Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.

Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.

Analyses of thymocyte commitment to regulatory T cell lineage in thymus of healthy subjects and patients with 22q11.2 deletion syndrome.

The Chromosome 22q11.2 deletion syndrome (22q11.2DS) results in an inborn error of immunity due to defective thymic organogenesis. Immunological abnormalities in 22q11.2DS patients are thymic hypoplasia, reduced output of T lymphocytes by the thymus, immunodeficiency and increased incidence of autoimmunity. While the precise mechanism responsible for increased incidence of autoimmunity is not completely understood, a previous study suggested a defect in regulatory T cells (Treg) cell lineage commitment during T cell development in thymus. Here, we aimed to analyze this defect in more detail. Since Treg development in human is still ill-defined, we first analyzed where Treg lineage commitment occurs. We performed systematic epigenetic analyses of the Treg specific demethylation region (TSDR) of the FOXP3 gene in sorted thymocytes at different developmental stages. We defined CD3+CD4+CD8+ FOXP3+CD25+ as the T cell developmental stage in human where TSDR demethylation first occurs. Using this knowledge, we analyzed the intrathymic defect in Treg development in 22q11.2DS patients by combination of TSDR, CD3, CD4, CD8 locus epigenetics and multicolor flow cytometry. Our data showed no significant differences in Treg cell frequencies nor in their basic phenotype. Collectively, these data suggest that although 22q11.2DS patients present with reduced thymic size and T cell output, the frequencies and the phenotype of Treg cell at each developmental stage are surprisingly well preserved.

Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation.

BACKGROUND: Forkhead box protein 3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution, and treatment choice. OBJECTIVE: We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. METHODS: We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n = 6) and "atypical" or asymptomatic (n = 9) patients with IPEX. RESULTS: Increased number of cells with Treg-cell-Specific Demethylated Region demethylation in FOXP3 is a consistent feature in patients with IPEX, with (1) highest values in those with typical IPEX, (2) increased values in subjects with pathogenic FOXP3 but still no symptoms, and (3) gradual increase over the course of disease progression. Large-scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and TH2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNF-α, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg-cell and Teff compartments, studied by Mass Cytometry by Time-Of-Flight, were skewed toward the TH2 compartment, especially in typical IPEX. CONCLUSIONS: Elevated TSDR-demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized type 2 inflammatory immune response, extends our understanding of IPEX diagnosis and heterogeneity.

Overexpression of Cu/Zn Superoxide Dismutase (Cu/Zn SOD) in Synechococcus elongatus PCC 7942 for Enhanced Azo Dye Removal through Hydrogen Peroxide Accumulation.

Discharge of recalcitrant azo dyes to the environment poses a serious threat to environmental health. However certain microorganisms in nature have developed their survival strategies by degrading these toxic dyes. Cyanobacteria are one such prokaryotic, photosynthetic group of microorganisms that degrade various xenobiotic compounds, due to their capability to produce various reactive oxygen species (ROS), and particularly the hydrogen peroxide (H2O2) when released in their milieu. The accumulation of H2O2 is the result of the dismutation of superoxide radicals by the enzyme superoxide dismutase (SOD). In this study, we have genetically modified the cyanobacterium Synechococcus elongatus PCC 7942 by integrating Cu/Zn SOD gene (sodC) from Synechococcus sp. PCC 9311 to its neutral site through homologous recombination. The overexpression of sodC in the derivative strain was driven using a strong constitutive promoter of the psbA gene. The derivative strain resulted in constitutive production of sodC, which was induced further during dye-treated growth. The genetically engineered Synechococcus elongatus PCC 7942 (MS-sodC+) over-accumulated H2O2 during azo dye treatment with a higher dye removal rate than the wild-type strain (WS-sodC-). Therefore, enhanced H2O2 accumulation through SODs overexpression in cyanobacteria may serve as a valuable bioremediation tool.

Preferential adsorption of uranium by functional groups of the marine unicellular cyanobacterium Synechococcus elongatus BDU130911.

This study reports the surface interaction of the chemically modified marine unicellular cyanobacterium Synechococcus elongatus BDU130911 with uranium. The selective functional groups of the control (dead biomass) for binding with uranium in unicellular marine cyanobacteria were identified as carboxyl groups. The adsorption capacity of the biomass in a 1 mM uranium solution was found to be 92% in the control, 85% in the amine-blocked treatments, and 20% in the carboxyl-blocked treatments. The Langmuir isotherm provided a good fit to the data, suggesting a monolayer of uranium adsorption on all the tested biomass. The functional groups involved in the adsorption of uranium by the control and modified biomass were assessed by Fourier transform infrared spectroscopy, energy dispersive X-ray fluorescence and X-ray diffractive analysis. The results of this study identify, carboxyl groups as the dominant anionic functional group involved in uranium adsorption, which validates an ionic interaction between the biomass and uranium, a cationic metal.

Cytomorphological and nitrogen metabolic enzyme analysis of psychrophilic and mesophilic Nostoc sp.: a comparative outlook.

Cyanobacterial diazotrophs play a significant role in environmental nitrogen economy despite their habitat either tropical or polar. However, the phenomenon by which it copes with temperature induced stress is poorly understood. Temperature response study of psychrophilic and mesophilic Nostoc strains explores their adaptive mechanisms. The selected psychrophilic and mesophilic strains were confirmed as Nostoc punctiforme and Nostoc calcicola respectively, by ultrastructure and 16S rDNA phylogeny. The psychrophilic strain has extensive glycolipid and polysaccharide sheath along with characteristic deposition of cyanophycin, polyhydroxybutyrate granules, and carboxysomes. This is possibly an adaptive strategy exhibited to withstand the freezing temperature and high intense of ultraviolet rays. The biomass measured in terms of dry weight, protein, and chlorophyll indicated a temperature dependant shift in both the psychrophilic and mesophilic strains and attained maximum growth in their respective temperature niches. At low temperature, psychrophilic organism exhibited nitrogenase activity, while mesophilic strains did not. The maximum glutamine synthetase activity was observed at 4 °C for psychrophilic and 37 °C for mesophilic strains. Activity at 4 °C in psychrophilic strains revealed their energetic mechanism even at low temperature. The nitrate and nitrite reductase of both psychrophilic and mesophilic strains showed maximum activity at 37 °C denoting their similar nitrogen assimilating mechanisms for combined nitrogen utilization. The activity studies of nitrogen fixation/assimilation enzymes have differential effects at varying temperatures, which provide valuable insights of physiological contribution and role of Nostoc strains in the biological nitrogen cycle.

SynRio: R and Shiny based application platform for cyanobacterial genome analysis.

UNLABELLED: SynRio is a Shiny and R based web analysis portal for viewing Synechocystis PCC 6803 genome, a cyanobacterial genome with data analysis capabilities. The web based user interface is created using R programming language powered by Shiny package. This web interface helps in creating interactive genome visualization based on user provided data selection along with selective data download options. AVAILABILITY: SinRio is available to download freely from Github - https://github.com/NFMC/SynRio or from http://www.nfmc.res.in/synrio/. In addition an online version of the platform is also hosted at nfmc.res.in/synrio, using shiny server (open source edition) installation.

Cyanobacterial KnowledgeBase (CKB), a Compendium of Cyanobacterial Genomes and Proteomes.

Cyanobacterial KnowledgeBase (CKB) is a free access database that contains the genomic and proteomic information of 74 fully sequenced cyanobacterial genomes belonging to seven orders. The database also contains tools for sequence analysis. The Species report and the gene report provide details about each species and gene (including sequence features and gene ontology annotations) respectively. The database also includes cyanoBLAST, an advanced tool that facilitates comparative analysis, among cyanobacterial genomes and genomes of E. coli (prokaryote) and Arabidopsis (eukaryote). The database is developed and maintained by the Sub-Distributed Informatics Centre (sponsored by the Department of Biotechnology, Govt. of India) of the National Facility for Marine Cyanobacteria, a facility dedicated to marine cyanobacterial research. CKB is freely available at http://nfmc.res.in/ckb/index.html.