2/3 in response to topoisomerase inhibitors: cell cycle stage and chromosome domain specificity.

Type 2 topoisomerases, in particular the alpha isoform in human cells, play a key role in cohesion and sister chromatid separation during mitosis. These enzymes are thus vital for cycling cells and are obvious targets in cancer chemotherapy. Evidence obtained in yeast and Xenopus model systems indicates that conjugation of topoisomerase 2 with small ubiquitin-like modifier (SUMO) proteins is required for its mitotic functions. Here, we provide biochemical and cytologic evidence that topoisomerase 2 alpha is conjugated to SUMO-2/3 during interphase and mitosis in response to topoisomerase 2 inhibitors and "poisons" (ICRF-187, etoposide, doxorubicin) that stabilize catalytic intermediates (cleavage complexes, closed clamp forms) of the enzyme onto target DNA. During mitosis, SUMO-2/3-modified forms of topoisomerase 2 alpha localize to centromeres and chromosome cores/axes. However, centromeres are unresponsive to inhibitors during interphase. Furthermore, formation of topoisomerase 2 alpha-SUMO-2/3 conjugates within mitotic chromosomes strongly correlates with incomplete chromatid decatenation and decreases progressively as cells approach the metaphase-anaphase transition. We also found that the PIASy protein, an E3 ligase for SUMO proteins, colocalizes with SUMO-2/3 at the mitotic chromosomal cores/axes and is necessary for both formation of SUMO-2/3 conjugates and proper chromatid segregation. We suggest that the efficacy of topoisomerase inhibitors to arrest cells traversing mitosis may relate to their targeting of topoisomerase 2 alpha-SUMO-2/3 conjugates that concentrate at mitotic chromosome axes and are directly involved in chromatid arm separation.

Mutagenic effects of poly (ADP-ribose) polymerase-1 deficiency in transgenic mice.

Poly (ADP-ribose) polymerase-1 (Parp1) plays a central role in the maintenance of genomic integrity and has been unequivocally associated to DNA base excision repair (BER) but its involvement in double-strand break (DSB) repair pathways remains unclear. In this work, using transgenic Parp1-deficient mice harbouring the lacZ reporter gene, we provide in vivo evidence that Parp1 contributes to the prevention of deletions/insertions in testis following an alkylation insult. In response to N-Methyl-N-Nitrosurea (MNU) treatment no significant difference in the mutant frequency (MF) in the liver and testis could be attributed to Parp1 status, given that both Parp1(+/+) and Parp1(-/-) mice showed a similar significant increase in the overall MF. However, restriction analysis of MNU-induced mutants evidenced a shift in the distribution of mutations between deletions/insertions and point mutations in testis, but not in the liver, dependent on the Parp1 status. A significant higher frequency of deletions/insertions was observed in testis from Parp1(-/-) in comparison to Parp1(+/+) mice, whereas point mutations were not significantly affected. Overall, our findings show that Parp1 participates in the prevention of deletions/insertions induced by methylating agents and that organ-specific factors may influence its capacity to protect against genotoxic damage.