Exploring the role of cell adhesion proteins in retrovirus transmission and establishing CRISPR/Cas9 target enrichment Nanopore sequencing for retrovirus integration analysis

Retroviruses like the human-pathogenic human immunodeficiency virus (HIV) and the mouse-pathogenic murine leukemia virus (MLV) can disseminate between leukocytes by cell contact-dependent transmission. Productively infected cells (donor) were shown to transmit virus particles to surrounding lymphocytes (target) across tight cell-cell contacts designated virological synapses (cis-infection). These structures resemble the immunological synapse between leukocytes during immune cell priming and support efficient virus spread in vitro. While intravital microscopy of MLV infection in the lymph nodes of wild-type (WT) C57BL/6 mice revealed stable cell-cell contacts between infected donor and target lymphocytes, their contribution to retrovirus spread in vivo has yet to be established. In this study, we examine the function of the cell adhesion-mediating proteins LFA1 (CD11a/CD18) and ICAM1 in cell contact-dependent transmission to assess the role of cis-infection for retroviral spread. Antibody-mediated blocking of LFA1 and ICAM1 in co-culture assays of MLV-infected (MLV+) primary CD19+ B1 cells with non-infected FoxP3+ T cells indicates a crucial function of LFA1 and ICAM1 in MLV cis-infection. To study the individual role of LFA1 and ICAM1 on donor and target cells, we combine primary lymphocytes isolated from ICAM1-knockout (KO) and CD11a-KO (LFA1- deficient) mice with WT-derived cells in the established in vitro co-culture assay. Interestingly, efficient MLV cell-to-cell transmission critically depends on expression of LFA1 on non-infected target cells, and expression of LFA1-ligand ICAM1 on MLV- infected donor cells. Adoptive transfer experiments with in vitro-transduced MLV+ FoxP3+ T cells are used to characterize the individual contribution of LFA1 and ICAM1 during cis-infection in vivo. Strikingly, consistent with our in vitro findings, LFA1 expression on target cells and the presence of ICAM1 on MLV-infected donor cells determine efficiency of MLV transmission in vivo. During in vitro HIV infection, cell-to-cell transmission from productively infected donor cells to target lymphocytes across a cell-cell interface was previously shown to exceed efficiency of cell-free infection by 100- to 10,000-fold. Cell contact-dependent spread protected HIV from some neutralizing antibodies and could overcome the effect of certain cellular restriction factors and anti-retroviral drugs. Importantly, cell-cell contacts supported simultaneous transmission of multiple retroviral genomes, resulting in multicopy integration of HIV genomes. With the objective to characterize the consequences of cell-to-cell transmission at a proviral level, previous studies applied provirus detection techniques, such as fluorescence in situ hybridization (FISH) and multifluorescent reporter systems. Unfortunately, all approaches have limitations in their performance to detect the total number of proviruses together with the exact chromosomal integration site. Here, we establish CRISPR/Cas9 target enrichment Nanopore sequencing as a novel provirus detection technique for localization and quantification of individual proviruses integrated in the genome of an infected host cell. This approach applies CRISPR/Cas9 activity for selective target enrichment without the requirement of PCR amplification and combines it with the long-read capacity of Nanopore sequencing. We validate our workflow on a library of individual, GFP-expressing (GFP+) cell clones, containing a GFP-encoding retrovirus reporter genome at distinct frequencies and locations. Purified genomic DNA of GFP+ cell clones is subjected to CRISPR/Cas9 target enrichment Nanopore sequencing. Genomic mapping of reads containing GFP sequences identifies retrovirus reporter genome integration sites within the host genome. Strikingly, the number of detected proviruses positively correlates with the GFP expression level (mean fluorescence intensity) and the relative GFP copy number for each analyzed GFP+ cell clone. Breakpoint-spanning PCR and Sanger sequencing validate accuracy of identified GFP-encoding retrovirus reporter integration sites. To implement our novel provirus detection technique for retroviral transmission studies, we combine CRISPR/Cas9 target enrichment Nanopore sequencing with in vitro co-culture assays. Interestingly, our first results indicate a contribution of both, provirus frequency and provirus integration site, to efficiency of MLV transmission from an MLV-infected donor cell to a non-infected target cell. In summary, the findings in our study reveal that cell adhesion-mediating proteins LFA1 and ICAM1 support efficient retroviral transmission in vitro and in vivo. In addition, with CRISPR/Cas9 target enrichment Nanopore sequencing, we established a long-read sequencing approach that allows for rapid and accurate identification of provirus integration sites. In future experiments, this technique can be applied to quantitatively and qualitatively assess the consequences of cell contact-dependent transmission during retrovirus infection.Retroviren wie das humanpathogene Humane Immundefizienz-Virus (HIV) und das mauspathogene Murine Leukämievirus (MLV) können Zellkontakt-abhängig zwischen Leukozyten übertragen werden. Produktiv infizierte Zellen (Donoren) transferieren dabei Viruspartikel über Zell-Zell-Kontakte, sogenannte virologische Synapsen (cis-Infektion), zu benachbarten Lymphozyten (Akzeptoren). Diese Strukturen ähneln der Immunologischen Synapse, die zwischen Leukozyten während des Primings von T-Zellen geformt wird, und ermöglichen hoch effiziente Virusübertragung. Intravitalmikroskopie der Lymphknoten MLV-infizierter Wildtyp (WT) C57BL/6 Mäuse erlaubte die Visualisierung stabiler Zell-Zell-Kontakte zwischen infizierten Donorzellen und nicht-infizierten Akzeptorzellen. Deren Bedeutung für die Ausbreitung von Retroviren in vivo ist allerdings unklar. In dieser Studie untersuchen wir die Funktion der Zelladhäsionsproteine LFA1 (CD11a/CD18) und ICAM1 während Zellkontakt-abhängiger retroviraler Übertragung, um die Rolle der cis-Infektion für die Ausbreitung von Retroviren zu verstehen. Kokulturen von MLV-infizierten (MLV+), primären CD19+ B1-Zellen mit nicht- infizierten FoxP3+ T-Zellen in Gegenwart von blockierenden Antikörpern deuten an, dass LFA1 und ICAM1 eine bedeutsame Rolle bei der cis-Infektion zukommt. Um den Beitrag von LFA1 und ICAM1 auf Donor- und Akzeptorzellen individuell zu untersuchen, kombinieren wir primäre Lymphozyten, isoliert von WT C57BL/6, ICAM1- knockout (KO) und CD11a-KO (LFA1-defizienten) Mäusen, in in vitro cis- Infektionsexperimenten. Interessanterweise determiniert die Expression von LFA1 auf der Oberfläche nicht-infizierter Akzeptorzellen und die Expression des LFA1-Liganden ICAM1 auf MLV-infizierten Donorzellen die Effizienz der Zell-Zell-Übertragung von MLV. Um den individuellen Beitrag von LFA1 und ICAM1 während der cis-Infektion in vivo zu charakterisieren, überführen wir mittels adoptivem Zelltransfer in vitro- transduzierte MLV+ FoxP3+ T-Zellen in lebende Mäuse. Im Einklang mit unseren Ergebnissen unter in vitro Bedingungen, beeinflussen die Präsenz von LFA1 auf Akzeptorzellen und ICAM1 auf Donorzellen die Effizienz der MLV Übertragung in vivo. Frühere in vitro Infektionsstudien haben gezeigt, dass die Zell-Zell-Übertragung von produktiv HIV-infizierten Donorzellen zu Akzeptor-Lymphozyten über Zell-Zell- Kontakte 100- bis 10,000-fach effizienter ist als eine zell-freie Infektion. Die Zellkontakt- abhängige Übertragung schützt HIV vor zahlreichen neutralisierenden Antikörpern und setzt die Wirkung bestimmter zellulärer Restriktionsfaktoren und anti-retroviraler Medikamente aus. Vor allem unterstützen Zell-Zell-Kontakte die simultane Übertragung multipler retroviraler Partikel, die in mehrfacher Integration einzelner HIV-Genome resultieren können. Mit dem Ziel die Konsequenzen retroviraler Zell-Zell Kontakte auf proviraler Ebene zu charakterisieren, haben vergangene Studien Provirusdetektionstechniken, wie Fluoreszenz-in-situ-Hybridisierung (FISH) und Multifluoreszenz-Reportersysteme, verwendet. Leider liefern diese Ansätze keine Erkenntnisse über die Gesamtzahl integrierter retroviraler Genome zusammen mit deren Integrationsstelle innerhalb des Wirtsgenoms. In dieser Studie etablieren wir CRISPR/Cas9 Zielanreicherung Nanopore-Sequenzierung als neue Provirusdetektionstechnik, um die Lokalisation und die Frequenz individueller, integrierter Proviren innerhalb des Genoms einer infizierten Wirtszelle zu bestimmen. Dieser Ansatz macht sich die Spezifizität des CRISPR/Cas9 Systems zu Nutze, um selektiv und ohne PCR-basierte Amplifikation Zielgenome anzureichern, und kombiniert dies mit der charakteristischen Eigenschaft von Nanopore-Sequenzierung, lange Reads zu generieren. Wir validieren unseren Ansatz anhand einer Bibliothek GFP- exprimierender (GFP+) Zellklone, die jeweils eine unterschiedliche Anzahl an GFP- kodierenden, retroviralen Reportergenomen an verschiedenen Integrationsstellen in ihrem Genom tragen. Die aufgereinigte, genomische DNA von GFP+ Zellklonen wird mit Hilfe von CRISPR/Cas9 Zielanreicherung Nanopore-Sequenzierung analysiert. Die Zuordnung von Reads, die Teile des GFP-kodierenden, retroviralen Reportergenoms enthalten, zu dem entsprechenden Referenzgenom erlaubt die Identifizierung von Integrationstellen des Reportergenoms innerhalb des Wirtsgenoms. Dabei korrelieren das GFP Expressionslevel (durchschnittliche Fluoreszenzintensität) und die relative GFP Kopienanzahl für jeden analysierten GFP+ Zellklon positiv mit der Anzahl detektierter Proviren. Anhand von PCR-basierter Amplifikation der Übergangsstellen von Wirtszellen- zu proviraler DNA und anschließender Analyse der amplifizierten Genfragmente mittels Sanger-Sequenzierung validieren wir die Präzision mit der einzelne Integrationsstellen des GFP-kodierenden, retroviralen Reportergenoms identifiziert wurden. Um unsere neue Provirusdetektionstechnik für erste Studien zur retroviralen Übertragung einzusetzen, kombinieren wir CRISPR/Cas9 Zielanreicherung Nanopore- Sequenzierung mit in vitro Kokultur Assays. Hierbei deuten unsere ersten Ergebnisse einen Einfluss der Provirusanzahl sowie der proviralen Integrationsstelle auf die Effizienz der MLV Übertragung von einer MLV-infizierten Donorzelle zu einer nicht-infizierten Akzeptorzelle an. Zusammenfassend zeigen die Ergebnisse unserer Studie, dass die Zelladhäsionsproteine LFA1 und ICAM1 zu effizienter, retroviraler Übertragung in vitro und in vivo beitragen. Zudem haben wir mit CRISPR/Cas9 Zielanreicherung Nanopore-Sequenzierung einen neuen Ansatz etabliert, der die schnelle und akkurate Identifikation von proviralen Integrationsstellen ermöglicht. In zukünftigen Experimenten kann diese Technik angewandt werden, um die quantitativen und qualitativen Konsequenzen von Zellkontakt-abhängiger retroviraler Transmission zu untersuchen

The Inclusive Semileptonic Decay Lepton Spectrum from B→Xeν‾B \to X e \overline{\nu}

In this talk, we review the QCD calculation of the lepton spectrum from inclusive semileptonic $B$ decay. We compare this prediction to that of the ACCMM model. This latter work was done in collaboration with Csaba Csaki.Comment: MIT-CTP-2333, uses LATEX. Invited Talk, Presented at WHEPP-3 Workshop in Madras,India, January, 199

How to assess technological developments in basic research? Enabling formative interventions regarding sustainability, ethics, and consumer issues at an early stage

In an era of ever faster and more momentous technological development, both technology assessment and transdisciplinary interventions are in danger of structurally lagging behind the speed of innovation. This paper proposes a new tiered approach to technology assessment at low Technology Readiness Levels that enables a both rapid and concerted interdisciplinary science response to this Great Acceleration. Covering sustainability, ethics, and consumer issues, this approach encourages and enables the innovators themselves to conduct assessments embedded in the innovation process as early as possible. Suitable tools for early engagement that help facilitate development-integrated assessments are introduced and described. The design and use of these instruments in the field of basic research is illustrated using the Cluster of Excellence livMatS as an example.In einer Ära immer rascherer und folgenreicherer technologischer Entwicklungen laufen sowohl die Technologiebewertung als auch transdisziplinäre Interventionen Gefahr, strukturell hinter der Dynamik von Innovationen zurückzubleiben. In diesem Beitrag wird ein neuer gestufter Ansatz für die Technikfolgenabschätzung für niedrige Technology Readiness Levels vorgeschlagen, der eine schnelle und konzertierte interdisziplinäre Reaktion der Wissenschaft auf diese "große Beschleunigung" ermöglicht. Dieser Ansatz, der Nachhaltigkeits-, Ethik- und Verbraucherfragen abdeckt, ermutigt und befähigt die Innovatoren, in den Innovationsprozess eingebettete Bewertungen so früh wie möglich selbst durchzuführen. Geeignete Instrumente für entwicklungsintegrierte Bewertungen werden vorgestellt und beschrieben. Die Konzeption und Anwendung dieser Instrumente im Bereich der Grundlagenforschung wird am Beispiel des Exzellenzclusters livMatS veranschaulicht

Chiral Perturbation Theory for B→D∗B \rightarrow D^* and B→DB \rightarrow D Semileptonic Transition Matrix Elements at Zero Recoil

Heavy quark symmetry predicts the value of $B \rightarrow D$ and $B \rightarrow D^*$ transition matrix elements of the current $\bar c \gamma_\mu (1 - \gamma_5)b$, at zero recoil (where in the rest frame of the $B$ the $D$ or $D^*$ is also at rest). We use chiral perturbation theory to compute the leading corrections to these predictions which are generated at low momentum, below the chiral symmetry breaking scale.Comment: 10 pages, 1 figure (not included), CALT-68-1844, MIT-CTP-217

Impact of Noisy Labels on Dental Deep Learning—Calculus Detection on Bitewing Radiographs

Supervised deep learning requires labelled data. On medical images, data is often labelled inconsistently (e.g., too large) with varying accuracies. We aimed to assess the impact of such label noise on dental calculus detection on bitewing radiographs. On 2584 bitewings calculus was accurately labeled using bounding boxes (BBs) and artificially increased and decreased stepwise, resulting in 30 consistently and 9 inconsistently noisy datasets. An object detection network (YOLOv5) was trained on each dataset and evaluated on noisy and accurate test data. Training on accurately labeled data yielded an mAP50: 0.77 (SD: 0.01). When trained on consistently too small BBs model performance significantly decreased on accurate and noisy test data. Model performance trained on consistently too large BBs decreased immediately on accurate test data (e.g., 200% BBs: mAP50: 0.24; SD: 0.05; p < 0.05), but only after drastically increasing BBs on noisy test data (e.g., 70,000%: mAP50: 0.75; SD: 0.01; p < 0.05). Models trained on inconsistent BB sizes showed a significant decrease of performance when deviating 20% or more from the original when tested on noisy data (mAP50: 0.74; SD: 0.02; p < 0.05), or 30% or more when tested on accurate data (mAP50: 0.76; SD: 0.01; p < 0.05). In conclusion, accurate predictions need accurate labeled data in the training process. Testing on noisy data may disguise the effects of noisy training data. Researchers should be aware of the relevance of accurately annotated data, especially when testing model performances

Lifestyle factors and experience of respiratory alarm symptoms in the general population

BACKGROUND: The first step in the diagnosis of lung cancer is for individuals in the general population to recognise respiratory alarm symptoms (RAS). Knowledge is sparse about RAS and factors associated with experiencing RAS in the general population. This study aimed to estimate the prevalence of RAS in the general population, and to analyse possible associations between lifestyle factors and experiencing RAS. METHODS: A web-based survey comprising 100 000 individuals randomly selected from the Danish Civil Registration System. Items regarding experience of RAS (prolonged coughing, shortness of breath, coughing up blood and prolonged hoarseness) and self-reported lifestyle factors (smoking status, alcohol intake and body mass index) were included in the analysis. RESULTS: A total of 49 706 individuals completed the questionnaire. 16 per cent reported at least one RAS. Prolonged coughing (8.4%) and shortness of breath (8%) were most prevalent, while coughing up blood was least prevalent (0.1%). More men than women reported RAS (p<0.001). Odds of reporting RAS increased with age (P(trend)<0.001). In men and women, former and current smoking was associated with reporting at least one RAS (former smoking: OR(men)=1.42, 95% CI 1.39 to 1.56; OR(women)=1.25, 95% CI 1.15 to 1.36; current smoking: OR(men)=2.58, 95% CI 2.35 to 2.83; OR(women)=2.45, 95% CI 2.25 to 2.68). Individuals who were underweight or obese were significantly more likely to report at least one RAS. Odds of reporting at least one RAS increased with increasing alcohol intake for both genders (P(trend)<0.001). CONCLUSIONS: RAS are common in the general population. Men experience more symptoms than women, and prevalence increases with age. Being a former or current smoker and being underweight or obese are positively associated with experiencing RAS. The likelihood of experiencing RAS increases with increasing alcohol intake. Future research should investigate healthcare seeking for RAS among individuals with different lifestyles

Comment on nonperturbative effects in B→XsγB\to X_s\gamma

Uncertainties in the theoretical prediction for the inclusive $B\to X_s\gamma$ decay rate are discussed. We emphasize that there is no operator product expansion for this process. Nonetheless, some nonperturbative effects involving a virtual $c\bar c$ loop are calculable using the operator product expansion. They give a contribution to the decay rate that involves the B meson matrix element of an infinite tower of operators. The higher dimension operators give effects that are only suppressed by powers of $m_b\Lambda_{QCD}/m_c^2 \sim 0.6$, but come with small coefficients.Comment: 9 pages revtex, 2 figures included; minor changes, to appear in Phys. Lett.

Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two- dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan- Brucella, B. abortus- and B. melitensis-specific antibodies