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In vitro

Mark J Bailey, Felix Broecker, Paul E Leon, Gene S Tan
Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-FcγR. Using this method, the contribution of specific Fc-FcγR interactions conferred by immunoglobulins can be determined using an in vitro assay...
February 23, 2018: Journal of Visualized Experiments: JoVE
Hao-Hsiang Hsu, John-Kevin Kracht, Laura Elisabeth Harder, Kerstin Rudnik, Gerd Lindner, Katharina Schimek, Uwe Marx, Ralf Pörtner
In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP))...
February 23, 2018: Journal of Visualized Experiments: JoVE
Jing Huang, Peng Guo, Marsha A Moses
The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., proliferation, migration, and others) and in vivo models have been developed to investigate and characterize angiogenic and non-angiogenic cell phenotypes, these methods are time and labor intensive, and often require expensive reagents and instruments, as well as significant expertise. In a recent study, we used a novel quantitative phase imaging (QPI) technique to conduct time-lapse and labeling-free characterizations of angiogenic and non-angiogenic human osteosarcoma KHOS cells...
February 16, 2018: Journal of Visualized Experiments: JoVE
Salma H Azam, Mitchell Smith, Vivek Somasundaram, Chad V Pecot
Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro...
February 16, 2018: Journal of Visualized Experiments: JoVE
Federica Cavalera, Mario Zanoni, Valeria Merico, Thi Thu Hien Bui, Martina Belli, Lorenzo Fassina, Silvia Garagna, Maurizio Zuccotti
Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure...
March 3, 2018: Journal of Visualized Experiments: JoVE
Jessica C Stark, Euan Wallace, Rebecca Lim, Bryan Leaw
Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury...
February 16, 2018: Journal of Visualized Experiments: JoVE
Calliandra Maria de-Souza-Silva, Fernanda Guilhelmelli, Daniel Zamith-Miranda, Marco Antônio de Oliveira, Joshua Daniel Nosanchuk, Ildinete Silva-Pereira, Patrícia Albuquerque
Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals...
February 14, 2018: Journal of Visualized Experiments: JoVE
Ju-Chun Chang, Se Jin Lee, Jae Su Kim, Chung-Hsiung Wang, Yu-Shin Nai
The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV)...
February 22, 2018: Journal of Visualized Experiments: JoVE
Monica Terracciano, Michela Napolitano, Luca De Stefano, Anna Chiara De Luca, Ilaria Rea
Diatomite is a fossil material made of amorphous porous silica. In this work, polyethylene glycol (PEG)-modified diatomite NPs (PEG-DNPs) are decorated with gold NPs (AuNPs) by one-pot liquid-phase synthesis. Nanocomplexes (PEG-DNPs@AuNPs), with an average size of about 450 nm, are characterized by dynamic light scattering, electron microscopy, nitrogen adsorption/desorption analysis, UV-VIS and photoluminescence spectroscopies. Preliminary studies on the use of the nanocomplex in nanomedicine are also presented...
March 19, 2018: Nanotechnology
Tomislav Rončević, Damir Vukičević, Nada Ilić, Lucija Krce, Goran Gajski, Marija Tonkić, Ivana Goić-Barišić, Larisa Zoranić, Yogesh Sonavane, Monica Benincasa, Davor Juretić, Ana Maravić, Alessandro Tossi
Antimicrobial peptides often show broad-spectrum activity due to a mechanism based on bacterial membrane disruption, which also reduces development of permanent resistance, a desirable characteristic in view of the escalating multidrug resistance problem. Host cell toxicity however requires design of artificial variants of natural AMPs to increase selectivity and reduce side effects. Kiadins were designed using rules obtained from natural peptides active against E. coli and a validated computational algorithm based on a training set of such peptides, followed by rational conformational alterations...
March 19, 2018: Journal of Medicinal Chemistry
Anna Żądło-Dobrowolska, Nina G Schmidt, Wolfgang Kroutil
Amide bond formation has considerable significance in synthetic chemistry. Although the C-acyltransferase from Pseudomonas protegens has been found to catalyze C-C bond formation in nature as well as in in vitro experiments with non-natural substrates, it is now shown that the enzyme is also able to catalyze amide formation using aniline derivatives as substrates with promiscuous activity. Importantly, the amide formation was enabled in aqueous buffer. Identifying phenyl acetate as the most suitable acetyl donor, the products were obtained with up to >99% conversion and up to 99% isolated yield...
March 19, 2018: Chemical Communications: Chem Comm
Chi Zhang, Huaigao Liu, Shangwu Chen, Yongkang Luo
Dipeptidyl peptidase-IV (DPP-IV) is a serine exo-peptidase that can inactivate incretins by removing N-terminal dipeptides. Currently, inhibiting the DPP-IV activity is a common treatment for type 2 diabetes (T2D). The goal of this study is to investigate whether IADHFL, a novel DPP-IV inhibitory peptide identified from bighead carp (Hypophthalmichthys nobilis), has the potential to modulate T2D. IADHFL remained stable after simulated gastrointestinal digestion and significantly decreased the activity and expression of both soluble and membrane-bound DPP-IV after 24 h and 48 h of treatment...
March 19, 2018: Food & Function
Gembu Maryu, Haruko Miura, Yoichi Uda, Akira T Komatsubara, Michiyuki Matsuda, Kazuhiro Aoki
Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions...
March 17, 2018: Cell Structure and Function
Rebecca R Payton, Louisa A Rispoli, Kimberly A Nagle, Cedric Gondro, Arnold M Saxton, Brynn H Voy, J Lannett Edwards
Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations...
March 18, 2018: Journal of Reproduction and Development
Jiyoung Kim, Gi-Ho Sung
Beauvericin is a mycotoxin which has insecticidal, anti-microbial, anti-viral and anti-cancer activities. Beauvericin biosynthesis is rapidly catalyzed by the beauvericin synthetase (BEAS) in Beauveria bassiana. Ca2+ plays crucial roles in multiple signaling pathways in eukaryotic cells. These Ca2+ signals are partially decoded by Ca2+ sensor calmodulin (CaM). In this report, we describe that B. bassiana BEAS (BbBEAS) can interact with CaM in a Ca2+ -dependent manner. A synthetic BbBEAS peptide, corresponding to the putative CaM-binding motif, formed a stable complex with CaM in the presence of Ca2+ ...
March 19, 2018: Journal of General and Applied Microbiology
C Incorvaia, C Cavaliere, F Frati, S Masieri
Allergic rhinitis (AR) was long considered a quite trivial disease, but the advance in epidemiological and clinical knowledge, with a major role for Allergic rhinitis and its Impact on Asthma (ARIA) initiative, substantially changed the scene. Now we know that AR has significant effects on patients’ quality of life and also has a relevant economic burden. The ARIA phenotypes related to the duration of symptoms and to the severity of AR are very useful in establishing the optimal strategy in each patient with AR, also according to the kind of allergens that cause rhinitis...
January 2018: Journal of Biological Regulators and Homeostatic Agents
E Savi, S Peveri, C Cavaliere, S Masieri, M Montagni
The introduction of highly purified natural and recombinant single allergenic molecules represented an important improvement in the diagnosis of IgE sensitization. The identification of specific IgE against cross-reacting molecules such as profilin, lipid transfer proteins, calcium binding proteins or against “genuine molecules”, represents an added value and allows to distinguish between true and false polysensitization. In vitro tests add information to recognize patients with sensitization to genuine molecules that cause allergic diseases and to evaluate in childhood the spreading of sensitization for each molecule in order to choose the best treatment and to identify the ideal patient for allergen immunotherapy...
January 2018: Journal of Biological Regulators and Homeostatic Agents
F Frati, C Incorvaia, C Cavaliere, G Di Cara, F Marcucci, S Esposito, S Masieri
The skin prick test (SPT) is the most common test for the diagnosis of allergy. SPT is performed by pricking the skin, usually in the volar surface of the forearm, with a lancet through a drop of an allergen extract and is usually the first choice test in the diagnostic workup for allergic diseases because of its reliability, safety, convenience and low cost. SPT is minimally invasive and has the advantage of testing multiple allergens in 15 to 20 min. In children, SPT is far less disturbing than venipuncture and is used to obtain a sample of serum to measure specific IgE through in vitro tests...
January 2018: Journal of Biological Regulators and Homeostatic Agents
Hao Tu, Qin Zhou, Xun Zhang, Ting Wang, Cheng-Fu Yuan, Chang-Cheng Zhang, Ding Yuan, Zhao-Qi Liu
This study aimed to investigate the molecular mechanism and protective effect of total saponins of Panax japonicas (TSPJ) on HepG2 cells apoptosis induced by palmitic acid (PA).The HepG2 cells were cultured in vitro , and divided into five groups: the control group, the model group, the high-dose group (50 mg·L⁻¹), the middle-dose group (25 mg·L⁻¹) and the low-dose group (12.5 mg·L⁻¹).The cells of the five groups were cultured continuously for 24 hours. The cell viability was measured with MTT. HepG2 cells apoptosis was detected by Hoechest staining and Annexin V-FITC/PI staining...
January 2018: Zhongguo Zhong Yao za Zhi, Zhongguo Zhongyao Zazhi, China Journal of Chinese Materia Medica
Meng-Die Qi, Jian Wang, Xiao-Jing Ma, Quan Zhang, Fang-Fang Wang, Ying Kang, Hui-Xin Lin, Yong Liu
Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata,with a variety of pharmacological activity,widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study,the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment,named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression...
January 2018: Zhongguo Zhong Yao za Zhi, Zhongguo Zhongyao Zazhi, China Journal of Chinese Materia Medica
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