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Viral variant and host vaccination status impact infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), yet how these factors shift cellular responses in the human nasal mucosa remains uncharacterized. We performed single-cell RNA sequencing (scRNA-seq) on nasopharyngeal swabs from vaccinated and unvaccinated adults with acute Delta and Omicron SARS-CoV-2 infections and integrated with data from acute infections with ancestral SARS-CoV-2. Patients with Delta and Omicron exhibited greater similarity in nasal cell composition driven by myeloid, T cell and SARS-CoV-2 hi cell subsets, which was distinct from that of ancestral cases. Delta-infected samples had a marked increase in viral RNA, and a subset of PER2 + EGR1 + GDF15 + epithelial cells was enriched in SARS-CoV-2 RNA + cells in all variants. Prior vaccination was associated with increased frequency and activation of nasal macrophages. Expression of interferon-stimulated genes negatively correlated with coronavirus disease 2019 (COVID-19) severity in patients with ancestral and Delta but not Omicron variants. Our study defines nasal cell responses and signatures of disease severity across SARS-CoV-2 variants and vaccination., Competing Interests: Competing interests: V.N.M. reports compensation from MPM Capital and RA Capital Management unrelated to this work. S.W.K. reports compensation for consulting services with Monopteros Therapeutics, Flagship Pioneering and Radera Biosciences. M.S. reports compensation for consulting services with GE Precision Healthcare. J.J.L. reports compensation for consulting services with Blueprint Medicines and Human Immunology Biosciences. J.O.-M. reports compensation for consulting services with Cellarity, Tessel Biosciences and Radera Biotherapeutics. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Hovione, Ochre Bio, Third Rock Ventures, Relation Therapeutics, Dahlia Biosciences, Bio-Rad Laboratories, IntrECate Biotherapeutics, FogPharma and Passkey Therapeutics. C.G.K.Z., V.N.M., A.H.O., A.W.N., Y.T., J.D.B., A.K.S., S.C.G., B.H.H. and J.O.-M. are co-inventors on a provisional patent application relating to methods of stratifying and treating viral infections. The other authors declare no competing interests., (© 2025. Springer Nature America, Inc.)

Tissue-specific T cell immune responses play a critical role in maintaining organ health but can also drive immune pathology during both autoimmunity and alloimmunity. The mechanisms controlling intratissue T cell programming remain unclear. Here, we leveraged a nonhuman primate model of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation to probe the biological underpinnings of tissue-specific alloimmune disease using a comprehensive systems immunology approach including multiparameter flow cytometry, population-based transcriptional profiling, and multiplexed single-cell RNA sequencing and TCR sequencing. Transcriptional profiling revealed substantial biological differences between T cells infiltrating the lung and liver during aGVHD. These included enrichment for transcriptional pathways controlling extracellular matrix remodeling and chemotaxis in the lung and enrichment for transcriptional pathways linked to nucleic acid metabolism and proliferation in the liver. Single-cell RNA sequencing and TCR sequencing substantiated divergent organ-specific transcriptional programing of tissue-infiltrating T cells, which was linked to clonal expansion, with expanded clones progressively enriched for C-X3-C motif chemokine receptor 1 ( CX3CR1 )-expressing CD8 effector T cells in the lung and eomesodermin ( EOMES )-expressing CD8 effector-memory T cells in the liver. This divergent evolution of T cells was maintained even for T cells sharing the same TCRs, indicating its independence from antigen specificity. Together, these results provide insights into the role that tissue microenvironment-derived signals play in local T cell transcriptional programming during alloimmune-mediated clonal expansion and suggest potential opportunities to develop tissue-specific therapeutics to curtail pathogenic immunity after transplant.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNA Pyl )/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO 2 -reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy., Competing Interests: MJ, NC, MJ, AH, BF, KK, AM, QC, JA, EH No competing interests declared, (© 2021, Jahn et al.)

Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.

During the larval stages, the visual system of the mosquito Aedes aegypti contains five stemmata, often referred to as larval ocelli, positioned laterally on each side of the larval head. Here we show that stemmata contain two photoreceptor types, distinguished by the expression of different rhodopsins. The rhodopsin Aaop3 (GPROP3) is expressed in the majority of the larval photoreceptors. There are two small clusters of photoreceptors located within the satellite and central stemmata that express the rhodopsin Aaop7 (GPROP7) instead of Aaop3. Electroretinogram analysis of transgenic Aaop7 Drosophila indicates that Aaop3 and Aaop7, both classified as long-wavelength rhodopsins, possess similar but not identical spectral properties. Light triggers an extensive translocation of Aaop3 from the photosensitive rhabdoms to the cytoplasmic compartment, whereas light-driven translocation of Aaop7 is limited. The results suggest that these photoreceptor cell types play distinct roles in larval vision. An additional component of the larval visual system is the adult compound eye, which starts to develop at the anterior face of the larval stemmata during the 1st instar stage. The photoreceptors of the developing compound eye show rhodopsin expression during the 4th larval instar stage, consistent with indications from previous reports that the adult compound eye contributes to larval and pupal visual capabilities., (© 2015. Published by The Company of Biologists Ltd.)