Methods Protoc., Volume 1, Issue 2 (June 2018) – 14 articles

Ionic liquids (ILs) have advanced a variety of applications, including matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). ILs can be used as matrices and solvents for analyte extraction and separation prior to analysis using laser desorption/ionization–mass spectrometry (LDI–MS). Most ILs show high stability with negligible sublimation under vacuum, provide high ionization efficiency, can be used for qualitative and quantitative analyses with and without internal standards, show high reproducibility, form homogenous spots during sampling, and offer high solvation efficiency for a wide range of analytes. Ionic liquids can be used as solvents and pseudo-stationary phases for extraction and separation of a wide range of analytes, including proteins, peptides, lipids, carbohydrates, pathogenic bacteria, and small molecules. This review article summarizes the recent advances of ILs applications using MALDI–MS. The applications of ILs as matrices, solvents, and pseudo-stationary phases, are also reviewed. Full article

The ability of a photocatalyst to degrade a target pollutant is a commonly used method to assess its effectiveness for environmental applications, while ultraviolet-visible (UV-vis) spectroscopy and spectroscopic ellipsometry are conventional techniques for the estimation of a semiconductor band gap. In this work, an array of six light-emitting diodes (LEDs), characterized by different emission peaks between 470–370 nm and absorbed power of 3 W, was implemented into an existing standard testing apparatus for the testing of nitrogen oxides degradation in air. The abatement indexes, obtained under different LEDs irradiation, were firstly compared to the ones determined according the standard and, secondly, correlated with the measured LED emission spectrum, in order to estimate the photocatalyst band gap. Results suggest that this expeditious technique can be easily implemented into existing testing apparatus for the estimation of the band gap and for the appraisal of photocatalytic materials under realistic conditions. Full article

The prevalence of non-alcoholic fatty liver disease (NAFLD) and its advanced form, nonalcoholic steatohepatitis (NASH), is increasing, and as such its contribution to the development of hepatocellular carcinoma is also rising. NAFLD has been shown to influence the immune tumor microenvironment. Therefore, development of pre-clinical mouse models in the context of NAFLD are increasingly important. Here, we describe a mouse model designed to recapitulate the findings of NAFLD followed by rapid induction of orthotopic liver tumors with intrahepatic tumor injection. Additionally, we utilized bioluminescent imaging to monitor tumor growth and response to therapy. The development of one dominant tumor nodule allows precise separation of tumor and liver tissue. This is useful for immunotherapy studies as mononuclear cells from the tumor and the surrounding liver tissue can be analyzed separately. Full article

Immunofluorescence staining has become an essential tool in pathology and biomedical sciences to identify rare cells, cell–cell interactions, and submicroscopic cellular components. Many experimental settings, however, suffer from the fact that traditional widefield fluorescence microscopy is usually restricted to imaging three or four fluorophores only. Due to a lack of morphological information and a high detection limit, even flow cytometry—which is capable of staining 20 or more fluorophores at the same time—is limited in its applicability, especially in areas such as rare cell detection. Other advanced imaging approaches, such as confocal laser scanning microscopy and imaging flow cytometry, may be addressing these shortcomings, but in turn require sophisticated downstream data processing and high capital outlay. Here, we describe a new method and filter set-up to routinely employ up to seven fluorophores on a traditional widefield fluorescence microscope equipped with a standard high-pressure mercury light source. Quantification of crosstalk between channels and actual seven-color imaging of cancer cells spiked into leukocytes demonstrate that there is no need for digital compensation correction algorithms. Our set-up thus permits a detailed analysis of rare cell populations, co-localization of antigens, and cell morphology in a standard research or routine laboratory setting. Full article

The chick chorioallantoic membrane (CAM) is an extra-embryonic membrane, comprised of a high density of blood and lymphatic vessels. CAM has a dense capillary network and is commonly used to study in vivo angiogenesis and anti-angiogenesis in response to potential biomolecules and drugs. Most of the earlier reported CAM assays described the in-ovo method—where the viability of the embryo is higher, but accessibility to the CAM is limited. Ex-ovo CAM methods were previously described that employed shell-less cultures of chick embryos, but the low viability of embryos reduced the overall robustness of the angiogenesis assays. We described a method (named as cup-CAM method) which is more economical, has better accessibility and has significantly improved the viability of the embryo till advanced developmental stages. We could perform this simple yet useful experimentation with the common tools available in the laboratory. We successfully used the cup-CAM method for showing the paracrine effects of conditioned media from tumor cells, on the angiogenesis. This method can be used to assay the angiogenic potential of a drug or protein and to observe the embryonic development of the chick embryo and other related scientific applications. Full article

Qβ is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qβ belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qβ is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (β) of a replicase, the maturation protein (A₂) and the read-through or minor coat protein (A₁), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qβ phage has attractive features for in vitro evolution. The A₁ protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein’s UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A₁ is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A₁ protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qβ phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A₁ proteins and exposed on the Qβ phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qβ system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qβ model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qβ phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses. Full article

Cas9 is a site-specific RNA-guided endonuclease (RGEN) that can be used for precise genome editing in various cell types from multiple species. Ribonucleoprotein (RNP) complexes, which contains the Cas9 protein in complex with a guide RNA, are sufficient for the precise editing of genomes in various cells. This DNA-free method is more specific in editing the target sites and there is no integration of foreign DNA into the genome. Also, there are ongoing studies into the interactions of Cas9 protein with modified guide RNAs, as well as structure-activity studies of Cas9 protein and its variants. All these investigations require highly pure Cas9 protein. A single-step metal affinity enrichment yielding impure Cas9 is the most common method of purification described. This is sufficient for many gene editing applications of this protein. However, to obtain Cas9 of higher purity, which might be essential for biophysical characterization, chemical modifications, and structural investigations, laborious multi-step protocols are employed. Here, we describe a two-step Cas9 purification protocol that uses metal affinity enrichment followed by cation exchange chromatography. This simple method can yield a milligram of highly pure Cas9 protein per liter of culture in a single day. Full article

CRISPR-Cas9 is revolutionizing the field of genome editing in non-model organisms. The robustness, ease of use, replicability and affordability of the technology has resulted in its widespread adoption among researchers. The African butterfly Bicyclus anynana is an emerging model lepidopteran species in the field of evo-devo, with a sequenced genome and amenable to germ line transformation. However, efficient genome editing tools to accelerate the pace of functional genetic research in this species have only recently become available with CRISPR-Cas9 technology. Here, we provide a detailed explanation of the CRISPR-Cas9 protocol we follow in the lab. The technique has been successfully implemented to knock-out genes associated with eyespot development and melanin pigmentation. Full article

Most natural DNA and RNA are devoid of long trinucleotide (TN) sequences that lack one specific nucleotide (missing nucleotide (MN)). Here we developed a novel method that is based on rolling circle amplification (RCA), in which the TN-information of short TN stretches is sequence-specifically recognized, transferred, extended, amplified and detected by padlock probes that consist entirely of nucleotides complementary to the three nucleotides present in the target sequence (complementary TN-information). Upon specific head-to-tail annealing and ligation to the TN-target sequence, these padlock probes represent extended complementary TN versions of the target sequence that can be further amplified by trinucleotide rolling circle amplification (TN-RCA). Since during TN-RCA the MN (as dNTP) is not added, background amplification is minimized with endogenous RNA/DNA (which mostly would require all four dNTP). Therefore, various labelled dNTP can be added to the TN-RCA reaction that enables the separation, isolation and detection of the amplified single-stranded DNA (ssDNA). Here the TN-RCA method is exemplified with RNA/DNA from Zika virus and from human papilloma virus (HPV). TN-RCA is a novel isothermal amplification technique that can be used for sensitive sequence-specific detection and diagnosis of natural and synthetic DNA or RNA containing TN stretches with low background in short time. Full article

Osteoblast/osteocyte cultures continue to emerge as essential tools for bone biology research in vitro. The change in cell number is an important parameter to be considered for investigating osteogenic differentiation. However, there is no reliable method for quantifying absolute cell count in differentiating osteoblast/osteocyte cultures because of their strongly adherent, multi-layered, super-confluent nature, and their accumulated extracellular matrix production which progressively mineralises in vitro. We developed a practical, simple and cost-effective method based on the fluorometric quantification of a nucleic dye, GelRed™, to enumerate cell number in osteoblast/osteocyte cultures. This method may also be suitable for quantifying cell numbers on other mammalian adherent cell types. Full article

Coomassie brilliant blue (CBB) dyes have been commonly used for the staining of protein bands in polyacrylamide gel electrophoresis (PAGE) gels. However, the staining and destaining of CBB dyes are time-consuming, and the use of methanol is hazardous to one’s health. I introduce a rapid electrophoretic destaining method using a semi-dry transfer unit and a high current power supply. In this method, ethanol was used instead of the hazardous methanol. Most of the protein bands became visible in 30 min. After a secondary destaining step, residual CBB was completely destained. The detection limit for a tested protein (5 ng) was higher than that of the conventional method. Therefore, this method is superior in its speed, safety, low cost, and sensitivity. Full article

In vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. However, these experiments must be complemented with in vitro and ex vivo experiments. Here, we describe a protocol for the preparation and ex vivo infection of lung slices from different mammalian host species with various respiratory paramyxoviruses expressing fluorescent reporter proteins, and suggest follow-up experiments including immunohistochemistry, flow cytometry and confocal microscopy. Full article

India has recently introduced a rotavirus vaccine under a universal immunization program. There is limited information on intussusception, an adverse event, following immunization in children from India. We are conducting sentinel surveillance for intussusception in children aged under two years at 19 hospitals. The sentinel sites’ selection followed a multistage process. The surveillance combines retrospective surveillance for 69 months and prospective surveillance for 18 months. The suspected intussusception cases shall be reviewed for capturing confirmed cases and detailed data collection and classification according to Brighton Collaboration criteria. Data shall be analysed to describe epidemiology, trends, regional and seasonal variations, clinical profiles, management modalities, and outcomes of intussusception. The combination of prospective and retrospective surveillance shall be informative about the trend of intussusception over the last seven years in India. At four sites where rotavirus vaccines have been introduced, the change in intussusception trends shall be documented. The potential association with rotavirus vaccines and other vaccines shall be assessed using case-control and self-controlled case series methodology. Results are forthcoming. The results shall support the national vaccine safety surveillance effort by providing baseline estimates of intussusception for continued monitoring. The surveillance protocol and site selection processes shall inform similar vaccine-safety surveillance in India and other developing countries. Full article

Epigenetic modifications enable cells to genetically respond to chemical inputs from environmental sources. These marks play a pivotal role in normal biological processes (e.g., differentiation, host defense and metabolic programs) but also contribute to the development of a wide variety of pathological conditions (e.g., cancer and Alzheimer’s disease). In particular, DNA methylation represents very stable epigenetic modification of cytosine bases that is strongly associated with a reduction in gene activity. Although High Performance Liquid Chromatography (HPLC) methodologies have been used to resolve methylated cytosine from unmodified cytosine bases, these represent only two of the five major cytosine analogs in the cell. Moreover, failure to resolve these other cytosine analogs might affect an accurate description of the cytosine methylation status in cells. In this present study, we determined the HPLC conditions required to separate the five cytosine analogs of the methylation/demethylation pathway. This methodology not only provides a means to analyze cytosine methylation as a whole, but it could also be used to more accurately calculate the methylation ratio from biological samples. Full article