A quantum coherent spin in hexagonal boron nitride at ambient conditions.

Solid-state spin-photon interfaces that combine single-photon generation and long-lived spin coherence with scalable device integration-ideally under ambient conditions-hold great promise for the implementation of quantum networks and sensors. Despite rapid progress reported across several candidate systems, those possessing quantum coherent single spins at room temperature remain extremely rare. Here we report quantum coherent control under ambient conditions of a single-photon-emitting defect spin in a layered van der Waals material, namely, hexagonal boron nitride. We identify that the carbon-related defect has a spin-triplet electronic ground-state manifold. We demonstrate that the spin coherence is predominantly governed by coupling to only a few proximal nuclei and is prolonged by decoupling protocols. Our results serve to introduce a new platform to realize a room-temperature spin qubit coupled to a multiqubit quantum register or quantum sensor with nanoscale sample proximity.

Two pathogen loci determine Blumeria graminis f. sp. tritici virulence to wheat resistance gene Pm1a.

Blumeria graminis f. sp. tritici (Bgt) is a globally important fungal pathogen of wheat that can rapidly evolve to defeat wheat powdery mildew (Pm) resistance genes. Despite periodic regional deployment of the Pm1a resistance gene in US wheat production, Bgt strains that overcome Pm1a have been notably nonpersistent in the United States, while on other continents, they are more widely established. A genome-wide association study (GWAS) was conducted to map sequence variants associated with Pm1a virulence in 216 Bgt isolates from six countries, including the United States. A virulence variant apparently unique to Bgt isolates from the United States was detected in the previously mapped gene AvrPm1a (BgtE-5612) on Bgt chromosome 6; an in vitro growth assay suggested no fitness reduction associated with this variant. A gene on Bgt chromosome 8, Bgt-51526, was shown to function as a second determinant of Pm1a virulence, and despite < 30% amino acid identity, BGT-51526 and BGTE-5612 were predicted to share > 85% of their secondary structure. A co-expression study in Nicotiana benthamiana showed that BGTE-5612 and BGT-51526 each produce a PM1A-dependent hypersensitive response. More than one member of a B. graminis effector family can be recognized by a single wheat immune receptor, and a two-gene model is necessary to explain virulence to Pm1a.

Shared neutrophil and T cell dysfunction is accompanied by a distinct interferon signature during severe febrile illnesses in children.

Severe febrile illnesses in children encompass life-threatening organ dysfunction caused by diverse pathogens and other severe inflammatory syndromes. A comparative approach to these illnesses may identify shared and distinct features of host immune dysfunction amenable to immunomodulation. Here, using immunophenotyping with mass cytometry and cell stimulation experiments, we illustrate trajectories of immune dysfunction in 74 children with multi-system inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, 30 with bacterial infection, 16 with viral infection, 8 with Kawasaki disease, and 42 controls. We explore these findings in a secondary cohort of 500 children with these illnesses and 134 controls. We show that neutrophil activation and apoptosis are prominent in multi-system inflammatory syndrome, and that this is partially shared with bacterial infection. We show that memory T cells from patients with multi-system inflammatory syndrome and bacterial infection are exhausted. In contrast, we show viral infection to be characterized by a distinct signature of decreased interferon signaling and lower interferon receptor gene expression. Improved understanding of immune dysfunction may improve approaches to immunomodulator therapy in severe febrile illnesses in children.

Diagnosis of Multisystem Inflammatory Syndrome in Children by a Whole-Blood Transcriptional Signature.

BACKGROUND: To identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections. METHODS: Children presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39). RESULTS: In the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV. CONCLUSIONS: MIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

RAPD PCR detects co-colonisation of multiple group B streptococcus genotypes: A practical molecular technique for screening multiple colonies.

Group B Streptococcus (GBS) is a leading cause of neonatal meningitis, pneumonia, and sepsis. The biggest contributing factor of neonatal infections is due to vertical transmission from maternal colonisation of GBS in the genitourinary tract. Multiple serotype colonisation is often not investigated in epidemiological studies, but it is an important consideration for serotype-based vaccine development and implementation to ensure less abundant serotypes are not under-represented. In this study, we show that RAPD PCR is a quick tool useful in screening the presence of genetically different strains using multiple colony picks from a single patient swab. We observed a maximum of five different GBS strains colonising a single patient at a specific time.

Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors.

We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.

The influence of vitamin B6 on the structure and function of the glucocorticoid receptor.

Pyridoxal phosphate influences several properties of steroid hormone receptors in vitro, but its role in vivo has not been clearly established. In an effort to address this issue, we have investigated the in vivo effects of vitamin B6 on the physical properties and biological function of the human glucocorticoid receptor. We demonstrate that vitamin B6 treatment of whole cells in culture produces an alteration in the isoelectric point of the receptor, as well as changes in the steroid and DNA binding capacities. Furthermore, glucocorticoid dependent transcriptional activation properties of the receptor are also altered by modulation of the vitamin B6 status. High concentrations of vitamin B6 suppress activation of transcription, while vitamin deficiency enhances responsiveness to steroid hormone. Together, these studies imply a physiological role for vitamin B6 in glucocorticoid hormone action.

Autoregulation of glucocorticoid receptor gene expression.

Glucocorticoid receptors are members of a highly conserved family of steroid receptor proteins, which are ligand-dependent transcription factors. Previous studies have shown that the presence of functional glucocorticoid receptors is a prerequisite for manifestation of cellular responses to hormone. Glucocorticoid receptors undergo down-regulation following treatment with glucocorticoids. To define the molecular mechanisms that are involved in this process we have analyzed the down-regulation of glucocorticoid receptors both in HeLa cells, which contain endogenous receptors, and in cells containing receptors that have been introduced by DNA transfection. Our results show that cells that contain glucocorticoid receptors--either endogenous or transfected--undergo down-regulation of steroid-binding capabilities, as well as reductions in receptor protein and mRNA levels, in a remarkably similar fashion. DNA sequences in the coding region of the human glucocorticoid receptor cDNA appear to be sufficient to account for down-regulation of receptor. This novel finding suggests that unique mechanisms are involved in controlling glucocorticoid receptor homeostasis.

Regulation of the human glucocorticoid receptor by long-term and chronic treatment with glucocorticoid.

HeLa S3 cells that contain endogenous glucocorticoid receptors (GR) were treated with dexamethasone (DEX) for periods of time ranging from 24 h to 2 weeks or chronically over a 2-year period. Regulation of GR protein and mRNA were examined by affinity labeling, Western blotting, and Northern blotting. Relatively short-term treatment of cells with DEX for 24 or 48 h revealed more profound down-regulation of GR protein than of GR mRNA. However, by 2 weeks of DEX treatment, the levels of both receptor protein and mRNA were both maximally down-regulated. Cells that had been chronically DEX treated (for up to 2 years) had no measurable GR protein or mRNA. The down-regulation of receptor protein and RNA that occurred after 2 weeks of DEX treatment is completely reversible upon DEX removal, whereas reversibility did not occur with cells that had been chronically treated with DEX. Furthermore, transfection of a glucocorticoid responsive reporter plasmid into these chronically DEX-treated cells demonstrated that these cells were no longer responsive to steroid treatment. However, cotransfection of a plasmid encoding the human GR into these chronically DEX-treated cells resulted in restored production of GR and responsiveness to hormone, indicating that the defect in these cells occurs only at the receptor level.

Beta 2-adrenoceptor stimulation increases the number of antigen-specific precursor B lymphocytes that differentiate into IgM-secreting cells without affecting burst size.

Previous studies have shown that early addition of a beta 2-adrenergic agonist to whole splenocyte cultures immunized with SRBC induced an increase in the number of cells secreting Ag-specific antibody. Because of the low frequency of Ag-specific B lymphocytes in these cultures, it has been difficult to determine the cellular mechanism by which this increase is produced. To gain insight into this cellular mechanism, the present study was designed to evaluate the responsiveness of TNP-specific B lymphocytes cultured at both high density and limiting dilution with keyhole limpet hemocyanin (KLH)-specific, IL-4-producing Th lymphocytes, TNP-KLH, and the beta 2-adrenergic agonist, terbutaline. The results showed that a maximal twofold increase in both the number of anti-TNP IgM-secreting cells and the amount of anti-TNP IgM secretion occurred in terbutaline-exposed lymphocytes after 5 days of bulk culture. This response occurred in a concentration-dependent manner and was inhibited by concomitant culture with beta-adrenoceptor antagonists. No appreciable change was measured in the level of either IgG1 secretion in terbutaline plus Ag-exposed bulk cultures or MHC class II expression on terbutaline plus Ag-exposed TNP-specific B lymphocytes as compared with Ag alone. These data raised the possibility that beta 2-adrenoceptor stimulation induced either the differentiation of a larger proportion of TNP-specific B lymphocyte precursors into anti-TNP IgM-secreting cells, or the extensive proliferation of a constant number of TNP-specific B lymphocyte precursors, or both. Limiting dilution results showed that beta 2-adrenoceptor stimulation induced a twofold increase in the number of TNP-specific B lymphocyte precursors that differentiated into anti-TNP IgM-secreting cells, without affecting the number of anti-TNP IgM-secreting cells produced by each precursor clone.

Vitamin B6 influences glucocorticoid receptor-dependent gene expression.

We have examined the influence of intracellular vitamin B6 concentration on glucocorticoid receptor function in HeLa S3 cells transfected with a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid. CAT activity is induced from this plasmid specifically by glucocorticoid hormones in a glucocorticoid receptor-dependent manner. The intracellular concentration of pyridoxal phosphate, the physiologically active form of the vitamin, was elevated by supplementation of the culture medium with the synthesis precursor pyridoxine and lowered by exposure to the pyridoxal phosphate synthesis inhibitor 4-deoxypyridoxine. Analysis of glucocorticoid responsiveness revealed that elevated concentrations of intracellular pyridoxal phosphate suppressed the amount of glucocorticoid-induced CAT activity whereas moderate deficiency enhanced the level of glucocorticoid receptor-mediated gene expression. In contrast, modulation of the intracellular pyridoxal phosphate concentration had no effect on either basal CAT activity derived from cells not stimulated with dexamethasone or on CAT activity derived from two glucocorticoid-insensitive reporter plasmids. The modulatory effects of pyridoxal phosphate concentration occur without changes in glucocorticoid receptor mRNA levels, glucocorticoid receptor protein concentration, or the steroid binding capacity of the receptor. These observations demonstrate that vitamin B6 selectively influences glucocorticoid receptor-dependent gene expression through a novel mechanism that does not involve alterations in glucocorticoid receptor concentration or ligand binding capacity.

Immune-associated toxicities induced by in vivo and in vitro exposure to interferon-alpha alone or in combination with nucleoside analogs.

The present studies examine whether immune-associated toxicities develop in rodents exposed to recombinant human interferon-alpha A/D (rHuIFN-alpha) alone or in combination with anti-retroviral nucleoside analogs. Four findings have emerged from these studies: (1) lymphocyte cell number and functional activity are suppressed after subchronic (1, 8 or 10 day) in vivo exposure to therapeutic doses of rHuIFN-alpha; (2) lymphocyte-associated toxicities lessen as in vivo exposure time to rHuIFN-alpha is extended; (3) T-cell-dependent antibody production is decreased after in vitro exposure of both antigen-specific T- and B-cells to rHuIFN-alpha; and (4) in vivo-induced leukocyte toxicities associated with either rHuIFN-alpha or the nucleoside analogs alone do not synergize when the therapies are combined. These data suggest that certain key immune parameters should be monitored carefully in the early stages of IFN-alpha therapy.