Subject: Social Sciences, Media Studies Keywords: live-streaming; video-conference; broadcast; scientific conferences; diversity; inclusion
Online: 10 March 2020 (02:29:22 CET)
Live streaming conferences increase the participation of a diverse audience, help defray travel costs and overcome problems related to travel restrictions. In this article, we lay out tips for implementing live-streaming in scientific meetings. We also cover legal, ethical, and technical aspects implicated with live-streaming scientific talks. To write this article, we leveraged knowledge from our experience in organizing the symposium “Deciphering the Denisovans,” presented at the 88th Annual Meeting of the American Association of Physical Anthropology (AAPA) in Cleveland, OH, in 2019, as well as literature on the topic.
ARTICLE | doi:10.20944/preprints202309.0409.v1
Subject: Medicine And Pharmacology, Reproductive Medicine Keywords: monopronuclear; embryo transfer; live birth
Online: 6 September 2023 (09:55:31 CEST)
Fertilized zygotes normally display 2 pronuclei (PN) but abnormal fertilization patterns (0, 1 or >2 PN) are daily observed in IVF labs. Multiple PN zygotes (>2) are generally discarded due to an increased risk of aneuploidy. However, the decision to transfer or not 1PN-derived embryos remains controversial. The aims of our study were to analyze the neonatal outcomes of fresh or frozen-thawed embryos derived from 1PN zygotes, and to evaluate the influence of the fertilization method. Data were retrospectively collected from cycles performed between January 2018 and December 2022. Fresh cycles were analyzed for the comparative fate of 1PN zygotes (n=1234) following conventional in vitro fertilization (cIVF; n=648) or intracytoplasmic sperm injection (ICSI; n=586), as well as the results of the 64 transfers of 1PN derived embryos (pregnancy rate (PR) and neonatal outcomes). This pregnancy follow-up was also applied to 167 transfers of frozen-thawed 1PN derived embryos. In fresh cycles, 46% of the 1PN zygotes in cIVF group gave rise to embryos of sufficient quality to be transferred or frozen (day 3 or 5/6). This rate decreased to 33% in the fresh ICSI cycles. Blastulation rate was also significantly higher in cIVF group (44%) in comparison to ICSI group (20%). The fresh embryo transfers (32 per group) allowed 7 pregnancies in the cIVF group (PR=21.9%) as compared to 4 pregnancies in the ICSI group (PR=12.5%). In the cIVF group, 4 deliveries of healthy newborns were achieved and only one in the ICSI group. In frozen/thawed cycles, 36 pregnancies were obtained out of the 167 transfers. A non-significative difference was observed between embryos derived from cIVF cycles (PR=26%) and ICSI cycles (PR=16%) with respectively 18 and 8 healthy babies born. In conclusion, we observed better outcomes for 1PN zygotes in cIVF cycles in comparison to ICSI cycles. Our center policy to transfer good quality 1PN-derived embryos allowed the birth of 31 healthy babies.
ARTICLE | doi:10.20944/preprints202304.0868.v1
Subject: Medicine And Pharmacology, Obstetrics And Gynaecology Keywords: IVF; blastocyst transfer; cryopreservation; live birth; age
Online: 25 April 2023 (03:01:55 CEST)
The ability to predict the likelihood of a live birth after single fresh embryo transfer is important for treatment planning and managing patient expectation, particularly in their first in vitro fertilization (IVF) cycle. Cryopreservation of supernumerary embryos is often regarded as an important prognostic variable and a surrogate marker of success for several reasons. While previous large studies have examined the association between the number of oocytes retrieved and cleavage-stage embryos available, and the odds of a live birth following a fresh embryo transfer, the relationship between the number of supernumerary blastocysts cryopreserved following a fresh embryo transfer has not been rigorously studied. We performed a retrospective analysis of data collected between 2006 and 2018 for all first time IVF patients with a fresh autologous day 5 single blastocyst transfer. The relationship between the likelihood of a live birth and number of supernumerary blastocysts cryopreserved was assessed according to patient age group. In patients aged <35 years and 35-39 years old, the likelihood of a live birth increased linearly from 0.33 (95%CI:0.31–0.34) to 0.80 (95%CI:0.74–0.86; P<.0001) and 0.30 (95%CI:0.28–0.32) to 0.82 (95%CI:0.73–0.91;P<.0001) between 1-6 blastocysts cryopreserved and then non-linearly to 0.95 (95% CI 0.92–0.97; P<.0001) and 0.96 (95%CI:0.93–0.99; P<.0001) if 10 or more blastocysts were cryopreserved, respectively. When aged 40 years and above, the likelihood of a live birth increased linearly from 0.26 (95%CI:0.19–0.32) to 0.83 (95%CI:0.68–0.97; P<.0001) between 1-4 blastocysts cryopreserved and then non-linearly to 0.99 (95%CI:0.98–0.99; P<.0001) if 10 or more blastocysts were cryopreserved. The present study demonstrated a non-linear relationship between the number of supernumerary blastocysts cryopreserved and the likelihood of a live birth after single blastocyst transfer in the first autologous fresh IVF/ICSI cycle across different age groups.
ARTICLE | doi:10.20944/preprints202305.2093.v1
Subject: Medicine And Pharmacology, Gastroenterology And Hepatology Keywords: NAFLD; Gut Microbiota; Inflammation; Metagenomics; Live Biotherapeutic Product
Online: 30 May 2023 (08:59:14 CEST)
Non-alcoholic fatty liver disease (NAFLD) affects about 20–40% of the adult population in high-income countries and is now a leading indication for liver transplantation and can drive to hepatocellular carcinoma. The link between gut microbiota dysbiosis and NAFLD is now clearly established. Through analyses of the gut microbiota with shotgun metagenomics, we observed that compared to healthy controls, Adlercreutzia equolifaciens is depleted in patients with liver diseases such as NAFLD. Its abundance also decreases as the disease progresses and eventually disappears in the last stages indicating a strong association with disease severity. Moreover, we show that A. equolifaciens possesses anti-inflammatory properties, both in vitro and in vivo in a humanized mouse model of NAFLD. Therefore, our results demonstrate a link between NAFLD and the severity of liver disease and the presence of A. equolifaciens and its anti-inflammatory actions. Counterbalancing dysbiosis with this bacterium may be a promising live biotherapeutic strategy of liver diseases..
ARTICLE | doi:10.20944/preprints201701.0137.v1
Subject: Biology And Life Sciences, Cell And Developmental Biology Keywords: cell culture; bioreactor; live cell imaging; tissue engineering
Online: 31 January 2017 (12:39:46 CET)
Here, we present a staged approach for an innovative repurposing of a portable infant humidicrib into a live cell growth, observation, and imaging system. Furthmore, humidicrib can support different variations of “umbilical” bioreactors, and can be used to conduct electrophysiology experiments and in situ immunohistochemistry. Modifications incorporate a closed loop carbon dioxide (CO2) concentration control system with umbilical CO2 and heating support for tailored bioreactors. The repurposing cost is inexpensive and allows for the continued observation and imaging of cells. This prototype unit has been used to continuously observe and image live primary neurons for up to 21 days. This demonstrates the repurposed units’ suitability for use in tissue culture based research, particularly where modifications to microscopes are required or where sensitive manipulation outside of a standard incubator is needed.
ARTICLE | doi:10.20944/preprints202203.0233.v2
Subject: Engineering, Control And Systems Engineering Keywords: UAV; UAS; SATCOM; ISR; Live aerial observations; HALE; HAPS
Online: 24 March 2022 (09:19:30 CET)
Unmanned Aerial vehicles (UAVs) can transmit critical Intelligence, surveillance, and reconnaissance data right from drones when operators need real-time access to information and find it hard to wait for the return of aircraft. Artificial satellites are applied in offering communication links between different points on earth. Satellite communication can be helpful in telecommunication systems globally. SATCOM is the data and voice service that allows aircraft to communicate using satellite using air traffic control and airline operations centers at high-frequency stations and when covering the conventional ground radar. Unmanned Aerial Vehicles Satcom are applied in different aspects of real-life applications like traffic monitoring, play load delivery, surveillance, and seemingly dangerous areas. They can go where one cannot go and experience experiences that were never seen before. UAV Satcom is used when transmitting the ISR, Intelligence, and reconnaissance data right from drones, especially in situations in which the decision-makers need near real-time. All current signs show the exciting and bright future for using unmanned aerial vehicle Satcom technology. The future generations are projected to feature enhanced autonomy and improve safety standards. SATCOM can be the future communication link for UAVs. SATCOM is being used increasingly in surveys in the aftermath of any disaster.
ARTICLE | doi:10.20944/preprints202210.0239.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: Microbiota; Immunity; Spermidine; Metabolic engineering; Probiotics; Live Biotherapeutic Product; Metabolomics
Online: 17 October 2022 (12:09:41 CEST)
Over the past decade, studies have demonstrated the importance of bioactive metabolites derived from the microbiota in the regulation of physiological processes essential for homeostasis and the maintenance of human health. Strategies to modulate the production of these metabolites in the gastrointestinal tract hold promise for combating dysbiosis or inflammatory bowel disease. Metabolic engineering of probiotics could be one of these solutions. In this work, we engineered Escherichia coli Nissle 1917 (EcN) to overproduce spermidine, a metabolite known for its anti-immunosenescence and anti-inflammatory properties. Using a rational synthetic biology approach coupled with analysis by high resolution mass spectrometry, we designed in several steps and validated engineered probiotics overproducing and excreting spermidine. Based on our results, we first added the enzyme substrate putrescine and showed the overproduction of spermidine and decided to add a transporter limiting the production of the acetylated form of spermidine. Next, we used untargeted metabolomics to study the impact of engineering on the central metabolism of E. coli Nissle. Untargeted metabolomics appears to be a good strategy to optimize the metabolic engineering of probiotic strains and thus accelerate their development for personalized medicine.
ARTICLE | doi:10.20944/preprints201811.0150.v2
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: meagre; Argyrosomus regius; protease activity; inhibitions; live foods; in vitro
Online: 9 November 2018 (10:43:38 CET)
The aim of this study was to gather preliminary data about the potential inhibitory effects and contributions of live foods used from 3 to 32 days after hatching (DAH) in routine feeding protocols on protease activities of meagre, Argyrosomus regius (Asso 1801) larvae, using in vitro techniques. Enriched rotifer, Artemia nauplii and Artemia metanauplii were tested in the present study. The highest values of protease activities of meagre larvae at 7 DAH in 2013 and 2014 years were established. The lowest values at 15 DAH in 2013 and at 20 DAH in 2014 were observed. Protease activities of enriched rotifer, Artemia nauplii, and Artemia metanauplii were 21.76±0.31, 36.00±1.48–29.33±0.93, and 416.44±19.7–403.53±11.85 U/mg protein, respectively (p< 0.05). The highest inhibitions of live foods were observed at 7 DAH. The positive contributions of live food Artemia metanauplii’s on protease activities of meagre larvae were significant (p< 0.05). The inhibitory effects and positive contributions of live foods on survival and growth rates of meagre larvae, should be taken into account in meagre and other marine fish larvae’s future studies. Cysteine protease activities of Artemia sp. should be investigated to provide the higher growth and survival ratio from the feeding protocols used in marine fish larvae.
ARTICLE | doi:10.20944/preprints202309.0490.v1
Subject: Biology And Life Sciences, Aquatic Science Keywords: nutrition; Hippocampus hippocampus; live feed enrichment; microalgae; essential fatty acids; copepods
Online: 7 September 2023 (07:11:45 CEST)
This study aimed to determine a nutritionally adequate feeding protocol for Hippocampus hippocampus juveniles. In the experimental trial, seahorses were fed copepods from 0-7 days post-parturition (DPP) and, from 8-28 DPP, four dietary treatments: (copepods (control diet), microalgae-enriched Artemia with a DHA/EPA ratio of 2:1, microalgae-enriched Artemia with a DHA/EPA ratio of 2:1 along with 5% copepods, and with 10% copepods) were tested. Results showed that juvenile seahorses fed on copepods attained a significantly higher (P<0.05) growth performance (5.1 mg d-1) than fish-fed diets with 5% and 10% copepod inclusion, (3.09 and 3.07 mg d-1, respectively), and fish-fed enriched Artemia (1.83 mg d-1) which performed poorly. Data suggests that feeding copepods during the first 7 DPP promotes maturation of the digestive tract of juvenile seahorses, and the addition of a limited amount of copepods to the diet improves H. hippocampus juvenile growth performance when compared to the use of Artemia as a single diet due to the improvement of the essential fatty acid profile in the diets.
ARTICLE | doi:10.20944/preprints202308.1770.v1
Subject: Medicine And Pharmacology, Reproductive Medicine Keywords: In vitro fertilization; gonadotropin dose; live birth rate; poor embryo quality
Online: 25 August 2023 (07:11:04 CEST)
We aimed to investigate the clinical results following poor-quality embryo transfer and the parameters to foresee the prognosis. In this study, 2123 cycles that had day 3 and day 5 single-fresh embryo with a poor-quality embryo transfers and a good-quality embryo transfers were compared. The cycles according to transfer day were evaluated by conducting a subgroup analysis. The correlation between all the obtained demographic characteristics, controlled ovarian stimulation parameters and cycle results were analysed. Clinical pregnancy was established in 53 patients that underwent transfer in the poor-quality embryo group (14.9%). Of these patients, 36 had live birth (live birth rate per clinical pregnancy 67.9%). In cleavage-stage embryos, live birth rates per clinical pregnancy were higher in poor-quality blastocyst transfer. When analysing the factors affecting live births in poor-quality embryo group, as the total gonadotropin dose increases, the probability of live birth decreases, as in the probability of hCG positivity. In conclusion; although the probability of pregnant is low, when clinical pregnancy is established, there is a high chance of having a live birth after poor-quality embryo transfers. This could be regarded as an acceptable option in cycles when only poor-quality embryos were available.
REVIEW | doi:10.20944/preprints202202.0011.v1
Subject: Medicine And Pharmacology, Pediatrics, Perinatology And Child Health Keywords: Rotavirus; off-target effects; neonatal; live attenuated; RV3-BB; epigenetic modulation
Online: 1 February 2022 (12:34:07 CET)
Following the introduction of live-attenuated rotavirus vaccines in many countries, a notable reduction in deaths and hospitalizations associated with diarrhoea in children <5 years of age has been reported. There is growing evidence to suggest that live-attenuated vaccines also provide protection against other infections beyond the vaccine-targeted pathogens. These so called off-target effects of vaccination have been associated with the tuberculosis vaccine Bacille Calmette Guérin (BCG), measles, oral polio and recently salmonella vaccines, and are thought to be mediated by modified innate and possibly adaptive immunity. Indeed, rotavirus vaccines have been reported to provide greater than expected reductions in acute gastroenteritis caused by other enteropathogens, that have mostly been attributed to herd protection and prior underestimation of rotavirus disease. Whether rotavirus vaccines also alter the immune system to reduce non targeted gastrointestinal infections has not been studied directly. Here we review the current understanding of the mechanisms underlying off-target effects of vaccines and propose a mechanism by which the live-attenuated neonatal rotavirus vaccine, RV3-BB, could promote protection beyond the targeted pathogen. Finally, we consider how vaccine developers may leverage these properties to improve health outcomes in children, particularly those in low-income countries where disease burden and mortality is disproportionately high relative to developed countries.
ARTICLE | doi:10.20944/preprints202008.0110.v1
Subject: Social Sciences, Sociology Keywords: physical activity; active live; motivational factors; health; Europe; structural equation modelling
Online: 5 August 2020 (05:18:39 CEST)
Low levels of physical activity (PA) and sedentarism are associated with the onset of different pathologies and health problems. Regular physical activity has been linked with being beneficial to the health of the general population. Within this framework of analysis, the aim of the present study is to analyse the existing association between the time spent doing physical activity and the expressed motives for doing so, from which the first innovative aspect of the paper emerges: the use of the time spent doing PA as a study variable of the phenomenon. The data analysed come from the latest special Eurobarometer survey about the sport and physical activity done in Europe. Using an exploratory factorial analysis and a structural equations model, we were able to find a six-dimensional factorial model that explains the reasons for doing PA, demonstrating that there is no relationship between the reasons for and time spent doing PA. Therefore, motivation is not a variable that explains the time spent doing PA and another type of variable must be used to explain the phenomenon if PA is to be incentivised.
ARTICLE | doi:10.20944/preprints202308.0620.v1
Subject: Chemistry And Materials Science, Analytical Chemistry Keywords: histidine; microbial fluorescence sensor; S. Typhimurium SL1344; live cell imaging; histidine deficiency
Online: 8 August 2023 (11:48:13 CEST)
Histidine is an essential amino acid with significant implications in human growth and neuromodulation. Its concentration within cells, when either elevated or reduced, can indicate different diseases. While various methods exist to measure increased histidine levels, there remains a significant lack of sensors capable of actively responding to histidine deficiency within cells and releasing strong signals. In this study, we exploited the high induction levels of the his operon in S. Typhimurium SL1344, a histidine auxotroph, within a histidine-deficient environment, to develop a specific bacterial sensor with sensitivity towards low histidine concentrations. The SL1344-pGEX sensor, with a high copy number, exhibited remarkable sensitivity and selectivity to histidine in the range of 0 to 50 μM. Notably, even a minute addition of histidine (approximately 2.5 μM) to the M9 medium led to observable fluorescence reduction, rendering it highly suitable for monitoring histidine-deficient cellular environments. This bacterial fluorescence sensor holds great potential for detecting changes in intracellular histidine concentrations during pathogenic bacterial infections and for assessing the impact of drugs on cellular metabolism.
ARTICLE | doi:10.20944/preprints202306.1798.v1
Subject: Biology And Life Sciences, Life Sciences Keywords: Ecological ice temperature; chilling stress; anhydrous live preservation; regulation mechanism; Patinopecten yessoensis
Online: 26 June 2023 (10:57:27 CEST)
In this paper, the quality change of scallop (Patinopecten yessoensis) in the process of anhydrous storage and transportation after cold-acclimation and induced dormancy was studied, and the regulation mechanism of quality degradation in the storage and transportation in the process of gradient chilling stress and drying exposure was further explored. The results show that while being transferred from anhydrous storage and transportation from hydrous storage and transportation, the breathing pattern of scallops were changed from aerobic to anaerobic. Their gill filaments were altered, and the apparent vitality was constantly declining, which was reflected by the edge shrinkage of pallium and the direct proportion between the edge reduction rate and the stimulus response period. After being in the anhydrous state for 4d, the AEC value dropped to 67.59%. At this time, if they were under hydration again, they would resume a nice growth state. By virtue of proteomics, it was revealed from the analysis that cold acclimation and dry exposure mainly led to changes in biological functions and pathways such as mitochondrial inner membrane and ATP hydrolysis activity. In addition, it can be seen from the functional annotation and enrichment analysis of the metabolite KEGG that cold acclimation promoted the purine metabolism of scallops, while dry exposure inhibited the metabolism of saturated fatty acids. Besides, in this study, the infrared sensing mode was used for the first time to record the heart rate changes of the scallops during circulation, which provides feasibility for the nondestructive vitality monitoring of Lamellibranchia.
ARTICLE | doi:10.20944/preprints202211.0208.v1
Subject: Computer Science And Mathematics, Analysis Keywords: social media; marketing; user engagement; brand analysis; long-running live event; fashion
Online: 11 November 2022 (02:08:43 CET)
The rapid penetration of social media has been redefining every facet of the old marketing and customer engagement tactics, not only for the low-end and mass products but also for luxury brands. In this context, brands are dealing with the challenge of keeping the balance between using mass marketing strategies concurrent with accentuating the exclusivity of their offerings. Social media can be considered a boon if brands employ them to reach the right audience and use the right platform by incorporating the right content. In this work, we propose a sector-specific, integrated, and holistic investigation of the social media strategies of luxury brands, together with the impact they generate in terms of the engagement level of the users as an indicator of their success. We provide empirical validation of the method in the sector of luxury fashion brands in the Italian market, providing qualitative and quantitative analysis of the content shared on social media, considering the type, timing, and modality of the sharing. We evaluate consumer-brand engagement in different contexts, including important live events in the field.
REVIEW | doi:10.20944/preprints202106.0018.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: emerging model organism; evo-devo; development; evolution; regeneration; genetic tools; live imaging
Online: 1 June 2021 (10:37:04 CEST)
Arthropods are the most abundant and diverse animals on earth. Among them, pancrustaceans are an ancient and morphologically diverse group, comprising a wide range of aquatic and semi-aquatic crustaceans as well as the insects, which emerged from crustacean ancestors to colonise most terrestrial habitats. Within insects, Drosophila stands out as one of the most powerful animal models, making major contributions to our understanding of development, physiology and behaviour. Given these attributes, crustaceans provide a fertile ground for exploring biological diversity through comparative studies. However, beyond insects, few crustaceans are developed sufficiently as experimental models to enable such studies. The marine amphipod Parhyale hawaiensis is currently the best established crustacean system, offering year-round accessibility to developmental stages, transgenic tools, genomic resources, and established genetics and imaging approaches. The Parhyale research community is small but diverse, investigating the evolution of development, regeneration, aspects of sensory biology, chronobiology, bioprocessing and ecotoxicology.
ARTICLE | doi:10.20944/preprints202103.0360.v1
Subject: Computer Science And Mathematics, Computer Science Keywords: music production, latent space, live system, recurrent autoencoder, dynamic time warping, compression
Online: 15 March 2021 (08:04:00 CET)
The onset of coronavirus disease 2019 (COVID-19), an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has sparked unprecedented change. Due to the public health guidelines imposed during the COVID-19 pandemic, there is no longer sufficient street traffic for remaining buskers to generate sufficient revenue, leading a majority of street musicians to pursue remote music production. However, real-time music production is notoriously difficult due to the excessively high latencies that current video call platforms such as Zoom and Google Meet harbor. In this paper, we propose an architecture for a platform with end-to-end, near-lossless audio transmission tailored specifically to online joint music production, called Latent Space. We discuss the usage of a recurrent autoencoder with sequence-aware encoding (RAES) and a 1D convolutional layer for audio compression, which we dub ClefNet, as well as propose a new evaluation metric for naive autoencoders (AEs), MSE-DTW loss, which combines the traditional mean square error (MSE) loss function with dynamic time warping (DTW) to prevent an increase in loss when the target sequence predicted by the AE is strictly a temporal variation of the source sequence. Moreover, we detail the logistics of a live system implementation which uses the Web Audio API to extract raw audio samples in real-time to feed into our client-side model before relaying the traffic using peer-to-peer WebRTC technology. The Latent Space platform can be accessed at https://latent-space.tech, and the code and data can be found under the MIT License at https://github.com/rvignav/ClefNet.
ARTICLE | doi:10.20944/preprints202009.0259.v1
Subject: Engineering, Industrial And Manufacturing Engineering Keywords: droplet breakup; microfluidic droplet generation; live image analysis; orifice; high pressure homogenization
Online: 11 September 2020 (12:46:49 CEST)
For our research on droplet deformation and breakup in scaled high-pressure homogenizing units we developed a pressure stable inline droplet generator. It consists of an optically accessible flow channel with a combination of stainless steel and glass capillaries and a 3D printed orifice. The droplet size is determined online by live image analysis. The influence of the orifice diameter, the mass flow of the continuous phase and the mass flow of the disperse phase on the droplet diameter was investigated. Furthermore, the droplet detachment mechanisms were identified. Droplet diameters with small diameter fluctuation between 175 µm and 500 µm could be realized, which allows a precise adjustment of the Ca and We Number in the subsequent scaled high pressure homogenizer disruption unit. The determined influence of geometry and process parameters on the resulting droplet size and droplet detachment mechanism agreed well with literature on microfluidics. Furthermore, droplet trajectories in an exemplary scaled high-pressure homogenizer disruption unit are presented which show that the droplets can be reinjected on a trajectory close to the center axis or close to the wall, which should result in different stresses on the droplets.
ARTICLE | doi:10.20944/preprints201902.0004.v2
Subject: Biology And Life Sciences, Cell And Developmental Biology Keywords: artificial intelligence; machine learning; live-cell imaging; super-resolution microscopy; classification; segmentation
Online: 19 February 2019 (12:20:04 CET)
Artificial Intelligence based on Deep Learning is opening new horizons in Biomedical research and promises to revolutionize the Microscopy field. Slowly, it now transitions from the hands of experts in Computer Sciences to researchers in Cell Biology. Here, we introduce recent developments in Deep Learning applied to Microscopy, in a manner accessible to non-experts. We overview its concepts, capabilities and limitations, presenting applications in image segmentation, classification and restoration. We discuss how Deep Learning shows an outstanding potential to push the limits of Microscopy, enhancing resolution, signal and information content in acquired data. Its pitfalls are carefully discussed, as well as the future directions expected in this field.
ARTICLE | doi:10.20944/preprints202309.0718.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: vNDV-VII.1.1 challenge; broiler chickens; live ND vaccines; V4; LaSota; PAMP-H9N2
Online: 12 September 2023 (03:43:10 CEST)
A comparative efficacy of apathogenic genotype I (V4) and lentogenic genotype II (LaSota) strains of live Newcastle disease virus (NDV) vaccines was done following vaccination with pathogen-associated molecular pattern (PAMP) H9N2 avian influenza vaccine and challenge with velogenic NDV genotype VII.1.1 (vNDV-VII.1.1). Eight groups (Gs) of day-old chicks were used (n=25). Groups 1-4 received a single dose of PAMP-H9N2 subcutaneously, while Gs (1, 5) and (2, 6) received eye drops of V4 and LaSota respectively as twice doses. All Gs except 4 and 8 were intramuscularly challenged with vNDV-VII.1.1 at 28th days of age. No signs were detected in Gs 1, 5, 4, and 8. The mortality rates were 0% in Gs 1, 4, 5, and 8; 40% in G2; 46.66% in G6; and 100% in Gs 3 and 7. Lesions were recorded as minimal in Gs 1 and 5, but mild to moderate in Gs 2 and 6. The lowest significant viral shedding was detected in Gs 1, 2, and 5. In conclusion, two successive vaccinations of broilers with a live V4 NDV vaccine provided a higher protection against vNDV-VII.1.1 challenge than LaSota. Besides, PAMP-H9N2 with live NDV vaccines induced more protection than live vaccine alone.
BRIEF REPORT | doi:10.20944/preprints202207.0083.v1
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: bioelectricity; ion flux; membrane potential; live sensor dyes; pH; serotonin; acetylcholine; GABA; hiNSC
Online: 6 July 2022 (03:52:01 CEST)
All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate cell behavior (such as migration, proliferation, differentiation) as well as higher-level tissue and organ patterning. Thus, voltage gradients represent an important parameter for diagnostics as well as a promising target for therapeutic interventions in birth defects, injury, and cancer. However, despite much progress in cell and molecular biology, little is known about bioelectric states in human stem cells. Here, we present simple methods to simultaneously track ion dynamics, membrane voltage, cell morphology, and cell activity (pH and ROS), using fluorescent reporter dyes in living human neurons derived from induced neural stem cells (hiNSC). We developed and tested functional protocols for manipulating ion fluxes, membrane potential, and cell activity, and tracking neural responses to wounding and re-innervation in vitro. Finally, using morphology sensor, we tested and quantified the ability of physiological actuators (neurotransmitters and pH) to manipulate nerve wound re-innervation. These methods are not specific to a particular cell type and should be broadly applicable to the study of bioelectrical controls across a wide range of combinations of models and endpoints.
ARTICLE | doi:10.20944/preprints201702.0006.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: Deformed cloacal region; Danio rerio; Live-Cell imaging; Salmonella infection; Immune response; Cloacitis
Online: 3 February 2017 (04:18:59 CET)
Pathogenic Salmonella strains have a set of virulence factors allowing them to generate systemic infections and damage in a variety of hosts. Among these factors, bacterial proteins secreted by specialized systems are used to penetrate the host’s intestinal mucosa, through the invasion and destruction of specialized epithelial M cells in the intestine. On the other hand, numerous studies have demonstrated that humans, as well as experimental animal hosts, respond to Salmonella infection by activating both innate and adaptive immune responses. Here, through live cell imaging of S. Typhimurium infection of zebrafish larvae, we showed that besides the intestinal colonization, a deformed cloacae region and a concomitant accumulation of S. Typhimurium cells was observed upon bacterial infection. The swelling led to a persistent inflammation of infected larvae, although the infection was non-lethal. The in vivo inflammation process was confirmed by the co-localization of GFP-tagged S. Typhimurium with mCherry-tagged neutrophils at 72 hours post exposition. Our live-cell analyses suggest that Salmonella Typhimurium induce cloacitis-like symptoms in zebrafish larvae. Typhimurium induce cloacitis-like symptoms in zebrafish larvae.
ARTICLE | doi:10.20944/preprints202308.1896.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: African swine fever; African swine fever virus; cell-adapted; live-attenuated; vaccine; immunity; protection
Online: 29 August 2023 (03:36:50 CEST)
African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. Currently, there is no ASF vaccine commercially available. All infected animals must be isolated and culled immediately upon confirmation of the presence of the virus. Works leading to the rational development of protective ASF vaccines are urgently needed. Here, we generated a safe and efficacious live-attenuated vaccine (LAV) VNUA-ASFV-LAVL2 by serial passaging of a field isolate (VNUA-ASFV-05L1, genotype II) in porcine alveolar macrophage (PAMs, 65 passages) and an immortalized porcine alveolar macrophage cell line (3D4/21, 55 passages). VNUA-ASFV-LAVL2 can efficiently replicate in both PAMs and 3D4/21 cells. It provides 100% protection, even with the low dose of 10^2 HAD50, for the vaccinated pigs against the challenge of contemporary pandemic ASFV field isolate. Pigs vaccinated with this LAV at a dose range of 10^2 to 10^5 HAD50 remained clinically healthy during both the 28-day observation period of immunization and the 28-day observation period of challenge. VNUA-ASFV-LAVL2 was completely eliminated from blood by 28 days post inoculation (DPI), and from feces or oral fluids by 17 DPI. It remained a safe, stable, and attenuated phenotype after five passages in pigs. Consistently high levels of ASFV-specific IgG antibodies and significant cellular immunity were detected in vaccinated pigs before the ASFV challenge. These results indicate that the VNUA-ASFV-LAVL2 strain is a safe and efficacious LAV against the genotype II ASFV strain responsible for current ASF outbreaks in Asia.
REVIEW | doi:10.20944/preprints202101.0581.v1
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: genetically encoded biosensors; live spatiotemporal imaging; multiparameter imaging; plant immune response; biotic stress; crops
Online: 28 January 2021 (12:33:42 CET)
Biosensors are indispensable tools to follow plant’s immunity as its spatiotemporal dimension is key in withstanding the complex plant immune signaling. The diversity of genetically encoded biosensors in plants is expanding, covering new analytes with ever higher sensitivity and robustness, but their assortment is limited in some aspects, such as their use to follow biotic stress response, employing more than one biosensor in the same chassis and their implementation into crops. In this review, we focused on the available biosensors that encompass these aspects. We show that in vivo imaging of calcium and reactive oxygen species is satisfactorily covered with the available genetically encoded biosensors, while on the other hand they are still underrepresented when it comes to imaging of the main three hormonal players of the immune response, salicylic acid, ethylene and jasmonic acid. Following more than one analyte in the same chassis, upon one or more conditions has so far been possible by using the most advanced genetically encoded biosensors in plants which allow to monitor calcium and two main hormonal pathways involved in plant development, auxin and cytokinin. These kinds of biosensors are also the most evolved in crops. In the last section, we gathered the challenges in the use of the biosensors and showed some strategies to overcome them.
ARTICLE | doi:10.20944/preprints202011.0641.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: entry; kinetics; luciferase; real-time; live assay, vesicular stomatitis virus; Ebola; Lassa; chikungunya; coronavirus.
Online: 25 November 2020 (13:05:59 CET)
Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare glycoprotein-mediated entry efficiencies of: VSV G, SARS-CoV-2 S, EBOV GP, LASV GP, and CHIKV E we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase-PEST (NLucP) reporter gene, which we used in combination with the live-cell substrate Endurazine™ to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.
REVIEW | doi:10.20944/preprints202003.0254.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: zebrafish; inflammation; tumour initiation; macrophage; neutrophil; cancer; pre-neoplastic; live imaging; tumourigenesis; tumour model
Online: 16 March 2020 (01:31:28 CET)
The zebrafish is now an important model organism for cancer biology studies and provides some unique and complementary opportunities in comparison to the mammalian equivalent. The translucency of zebrafish has allowed in vivo live imaging studies of tumour initiation and progression at the cellular level thus providing novel insights into our understanding of cancer. Here we summarise and discuss available transgenic zebrafish tumour models and what we have gleaned from them with respect to cancer inflammation. In particular, we focus on the host inflammatory response toward transformed cells during the pre-neoplastic stage of tumour development. We discuss features of tumour associated macrophages and neutrophils in mammalian models and present evidence which supports the idea that these inflammatory cells promote early stage tumour development and progression. Direct live imaging of tumour initiation in zebrafish models has shown that the intrinsic inflammation induced by pre-neoplastic cells is tumour promoting. Signals mediating leukocyte recruitment to pre-neoplastic cells in zebrafish correspond to signals mediating leukocyte recruitment in mammalian tumours. The activation state of macrophages and neutrophils recruited to pre-neoplastic cells appears to be heterogenous, as seen in mammalian models, which provides an opportunity to study the plasticity of innate immune cells during tumour initiation. Although several potential mechanisms are described that might mediate the trophic function of innate immune cells during tumour initiation in zebrafish, there are several unknowns that are yet to be resolved. Rapid advancement of genetic tools and imaging technologies for zebrafish will facilitate research into the mechanisms that modulate leukocyte function during tumour initiation and identify targets for cancer prevention.
ARTICLE | doi:10.20944/preprints201812.0317.v1
Subject: Biology And Life Sciences, Virology Keywords: universal influenza vaccine; chimeric hemagglutinin; nucleoprotein; live attenuated influenza vaccine; sequential immunization; ferret model
Online: 27 December 2018 (10:14:21 CET)
The development of universal influenza vaccines, i.e. vaccines that can provide broad protection against seasonal and potentially pandemic influenza viruses, has been a priority for more than 20 years. Several approaches have been proposed that redirect the adaptive immune responses from immunodominant hypervariable regions to low-immunogenic but highly conserved regions of viral proteins. Here we induced broadly reactive anti-hemagglutinin (HA) stalk antibody by sequential immunizations with live attenuated influenza vaccines (LAIVs) expressing chimeric HA (cHA). These vaccines contained the HA stalk domain from H1N1pdm09 virus but antigenically unrelated globular head domains from avian influenza viruses H5N1, H8N4 and H9N2. In addition, the source of the viral nucleoprotein (NP) of the LAIV strains was changed from A/Leningrad/17 master donor virus (MDV) to wild-type (WT) H1N1pdm09 virus, in order to induce CD8 T-cell immune responses more relevant to current infections. To avoid any difference in protective effect of the various anti-neuraminidase (NA) antibodies, all LAIVs were engineered to contain the NA gene of Len/17 MDV. Naïve ferrets were immunized with three doses of (i) classical LAIVs containing non-chimeric HA and NP from MDV (LAIVs (NP-MDV)); (ii) cHA-based LAIVs containing NP from MDV (cHA LAIVs (NP-MDV)); and (iii) cHA-based LAIVs containing NP from H1N1pdm09 virus (cHA LAIVs (NP-WT)). A high-dose challenge with H1N1pdm09 virus induced significant pathology in the control, non-immunized ferrets, including high virus titers in respiratory tissues, clinical signs of disease and histopathological changes in nasal turbinates and lung tissues. All three vaccination regimens protected animals from clinical manifestations of disease: immunized ferrets did not lose weight or show clinical symptoms, and their fever was significantly lower than in the control group. Further analysis of virological and pathological data revealed the following hierarchy in the cross-protective efficacy of the vaccines: cHA LAIVs (NP-WT) > cHA LAIVs (NP-MDV) > LAIVs (NP-MDV). This ferret study showed that prototype universal LAIVs that combine the two approaches of inducing anti-HA stalk antibody and more relevant CD8 T-cell immune responses are highly promising candidates for further clinical development.
ARTICLE | doi:10.20944/preprints201706.0077.v1
Subject: Social Sciences, Education Keywords: zoo; mobile zoo; mobile animal exhibits; animal display; mobile live animal programs; negative education
Online: 16 June 2017 (05:32:18 CEST)
This paper assesses whether there is intrinsic positive educational value in travelling animal presentations and exhibits, referred to here as Mobile Live Animal Programs (MLAPs). Given that educational claims serve as the basis for allowing MLAPs to operate in many jurisdictions throughout Canada and the United States, it is essential to examine whether these purported claims are valid. This study takes a twofold approach of examining first, what constitutes an MLAP and how such programs are situated within the larger context of animal observation and tourism, and second, what constitutes both positive and negative education, and how such learning can empirically be measured in these settings. This approach provokes the ethical question of whether or not MLAPs should be allowed to operate given the high price paid not only by the individual animals used, but also to our psychological, emotional, and intellectual relationship with other species when we use non-human animals for our own knowledge, pleasure or comfort. The paper concludes that we must consider that the pervasive problem of negative education, that using displaced captive wild animals as learning tools that highlights human control over them, their objectification and their exploitation, is not justified by the purported positive educational claims of MLAPs.
ARTICLE | doi:10.20944/preprints202306.0389.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: body composition; body condition score; body parameters; fat; live weight; ewes condition; im-age analysis
Online: 6 June 2023 (05:54:38 CEST)
Farmers are continually looking for new reliable, objective and non-invasive methods for evaluation of ewe condition. Live weight (LW) and body condition score (BCS) are used by farmers as a basis to determine the condition of the animal. Body composition is an important aspect of monitoring animal condition. The body composition is the amount of fat, lean and bone; knowing the amount of each is important because the information can be used for better strategic management interventions. Experiments were conducted to establish the relationship between body composition and body parameters, at key life’s stages (weaning and pre-mating), using measurements automatically determined by an image processing ap-plication at Lincoln University sheep farm for 88 Coopworth ewes. Computerized Tomography technology was used to develop relationship with body parameters and a subset was used to validate the predicted model. Multivariate linear regression (MLR), artificial neural network (ANNs) and regression tree (RT) statistical analysis methods were evaluated to determine their efficacy to predict body fat, lean and bone. The results showed a correlation between fat, lean and bone determined by CT and the fat, lean, bone weight estimated by live weight and body parameters calculated using the image processing application with R2 values of 0.90 for fat, 0.72 for lean and 0.50 for bone using ANNs statistical model. From these results, farmers can utilize accurate measures of fat which will enhance nutritional and management practices.
ARTICLE | doi:10.20944/preprints202108.0177.v1
Subject: Medicine And Pharmacology, Dentistry And Oral Surgery Keywords: Antibacterial composites; Antiadhesive composites; Poly-Pore; Split-mouth; Clinical trial; Live/dead staining; Bacterial viability
Online: 9 August 2021 (07:53:57 CEST)
The purpose of the study was to investigate the bacterial viability of the initial biofilm on the surface of experimental modified dental resin composites. Twenty-five healthy individuals with good oral hygiene were included in this study. In a split-mouth design, they received acrylic splints with five experimental composite resin specimens. Four of them were modified with either a novel polymeric hollow beads delivery system or methacrylated polymerizable Irgasan (Antibacterial B), while one specimen served as unmodified control (ST). The delivery system based on Poly-Pore® was loaded with one of the active agents Tego® Protect 5000 (Antiadhesive A), Dimethicone (Antiadhesive B) or Irgasan (Antibacterial A). All study subjects refrained from toothbrushing during the study period. Specimens were detached from the splints after 8h and given a live/dead staining before fluorescence microscopy. Friedman test and post-hoc Nemenyi test were applied with significance level at p < 0.05. In summary all materials but Antibacterial B showed a significant antibacterial effect compared to ST. In conclusion dental resin composites with Poly-Pore loaded active agents show antibacterial effectiveness in situ.
ARTICLE | doi:10.20944/preprints201811.0098.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: PUFA; oil; prime lamb; feedlot; carcass characteristics; live performance; oils; canola; flaxseed; safflower; rice bran
Online: 5 November 2018 (10:16:48 CET)
This study investigated live animal performance and carcass characteristics of Australian prime lambs fed oil based polyunsaturated fatty acid (PUFA) enriched pellets in a feedlot system. The tested hypothesis was that supplementation of lambs with a variety of dietary oil based PUFA enriched pellets would enhance growth and carcass characteristics compared with the control lambs on lucerne only. Seventy-two, 6 months old White Suffolk x Corriedale first-cross prime lambs with an average liveweight (LWT) of 35.7 ± 0.9 kg were allocated to six treatment groups in a completely randomised experimental design. The treatments were: (1) control: lucerne hay only; or lucerne hay plus wheat-based pellets infused with 50 ml/kg DM of oils from (2) rice bran (RBO); (3) canola (CO); (4) rumen protected (RPO); (5) flaxseed (FO) and (6) safflower (SO) dietary sources. All lambs had ad libitum access to lucerne hay and clean fresh water. Supplemented lambs were fed 1kg of pellet/head/day for 10 weeks. Feed intake, final LWT, average daily gain (ADG), body conformation and carcass characteristics of lambs in the supplemented groups were all greater than for the control group. SO lambs had the lowest ADG of 190.3 g/day. RBO and CO treatments had the lowest feed cost per unit gain of AU$ 3.0/kg. Supplemented lambs had similar over the hooks (OTH) incomes that were all higher than that of the control group. This empirical evidence-based data demonstrated that supplementation of lambs with RBO and CO had comparatively lower feed costs without compromising ADG, carcass characteristics and OTH income.
HYPOTHESIS | doi:10.20944/preprints202211.0547.v1
Subject: Social Sciences, Psychology Keywords: twitch.tv; twitch; live streaming; online gambling; addiction; gambling disorder; internet use; slotstreams; online casino; online slots.
Online: 29 November 2022 (09:23:05 CET)
(1) Background: Twitch.tv is a live video content website. As of 2022, Twitch users are generally adolescents and young adults, with estimates of the percentage of users aged 16-24 between 22.3% and 41%, predominantly males with estimates ranging from 65% to 78.36%. In recent years, “slotstream” content has become increasingly popular, where streamers gamble online while users watch them. (2) Methods: from July 2022 to November 2022, we researched articles related to Twitch, live streaming, gambling, casino, slot, gambling online, modeling, social media influencers, conditioning, and celebrities, searching for relevant studies in the databases LexisNexis Academic, Business Source Complete, PubMed, Web of Science, Freedom Collection, Health & Medical Collection, Elsevier Journal, Springer, APA PsycARTICLES, Wiley, and other single journals. (3) Results: we took into consideration Gambling Disorder, Online Gambling Disorder, and Internet Gaming Disorder on the one hand, and consumer components such as persuasive communication and influencer-audience relationship on the other; we then hypothesized that the presence of these variables within the slotstream format is positively correlated with the development of pathological gambling, especially in an adolescent audience. (4) Conclusion: we assume that there is a positive correlation between watching slotstream content and the probability of developing both pathological and non-pathological gambling behavior.
REVIEW | doi:10.20944/preprints202009.0649.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: Infertile women; Hysteroscopy; Clinical pregnancy rate; Live birth rate; No Intrauterine pathology; endometrial stimulation; Systematic review
Online: 26 September 2020 (16:39:39 CEST)
(1) Background: The aim of this work was to systematically review existing studies on whether hysteroscopy improves the reproductive outcomes of women with infertility even in the absence of intrauterine pathologies when compared to women who did not receive a hysteroscopy. (2) Methods: We established the Participant-Intervention-Comparison-Outcome strategy and used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement to conduct a systematic review of 11 studies which were retrieved from 3 electronic databases: Ovid-Medline, Ovid-Embase, and the Cochrane Library. Two independent investigators extracted the data from the included studies and used the Cochrane risk-of-bias tool to assess their quality. (3) Results: The primary outcome measures were the clinical pregnancy rates (CPRs) and live birth rates (LBRs) in the in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Hysteroscopy in infertile women without intrauterine pathologies showed higher CPRs and LBRs than those in the same population who did not receive hysteroscopy in cases of recurrent implantation failure and IVF (odds ratio: 1.79 and 1.46, 95% confidence interval: 1.46-2.30 and 1.08-1.97 for CPR and LBR, respectively); however, the degree of significance was not as high for LBR. (4) Conclusions: Hysteroscopy before IVF/ICSI in infertile women without intrauterine pathologies may potentially be effective in improving the CPRs and LBRs in patients with RIF. Robust and high-quality randomized trials are warranted to confirm this finding.
REVIEW | doi:10.20944/preprints202108.0453.v1
Subject: Biology And Life Sciences, Biophysics Keywords: Hybrid epithelial/mesenchymal phenotypes; Collective cell migration; epithelial-mesenchymal heterogeneity; mathematical modeling; cell-state transition; live-cell imaging
Online: 23 August 2021 (14:32:04 CEST)
The Epithelial- Mesenchymal Transition (EMT) is a biological phenomenon associated with explicit phenotypic and molecular changes in cellular traits. Unlike the earlier-held popular belief of it being a binary process, EMT is now thought of as a landscape including diverse hybrid E/M phenotypes manifested by varying degrees of the transition. These hybrid cells can co-express both epithelial and mesenchymal markers and/or functional traits, and can possess the property of collective cell migration, enhanced tumor-initiating ability, and immune/targeted therapy-evasive features, all of which are often associated with worse patient outcomes. These characteristics of the hybrid E/M cells have led to a surge in studies that map their biophysical and biochemical hallmarks that can be helpful in exploiting their therapeutic vulnerabilities. This review discusses recent advances made in investigating hybrid E/M phenotype(s) from diverse biophysical and biochemical aspects by integrating live cell-imaging, cellular morphology quantification and mathematical modeling, and highlights a set of questions that remain unanswered about the dynamics of hybrid E/M states.
ARTICLE | doi:10.20944/preprints201904.0304.v1
Subject: Engineering, Civil Engineering Keywords: assessment; bridge evaluation; compressive membrane action; concrete bridges; fatigue; fatigue assessment; live loads; prestressed concrete; punching shear; scale model
Online: 28 April 2019 (08:38:27 CEST)
In the Netherlands, the assessment of existing prestressed concrete slab-between-girder bridges showed that the thin, transversely prestressed slabs may be critical for static and fatigue punching when evaluated using the recently introduced Eurocodes. On the other hand, compressive membrane action increases the capacity of these slabs and changes the failure mode from bending to punching shear. To improve the assessment of the existing prestressed slab-between-girder bridges in the Netherlands, two 1:2 scale models of an existing bridge, the Van Brienenoord Bridge, were built in the laboratory and tested monotonically as well as under cycles of loading. The result of these experiments is: 1) the static strength of the decks, showing that compressive membrane action significantly enhances the punching capacity, and 2) the Wöhler curve of the decks, showing that compressive membrane action remains under fatigue loading. The experimental results can then be used for the assessment of the most critical existing slab-between-girder bridge. The outcome is that the bridge has sufficient punching capacity for static and fatigue loads, and thus that the existing slab-between-girder bridges in the Netherlands fulfil the code requirements for static and fatigue punching.
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: super-resolution microscopy; advanced light microscopy; quantitative microscopy; live-cell microscopy; SMLM; STORM; SIM; STED; expansion microscopy; influenza virus; viral replication
Online: 6 January 2021 (10:40:59 CET)
With an estimated 3 to 5 million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society  and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are shedding new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies.
ARTICLE | doi:10.20944/preprints201809.0065.v1
Subject: Physical Sciences, Applied Physics Keywords: AFM, photothermal excitation, off-resonance tapping, high-speed atomic force microscopy, live cell imaging, antimicrobial peptide, thrombocytes, bacterial imaging, cell lysis
Online: 4 September 2018 (12:25:20 CEST)
Imaging living cells by atomic force microscopy (AFM) promises not only high-resolution topographical data, but additionally, mechanical contrast, which are not obtainable with other microscopy techniques. Such imaging is however challenging, as cells need to be measured with low interaction forces to prevent either deformation or detachment from the surface. Off-resonance modes which periodically probe the surface have been shown to be advantageous, as they provide excellent force control combined with large amplitudes, which help reduce lateral force interactions. However, the low actuation frequency in traditional off-resonance techniques limits the imaging speed significantly. Using photothermal actuation, we probe the surface by directly actuating the cantilever. Due to the much smaller mass that needs to be actuated, the achievable measurement frequency is increased by two orders of magnitude. Additionally, photothermal off-resonance tapping retains the precise force control of conventional off-resonance modes and is therefore well suited to gentle imaging. Here we show how photothermal off-resonance tapping can be used to study live cells by AFM. As an example of imaging mammalian cells, the initial attachment, as well as long term detachment of a human thrombocytes are presented. The membrane disrupting effect of the antimicrobial peptide CM-15 is shown on the cell wall of E. coli. Finally, the dissolution of the cell wall of B. subtilis by lysozyme is shown. Taken together, these evolutionarily disparate forms of life exemplify the usefulness of PORT for live cell imaging in a multitude of biological disciplines.
ARTICLE | doi:10.20944/preprints202106.0020.v1
Subject: Biology And Life Sciences, Virology Keywords: Vesicular Stomatitis; Herpes Simplex; Yellow Fever; Animal Viruses; Plaque Assay; Real-time; Live Cell Imaging, Automated Image Analysis; DNA Fluorescent Dyes, Antiviral Screening
Online: 1 June 2021 (10:40:37 CEST)
Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to counta-ble plaques that are visualized with standard techniques such as crystal violet, neutral red or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluores-cent dyes with differential cell permeability to visualize them by live cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applica-tions in virology.
ARTICLE | doi:10.20944/preprints202301.0426.v1
Subject: Medicine And Pharmacology, Emergency Medicine Keywords: Dispatch; Emergency Medical Dispatch; Emergency Medical Communication Centre; Video Live; COVID19; Emergency Call; Video triage; Public Safety Answering Point; Telemedecine; Emergency Medical Services; Remote assessment; Triage
Online: 24 January 2023 (08:20:00 CET)
The COVID19 pandemic had a major impact on emergency medical communication centres (EMCC). A live video facility was made available to second-line physicians in an EMCC with a first-line paramedic to receive emergency calls. The objective of this study was to measure the contribution of live video to remote medical triage. The single-centre retrospective study included all telephone assessments of patients with suspected COVID19 symptoms from 01.04.2020 to 30.04.2021 in Geneva, Switzerland. The organisation of the EMCC and the characteristics of patients who called the two emergency lines (official emergency number and COVID19 number) with suspected COVID19 symptoms were described. A prospective web-based survey of physicians was conducted during the same period to measure the indications, limitations and impact of live video on their decisions. 8,957 patients were included. 2,157 (48.0%) of the 4,493 patients assessed on the official emergency number had dyspnoea. 4,045 (90.6%) of 4,464 patients assessed on the COVID19 number had flu-like symptoms. 1,798 (20.1%) patients were reassessed remotely by a physician, including 405 (22.5%) with live video, successfully in 315 (77.8%) attempts. The web-based survey (107 forms) showed that physicians used live video to assess mainly the breathing (81.3%) and general condition (78.5%) of patients. They felt that their decision was modified in 75.7% (n=81) of cases, and caught 7 (7.7%) patients in life-threatening emergency. Medical triage decisions for suspected COVID19 patients are strongly influenced by the use of live video.
HYPOTHESIS | doi:10.20944/preprints202004.0153.v1
Subject: Biology And Life Sciences, Virology Keywords: Severe acute respiratory syndrome coronavirus 2; 2019-nCoV; COVID-19; angiotensin converting enzyme 2; Receptor; Live Attenuated Oral Vaccine; Intestine; proximal and distal enterocytes; herd immunity
Online: 9 April 2020 (13:10:50 CEST)
Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2/2019-nCoV) infection is an emerging pandemic. The virus binds to angiotensin converting enzyme 2 (ACE2) and TMPRSS2 which are abundantly expressed on various human cells including lung epithelial cells and intestinal cells and the virus can infect these cells. Currently no specific treatments or vaccines are available for this disease. A per oral live attenuated vaccine can be beneficial in SARS-Cov-2 infection because the attenuated virus initially infects the gut, stimulates the mucosa associated immune system sparing the respiratory system during the initial immune response. The live virus can also spread in the community boosting herd immunity.