Visualization of the HIV-1 Nuclear Preintegration Complex Structure by High Precision Correlative Light - and Electron Microscopy and - Tomography

Upon fusion of the viral envelope with the host cell membrane, the capsid of the human immunodeficiency virus 1 (HIV-1) is released into the host cell cytoplasm. To productively infect a cell, the viral RNA genome needs to be reverse transcribed into viral DNA. This in turn needs to become integrated into the host cell genome. Integration can, however, only happen, after the viral genome is released from its capsid-container, in a process called uncoating. This is a vital process and needs to be regulated and orchestrated in certain ways – which are still elusive and controversially discussed. Some studies suggest that uncoating takes place soon after -, or concomitant with viral entry. Other researchers came to the result that the capsid needs to retain its structure to shield the viral components from being sensed by the innate cellular immune system. Both hypotheses, early uncoating and prolonged structural retention, are solidly supported by experimental data. Therefore, the timing and kinetics of uncoating remain unresolved. Based on previous results from our group, we had reason to believe that the capsid might indeed be retained, possibly even within the nucleus. A method was developed, that allows the detection of viral DNA. The presence of viral DNA was used as a criterion to discriminate between productive and nonproductive subviral particles in infected cells. Surprisingly, productive subviral particles displayed an intense, stable signal for capsid protein in immunofluorescence experiments, throughout the cytoplasm and even within the nuclei of infected cells. A strong signal is can be understood as a high concentration of labeled protein, which in turn might indicate the presence of a retained structure. However, intense immunofluorescence signals can also mean more efficient binding of antibodies due to structural rearrangements (such as uncoating), and a high spatial concentration of proteins cannot be directly interpreted as structure retention. In this study, we present a unique way to address and solve this important question. We specifically focused on the small fraction of productive particles. Light Microscopy allows specific labeling but has low resolution. Electron Microscopy yields much higher resolution, but specific (immuno)labeling is difficult and often detrimental to ultrastructural retention. We overcame both limitations by correlative light – and electron microscopy: Regions of interest were identified by specific nuclear subviral particle surrogate markers in light microscopy. On these regions, tilt series electron tomography was performed, to visualize the subviral particles’ structure, as well as the subcellular environment, around the region of interest. Performing high resolution tilt series electron tomography, we could repeatedly and convincingly visualize a capsid-reminiscent structure that underlies HIV-1 nuclear preintegration complexes. This apparent structure is very similar in shape, but smaller in size compared to capsids of virus particles of mostly identical preparations. The discovery of a retained capsid structure in the nucleus of an infected cell will advance on our understanding of nuclear entry and provides whole new insights into the overall understanding of HIV-1 in early steps of infection

When Optimal Isn’t Optimal

Whether you’re an operations manager, a financial executive, an engineer, or an analyst, chances are you’ve been taught to optimize the outcomes of your decisions. And taken in a vacuum, that makes a good deal of sense. But as the new discipline of complex adaptive networks shows, in our heavily interconnected, interdependent world optimizing can introduce hidden vulnerabilities to surprisingly expensive failures. In this webinar we’ll discuss examples of such failures and identify conditions for which a satisfactory but not optimal solution is the best approach overall

Profluorogenic reductase substrate for rapid, selective, and sensitive visualization and detection of human cancer cells that overexpress NQO1

Achieving the vision of identifying and quantifying cancer-related events and targets for future personalized oncology is predicated on the existence of synthetically accessible and economically viable probe molecules fully able to report the presence of these events and targets in a rapid and highly selective and sensitive fashion. Delineated here are the design and evaluation of a newly synthesized turn-on probe whose intense fluorescent reporter signature is revealed only through probe activation by a specific intracellular enzyme present in tumor cells of multiple origins. Quenching of molecular probe fluorescence is achieved through unique photoinduced electron transfer between the naphthalimide dye reporter and a covalently attached, quinone-based enzyme substrate. Fluorescence of the reporter dye is turned on by rapid removal of the quinone quencher, an event that immediately occurs only after highly selective, two-electron reduction of the sterically and conformationally restricted quinone substrate by the cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1). Successes of the approach include rapid differentiation of NQO1-expressing and -nonexpressing cancer cell lines via the unaided eye, flow cytometry, fluorescence imaging, and two-photon microscopy. The potential for use of the turn-on probe in longer-term cellular studies is indicated by its lack of influence on cell viability and its in vitro stability. © 2012 American Chemical Society

Equine nutrition: a survey of perceptions and practices of horse owners undertaking a massive open online course in equine nutrition

An online survey was designed to ascertain the following information: demographics, current feeding practices, and perceptions and knowledge of equine nutrition, including nutrition-related disorders. Response rate was 34% (6,538 respondents). More than 80% of respondents were horse owners or caretakers, with the majority owning between one and five horses (75%) aged 5 years and older (74%). Most kept their horses for pleasure (54%), with 33% using them mostly for competition and 13% using them for an equal mix of both pleasure and competition. Concentrates were fed by the majority (87%), and more than 70% stated that their horses had some access to pasture. Over half of respondents (60%) regularly monitored their horses' weight, with most doing this monthly. Weight tapes were most commonly used (62%), although many reported to guess the weight of their horse(s) with very few (5%) using weight scales. Under half (46%) stated that they regularly used body condition scoring (BCS), many did not use BCS at all (24%), and some did not know what BCS was (10%). Of those that did use BCS, most (36%) did this monthly, with others weekly (25%), daily (14%), and when they remembered (15%). Overall knowledge of nutrition was reported by most as average (median, 3 on Likert scale—average); however, respondents were less knowledgeable on the management of nutrition-related disorders

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): study protocol for a two-factorial randomized controlled multi-arm trial with cluster sampling

Background: To achieve higher effectiveness in population-based SARS-CoV-2 surveillance and to reliably predict the course of an outbreak, screening, and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic. Methods: This trial implements a two-factorial, randomized, controlled, multi-arm, prospective, interventional, single-blinded design with cluster sampling and four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy based on individuals' self-collection of saliva samples which are then sent to and analyzed by a laboratory. The targeted sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (2500 participants per arm). Strategies differ with respect to tested population groups (individuals vs. all household members) and testing approach (without vs. with pre-screening survey). The trial is complemented by an economic evaluation and qualitative assessment of user experiences. Primary outcomes include costs per completely screened person, costs per positive case, positive detection rate, and precision of positive detection rate. Discussion: Systems for active surveillance of the general population will gain more importance in the context of pandemics and related disease prevention efforts. The pandemic parameters derived from such active surveillance with routine population monitoring therefore not only enable a prospective assessment of the short-term course of a pandemic, but also a more targeted and thus more effective use of local and short-term countermeasures. Trial registration: ClinicalTrials.gov DRKS00023271. Registered November 30, 2020, with the German Clinical Trials Register (Deutsches Register Klinischer Studien

From disgusting and complicated to simple and brilliant: Implementation perspectives and lessons learned from users and rejectors of mail-in SARS-CoV-2 gargle tests

BackgroundDespite the important role of testing as a measure against the COVID-19 pandemic, user perspectives on SARS-CoV-2 tests remain scarce, inhibiting an improvement of testing approaches. As the world enters the third year of the pandemic, more nuanced perspectives of testing, and opportunities to expand testing in a feasible and affordable manner merit consideration.MethodsConducted amid the second pandemic wave (late 2020–early 2021) during and after a multi-arm trial evaluating SARS-CoV-2 surveillance strategies in the federal state Baden-Württemberg, Germany, this qualitative sub-study aimed to gain a deeper understanding of how test users and test rejectors perceived mail-in SARS-CoV-2 gargle tests. We conducted 67 semi-structured in-depth interviews (mean duration: 60 min) via telephone or video call. Interviews were audio-recorded, transcribed verbatim and analyzed inductively using thematic analysis. The Consolidated Framework for Implementation Research guided the findings' presentation.ResultsRespondents generally described gargle sampling as simple and comfortable. However, individual perceptions of the testing method and its feasibility varied widely from disgusting and complicated to simple and brilliant. Self-sampling was appreciated for lowering infection risks during testing, but also considered more complex. Gargle-sampling increased participants' self-efficacy to sample correctly. Communication (first contact, quantity and content of information, reminders, support system) and trust (in the study, its institutional affiliation and test method) decisively influenced the intervention's acceptability.ConclusionUser-driven insights on how to streamline testing include: consider communication, first impressions of tests and information as key for successful mail-in testing; pay attention to the role of mutual trust between those taking and administering tests; implement gargle self-sampling as a pleasant alternative to swab testing; offer multiple test methods to increase test up-take

Genes in S and T Subgenomes Are Responsible for Hybrid Lethality in Interspecific Hybrids between Nicotiana tabacum and Nicotiana occidentalis

Many species of Nicotiana section Suaveolentes produce inviable F(1) hybrids after crossing with Nicotiana tabacum (genome constitution SSTT), a phenomenon that is often called hybrid lethality. Through crosses with monosomic lines of N. tabacum lacking a Q chromosome, we previously determined that hybrid lethality is caused by interaction between gene(s) on the Q chromosome belonging to the S subgenome of N. tabacum and gene(s) in Suaveolentes species. Here, we examined if hybrid seedlings from the cross N. occidentalis (section Suaveolentes)×N. tabacum are inviable despite a lack of the Q chromosome.Hybrid lethality in the cross of N. occidentalis×N. tabacum was characterized by shoots with fading color. This symptom differed from what has been previously observed in lethal crosses between many species in section Suaveolentes and N. tabacum. In crosses of monosomic N. tabacum plants lacking the Q chromosome with N. occidentalis, hybrid lethality was observed in hybrid seedlings either lacking or possessing the Q chromosome. N. occidentalis was then crossed with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT), to reveal which subgenome of N. tabacum contains gene(s) responsible for hybrid lethality. Hybrid seedlings from the crosses N. occidentalis×N. tomentosiformis and N. occidentalis×N. sylvestris were inviable.Although the specific symptoms of hybrid lethality in the cross N. occidentalis×N. tabacum were similar to those appearing in hybrids from the cross N. occidentalis×N. tomentosiformis, genes in both the S and T subgenomes of N. tabacum appear responsible for hybrid lethality in crosses with N. occidentalis

Independent, Rapid and Targeted Loss of Highly Repetitive DNA in Natural and Synthetic Allopolyploids of Nicotiana tabacum

Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations. We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor). Using next generation sequencing, a recently developed graph-based repeat identification pipeline, Southern blot and fluorescence in situ hybridisation (FISH) we characterise two highly repetitive DNA sequences (NicCL3 and NicCL7/30). Analysis of two independent high-throughput DNA sequencing datasets indicates NicCL3 forms 1.6–1.9% of the genome in N. tomentosiformis, sequences that occur in multiple, discontinuous tandem arrays scattered over several chromosomes. Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum. Surprisingly elimination of NicCL3 is repeated in some synthetic lines of N. tabacum in their forth generation. The retroelement NicCL7/30, which occurs interspersed with NicCL3, is also under-represented but to a much lesser degree, revealing targeted elimination of the latter. Analysis of paired-end sequencing data indicates the tandem component of NicCL3 has been preferentially removed in natural N. tabacum, increasing the proportion of the dispersed component. This occurs across multiple blocks of discontinuous repeats and based on the distribution of nucleotide similarity among NicCL3 units, was concurrent with rounds of sequence homogenisation

Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity

The epithelial-to-mesenchymal transition (EMT) is a critical developmental process that has recently come to the forefront of cancer biology. In breast carcinomas, acquisition of a mesenchymal-like phenotype that is reminiscent of an EMT, termed oncogenic EMT, is associated with pro-metastatic properties, including increased motility, invasion, anoikis resistance, immunosuppression and cancer stem cell characteristics. This oncogenic EMT is a consequence of cellular plasticity, which allows for interconversion between epithelial and mesenchymal-like states, and is thought to enable tumor cells not only to escape from the primary tumor, but also to colonize a secondary site. Indeed, the plasticity of cancer cells may explain the range of pro-metastatic traits conferred by oncogenic EMT, such as the recently described link between EMT and cancer stem cells and/or therapeutic resistance. Continued research into this relationship will be critical in developing drugs that block mechanisms of breast cancer progression, ultimately improving patient outcomes