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We applied a one-step reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs). The cold-adapted reassortant influenza viruses A/H1N1 pdm09, A/H3N2, B/Yamagata and B/Victoria, included in the composition of the LAIV in 2015-2016, 2017-2018 and 2018-2019 flu season were studied for reproductive activity in MDCK cells as part of a mono-vaccine and tri-vaccine. For this we have developed a set of specific primers and probes. Method validation was performed using ELISA-test after mouse monoclonal antibodies to hemagglutinin (HA) staining of MDCK monolayer. Influenza B viruses B/Yamagata and B/Victoria were studied in MDCK cells in mono-infection and coinfection with different multiplicity of infection (MOI) using quantitative rRT-PCR.•RT-PCR analysis was adjusted to assess the growth characteristics of cold-adapted reassortant influenza viruses in MDCK cell line. The greatest suppression in the composition of the tri-vaccine was exposed to the H1N1 pdm09 LAIV component.•Influenza B viruses are least suppressed in trivalent LAIV. Influenza viruses B/Yamagata and B/Victoria reproduced as part of a mixed preparation not lower, if not better than as a mono-preparation at an MOI of 0.1. At an MOI of 0.01, the reproduction of both B/Yamagata and B/Victoria in the mixture was reduced compared to mono-vaccine.•The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. This indicates that it is undesirable to reduce the titers of vaccine viruses, including at the stages of transportation and storage of LAIV, Graphical abstract Image, graphical abstract

Graphical abstract, We describe a protocol for glycomic and proteomic profiling that uses serial enzyme digestions from the surface of fresh frozen or fixed tissue slides. The abundances of the extracted glycans and peptides are determined using liquid chromatography-mass spectrometry. In a typical experiment, our method quantifies 14 heparan sulfate disaccharides, 11 chondroitin sulfate disaccharides, 50 N-glycan compositions and approximately 1200 proteins from a 1.8 mm circle, on the surface of a fresh frozen tissue slide from rat brain. Each enzymatic digestion is incubated overnight with direct application of enzyme on the tissue surface. Overall, the sample preparation process for multiple tissue slides takes a day per biomolecule class. This protocol saves time by simultaneous digestion of large N-glycans and small HS disaccharides and subsequent separation using size exclusion chromatography. Compared to wet tissue analysis, this method requires less time by a factor of two. By comparison, MALDI-imaging provides higher spatial resolution of glycans and proteins but lower depth of coverage. MALDI dissociates fragile glycan substituents including sulfates and is not recommended for analysis of glycosaminoglycans (GAGs).

Bevacizumab or Avastin® (Av), the recombinant antibody targeting VEGF, improves progression-free but not overall survival of metastatic breast cancer patients due to development of Av resistance. We showed that Av-therapy-induced inflammatory microenvironment contributes to the refractoriness to Av treatment. Here we present data regarding the effect of Av treatment on migration of a non-invasive breast cancer cell line, MCF-7. The data presented hereis related to the research article “Bevacizumab induces inflammation in MDA-MB-231 breast cancer cell line and in a mouse model” (Hajjar et al., 2018). Keywords: Bevacizumab, MCF-7, Migration, RTCA

The data presented in this article are related to the research paper entitled “Intensified mitophagy in skeletal muscle with aging is downregulated by PGC-1alpha overexpression in vivo” (Yeo et al., 2019). The data explained the surgical procedure of in vivo local transfection by electroporation method in aged mouse tibialis anterior muscle, and plasmid DNA preparation and verification protocol. The data also showed the transfection efficiency levels of GFP or GFP-tagged PGC-1alpha through immunohistochemistry method for frozen muscle cross-sections.

In this article, using human pancreatic cancer cell lines and tumor specimens, we analyze the expression and localization of the invadopodia-related proteins TKS5 and Cortactin. Specifically, we present data on: a) TKS5 expression and localization by immunofluorescence in human pancreatic tumors, b) Cortactin expression by western blotting in various human pancreatic adenocarcinoma cell lines, c) TKS5 and Cortactin localization at invadopodia in BxPC-3 pancreatic adenocarcinoma cells, and d) TKS5 and Cortactin localization by co-immunofluorescence in human pancreatic cancer specimens. Data presented here is related to and supportive of the research article by Chen et al., "TKS5-positive invadopodia-like structures in human tumor surgical specimens" (Chen et al., 2019), where interpretation of the research data presented here is available.

This data is a curated collection of visual images of gene expression patterns from the pre- and post-natal mouse lung, accompanied by associated mRNA probe sequences and RNA-Seq expression profiles. Mammalian lungs undergo significant growth and cellular differentiation before and after the transition to breathing air. Documenting normal lung development is an important step in understanding abnormal lung development, as well as the challenges faced during a preterm birth. Images in this dataset indicate the spatial distribution of mRNA transcripts for over 500 different genes that are active during lung development, as initially determined via RNA-Seq. Images were systematically acquired using high-throughput in situ hybridization with non-radioactive digoxigenin-labeled mRNA probes across mouse lungs from developmental time points E16.5, E18.5, P7, and P28. The dataset was produced as part of The Molecular Atlas of Lung Development Program (LungMAP) and is hosted at https://lungmap.net. This manuscript describes the nature of the data and the protocols for generating the dataset.

Graphical abstract, Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. Alternative ways to provide the replacement DNA sequence, method of Cas9 delivery, and tethering the template sequence to Cas9 or the guide RNA (gRNA) have all been tested in an effort to maximize homology-directed repair for precise modification of the genome. We present two CRISPR/Cas9 methods that have been used to successfully generate large and complex gene-edits in mouse. In the first method, the Cas9 enzyme is used in conjunction with two sgRNAs and a long single-stranded DNA (lssDNA) template prepared by an alternative protocol. The second method utilizes a tethering approach to couple a biotinylated, double-stranded DNA (dsDNA) template to a Cas9-streptavidin fusion protein. • Alternative method for generating long, single-stranded DNA templates for CRISPR/Cas9 editing. • Demonstration that using two sgRNAs with Cas9-streptavidin/biotinylated-dsDNA is feasible for large DNA modifications.

Graphical abstract, RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5′ triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5′PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5′ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. • We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5′ triphosphorylated small RNAs; • RppH treatment is a valid alternative to TAP treatment.

Graphical abstract, Polymorphonuclear leukocytes (PMNLs) or neutrophils play an important role in the innate immune response. Working with human neutrophils is challenging because these cells are sensitive to changes in the surrounding media and quickly become apoptotic. Meanwhile the experiments with mature neutrophils may be very important for studies of blood function. In this paper we propose an improved technique of flow cytometry nuclear protein analysis with double antibody labeling, which allows direct comparison of protein quantity (overlay histograms) in the primary cells (neutrophils) and progenitor cell lines (line HL-60), to study differentiation process and for other research purposes. We suggest improved technique to analyze and compare nuclear proteins levels in the myeloid differentiation model system (HL-60 cell line) and / or primary human neutrophils. This method was justified with measurement of GFI1 protein expression level, as well-known transcription factor, typical and essential for mature neutrophils. The key protocol features are as follows: • Suggested protocol allows simply, direct and correct visual comparison of flow cytometry data in overlay diagrams for myeloid blood cells on various stages of differentiation. • 70% ethanol permeabilization of neutrophils and HL-60 cells results in lower background fluorescence and better peak resolution than MeOH and Saponin permeabilization. • Non-specific antibody binding in neutrophils can be efficiently blocked by using 1% BSA and non-immune goat serum.

Graphical abstract, DNA cloning remains the primary step before the further investigation of gene function. Restriction enzyme-based cloning methods are still widely used and numerous restriction-free cloning techniques are available as alternatives. Here we describe a PCR-based cloning method named ABC cloning. This method uses PCR to combine three overlapping DNA fragments into a recombinant vector that can be immediately transformed into competent cells. This technique uses only a thermostable DNA polymerase and is more rapid and efficient than previously described methods.

Graphical abstract, Protein kinases play a crucial role in cellular functions by adding phosphate group to the protein substrates. It is an indispensable post-translational modification that regulates intracellular signaling and key cellular processes. They thus serve as an excellent target for chemotherapeutic interventions. A vast repertoire of protein kinases is present in a cell with diverse substrates as well as phosphorylation sites. To study full kinome for its activity, there is an urgent need of designing a comprehensive, in vitro assay which itself is an impractical task. However, in this study, we have attempted to develop a robust assay that not only mimics the in vivo nature of the kinases but can also be used in a high throughput drug-screening platform. Herein, the Leishmania donovani parasites are lysed and the total protein content is extracted. This extracted proteome is further sub divided into two parts: one active fraction containing cellular kinases and the substrate is heat-denatured fraction that loses all the enzymatic activity but retains the potential phosphorylation sites. These fractions are then co-incubated in the presence of ATP to initiate the kinase reaction and the total kinase activity is measured using ADP-glo kinase assay. Overall, this method • Presents a simple and robust approach to understand the participation of kinases in signaling networks. • Presents a high-throughput platform for ex-vivo drug screening.

Graphical abstract Abstract The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - • DNA sequencing, even with small indels that can be difficult to discern on a gel. • additional enzymatic reaction steps, such as with the T7EI/Cel-I assay. • specialized equipment and analysis tools, such as with HRM analysis.

Graphical abstract, Extraction of high-quality DNA from Gossypium (Cotton) species is notoriously difficult due to high contents of polysaccharides, quinones and polyphenols other secondary metabolites. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from cotton, which is amenable for downstream analyses. In contrast to other CTAB methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, CsCl gradient ultracentrifugation, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use of PVP-10 to rid the problems associated with polysaccharides and polyphenols, respectively. The average yield was approximately 10–15 μg of good quality DNA from 100 mg of tissue weight, which is adequate for projects, like genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation. • Buffers A and B were redesigned from Paterson et al. (1993) and Porebski et al. (1997), respectively. • Ribonuclease A was added before chloroform extraction. • A simple, rapid and inexpensive DNA extraction method is described.

Graphical abstract, Advances in contactless acoustic liquid transfer technologies have unlocked opportunities to substantially increase the throughput of crystallization screens and decrease the consumption of reagents and consumables. Acoustic energy transfer enables crystallization experiments to be set up precisely and rapidly on a nanoliter scale. Nonetheless, adapting acoustic transfer methods to a diverse range of crystallization conditions and their physicochemical idiosyncrasies remains a major bottleneck for true universality of this technique. Even though the reagent limitations still remain an issue, we present a straightforward protocol for setting up crystallization experiments by acoustic transfer using a Labcyte Echo 550 instrument, with a focus on the technical limitations of this method, including reagent compatibilities, spatial resolution and downscaling limits. • Set up crystallization screens in a small scale with reliable drop volumes as low as 50 nl • Overview of commonly used crystallographic screen compatibility with acoustic dispensing • Comparison of instrument calibrations and settings and its effects on error rate and screen reproducibility

Graphical abstract, Cellular migration plays a crucial role within multicellular organisms enabling organ development, wound healing and efficient immune responses, but also metastasis. Therefore, it is crucial to dissect the underlying mechanisms. Directed migration and invasion are most efficient in response to chemotactic signals. To study cell migration and chemotactic responses, current experimental setups use either simplified 2D, tissue-mimetic 3D (e.g. collagen matrices) or in vivo environments. While the in vivo experiments are closest to the real physiological situation, they require animal experiments and are thus ill-suited for screening purposes. 3D matrices, on the other hand, can mimic in vivo conditions in many respects thus serving as instructive settings for the initial dissection of cell migration and chemotaxis. However, performing 3D chemotaxis assays has its limitations due to the delicate nature of most available setups and the tedious and time-consuming manual quantification process. Here, we present • A method for the easy construction of a chemotaxis chamber suitable for the analysis of large cell numbers. • A procedure to quantify their migration automatically with minimal input required by the experimenter. • Both successfully validated by analyzing the 3D chemotaxis of highly migratory primary dendritic cells and the invasive MDA-MB-231 cancer cells.

Molecular Interaction Network Approach (MINA) was used to elucidate candidate disease genes. The approach was implemented to identify novel gene association with commonly known autoimmune diseases [1]. In MINA, we evaluated the hypothesis that “network proximity” within a whole genome molecular interaction network can be used to inform the search for multigene inheritance. There are now numerous examples of gene discoveries based upon network proximity between novel and previously identified disease genes (Yin et al., 2017 [2], Wang et al., 2011 [3], and Barrenas et al., 2009 [4]). This study extends the application of interaction networks to the interrogation of Genome Wide Association studies: first, by showing that a group of nine autoimmune diseases (AuD) genes “seed genes”, are connected in a highly non-random manner within a whole genome network; and second, by showing that the minimal number of connecting genes required to connect a maximal number of AuD candidate genes are highly enriched as candidate genes for AuD predisposing mutations. The findings imply that a threshold number of candidate genes for any heritable disorder can be used to “seed” a molecular interaction network that • Serves to validate the disease status of closely associated seed genes • Identifies genes that are highly enriched as novel candidate disease genes • Provides a strategy for elucidation of epistatic gene x gene interactionsThe method could provide a critical toll for understanding the genetic architecture of common traits and disorders. Method name: MINA, Molecular Interaction Network Approach, Keywords: Autoimmune diseases, Crohn’s disease (CD), Systemic lupus erythematosus (SLE), Rheumatoid arthritis (RA), Psoriasis (PSO), Type-1 diabetes (T1D), Type-2 diabetes (T2D), Celiac disease (CeD), Multiple sclerosis (MS), Association, Molecular network

Graphical abstract, Microscopy of multiple samples using slides and coverslips is time consuming and good images are sometimes difficult to obtain due to cell movement. Our method involves manual spotting of multiple cell samples onto solid-medium pads, which creates the following benefits: • Rapid, high-quality imaging of multiple samples (hundreds per day) by visible and fluorescence microscopy. • No need for expensive automated equipment, multi-well plates or large amounts of consumables. • Wide range of working cell concentrations. The method was implemented for S. cerevisiae yeast, however it is likely to be applicable to all types of microorganisms and possibly other microscopic samples such as pollen. The lack of need for automated equipment may also make this method useful for field work.

The detection and molecular analysis of circulating tumour cells (CTCs) potentially provides a significant insight to the characterisation of disease, stage of progression and therapeutic options for cancer patients. Following on from the protocol by Warkiani et al. 2016, which describes a method of enriching CTCs from cancer patient blood with inertial microfluidics, we describe a method to measure the CTC RNA expression in the enriched fraction using droplet digital PCR and compare transcript detection with and without RNA pre-amplification. • Inertial microfluidics combined with droplet digital PCR is advantageous as it allows for CTC enrichment and subsequent RNA analysis from patient blood. This allows for patient tumour analysis with increased sensitivity and precision compared to quantitative Real Time PCR and enables the direct quantification of nucleic acids without the need for tumour biopsy. • This method demonstrates an efficient approach providing important insights into the analysis of colorectal cancer patients’ CTCs using a specific gene subset or biomarkers, an approach that may be tailored to tumour type or expanded to larger panels. Method name: Circulating tumour cells isolation and analysis with droplet digital PCR, Keywords: Circulating tumour cell, Droplet digital PCR, Colorectal cancer

Graphical abstract, Luciferase-based reporter assays are one of the most common cell-based screening formats for drug discovery, and simultaneous evaluation of the cytotoxic effect of test compounds is of great value in reducing false-positives. Here we share a multiplex assay protocol that allows sequential measurement of cell viability (cell number) and luciferase activity of the same sample in a multi-well-plate format. The viability assay employs a fluorogenic esterase substrate, CytoRed. • This protocol allows sequential measurement of endogenous esterase activity (as a surrogate for cell number) and then luciferase activity in a single sample. • The protocol eliminates the need for parallel viability assay or protein assay using separate aliquots of the lysate. • This protocol is especially useful for assays with cells stably expressing a luciferase construct, for which co-transfection of another reporter gene is not a viable option.

Graphical abstract, Acyl-CoA:cholestereol acyltransferase 1 (ACAT1) is a two-fold dimer (homotetramer) and has two distinct dimerization domains. One domain is in an alpha-helical rich region near the cytoplasmic N-terminus. The other is proposed to be near the C-terminus where multiple transmembrane domains promote hydrophobic interactions between two ACAT1 subunits. The truncation of the ACAT1 N-terminal dimerization domain, Δ1-65, creates a dimer which is fully enzymatically active. It is currently not known how the C-terminal dimerization domain contributes to ACAT1 enzymatic activity. Here we describe a simple method that dissociates ACAT1 dimers through the addition of the non-ionic detergents Triton X-100 or octyl glucoside which disrupt the C-terminal dimerization domain. We also document the protocols for a method to exchange Triton X-100 with CHAPS to restore C-terminal dimerization of the ACAT1 protein, and an optimized liposomal assay to assess ACAT enzymatic activity. • This method can be applied to dissociate ACAT1 subunits by using Triton X-100 or octyl glucoside. • ACAT1 dimerization can be restored by exchanging Triton X-100 with CHAPS. • The liposomal ACAT activity assay conditions have been optimized.

Graphical abstract, Genome-scale species tree inference is largely restricted to heuristic approaches that use estimated gene trees to reconstruct species-level relationships. Central to these heuristic species tree methods is the assumption that the gene trees are estimated without error. To increase the accuracy of input gene trees used to infer species trees, several techniques have recently been developed for constructing longer “supergenes” that represent sets of loci inferred to share the same genealogical history. While these supergene methods are designed to increase the amount of data for gene tree estimation by concatenating several loci into “supergenes” to increase gene tree accuracy, no formal protocols have been proposed to validate this key “supergene” concatenation step. In a recent study, we developed several supergene validation strategies for assessing the accuracy of a popular supergene method: the so-called “statistical binning” pipeline. In this article, we describe a more generalizable and model-based “supergene validation” protocol for assessing the accuracy of supergenes and supergene methods using model-based tests of phylogenetic congruency. • Supergenes are validated by adopting model-based tests of topological congruence • These model-based procedures out preform non-model based methods for supergene construction • The results of this protocol can be used to assess the overall performance of a supergene method across a phylogenomic dataset

The data presented in this article are connected to our research article entitled “D2A-Ala peptide derived from the urokinase receptor exerts anti-tumoural effects in vitro and in vivo” (Furlan et al., 2018). These data further extend our understanding of the inhibitory effects of D2A-Ala peptide. Dose-response curve using a wide range of concentrations of D2A-Ala shows that this peptide has no effects per se on proliferation of rat smooth muscle cells (RSMC). However, D2A-Ala dose-dependently inhibits epidermal growth factor (EGF)-induced RSMC proliferation. Kinetics lasting up to seven days revealed that D2A-Ala peptide completely blocked EGF-promoted RSMC proliferation. Moreover, D2A-Ala peptide inhibited invasion of HT 1080 cells towards RSMC. Keywords: Peptide, Urokinase receptor, Cell proliferation, Cell invasion

Optogenetic systems use genetically-encoded light-sensitive proteins to control and study cellular processes. As the number and quality of these systems grows, there is an increasing need for user-friendly and flexible hardware to provide programmed illumination to cultures of cells. One platform which satisfies this need for a variety of optogenetic systems and organisms is the Light Plate Apparatus (LPA), which delivers a controlled light dose to each well of a 24-well plate. Experimental reproducibility requires appropriate calibration to produce accurate light doses within individual wells of the LPA and between LPAs. In this study, we present an easy and accurate method for calibrating the LPA. In particular, we: • developed a 3D printed adaptor and MATLAB code to allow rapid measurement of irradiance produced by the LPA and subsequent calibration • provide appropriate code and methodology for generating a standard curve for each LPA • demonstrate the utility and accuracy of this method between users and LPAs Method name: Easy calibration of the Light Plate Apparatus for optogenetic experiments, Keywords: Optogenetics, Light Plate Apparatus, Calibration, Saccharomyces cerevisiae, Microbes, Optical power meter

The interaction of lipids (entry mechanism) with respect to both oxy- and deoxy-myoglobin was explored using unrestrained Molecular Dynamics simulations. The results indicated a spontaneous entry of both palmitic and palmitoylcarnitine molecules into the oxy-Mb structure at the main binding site, whereas in deoxy-Mb, both the lipid ligands move away from the protein surface. For the alternative binding locations, entry of the ligands was independent of the oxygenation state. Presented here are the tables with the myoglobin binding energies for palmitic acid and palmitoylcarnitine estimated using Alchemical Free Energy Perturbation approach for the key structures obtained in unrestrained Molecular Dynamics simulations. These data are referenced in the original article "Exploring the entry route of palmitic acid and palmitoylcarnitine into myoglobin", reference number YABBI7787.

This article contains microbiome data from the upper respiratory tract of patients living with HIV/TB, HIV and TB from Meiktila, a town in Myanmar where there is a high incidence of HIV and TB. Microbiomes were compared for HIV/TB infected and healthy adults from the same population. We collected nasopharyngeal and oropharyngeal swabs from a total of 33 participants (Healthy {5}, HIV/TB {8}, HIV {14}, and TB {6}). DNA was extracted from the swabs and subjected to custom single step 16s rRNA sequencing on an Illumina MiSeq platform. The sequencing data is available via http://www.ncbi.nlm.nih.gov/bioproject/ PRJNA432583.

The data provided in this article are related to the research article entitled “Effect of Super Magnetic Nanoparticles Coated with Various Electric Charges on α-Synuclein Protein Fibrillation Process” (Javdani et al.). This article describes how electrically different charged and concentrated iron oxide nanoparticles synthesized using reverse co-precipitation method affects the fibrillation of albumin protein. Keywords: Nanoparticle, Iron Oxide, Albumin, Fibrillation

The functional interrogation of factors underlying early mammalian development is necessary for the understanding and amelioration of human health conditions. The associated article [1] reports on the molecular characterization of markers of neural crest cells in gastrula and neurula stage rabbit embryos. This article presents survival data of rabbit embryos cultured in vitro, as well as immunofluorescence data for molecular markers of neural crest cells following approximately 24-h of culture. Lastly, towards the functional analysis of early neural crest and other developmental genes, this article provides data on the introduction of exogenous DNA into early stage rabbit embryos using electroporation.

The data presented here pertain to a research article entitled “Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation” (Okuda et al., 2010). The present article provides additional structural and gene expression data for the characterization of a TNF-α–inducible glycosphingolipid, globotetraosylceramide (Gb4), in vascular endothelial cells. (i) Structural details of Gb4 in lipid raft–enriched cell membranes were determined by MALDI-TOF MS. These analyses identified Gb4 with very-long-chain fatty acids as the major molecular species in this fraction, and the expression levels of whole molecular species of Gb4 with different fatty acid structures in the membrane are uniformly upregulated by TNF-α stimulation. (ii) The expression levels of genes encoding enzymes for synthesis of the ceramide portion of Gb4 were analyzed by real-time PCR. These assays revealed that TNF-α stimulation promotes transcription of the Elovl1 and Cers5 genes, which are involving in the synthesis of Gb4 with very-long-chain fatty acids. Collectively, these results indicate that TNF-α regulates glycosphingolipid synthesis and lipid raft formation in vascular endothelial cells via transcriptional up-regulation of related genes. These data thus provide new insights useful for understanding the molecular basis of inflammation-associated pathology in vascular endothelia.

Retinal vein occlusion (RVO) is a common vascular disease of the retina; however, the pathogenesis of RVO is still unclear. Branch RVO (BRVO) commonly occurs at the arteriovenous crossing and it was formerly believed that the diseased artery mechanically compresses the vein. However, it has been reported that the retinal vein runs deep beneath the artery at the arteriovenous crossing in eyes with an arterial overcrossing, and the venous lumen often appears to be preserved, even at the arteriovenous crossing, as shown by optical coherence tomography. Paques et al. [1] found venous nicking without arteriovenous contact using adaptive optics imaging. Thus, we investigated the potential role of a dysregulation of the retinal vein. While the pathogenesis of retinal vein occlusion (RVO) is still unclear, systemic hypertension and increased level of endothelin-1 (ET-1) are known risk factors (Flammer and Konieczka, 2015) [2]. We focused on the behavior of retinal veins in spontaneous hypertensive rats (SHR). Then, one of the retinal veins became exceptionally constricted and was nearly occluded (Fig. 1), and the chorioretinal blood flow significantly decreased in the retinas of SHRs following the intravenous injection of ET-1. In addition, immunoreactivity to ET-A receptor was higher in SHR retinas than in control (WKY; Wistar Kyoto rat) retinas (Fig. 2). The protein levels of ET-A receptor and HIF-1 were also significantly higher in SHR retinas than in WKY retinas (Fig. 3). We observed vasoactivity of retinal veins; a retinal venous constriction (Kida et al., 2018) [3]. This supports the hypothesis that ET-1 can constrict retinal veins, thus increasing retinal venous pressure, and that ET-1 may even contribute to the pathogenesis of RVO.

This data article indicates the in vitro cytotoxicity of kaempferol and gallic acid across different cancer cell lines including A2780 (ovarian), H460 (lung), A431 (skin), MIA PaCa-2 (pancreas), Du145 (prostate), HT29 (colon), MCF-7 (breast), BE2-C (neuroblastoma), SJ-G2, U87 and SMA (glioblastoma). The dataset showed that the inhibitory activity of kaempferol was comparatively stronger than gallic acid. Thereby, kaempferol is offered as a potent anticancer agent for further investigation and beneficial as a dietary supplement. The data within this article relates to the research article entitled “Screening phytochemical content, antioxidant, antimicrobial and cytotoxic activities of Catharanthus roseus (L.) G. Don stem extract and its fractions” (Pham et al., 2018).

The data described here support the research article “4-HNE carbonylation induces local conformational changes on bovine serum albumin and thioredoxin. A molecular dynamics study” (Alviz-Amador et al., 2018) . Dataset on Gaff force field parameters of AMBER is provided for assembled three non-standard amino acids resulting of the 4-HNE Michael addition, the main end product of lipids peroxidation. Data include a framework for derivation of missing bonds, angles and dihedral parameters for Cys, His, and Lys modified amino acids, alongside optimized partial charges derived with Restrained Electrostatic Potential (RESP) method and the new force field parameters obtained by quantic mechanical (QM) using HF/6-31G** level of theory. Benchmark as a graphics tutorial summary steps to obtained new parameters and the validation of non-standard amino acids is presented. The new residues constructed are put available to the scientific community to perform molecular dynamics simulations of modified 4-HNE proteins. Keywords: AMBER, Gaff, Force field parameterization, Mechanical quantic, Molecular dynamics, Geometry optimization, Validation

The data presented in this article shows the microarray based transcriptome profiles of ∆lon and ∆lon rpoB12 strains of Escherichia coli. The rif mutation namely, rpoB12 was isolated spontaneously in the background of ∆lon strain (over-produces colanic acid capsular polysaccharide) as a suppressor for over-production of colanic acid capsular polysaccharide (Meenakshi and Munavar, 2015) [1]. The E. coli strains were grown in LB medium at 30 °C overnight in duplicates. Total RNA from each samples were isolated and microarray based transcriptome profiles were studied and compared. The detailed methodology and data are given in this article. The interpretation of these data are discussed in the research article, “Evidence for Up and Down Regulation of 450 genes by rpoB12 (rif) Mutation and their Implications in Complexity of Transcription Modulation in Escherichia coli” (Meenakshi and Munavar, 2018) [2]. Keywords: ∆lon, rpoB12, Capsule, Biofilm, Motility, Stress response

We screened 583 chemicals for receptor binding activity to the human estrogen receptor (hER), the Japanese medaka estrogen receptor (medER), and the aryl hydrocarbon receptor (AhR) using the yeast two-hybrid assay. The substances tested included substances that could potentially be produced unintentionally by industrial processes, such as halogenated steroids and phenols. Antagonistic effects on hER and the androgen receptor were also screened. The test chemicals were selected for screening on the basis of chemical structure associated with possible estrogen receptor binding activity. The current study presents the report on the screening of 583 chemicals for different kinds of endocrine disrupting activity.

Human embryonic stem cells (hESCs) were used as a model of embryonic neurogenesis to identify the effect of excess fat uptake on neurodevelopment (Ardah et al., 2018). Herein, by directed differentiation of hESCs into neurons using established protocols, this data was generated for expression profiles of select lncRNAs during in vitro embryonic neurogenesis and their differential expression due to excess fat (palmitate) uptake. The undifferentiated hESCs were treated with 250 µM palmitate after identifying it as the highest concentration which is non-toxic to these cells. The palmitate treated hESCs were differentiated towards neurons keeping the levels of palmitate consistent throughout the differentiation process and fat uptake was confirmed by Oil Red O staining. The expression analysis of lncRNAs was performed by RT-qPCR on vehicle control and palmitate treated cells from 4 stages of differentiation, D0 (undifferentiated hESCs), D12 (neural stem cells), D44 (neural progenitors) and D70 (neurons) using lncRNAs array plates from Arraystar Inc. which contains 372 functionally identified lncRNAs found to be associated with lipid metabolism and other pathways (Cat# AS-NR-004).

This study investigated the effect of cupuassu butter on the cell number of human skin fibroblasts, as well as the gene expression profiles of certain growth factors in these fibroblasts. Cupuassu butter is a triglyceride composed of saturated and unsaturated fatty acids extracted from the fruit of Theobroma grandiflorum. The dataset includes expression profiles for genes encoding basic fibroblast growth factor (bFGF), stem cell factor (SCF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factor-7 (FGF7), and epidermal growth factor (EGF). Cell viability profile is presented as a line graph, and the expression profiles are shown as bar graphs. Furthermore, this article also describes the effects of cupuassu butter on wound healing in vitro. The wound healing effects are shown as a bar graph accompanied with representative microscopic images. Keywords: Cell number, Calcein assay, Real-time PCR, Gene expression profile, Fibroblast, Wound scratch assay

Yarrowia lipolytica, which is model oleaginous yeast with high industrial interest, was cultivated on fatty substrates. Data concerning fatty acid composition of both substrate and yeast lipids and comparisons of the experimental data with model predictions presented in “Biomodification of fats and oils and scenarios of adding value on renewable fatty materials through microbial fermentations: Modelling and trials with Yarrowia lipolytica” (Vasiliadou et al., 2018) were provided. Furthermore, the total yeast lipids were fractionated into their main fractions, that is, phospholipids, glucolipids plus sphingolipids and neutral lipids, and the fatty acid composition of each lipid fraction was reported.

Cerebrospinal fluid (CSF) is a key body fluid that maintains the homeostasis in central nervous system (CNS). As a biofluid whose content reflects the brain metabolic activity, the CSF is analyzed in the context of neurological diseases and is rarely collected from healthy subjects. For this reason, the metabolite variation associated with general phenotypic characteristics such as gender and age have hardly ever been studied. Here we present the hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) data as a result of untargeted metabolomics analysis of a cohort of elderly cognitively healthy volunteers ( n = 32). 146 unambiguously identified water soluble metabolites (using accurate mass, retention time and MS/MS matching against spectral libraries) were measured and their abundances across all the subjects depending on their gender are provided in this article. Data tables are available at https://data.mendeley.com/datasets/c73xtsd4s5/1. it's published on mendeley, the DOI is DOI:10.17632/c73xtsd4s5.1. The data presented in this article are related to the research article entitled "A global HILIC-MS approach to measure polar human cerebrospinal fluid metabolome: Exploring gender-associated variation in a cohort of elderly cognitively healthy subjects" (Gallart-Ayala et al., 2018, In press).

These data and analyses support the research article “Re-evaluating the LD50 requirements in the codified potency testing of veterinary vaccines containing Leptospira serogroups Icterohaemorrhagiae and Canicola in the United States” (Walker et al., 2018). Validity and disposition requirements submitted to the Center for Veterinary Biologics (CVB) are provided for serials (numbered lots) of commercial product potency tested for serogroups Canicola and Icterohaemorrhagiae in support of the Virus-Serum-Toxin Act (VSTA). Time course data for hamster loss after challenge with various concentrations of Leptospira during codified potency testing are also presented. The dose of Leptospira lethal to 50% of hamsters (LD50) was calculated by the Dragstedt-Behrens method for the in vivo data collected, and the equation is described here.

We present a transcriptome dataset generated from migratory chick trunk neural crest cells, which are destined to form components of the peripheral nervous system. Using the Sox10E1 enhancer, which specifically labels neural crest cells migrating on the trunk ventral pathway, we performed fluorescence activated cell sorting (FACS) of electroporated embryos to obtain a pure population of these cells for library preparation and Illumina sequencing. The results provide a list of genes that are enriched in the trunk neural crest. To validate the data, we performed in situ hybridization to visualize expression of selected transcripts.

Carbohydrate-binding modules (CBMs) are auxiliary domains into glycoside-hydrolases that allow the interaction between the insoluble substrate and the solubilized enzyme, through hydrophobic, CH-π interactions and hydrogen bonds. Here, we present the data article related to the interaction of one LaCBM26 and some mutated proteins with soluble α-glucans determined by enzyme-linked carbohydrate-binding assay, isothermal titration calorimetry (ITC), and affinity gel electrophoresis (AGE). The data of the behavior of proteins in presence and absence of substrate analyzed by circular dichroism CD and thermofluor are also presented. These results are complementary to the research article “The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction” (Armenta et al., 2019). Keywords: Carbohydrate-binding module, Starch, α-Glucans recognition

A single center, prospective cohort study recruiting 107 burn patients and 62 control patients was conducted to characterize the early phase of acute hyperglycemia in patients with burns. A total of 1643 blood samples were collected, and blood glucose and serum proinflammatory cytokines were detected and analyzed. A mouse severe burn model was used to study the underlying mechanisms of acute hyperglycemia postburn. An expression profile of IL-1 receptor, serum IL-1β and pancreatic islet function were detected in both control mice and burned mice. The data in this article is associated with the research article published in Biochim Biophys Acta "Acute pancreatic beta cell apoptosis by IL-1β is responsible for postburn hyperglycemia: evidence from humans and mice" [1].

Graphical abstract, DNA-fluorescence in situ hybridisation (DNA-FISH) allows visualisation of chromosome organisation and rearrangement. FISH probes are pools of short fluorescently labelled DNA fragments that are often produced from template plasmids that contain large genomic inserts. For effective sample penetration and target hybridisation it is critical that probe fragments are between 200 and 500bp. Production of these short probes requires significant optimisation and can be confounded access to expensive sonication equipment or inherent sequence features that influence enzymatic fragmentation or amplification. Here we demonstrate that effective FISH probes can be prepared without the need for optimisation of fragmentation using a cocktail of two the 4bp recognition sequence restriction enzymes CviQI and AluI.

This data article contains experimental data on the preparation and semi-preparative isolation of S -Alk(en)ylmercaptocysteine (CySSRs, R = allyl, “A”, 1-propenyl, “Pe” or methyl, “Me”) generated through conjugation reactions between allyl and 1-propenyl enriched thiosulfinates (TS) and cysteine. The data presented are related to the research article “ S- Alk(en)ylmercaptocysteine suppresses LPS-induced pro-inflammatory responses in murine macrophages through inhibition of NF-κB pathway and modulation of thiol redox status” (Tocmo and Parkin, in press). In this data article, we included a detailed procedure for CySSR preparation, their purification through semi-preparative chromatography and their toxicity profiles in RAW 264.7 macrophages. Data included also highlight, the ability of CySSRs to modulate intracellular thiol redox status.

Bamboos are known for their economical, ecological, and cultural importance. The plants can be annual or perennial and can be herbs, shrubs or trees and can also show different growth habits. The cell wall is the main component of the mechanical properties of the tissues. Data set presented here contains the results of cell walls fractioning of culms from six neotropical bamboo species: Apoclada simplex, Chusquea capituliflora, Filgueirasia arenicola, Filgueirasia cannavieira, Guadua tagoara, Merostachys riedeliana and Parodiolyra micrantha. The cell walls were fractionated with oxalate and increasing NaOH concentrations sequentially. The yield and the monosaccharide compositions showed a small amount of pectin as expected for Poaceae and arabinoxylan as the main hemicellulose. The digestion of the hemicellulose fraction with xylanase produced an oligosaccharide profile that could be used to compare the similarity of the arabinoxylan from different species without identifying each individual oligosaccharide. Our data showed that the differences in cell wall composition do not vary according to the growth habit, but are in close association with the phylogenetic relations within the family. The differences in load capacity in plants with different habits (trees and herbs, for example) are more associated with the amount of support tissues than with different cell wall compositions. The importance of evaluate the cell wall of tropical bamboo species aimed at improving resources for biotechnology was discussed by Tine et al. 2020 [1] .

We provide a 16S rRNA gene dataset of prokaryotic assemblages of a subantarctic marine ecosystem. Samples were collected at 2 stations (one near Ushuaia Bay and the other close to Bridges islands in the Beagle Channel). At each station, 2 depths (subsurface and bottom waters) were sampled in february, march, may and september during 2018, giving a total of 10 samples. 16S rRNA gene amplicon sequencing (V4 hypervariable region) was performed with the Illumina MiSeq platform. A total of 1116 amplicon sequence variants (ASVs) were recovered from the dataset. The sequences were taxonomically assigned to Alphaproteobacteria (23 ± 2%, mean ± standard error), Gammaproteobacteria (17 ± 1.5%), Flavobacteriia (8 ± 2%), Deltaproteobacteria (3.7 ± 0.5%), Acidimicrobiia (1.7 ± 0.1%), Planctomycetia (1.9 ± 0.4%), and AB16 group (1.7 ± 0.3%). Sequences affiliated with Archaea were abundant, reaching one third of analyzed sequences, mainly Thaumarchaeota (22 ± 3%), and Thermoplasmata (10 ± 1%). Together, sequences assigned to all these groups accounted for more than 90% of the sequences. This dataset constitutes a valuable resource for future scientific research aiming to unveil the role of these communities in ecosystem services such as carbon and nutrient cycling, and pollutants degradation. This will turn into benefits for future environmental monitoring and preservation actions, considering the tangible heritage of Ushuaia Bay and surrounding waters. Fil: Rodriguez Florez, Clara Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Malits, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Lozada, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina

Pseudomonas aeruginosa generally forms strong biofilm during chronic condition of wound. The whole mechanism of biofilm formation works in tandem with quorum sensing circuit of the organism in order to produce virulence. Here we report the draft genome sequence of two diabetic foot ulcer Pseudomonas aeruginosa isolates (VIT PC 7 and VIT PC 9) displaying homoserine lactone, rhamnolipid producing, biofilm phenotype and antibiotic resistance genes related to carbapenem, aminoglycoside, beta- lactamase and tetracycline resistance. The whole genome sequencing library was prepared according to the Oxford Nanopore's SQK-LSK108 kit protocol on Oxford Nanopore's Minion platform. The 7.1 Mb and 6.3-Mb draft genome sequence with GC content of 65.8% and 66.4% respectively provides insight into their resistance mechanism and virulence factors.

Reactive oxygen species (ROS) generated by the NADPH oxidase are crucial for antimicrobial host defense and also play a role in the regulation of inflammatory processes. Signals generated by formyl peptide receptor 2 (FPR2) activate the neutrophil ROS generating NADPH oxidase; such signals are mediated when the receptors bind an activating agonist, as well as when agonist desensitized FPR2 are reactivated by the receptor for platelet-activating factor (PAF). We present data on the effects of Idelalisib, a specific inhibitor for the PI3Kδ isoform, on ROS production during FPR2 activation and reactivation by PAF, respectively. Neutrophils were isolated from peripheral blood of healthy adults obtained from the blood bank at Sahlgrenska University Hospital and ROS release was measured using isoluminol-amplified chemiluminescence.

Large-scale gene expression analysis of legacy* and emerging** brominated flame retardants were conducted in the male Harlan Sprague Dawley rat [1] . Each animal was dosed for 5 days with the chemical at concentrations of 0.1 – 1000 μmol/kg body weight per day. Following the last dose, a specimen of the left liver was removed for RNA extraction. The amplified RNA (aRNA) was fragmented and then hybridized to Affymetrix Rat Genome 230 2.0 Arrays. Each GeneChip® array was scanned using an Affymetrix GeneChip® Scanner 3000 7 G to generate raw expression level data (.CEL files). Statistical contrasts were used to find pairwise gene expression differences between the control group and each dose group using the R/maanova package [2] . The transcriptomic data can be used to provide insights into the degree of toxicity, toxic mechanisms, disease pathways activated by exposure, and for benchmark dose analysis. The gene expression data for each of the nine flame retardants discussed here accompanies the research article entitled, “Comparative Toxicity and Liver Transcriptomics of Legacy and Emerging Brominated Flame Retardants following 5-Day Exposure in the Rat” [1] . * polybrominated diphenyl ether 47 (PBDE 47), decabromodiphenyl ether (decaBDE), hexabromocyclododecane (HBCD); ** 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB); bis(2-ethylhexyl) tetrabromophthalate (TBPH); tetrabromobisphenol A-bis(2,3-dibromopropyl ether (TBBPA-DBPE); 1,2-bis(tribromophenoxy)ethane (BTBPE); decabromodiphenylethane (DBDPE); hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO).

We provide the dataset supporting the research article “Nematodes as bioindicators of polluted sediments using metabarcoding and microscopic taxonomy” [1]. Nematodes are frequently used as bioindicators and the NemaSPEAR[%] is an validated index that is originally based on morphological data. The index was compared to molecular sequence data for the 28S rDNA, 18S rDNA and COI gene for 7 locations. This dataset includes chemical analyses of the sediments for 33 different substances. The sequence data for OTU-based analyses for the 28S rDNA, 18S rDNA and COI gene is given, together with the read distribution during bioinformatics processing. We furthermore include alternative ASV data, based on a cluster-independent approach. The morphological data is presented, including the biomass for each species, as well as an overview about whether the species is represented in the NCBI database. Furthermore, rarefaction analysis is given for the morphological data, and furthermore NMDS plots for the species and genus level based on morphological and molecular data. The correlation between the mean PEC-Q and the NemaSPEAR[%] values is given in order to compare the efficiency of the index, based on morphological and molecular data.

Chickpea (Cicer arietinum L.) is the second most important grain legume worldwide. Recent advances in the sequencing of the chickpea genome has provided a new and valuable resource to aid efforts in gene discovery and crop trait improvement. Technical difficulties in stable chickpea transgenics and the lack of a transient expression system for rapid analysis of gene expression and function; however, has limited the usefulness of this genomic resource. As a step toward alleviating this limitation, we report here the development of a simple and efficient transient gene expression protocol. Using leaves from chickpea seedlings, we have established a procedure that enables the generation of large quantities of vital chickpea protoplasts within only a few hours. In addition, we have optimized a PEG-calcium-mediated transfection method to efficiently deliver exogenous DNA into the chickpea protoplast. The current study is the first to present a detailed step-by-step procedures for protoplast isolation, evaluation, transfection, and application in chickpea. In addition, we optimize the transfection efficiency which has not been previously reported. Our protoplast transfection approach provides a platform that will allow rapid high-throughput screening and systematic characterization of gene expression and function. Knowledge gained through such studies will benefit current efforts to improve chickpea production and quality.•Modified enzymatic digestion solution for higher yield and viability.•Optimize transfection of chickpea protoplasts., Graphical abstract Image, graphical abstract