Publisher Correction: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

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Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.

DNA Polymerase θ: A Unique Multifunctional End-Joining Machine.

The gene encoding DNA polymerase θ (Polθ) was discovered over ten years ago as having a role in suppressing genome instability in mammalian cells. Studies have now clearly documented an essential function for this unique A-family polymerase in the double-strand break (DSB) repair pathway alternative end-joining (alt-EJ), also known as microhomology-mediated end-joining (MMEJ), in metazoans. Biochemical and cellular studies show that Polθ exhibits a unique ability to perform alt-EJ and during this process the polymerase generates insertion mutations due to its robust terminal transferase activity which involves template-dependent and independent modes of DNA synthesis. Intriguingly, the POLQ gene also encodes for a conserved superfamily 2 Hel308-type ATP-dependent helicase domain which likely assists in alt-EJ and was reported to suppress homologous recombination (HR) via its anti-recombinase activity. Here, we review our current knowledge of Polθ-mediated end-joining, the specific activities of the polymerase and helicase domains, and put into perspective how this multifunctional enzyme promotes alt-EJ repair of DSBs formed during S and G2 cell cycle phases.

Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice kill B cells, suppress humoral immunity and expedite early mortality.

Catenation and encapsulation induce distinct reconstitutions within a dynamic library of mixed-ligand Zn4L6 cages.

Two new Zn4L6 cages composed of diamine subcomponents containing either naphthalene diimide (NDI) or porphyrin moieties are described. Their structural differences allow these cages to exhibit distinct interactions with different chemical stimuli, yielding different supramolecular products. The electron-poor NDI subunits of the first cage were observed to thread through electron-rich aromatic crown-ether macrocycles, forming mechanically-interlocked species up to a [3]catenane, whereas the porphyrin ligands of the second cage interacted favourably with C70, causing it to be bound as a guest. When mixed, the two cages were observed to form a dynamic combinatorial library (DCL) of seven constitutionally distinct mixed-ligand Zn4L6 cages. The DCL was observed to reconstitute in opposing ways when treated with either the crown ether or C70: the electron-rich macrocycle templated the formation of heteroleptic catenanes, whereas C70 caused the DCL to self-sort into homoleptic structures.

Stacking Interactions Drive Selective Self-Assembly and Self-Sorting of Pyrene-Based M(II)4L6 Architectures.

Subcomponent self-assembly of two isomeric bis(3-aminophenyl)pyrenes, 2-formylpyridine and the metal ions Fe(II), Co(II), and Zn(II) led to the formation of two previously unidentified structure types: a C2-symmetric M(II)4L6 assembly with meridionally coordinated metal centers, and a C3-symmetric self-included M(II)4L6 assembly with facially coordinated metal centers. In both structures the meta linkages within the ligands facilitate π-stacking between the pyrene panels of the ligands. A C2h-symmetric M(II)2L2 box was also obtained, which was observed to selectively bind electron-deficient aromatic guests between two parallel pyrene subunits. Similar donor-acceptor interactions drove the selective self-assembly of a singular M(II)4L4L'2 architecture incorporating both a pyrene-containing diamine and an electron-deficient NDI-based diamine. This heteroleptic architecture was shown to be thermodynamically favored over the corresponding homoleptic M(II)4L6 and M(II)4L'6 complexes, which were nonetheless stable in each others' absence. By contrast, an isomeric pyrene-based diamine was observed to undergo narcissistic self-sorting in the presence of the NDI-based diamine.

IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens.

IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans.

Disulfide exchange: exposing supramolecular reactivity through dynamic covalent chemistry.

A Tutorial Review of the subtle supramolecular interactions influencing the outcomes of equilibrating systems, focusing on the dynamic covalent chemistry (DCC) of disulfide exchange reactions, is presented. We discuss the topics of cation-π interactions (2.1), hydrophobic effects (2.2), hydrogen bonding interactions (2.3) aromatic donor-acceptor interactions (2.4), and metal-ligand interactions (2.5) in the context of dynamic disulfide chemistry.

An in vitro model of the horse gut microbiome enables identification of lactate-utilizing bacteria that differentially respond to starch induction.

Laminitis is a chronic, crippling disease triggered by the sudden influx of dietary starch. Starch reaches the hindgut resulting in enrichment of lactic acid bacteria, lactate accumulation, and acidification of the gut contents. Bacterial products enter the bloodstream and precipitate systemic inflammation. Hindgut lactate levels are normally low because specific bacterial groups convert lactate to short chain fatty acids. Why this mechanism fails when lactate levels rapidly rise, and why some hindgut communities can recover is unknown. Fecal samples from three adult horses eating identical diets provided bacterial communities for this in vitro study. Triplicate microcosms of fecal slurries were enriched with lactate and/or starch. Metabolic products (short chain fatty acids, headspace gases, and hydrogen sulfide) were measured and microbial community compositions determined using Illumina 16S rRNA sequencing over 12-hour intervals. We report that patterns of change in short chain fatty acid levels and pH in our in vitro system are similar to those seen in in vivo laminitis induction models. Community differences between microcosms with disparate abilities to clear excess lactate suggest profiles conferring resistance of starch-induction conditions. Where lactate levels recover following starch induction conditions, propionate and acetate levels rise correspondingly and taxa related to Megasphaeraelsdenii reach levels exceeding 70% relative abundance. In lactate and control cultures, taxa related to Veillonellamontpellierensis are enriched as lactate levels fall. Understanding these community differences and factors promoting the growth of specific lactate utilizing taxa may be useful to prevent acidosis under starch-induction conditions.

Requirement for active glycogen synthase kinase-3ß in TGF-ß1 upregulation of connective tissue growth factor (CCN2/CTGF) levels in human gingival fibroblasts.

Connective tissue growth factor (CCN2/CTGF) mediates transforming growth factor-ß (TGF-ß)-induced fibrosis. Drug-induced gingival overgrowth is tissue specific. Here the role of the phosphoinositol 3-kinase (PI3K) pathway in mediating TGF-ß1-stimulated CCN2/CTGF expression in primary human adult gingival fibroblasts and human adult lung fibroblasts was compared. Data indicate that PI3K inhibitors attenuate upregulation of TGF-ß1-induced CCN2/CTGF expression in human gingival fibroblasts independent of reducing JNK MAP kinase activation. Pharmacologic inhibitors and small interfering (si)RNA-mediated knockdown studies indicate that calcium-dependent isoforms and an atypical isoform of protein kinase C (PKC-δ) do not mediate TGF-ß1-stimulated CCN2/CTGF expression in gingival fibroblasts. As glycogen synthase kinase-3ß (GSK-3ß) can undergo phosphorylation by the PI3K/pathway, the effects of GSK-3ß inhibitor kenpaullone and siRNA knockdown were investigated. Data in gingival fibroblasts indicate that kenpaullone attenuates TGF-ß1-mediated CCN2/CTGF expression. Activation of the Wnt canonical pathways with Wnt3a, which inhibits GSK-3ß, similarly inhibits TGF-ß1-stimulated CCN2/CTGF expression. In contrast, inhibition of GSK-3ß by Wnt3a does not inhibit, but modestly stimulates, CCN2/CTGF levels in primary human adult lung fibroblasts and is ß-catenin dependent, consistent with previous studies performed in other cell models. These data identify a novel pathway in gingival fibroblasts in which inhibition of GSK-3ß attenuates CCN2/CTGF expression. In adult lung fibroblasts inhibition of GSK-3ß modestly stimulates TGF-ß1-regulated CCN2/CTGF expression. These studies have potential clinical relevance to the tissue specificity of drug-induced gingival overgrowth.

Impact of laminitis on the canonical Wnt signaling pathway in basal epithelial cells of the equine digital laminae.

The digital laminae is a two layer tissue that attaches the distal phalanx to the inner hoof wall, thus suspending the horse's axial skeleton in the hoof capsule. This tissue fails at the epidermal:dermal junction in laminitic horses, causing crippling disease. Basal epithelial cells line the laminar epidermal:dermal junction, undergo physiological change in laminitic horses, and lose versican gene expression. Versican gene expression is purportedly under control of the canonical Wnt signaling pathway and is a trigger for mesenchymal-to-epithelial transition; thus, its repression in laminar epithelial cells of laminitic horses may be associated with suppression of the canonical Wnt signaling pathway and loss of the epithelial cell phenotype. In support of the former contention, we show, using laminae from healthy horses and horses with carbohydrate overload-induced laminitis, quantitative real-time polymerase chain reaction, Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis, and immunofluorescent tissue staining, that positive and negative regulatory components of the canonical Wnt signaling pathway are expressed in laminar basal epithelial cells of healthy horses. Furthermore, expression of positive regulators is suppressed and negative regulators elevated in laminae of laminitic compared to healthy horses. We also show that versican gene expression in the epithelial cells correlates positively with that of ß-catenin and T-cell Factor 4, consistent with regulation by the canonical Wnt signaling pathway. In addition, gene and protein expression of ß-catenin correlates positively with that of integrin ß4 and both are strongly suppressed in laminar basal epithelial cells of laminitic horses, which remain E-cadherin(+)/vimentin(-), excluding mesenchymal transition as contributing to loss of the adherens junction and hemidesmosome components. We propose that suppression of the canonical Wnt signaling pathway, and accompanying reduced expression of ß catenin and integrin ß4 in laminar basal epithelial cells reduces cell:cell and cell:basement membrane attachment, thus, destabilizing the laminar epidermal:dermal junction.

Metal-organic container molecules through subcomponent self-assembly.

A variety of different three-dimensional metal-organic container molecules have recently been prepared using subcomponent self-assembly, which relies upon metal template effects to generate complex structures from simple molecular precursors and metal salts. Many of these structures have well defined internal pockets, allowing guest species to be bound and the chemical reactivity of these guests to be modified. Such host molecules have potential applications ranging from the protection of sensitive chemical species to the separation and purification of substrates as diverse as gases, gold compounds, and fullerenes.

Behavioral and physiological female responses to male sex ratio bias in a pond-breeding amphibian.

INTRODUCTION: The phenomenon of sexual conflict has been well documented, and in populations with biased operational sex ratios the consequences for the rarer sex can be severe. Females are typically a limited resource and males often evolve aggressive mating behaviors, which can improve individual fitness for the male while negatively impacting female condition and fitness. In response, females can adjust their behavior to minimize exposure to aggressive mating tactics or minimize the costs of mating harassment. While male-male competition is common in amphibian mating systems, little is known about the consequences or responses of females. The red-spotted newt (Notophthalmus viridescens) is a common pond-breeding amphibian with a complex, well-studied mating system where males aggressively court females. Breeding populations across much of its range have male-biased sex ratios and we predicted that female newts would have behavioral mechanisms to mitigate mating pressure from males. We conducted four experiments examining the costs and behavioral responses of female N. viridescens exposed to a male-biased environment. RESULTS: In field enclosures, we found that female newts exposed to a male-biased environment during the five-month breeding season ended with lower body condition compared to those in a female-biased environment. Shorter-term exposure to a male-biased environment for five weeks caused a decrease in circulating total leukocyte and lymphocyte abundance in blood, which suggests females experienced physiological stress. In behavioral experiments, we found that females were more agitated in the presence of male chemical cues and females in a male-biased environment spent more time in refuge than those in a female-biased environment. CONCLUSIONS: Our results indicate that male-biased conditions can incur costs to females of decreased condition and potentially increased risk of infection. However, we found that females can also alter their behavior and microhabitat use under a male-biased sex ratio. Consistent with surveys showing reduced detection probabilities for females, our research suggests that females avoid male encounters using edge and substrate habitat. Our work illustrates the integrated suite of impacts that sexual conflict can have on the structure and ecology of a population.

Distribution and processing of a disintegrin and metalloproteinase with thrombospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae.

OBJECTIVE: To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses. SAMPLE: Laminae from the forelimb hooves of 8 healthy horses. PROCEDURES: Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization. RESULTS: Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae. CONCLUSIONS AND CLINICAL RELEVANCE: Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.

Effects of cleavage by a disintegrin and metalloproteinase with thrombospondin motifs-4 on gene expression and protein content of versican and aggrecan in the digital laminae of horses with starch gruel-induced laminitis.

OBJECTIVE: To determine whether increased gene expression of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) in laminae of horses with starch gruel-induced laminitis was accompanied by increased enzyme activity and substrate degradation. SAMPLE: Laminae from the forelimb hooves of 8 healthy horses and 17 horses with starch gruel-induced laminitis (6 at onset of fever, 6 at onset of Obel grade 1 lameness, and 5 at onset of Obel grade 3 lameness). PROCEDURES: Gene expression was determined by use of cDNA and real-time quantitative PCR assay. Protein expression and processing were determined via SDS-PAGE and quantitative western blotting. Protein distribution and abundance were determined via quantitative immunofluorescent staining. RESULTS: ADAMTS-4 gene expression was increased and that of versican decreased in laminitic laminae, compared with expression in healthy laminae. Catalytically active ADAMTS-4 also was increased in the tissue, as were ADAMTS-4-cleavage fragments of versican. Immunofluorescent analyses indicated that versican was depleted from the basal epithelia of laminae of horses at onset of Obel grade 3 lameness, compared with results for healthy laminae, and this was accompanied by regional separation of basal epithelial cells from the basement membrane. Aggrecan gene and protein expression were not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in gene and protein expression of ADAMTS-4 and versican in the basal epithelium of laminitic laminae indicated a fundamental change in the physiology of basal epithelial cells. This was accompanied by and may have caused detachment of these cells from the basement membrane.

B Lymphocytes provide an infection niche for intracellular bacterium Brucella abortus.

BACKGROUND: Brucella spp. are intracellular bacteria that establish lifelong infections whose mechanisms of chronicity are poorly understood. Notably, B cells facilitate the establishment of the high infection plateau that persists for months. METHODS: We evaluated the contribution of murine B cells toward providing infection niches for Brucella by using flow cytometry and microscopy and by determining live bacterial counts associated with B cells both in vivo and in vitro. RESULTS: Herein we demonstrate that immunoglobulin M and complement-opsonized Brucella abortus infects and survives inside primary murine B cells protected from bactericidal effects of gentamicin. The entry was dependent on microfilaments for internalization and subsequently brucellae reside in a late endosomal/lysosomal compartment. Throughout the infection, 10% of colony-forming units from infected mice was associated with B cells, and these cells transferred disease to naive hosts. Furthermore, Brucella-positive cells were positive for transforming growth factor (TGF) ß1, and about 10% of such cells were B cells, similar to rates found for other intracellular pathogens that induce their hosts cells to produce TGF-ß1. CONCLUSIONS: To conclude, infected B cells contribute to chronic bacterial infections by providing an intracellular niche that may exert an immunoregulatory role. Although professional phagocytic cells harbor intracellular bacteria including Brucella, infection of lymphocytes by bacteria has not been previously appreciated.

Laminar chemokine mRNA concentrations in horses with carbohydrate overload-induced laminitis.

Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis.

T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis.

African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG) coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB) and Follicular B (FoB) cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves.