Scientific Video Research Articles

Presynapse Formation Assay Using Presynapse Organizer Beads and “Neuron Ball” Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.

A Case Series of Successful Abdominal Closure Utilizing a Novel Technique Combining a Mechanical Closure System with a Biologic Xenograft that Accelerates Wound Healing

A Case Series Of Successful Abdominal Closure Utilizing A Novel Technique Combining A Mechanical Closure System With A Biologic Xenograft That Accelerates Wound Healing - Video. JoVE publishes peer-reviewed scientific video protocols to accelerate biological, medical, chemical and physical research. Watch our scientific video articles. JoVE is the world-leading producer and provider of science videos with the mission to improve scientific research, scientific journals, and education. Millions of scientists, educators and students at thousands of universities, colleges, hospitals and biopharmaceutical companies worldwide use JoVE for their research, teaching and learning.

A Case Series of Successful Abdominal Closure Utilizing a Novel Technique Combining a Mechanical Closure System with a Biologic Xenograft that Accelerates Wound Healing.

In the acute setting, once intra-abdominal injuries have been addressed, the next great hurdle is restoring a functional and intact abdominal compartment. The short and long-term consequences of living with a chronically open abdominal compartment include pulmonary, musculoskeletal, gastrointestinal, and emotional disability. The closure of catastrophic open abdomens presents a challenge to the surgeon. We present a technique utilizing a mechanical abdominal closure device in conjunction with biologic xenograft in closing complex open abdomens. This technique offers another option for definitive fascial closure and accelerated wound healing in this difficult patient population. The dynamic tissue system (DTS) is installed after control of original intraabdominal pathology. A porcine urinary bladder matrix (PUBM) is then placed in the subcutaneous space once fascial closure is achieved. Overall, primary myofascial closure was achieved in 100% of patients at a mean of 9.36 days.

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

A Popular Science Video Program Proposed for Promoting Scientific Literacy in Water Disaster Solutions Adapting Climate Change

A Popular Science Video Program Proposed for Promoting Scientific Literacy in Water Disaster Solutions Adapting Climate Change

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

Isolation And Characterization Of Tumor-initiating Cells From Sarcoma Patient-derived Xenografts - Video. JoVE publishes peer-reviewed scientific video protocols to accelerate biological, medical, chemical and physical research. Watch our scientific video articles. JoVE is the world-leading producer and provider of science videos with the mission to improve scientific research, scientific journals, and education. Millions of scientists, educators and students at thousands of universities, colleges, hospitals and biopharmaceutical companies worldwide use JoVE for their research, teaching and learning.

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy.

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals' leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick's immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female's body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gené's organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes' disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,however, require direct quantification of these forces. We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems. In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-β, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-β exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

ESTUDIO DE LA CALIDAD DEL VÍDEO ONLINE EN LA COMUNICACIÓN DE LA CIENCIA

Communicators and scientific journalists who work in the communication of popular science face certain peculiarities typical of this specialized area: communicating complex concepts and making them attractive to the general public. To achieve this goal, and taking into account that the internet and online video have become great allies of science divulgation, the journalist needs to know and master the peculiarities of online scientific video. The objective of this research is to determine what features make online video an effective tool to communicate science. The methodology used combines qualitative and quantitative techniques. On the one hand, it carries out a content analysis on a sample of 900 online videos on science, specifically on three current scientific topics: vaccines, nanotechnology and climate change. And, in parallel, interviews have also been conducted with a panel of nine experts: scientific journalists, producers, academics and web video experts. The conclusions suggest that online videos about science, of very various types, are usually brief and easy to understand, with a mainly informative objective. Despite the recommendations of the experts, they do not usually have elements of entertainment.

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Genetic Engineering Of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors For Frozen Sectioning - Video. JoVE publishes peer-reviewed scientific video protocols to accelerate biological, medical, chemical and physical research. Watch our scientific video articles. JoVE is the world-leading producer and provider of science videos with the mission to improve scientific research, scientific journals, and education. Millions of scientists, educators and students at thousands of universities, colleges, hospitals and biopharmaceutical companies worldwide use JoVE for their research, teaching and learning.

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

Development & validation of scientific video vignettes to promote perception of authentic science in student laboratories

ABSTRACTBackground: Many students have incomplete or incorrect perceptions of science and scientists. These simplified images, mediated by media or influential agents of socialisation, result in common stereotypes. Especially for occupational choices it is important to convey an authentic image about science and scientists.Purpose: One manner to convey an authentic image and thus the aim of this study is the development and validation of scientific videos including collected activities of scientists.Program description: Professors were interviewed regarding their typical scientific activities. This was followed by the development of a questionnaire which was answered by junior scientists. Authentic scientific videos were developed and finally validated in a science lab for school-students based on qualitative and quantitative results.Sample: 92 junior scientists answered the questionnaire and eight professors and 96 students (31 girls and 65 boys; grade 10 to 13) were interviewed.Design and methods: The scientists were surveyed before the development of the videos. The RIASEC+N model was used to categorise the collected activities of scientists. Finally, students were interviewed for the video validation.Results: A number of different scientific activities of each RIASEC+N dimension could be detected, which were then integrated into four videos. The interviewed students who watched those videos successfully identified all of the activities.Conclusion: The working day of scientists contains more than stereotypical aspects and well-considered/planned videos are one suitable option to promote an authentic overview about science and scientists.

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant's genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressedin roots.

The Role of Journalism on YouTube: Audience Engagement with ‘Superbug’ Reporting

Journalism has gradually become ‘normalized into social media’, and most journalists use social media platforms to publish their work (Bruns, 2018). YouTube is an influential social media platform, reaching over a billion users worldwide. Its extensive reach attracts professional and amateur video producers who turn to YouTube to inform, entertain and engage global publics. Focusing on YouTube, this study explores the place for journalism within this media ecology. This study uses a mixed-method approach to examine forms of audience engagement to YouTube videos about antimicrobial resistance (AMR), or so called “superbugs”, caused by overuse and misuse of antibiotics. The analysis focuses on the most viewed YouTube videos about AMR between 2016 and 2018, and compares engagement themes expressed in comments to journalistic videos with popular science videos. The most viewed videos about AMR on YouTube are professionally produced educational popular science videos. The qualitative analysis of 3,049 comments identifies seven main forms of high-level engagement, including expressions of emotions, blame and calls for action. This study shows that journalism plays an important role on YouTube by generating audience discussions about social and political accountability. Our findings demonstrate that journalism videos were associated with propositions for political, economic and social/lifestyle actions, while popular science videos were associated with medicines, scientific or pseudo-scientific, and medical practice changes.