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Oxidation of the epigenetic DNA mark 5-methylcytosine by Tet dioxygenases is an established route to diversify the epigenetic information, modulate gene expression and overall cellular (patho-)physiology. Here, we demonstrate that Tet1 and its short isoform Tet1s exhibit distinct nuclear localization during DNA replication resulting in aberrant cytosine modification levels in human and mouse cells. We show that Tet1 is tethered away from heterochromatin via its zinc finger domain, which is missing in Tet1s allowing its targeting to these regions. We find that Tet1s interacts with and is ubiquitinated by CRL4(VprBP). The ubiquitinated Tet1s is then recognized by Uhrf1 and recruited to late replicating heterochromatin. This leads to spreading of 5-methylcytosine oxidation to heterochromatin regions, LINE 1 activation and chromatin decondensation. In summary, we elucidate a dual regulation mechanism of Tet1, contributing to the understanding of how epigenetic information can be diversified by spatio-temporal directed Tet1 catalytic activity. A short isoform of the Tet1 enzyme (Tet1s) that oxidizes the DNA 5-methylcytosine (5mC) mark is overexpressed in tumors. Here the authors show Tet1s, but not full length Tet1, changes localization over the cell cycle upon ubiquitination and Uhrf1 interaction and is targeted to heterochromatin during S-phase. This leads to 5mC oxidation and loss of DNA methylation in heterochromatin.

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein–protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets. Delivery of antibodies into living cells enables the labelling and manipulation of intracellular antigens; however, transporting antibodies into the cytosol in a functional state is difficult. Now, a modular strategy for creating cell-permeable nanobodies capable of targeting intracellular antigens has been developed. The cell-permeable nanobodies are formed by site-specific attachment of cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain antibody fragments.

Abstract DNA base modifications and mutations are observed in all genomes throughout the kingdoms of life. Proteins involved in their establishment and removal were shown to use a base flipping mechanism to access their substrates. To better understand how proteins flip DNA bases to modify or remove them, we optimized and developed a pipeline of methods to step-by-step detect the process starting with protein–DNA interaction, base flipping itself and the ensuing DNA base modification or excision. As methylcytosine is the best-studied DNA modification, here we focus on the process of writing, modifying and reading this DNA base. Using multicolor electrophoretic mobility shift assays, we show that the methylcytosine modifier Tet1 exhibits little DNA sequence specificity with only a slight preference for methylated CpG containing DNA. A combination of chloroacetaldehyde treatment and high-resolution melting temperature analysis allowed us to detect base flipping induced by the methylcytosine modifier Tet1 as well as the methylcytosine writer M.HpaII. Finally, we show that high-resolution melting temperature analysis can be used to detect the activity of glycosylases, methyltransferases and dioxigenases on DNA substrates. Taken together, this DNA base flipping analytical pipeline (BaFAP) provide a complete toolbox for the fast and sensitive analysis of proteins that bind, flip and modify or excise DNA bases.

In two identical trials involving a total of 1882 patients with multiple sclerosis, the human anti-CD20 monoclonal antibody ofatumumab was associated with a lower annualized relapse rate than the pyrimidine-synthesis inhibitor teriflunomide, as well as fewer lesions on MRI.

Background and ObjectivesWe examined whether liposome bupivacaine (Exparel) given in the interscalene brachial plexus block lowers pain in the setting of multimodal postoperative pain management for major shoulder surgery.MethodsFifty-two adult patients were randomized to receive either 5 mL of 0.25% bupivacaine HCl immediately followed by 10 mL of liposome bupivacaine 133 mg (n = 26) or 15 mL of 0.25% standard bupivacaine alone (n = 26) in interscalene brachial plexus block. The primary outcome (worst pain in the first postoperative week) was assessed by the Modified Brief Pain Inventory short form. Secondary outcomes were overall satisfaction with analgesia (OBAS), functionality of the surgical arm, sleep duration, time to first opioid (tramadol) request and opioid consumption (mEq), sensory-motor block characteristics, and the occurrence of adverse effects.ResultsWorst pain was lower in patients given liposome bupivacaine added to standard bupivacaine than in patients given standard bupivacaine alone (generalized estimating equation [GEE] estimated marginal mean values, 3.6 ± 0.3 vs 5.3 ± 0.4 points on the Numeric Rating Scale, respectively, although the effect was modest, 1.6 ± 0.5; 95% confidence interval, 0.8–2.5). Total OBAS scores indicated greater satisfaction (GEE estimated marginal mean values, 1.8 ± 0.3 vs 3.3 ± 0.4 on total OBAS, respectively, with modest effect, difference, 1.4 ± 0.5; 95% confidence interval, 0.5–2.4). There were no differences in any of the other secondary outcomes.ConclusionsLiposome bupivacaine added to standard bupivacaine may lower pain and enhance patient's satisfaction in the first postoperative week even in the setting of multimodal analgesia for major shoulder surgery.This study was registered with clinicaltrials.gov (NCT02554357) on July 11, 2015, by Principal Investigator Catherine Vandepitte, MD.

ObjectivesThe COVID-19 pandemic has resulted in widespread morbidity and mortality with the consequences expected to be felt for many years. Significant variation exists in the care even of similar patients with COVID-19, including treatment practices within and between institutions. Outcome measures vary among clinical trials on the same therapies. Understanding which therapies are of most value is not possible unless consensus can be reached on which outcomes are most important to measure. Furthermore, consensus on the most important outcomes may enable patients to monitor and track their care, and may help providers to improve the care they offer through quality improvement. To develop a standardised minimum set of outcomes for clinical care, the International Consortium for Health Outcomes Measurement (ICHOM) assembled a working group (WG) of 28 volunteers, including health professionals, patients and patient representatives.DesignA list of outcomes important to patients and professionals was generated from a systematic review of the published literature using the MEDLINE database, from review of outcomes being measured in ongoing clinical trials, from a survey distributed to patients and patient networks, and from previously published ICHOM standard sets in other disease areas. Using an online-modified Delphi process, the WG selected outcomes of greatest importance.ResultsThe outcomes considered by the WG to be most important were selected and categorised into five domains: (1) functional status and quality of life, (2) mental functioning, (3) social functioning, (4) clinical outcomes and (5) symptoms. The WG identified demographic and clinical variables for use as case-mix risk adjusters. These included baseline demographics, clinical factors and treatment-related factors.ConclusionImplementation of these consensus recommendations could help institutions to monitor, compare and improve the quality and delivery of care to patients with COVID-19. Their consistent definition and collection could also broaden the implementation of more patient-centric clinical outcomes research.

Background The first years of life are a significant period for child development, when children are particularly sensitive and prone to crises. This early phase lays the foundation for healthy growth. Clinical assessment of psychological symptoms in early infancy and adequate treatment are both important in improving the diagnostic outcome and preventing later long-term developmental consequences. The most common psychological problems in the first 3 years of life are regulatory disorders. The aim of this trial is to investigate the efficacy of Parent-Infant Psychotherapy (PIP) for infants and young children (aged 0–36 months, diagnosed with at least one regulatory disorder) and their mothers, compared to care as usual (CAU). Methods In this open multicentre randomised controlled trial, 160 mother-infant dyads are randomised to receive PIP or CAU for 6 weeks of intervention in clinical or outpatient (including home treatment) settings. The primary outcome is the maternal sensitivity (sensitivity scale of the Emotional Availability Scales (EAS)) after 6 weeks. Secondary outcomes include assessment of interaction, mental health problems, attachment, development, psychological factors, treatment adherence, health care system utilisation, and costs, after 6 weeks and 12 months. Discussion This study will evaluate whether a manualised focus-based short-term psychodynamic psychotherapeutic intervention in mother-child dyads improves the care situation for families of children diagnosed with regulatory disorders, and helps prevent long-term psychopathologies. Assessment of the intervention in different settings will support the development of more tailored interventions for affected infants and their mothers. Trial registration German Clinical Trial Register, ID: DRKS00017008 . Registered 03/20/2019.

In patients ineligible for transplantation, the combination of lenalidomide and dexamethasone with lenalidomide maintenance until disease progression achieved significantly improved progression-free survival, as compared with melphalan, prednisone, and thalidomide therapy. For patients with newly diagnosed multiple myeloma who are ineligible for autologous stem-cell transplantation, the standard therapy is melphalan and prednisone (MP) combined with either thalidomide (MPT) or bortezomib (VMP). 1 – 10 Lenalidomide (Revlimid, Celgene) is an immunomodulatory drug that, in combination with dexamethasone, is a standard treatment option for patients with multiple myeloma who have received at least one prior therapy as approved by the Food and Drug Administration and the European Medicines Agency. 7 – 13 In a randomized trial that included both younger and older patients with newly diagnosed multiple myeloma, lenalidomide plus low-dose dexamethasone was associated with fewer adverse . . .