FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

Visualization of uracils created by APOBEC3A using UdgX shows colocalization with RPA at stalled replication forks.

The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in U•G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.

Photoactivatable Glycolipid Probes for Identifying Mycolate-Protein Interactions in Live Mycobacteria.

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

Bioorthogonal Chemical Reporters for Selective In†Situ Probing of Mycomembrane Components in Mycobacteria.

The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.

Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides.

Celiac disease (CD) is a common CD4(+) T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin-specific T cell response in vivo. We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLA-DQ2.5(+) CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye. Avenin-specific responses were observed in 6/73 HLA-DQ2.5(+) CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability. Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.

A redox-sensitive iron-sulfur cluster in murine FAM72A controls its ability to degrade the nuclear form of uracil-DNA glycosylase.

Murine FAM72A, mFAM72A, binds the nuclear form of uracil-DNA glycosylase, mUNG2, inhibits its activity and causes its degradation. In immunoprecipitation assays the human paralog, hFAM72A, binds hUNG2 and is a potential anti-cancer drug target because of its high expression in many cancers. Using purified mFAM72A, and mUNG2 proteins we show that mFAM72A binds mUNG2, and the N-terminal 25 amino acids of mUNG2 bind mFAM72A at a nanomolar dissociation constant. We also show that mFAM72A is present throughout the cells, and mUNG2 helps localize it to nuclei. Based on in silico models of mFAM72A-mUNG2 interactions, we constructed several mutants of mFAM72A and found that while they have reduced ability to deplete mUNG2, the mutations also destabilized the former protein. We confirmed that Withaferin A, a predicted lead molecule for the design of FAM72A inhibitors, binds mFAM72A with micromolar affinity but has little affinity to mUNG2. We identified two potential metal-binding sites in mFAM72A and show that one of the sites contains an Fe-S cluster. This redox-sensitive cluster is involved in the mFAM72A-mUNG2 interaction and modulates mFAM72A activity. Hydrogen peroxide treatment of cells increases mUNG2 depletion in a FAM72A-dependent fashion suggesting that mFAM72A activity is redox-sensitive.

PPE51 mediates uptake of trehalose across the mycomembrane of Mycobacterium tuberculosis.

The disaccharide trehalose is essential for viability of Mycobacterium tuberculosis, which synthesizes trehalose de novo but can also utilize exogenous trehalose. The mycobacterial cell wall encompasses two permeability barriers, the cytoplasmic membrane and the outer mycolic acid-containing mycomembrane. The ABC transporter LpqY-SugA-SugB-SugC has previously been demonstrated to mediate the specific uptake of trehalose across the cytoplasmic membrane. However, it is still unclear how the transport of trehalose molecules across the mycomembrane is mediated. In this study, we harnessed the antimycobacterial activity of the analogue 6-azido trehalose to select for spontaneous resistant M. tuberculosis mutants in a merodiploid strain harbouring two LpqY-SugA-SugB-SugC copies. Mutations mediating resistance to 6-azido trehalose mapped to the proline-proline-glutamate (PPE) family member PPE51 (Rv3136), which has recently been shown to be an integral mycomembrane protein involved in uptake of low-molecular weight compounds. A site-specific ppe51 gene deletion mutant of M. tuberculosis was unable to grow on trehalose as the sole carbon source. Furthermore, bioorthogonal labelling of the M. tuberculosis Δppe51 mutant incubated with 6-azido trehalose corroborated the impaired internalization. Taken together, the results indicate that the transport of trehalose and trehalose analogues across the mycomembrane of M. tuberculosis is exclusively mediated by PPE51.

Human Herpes Simplex Virus-1 depletes APOBEC3A from nuclei.

APOBEC3 family of DNA-cytosine deaminases inactivate and mutate several human viruses. We constructed a human cell line that is inducible for EGFP-tagged APOBEC3A and found A3A predominantly in the nuclei. When these cells were infected with Herpes Simplex Virus-1, virus titer was unaffected by A3A expression despite nuclear virus replication. When A3A expression and virus infection were monitored, A3A was found predominantly to be nuclear in infected cells up to 3 h post-infection, but was predominantly cytoplasmic by 12 h. This effect did not require the whole virus, and could be reproduced using the UL39 gene of the virus which codes for a subunit of the viral ribonucleotide reductase. These results are similar to the reported exclusion of APOBEC3B by Epstein Barr virus ortholog of UL39, BORF2, but HSV1 UL39 gene product appears better at excluding A3A than A3B from nuclei.

Treatment of Maternal Depression in a Medication Clinical Trial and Its Effect on Children.

(Reprinted from the American Journal of Psychiatry 2015; 172:450-459, with permission from American Psychiatric Association Publishing).

Investigations into the influence of host genetics on the predominant eubacteria in the faecal microflora of children.

The eubacterial population was studied in faecal samples of related and unrelated children. Temporal temperature gradient gel electrophoresis (TTGE) provided a snapshot of the bacterial population and allowed calculation of the degree of similarity in the predominant faecal microflora of identical twin pairs, fraternal twin pairs and unrelated paired controls. The highest levels of similarity were found in genetically identical twins. Significant differences were observed between the identical and fraternal twins (P = 0.037), strongly suggesting a genetic influence over the composition of the faecal microflora. The unrelated control group had the lowest similarity and was significantly different from the twins (P = 0.001). The results of this study indicate that host genetics influence the composition of the dominant eubacterial population in children.

A trifunctional cyclooctyne for modifying azide-labeled biomolecules with photocrosslinking and affinity tags.

A bicyclo[6.1.0]nonyne (BCN)-based cyclooctyne reagent bearing a photocrosslinking diazirine (DAz) group and a biotin affinity handle, BCN-DAz-Biotin, is reported. BCN-DAz-Biotin is capable of simultaneously delivering photocrosslinking and affinity tags to azide-labeled biomolecules, enabling photoactivated capture and enrichment/detection of interacting species in native contexts.

Treatment of maternal depression in a medication clinical trial and its effect on children.

OBJECTIVE: Observational studies show that when a depressed mother's symptoms remit, her children's psychiatric symptoms decrease. Using randomized treatment assignment, the authors sought to determine the differential effects of a depressed mother's treatment on her child. METHOD: The study was a randomized double-blind 12-week trial of escitalopram, bupropion, or the combination of the two in depressed mothers (N=76), with independent assessment of their children (N=135; ages 7-17 years). RESULTS: There were no significant treatment differences in mothers' depressive symptoms or remission. Children's depressive symptoms and functioning improved significantly among those whose mothers were in the escitalopram group (compared with those whose mothers were in the bupropion and combination treatment groups). Only in the escitalopram group was significant improvement of mother's depression associated with improvement in the child's symptoms. Exploratory analyses suggested that this may be due to changes in parental functioning: Mothers in the escitalopram group reported significantly greater improvement, compared with the other groups, in their ability to listen and talk to their children, who as a group reported that their mothers were more caring over the 12 weeks. Maternal baseline negative affectivity appeared to moderate the effect of maternal treatment on children, although the effect was not statistically significant. Children of mothers with low negative affectivity improved in all treatment groups. Children of mothers with high negative affectivity improved significantly only for those whose mothers were in the escitalopram group. CONCLUSIONS: The effects of the depressed mother's improvement on her children may depend on her type of treatment. Depressed mothers with high anxious distress and irritability may require medications that reduce these symptoms in order to show the effect of her remission on her children.

Can people with nonsevere major depression benefit from antidepressant medication?

BACKGROUND: Several meta- or mega-analyses suggest antidepressant medications should be given only to severely depressed patients. In our experience, mild depression benefits from medication. We reanalyzed 1 clinic's randomized placebo-controlled antidepressant studies, limiting analyses to patients with major depressive disorder (MDD) without severe illness, to determine whether nonsevere depression responds to antidepressant medication. DATA SOURCES: Archives of the Depression Evaluation Service outpatient clinic of the New York State Psychiatric Institute were searched for randomized, placebo-controlled antidepressant studies that were conducted between 1977 and 2009 and included patients having MDD and pretreatment Hamilton Depression Rating Scale (HDRS) scores < 23. STUDY SELECTION: Six placebo-controlled studies were found, including 8 active treatment arms and 1,440 patients. 825 patients were randomized and had MDD and an HDRS score < 23. DSM-III, DSM-III-R, or DSM-IV diagnostic criteria contemporary to each study were employed. DATA EXTRACTION: Treatments were compared within study and via a patient-level meta-analysis using analysis of covariance (ANCOVA) of HDRS end point scores adjusted for pretreatment score. The number needed to treat (NNT) was calculated from remission rates (HDRS end point score ≤ 7), which were compared by χ². Effect sizes were calculated from change in HDRS scores. Secondary analyses investigated the effect of chronicity and atypical features on treatment response. DATA SYNTHESIS: Three of 6 studies showed significant (P < .001) treatment effects by ANCOVA, and 4 of 6 studies showed significant (P < .04) differences in remission. The NNT ranged from 3 to 8. Effect sizes ranged from -0.04 to 0.8, with 4 of 8 greater than 0.4. The patient-level meta-analysis confirmed these results; neither chronicity nor atypical features significantly affected outcome. Secondary analyses utilizing global ratings and self-report mimicked the main findings. CONCLUSIONS: Several studies demonstrated significant antidepressant efficacy for patients having nonsevere MDD. Efficacy was not trivial, as NNT ranged from 3 to 8, a range accepted by researchers as sufficiently robust to recommend treatment. These findings suggest mild-moderate MDD can benefit from antidepressants, contrary to findings by several other meta- or mega-analyses.

Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease.

Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4(+ )T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat alpha-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from omega-gliadin (wheat) and C-hordein (barley) but not alpha-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.