Quality control in the IVF laboratory: in-vitro and in-vivo development of mouse embryos is unaffected by the quality of water used in culture media

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

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Tyrosol-Enriched Tomatoes by Diffusion across the Fruit Peel from a Chitosan Coating: A Proposal of Functional Food

Foods ◽

10.3390/foods10020335 ◽

2021 ◽

Vol 10 (2) ◽

pp. 335

Author(s):

Silvia Tampucci ◽

Antonella Castagna ◽

Daniela Monti ◽

Clementina Manera ◽

Giuseppe Saccomanni ◽

...

Keyword(s):

Tomato Fruit ◽

Storage Period ◽

Food Matrix ◽

Fruit Peel ◽

Fresh Food ◽

In Vitro Permeation ◽

Active Transfer

Chitosan is receiving increasing attention from the food industry for being a biodegradable, non-toxic, antimicrobial biopolymer able to extend the shelf life of, and preserve the quality of, fresh food. However, few studies have investigated the ability of chitosan-based coatings to allow the diffusion of bioactive compounds into the food matrix to improve its nutraceutical quality. This research is aimed at testing whether a hydrophilic molecule (tyrosol) could diffuse from the chitosan-tyrosol coating and cross the tomato peel. To this end, in vitro permeation tests using excised tomato peel and an in vivo application of chitosan-tyrosol coating on tomato fruit, followed by tyrosol quantification in intact fruit, peel and flesh during a seven-day storage at room temperature, were performed. Both approaches demonstrated the ability of tyrosol to permeate across the fruit peel. Along with a decreased tyrosol content in the peel, its concentration within the flesh was increased, indicating an active transfer of tyrosol into this tissue. This finding, together with the maintenance of constant tyrosol levels during the seven-day storage period, is very promising for the use of chitosan formulations to produce functional tomato fruit.

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Influence of Light Conditions and Medium Composition on Morphophysiological Characteristics of Stevia rebaudiana Bertoni In Vitro and In Vivo

Horticulturae ◽

10.3390/horticulturae7070195 ◽

2021 ◽

Vol 7 (7) ◽

pp. 195

Author(s):

Alla A. Shulgina ◽

Elena A. Kalashnikova ◽

Ivan G. Tarakanov ◽

Rima N. Kirakosyan ◽

Mikhail Yu. Cherednichenko ◽

...

Keyword(s):

Culture Media ◽

Stevia Rebaudiana ◽

Adventitious Shoot ◽

Optimal Growth ◽

Medium Composition ◽

Light Spectra ◽

Stevia Rebaudiana Bertoni ◽

Multiplication Coefficient

We investigated the influence of different conditions (light composition and plant growth regulators (PGRs) in culture media) on the morphophysiological parameters of Stevia rebaudiana Bertoni in vitro and in vivo. Both PGRs and the light spectra applied were found to significantly affect plant morphogenesis. During the micropropagation stage of S. rebaudiana, optimal growth, with a multiplication coefficient of 15, was obtained in an MS culture medium containing 2,4-epibrassinolide (Epin) and indole-3-acetic acid (IAA) at concentrations of 0.1 and 0.5 mg L−1, respectively. During the rooting stage, we found that the addition of 0.5 mg L−1 hydroxycinnamic acid (Zircon) to the MS medium led to an optimal root formation frequency of 85% and resulted in the formation of strong plants with well-developed leaf blades. Cultivation on media containing 0.1 mg L−1 Epin and 0.5 mg L−1 IAA and receiving coherent light irradiation on a weekly basis resulted in a 100% increase in the multiplication coefficient, better adventitious shoot growth, and a 33% increase in the number of leaves. S. rebaudiana microshoots, cultured on MS media containing 1.0 mg L−1 6-benzylaminopurine (BAP) and 0.5 mg L−1 IAA with red monochrome light treatments, increased the multiplication coefficient by 30% compared with controls (white light, media without PGRs).

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In vitro characteristics and in vivo platelet quality of whole blood treated with riboflavin and UVA / UVB light and stored for 24 hours at room temperature

Transfusion ◽

10.1111/trf.16500 ◽

2021 ◽

Vol 61 (S1) ◽

Author(s):

Turid Helen Felli Lunde ◽

Lindsay Hartson ◽

Shawn Lawrence Bailey ◽

Tor Audun Hervig

Keyword(s):

Whole Blood ◽

Room Temperature

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Humanin is an endogenous activator of chaperone-mediated autophagy

The Journal of Cell Biology ◽

10.1083/jcb.201606095 ◽

2017 ◽

Vol 217 (2) ◽

pp. 635-647 ◽

Cited By ~ 33

Author(s):

Zhenwei Gong ◽

Inmaculada Tasset ◽

Antonio Diaz ◽

Jaime Anguiano ◽

Emir Tas ◽

...

Keyword(s):

Quality Control ◽

Cell Death ◽

Heat Shock Protein 90 ◽

Protective Effects ◽

Neuroprotective Effects ◽

Selective Degradation ◽

Cytosolic Side ◽

Chaperone Mediated Autophagy

Chaperone-mediated autophagy (CMA) serves as quality control during stress conditions through selective degradation of cytosolic proteins in lysosomes. Humanin (HN) is a mitochondria-associated peptide that offers cytoprotective, cardioprotective, and neuroprotective effects in vivo and in vitro. In this study, we demonstrate that HN directly activates CMA by increasing substrate binding and translocation into lysosomes. The potent HN analogue HNG protects from stressor-induced cell death in fibroblasts, cardiomyoblasts, neuronal cells, and primary cardiomyocytes. The protective effects are lost in CMA-deficient cells, suggesting that they are mediated through the activation of CMA. We identified that a fraction of endogenous HN is present at the cytosolic side of the lysosomal membrane, where it interacts with heat shock protein 90 (HSP90) and stabilizes binding of this chaperone to CMA substrates as they bind to the membrane. Inhibition of HSP90 blocks the effect of HNG on substrate translocation and abolishes the cytoprotective effects. Our study provides a novel mechanism by which HN exerts its cardioprotective and neuroprotective effects.

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Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000443040 ◽

2016 ◽

Vol 38 (3) ◽

pp. 859-870 ◽

Cited By ~ 27

Author(s):

Mingfeng He ◽

Hongquan Dong ◽

Yahui Huang ◽

Shunmei Lu ◽

Shu Zhang ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Microglial Activation ◽

Culture Media ◽

Microglial Cells ◽

Migration Ability ◽

Migration Assay ◽

Tnf Α

Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

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Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02089-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Gabriel Peinkofer ◽

Martina Maass ◽

Kurt Pfannkuche ◽

Agapios Sachinidis ◽

Stephan Baldus ◽

...

Keyword(s):

Extracellular Matrix ◽

Stem Cell ◽

Cell Adhesion ◽

Developmental Stage ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Induced Pluripotent

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

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Pharmacological Effects and Potential Clinical Usefulness of Polyphenols in Benign Prostatic Hyperplasia

Molecules ◽

10.3390/molecules26020450 ◽

2021 ◽

Vol 26 (2) ◽

pp. 450

Author(s):

Kensuke Mitsunari ◽

Yasuyoshi Miyata ◽

Tomohiro Matsuo ◽

Yuta Mukae ◽

Asato Otsubo ◽

...

Keyword(s):

Benign Prostatic Hyperplasia ◽

Molecular Mechanisms ◽

Prostatic Hyperplasia ◽

Pathological Conditions ◽

Urinary Tract Symptoms ◽

Anti Inflammatory ◽

Lower Urinary

Benign prostatic hyperplasia (BPH) is arguably the most common benign disease among men. This disease is often associated with lower urinary tract symptoms (LUTS) in men and significantly decreases the quality of life. Polyphenol consumption reportedly plays an important role in the prevention of many diseases, including BPH. In recent years, in addition to disease prevention, many studies have reported the efficacy and safety of polyphenol treatment against various pathological conditions in vivo and in vitro. Furthermore, numerous studies have also revealed the molecular mechanisms of the antioxidant and anti-inflammatory effects of polyphenols. We believe that an improved understanding of the detailed pharmacological roles of polyphenol-induced activities at a molecular level is important for the prevention and treatment of BPH. Polyphenols are composed of many members, and their biological roles differ. In this review, we first provide information regarding the pathological roles of oxidative stress and inflammation in BPH. Next, the antioxidant and anti-inflammatory effects of polyphenols, including those of flavonoids and non-flavonoids, are discussed. Finally, we talk about the results and limitations of previous clinical trials that have used polyphenols in BPH, with particular focus on their molecular mechanisms of action.

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Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

Journal of Clinical Medicine ◽

10.3390/jcm10010078 ◽

2020 ◽

Vol 10 (1) ◽

pp. 78

Author(s):

April Nettesheim ◽

Myoung Sup Shim ◽

Angela Dixon ◽

Urmimala Raychaudhuri ◽

Haiyan Gong ◽

...

Keyword(s):

Trabecular Meshwork ◽

Cathepsin B ◽

Molecular Mechanisms ◽

Culture Media ◽

Ecm Remodeling ◽

Trabecular Meshwork Cells ◽

Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

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Preparation of fibrils for intracerebral injection v1

10.17504/protocols.io.bws4pegw ◽

2021 ◽

Author(s):

The Michael J Fox Foundation Pff Standardization Consortium

Keyword(s):

Quality Control ◽

Best Practices ◽

External Validation ◽

Alpha Synuclein ◽

Intracerebral Injection ◽

Primary Neuron ◽

Consensus Protocol ◽

Reference Section

This is a consensus protocol developed through discussions with Laura Volpicelli-Daley, Caryl Sortwell, Kelvin Luk, Lindsey Gottler, and Virginia Lee. This protocol is intended for research purposes only, using specially-formulated monomeric alpha-synuclein protein available for purchase at Proteos, Inc as the result of efforts by The Michael J. Fox Foundation (MJFF). Each batch of the “Alpha-Synuclein Monomer Protein for Making Pre- Formed Fibrils” has undergone internal purification and quality control at Proteos in addition to external validation to confirm successful generation of pathogenic aSyn PFFs. See Reference section for methods and results from application of alpha-synuclein pre-formed fibrils (aSyn PFFs) in primary neuron cultures in vitro or in mice in vivo. This protocol is referenced in the Polinski et al 2018 paper entitled "Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents" (doi: 10.3233/JPD-171248).

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