Two Forms of Activation-Induced Cytidine Deaminase Differing in Their Ability to Bind Agarose

Background: Activation-induced cytidine deaminase (AID) is a B-cell-specific DNA mutator that plays a key role in the formation of the secondary antibody repertoire in germinal center B cells. In the search for binding partners, protein coimmunoprecipitation assays are often performed, generally with agarose beads. Methodology/Principal Findings: We found that, regardless of whether cell lysates containing exogenous or endogenous AID were examined, one of two mouse AID forms bound to agarose alone. Conclusions/Significance: These binding characteristics may be due to the known post-translational modifications of AID; they may also need to be considered in coimmunoprecipitation experiments to avoid false-positive results

AID Komplexe im Zytoplasma und ihre Funktion in angeborener Immunität

Aktivierungsinduzierte Cytidin-Deaminase (AID) gehört zur APOBEC-Protein-Familie und ist ein B-Zell-spezifischer Mutator, der Cytosin zu Uracil in DNA, und möglicherweise auch RNA desaminiert. Familienmitglieder von AID vermitteln angeborene Immunität, indem sie vor Retroviren und Retroelementen schützen; AID hingegen ist bekannt für seine Funktion in adaptiver Immunität bei der Erzeugung des sekundären Antikörperrepertoires in B-Zellen des Keimzentrums. Nach Antigen-vermittelter B-Zell-Aktivierung leitet AID somatische Hypermutation sowie den Klassenwechsel von Immunglobulingenen ein – das bringt hoch spezifische Antikörper mit unterschiedlichen Effektorfunktionen hervor. Das Ziel meiner Doktorarbeit bestand darin zu untersuchen, ob AID seinen APOBEC3 Familienmitgliedern funktionell ähnlich ist in Bezug auf das Vermitteln von angeborener Immunität. APOBEC3G bildet zytoplasmatische Komplexe aus, die Retroelemente enthalten und inaktivieren. Die spezifische Aufgabenstellung war es zu untersuchen, ob AID auch Komplexe bildet, und wenn ja, welche Funktion und Zusammensetzung diese haben. Um diese Fragestellung zu beantworten, habe ich Immunpräzipitationen mit Agarose-Beads durchgeführt und kam zu meinem ersten Ergebnis: es gibt zwei Formen von AID – eine bindet an Agarose, die andere nicht. In anschließenden Experimenten habe ich gezeigt, dass AID in primären B-Zellen endogene L1-Retroelemente inhibiert, indem es hochmolekulare Komplexe mit deren mRNA- und Proteinprodukten bildet. Abwesenheit von AID hat eine stark erhöhte Expression von L1-Retroelementen zur Folge. Im Einklang mit Daten aus den letzten Jahren, nach denen endogene Retroelemente Autoimmunerkrankungen verursachen können, erklären meine Ergebnisse möglicherweise, warum AID-defiziente Mäuse und Patienten unter lymphoider Hyperplasie und Autoimmunerkrankungen leiden. Ich postuliere, dass AID angeborene Immunität vermittelt, indem es B-Zellen vor nachteiligen Retroelement-Effekten schützt, und zwar vor (1) Insertionsmutagenese, die lymphoproliferative Erkrankung verursachen kann, und (2) intrazellulärer Akkumulation von Retroelementen, die Autoimmunerkrankung auslösen kann. In meiner Arbeit habe ich eine zusätzliche Funktion von AID nachgewiesen, nämlich, dass AID angeborene Immunität gegen endogene Retroelemente vermittelt.A member of the APOBEC protein family, activation-induced cytidine deaminase (AID) is a B cell-specific mutator that deaminates cytosine to uracil in DNA and possibly RNA. Family members of AID function in innate immunity by protecting from retroviruses and retroelements, whereas AID has been known for its key function in adaptive immunity by shaping the secondary antibody repertoire in germinal center B cells. After B cell activation by antigen, AID initiates somatic hypermutation and class switching of immunoglobulin genes, which generates highly specific antibodies and changes the effector functions of antibodies, respectively. The objective of my thesis was to investigate whether AID functionally resembles its APOBEC3 family members in regard to mediating innate immunity. APOBEC3G forms large cytoplasmic complexes containing and thus inactivating retroelements. The primary specific aim was to examine whether AID also forms complexes, and if so, their function and composition. To address this, I performed agarose bead based immunoprecipitations and found that there are two forms of AID with different agarose binding abilities – one that binds to agarose and one that clearly does not. In subsequent experiments I demonstrated in primary B cells that AID restricts endogenous L1 retroelements by forming cytoplasmic high molecular mass complexes with their mRNA and their protein products. I found that absence of AID greatly increased the expression of L1 retrotransposons. Along with recent data that endogenous retroelements can cause autoimmune disease, my findings may explain why AID-deficient mice and patients suffer from both lymphoid hyperplasia and autoimmune disease. I propose that AID provides innate immunity by protecting B cells from the adverse effects of retroelements – (i) insertional mutagenesis causing lymphoproliferative disease and (ii) intracellular accumulation of retroelements causing autoimmune disease. My thesis thus uncovered another function of AID, i.e., mediating innate immunity to endogenous retroelements

LINE-1 retroelements complexed and inhibited by activation induced cytidine deaminase.

LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus

Exploring communication preferences of trans and gender diverse individuals-A qualitative study.

BackgroundTrans and gender-diverse individuals experience adverse health outcomes that might be due, in addition to other factors, to stigma and discrimination in the health care sector. At the same time, the concept of person-centred care acknowledges the role of patient-physician communication in health care outcomes. This study aims to explore patient-physician communication preferences in trans and gender-diverse individuals.MethodA qualitative interview study was conducted, including N = 10 participants between February and March 2022. Participants were interviewed using a semi-structured interview guideline, based on previous knowledge in person-centred care and sample specific communication. Participants were asked about their experiences and wishes in patient-physician centeredness. Analyses were conducting using a qualitative content analysis strategy.ResultsMean age was 29.3 years; n = 6 participants identified themselves within the binary gender concept, while n = 4 identified themselves with a non-binary gender. Communication preferences for patient-physician communication were categorised into four themes: general communication aspects (e.g. active listening); the role of gender during appointments (e.g. appropriate/inappropriate addressing); gender-neutral language (e.g. experiences use of gender neutral language by physicians); own communication style (e.g. early outing and justification). Furthermore, possible contextual factors of patient-physician communication where found (e.g. trusting relationship).ConclusionAdding knowledge to communication preferences of trans and gender-diverse individuals, this study was able to identify preferences that are specific to the sample as well as preferences that differ from the cis-gendered population. However, it remains unclear how the patient-physician communication preferences affects health care utilization and outcomes.Trial registrationGerman Clinical Trial Register (DRKS00026249)

AID inhibits L1 retrotransposition.

<p>(<b>A</b>) L1 retrotransposition reporter construct. The plasmid contains the human L1_RP element with its proteins, ORF1 and ORF2. The construct also contains a puromycin resistance marker to select for transfected cells (not shown). The intron (in red) in the EGFP is in the same transcriptional orientation as ORF1 and ORF2 (indicated by the arrowhead of the ORF2 exon). When transcribed from the L1 promoter, the GFP gene is spliced, but it cannot be translated, as it is in the inverse orientation and does not encode functional protein. When transcribed from the EGFP promoter (opposite orientation; indicated by the arrowhead of the second EGFP exon), it cannot be spliced and thus not translated. Only when the L1 transcript is spliced, reverse-transcribed and inserted into the genome (a retrotransposition event) is GFP protein expressed. (<b>B</b>) Retrotransposition assay in HEK293 cells. Above, GFP-positive cells as determined by flow cytometry on days 3 and 6 after transfection of the reporter construct (L1) and/or AID. L1 mut is a reporter construct in which the ORF1 contains two missense mutations. Scales on the y-axis differ for day 3 and day 6. Values represent mean ± SD; n = 2 transfection samples. Student's t test; *p≤0.01 and **p≤0.001. Below, Western blots of lysates from cells 3 days after transfection, developed with anti-AID, anti-ORF1, or anti-actin antibody. The position of the molecular mass standard (in kDa) is indicated next to the blots. (<b>C</b>) Retrotransposition assay in WEHI-231 cells. GFP-positive cells as determined by flow cytometry on day 6 after transfection of the reporter constructs L1 and L1 mut. +, AID-positive WEHI-231 subclone; –, AID-negative WEHI-231 subclone. Values represent mean ± SD; n = 2 transfection samples. Student's t test; *p≤0.01.</p

AID binds to L1 mRNA.

<p>(<b>A and B</b>) Agarose gels of products from RT-PCR on RNA-immunoprecipitates (RNA-IP) showing RNA binding for endogenous (A) and exogenous (B) AID from murine B lymphoblasts. Proteins and nucleic acids contained in 1×10⁸ LPS- plus IL-4-stimulated AID-deficient (AID −) and wild-type (AID +) B cells (A) or 7.5×10⁷ LPS- plus IL-4-stimulated AID-deficient B cells, transduced with Flag-GFP (AID −) or Flag-AID (AID +) (B), were cross-linked via treatment with UV (UV +), or not treated (UV−); the cells were then lysed, followed by immunoprecipitation with monoclonal anti-AID (A) or monoclonal anti-Flag (B) antibody. After DNase digest, RT-PCR using oligo(dT) primers, followed by L1-, germ line transcript (GLT)-, Ig κ-light chain (κ chain)-, Ig µ-heavy chain (µ chain)- or GAPDH-specific primers, was performed on the immunoprecipitates (“RT-PCR” panel, “RNA-IP”). To monitor the amount of AID in lysate and immunoprecipitates, we analyzed aliquots of lysates (WB panel, “input”) and immunoprecipitates (WB panel, “IP”), both equivalent to 5×10⁶ cells, and electrophoresed, Western blotted and developed them with an anti-actin, anti-Flag or anti-AID antibody. Lanes 1–4, RNA-IP samples; lanes 5–7, cDNA synthesis and PCR controls, i.e., total RNA of LPS- plus IL-4-stimulated AID-deficient and wild-type B cells with (+ RT) or without (− RT) reverse transcriptase. (<b>C</b>) Left: identity of L1 elements in the immunoprecipitates confirmed by cDNA sequencing. Sequences (nt 151–200) of the 300-bp L1 ORF2 fragments that were amplified from RNA-IP shown in panel A. M13002, L1 reference sequence of BALB/c strain origin; L1.1 and L1.2, sequences obtained from RNA-IP of lane 2 in panel A; L1.3 to L1.5, sequences obtained from lane 4 in panel A. Right: schematic of a full-length L1 element. Arrows indicate the position of the primers used to amplify the 300-bp L1 ORF2 fragment from RNA-IP shown in panel A and B. The numbers represent the nucleotide positions according to the L1Md-A2 sequence [L1 sequence, Genbank:M13002] <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049358#pone.0049358-Loeb1" target="_blank">[68]</a>.</p

Activated B lymphocytes express L1 mRNA, but not L1 protein.

<p>(<b>A and B</b>) L1, IAP and MLV mRNA expression. After digestion with DNase, mRNA was reverse-transcribed into cDNA using oligo(dT) primers, followed by L1-, IAP- and MLV-specific quantitative real-time PCR. PCR data were quantified according to the comparative <i>C</i>_T method. The amount of target mRNA was normalized to the endogenous control 18S ribosomal RNA. Values represent mean ± SD; y-axis, fold change in mRNA expression. (A) LPS- plus IL-4-stimulated (LPS) and unstimulated B lymphocytes from the spleens of AID-deficient (AID KO; n = 12 mice) and wild-type (WT; n = 12 mice) BALB/c mice. Student's t test, *p≤0.05 and **p≤0.001. (B) Tissues (pancreas, testis) and activated B cells (spleen LPS) from a wild-type BALB/c mouse and mouse cell lines F9 and MOPC104E; F9, mouse embryonal carcinoma cell line expressing high levels of L1 mRNA; spleen LPS, LPS- plus IL-4-stimulated B lymphocytes, as in panel A; n = 2 independent experiments. (<b>C and D</b>) ORF1 protein expression. Cell lysates were electrophoresed, Western blotted and developed with anti-ORF1 or anti-actin antibody. The position of the molecular mass standard (in kDa) is indicated to the left of the blots; the positions of the exogenous (exo) and endogenous (endo) human (hORF1) and mouse (mORF1) ORF1 bands are indicated to the right of the blots; LPS blasts, lysates of LPS- plus IL-4-activated B lymphocytes from BALB/c mice; + AID and – AID, AID-sufficient and AID-deficient, respectively. (C) anti-mORF1 staining; testis, whole testis lysates; IR, cells gamma irradiated 24 h before lysis with 0, 1 or 50 Gy. (D) anti-hORF1 staining; 2102Ep, lysate from a human embryonal carcinoma cell line expressing high levels of ORF1; Phoenix and LPS blasts, lysates of cells transfected (Phoenix) or transduced (LPS Blasts) with a retroviral human ORF1 (+ exo hORF1) or a GFP-only construct (− exo hORF1).</p

AID decreases the steady-state level of L1 protein.

<p>The doxycycline (Dox)-inducible construct encodes AID and GFP, expressed as two proteins; HeLa, untransfected HeLa cells; HeLa-AID, HeLa clone stably transfected with the inducible AID construct; – Dox, uninduced cells; + Dox, induced cells. (<b>A</b>) AID induction in HeLa cells: flow cytometry analysis. GFP expression (x-axis) as an indicator of AID expression. Numbers represent the percentage of GFP-positive cells; empty and black parts of the histogram are – Dox. (<b>B</b>) AID induction in HeLa cells: Western blot analysis. HeLa cell lysates were electrophoresed, Western blotted and developed with monoclonal anti-AID antibody. The position of the molecular mass standard (in kDa) is indicated next to the blot. (<b>C</b>) L1 mRNA levels in HeLa cells. After digestion with DNase, mRNA of HeLa cells was reverse-transcribed into cDNA using oligo(dT) primers, followed by L1-specific quantitative real-time PCR. PCR data were quantified according to the comparative <i>C</i>_T method. The amount of L1 target mRNA was normalized to the endogenous control GAPDH. Values represent mean ± SD; n = 3 independent experiments; y-axis, fold change in L1 mRNA expression (HeLa-AID set to 1.0). (<b>D</b>) ORF1 protein levels in HeLa cells. HeLa cell lysates were electrophoresed, Western blotted and developed with anti-ORF1, anti-AID or anti-actin antibody. 2102Ep, lysate from a human embryonal carcinoma cell line expressing high levels of ORF1. The position of the molecular mass standard (in kDa) is indicated next to the blots. (<b>E</b>) Western blot developed with ORF1 (above) and AID (below). ORF1 protein was co-precipitated with AID in lysates of Flag-AID (+)- and Flag-GFP (−)-transfected Phoenix cells (derived from HEK293 cells). Lysate, input used for IP; IP, immunoprecipitates; anti-Flag, immunoprecipitation with Flag-specific antibody; –, immunoprecipitation control without antibody. The position of the molecular mass standard (in kDa) is indicated next to the blots.</p

AID forms high-molecular-mass complexes in the cytoplasm.

<p>Fractionation according to size by gel filtration of A3G (A) and AID (B and C), followed by Western blot analysis. Numbers above panels, fractions of separation by FPLC; numbers to the left of panels, molecular mass standards (in kDa) of the SDS gel run; numbers below panels, molecular mass standards (in kDa) of the fractions of the FPLC run. (<b>A</b>) Western blots of fractions of FPLC eluates were developed with polyclonal antibody to A3G. Input, untreated lysate of A3G-positive cells; input + RNase, RNase A-treated lysate of A3G-positive cells; A3G minus, lysate of A3G-negative cells; H9, A3G-positive cell line; fractions 3–16, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from human A3 transgenic mice. Upper panel, untreated cell lysate (devoid of nuclei); lower panel, treated with RNase A before fractionation on FPLC. (<b>B and C</b>) Western blots of fractions of FPLC eluates developed with monoclonal antibody to AID. AID minus, lysate of AID-negative HeLa cells; AID plus, lysate of AID-positive HeLa cells; fractions 3–15, fractionated lysates of LPS- plus IL-4-activated B lymphocytes from AID-sufficient mice (B) and of AID-positive HeLa cells (C). Top panel, untreated cell lysate (devoid of nuclei); middle panel, treated with RNase A before fractionation on FPLC; bottom panel, treated with RNase A and RNase inhibitors before fractionation. Concomitant with RNase A treatment, proteins were somewhat digested for unknown reasons.</p

Protective mental health factors in children of parents with alcohol and drug use disorders: A systematic review

<div><p>Children of parents with drug and alcohol use disorders often grow up under severe stress and are at greater risk of developing psychological and social problems. However, a substantial proportion of affected children adapt to their distressing life conditions and show positive development in terms of their mental health. These children are described as resilient. One difference between resilient and maladapted children is the presence of protective factors. The aim of this systematic review is to provide an overview of the current state of the research concerning protective mental health factors in children of parents with alcohol or drug use disorders (COPAD). For that purpose, the PsychInfo, PubMed, CINAHL and ISI Web of Science databases were searched through January 2017. All the identified publications were screened using previously developed inclusion criteria. The search yielded 3,402 articles. Eleven of these publications (2003–2013) met the criteria for inclusion in the present review. Information on the studies was extracted using an extraction form. A narrative analysis was performed, and the methodological quality was examined using a checklist based on the Mixed Methods Appraisal Tool. The research identified familial, parental, child-related and biological factors that influenced mental health outcomes in affected children (N = 1,376, age range = 1–20 years). Overall, protective mental health factors are understudied in this target group. Most of the included studies were conducted in the United States and employed a cross-sectional design. A comparison of the included cross-sectional and longitudinal studies indicated consistent results related to a secure parent-child attachment. Based on the current state of the research, no causal conclusions with regard to the effectiveness of protective factors can be drawn. To develop effective prevention programs, further longitudinal studies and studies assessing the interactions between risk and protective factors are needed.</p></div