How to Apply Social Network Analysis to Evaluate Professional Development Programs in Biomedical Research.

SPAdes-St. Petersburg genome Assembler-was originally developed for de novo assembly of genome sequencing data produced for cultivated microbial isolates and for single-cell genomic DNA sequencing. With time, the functionality of SPAdes was extended to enable assembly of IonTorrent data, as well as hybrid assembly from short and long reads (PacBio and Oxford Nanopore). In this article we present protocols for five different assembly pipelines that comprise the SPAdes package and that are used for assembly of metagenomes and transcriptomes as well as assembly of putative plasmids and biosynthetic gene clusters from whole-genome sequencing and metagenomic datasets. In addition, we present guidelines for understanding results with use cases for each pipeline, and several additional support protocols that help in using SPAdes properly. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Assembling isolate bacterial datasets Basic Protocol 2: Assembling metagenomic datasets Basic Protocol 3: Assembling sets of putative plasmids Basic Protocol 4: Assembling transcriptomes Basic Protocol 5: Assembling putative biosynthetic gene clusters Support Protocol 1: Installing SPAdes Support Protocol 2: Providing input via command line Support Protocol 3: Providing input data via YAML format Support Protocol 4: Restarting previous run Support Protocol 5: Determining strand-specificity of RNA-seq data.

Entamoeba histolytica is a parasitic protozoan and the causative agent of amoebiasis in humans. Amoebiasis has a high incidence of disease, resulting in ∼67,900 deaths per year, and it poses a tremendous burden of morbidity and mortality in children. Despite its importance, E. histolytica is an understudied parasite. These protocols describe the in vitro growth, maintenance, cryopreservation, genetic manipulation, and cloning of axenic E. histolytica trophozoites. There has been significant progress in genetic manipulation of this organism over the past decade, and these protocols outline the ways in which these advances can be implemented. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culturing E. histolytica trophozoites Support Protocol 1: Preparation of TYI-S-33 medium Support Protocol 2: Lot testing of Biosate peptone and adult bovine serum for TYI-S-33 medium Basic Protocol 2: Cryopreservation of E. histolytica trophozoites Support Protocol 3: Preparation of cryoprotectant solutions Basic Protocol 3: Transfection of E. histolytica trophozoites with Attractene reagent Basic Protocol 4: Creating clonal lines using limiting dilution Basic Protocol 5: Knockdown of one to two genes with trigger-induced RNA interference Support Protocol 4: Evaluation of RNA interference knockdown with reverse transcriptase PCR Basic Protocol 6: E. histolytica growth curves.

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions.

Cell‐cell interactions are required for development and homeostasis in multicellular organisms from insects to mammals. A critical process governed by these interactions is cell competition, which functions throughout development to control tissue composition by eliminating cells that possess a lower fitness status than their neighbors. Human pluripotent stem cells (hPSCs) are a key biological tool in modeling human development and offer further potential as a source of clinically relevant cell populations for regenerative medicine applications. Recently, cell competition has been demonstrated in hPSC cultures and during induced pluripotent stem cell reprogramming. In turn, these findings suggest that hPSCs can be used as a tool to study and model cell‐cell interactions during different stages of development and disease. Here, we provide a panel of protocols optimized for hPSCs to investigate the potential role that cell competition may have in determining the fate and composition of cell populations during culture. The protocols entail assessment of the competitive phenotype and the mode through which cell competition may lead to elimination of less‐fit cells from mosaic cultures with fitter counterparts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Electroporation of hPSCs to establish a fluorescent reference cell line Support Protocol 1: Single‐cell dissociation of hPSCs Support Protocol 2: Single‐cell cloning of fluorescently labeled hPSCs Basic Protocol 2: Separate culture and co‐culture proliferation assays Basic Protocol 3: Assessing levels of apoptosis in hPSC cultures using flow cytometry Basic Protocol 4: Transwell assay Support Protocol 3: Immunohistochemistry and image quantification of cleaved caspase‐3 Basic Protocol 5: Cell confrontation assay Basic Protocol 6: Cell compression assay Basic Protocol 7: Time‐lapse imaging to assess mechanical extrusion

The development in technology has led to the generation of huge amounts of data from various sources, including biological data, social networking data, etc. Accordingly, social network analysis has received considerable attention with the availability of more raw datasets which could be realized using a network structure. Most of the datasets can be represented as a social network which is a graph consisting of actors having relationships. Many tools exist for social network analysis inspired to extract knowledge from the networks. NetDriller has been developed as a social network extraction, manipulation and analysis tool to cover the lack that exists in other tools. It is capable of constructing social networks from raw data by employing a variety of data mining and machine learning techniques. In this paper, we describe an extend version of NetDriller, which has some new essential functions, including social network construction using data collection from Twitter, DBLP and IEEE. We also added (1) a new chart for viewing the network property and metrics, and (2) new graph manipulation techniques using GUI to keep the tool up to date with the huge volume of networks and the different types of raw data available on the web.

Spam community detection & influence minimization using NRIM algorithm

This article details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Additionally, active induction of EAE in the C57BL/6 strain using myelin oligodendrocyte glycoprotein (MOG) peptide is also discussed. Detailed materials and methods required for the purification of both PLP and MBP are described, and a protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains. © 2021 Wiley Periodicals LLC. Basic Protocol: Active induction of EAE with PLP, MBP, and MOG protein or peptide Alternate Protocol: Adoptive induction of EAE with PLP-, MBP-, or MOG-specific lymphocytes Support Protocol 1: Purification of proteolipid protein Support Protocol 2: Purification of myelin basic protein Support Protocol 3: Isolation of CNS-infiltrating lymphocytes.

Spatial transcriptomic technologies have been developed rapidly in recent years. The addition of spatial context to expression data holds the potential to revolutionize many fields in biology. However, the lack of computational tools remains a bottleneck that is preventing the broader utilization of these technologies. Recently, we have developed Giotto as a comprehensive, generally applicable, and user-friendly toolbox for spatial transcriptomic data analysis and visualization. Giotto implements a rich set of algorithms to enable robust spatial data analysis. To help users get familiar with the Giotto environment and apply it effectively in analyzing new datasets, we will describe the detailed protocols for applying Giotto without any advanced programming skills. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Getting Giotto set up for use Basic Protocol 2: Pre-processing Basic Protocol 3: Clustering and cell-type identification Basic Protocol 4: Cell-type enrichment and deconvolution analyses Basic Protocol 5: Spatial structure analysis tools Basic Protocol 6: Spatial domain detection by using a hidden Markov random field model Support Protocol 1: Spatial proximity-associated cell-cell interactions Support Protocol 2: Assembly of a registered 3D Giotto object from 2D slices.

Basophils represent the rarest type of granulocyte in human peripheral blood. Thus, researching basophils has historically been challenging and has often been reliant on enrichment protocols using density gradient centrifugation. This article describes a novel, fast, and cost-effective method to purify highly viable human basophils from peripheral blood through negative immunomagnetic selection, foregoing the density centrifugation step in the Basic Protocol. The technique is easy to use and consistently produces purities >96%. Furthermore, the Support Protocols describe procedures to determine basophil yield, purity, and viability, and how to investigate functional activity of the purified basophils through flow cytometry and visualize the basophils through microscopy. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Gradient centrifugation-independent basophil isolation Support Protocol 1: Flow cytometry staining to assess basophil yield, purity, and viability Support Protocol 2: Giemsa staining Support Protocol 3: Calcium flux analysis Support Protocol 4: Basophil activation test.

The aim of the study is a practical analysis of social network services as a place to store multimedia resources that allow you to store and share digital photos, audio and video recordings, text files, presentations, and organize resource discussions for free. They can be used as educational materials (educational videos, photos on various topics, audio and video recordings of scientists' speeches, eyewitnesses of events, etc.), as well as means to store student video, photo, audio files and creative works. Method. Theoretical materials of the research are the best practices of foreign and domestic scientists in the field of network communications and their impact on the future specialists preparing in geography and tourism, namely articles, monographs, abstracts, dissertations. The study uses the method of theoretical materials analysis, as well as methods of observation, abstraction in identifying the qualification characteristics of social Internet networks. Content analysis (analysis of the social networks community messages content); statistical and comparative information analysis was carried out. Empirical research on the social Internet networks lies in creating and filling a community on the social Internet network, as well as engaging students in that community. Results. In the research it was found out that social networks can be used to solve the following tasks: to organize the collective work of students in the classroom (and outside it), which will be favourable to the communication, to gain the team experience; to ensure the development of the students personalized learning environment; to promote students independent learning, as everyone works in his own rhythm; to promote informal communication between the tutor and the students; to change the tutor's role, who will become an assistant, friend, mentor; to create digital educational content of the disciplines. The scientific novelty of the obtained results lies in the fact that on the comprehensive analysis of social internet networks, internet blogs, tourist internet resources basis, their place and role in the information and educational environment of higher education institutions are determined, in particular: the specifics of the content, prospects and mechanisms of the development of the social Internet networks from the point of view of training future tourists are determined; the dependence of the functions of the Social Internet Network, Internet blogs on their structural elements is established. Practical significance. The obtained scientific results are used in the educational process of future specialists in the field of training "Tourism", with the educational qualification "tourism expert, organizer of excursion and animation activities" when teaching the disciplines "study of local lore and tourism", "tourist work", "recreational geography", "geography of Ukraine", "professional competencies in tourism business", "organization of tourist activities". The research practices can be used to conduct tourist, museum, local history, excursion and other types of student's practice.

The global spread of the arbovirus vector Aedes albopictus has become a major public health concern. In tropical regions, competitive interactions with native species such as Aedes aegypti may have influenced its invasive success. Understanding the bioecological drivers behind its establishment and spread is crucial for informing targeted control measures. Here, we propose experimental settings, based on life history traits, to elucidate the factors that allow Ae. albopictus to colonize new environments, outcompete native species, expand geographically, and ultimately subject invaded regions to risks of arbovirus diseases. To this effect, we designed a series of Basic Protocols listed below. Each protocol simulates environmental conditions in which Ae. albopictus and Ae. aegypti coexist, allowing comparison of their bioecological responses and adaptability to shared resources. The validated results, showing the reproducibility of these methods, provide a foundation for evidence-based strategies to mitigate the public health risks posed by the spread of Ae. albopictus in invaded territories. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Support Protocol: Essential preliminary procedures Basic Protocol 1: Evaluation of larval habitat occupancy rates Basic Protocol 2: Evaluation of the female oviposition rate and reproductive potential Basic Protocol 3: Evaluation of embryonic kinetics Basic Protocol 4: Evaluation of hatching rate Basic Protocol 5: Evaluation of longevity.

Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium commonly found in the human intestine. Although it typically exists as part of the normal flora, it can also cause healthcare-associated infections with severe consequences. Understanding the specific genes responsible for its virulence through genetic manipulation is crucial for potential therapeutic interventions. However, manipulating K. pneumoniae presents challenges due to its exopolysaccharide capsule. This article presents a comprehensive collection of protocols designed to facilitate the genetic manipulation of K. pneumoniae. By following these protocols, researchers will acquire the necessary skills to prepare electrocompetent cells, utilize electroporation for efficient plasmid DNA introduction, construct isogenic mutants using the λ Red recombinase system, and generate a complementation vector for restoring the phenotypic traits of knockout strains. These protocols provide valuable tools and techniques to navigate the intricacies associated with studying and modifying K. pneumoniae. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing electrocompetent K. pneumoniae cells Alternate Protocol 1: Preparing electrocompetent K. pneumoniae cells for recombineering Basic Protocol 2: Transforming K. pneumoniae using electroporation Basic Protocol 3: Constructing isogenic mutants in K. pneumoniae using the λ Red recombinase system Support Protocol 1: Confirming a knockout via colony PCR Support Protocol 2: Verifying absence of secondary mutations Basic Protocol 4: Generating unmarked knockout mutants in K. pneumoniae using the pFLP plasmid Basic Protocol 5: Constructing a complementation vector for K. pneumoniae.

The dynamics of the cellular actomyosin cytoskeleton are crucial to many aspects of cellular function. Here, we describe techniques that employ active micropost array detectors (AMPADs) to measure cytoskeletal rheology and mechanical force fluctuations. The AMPADS are arrays of flexible poly(dimethylsiloxane) (PDMS) microposts with magnetic nanowires embedded in a subset of microposts to enable actuation of those posts via an externally applied magnetic field. Techniques are described to track the magnetic microposts’ motion with nanometer precision at up to 100 video frames per second to measure the local cellular rheology at well‐defined positions. Application of these high‐precision tracking techniques to the full array of microposts in contact with a cell also enables mapping of the cytoskeletal mechanical fluctuation dynamics with high spatial and temporal resolution. This article describes (1) the fabrication of magnetic micropost arrays, (2) measurement protocols for both local rheology and cytoskeletal force fluctuation mapping, and (3) special‐purpose software routines to reduce and analyze these data. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fabrication of magnetic micropost arrays Basic Protocol 2: Data acquisition for cellular force fluctuations on non‐magnetic micropost arrays Basic Protocol 3: Data acquisition for local cellular rheology measurements with magnetic microposts Basic Protocol 4: Data reduction: determining microposts’ motion Basic Protocol 5: Data analysis: determining local rheology from magnetic microposts Basic Protocol 6: Data analysis for force fluctuation measurements Support Protocol 1: Fabrication of magnetic Ni nanowires by electrodeposition Support Protocol 2: Configuring Streampix for magnetic rheology measurements

Targetron technology, a gene-editing approach based on the use of mobile group II introns, is particularly useful for bacterial strains deficient in homologous recombination. Specifically, the Ll.LtrB intron from Lactococcus lactis can be used in a wide range of species and can be easily retargeted, that is, modified for integration into any locus of interest. Targetron technology is thus a powerful tool for generating genomic insertions in a broad range of genetic backgrounds, mainly when no other techniques can be efficiently employed. Notably, the approach can be coupled to CRISPR/Cas9 counterselection of wildtype DNA sequences to decrease the population of unmodified cells and ultimately improve Ll.LtrB insertion efficiency. Here, we describe a step-by-step protocol for delivering exogenous sequences into the genome of Gram-negative bacteria by means of targetron technology and CRISPR/Cas9 counterselection using Pseudomonas putida as a model. We describe the retargeting of the Ll.LtrB intron to the locus selected for insertion, the design of specific spacers for eliminating unmutated cells through CRISPR/Cas9 counterselection, and the cloning of exogenous sequences into Ll.LtrB. We also provide a protocol for delivering a specific cargo to the locus of choice once all necessary components of the system are ready. Lastly, we describe a general protocol for curing the engineered strain of all plasmids. CRISPR/Cas9-enhanced Ll.LtrB insertion can be an efficient alternative for overcoming low recombination-based editing efficiency and can be used in numerous bacterial species. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retargeting the Ll.LtrB intron to the target locus Support Protocol 1: Preparation of competent E. coli Basic Protocol 2: Design and cloning of CRISPR spacers to counterselect Ll.LtrB insertions Support Protocol 2: Interference assay to check efficiency of selected spacers Basic Protocol 3: Cloning cargos into Ll.LtrB Basic Protocol 4: Ll.LtrB/CRISPR/Cas9-mediated insertion Basic Protocol 5: Curing the engineered strain of plasmids.

Software tools for Social Network Analysis (SNA) are being developed which support various types of analysis of social networks extracted from social media websites (e.g., Twitter). Once extracted and stored in a database such social networks are amenable to analysis by SNA software. This data analysis often involves searching for occurrences of various subgraph patterns (i.e., graphical representations of entities and relationships). The authors have developed the Graph Matching Toolkit (GMT) which provides an intuitive Graphical User Interface (GUI) for a heuristic graph matching algorithm called the Truncated Search Tree (TruST) algorithm. GMT is a visual interface for graph matching algorithms processing large social networks. GMT enables an analyst to draw a subgraph pattern by using a mouse to select categories and labels for nodes and links from drop-down menus. GMT then executes the TruST algorithm to find the top five occurrences of the subgraph pattern within the social network stored in the database. GMT was tested using a simulated counter-insurgency dataset consisting of cellular phone communications within a populated area of operations in Iraq. The results indicated GMT (when executing the TruST graph matching algorithm) is a time-efficient approach to searching large social networks. GMT’s visual interface to a graph matching algorithm enables intelligence analysts to quickly analyze and summarize the large amounts of data necessary to produce actionable intelligence.