Monitoring molecular events during photo-driven ubiquinone pool reduction in PufX+ and PufX- membranes from Rhobobacter capsulatus by time-resolved FTIR difference spectroscopy.

The PufX protein is found in the photosynthetic membranes of several purple bacteria and is involved in ubiquinol-ubiquinone exchange at the QB site of the reaction center. We have studied quinone pool reduction in chromatophores from PufX+ and PufX- strains of Rhodobacter capsulatus by time-resolved FTIR difference spectroscopy under and after continuous illumination. To our knowledge, it is the first time that quinone pool reduction has been directly followed in real time in Rba. capsulatus membranes. Thanks to the availability in the literature of IR marker bands for protein conformational changes, ubiquinone consumption, ubiquinol production, Q---QH2 quinhydrone complex formation, as well as for RC-bound QA- and QB- semiquinone species, it is possible to follow all the molecular events associated with light-induced quinone pool reduction. In Rba. capsulatus PufX + chromatophores, these events resemble the ones found in Rba. sphaeroides wild-type membranes. In PufX- chromatophores the situation is different. Spectra recorded during 22.7 s of illumination showed a much smaller amount of photoreduced quinol, consistent with previous observations that PufX is required for efficient QH2/Q exchange at the QB site of the RC. Q consumption and QH2 formation are rapidly associated with QA- formation, showing that the structure of the RC-LH1 complex in PufX- membranes does not provide efficient access to the QB site of the RC to a large fraction of the quinone pool, evidently because the LH1 ring increases in size to impair access to the RC. The presence of a positive band at 1560 cm-1 suggests also the transient formation, in a fraction of chromatophores or of RC-LH1 complexes, of a Q---QH2 quinhydrone complex. Experiments carried out after 2-flash and 10-flash sequences make it possible to estimate that the size of the quinone pool with access to the QB site in PufX- membranes is ≥ 5 ubiquinone molecules per RC. The results are discussed in the framework of the current knowledge of protein organization and quinone pool reduction in bacterial photosynthetic membranes.

Hypoxia induces ROS-resistant memory upon reoxygenation in vivo promoting metastasis in part via MUC1-C.

Hypoxia occurs in 90% of solid tumors and is associated with metastasis and mortality. Breast cancer cells that experience intratumoral hypoxia are 5x more likely to develop lung metastasis in animal models. Using spatial transcriptomics, we determine that hypoxic cells localized in more oxygenated tumor regions (termed 'post-hypoxic') retain expression of hypoxia-inducible and NF-kB-regulated genes, even in the oxygen-rich bloodstream. This cellular response is reproduced in vitro under chronic hypoxic conditions followed by reoxygenation. A subset of genes remains increased in reoxygenated cells. MUC1/MUC1-C is upregulated by both HIF-1α and NF-kB-p65 during chronic hypoxia. Abrogating MUC1 decreases the expression of superoxide dismutase enzymes, causing reactive oxygen species (ROS) production and cell death. A hypoxia-dependent genetic deletion of MUC1, or MUC1-C inhibition by GO-203, increases ROS levels in circulating tumor cells (CTCs), reducing the extent of metastasis. High MUC1 expression in tumor biopsies is associated with recurrence, and MUC1+ CTCs have lower ROS levels than MUC1- CTCs in patient-derived xenograft models. This study demonstrates that therapeutically targeting MUC1-C reduces hypoxia-driven metastasis.

Cytokines drive the formation of memory-like NK cell subsets via epigenetic rewiring and transcriptional regulation.

Activation of natural killer (NK) cells with the cytokines interleukin-12 (IL-12), IL-15, and IL-18 induces their differentiation into memory-like (ML) NK cells; however, the underlying epigenetic and transcriptional mechanisms are unclear. By combining ATAC-seq, CITE-seq, and functional analyses, we discovered that IL-12/15/18 activation results in two main human NK fates: reprogramming into enriched memory-like (eML) NK cells or priming into effector conventional NK (effcNK) cells. eML NK cells had distinct transcriptional and epigenetic profiles and enhanced function, whereas effcNK cells resembled cytokine-primed cNK cells. Two transcriptionally discrete subsets of eML NK cells were also identified, eML-1 and eML-2, primarily arising from CD56bright or CD56dim mature NK cell subsets, respectively. Furthermore, these eML subsets were evident weeks after transfer of IL-12/15/18-activated NK cells into patients with cancer. Our findings demonstrate that NK cell activation with IL-12/15/18 results in previously unappreciated diverse cellular fates and identifies new strategies to enhance NK therapies.

Inferring cellular and molecular processes in single-cell data with non-negative matrix factorization using Python, R and GenePattern Notebook implementations of CoGAPS.

Non-negative matrix factorization (NMF) is an unsupervised learning method well suited to high-throughput biology. However, inferring biological processes from an NMF result still requires additional post hoc statistics and annotation for interpretation of learned features. Here, we introduce a suite of computational tools that implement NMF and provide methods for accurate and clear biological interpretation and analysis. A generalized discussion of NMF covering its benefits, limitations and open questions is followed by four procedures for the Bayesian NMF algorithm Coordinated Gene Activity across Pattern Subsets (CoGAPS). Each procedure will demonstrate NMF analysis to quantify cell state transitions in a public domain single-cell RNA-sequencing dataset. The first demonstrates PyCoGAPS, our new Python implementation that enhances runtime for large datasets, and the second allows its deployment in Docker. The third procedure steps through the same single-cell NMF analysis using our R CoGAPS interface. The fourth introduces a beginner-friendly CoGAPS platform using GenePattern Notebook, aimed at users with a working conceptual knowledge of data analysis but without a basic proficiency in the R or Python programming language. We also constructed a user-facing website to serve as a central repository for information and instructional materials about CoGAPS and its application programming interfaces. The expected timing to setup the packages and conduct a test run is around 15 min, and an additional 30 min to conduct analyses on a precomputed result. The expected runtime on the user's desired dataset can vary from hours to days depending on factors such as dataset size or input parameters.

Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses.

Assays linking cellular phenotypes with T cell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between T cell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After ex vivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.

Digitize your Biology! Modeling multicellular systems through interpretable cell behavior.

Cells are fundamental units of life, constantly interacting and evolving as dynamical systems. While recent spatial multi-omics can quantitate individual cells' characteristics and regulatory programs, forecasting their evolution ultimately requires mathematical modeling. We develop a conceptual framework-a cell behavior hypothesis grammar-that uses natural language statements (cell rules) to create mathematical models. This allows us to systematically integrate biological knowledge and multi-omics data to make them computable. We can then perform virtual "thought experiments" that challenge and extend our understanding of multicellular systems, and ultimately generate new testable hypotheses. In this paper, we motivate and describe the grammar, provide a reference implementation, and demonstrate its potential through a series of examples in tumor biology and immunotherapy. Altogether, this approach provides a bridge between biological, clinical, and systems biology researchers for mathematical modeling of biological systems at scale, allowing the community to extrapolate from single-cell characterization to emergent multicellular behavior.

Immunologic profiling in schizophrenia and rheumatoid arthritis.

The negative relationship between schizophrenia (SCZ) and rheumatoid arthritis (RA) has been observed for 85 years, but the mechanisms driving this association are unknown. This study analyzed differences in profiles of cytokines (IL-1ß, IL-Ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα), selected genes (HLA-DRB1, IL1RN, HP2), and antibodies related to gluten sensitivity (AGA-IgG, AGA-IgA), celiac disease (tTG), and systemic autoimmunity (ANA, anti-CCP, RF) in 40 subjects with SCZ, 40 with RA, and 40 healthy controls (HC). HLA-DRB1*04:01 alleles were enriched in persons with SCZ and RA compared with HC, and the HP2/HP2 genotype was 2-fold more prevalent in AGA/tTG-positive versus negative SCZ patients. Patients with SCZ demonstrated 52.5% positivity for any of the antibodies tested, compared to 90% of RA patients and 30% of HC. Cluster analysis of the cytokines revealed three clusters: one associated with SCZ marked by high levels of IL-1Ra, one associated with HC, and one associated with both SCZ and RA marked by elevated levels of IFNγ, TNFα, and IL-6. These analyses suggest that stratification of SCZ patients by cytokine profile may identify unique SCZ subgroups and enable the use of currently available cytokine-targeted treatment strategies.

Resection of an ameloblastoma in a pediatric patient and immediate reconstruction using a combination of tissue engineering and costochondral rib graft: A case report.

BACKGROUND AND OVERVIEW: Ameloblastoma is an odontogenic tumor predominantly occurring in patients who are in their 20s and 30s. Approximately 10% to 15% of ameloblastomas occur in patients younger than 18 years. Although it is a benign tumor, an ameloblastoma can have a devastating effect on children both physically and emotionally. The aim of this case report is to demonstrate how tissue engineering and surgical techniques can minimize morbidity and recovery time after extirpation and immediate reconstruction of a mandibular ameloblastoma. CASE DESCRIPTION: An 11-year-old girl was referred for surgical evaluation of a lesion found on a routine dental radiograph. Resection of a mandibular unicystic ameloblastoma resulted, including immediate reconstruction using a costochondral rib graft, allogeneic bone, bone marrow aspirate concentrate, and recombinant human morphogenetic protein-2. One year postoperatively, the patient had no evidence of recurrence as well as excellent mandibular bone height and width with good facial form. The patient has returned to her daily life without any disabilities or disfigurement. CONCLUSIONS AND PRACTICAL IMPLICATIONS: Dentists are typically the first health care providers to discover oral pathology in patients. The coordination of care by the dental care providers and the oral and maxillofacial specialist was key to the successful outcome for this patient. With biotechnology and surgical techniques, the dental surgeon can extirpate an ameloblastoma and reconstruct the mandible defect to the ideal shape and size with minimal morbidity and recovery time.

Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA.

UNLABELLED: Gene transfer agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated gene acquisition. This paper gives genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated gene transfer combines key aspects of two bacterial horizontal gene transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCE: Horizontal gene transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by the R. capsulatus RcGTA into recipient cells requires key genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.

The O2-responsive repressor PpsR2 but not PpsR1 transduces a light signal sensed by the BphP1 phytochrome in Rhodopseudomonas palustris CGA009.

Regulatory properties of bacteriophytochrome BphP1 were evaluated with respect to the photosynthesis gene transcription repressors PpsR1 and PpsR2 of Rhodopseudomonas palustris strain CGA009 in gene complementation, replacement and deletion experiments. The results indicate that 750 nm wavelength light activates BphP1 to antagonize repression of photosynthesis gene expression by PpsR2, but not PpsR1. It is suggested that an equilibrium exists between BphP1-active and -inactive conformations, with 750 nm light shifting the equilibrium to an active conformation, although a phenotypically detectable component of BphP1 is in the active conformation in the absence of illumination.

Dynamics of fluxes through photosynthetic complexes in response to changing light and inorganic carbon acclimation in Synechococcus elongatus.

Cyanobacteria acclimate to environmental inorganic carbon (C(i)) concentrations through re-organisations of photosynthetic function and the induction of carbon concentrating mechanisms (CCMs), which alter and constrain their subsequent acclimation to changing light. We grew cells acclimated to high C(i) (4 mM) or low C(i) (0.02 mM), shifted them from 50 micromol m(-2) s(-1) to 500 micromol m(-2) s(-1), and quantified their photosynthetic performance in parallel with quantitation of allocations to key indicator macromolecules. Pigments cell(-1) declined, PsbA (PS II), AtpB (ATP Synthase), RbcL (Rubisco) and GlnA (Glutamine Synthetase) increased, and PsaC (PS I) remained stable through the light shift. The increase in these protein pools was slower and smaller in low C(i) cells, but acted in both cell types to re-normalise the electron fluxes through the catalytic complexes back toward values before the light shift (for PsbA and GlnA) or even below the initial flux per complex (for RbcL). In contrast, an increased electron flux per PsaC was sustained for at least 6 h after the increase in light. Initially, high levels of PS II cell(-1) and PS II connectivity in high C(i) cells caused a more rapid net photoinactivation of PS II in high C(i) cells than in low C(i) cells, depressing the rate of PS II-specific electron transport (PS II ETR) to levels similar to linear ETR (net O(2) evolution minus respiration). In low C(i) cells, PS II ETR remained in excess of linear ETR and may have helped maintain CCM activity. The pool sizes of PsbA, AtpB and GlnA correlated with cellular growth rate, and changed at similar rates in high C(i) and low C(i) cells when expressed on a generational rather than chronological timescale, which has implications for differing ecology of high and low C(i) cells under variable natural light.

Large reallocations of carbon, nitrogen, and photosynthetic reductant among phycobilisomes, photosystems, and Rubisco during light acclimation in Synechococcus elongatus strain PCC7942 are constrained in cells under low environmental inorganic carbon.

Synechococcus elongatus strain PCC7942 cells were grown in high or low environmental concentrations of inorganic C (high-C(i), low-C(i)) and subjected to a light shift from 50 micromol m(-2) s(-1) to 500 micromol m(-2) s(-1). We quantified photosynthetic reductant (O(2) evolution) and molar cellular contents of phycobilisomes, PSII, PSI, and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through the light shift. Upon the increase in light, small initial relative decreases in phycobilisomes per cell resulted from near cessation of phycobilisome synthesis and their dilution into daughter cells. Thus, allocation of reductant to phycobilisome synthesis dropped fivefold from pre- to post-light shift. The decrease in phycobilisome synthesis liberated enough material and reductant to allow a doubling of Rubisco and up to a sixfold increase in PSII complexes per cell. Low-C(i) cells had smaller initial phycobilisome pools and upon increased light; their reallocation of reductant from phycobilisome synthesis may have limited the rate and extent of light acclimation, compared to high-C(i) cells. Acclimation to increased light involved large reallocations of C, N, and reductant among different components of the photosynthetic apparatus, but total allocation to the apparatus was fairly stable at ca. 50% of cellular N, and drew 25-50% of reductant from photosynthesis.