Cell-intrinsic Wnt4 promotes hematopoietic stem and progenitor cell self-renewal.

Although intracellular Wnt signaling pathways need to be tightly regulated to promote hematopoietic stem cell self-renewal, the source and identity of important Wnt ligands in the bone marrow is still largely unknown. The noncanonical ligand Wnt4 is expressed in the bone marrow as well as in the stroma, and its overexpression in fetal liver cells facilitates thymic recovery; however, its impact on adult hematopoietic stem cell function remains unclear. Here, we report that the deletion of Wnt4 from hematopoietic cells in mice (Wnt4Δ/Δ ) resulted in decreased lymphopoiesis at steady state. This was likely at least in part due to the increased proinflammatory environment present in the bone marrow of Wnt4Δ/Δ mice. Wnt4Δ/Δ hematopoietic stem cells displayed reduced reconstitution capacity in serial transplants, thus demonstrating defective self-renewal, and they expanded poorly in response to lipopolysaccharide stimulation. This appeared to be the result of the absence of Wnt4 in stem/progenitor cells, as myeloid-restricted Wnt4 deletion had no notable effect. Finally, we observed that Wnt4Δ/Δ stem/progenitor cells were more quiescent, presenting enhanced levels of stress-associated JNK phosphorylation and p16INK4a expression, likely contributing to the reduced expansion observed in transplants. In conclusion, our results identify a new, largely autocrine role for Wnt4 in hematopoietic stem cell self-renewal, suggesting that regulation of Wnt signaling in hematopoiesis may not need Wnt secretion and could be independent of morphogen gradients.

CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees.

Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose, and fructose are the main components of plant nectars. Intriguingly, honeybees express only 3 putative sugar receptors (AmGr1, AmGr2, and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioral change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterization in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.

Persistent Cutaneous Leishmania major Infection Promotes Infection-Adapted Myelopoiesis.

Hematopoietic stem/progenitor cells (HSPC) are responsible for the generation of most immune cells throughout the lifespan of the organism. Inflammation can activate bone marrow HSPCs, leading to enhanced myelopoiesis to replace cells, such as neutrophils, which are attracted to inflamed tissues. We have previously shown that HSPC activation promotes parasite persistence and expansion in experimental visceral leishmaniasis through the increased production of permissive monocytes. However, it is not clear if the presence of the parasite in the bone marrow was required for infection-adapted myelopoiesis. We therefore hypothesized that persistent forms of Leishmania major (cutaneous leishmaniasis) could also activate HSPCs and myeloid precursors in the C57Bl/6 mouse model of intradermal infection in the ear. The accrued influx of myeloid cells to the lesion site corresponded to an increase in myeloid-biased HSPCs in the bone marrow and spleen in mice infected with a persistent strain of L. major, together with an increase in monocytes and monocyte-derived myeloid cells in the spleen. Analysis of the bone marrow cytokine and chemokine environment revealed an attenuated type I and type II interferon response in the mice infected with the persistent strain compared to the self-healing strain, while both strains induced a rapid upregulation of myelopoietic cytokines, such as IL-1ß and GM-CSF. These results demonstrate that an active infection in the bone marrow is not necessary for the induction of infection-adapted myelopoiesis, and underline the importance of considering alterations to the bone marrow output when analyzing in vivo host-pathogen interactions.

Sugar perception in honeybees.

Honeybees (Apis mellifera) need their fine sense of taste to evaluate nectar and pollen sources. Gustatory receptors (Grs) translate taste signals into electrical responses. In vivo experiments have demonstrated collective responses of the whole Gr-set. We here disentangle the contributions of all three honeybee sugar receptors (AmGr1-3), combining CRISPR/Cas9 mediated genetic knock-out, electrophysiology and behaviour. We show an expanded sugar spectrum of the AmGr1 receptor. Mutants lacking AmGr1 have a reduced response to sucrose and glucose but not to fructose. AmGr2 solely acts as co-receptor of AmGr1 but not of AmGr3, as we show by electrophysiology and using bimolecular fluorescence complementation. Our results show for the first time that AmGr2 is indeed a functional receptor on its own. Intriguingly, AmGr2 mutants still display a wildtype-like sugar taste. AmGr3 is a specific fructose receptor and is not modulated by a co-receptor. Eliminating AmGr3 while preserving AmGr1 and AmGr2 abolishes the perception of fructose but not of sucrose. Our comprehensive study on the functions of AmGr1, AmGr2 and AmGr3 in honeybees is the first to combine investigations on sugar perception at the receptor level and simultaneously in vivo. We show that honeybees rely on two gustatory receptors to sense all relevant sugars.

Head-to-Head Comparison of Consensus-Recommended Platelet Function Tests to Assess P2Y12 Inhibition-Insights for Multi-Center Trials.

The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by Multiplate® whole-blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR-C 66096). Platelet function testing was carried out simultaneously by Multiplate® aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA- and flow cytometry-based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with Multiplate® was slightly higher for the flow cytometry-based VASP assay (r = 0.79, p < 0.0001) than for the ELISA-based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (κ between 0.86 and 0.92). In conclusion, the consensus-recommended assays with their standardized cut-offs should not be used interchangeably in multi-center clinical studies but, rather, they should be standardized throughout sites.

Manejo de cárie radicular: um guia para o dentista brasileiro baseado na tradução e adaptação cultural do consenso internacional/ORCA E EFCD / Management of root caries: a guide for the Brazilian dentist based on the translation and cultural adaptation of the international consensus/ORCA and EFCD

Objetivo: A cárie radicular é um problema da Odontologia moderna, porém é notável a falta de diretrizes sobre o seu manejo. Objetivamos elaborar e adaptar um guia a partir da tradução das recomendações do consenso in-ternacional European Organization for Caries Research (ORCA) e European Federation of Conservative Dentistry (EFCD) para as tomadas de decisão clínica na intervenção do processo de cárie na pessoa idosa, com foco na cárie radicular. Materiais e métodos: O protocolo de tradução das recomendações do consenso internacional consistiu nas etapas: (1) tradução inicial, (2) síntese da tradução, (3) retradução, (4) revisão por comitê de especialistas, com adaptação cultural. A partir da tradução, foi desenvolvido um guia com diretrizes para tratamento de cárie radicular no Brasil. Resultados: Para prevenção de novas lesões é recomendada a escovação diária com dentifrício >1.500ppm/F. Dentifrícios com 5.000ppm/F ou vernizes (>20.000ppm/F) podem ser indicados para paralisar lesões radiculares ativas e para prevenção em pessoas idosas com alta suscetibilidade à cárie radicular, e o Diamino Fluoreto de Prata (>30%) para paralisar lesões ativas. Intervenções invasi-vas diretas são indicadas dependendo da situação clínica. Discussão: Nota-se uma falta de interesse em estudos primários sobre tratamentos para cárie radicular, criando assim uma lacuna em relação ao seu manejo, que reflete no nível de evidência detectado pelo consenso. Conclusão: Guias clíni-cos são importantes para reduzir a lacuna entre a pesquisa e a prática clínica. Essa tradução para o português facilitará o acesso dos dentistas bra-sileiros em relação a evidência consolidada até o momento para o manejo de cárie radicular.

Aim: Root caries are a problem in modern dentistry, but the lack of guidelines regarding their management is notable. We aim to develop and adapt a guide based on the translation of the recommendations of the international consensus as outlined by the European Organization for Caries Research (ORCA) and the European Federation of Conservative Dentistry (EFCD) for clinical decision-making in the intervention of the caries process in the elderly, with a focus on root caries. Materials and methods: The protocol for translating the recommendations of the international consensus consisted of the following steps: (1) initial translation, (2) synthesis of the translation, (3) back-translation, (4) review by an expert committee with cultural adaptation. Based on the translation, a guide was developed with guidelines for the treatment of root caries in Brazil. Results: To prevent new lesions, daily brushing with toothpaste >1,500ppm/F is recommended. Toothpaste with 5,000ppm/F or varnishes (>20,000ppm/F) may be recommended to paralyze active root lesions and for prevention in elderly people with high susceptibility to root caries, and Silver Diamine Fluoride (>30%) to paralyze active lesions. Direct invasive interventions are indicated depending on the clinical situation. Discussion: There is a lack of interest in primary studies on treatments for root caries, thus creating a gap in relation to its management, which is reflected by the level of evidence detected in the consensus. Conclusion: Clinical guidelines are important to reduce the gap between research and clinical practice. This translation into Portuguese will facilitate access by Brazilian dentists to the consolidated evidence gathered to date for the management of root caries.