Gene expression down-regulation in CD90+ prostate tumor-associated stromal cells involves potential organ-specific genes

<p>Abstract</p> <p>Background</p> <p>The prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THY1. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment.</p> <p>Methods</p> <p>Prostate CD90⁺stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder.</p> <p>Results</p> <p>The tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes.</p> <p>Conclusion</p> <p>CD90⁺prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development.</p

Resolving the homology-function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

The effects of interleukin-17A on T helper 2 responses in allergic airways disease

IL-17A (IL-17), a cytokine associated with severe asthma in humans, both positively and negatively regulates Th2 responses in murine models of allergic airways disease. In addition to Th17 cells, γδ T cells have also been described as a source of IL-17 in some murine models of asthma. While Th17 cells appear to act as positive regulators, some evidence suggests that IL-17-γδ T cells act as negative regulators of Th2-induced allergic airway responses. In order to better understand the effect of IL-17 on Th2-induced immune responses, we addressed the role of IL-17 sourced from CD4+ T cells and γδ T cells, and as a recombinant protein, using different murine models of allergic airways disease.We first determined the primary source of IL-17 and the role of the adjuvants, alum and complete Freund’s adjuvant (CFA) on IL-17 and Th2 inflammatory responses using an intraperitoneal ovalbumin (OVA) sensitization protocol. Following airway challenge, OVA/CFA sensitized mice had significantly greater serum levels of OVA-specific IgE and airway inflammation compared to OVA/sal and OVA/alum sensitized mice, but were comparatively airway hyporesponsive. Additionally, alum and CFA skewed the frequency of IL-17-producing bronchoalveolar lavage fluid (BALF) cells from Th17 toward IL-17-γδ T cells. Increasing the frequency of IL-17-γδ T cells in OVA/CFA sensitized mice, using a γδ TCR stimulatory antibody, inhibited airway hyperresponsiveness (AHR) and eosinophilia. Together these data provide evidence that IL-17-γδ T cells negatively regulate allergic airway responses.We next examined how recombinant IL-17 modulated airway inflammatory responses induced by IL-13, considered the major effector cytokine of Th2 cells. Two different doses of recombinant IL-17 were co-administered intranasally with IL-13. The lower dose of IL-17 enhanced IL-13-induced airway eosinophilic and lymphocytic inflammation, whereas the higher dose of IL-17 attenuated IL-13-induced AHR and eosinophilic inflammation. The adoptive transfer of γδ T cells, stimulated to express IL-17, similarly attenuated IL-13-induced eosinophilic and lymphocytic inflammation. Thus, the dose of IL-17 influences its role as a positive or negative regulator, while IL-17-γδ T cells act as a negative regulator of IL-13-induced airway responses.Finally, we used a T cell adoptive transfer model of allergic airways disease to examine how overexpressing IL-17, specifically by CD4+ T cells, modulated airway inflammatory responses. Populations of OVA-stimulated, gene-modified T cells overexpressing IL-17 were generated by transduction with recombinant retroviruses encoding IL-17. Following adoptive transfer and airway OVA challenge, our data demonstrated that BALF levels of IL-17 positively correlated with AHR. Moderate increases in BALF IL-17 were sufficient to induce airway neutrophilia, while higher BALF levels of IL-17 were associated with AHR, inflammation and enhanced Th2 responses. Thus, antigen-induced IL-17 production by CD4+ T cells positively regulates Th2-induced allergic airway responses.L’IL-17A (IL-17), une cytokine associée à l’asthme sévère chez les êtres humains, régule à la fois positivement et négativement les réponses des cellules Th2 chez les modèles murins de la maladie respiratoire allergique. En plus des cellules Th17, les cellules T γδ ont aussi été décrites comme étant une source d’IL-17 chez certains modèles murins de l’asthme. Alors que les cellules Th17 semblent agir en tant que régulateurs positifs, certaines données suggèrent que les cellules T IL-17-γδ agissent comme régulateurs négatifs aux réponses allergiques des voies respiratoires induites par les cellules Th2. Afin de mieux comprendre l’effet de l’IL-17 sur les réponses immunitaires induites par les cellules Th2, nous avons abordé le rôle de l’IL-17 provenant des cellules T CD4+ et cellules T γδ, et en tant que protéine recombinante, en utilisant divers modèles murins de la maladie allergique des voies respiratoires. Premièrement, nous avons déterminé la source primaire de l’IL-17 et le rôle des adjuvants, l’alum et l’adjuvant complet de Freund (ACF) sur l’IL-17 et les réponses inflammatoires des cellules Th2 en utilisant un protocole de sensibilisation intra-péritonéale avec de l’ovalbumine (OVA). Suite à l’épreuve sur les voies respiratoires, l’inflammation respiratoire et les niveaux sériques d’IgE spécifique à l’OVA étaient significativement plus élevés chez les les souris sensibilisées à l’OVA/ACF en comparaison à celles sensibilisées à l’OVA/salin ou à l’OVA/alum, même si la réponse comparative des voies respiratoires était hyporéactive. De plus, l’alum et l’ACF ont influé la fréquence de la production d’IL-17 par les cellules de fluide de lavage broncho-alvéolaire (FLBA) des cellules Th17 vers les cellules T IL-17-γδ. L’augmentation de la fréquence des cellules T IL-17-γδ chez les souris sensibilisées à l’OVA/ACF en utilisant un anticorps stimulant TCR γδ a inhibé l’hyperréactivité des voies respiratoires (HVR) et l’éosinophilie. L’ensemble de ces données fournissent la preuve que les cellules T IL-17-γδ régulent négativement les réponses allergiques des voies respiratoires. Nous avons ensuite examiné comment l’IL-17 recombinante modulait la réponse inflammatoire des voies respiratoires induite par l’IL-13, qui est considérée comme étant une cytokine effectrice principale des cellules Th2. Deux doses différentes d’IL-17 recombinante ont été co-administrées par voie intranasale avec l’IL-13. La plus faible dose d’IL-17 a renforcé l’inflammation éosinophilique et lymphocytaire des voies respiratoires induite par l’IL-13 alors que la dose la plus élevée d’IL-17 a atténué l’HVR induite par l’IL-13 et l’inflammation éosinophilique. Le transfert adoptif des cellules T γδ, stimulées pour exprimer l’IL-17, a atténué l’inflammation éosinophilique et lymphocytaire induite par l’IL-13 de façon similaire. Ainsi, la dose d’IL-17 influence son rôle en tant que régulateur positif ou négatif tandis que les cellules T IL-17-γδ agissent en tant que régulateurs négatifs des réponses des voies respiratoires induites par IL-13. Finalement, des populations de cellules T stimulées par de l’OVA, génétiquement modifiées et surexprimant IL-17 ont été générées par transduction avec des rétrovirus recombinants codant pour l’IL-17. Suite au transfert adoptif et à l’épreuve de l’OVA sur les voies respiratoires, nos données ont démontré que les niveaux d’IL-17 des cellules FLBA corrélaient positivement avec l’HVR. Des augmentations modérées d’IL-17 dans les cellules FLBA ont été suffisantes pour induire la neutrophilie dans les voies respiratoires alors que des niveaux plus élevés d’IL-17 dans les FLBA ont été associés à l’HVR, l’inflammation et les réponses renforcées par les cellules Th2. Ainsi, la production d’IL-17 induite par antigènes by les cellules T CD4+ régulent positivement les réponses allergiques des voies respiratoires induites par les cellules Th2

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3−/− in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies

Detection and genomic analysis of BRAF fusions in Juvenile Pilocytic Astrocytoma through the combination and integration of multi-omic data

Abstract Background Juvenile Pilocytic Astrocytomas (JPAs) are one of the most common pediatric brain tumors, and they are driven by aberrant activation of the mitogen-activated protein kinase (MAPK) signaling pathway. RAF-fusions are the most common genetic alterations identified in JPAs, with the prototypical KIAA1549-BRAF fusion leading to loss of BRAF’s auto-inhibitory domain and subsequent constitutive kinase activation. JPAs are highly vascular and show pervasive immune infiltration, which can lead to low tumor cell purity in clinical samples. This can result in gene fusions that are difficult to detect with conventional omics approaches including RNA-Seq. Methods To this effect, we applied RNA-Seq as well as linked-read whole-genome sequencing and in situ Hi-C as new approaches to detect and characterize low-frequency gene fusions at the genomic, transcriptomic and spatial level. Results Integration of these datasets allowed the identification and detailed characterization of two novel BRAF fusion partners, PTPRZ1 and TOP2B, in addition to the canonical fusion with partner KIAA1549. Additionally, our Hi-C datasets enabled investigations of 3D genome architecture in JPAs which showed a high level of correlation in 3D compartment annotations between JPAs compared to other pediatric tumors, and high similarity to normal adult astrocytes. We detected interactions between BRAF and its fusion partners exclusively in tumor samples containing BRAF fusions. Conclusions We demonstrate the power of integrating multi-omic datasets to identify low frequency fusions and characterize the JPA genome at high resolution. We suggest that linked-reads and Hi-C could be used in clinic for the detection and characterization of JPAs