bluuebunny/crossref_metadata_2025 huggingface dataset:
ATLANTIS: An adaptive multi-arm phase II trial of maintenance targeted therapy after chemotherapy in advanced/metastatic urothelial cancer - Enzalutamide arm v1
Authors: J Jones Robert , Powles Thomas , Crabb Simon
Published: May 2022

Background: Metastatic urothelial cancer (MUC) is the eighth most common cause of cancer death in the UK. Chemotherapy is standard treatment for MUC. Although MUC is initially sensitive to chemotherapy, patients usually relapse within 6 months of completion. The outcome, once relapse has occurred, is very poor (median overall survival 8 months). Therefore, maintaining response to chemotherapy is an attractive goal, and a good environment for testing active single agents in MUC. ATLANTIS is a multi-arm, multi-stage signal searching trial. Patients will be allocated to treatment (vs observation or placebo where possible) based on biomarker expression. Design: ATLANTIS is a randomised phase II biomarker directed umbrella screening trial of maintenance therapy in biomarker defined subgroups of patients with advanced or metastatic urothelial cancer. The primary endpoint is progression-free survival. The protocol consists of a generic main section plus novel drug-specific appendices. The relevant novel drug-specific appendices will be amended when each specific subgroup trial completes or when a further novel drug subgroup is introduced. Central tissue screening for biomarker will occur prior to randomisation. Objectives: The trial will investigate whether maintenance targeted therapy after chemotherapy, with treatment selection based on biomarker expression, delays time to progression and overall survival for patients with MUC. Endpoints: Primary: To compare progression-free survival (PFS) of trial drug and placebo/observation. Different drugs will be tested in different biomarker defined subgroups in an adaptive design. PFS has been chosen as the primary end-point as it is largely objective (the majority of urothelial cancer patients will display progression in accordance with RECIST1.1 criteria) and clinically meaningful - progression after first line therapy represents the transition to the lethal stage of the disease and often the requirement for further cytotoxic therapy. Secondary: Overall survival, response rate, maximum percentage decrease in measurable disease, safety and tolerability. Exploratory: Progression free survival in subgroups defined by biomarkers other than those used for treatment allocation. Translational: Archival tissue, plasma and circulating tumor DNA will be stored and used for future research.
Proteogenomics Reference Database Protocol v1
Authors: Manna Kanishka , Dharanipragada Prashanthi , Alkam Duah , L. Avaritt Nathan , L. Washam Charity , Chiang Tung-chin , S Robeson Michael , D. Edmondson Ricky , G. Mackintosh Samuel , Yang Zhentao , Wang Yan , H. Lomeli Shirley , Moriceau Gatien , S. Lo Roger , J. Tackett Alan , D. Byrum Stephanie
Published: May 2022

Therapeutic approaches to treat melanoma include small molecule drugs that target activating protein mutations in pro-growth signaling pathways like the MAPK pathway. While beneficial to the approximately 50% of patients with activatingBRAFV600mutation, mono- and combination therapy with MAPK inhibitors is ultimately associated with acquired resistance. To better characterize the mechanisms of MAPK inhibitor resistance in melanoma, we utilize patient-derived xenografts and apply proteogenomic approaches leveraging genomic, transcriptomic, and proteomic technologies that permit the identification of resistance-specific alterations and therapeutic vulnerabilities. A specific challenge for proteogenomic applications comes at the level of data curation to enable multi-omics data integration. Here, we present a proteogenomic approach that uses custom curated databases to identify unique resistance-specific alternations in melanoma PDX models of acquired MAPK inhibitor resistance. We demonstrate this approach with aNRASQ61Lmelanoma PDX model from which resistant tumors were developed following treatment with a MEK inhibitor. Our multi-omics strategy addresses current challenges in bioinformatics by leveraging development of custom curated proteogenomics databases derived from individual resistant melanoma that evolves following MEK inhibitor treatment and is scalable to comprehensively characterize acquired MAPK inhibitor resistance across patient-specific models and genomic subtypes of melanoma. The computational workflow for curation of a proteogenomics database is described here.
Intracellular Cytokine (ICS) Staining Protocol v1
Authors: P. Williams Gregory , not provided cecilia
Published: May 2022

This protocol details about intracellular cytokine (ICS) staining.
JAX - Nuclei Isolation for 10x Genomics A v1
Authors: F F Flynn William , Courtois Elise , A Perry Greggory , L Daigle Sandra , not provided Paul.gabriel , Luo Diane , Grassmann Jessica
Published: May 2022

The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the 10x Genomics Multiome assay. This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma. This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).
Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, del143-145, and LPPA24S) in settled solids using digital RT-PCR v4
Authors: Hughes Bridgette , J. White Bradley , K. Wolfe Marlene , B B Boehm Alexandria
Published: May 2022

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, del143-145, and LPPA24S) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.
FLASH-seq Low-Amplification protocol v3
Authors: Picelli Simone , Hahaut Vincent
Published: May 2022

Building upon the existing Smart-seq2/3 workflows, we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than both previous versions, requiring limited hands-on time and with a great potential for customization. FLASH-seq Low-Amplification (FS-LA), represents FS quickest iteration, generating sequencing-ready libraries in 4.5 hours by removing intermediate cleanups and QC steps and without sacrificing performance. FS-LA is the best choice when a large number of plates need to be processed in parallel.
DNA metabarcoding protocol for siphonophore gut contents v2
Authors: Damian Serrano Alejandro
Published: May 2022

Protocol for the SiphWeb DNA metabarcoding of siphonophore gut content. Starting with DNA extractions from pooled frozen gastrozooids, this protocol takes the user through the steps of PCR amplification, PCR cleanup and pooling of amplicons from six complementary 18S barcodes that can be submitted into an Illumina MiSeq Lane.
Protocol:Resin-based adhesives, composites, and luting agents: A cross-sectional study of citations and altmetrics v1
Authors: Lauer Fernanda
Published: May 2022

The aim of this cross-sectional, scientometric study is to investigate factors associated with journal citations and altmetrics of dental articles reporting original research on resin-based materials.
Detection of recombinant and endogenous LPPR3 by western blot v2
Authors: not provided cristina.kroon
Published: May 2022

This is a protocol for detection of overexpressed and endogenous LPPR3 from N1E-115 cells and primary hippocampal neurons.
Detection and Quantification of Calcitonin Gene-Related Peptide in Human Blood Plasma using a Modified Enzyme-Linked Immunosorbent Assay v2
Authors: S. Krishnan Pavan , T. Zamuner Fernando , M. Jenks Carolyn , Xie Yanjun , Zhang Lisa , Lehar Mohammed , Iglesias Marcos , S. Fedarko Neal , Brait Mariana , P. Carey John
Published: May 2022

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. Here, we present a protocol for purification and quantification of CGRP in human plasma. This protocol involves sample collection and preparation, extraction as a means of purification, and quantification via enzyme-linked immunosorbent assay (ELISA). After finding unrealistically high CGRP concentrations in spike-and-recovery experiments using a commercial CGRP ELISA kit protocol, we modified the existing protocol to produce satisfactory percent recoveries. This protocol can theoretically be used to quantify CGRP concentrations in plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.
Staphilococcus Aureus Sampling v6
Authors: Roca Cugat Pol , Sánchez Olga
Published: May 2022

This protocol is intended to study the affectation of Staphilococcus Aureus, including the MRSA variant. It outlines the basic protocol for a multi-subject study.
Wastewater QC workflow in GalaxyTrakr (SSQuAWK3) v4
Authors: Amirzadegan Jasmine , Kayikcioglu Tunc , not provided hugh.rand , Timme Ruth , Balkey Maria
Published: May 2022

PURPOSE: Step-by-step instructions for checking sequence quality for SARS-CoV-2 wastewater samples using SSQuAWK3: SARS - CoV - 2 Sequence Quality Assurance Workflow and Kontraption, version 3. The SSQuAWK3 workflow, implemented in CFSAN's custom Galaxy instance (GalaxyTrakr) will produce quality assessments for raw reads (Illumina MiSeq paired-end fastq files). SCOPE: This protocol covers the following tasks: 1. Set up an account in GalaxyTrakr 2. Create a new history 3. Upload data and reference files 4. Execute the SSQuAWK3 workflow 5. Interpret the results Protocol and SSQuAWK workflow version history: Protocol V1, SSQuAWK version 1: Basic protocol steps with screenshots Protocol V2, SSQuAWK version 1: Addition of a detailed 12 minute video tutorial Protocol V3, SSQuAWK version 2: Addition of 5 new genome mapping metrics Protocol V4 SSQuAWK version 3: Metrics now reported with fewer softwares, fewer underlying GalaxyTrakr jobs,and about 50% fewerunderlying GalaxyTrakr steps. Cleaner output table formats now include QC placeholder columns for SRA metadata template.
P2 sharing is enable v1
Authors: not provided maria
Published: May 2022

test
Golden Gate Assembly v1
Authors: Núñez Isaac , Matute Tamara , Federici Fernan
Published: May 2022

The Golden Gate technique allows the assembly of genetic sequences from libraries of standardized basic components, which are cleaved from their donor vectors and concatenated in the acceptor vector in a defined order. This reaction is subsequently transformed into competent bacterial cells that are grown overnight to reveal positive colonies carrying the correctly assembled vector. The cleavage of the components is done by a TypeIIS restriction enzyme, which recognizes asymmetric DNA sequences and cleaves outside of this site, leaving overhangs in a directional way (Figure 1A). This property allows the creation of ordered assembly positions by defining synthetic syntaxes of these overhangs (Figure 1B). Within the different methodologies and libraries based on Golden Gate, some methods, such as uLoop Assembly (Pollak et al, 2020), allow the cyclic assembly of increasingly large and complex vectors in which the products of a reaction are the substrates of the next level reactions (Figure 2). By this means it is possible to build libraries of easily reusable components to perform assemblies in a combinatorial way of odd and even assembly levels. The standardized DNA components have to be flanked by the enzyme recognition sites and the proper syntax according to the desired position of assembly. These DNA pieces can be obtained from previously created libraries or custom made. The reaction operates cycling between 37°C and 16°C. At the first temperature, the restriction enzyme cuts the DNA pieces. These pieces are concatenated by their overhangs sequence homology and sealed by the catalysis of a ligase enzyme at the second temperature. Once a piece is correctly assembled it cannot be cutted again, and cycle by cycle, the pieces are concatenated in the final assembly product.
Preparation of 100mg/ml kanamycin solution v1
Authors: Molloy Jenny , Mowoh Nadine
Published: May 2022

Kanamycin is an aminoglycoside antibiotic which binds the 30S ribosomal subunit; causes mis-translation. It is used for the selection of cells containing Kanamycin-resistant plasmids on plates and on liquid media at a working concentration of 50-100 µg/mL. It is most often prepared as a 1000x working stock of 50 -100 mg/mL that is stored at -20 ºC. The kanamycin solution is measured and then mixed into a container of liquid or plate media before it has solidified. Cells are then streaked onto the plates or grown in the liquid media. Only cells containing the plasmids resistant to kanamycin will grow.
WU sn-prep Protocol for Solid Tumors - snRNA protocol v2.8 v1
Authors: Caravan Wagma , Jayasinghe Reyka , Naser Al Deen Nataly
Published: May 2022

WU sn-prep Protocol for Solid Tumors -snRNA protocol v2.7
Sequencing of construct v1
Authors: Baden Pascale , not provided michela.deleidi
Published: May 2022

This protocol describes sequencing of construct.
Creating Differential Transcript Expression Results with DESeq2 v2
Authors: A Eccles David
Published: May 2022

Differential expression analysis of transcript count tables using DESeq2
USER MANUAL of U-uF-BM# Bifurcating Mixing Microfluidic Chips v2
Authors: S Serhat
Published: May 2022

This is the user manual for U-uF-BM# Bifurcating Mixing Microfluidic Chips of uFluidic.com. It is designed to make nanoencapsulation liposomes and polimeric nanoparticles.
Scorpion metabarcoding v1
Authors: not provided yurisimone
Published: May 2022

The following steps include the protocols of the 1. Diet Regime 2. Dissection procedure 3. Beads extraction 4. Salt-out DNA extraction
Natures Only CBD Gummies Mayim Bialik - Reviews v1
Authors: not provided naturesonlymmiesmayim
Published: May 2022

Natures Only CBD Gummies Mayim Bialik - Reviews
H&E Staining for Pancreas or Eye Cryosections v2
Authors: Saunders Diane , Kruse Angela , Allen Jamie , Romer Carrie , Gutierrez Danielle , Powers Alvin , Spraggins Jeff
Published: May 2022

This protocol for H&E staining can be applied to either fixed or unfixed frozen cryosections.
DNA extraction and Nanopore library prep from single wild-caught drosophilids v1
Authors: Y Kim Bernard
Published: May 2022

We have been assembling the genomes of many Drosophila species. This protocol fork allows for the sequencing of single wild-caught flies using Nanopore MinION sequencers, in contrast to our previous protocol that originally used gDNA extracted from tens or hundreds of flies from an inbred line. While read lengths are not nearly close to what we were seeing before, libraries range from 2-10kb read N50 and typically produce genome assemblies with contig N50 >1 Mbp. The protocol for ultra-long read sequencing from large pools of flies can be found here: dx.doi.org/10.17504/protocols.io.bdfqi3mw Please cite the associated paper if you use this protocol: https://doi.org/10.7554/eLife.66405 An update to the original paper that includes many single wild-caught fly genomes is in progress.
Keller (K) Optimized Medium for Culturing Microalgae v1
Authors: Culture Collection Roscoff , Doran Lynn , J Burgess Steven
Published: May 2022

Medium to growmost phytoplankton species insmall eukaryotes (Mamiellophyceae, Pelagophyceae etc...). Optimized for Ostreococcus tauri. Keller Optimized Medium was developed by Smallwood et al. (https://doi.org/10.1101/291211) which includes additions of 17 mM sodium nitrate and 0.37 mM NaH2PO4
Protocolo Tcg4 v1
Authors: not provided dgalvana
Published: May 2022

Protocolo para realizar PCR de Tcg4
Prescribing pattern of analgesics in Colombia. Are there differences between capital cities and municipalities? v1
Authors: Machado Alba Jorge
Published: May 2022

Background. Analgesics provide multiple clinical benefits, but they are not without risks. Objective. To compare the outpatient prescribing pattern of analgesics between cities and municipalities in Colombia and to identify the variables associated with prescribing opioid analgesics. Methods. A cross-sectional study that identified the prescription of analgesics for outpatient use from a population database of 8.5 million Colombians. A descriptive, bivariate and multivariate analysis was performed. Results. A total of 573,248 patients were identified who had received prescriptions for some analgesic; they had a mean age of 46.5±23.6 years, and 65.7% were women. The most commonly prescribed analgesics were nonopioid analgesics, antispasmodics and opioid analgesics. The average milligram equivalent of morphine was higher in capital cities than in municipalities. Age ≥65 years (OR:2.60, 95%CI:2.54-2.67), being male (OR:1.09, 95%CI:1.07-1.11), dispensing in cities (OR:2.25; 95%CI:2.20-2.30) and suffering from chronic pain (OR:13.25; 95%CI:10.89-16.14) were associated with an increased risk of receiving an opioid analgesic. Conclusions. Differences were found in the prescription of analgesics between capital cities and municipalities. The use of opioids does not appear to be in line with the recommendations for clinical practice, and they were mainly prescribed for elderly men with chronic nononcological pain and for residents of capital cities.
High coverage and timeliness of vaccination of children under 6 years of age in Risaralda, Colombia v1
Authors: Machado Alba Jorge
Published: May 2022

Introduction. Child vaccination is one of the most cost-effective processes and interventions in public health. Objective. To determine coverage and timeliness of immunization in a sample of children under 6 years of age from a region of Colombia. Methods. Retrospective cohort study that evaluated the vaccination coverage and timeliness verification survey conducted in 2019 to a population of 2457 children under 6 years of age from Risaralda, Colombia. Variables such as age, municipality of residence, record of vaccines included in the national expanded program of immunization (EPI: BCG, hepatitis-B [HBV], diphtheria/pertussis/tetanus [DTP], Haemophilus influenzae [Hib], polio, rotavirus, pneumococcus, influenza, measles/mumps/rubella [MMR], Varicella, hepatitis-A [HAV], yellow fever), date of immunization were considered. Vaccination was defined as timely until 29 days after the day established by the EPI. Results. Survey evaluated children from 14 municipalities, with a mean age of 27.1±21.4 months. Respective coverage and timeliness by age group and vaccine were: newborns: BCG (100% -99.4%), HBV (99.7% -99.7%); 2 months: pentavalent -1 (DPT + Hib + HBV: 91.3%), polio, rotavirus and pneumococcus: 91.3%; 4 months: pentavalent -2 (88.0%), rotavirus, pneumococcus (97.0% -79.4%); 6 months: pentavalent -3 (95.0% -79.4%), polio (95.0% -79.4%), seasonal influenza (85.6% -85.6%), 12 months: MMR (98.0% -94.1%), Varicella (98.0% -94.3%), pneumococcus (98.0% -94.1%), HAV (98.0% -98.9%); 18 months: DPT Booster (91.0%-89.1%), polio (91.0% -89.1%), yellow fever (49.2% -49.2%); 60 months: DPT (95.0%-91.9%) and polio (95.0% -91.9%). Conclusions. Before COVID-19 pandemic, EPI demonstrated high coverage and timeliness in vaccination of children under 5 years of age; however, timeliness for third dose of DTP-Hib-HBV and polio identified opportunities for improvement.
Isolation of Nuclei from Adult_Human_Brain_Tissue v2
Authors: Institute for Brain Science Allen
Published: May 2022

Isolation of nuclei from frozen adult human brain tissue or thawed and microdissected brain tissue sections for FPCR and/or RNA-seq analysis. Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
SH-SY5Y culturing v1
Authors: Smith Laura
Published: May 2022

SH-SY5Y neuroblastoma cells were cultured in a 1:1 mixture of Ham’s F-12 and DMEM growth medium supplemented with 10% FBS, non-essential amino acids (NEAA: 0.1 mM of: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine) and penicillin/streptomycin antibiotic cocktail (50ng/ml) at 37°C and 5% CO2. Cells were passaged at around 80% confluency using trypsin digestion. Culture medium was removed from cells, centrifuged at 1000 RPM for 5 minutes to remove floating cells and stored at 80°C for analysis. Cells were washed once in PBS and trypsin was added (0.25% trypsin in Versene). Following an incubation of 2 minutes, the plates were tapped to detach cells. The trypsin was neutralised in 9 volumes of normal growth medium. For continued culturing, a fraction of cells was transferred to a falcon tube and pelleted for 10 minutes at 1000 RPM (200 x g). Pellets were resuspended in the appropriate volume of normal medium and split into new plates. If required, cells were counted prior to seeding using the C-Chip NanoEnTek DHC-N01 counter. Cell line stocks were generated by resuspending cell pellets in 1 mL of freezing medium (10% DMSO in FBS).
In Vitro GCase Activity Assay (Total Cell Lysate) v1
Authors: Smith Laura
Published: May 2022

Taurocholate inhibits the cytosolic B–glucosidase (GBA2) and activates lysosomal B–glucosidase (GBA1, a.k.a glucocerebrosidase (GCase)).In cultured cells the pH optimum is increased in the presence of taurocholate.Cultured cells are assayed without taurocholate at pH 4.5 and with taurocholate at pH 5.4.In normal controls and heterozygotes the B-glucosidase activity in the presence of taurocholate is approximately 2-10 times higher than without taurocholate.In cases of Gaucher disease the -glucosidase activity may be lower in the presence of the taurocholate.However, in fibroblasts there can be as much as a threefold increase in activity but the specific activity is very low compared with controls and heterozygotes. At acid pH B–glucosidase hydrolyses the substrate 4–methylumbelliferyl–B–D–glucopyranoside to 4–methylumbelliferone and glucose.Adding alkaline buffer stops the enzyme reaction and causes 4–methylumbelliferone to fluoresce at a different wavelength from unhydrolysed substrate, thereby permitting its measurement in the presence of a vast excess of unhydrolysed substrate.
In Vitro B-galactosidase and B-hexosaminidase Activity Assay (Total Cell Lysate) v1
Authors: Smith Laura
Published: May 2022

Synthetic substrate can be used at acidic pH (pH 4.1) to assess activity of lysosomal hydrolases, β-Galactosidase and β-Hexosaminidase. The substrate is cleaved by β-Galactosidase and β-Hexosaminidase and produces a fluorescent product proportional to activity.
Diagnosis of hypertension based on TCM (Traditional Chinese Medicine) constitution and wrist pulse wave signal v1
Authors: not provided Anonymous , Fan Lin , Yao Rui-Ling , Wang Zhong-Ming , zhang Rong , Li Yan , Zhang Xiao-Kang
Published: May 2022

In previous studies, pulse signals have been analysed primarily to standardise and objectify pulse diagnosis, but the influence of TCM constitution on pulse signals and disease has been neglected. Our study links the wrist pulse wave signal to TCM constitution to find TCM constitution factors that predispose to disease while improving the accuracy of disease classification. Our specific approach is set out below. A wrist pulse signal sampler based on a piezoelectric sensor was designed and 95 healthy subjects and 20 hypertensive patients were invited to complete the CCMQ (Constitution in the Chinese Medicine Questionnaire), of which 48 completed the pulse wave signal. Meanwhile, we improved the Butterworth filtering algorithm to denoise the pulse wave signals, and after smoothing and period segmentation, we finally extracted 27 time-domain features and 8 wavelet packet energy features. Then, we used the independent sample t-test and binary logistic regression analysis to analyze the correlation between the prevalence of hypertension and TCM constitution and found that the three constitutions of YaD (Yang-deficiency), YiD (Yin-deficiency), and PW (Phlegm-wet) were significantly associated with whether the subjects suffered from hypertension. The final classification results showed that the fusion features after adding the three constitution features with a high correlation with hypertension had a 17.46% higher F-score than the classification model without the constitution features. It can be seen that the incorporation of constitution characteristics into pulse characteristics in this paper has important implications for disease classification. This also provides a basis for studying the correlation between TCM physical characteristics and certain diseases, and patients can adopt targeted TCM management techniques to prevent diseases in advance.
High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v5
Authors: Topol (Verily Life Sciences) Aaron , not provided marlene.wolfe , White (Verily Life Sciences) Brad , Wigginton Krista , B B Boehm Alexandria
Published: May 2022

v3 updates: changes to the S gene primers and probes for detection of all SARS-CoV-2 variants This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.
Peptide N-Terminal Modification v1
Authors: Miller Cathy
Published: May 2022

Although many of the most widely recognized post-translational modifications are characteristic of secretory or cell-surface proteins, most proteins, whatever their ultimate cellular desination, undergo some modification. For proteins synthesized completely within the cytoplasm, the earliest and most widespread are removal or modification of the N-terminal residue. In many proteins the N-terminal α-ammonium group (PK=8) undergoes secondary modification. N-terminal modification reduces overall solubility of the peptide by reducing its overall charges. However, the stability of the peptide could also be increased because N-terminal modification generates a closer mimic of the native protein. Therefore, these modifications might increase the biological activity of a peptide and prevent degradation by enzymes. Creative Peptides is specialized in the custom synthesis of N-terminal modification peptides, providing a confidential and efficient service at competitive prices. Every step of peptide synthesis is subject to Creative Peptides' stringent quality control. Typical delivery specifications include HPLC chromatogram Mass spec analysis Synthesis report Certificate of analyses
Peptide C-Terminal Modification v1
Authors: Miller Cathy
Published: May 2022

The post-translation modification of eukaryotic proteins by the addition of isoprenyl lipids at their C-termini was first observed in the 1970s and 1980s. Since then, more than a hundred proteins have been shown to be modified by C15 farnesyl or C20 geranylgeranyl groups, including most members of the Ras, Rho, and Rab families of G proteins. The C-terminal of the peptide is synthesized as an amide to neutralize the negative charge created by the C-terminal COOH. This modification is added to prevent enzyme degradation, to mimic native proteins, and in some cases to remove hydrogen bonding at the C-terminal of the peptides which may interfere with the assays. Creative Peptides specialized in the custom synthesis of C-terminal modification peptides, providing a confidential and efficient service at competitive prices. Every step of peptide synthesis is subject to Creative Peptides' stringent quality control. Typical delivery specifications include HPLC chromatogram Mass spec analysis Synthesis report Certificate of analyses
Taxon group: Larval stage insects (TSS2) v1
Authors: Pereira da Conceicoa Lyndall , Sivell Olga , Sivess Laura , Fletcher Chris , R. Broad Gavin , Crowley Liam , Januszczak Inez
Published: May 2022

This is part of the collection "DToL Taxon-specific Standard Operating Procedure (SOP) for the Terrestrial and Freshwater Arthropods Working Group". The SOP collection contains guidance on how to process the various terrestrial and freshwater arthropod taxa within the scope of the Darwin Tree of Life project. The guidance specifically refers to the tissue samples needed for DNA barcoding (which takes place at the Natural History Museum (NHM)) and outlines the dissected tissues required for whole genome sequencing (WGS), which takes place at the Wellcome Sanger Institute . Every specimen is submitted for DNA barcoding first before potentially being sent to the Wellcome Sanger institute. TSS2: Taxon group: Larval stage insects Definition: Holometabolous insect larvae and pupae Including: Coleoptera, Diptera, symphytan Hymenoptera, Lepidoptera, Mecoptera, Megaloptera,Neuroptera, Raphidioptera, Trichoptera. Excluding: Specimens smaller than 5mm. See the Guidelines for important details and checklist.
Solanum pimpinellifolium seed propagation protocol v1
Authors: Sussman Hayley , M Julkowska Magdalena
Published: May 2022

This protocol describes S. pimpinellifolium propagation from seed to seed. It describes seed germination, plant care and growth. It also covers pollination, fruit harvesting, seed cleaning and seed storage.
Low-cost recombinase polymerase amplification (RPA) v1
Authors: Hegde Smitha
Published: May 2022

This protocol describes the expression of enzymes and creation of a master mix for recombinase polymerase amplification (RPA) assays.
Cryosectioning mouse brain v1
Authors: Srivastava Pranay
Published: May 2022

This protocol details the cryosectioning of mouse brain.
Glucagon measurement from islet populations using a TR-FRET assay v1
Authors: Eaton Wesley , Roper Michael
Published: May 2022

TR-FRET homogenous assay method using a microfluidic device for quantitation of glucagon secretion from human islet populations is described. A PDMS microfluidic device was used to perfuse a population of islets and collect 10µL fractions of perfusate every two minutes. The method was used to measure glucagon secretion from human islet populations from 5 donors.
Ectopic receptor & endosome size and count image analysis and quantification v1
Authors: G G Hanna Michael , De Camilli Pietro
Published: May 2022

This protocol is to help with the imaging analysis of ectopic receptor localization and endosome size.
Generation of human colon organoids from healthy and inflammatory bowel disease mucosa v1
Authors: Dotti Isabella , Mayorgas Aida , Salas Azucena
Published: May 2022

Ulcerative colitis and Crohn’s Disease are chronic inflammatory bowel diseases (IBD) of unknown cause characterized by a relapsing-remitting behavior. Growing evidence supports the idea that the epithelial barrier plays a central role in the pathogenesis of IBD as well as in its evolution over time, thus representing a potential target for novel therapeutic options. In the last decade, the introduction of 3D epithelial cultures from ex vivo-expanded intestinal adult stem cells (ASCs) has impacted our ability to study the function of the epithelium in several gastrointestinal disorders, including IBD. Here, we describe in detail a reproducible protocol to generate Matrigel-embedded epithelial organoids from ASCs of non-IBD and IBD donors using small colonic biopsies, including steps for its optimization. A slightly modified version of this protocol is also provided in case surgical samples are used. With this method, epithelial organoids can be expanded over several passages, thereby generating a large quantity of viable cells that can be used in multiple downstream analyses including genetic, transcriptional, proteomic and/or functional studies. In addition, 3D cultures generated using our protocol are suitable for the establishment of 2D cultures, which can model relevant cell-to-cell interactions that occur in IBD mucosa.
Engineering brain assembloids to interrogate human neural circuits v1
Authors: Miura Yuki , Li Min-Yin , Revah Omer , Yoon Se-Jin , Narazaki Genta , Pasca Sergiu
Published: May 2022

The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to the tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into regionalized neural organoids that can be fused and integrated to form assembloids and study neuronal migration. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.
Dead-end Ultrafiltration Water Collection v1
Authors: Ottesen Andrea , Kocurek Brandon
Published: May 2022

Dead end ultrafiltration is the most basic form of filtration. In this case a Rexeed #25S Filter is used with a peristaltic pump to pull volumes from 10L to 100 L through nanometer sized pores ( approximately 30 kDa). This size selection is perfect for capture of viruses, bacteria, protists and even larger particles and organisms. The large collection volumes made possible by this method have great value if there is a need to examine rare members of a community such as pathogens.
Investigating EFL Learners’ Ability to Analyse Poetic Language: A Pedagogical Corpus Stylistic Approach
Authors: Jaafar Eman Adil , Ganapathy Malini
Published: May 2022

Pedagogical stylistics refers to the application of the tools of stylistics in the teaching of the English language as a foreign or a second language.  Teaching and learning poetry is challenging. Thus, pedagogical corpus stylistics (Henceforth, PCS) approach has been introduced to Iraqi undergraduate foreign language learners (EFL) to guide them to analyze poetic language.  The study aims to make students interact with authentic examples of poetic language and answer questions about it. The main objective of the study is to examine whether PCS tools enable the learners to provide linguistic evidence from the poetic texts they are exposed to. This in turn ensures objective poetic analyses. Moreover, it aims to enable EFL Iraqi students to discover salient norms related to the Standard English language. The study reveals that the pedagogical corpus stylistic approach helps the students to discover language patterns using corpus tools with the help of the teacher’s guidance at the beginning but this eventually leads to independent learning and discoveries about poetic language.
Conceptos sociales, etiquetas y cambio conceptual: un enfoque semántico de la injusticia hermenéutica
Authors: Giromini José , Vilatta Emilia
Published: May 2022

El propósito del presente trabajo es analizar algunos aspectos semánticos del fenómeno de la injusticia hermenéutica que no han sido suficientemente considerados en la literatura reciente. En primer lugar, examinaremos diversos casos de injusticias hermenéuticas y propondremos clasificarlos de acuerdo a su estructura semántica. El núcleo de la clasificación que ofrecemos consiste en distinguir casos vinculados a problemas de contenido y casos vinculados a problemas de circulación de los conceptos sociales. En segundo lugar, cuestionaremos una concepción semántica, implícita en muchos trabajos acerca de la injusticia hermenéutica, de acuerdo a la cual los conceptos son simples etiquetas. Mostraremos que esta concepción impide comprender adecuadamente los diferentes casos de injusticia hermenéutica que identificamos debido a que, por un lado, no permite capturar las dinámicas de cambio o refinamiento conceptual que éstos involucran y, por otro, lleva a diagnosticarlos como meros problemas de aplicación de conceptos.
FUNCTIONAL ZONING OF TERRITORIES OF NATIONAL PARKS – THE BACKGROUND OF ITS PLANNING ORGANIZATION OF THE WORLD AND UKRAINE
Authors:   State Ecological Academy of Postgraduate Education and Management of the Ministry of Energy and Environmental Protection of Ukraine, Getman V.
Published: May 2022

The issues of functional zoning of the territories of national natural parks of Ukraine and national parks of the world as the key problem of their territorial organization or planning are considered. In a comparative analysis, both positive phenomena and shortcomings of local and world environmental planning, primarily in recreational areas, are shown. The issue of recreational pressure within local and foreign national (natural) parks and their standards is also raised. It should be noted that the literature does not yet contain sufficiently scientifically substantiated methodological recommendations for the allocation of functional zones, their optimal areas, configurations, permissible anthropogenic (recreational) loads on parts of nature protected areas and sites. The practical experience of the creation and functioning of the Ukrainian NPP assures that the problem of planning their territory is of fundamental importance. Its practical solution is a basic prerequisite for ensuring the balanced and conflict-free functioning of these multifunctional nature protected areas. Functional zoning of the NPP territory can also be considered as a target spatial-functional model of the protected area. Accordingly, the practical activities for its protection are territorially differentiated by the allocated functional zones. It is the way to implement this model. The planning organization is based on the features of the functional zoning of national parks, as well as on the basis of the territorial configuration of the landscape sites (forest areas, systems of lakes, groups of islands, river deltas, and banks of large reservoirs). The planning organization of national parks is the functional zoning of their territory, which is conditioned by natural factors, on the basis of which different types of spatial planning structures are formed, a kind of “frames” along with the directions of natural axes taking into account the existing systems of settlement, production, transport, and engineering structures.
PREREQUISITES FOR THE DEVELOPMENT OF OUTSOURCING IN UKRAINE: SOCIO-GEOGRAPHICAL ANALYSIS
Authors:   Taras Shevchenko National University of Kyiv, Razumova O.
Published: May 2022

Outsourcing, particularly in the field of IT, is considered one of the key economic activities in today’s globalized economy. It can be called a special form of doing business. The article includes the definition of the process of outsourcing, and considers the reasons for its formation. The consequences of the outsourcing practice were briefly described. The article considers the origin of outsourcing in its modern form and provides a brief analysis of its features. The most prominent business practices, that are usually associated with outsourcing (as opposed to its perception only as “offshore programming”) are described. Typical outsourcing services were named. The process of choosing the outsource destination, from the standpoint of a client company, is outlined. The study analyzes the factors that led to the attractiveness of Ukraine as a market for IT outsourcing services. The importance of geographical location as a factor that can influence the choice of a country as a location for the productive forces of foreign companies is highlighted. At the same time, natural resources play a secondary role in relation to the actual geographical location. The geographical position of Ukraine is considered a factor that significantly increases the attractiveness of working with Ukrainian IT companies from the customer’s point of view. Possible negative consequences of outsourcing choices, based purely on geographical location as the most prominent factor were mentioned, in the context of possible cultural and language incompatibility in amongst the very distant cultures in question. Work schedule compatibilities between Ukraine and the greatest outsource spenders in the world – countries of Western Europe and Northern America are highlighted. The main reasons for the successful growth of demand for the services of Ukrainian IT companies are outlined. The image of Ukraine as a popular outsourcing destination is highlighted, and its growth in the last three years is briefly considered. The income of IT workers in Ukraine, Europe and the world was briefly analyzed as a factor that directly affects the attractiveness of outsourcing in Ukraine. The issue of staff rotation in IT projects is briefly considered a factor that increases the competitiveness of Ukrainian companies in the global market. Statistical data on the growth of the Ukrainian IT sphere was briefly analyzed, along with its projected growth.
GEOGRAPHY OF CRIME IN UKRAINIAN REGIONAL CENTERS
Authors:   Taras Shevchenko National University of Kyiv, Glybovets V.
Published: May 2022

The article considers the issue of the spread of criminal offenses in the regional centers of Ukraine in 2015–2019, as well as in the first half of 2020. The purpose of the article is to reveal the topic of crime in the regional centers of Ukraine, as one of the most important problems for the further development of Ukraine as a European state. The author focuses on the place of some Ukrainian regional centers in the ranking of criminal cities in the world. Using the statistical data of the State Statistics Service of Ukraine, the author calculated the intensity of crime in the regional centers of Ukraine for 2015–2019, as well as the intensity of crime in the regional centers of Ukraine for six months of 2020. Based on the data calculated by the formula of crime intensity, the author grouped regional cities of Ukraine by low, below medium, medium and above medium crime rate. The research identified the cities with the largest and smallest number of recorded serious and especially serious criminal offenses per 10 thousand inhabitants; cities with the largest and smallest number of recorded criminal offenses committed in the field of trafficking in narcotic drugs, psychotropic substances, their analogues or precursors and other crimes against public health per 10 thousand inhabitants; cities with the largest and smallest number of recorded criminal offenses committed in the field of economic activity per 10 thousand inhabitants; cities with the largest and smallest number of recorded criminal offenses committed in the field of official activities and professional activities related to the provision of public services per 10 thousand inhabitants; cities with the largest and smallest number of identified persons who committed criminal offenses per 10 thousand inhabitants in 2018. The article also calculates the number of detected 2018 minors and juveniles who committed criminal offenses per 10 thousand inhabitants. The author touches on the issue of latent crime, a victimological opinion polls and provides a list of measures that can prevent an increase in the number of criminal offenses in the regional centers of Ukraine.