Biophysical characterization and design of a minimal version of the Hoechst RNA aptamer.

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.

Male and female red-cheeked cordon bleus sing similar yet individualistic songs.

Birdsong is an excellent system for studying complex vocal signaling in both males and females. Historically, most research in captivity has focused only on male song. This has left a gap in our understanding of the environmental, neuroendocrine, and mechanistic control of female song. Here, we report the overall acoustic features, repertoire, and stereotypy of both male and female Red-Cheeked Cordon Bleus (Uraeginthus bengalus) (RCCBs) songs in the lab. We found few sex differences in the acoustic structure, song repertoire, and song stereotypy of RCCBs. Both sexes had similar song entropy, peak frequency, and duration. Additionally, individuals of both sexes sang only a single song type each and had similar levels of song and syllable stereotypy. However, we did find that female RCCBs had higher song bandwidth but lower syllable repertoires. Finally, and most strikingly, we found highly individualistic songs in RCCBs. Each individual produced a stereotyped and unique song with no birds sharing song types and very few syllable types being shared between birds of either sex. We propose that RCCBs represent a promising species for future investigations of the acoustic sex differences in song in a lab environment, and also for understanding the evolutionary driving forces behind individualistic songs.

Streamlined quantitative BOLD for detecting visual stimulus-induced changes in oxygen extraction fraction in healthy participants: toward clinical application in human glioma.

OBJECTIVE: Monitoring brain oxygenation is critical in brain tumors, as low oxygenation influences tumor growth, pathological angiogenesis, and treatment resistance. This study examined the ability of the streamlined quantitative (sq)BOLD MRI technique to detect oxygenation changes in healthy individuals, as well as its potential application in a clinical setting. METHODS: We used the asymmetric spin echo (ASE) technique with FLAIR preparation, along with model-based Bayesian inference to quantify the reversible transverse relaxation rate (R2') and oxygen extraction fraction (OEF) across the brain at baseline and during visual stimulation in eight healthy participants at 3T; and two patients with glioma at rest only. RESULTS: Comparing sqBOLD-derived parameters between baseline and visual stimulation revealed a decrease in OEF from 0.56 ± 0.09 at baseline to 0.54 ± 0.07 at the activated state (p = 0.04, paired t test) within a functional localizer-defined volume of interest, and a decline in R2' from 6.5 ± 1.3s-1 at baseline to 6.2 ± 1.4s-1 at the activated state (p = 0.006, paired t test) in the visual cortex. CONCLUSION: The sqBOLD technique is sensitive enough to detect and quantify changes in oxygenation in the healthy brain and shows potential for integration into clinical settings to provide valuable information on oxygenation in glioma.

Acute Recurrent Pancreatitis in a Pediatric Patient in the Setting of Viral Infection and COVID-19 Vaccination.

Acute pancreatitis within pediatric populations is predominately caused by mechanical obstruction, trauma, medications, and infections. We present a case of an adolescent female without any known anatomic or metabolic pre-disposition, developing recurrent acute pancreatitis that is seemingly related to acute viral infection and COVID-19 vaccination.

Methionine-producing tumor micro(be) environment fuels growth of solid tumors.

BACKGROUND: Recent studies have uncovered the near-ubiquitous presence of microbes in solid tumors of diverse origins. Previous literature has shown the impact of specific bacterial species on the progression of cancer. We propose that local microbial dysbiosis enables certain cancer phenotypes through provisioning of essential metabolites directly to tumor cells. METHODS: 16S rDNA sequencing of 75 patient lung samples revealed the lung tumor microbiome specifically enriched for bacteria capable of producing methionine. Wild-type (WT) and methionine auxotrophic (metA mutant) E. coli cells were used to condition cell culture media and the proliferation of lung adenocarcinoma (LUAD) cells were measured using SYTO60 staining. Further, colony forming assay, Annexin V Staining, BrdU, AlamarBlue, western blot, qPCR, LINE microarray and subcutaneous injection with methionine modulated feed were used to analyze cellular proliferation, cell-cycle, cell death, methylation potential, and xenograft formation under methionine restriction. Moreover, C14-labeled glucose was used to illustrate the interplay between tumor cells and bacteria. RESULTS/DISCUSSION: Our results show bacteria found locally within the tumor microenvironment are enriched for methionine synthetic pathways, while having reduced S-adenosylmethionine metabolizing pathways. As methionine is one of nine essential amino acids that mammals are unable to synthesize de novo, we investigated a potentially novel function for the microbiome, supplying essential nutrients, such as methionine, to cancer cells. We demonstrate that LUAD cells can utilize methionine generated by bacteria to rescue phenotypes that would otherwise be inhibited due to nutrient restriction. In addition to this, with WT and metA mutant E. coli, we saw a selective advantage for bacteria with an intact methionine synthetic pathway to survive under the conditions induced by LUAD cells. These results would suggest that there is a potential bi-directional cross-talk between the local microbiome and adjacent tumor cells. In this study, we focused on methionine as one of the critical molecules, but we also hypothesize that additional bacterial metabolites may also be utilized by LUAD. Indeed, our radiolabeling data suggest that other biomolecules are shared between cancer cells and bacteria. Thus, modulating the local microbiome may have an indirect effect on tumor development, progression, and metastasis.

Best Practice Recommendations for Stroke Vascular Imaging During Iodinated Contrast Shortage.

GE Healthcare© announced on April 19, 2022, that their main factory and distributor of iodinated contrast had experienced a temporary shutdown because of COVID-19 outbreak in Shanghai, China. This, along with other supply chain issues, led to a worldwide shortage of iodinated contrast agents, Omnipaque and Visipaque. Our Comprehensive Stroke Center was confronted with the cascading effect of this iodinated contrast material shortage. We took immediate steps to revise our protocols and processes to continue to provide high-quality care to our stroke patients. A multidisciplinary working group comprised of representatives of our stroke center, including vascular neurology, diagnostic neuroradiology, and neurovascular surgery, urgently met to brainstorm how to mitigate the shortage. We established parameters and local guidelines for the use of CT angiography, CT perfusion, and digital subtraction angiography for stroke patients. In this article, we propose "best practice" recommendations from a single Joint Commission approved Comprehensive Stroke Center that can be used as blueprint by other hospital systems when navigating potential future supply chain issues, to provide consistent high-quality stroke care.

Allosteric modulation of GluN1/GluN3 NMDA receptors by GluN1-selective competitive antagonists.

NMDA-type ionotropic glutamate receptors are critical for normal brain function and are implicated in central nervous system disorders. Structure and function of NMDA receptors composed of GluN1 and GluN3 subunits are less understood compared to those composed of GluN1 and GluN2 subunits. GluN1/3 receptors display unusual activation properties in which binding of glycine to GluN1 elicits strong desensitization, while glycine binding to GluN3 alone is sufficient for activation. Here, we explore mechanisms by which GluN1-selective competitive antagonists, CGP-78608 and L-689,560, potentiate GluN1/3A and GluN1/3B receptors by preventing glycine binding to GluN1. We show that both CGP-78608 and L-689,560 prevent desensitization of GluN1/3 receptors, but CGP-78608-bound receptors display higher glycine potency and efficacy at GluN3 subunits compared to L-689,560-bound receptors. Furthermore, we demonstrate that L-689,560 is a potent antagonist of GluN1FA+TL/3A receptors, which are mutated to abolish glycine binding to GluN1, and that this inhibition is mediated by a non-competitive mechanism involving binding to the mutated GluN1 agonist binding domain (ABD) to negatively modulate glycine potency at GluN3A. Molecular dynamics simulations reveal that CGP-78608 and L-689,560 binding or mutations in the GluN1 glycine binding site promote distinct conformations of the GluN1 ABD, suggesting that the GluN1 ABD conformation influences agonist potency and efficacy at GluN3 subunits. These results uncover the mechanism that enables activation of native GluN1/3A receptors by application of glycine in the presence of CGP-78608, but not L-689,560, and demonstrate strong intra-subunit allosteric interactions in GluN1/3 receptors that may be relevant to neuronal signaling in brain function and disease.

Corrigendum: The survivor strain: Isolation and characterization of Phormidium yuhuli AB48, a filamentous phototactic cyanobacterium with biotechnological potential.

[This corrects the article DOI: 10.3389/fbioe.2022.932695.].

Complete Genome Sequence of Phormidium yuhuli AB48, Isolated from an Industrial Photobioreactor Environment.

We report the genome of Phormidium yuhuli AB48, which includes a circular chromosome and a circular plasmid (4,747,469 bp and 51,599 bp, respectively). This is currently the only closed reference genome of an isolate of the Phormidium genus, based on the Genome Taxonomy Database (GTDB), providing a potential model system for sustainable biotechnology innovation.

MRI-Based Assessment of Brain Tumor Hypoxia: Correlation with Histology.

Cerebral hypoxia significantly impacts the progression of brain tumors and their resistance to radiotherapy. This study employed streamlined quantitative blood-oxygen-level-dependent (sqBOLD) MRI to assess the oxygen extraction fraction (OEF)-a measure of how much oxygen is being extracted from vessels, with higher OEF values indicating hypoxia. Simultaneously, we utilized vessel size imaging (VSI) to evaluate microvascular dimensions and blood volume. A cohort of ten patients, divided between those with glioma and those with brain metastases, underwent a 3 Tesla MRI scan. We generated OEF, cerebral blood volume (CBV), and vessel size maps, which guided 3-4 targeted biopsies per patient. Subsequent histological analyses of these biopsies used hypoxia-inducible factor 1-alpha (HIF-1α) for hypoxia and CD31 for microvasculature assessment, followed by a correlation analysis between MRI and histological data. The results showed that while the sqBOLD model was generally applicable to brain tumors, it demonstrated discrepancies in some metastatic tumors, highlighting the need for model adjustments in these cases. The OEF, CBV, and vessel size maps provided insights into the tumor's hypoxic condition, showing intertumoral and intratumoral heterogeneity. A significant relationship between MRI-derived measurements and histological data was only evident in the vessel size measurements (r = 0.68, p < 0.001).

Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells.

Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. This protocol was validated in: Sci Transl Med (2020), DOI: 10.1126/scitranslmed.aaz3866 Graphical abstract.

Metagenome-Assembled Genomes for "Candidatus Phormidium sp. Strain AB48" and Co-occurring Microorganisms from an Industrial Photobioreactor Environment.

Here, we report metagenome-assembled genomes for "Candidatus Phormidium sp. strain AB48" and three cooccurring microorganisms from a biofilm-forming industrial photobioreactor environment, using the PacBio sequencing platform. Several mobile genetic elements, including a double-stranded DNA phage and plasmids, were also recovered, with the potential to mediate gene transfer within the biofilm community.

PHLPP Signaling in Immune Cells.

Pleckstrin homology domain leucine-rich repeat protein phosphatases (PHLPP) belong to the protein phosphatase magnesium/manganese-dependent family of Ser/Thr phosphatases. Their general role as tumor suppressors has been documented for over a decade. In recent years, accumulating evidence suggests that PHLPP isozymes have key regulatory roles in both innate and adaptive immunity. In macrophages, PHLPP1 dampens signaling through TLR4 and the IFN-γ receptor by altering cytosolic signaling pathways. Additionally, nuclear-localized PHLPP1 inhibits STAT1-mediated inflammatory gene expression by direct dephosphorylation at Ser 727. PHLPP1 also regulates the migratory and inflammatory capacity of neutrophils in vivo. Furthermore, PHLPP1-mediated dephosphorylation of AKT on Ser 473 is required for both the suppressive function of regulatory T cells and for the pro-apoptotic effects of PHLPP1 in B cell chronic lymphocytic leukemia. In the context of immune homeostasis, PHLPP1 expression is modulated in multiple cell types by inflammatory signals, and the dynamics of its expression have varying effects on the pathogenesis of inflammatory bowel disease and septic shock. In this review, we summarize recent findings on the functions of PHLPP in inflammatory and regulatory signaling in the context of both innate and adaptive immunity.

The survivor strain: isolation and characterization of Phormidium yuhuli AB48, a filamentous phototactic cyanobacterium with biotechnological potential.

Despite their recognized potential, current applications of cyanobacteria as microbial cell factories remain in early stages of development. This is partly due to the fact that engineered strains are often difficult to grow at scale. This technical challenge contrasts with the dense and highly productive cyanobacteria populations thriving in many natural environments. It has been proposed that the selection of strains pre-adapted for growth in industrial photobioreactors could enable more productive cultivation outcomes. Here, we described the initial morphological, physiological, and genomic characterization of Phormidium yuhuli AB48 isolated from an industrial photobioreactor environment. P. yuhuli AB48 is a filamentous phototactic cyanobacterium with a growth rate comparable to Synechocystis sp. PCC 6803. The isolate forms dense biofilms under high salinity and alkaline conditions and manifests a similar nutrient profile to Arthrospira platensis (Spirulina). We sequenced, assembled, and analyzed the P. yuhuli AB48 genome, the first closed circular isolate reference genome for a member of the Phormidium genus. We then used cultivation experiments in combination with proteomics and metabolomics to investigate growth characteristics and phenotypes related to industrial scale cultivation, including nitrogen and carbon utilization, salinity, and pH acclimation, as well as antibiotic resistance. These analyses provide insight into the biological mechanisms behind the desirable growth properties manifested by P. yuhuli AB48 and position it as a promising microbial cell factory for industrial-scale bioproduction[221, 1631].

Masculinized Sexual Ornaments in Female Lizards Correlate with Ornament-Enhancing Thermoregulatory Behavior.

The adaptive significance of colorful or exaggerated traits (i.e., ornaments) expressed in females is often unclear. Competing hypotheses suggest that expression of female ornaments arises from maladaptive (or neutral) genetic inheritance from males along with incomplete epigenetic regulation, or from positive selection for ornaments in females under social competition. Whether costly or advantageous, the visibility of such traits can sometimes be behaviorally modulated in order to maximize fitness. Female eastern fence lizards express blue badges that are variable in size and color saturation. These are rudimentary compared to those seen in males and carry important costs such as reduced mating opportunities. Body temperature is a well-established enhancer of badge color, and thus thermoregulation may be one way these animals modulate badge visibility. We quantified realized body temperatures of female lizards paired in laboratory trials and observed that females with larger badges attained higher body temperatures when freely allowed to thermoregulate, sometimes beyond physiological optima. In this association between phenotype and behavior, females with larger badges exhibited thermoregulatory patterns that increase their badges' visibility. This signal-enhancing behavior is difficult to reconcile with the widely held view that female ornaments are maladaptive, suggesting they may carry context-dependent social benefits.

É muitas vezes incerta a significância adaptativa de caracteres vívidos e coloridos em fêmeas. Hipóteses para esse fenômeno sugerem uma herança maladaptativa (ou neutra) de caracteres selecionados em machos aliada à regulação epigenética incompleta em fêmeas, ou ainda seleção positiva em fêmeas imposta por competição social. Vantajosos ou não, a visibilidade de ornamentos muitas vezes é modulada por vias comportamentais do portador de modo a balancear seus custos e benefícios. Fêmeas Sceloporus undulatus possuem um par de marcas verde-azuis na parte ventral do pescoço que são variáveis em área e saturação entre indivíduos. Esses ornamentos são rudimentares em relação aos vistos em machos, mas ainda assim estão associados a custos reprodutivos importantes. Nessa espécie, temperatura corporal aumenta a visibilidade desses ornamentos significativamente. Portanto, a termorregulação é um comportamento que fêmeas poderiam empregar para modular a visibilidade de ornamentos. Nós quantificamos temperaturas corporais obtidas por pares de lagartos fêmeas em testes comportamentais e observamos que fêmeas com os maiores ornamentos também obtiveram temperaturas corporais mais altas, às vezes além do ótimo fisiológico. Nesta associação entre fenótipo e comportamento, fêmeas com os maiores ornamentos exibiram padrões de termoregulação que aumentaram sua visibilidade. Este padrão é difícil de conciliar com a perspectiva de que ornamentos são maladaptativos em fêmeas, sugerindo benefícios que são dependentes do contexto social.

PTEN is required for human Treg suppression of costimulation in vitro.

Regulatory T-cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signaling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T-cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.

BHLHE40 Regulates the T-Cell Effector Function Required for Tumor Microenvironment Remodeling and Immune Checkpoint Therapy Efficacy.

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell-dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti-PD-1 and anti-CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen-specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti-PD-1 and anti-CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome-tumor outgrowth-several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.

Helios is a marker, not a driver, of human Treg stability.

Treg therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T-cells and, thus, exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Treg lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part, due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid )-expressing population correlated with Treg destabilization. We used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-KO and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T-cells.

Suppression of Human Dendritic Cells by Regulatory T Cells.

Regulatory T cells (Tregs) suppress immune responses via a variety of mechanisms and can be used as a cellular therapy to induce tolerance. The function of Tregs is commonly assessed in vitro using assays that measure suppression of effector T cell proliferation and/or cytokine production. However, Tregs can also suppress the function of antigen presenting cells, creating a need for methodology to routinely measure this aspect of their function. This protocol describes a method to measure human Treg-mediated suppression of CD80 and CD86 expression on mature, monocyte-derived dendritic cells. Representative data show suppression mediated by polyclonal Tregs as well as antigen-specific Tregs generated using chimeric antigen receptor (CAR) technology. This method can be used in parallel to T cell suppression assays to measure the functional activity of human Tregs.