scholarly journals Composite Bioinks With Mesoporous Bioactive Glasses—A Critical Evaluation of Results Obtained by In Vitro Experiments

2022 ◽  
Vol 9 ◽  
Author(s):  
Vera Guduric ◽  
Johannes Wieckhusen ◽  
Anne Bernhardt ◽  
Tilman Ahlfeld ◽  
Anja Lode ◽  
...  
Keyword(s):  
Cell Viability ◽  
Critical Evaluation ◽  
High Specific Surface Area ◽  
Channel Structure ◽  
The Other ◽  
Burst Release ◽  
Bioactive Glasses ◽  
Other Hand ◽  
Common Cell

Besides osteoconductivity and a high degradation rate, mesoporous bioactive glasses (MBGs) are specific for their highly ordered channel structure and high specific surface area, making them suitable as drug and/or growth factor delivery systems. On the other hand, the mesoporous channel structure and MBG composition can have an effect on common cell evaluation assays, leading to inconclusive results. This effect is especially important when MBG is mixed in composite bioinks, together with cells. Additionally, the hydrogel component of the ink can influence the degradation of MBG, leading to different ion releases, which can additionally affect the analyses. Hence, our aim here was to show how the MBG structure and composition influence common cell viability and differentiation assays when calcium (Ca)- or magnesium (Mg)-containing glass is part of an alginate-based composite bioink. We suggested pre-labeling of cells with DiI prior to bioprinting and staining with calcein-AM to allow identification of metabolically active cells expressing signals in both green and red channels, allowing the use of fluorescence imaging for cell viability evaluations in the presence of high amounts (7 wt %) of MBGs. The release and uptake of ions during degradation of CaMBG and MgMBG were significantly changed by alginate in the composite bioinks, as confirmed by higher release and uptake from bulk glasses. Additionally, we detected a burst release of Mg2+ from composites only after 24 h of incubation. Furthermore, we demonstrated that released ions and the mesoporous channel structure affect the measurement of lactate dehydrogenase (LDH) and alkaline phosphatase activity (ALP) in bioprinted composite scaffolds. Measured LDH activity was significantly decreased in the presence of CaMBG. On the other hand, the presence of MgMBG induced increased signal measured for the ALP. Taken together, our findings show how composite bioinks containing MBGs can interfere with common analyses, obtaining misleading results.

scholarly journals Analysis Of Candidate Genes Expected To Be Essential For Melanoma Surviving

2020 ◽  
Author(s):  
Irina Krivosheeva ◽  
Alexandra Filatova ◽  
Sergei Moshkovskii ◽  
Ancha Baranova ◽  
Mikhail Skoblov
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Computational Models ◽  
Target Genes ◽  
The Other ◽  
Other Hand ◽  
Before And After

Abstract Cancers may be treated by selective targeting of the genes vital for their survival. A number of attempts have led to discovery of several genes essential for surviving of tumor cells of different types. In this work, we tried to analyze genes that were previously predicted to be essential for melanoma surviving. Here we present the results of transient siRNA-mediated knockdown of the four of such genes, namely, UNC45A, STK11IP, RHPN2 and ZNFX1, in melanoma cell line A375, then assayed the cells for their viability, proliferation and ability to migrate in vitro. In our study, the knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. Possible explanation for such counterintuitive results may include insufficiency of the predicting computational models or the necessity of a multiplex knockdown of the genes.AimsTo examine the hypothesis of essentiality of hypomutated genes for melanoma surviving we have performed knockdown of several genes in melanoma cell line and analyzed cell viability and their ability to migrate.MethodsKnockdown was performed by siRNAs transfected by Metafectene PRO. The levels of mRNAs before and after knockdown were evaluated by RT-qPCR analysis. Cell viability and proliferation were assessed by MTT assay. Cell migration was assessed by wound healing assay.ResultsThe knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. ConclusionOur results do not confirm initial hypothesis that the genes predicted essential for melanoma survival as a matter of fact support the survival of melanoma cells.

scholarly journals Rescue and Conservation of Male Adult Alpacas (Vicugna pacos) Based on Spermatogonial Stem Cell Biotechnology Using Atomized Black Maca as a Supplement of Cryopreservation Medium

2021 ◽  
Vol 8 ◽  
Author(s):  
Martha Valdivia ◽  
Zezé Bravo ◽  
Jhakelin Reyes ◽  
Gustavo Gonzales
Keyword(s):  
Cell Viability ◽  
Antioxidant Properties ◽  
Testicular Tissue ◽  
The Other ◽  
Mitochondrial Activity ◽  
Isolated Cells ◽  
Male Adult ◽  
Testicular Cells ◽  
Other Hand

This is the first time that testicular tissue (n = 44) and isolated testicular cells (n = 51) were cryopreserved from alpaca testes 24 h postmortem. For this purpose, internally designed freezing media and cryopreservation protocols were used. Testicular tissue fragments (25 mg) and isolated testicular cells were frozen in MTDB (trehalose and black maca), MTD (trehalose), MSDB (sucrose and black maca), and MSD (sucrose) media. Isolated spermatogonial cells were cryopreserved in two ways, before and after proliferation in vitro. After cryopreservation, the percentage of cell viability in Group 1 (>50% of cell viability) by trypan blue did not show differences within each group (p > 0.05) but showed significant differences when comparing fragments with isolated cells (p < 0.05). Spermatogonial stem cells (SSC) were identified by flow cytometry as strong Dolichos biflorus agglutinin (sDBA) and mitochondrial activity of SSC as strongly positive for MitoSense (sMitoSense+) in intact mitochondria cells, weakly positive for MitoSense (wMitoSense+) in early apoptosis, and necrosis with 7-Aminoactinomycin-D positive (7-AAD). After freezing, in Group 1M (≥30% sMitoSense+), the fragments did not show differences between the media (p > 0.05), but in the isolated cells frozen in MSDB medium, 63.68 ± 8.90% (p < 0.05). In Group 2M (<30% sMitoSense+), necrosis (7AAD+) in MSDB medium was 27.03 ± 5.80%, and necrosis in isolated cells was 14.05 ± 9.3% with significant differences between these groups (p < 0.05); in sMitoSense+, the isolated cells (34.40 ± 23%) had a higher percentage than the fragments (12.4 ± 5.2) (p < 0.05). On the other hand, MSDB and MSD media were significantly higher for isolated cells than for fragments in sDBA+ (p < 0.05). On the other hand, the SSC (sDBA+) had significant differences (p < 0.05) between fresh cells 7.43 ± 1.3% (sDBA+) compared with those cryopreserved in MSDB medium 1.46 ± 0.34% (sDBA+). Additionally, the proliferated and cryopreserved SSC 6.29 ± 1.17% (sDBA+) did not show significant differences concerning the fresh cells (p > 0.05). In conclusion, the black maca showed antioxidant properties when it was included in the freezing medium and, therefore, improved the SSC's conservation of the alpaca. Furthermore, the proliferation of isolated cells in vitro produces a higher amount of SSC after thawing them for further preclinical or clinical work.

scholarly journals Analysis of candidate genes expected to be essential for melanoma surviving

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Irina A. Krivosheeva ◽  
Alexandra Yu. Filatova ◽  
Sergei A. Moshkovskii ◽  
Ancha V. Baranova ◽  
Mikhail Yu. Skoblov
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Computational Models ◽  
Target Genes ◽  
The Other ◽  
Other Hand ◽  
Before And After

Abstract Introduction Cancers may be treated by selective targeting of the genes vital for their survival. A number of attempts have led to discovery of several genes essential for surviving of tumor cells of different types. In this work, we tried to analyze genes that were previously predicted to be essential for melanoma surviving. Here we present the results of transient siRNA-mediated knockdown of the four of such genes, namely, UNC45A, STK11IP, RHPN2 and ZNFX1, in melanoma cell line A375, then assayed the cells for their viability, proliferation and ability to migrate in vitro. In our study, the knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. Possible explanation for such counterintuitive results may include insufficiency of the predicting computational models or the necessity of a multiplex knockdown of the genes. Aims To examine the hypothesis of essentiality of hypomutated genes for melanoma surviving we have performed knockdown of several genes in melanoma cell line and analyzed cell viability and their ability to migrate. Methods Knockdown was performed by siRNAs transfected by Metafectene PRO. The levels of mRNAs before and after knockdown were evaluated by RT-qPCR analysis. Cell viability and proliferation were assessed by MTT assay. Cell migration was assessed by wound healing assay. Results The knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. Conclusion Our results do not confirm initial hypothesis that the genes predicted essential for melanoma survival as a matter of fact support the survival of melanoma cells.

EFFECT OF ACTINOMYCIN D ON TSH-INDUCED STIMULATION OF THYROIDAL PROTEIN SYNTHESIS IN THE RAT

1974 ◽  
Vol 77 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Gustav Wägar
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
The Other ◽  
Leucine Incorporation ◽  
Short Term ◽  
Other Hand ◽  
Thyroid Glands ◽  
Translational Level ◽  
Stimulation Of

ABSTRACT Whether the short-term regulation of thyroidal protein synthesis by TSH occurs at the transcriptional or the translational level was tested by measuring the effect of actinomycin D (act D) on the TSH-induced stimulation of L-14C-leucine incorporation into the thyroidal proteins of rats. TSH was injected 6 h before the rats were killed. The thyroid glands were then removed and incubated in vitro in the presence of L-14C-leucine for 2 h. The pronounced stimulation of leucine incorporation in the TSH-treated animals was depressed as compared with controls but still significant even when the animals had been pre-treated with 100 μg act D 24 and 7 h before sacrifice. On the other hand, act D strongly decreased incorporation of 3H-uridine into RNA. Short-term regulation of thyroidal protein synthesis by TSH appears to be partly but not wholly dependent on neosynthesis of RNA. Hence regulation may partly occur at the translation level of protein synthesis.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

1997 ◽  
Vol 62 (11) ◽  
pp. 1804-1814 ◽  
Author(s):  
Marie Stiborová ◽  
Hana Hansíková
Keyword(s):  
Cytochromes P450 ◽  
Kinetic Studies ◽  
The Other ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Other Hand ◽  
Plant Peroxidases ◽  
Tulip Bulbs

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

scholarly journals Fetal erythropoiesis in steel mutant mice. III. Defect in differentiation from BFU-E to CFU-E during early development

Blood ◽  
1978 ◽  
Vol 51 (3) ◽  
pp. 539-547 ◽  
Author(s):  
DH Chui ◽  
SK Liao ◽  
K Walker
Keyword(s):  
Progenitor Cells ◽  
Early Development ◽  
The Other ◽  
Mutant Mice ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Other Hand ◽  
Colony Forming Units ◽  
Fetal Erythropoiesis

Abstract Erythroid progenitor cells in +/+ and Sl/Sld fetal livers manifested as burst-forming units-erythroid (BFU-E) and colony-forming units- erythroid (CFU-E) were assayed in vitro during early development. The proportion of BFU-E was higher as mutant than in normal fetal livers. On the other hand, the proportion of CFU-E was less in the mutant than in the normal. These results suggest that the defect in Sl/Sld fetal hepatic erythropoiesis is expressed at the steps of differentiation that effect the transition from BFU-E to CFU-E.

scholarly journals THE PATHOLOGIC EFFECTS OF STREPTOCOCCI FROM CASES OF POLIOMYELITIS AND OTHER SOURCES

1917 ◽  
Vol 25 (4) ◽  
pp. 557-580 ◽  
Author(s):  
Carroll G. Bull
Keyword(s):  
Spinal Cord ◽  
Guinea Pigs ◽  
The Other ◽  
Laboratory Animals ◽  
Histological Changes ◽  
Other Hand ◽  
Brain And Spinal Cord ◽  
The Brain

Streptococci cultivated from the tonsils of thirty-two cases of poliomyelitis were used to inoculate various laboratory animals. In no case was a condition induced resembling poliomyelitis clinically or pathologically in guinea pigs, dogs, cats, rabbits, or monkeys. On the other hand, a considerable percentage of the rabbits and a smaller percentage of some of the other animals developed lesions due to streptococci. These lesions consisted of meningitis, meningo-encephalitis, abscess of the brain, arthritis, tenosynovitis, myositis, abscess of the kidney, endocarditis, pericarditis, and neuritis. No distinction in the character or frequency of the lesions could be determined between the streptococci derived from poliomyelitic patients and from other sources. Streptococci isolated from the poliomyelitic brain and spinal cord of monkeys which succumbed to inoculation with the filtered virus failed to induce in monkeys any paralysis or the characteristic histological changes of poliomyelitis. These streptococci are regarded as secondary bacterial invaders of the nervous organs. Monkeys which have recovered from infection with streptococci derived from cases of poliomyelitis are not protected from infection with the filtered virus, and their blood does not neutralize the filtered virus in vitro. We have failed to detect any etiologic or pathologic relationship between streptococci and epidemic poliomyelitis in man or true experimental poliomyelitis in the monkey.

In vitro insulin-mimetic activity and in vivo metallokinetic feature of oxovanadium(IV)porphyrin complexes in healthy rats

2012 ◽  
Vol 16 (01) ◽  
pp. 114-121 ◽  
Author(s):  
Tapan K. Saha ◽  
Yutaka Yoshikawa ◽  
Hirouki Yasui ◽  
Hiromu Sakurai
Keyword(s):  
The Other ◽  
Insulin Mimetic ◽  
Other Hand ◽  
Better Than

We prepared [meso-tetrakis(4-carboxylatophenyl)porphyrinato]oxovanadium(IV) tetrasodium, ([VO(tcpp)]Na4), and investigated its in vitro insulin-mimetic activity and in vivo metallokinetic feature in healthy rats. The results were compared with those of previously proposed insulin-mimetic oxovanadium(IV)porphyrin complexes and oxovanadium(IV) sulphate. The in vitro insulin-mimetic activity and bioavailability of [VO(tcpp)]Na4 were considerably better than those of [meso-tetrakis (1-methylpyridinium-4-yl)porphyrinato]oxovanadium(IV)(4+) tetraperchlorate ([VO(tmpyp)](ClO4)4) and oxovanadium(IV) sulphate. On the other hand, [VO(tcpp)]Na4 and [meso-tetrakis(4-sulfonatophenyl) porphyrinato]oxidovanadate(IV)(4-)([VO(tpps)]) showed very similar in vitro insulin-mimetic activity and in vivo metallokinetic feature in healthy rats. In particular, the order of in vitro insulin-mimetic activity of the complexes was determined to be: [VO(tcpp)]Na4 ≈ [VO(tpps)] > ([VO(tmpyp)](ClO4)4 > oxovanadium(IV) sulphate.

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