Shared inflammatory glial cell signature after stab wound injury, revealed by spatial, temporal, and cell-type-specific profiling of the murine cerebral cortex.

Traumatic brain injury leads to a highly orchestrated immune- and glial cell response partially responsible for long-lasting disability and the development of secondary neurodegenerative diseases. A holistic understanding of the mechanisms controlling the responses of specific cell types and their crosstalk is required to develop an efficient strategy for better regeneration. Here, we combine spatial and single-cell transcriptomics to chart the transcriptomic signature of the injured male murine cerebral cortex, and identify specific states of different glial cells contributing to this signature. Interestingly, distinct glial cells share a large fraction of injury-regulated genes, including inflammatory programs downstream of the innate immune-associated pathways Cxcr3 and Tlr1/2. Systemic manipulation of these pathways decreases the reactivity state of glial cells associated with poor regeneration. The functional relevance of the discovered shared signature of glial cells highlights the importance of our resource enabling comprehensive analysis of early events after brain injury.

In the mouse cortex, oligodendrocytes regain a plastic capacity, transforming into astrocytes after acute injury.

Acute brain injuries evoke various response cascades directing the formation of the glial scar. Here, we report that acute lesions associated with hemorrhagic injuries trigger a re-programming of oligodendrocytes. Single-cell RNA sequencing highlighted a subpopulation of oligodendrocytes activating astroglial genes after acute brain injuries. By using PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice, we visualized this population of oligodendrocytes that we termed AO cells based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and repeated chronic in vivo imaging with two-photon laser-scanning microscopy, we observed the conversion of oligodendrocytes into astrocytes via the AO cell stage. Such conversion was promoted by local injection of IL-6 and was diminished by IL-6 receptor-neutralizing antibody as well as by inhibiting microglial activation with minocycline. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma due to microglia-derived IL-6.

Innate Immune Pathways Promote Oligodendrocyte Progenitor Cell Recruitment to the Injury Site in Adult Zebrafish Brain.

The oligodendrocyte progenitors (OPCs) are at the front of the glial reaction to the traumatic brain injury. However, regulatory pathways steering the OPC reaction as well as the role of reactive OPCs remain largely unknown. Here, we compared a long-lasting, exacerbated reaction of OPCs to the adult zebrafish brain injury with a timely restricted OPC activation to identify the specific molecular mechanisms regulating OPC reactivity and their contribution to regeneration. We demonstrated that the influx of the cerebrospinal fluid into the brain parenchyma after injury simultaneously activates the toll-like receptor 2 (Tlr2) and the chemokine receptor 3 (Cxcr3) innate immunity pathways, leading to increased OPC proliferation and thereby exacerbated glial reactivity. These pathways were critical for long-lasting OPC accumulation even after the ablation of microglia and infiltrating monocytes. Importantly, interference with the Tlr1/2 and Cxcr3 pathways after injury alleviated reactive gliosis, increased new neuron recruitment, and improved tissue restoration.

Design of cross-linked RNA/protein complexes for structural studies.

Crystallographic studies of RNA/protein complexes are primordial for the understanding of recognition determinants and catalytic mechanisms in the case of enzymes. However, due to the flexibility and propensity to conformational heterogeneity of RNAs, as well as the mostly electrostatic interactions of RNA/protein complexes, they are difficult to crystallize. We present here a method to trap the two interacting partners in a covalent complex, based on a modified reactive RNA allowing the use of the full range of common crystallogenesis tools. We demonstrate the practicability of our approach with the production of a covalent complex of the Thermus thermophilus m1A58 tRNA modification enzyme, and a modified stem loop mimicking the natural substrate of the enzyme.

Role of the K+-Cl- Cotransporter KCC2a Isoform in Mammalian Respiration at Birth.

In central respiratory circuitry, synaptic excitation is responsible for synchronizing neuronal activity in the different respiratory rhythm phases, whereas chloride-mediated inhibition is important for shaping the respiratory pattern itself. The potassium chloride cotransporter KCC2, which serves to maintain low intraneuronal Cl- concentration and thus render chloride-mediated synaptic signaling inhibitory, exists in two isoforms, KCC2a and KCC2b. KCC2 is essential for functional breathing motor control at birth, but the specific contribution of the KCC2a isoform remains unknown. Here, to address this issue, we investigated the respiratory phenotype of mice deficient for KCC2a. In vivo plethysmographic recordings revealed that KCC2a-deficient pups at P0 transiently express an abnormally low breathing rate and a high occurrence of apneas. Immunostainings confirmed that KCC2a is normally expressed in the brainstem neuronal groups involved in breathing (pre-Bötzinger complex, parafacial respiratory group, hypoglossus nucleus) and is absent in these regions in the KCC2a-/- mutant. However, in variously reduced in vitro medullary preparations, spontaneous rhythmic respiratory activity is similar to that expressed in wild-type preparations, as is hypoglossal motor output, and no respiratory pauses are detected, suggesting that the rhythm-generating networks are not intrinsically affected in mutants at P0. In contrast, inhibitory neuromodulatory influences exerted by the pons on respiratory rhythmogenesis are stronger in the mutant, thereby explaining the breathing anomalies observed in vivo. Thus, our results indicate that the KCC2a isoform is important for establishing proper breathing behavior at the time of birth, but by acting at sites that are extrinsic to the central respiratory networks themselves.

Randomized placebo-controlled trial of hen's egg consumption for primary prevention in infants.

BACKGROUND: Hen's egg is the most common cause of food allergy in early childhood. OBJECTIVE: We investigated the efficacy and safety of early hen's egg introduction at age 4 to 6 months to prevent hen's egg allergy in the general population. METHODS: This randomized, placebo-controlled trial included 4- to 6-month-old infants who were not sensitized against hen's egg, as determined based on specific serum antibodies (IgE). These infants were randomized to receive either verum (egg white powder) or placebo (rice powder) added to the first weaning food 3 times a week under a concurrent egg-free diet from age 4 to 6 until 12 months. The primary outcome was sensitization to hen's egg (increased specific serum IgE levels) by age 12 months. Hen's egg allergy (secondary outcome) was confirmed by double-blind, placebo-controlled food challenges. RESULTS: Among 406 screened infants, 23 (5.7%) had hen's egg-specific IgE before randomization. Seventeen of 23 underwent subsequent double-blind, placebo-controlled food challenges, and 16 were confirmed as allergic, including 11 with anaphylactic reactions. Of the 383 nonsensitized infants (56.7% male), 184 were randomized to verum and 199 to placebo. At 12 months of age, 5.6% of the children in the verum group were hen's egg sensitized versus 2.6% in the placebo group (primary outcome; relative risk, 2.20; 95% CI, 0.68-7.14; P = .24), and 2.1% were confirmed to have hen's egg allergy versus 0.6% in the placebo group (relative risk, 3.30; 95% CI, 0.35-31.32; P = .35). CONCLUSION: We found no evidence that consumption of hen's egg starting at 4 to 6 months of age prevents hen's egg sensitization or allergy. In contrast, it might result in frequent allergic reactions in the community considering that many 4- to 6-month-old infants were already allergic to hen's egg.

Expanding the Scope of 2'-SCF3 Modified RNA.

The 2'-trifluoromethylthio (2'-SCF3 ) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2'-SCF3 adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc-protecting group strategy, are reported. RNA solid-phase synthesis provided site-specifically 2'-SCF3 -modified oligoribonucleotides that were investigated intensively. Their excellent behavior in (19) F NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule-RNA interaction. Moreover, comparably to the 2'-SCF3 pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off-target effects and their performance in silencing of the BASP1 gene was demonstrated.

Molecular sIgE profile in infants and young children with peanut sensitization and eczema.

BACKGROUND: Many children develop a sensitization to peanut in early infancy, even before peanut is introduced in their diet. Sensitization is particularly common in young children with eczema. There have been scant data available to date on the sensitization pattern for specific peanut allergens in this patient group. The aim of this study was to investigate the allergen profile of infants and young children with peanut sensitization and eczema. METHODS: Sera from 53 children aged ≤ 20 months with eczema and sensitization to peanut but who had not yet consumed products containing peanuts were included in the analysis. Sera were analyzed using microarray immunoassay (ImmunoCAP ISAC). RESULTS: In total, 63 % of peanut-sensitized children showed specific immunoglobulin E (sIgE) against at least one peanut allergen on the microarray. Specific IgE to the 7S globulin Ara h 1 was detected in 40 % of the children, to the 2S albumin Ara h 2 in 30 % and to the 11S globulin Ara h 3 in 23 %. Only one child had sIgE to Arah 8, the homologoue of Bet-v-1. Data on clinical relevance were available for 24 of 53 children: 14 of 24 patients had objective allergic reactions to peanut, while 10 children were peanut-tolerant. The seed storage protein Ara h 2 was not detected on microarray in 43 % (6 of 14) of children with peanut allergy. Two of these six children were mono-sensitized to Ara h 1 and two to Ara h 3, while in three children none of these seed storage proteins was detected. DISCUSSION: It could be shown that infants and young children with eczema and sensitization to peanut recognize predominantly seed storage proteins from peanut, even before the introduction of peanut into their diet. Sensitization to pollen-related food allergens seems to be rare at this age. At this age not only Ara h 2, but also Ara h 1 seems to be related to clinical relevance.

Tuning a riboswitch response through structural extension of a pseudoknot.

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ1 class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem-loop structure within its 3'-terminal region immediately upstream of the Shine-Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ1 riboswitch folding from multiple structural perspectives, we reveal that the extra stem-loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem-loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.