Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.

Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

Ultrastructure of the Dentin Pellicle and the Impact of Erosion.

While the ultrastructure of the enamel pellicle and its erosion protective properties are well studied, the dentin pellicle is still neglected in dental research. Therefore, the ultrastructure and erosion protective properties of a pellicle formed on bovine dentin specimens were investigated in the present study. The dentin pellicle was formed in situ for 3, 30, 120, and 360 min at buccal or palatal oral sites of 3 subjects and analyzed by transmission electron microscopy. In order to clarify the impact of an erosive challenge to the ultrastructure of the pellicle and the underlying dentin, specimens were exposed to the oral cavity and eroded in vivo with 0.1% or 1% citric acid either immediately or after 30 min of pellicle formation. Specimens that were eroded without exposure to the oral cavity served as control. In another trial, specimens with a 30-min pellicle were exposed to the oral cavity for a further 60 min after the erosive challenge to investigate the effect of saliva on the impaired pellicle and dentin. Transmission electron micrographs reveal a globular and granular structured pellicle layer, which was thicker when the pellicle was formed buccally or with longer formation times. Erosion with citric acid reduced the thickness of the pellicle and interrupted its continuity. The dentin was also affected by erosion, which was represented by a lower electron density and formation of demineralized lacunae. These were infiltrated by a granular structured material when specimens were exposed to the oral cavity. After further intraoral exposure, the infiltration was more pronounced, indicating a significant impact of saliva on the demineralized dentin. A reformation of the dentin pellicle on the other hand did not occur. In conclusion, the dentin pellicle is neither acid-resistant nor able to effectively protect dentin from erosion.

Consequences of Autophagy Deletion on the Age-Related Changes in the Epidermal Lipidome of Mice.

Autophagy is a controlled mechanism of intracellular self-digestion with functions in metabolic adaptation to stress, in development, in proteostasis and in maintaining cellular homeostasis in ageing. Deletion of autophagy in epidermal keratinocytes does not prevent the formation of a functional epidermis and the permeability barrier but causes increased susceptibility to damage stress and metabolic alterations and accelerated ageing phenotypes. We here investigated how epidermal autophagy deficiency using Keratin 14 driven Atg7 deletion would affect the lipid composition of the epidermis of young and old mice. Using mass spectrometric lipidomics we found a reduction of age-related accumulation of storage lipids in the epidermis of autophagy-deficient mice, and specific changes in chain length and saturation of fatty acids in several lipid classes. Transcriptomics and immunostaining suggest that these changes are accompanied by changes in expression and localisation of lipid and fatty acid transporter proteins, most notably fatty acid binding protein 5 (FABP5) in autophagy knockouts. Thus, maintaining autophagic activity at an advanced age may be necessary to maintain epidermal lipid homeostasis in mammals.

P067 Immune Checkpoint Inhibitor Colitis in a Community-Based Hospital System.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have significantly changed the oncologic treatment landscape for a wide array of cancers over the past few decades. While these medications can produce a durable remission for patients they also come with a risk of inflammatory toxicities. With the widespread use of immune checkpoint inhibitors there have been increased reports of immune-related adverse events (irAEs) including severe gastrointestinal toxicities such as colitis. While multiple studies have assessed ICI colitis in tertiary care settings little is reported on the characteristics and treatment outcomes of ICI colitis in a community-based population. Additionally, the utility of gastroenterologists in the diagnosis and management of ICI colitis is poorly defined. The aim of this study is to report the characteristics and treatment outcomes of ICI colitis in a community-based hospital system. METHODS: This is a single-center retrospective case-series of patients with ICI colitis. Charts were reviewed from patients in the Prisma Health Upstate Network from 2/1/2016 to 12/31/2020. Potential cases of ICI colitis were identified using a diagnosis code of "toxic colitis", "indeterminate colitis", "unspecified colitis", and "noninfective gastroenteritis and colitis" (ICD-10 K52.1, K 52.3, K 52.89 and K52.9) as well as a prior infusion (within 12 months) of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, or ipilimumab. Charts were abstracted for demographics, tumor type, checkpoint inhibitor used, mode of colitis diagnosis, endoscopic severity of inflammation, and treatments used. Study was approved by the Institutional Review Board at Prisma Health Upstate. RESULTS: The mean (±SD) age for the 37 patients was 64.1 ± 13.2 years. The patient population was predominantly male (62%) and Caucasian (100%). The cancer type in the patient population were melanoma (43.2%), non-small cell lung cancer (18.9%), renal cell carcinoma (10.8%), and other (27%). Patients with ICI colitis were most-commonly treated with ipilimumab (35.1%), nivolumab (35%), pembrolizumab (10.8%), and other (13.5%). The mean time to colitis onset was 25.7 ± 29.7 weeks. Over 56% of the patient population reported abdominal pain and 45.9% were hospitalized. Only 64.8% were evaluated by a gastroenterologist. Cross sectional abdominal imaging by computed tomography (CT) was obtained in 20 (54.0%) and showed colitis in 13. 54% obtained a colonoscopy with inflammation severity stratified into normal (25.0%), mild (40.0%), moderate (20.0%) and severe (15.0%). Endoscopic extent showed proctitis (0%), left-sided disease (5.0%), right-sided disease (5.0%), pancolitis (55.0%), and isolated ileal disease (25%). Histology showed 90.0% with active inflammation and 10.0% with microscopic colitis. Outcomes were resolution with steroids (64.8%), antibiotics (5.4%), biologic (10.8%), surgery (5.4%), hospice (8.1%). Of those that received biologic therapy, 6 received infliximab and 1 received vedolizumab. CONCLUSION: As immune checkpoint inhibitors gain approval in more cancer treatment ICI-colitis is being seen in a community-based setting. The most common treatment were steroids and often required a prolonged course. Biologic use in our series (18.9%) was much lower than prior reports from academic tertiary referral centers (>50%). Only 64.8% of patients saw a gastroenterologist for ICI-colitis symptoms. Early gastroenterology referral in the community setting could help to identify more severe disease and patients necessitating escalation to biologic therapy.

Resourceful Actors, Not Weak Victims: Reframing Refugees' Stigmatized Identity Enhances Long-Term Academic Engagement.

Refugees suffer from a stigmatized identity portraying them as weak, unskilled victims. We developed a brief (~10-min) intervention that reframed refugees' identity as being, by its very nature, a source of strength and skills. Reading and writing exercises, provided by a university, highlighted how refugees' experiences helped them acquire skills such as perseverance and the ability to cope with adversity, which could help them succeed in a new country. In Experiment 1 (N = 93), the intervention boosted refugees' (a) confidence in their ability to succeed at an imagined university and (b) challenge seeking: Participants were 70% more likely to take on an academic exercise labeled as difficult. In Experiment 2, the intervention, delivered to refugees entering an online university (N = 533), increased engagement in the online-learning environment by 23% over the subsequent year. There was also evidence of greater course completion. It is possible to reframe stigmatized individuals' identity as inherently strong and resourceful, helping them put their strengths to use.

Environmentally Friendly, Photochemical Access to [ N ∧ C ∧ N ]AuIII Pincer Complexes By Oxidative Addition.

Starting from commercially available DMSAuCl and diazonium salts, cationic [ N ∧ C ∧ N ]AuIII complexes were synthesized in a selective, photosensitizer-free, photochemical reaction by irradiation with blue LED light. This new protocol represents the first easy synthesis of these types of pincer complexes in moderate to excellent yield starting from a readily available gold(I) precursor with nitrogen as the only by-product. Owing to the disadvantages of known protocols, especially the toxicity in the case of a transmetalation with mercury or the necessity for a mostly twofold excess of a gold precursor, this method offers an attractive alternative towards this kind of gold(III) complexes. In addition, the first arylated [ N ∧ C ∧ N ]Au(III) pincer complex was synthesized by using this technology.

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

BACKGROUND: Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. METHODS: We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. RESULTS: HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. CONCLUSIONS: HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV.

Control of Grafting Density and Distribution in Graft Polymers by Living Ring-Opening Metathesis Copolymerization.

Control over polymer sequence and architecture is crucial to both understanding structure-property relationships and designing functional materials. In pursuit of these goals, we developed a new synthetic approach that enables facile manipulation of the density and distribution of grafts in polymers via living ring-opening metathesis polymerization (ROMP). Discrete endo,exo-norbornenyl dialkylesters (dimethyl DME, diethyl DEE, di-n-butyl DBE) were strategically designed to copolymerize with a norbornene-functionalized polystyrene (PS), polylactide (PLA), or polydimethylsiloxane (PDMS) macromonomer mediated by the third-generation metathesis catalyst (G3). The small-molecule diesters act as diluents that increase the average distance between grafted side chains, generating polymers with variable grafting density. The grafting density (number of side chains/number of norbornene backbone repeats) could be straightforwardly controlled by the macromonomer/diluent feed ratio. To gain insight into the copolymer sequence and architecture, self-propagation and cross-propagation rate constants were determined according to a terminal copolymerization model. These kinetic analyses suggest that copolymerizing a macromonomer/diluent pair with evenly matched self-propagation rate constants favors randomly distributed side chains. As the disparity between macromonomer and diluent homopolymerization rates increases, the reactivity ratios depart from unity, leading to an increase in gradient tendency. To demonstrate the effectiveness of our method, an array of monodisperse polymers (PLAx-ran-DME1-x)n bearing variable grafting densities (x = 1.0, 0.75, 0.5, 0.25) and total backbone degrees of polymerization (n = 167, 133, 100, 67, 33) were synthesized. The approach disclosed in this work therefore constitutes a powerful strategy for the synthesis of polymers spanning the linear-to-bottlebrush regimes with controlled grafting density and side chain distribution, molecular attributes that dictate micro- and macroscopic properties.

A modular open platform for systematic functional studies under physiological conditions.

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.

Anchoring interprofessional education in undergraduate curricula: The Heidelberg story.

The ability of health professionals to collaborate effectively has significant potential impact on patient safety and quality-care outcomes, especially given the increasingly complex and dynamic clinical practice environments of today. Educators of the health professions are faced with an immediate challenge to adapt curricula and traditional teaching methods to ensure graduates are equipped with the necessary interprofessional competencies and (inter)professional values for their future practice. The World Health Organization's "Framework for action in interprofessional education (IPE) and collaborative practice" promotes IPE as a key strategy to enhance patient outcomes by preparing a "collaborative practice-ready health workforce." Logistical and attitudinal barriers can hinder integration of IPE into curricula. Lessons learned through the implementation of a planned change to establish four interprofessional seminars (team communication, medical error communication, healthcare English, and small business management) at Heidelberg University Medical Faculty, Germany, are described. A key factor in successfully anchoring IPE seminars in the undergraduate curricula was the structured approach drawing on change management concepts.

Phosphorylation of TET proteins is regulated via O-GlcNAcylation by the O-linked N-acetylglucosamine transferase (OGT).

TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli.

In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides.

BACKGROUND & AIMS: Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting. METHODS: A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively. RESULTS: ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro. CONCLUSION: Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation.

The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes.

PD-BAT: A novel approach of pooling basophil donors for expansion of commercial laboratory testing of Chronic Spontaneous Urticaria.

The type II autoimmune subtype of Chronic Spontaneous Urticaria (CSU) is characterized by the presence of IgG autoantibodies targeting IgE or the IgE high-affinity receptor (FcεRI) on mast cells and basophils. In evaluation of CSU patients, indirect basophil activation testing (BAT), has been utilized, involving the mixing of patient serum with heterologous peripheral blood donors, followed by flow cytometric assessment of basophil markers. However, the reliability of the indirect BAT results hinges on the quality of the donor basophils utilized. In this study, we introduce an innovative approach where multiple potential basophil donors undergo rigorous BAT characterization alongside control samples. By selecting and pooling donors with optimal performance, we significantly enhance the inter-assay reproducibility of the indirect BAT test.

Light harvesting enhancement in solar cells with quasicrystalline plasmonic structures.

Solar cells are important in the area of renewable energies. Since it is expensive to produce solar-grade silicon [Electrochem. Soc. Interface 17, 30 (2008)], especially thin-film solar cells are interesting. However, the efficiency of such solar cells is low. Therefore, it is important to increase the efficiency. The group of Polman has shown that a periodic arrangement of metal particles is able to enhance the absorbance of light [Nano Lett. 11, 1760 (2011)]. However, a quasicrystalline arrangement of the metal particles is expected to enhance the light absorbance independent of the incident polar and azimuthal angles due to the more isotropic photonic bandstructure. In this paper, we compare the absorption enhancement of a quasiperiodic photonic crystal to that of a periodic photonic crystal. We indeed find that the absorption enhancement for the quasicrystalline arrangement shows such an isotropic behavior. This implies that the absorption efficiency of the solar cell is relatively constant during the course of the day as well as the year. This is particularly important with respect to power distribution, power storage requirements, and the stability of the electric grid upon massive use of renewable energy.

Unbiased probing of the entire hepatitis C virus life cycle identifies clinical compounds that target multiple aspects of the infection.

Over 170 million people are chronically infected by the hepatitis C virus (HCV) and at risk for dying from liver cirrhosis and hepatocellular carcinoma. Current therapy is expensive, associated with significant side effects, and often ineffective. Discovery of antiviral compounds against HCV traditionally involves a priori target identification followed by biochemical screening and confirmation in cell-based replicon assays. Typically, this results in the discovery of compounds that address a few predetermined targets and are prone to select for escape variants. To attempt to identify antiviral compounds with broad target specificity, we developed an unbiased cell-based screening system involving multiple rounds of infection in a 96-well format. Analysis of a publicly available library of 446 clinically approved drugs identified 33 compounds that targeted both known and previously unexplored aspects of HCV infection, including entry, replication, and assembly. Discovery of novel viral and cellular targets in this manner will broaden the therapeutic armamentarium against this virus, allowing for the development of drug mixtures that should reduce the likelihood of mutational escape.

Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning.

Since its development in the 1980s, the Nobel Prize-awarded phage display technology has been one of the most commonly used in vitro selection technologies for the discovery of therapeutic and diagnostic antibodies. Besides the importance of selection strategy, one key component of the successful isolation of highly specific recombinant antibodies is the construction of high-quality phage display libraries. However, previous cloning protocols relied on a tedious multistep process with subsequent cloning steps for the introduction of first heavy and then light chain variable genetic antibody fragments (VH and VL). This resulted in reduced cloning efficiency, higher frequency of missing VH or VL sequences, as well as truncated antibody fragments. With the emergence of Golden Gate Cloning (GGC) for the generation of antibody libraries, the possibility of more facile library cloning has arisen. Here, we describe a streamlined one-step GGC strategy for the generation of camelid heavy chain only variable phage display libraries as well as the simultaneous introduction of heavy chain and light chain variable regions from the chicken into a scFv phage display vector.

Generation and engineering of potent single domain antibody-based bispecific IL-18 mimetics resistant to IL-18BP decoy receptor inhibition.

Here, we generated bispecific antibody (bsAb) derivatives that mimic the function of interleukin (IL)-18 based on single domain antibodies (sdAbs) specific to IL-18 Rα and IL-18 Rß. For this, camelids were immunized, followed by yeast surface display (YSD)-enabled discovery of VHHs targeting the individual receptor subunits. Upon reformatting into a strictly monovalent (1 + 1) bispecific sdAb architecture, several bsAbs triggered dose-dependent IL-18 R downstream signaling on IL-18 reporter cells, as well as IFN-γ release by peripheral blood mononuclear cells in the presence of low-dose IL-12. However, compared with IL-18, potencies and efficacies were considerably attenuated. By engineering paratope valencies and the spatial orientation of individual paratopes within the overall design architecture, we were able to generate IL-18 mimetics displaying significantly augmented functionalities, resulting in bispecific cytokine mimetics that were more potent than IL-18 in triggering proinflammatory cytokine release. Furthermore, generated IL-18 mimetics were unaffected from inhibition by IL-18 binding protein decoy receptor. Essentially, we demonstrate that this strategy enables the generation of IL-18 mimetics with tailor-made cytokine functionalities.

Cerebrospinal fluid in Borna disease virus 1 (BoDV-1) encephalitis.

Borna disease virus 1 (BoDV-1) has been recognized as a rare cause of very severe encephalitis with rapid onset in central Europe. Data on cerebrospinal fluid (CSF) analysis have not yet been analyzed in detail. Here, we present the first study on CSF changes in BoDV-1 encephalitis. We retrospectively analyzed CSFs from 18 BoDV-1 encephalitis cases from Bavaria, Germany, an endemic region, from 1996 to 2021. Data were obtained through review of medical records and institutional databases. We found that white blood cell count (WBC) in CSF is elevated in 13 of our 18 patients at first examination (average 83.2 ± 142.3 leukocytes/µl) and cytology showed predominance of lymphocytes. Patients with typical symptoms of meningoencephalitis had higher WBC in first CSF analyzation (133.5 ± 163.1 vs 4.0 ± 3.2/µl; p = 0.065). BoDV-1 PCR of CSF is not always positive when tested (7 of 9 cases). Four of five patients tested showed a polyvalent reaction against multiple viruses in the CSF suggesting that BoDV-1 may trigger autoimmune mechanisms. CSF changes in BoDV-1 encephalitis seem similar to those of other viral encephalitis and at the beginning WBC can be normal in up to 28%, making the diagnosis even more challenging. All in all, BoDV-1 should be included in the diagnostic workup of patients with rapidly evolving and/or severe encephalitis and patients with severe neuropathy and secondary encephalopathy with and without CSF changes. Repeated CSF examinations as well as BoDV-1 serology and CSF PCR have to be considered in endemic areas.