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2023 ◽  
Vol 83 ◽  
V. A. Nascimento ◽  
S. M. Malmonge ◽  
A. R. Santos Jr.

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

2023 ◽  
Vol 83 ◽  
L. O. Correa ◽  
A. F. M. Bezerra ◽  
L. R. S. Honorato ◽  
A. C. A. Cortez ◽  
J. V. B. Souza ◽  

Abstract Pesticide residues that contaminate the environment circulate within the hydrological cycle can accumulate within the food chain and cause problems to both environmental and human health. Microbes, however, are well known for their metabolic versatility and the ability to degrade chemically stable substances, including recalcitrant xenobiotics. The current study focused on bio-prospecting within Amazonian rainforest soils to find novel strains fungi capable of efficiently degrading the agriculturally and environmentally ubiquitous herbicide, glyphosate. Of 50 fungal strains isolated (using culture media supplemented with glyphosate as the sole carbon-substrate), the majority were Penicillium strains (60%) and the others were Aspergillus and Trichoderma strains (26 and 8%, respectively). All 50 fungal isolates could use glyphosate as a phosphorous source. Eight of these isolates grew better on glyphosate-supplemented media than on regular Czapek Dox medium. LC-MS revealed that glyphosate degradation by Penicillium 4A21 resulted in sarcosine and aminomethylphosphonic acid.

2024 ◽  
Vol 84 ◽  
M. C. S. Virtuoso ◽  
E. H. C. Silva ◽  
E. M. Silva ◽  
T. S. Valente ◽  
P. F. Vargas ◽  

Abstract The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus’ survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores’ size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

2024 ◽  
Vol 84 ◽  
A. Azam ◽  
R. Ejaz ◽  
S. Qadeer ◽  
S. Irum ◽  
A. Ul-Husna ◽  

Abstract The objective of the current study was to investigate the synergistic impact of α-Tocopherol and α-Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50μM α-Tocopherol, GROUP 3: MM + 100 µM ALA + 100μM α-Tocopherol, GROUP 4: MM + 100 µM ALA + 200 μM α-Tocopherol and GROUP 5: MM + 100 µM ALA + 300 μM α-Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of α-Linolenic acid (100 µM) and α-Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of α-Linolenic acid (100 µM) in IVM media and increasing concentration of α-tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of α-tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of α-tocopherol in maturation media having α-Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

2022 ◽  
Vol 43 (2) ◽  
pp. 739-750
Patricia Rodrigues Condé ◽  
Cláudia Lúcia de Oliveira Pinto ◽  
Scarlet Ohana Gandra ◽  
Renata Cristina Almeida Bianchini Campos ◽  

This work aimed to characterize, identify, and determine the deteriorating potential of the contaminating psychrotrophic bacteria in refrigerated raw milk. Samples were submitted to serial dilutions and plated in specific culture media to form a bacterial culture collection. The isolates were characterized for their morphology and biochemical characteristics. The deteriorating potential of the isolates was determined according to the proteolytic, lipolytic and lecithinase activities at 4.0 ºC, 6.5 ºC, and 25.0 ºC. The results obtained for deterioration potential were assessed by the multivariate statistical method and by the principal components analysis (PCA). A total of 159 isolates were characterized, and of these, 46 strongly proteolytic Gram-negative isolates were selected for identification using the API 20 NE kit. The predominant bacteria were Gram-negative and oxidase and catalase positive, with a predominance of bacteria of the genus Pseudomonas. Using PCA, it was shown that the bacteria with the greatest deterioration potential were lecithinase producers, and that, in the autumn, proteolytic bacteria predominated at 4.0 ºC. Of the 46 isolates identified, more than 80% belonged to the species Pseudomonas fluorescens. Thus, attention should be given to the importance of implementing microbial contamination prevention measures in the bulking process, since, even under refrigeration, psychrotrophic bacteria multiply and produce enzymes that deteriorate lipids and proteins, with consequent quality losses of the milk and its derivatives, yield losses in the production of dairy products, and economic losses.

2022 ◽  
Vol 8 ◽  
Jamie L. Stewart ◽  
Liying Gao ◽  
Jodi A. Flaws ◽  
Vitor R. G. Mercadante ◽  
Nicholas W. Dias ◽  

Nerve growth factor-β (NGF) is critical for ovulation in the mammalian ovary and is luteotrophic when administered systemically to camelids and cattle. This study aimed to assess the direct effects of purified bovine NGF on steroidogenesis and angiogenic markers in the bovine pre-ovulatory follicle. Holstein heifers (n = 2) were synchronized with a standard protocol, and heifers with a preovulatory follicle (≥ 12 mm) had the ovary containing the dominant follicle removed via colpotomy. Pre-ovulatory follicles were dissected into 24 pieces containing theca and granulosa cells that were randomly allocated into culture media supplemented with either purified bovine NGF (100 ng/mL) or untreated (control) for 72 h. The supernatant media was harvested for quantification of progesterone, testosterone, and estradiol concentrations, whereas explants were subjected to mRNA analyses to assess expression of steroidogenic and angiogenic markers. Treatment of follicle wall pieces with NGF upregulated gene expression of steroidogenic enzyme HDS17B (P = 0.04) and increased testosterone production (P < 0.01). However, NGF treatment did not alter production of progesterone (P = 0.81) or estradiol (P = 0.14). Consistently, gene expression of steroidogenic enzymes responsible for producing these hormones (STAR, CYP11A1, HSD3B, CYP17A1, CYP19A1) were unaffected by NGF treatment (P ≥ 0.31). Treatment with NGF downregulated gene expression of the angiogenic enzyme FGF2 (P = 0.02) but did not alter PGES (P = 0.63), VEGFA (P = 0.44), and ESR1 (P = 0.77). Collectively, these results demonstrate that NGF from seminal plasma may interact directly on the theca and granulosa cells of the bovine pre-ovulatory follicle to stimulate testosterone production, which may be secondary to theca cell proliferation. Additionally, decreased FGF2 expression in NGF-treated follicle wall cells suggests hastened onset of follicle wall cellular remodeling that occurs during early luteal development.

Science ◽  
2022 ◽  
Vol 375 (6577) ◽  
pp. 143-144
Jan van der Valk

Ethical and possible reproducibility issues arise when using fetal bovine serum in cell culture media

Plants ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 198
Dragana Stojičić ◽  
Svetlana Tošić ◽  
Gordana Stojanović ◽  
Bojan Zlatković ◽  
Snežana Jovanović ◽  

Clinopodium pulegium (Rochel) Bräuchler (Lamiaceae) is an endangered species endemic to the Southern Carpathians. It is characterized by the production of high amounts of essential oils, which emit volatile organic compounds (VOCs) that have an essential role in biotic and abiotic stress responses and in plant–plant and plant–insect interactions. The present study was initiated to phytochemically examine the influence of different carbon sources in the nutrition medium on VOC emissions of micropropagated C. pulegium plants, using gas chromatography–mass spectrometry analysis of headspace VOCs. The volatile profiles were subjected to multivariate analysis with respect to the presence, concentration and type of carbon source in the nutrient medium. In addition, the effect of different carbohydrates on the density and size of the leaf glandular trichomes, the main structures involved in the emission of VOCs, was determined. A total of 19 VOCs, primarily belonging to mono- and sesquiterpenes previously described in plants, were tentatively identified. Six VOCs were produced at levels higher than 2% of the total VOC emission, dominated by pulegone, ß-pinene and menthone. Inclusion of the carbohydrates in the culture media affected the production of the main leaf trichome-associated volatile allelochemicals although the qualitative composition of the volatiles changed only slightly. Multivariate analysis showed that the concentration, rather than the carbohydrate type, influenced the VOC profile.

2022 ◽  
Filipe Magnum Silva Dos Santos ◽  
Kah Hin Low ◽  
Lay Ching Chai

Abstract Bacteria emits a multitude of volatile organic compounds (VOCs) into the headspace as a mean of interactions with the environments, as well as intra- and interkingdom communication for survival and persistence in the nature and within their hosts. Campylobacter, which is often found in poultry and ruminants, has shown great persistence in aquatic environments, making it one of the world's most dangerous foodborne pathogens, killing thousands of people annually. In this study, the VOCs emitted by both thermophilic (C. jejuni, C. coli and C. lari) and non-thermophilic Campylobacter (C. fetus) of clinical concerns, impacted by nutrients composition (media) and growth phase were identified. Most thermophilic Campylobacter were shown to release volatile alcohols and ketones (1s,4R,7R,11R-1,3,4,7-Tetramethyltricyclo [,11)] undec-2-en-8-one and Isophorone) during early stationary and stationary phases using active sampling with active charcoal adsorbent and GC-MS analysis. C. jejuni cultured in the Brain Heart Infusion had 1-Heptadecanol in its headspace gas, but not in Bolton Broth. The non-thermophilic C. fetus did not produce alcohols or ketones, but rather a variety of unidentified chemicals that will require further investigation in the future. Overall, PCA analysis revealed that the five Campylobacter strains studied created distinct volatilomes, allowing for future Campylobacter identification based on VOCs.

2022 ◽  
Kelly T. Rios ◽  
Taylor M. Dickson ◽  
Scott E Lindner

Some early antimalarial drugs have been repurposed for experimental applications, thus extending their utility well beyond the point when resistance becomes prevalent in circulating parasite populations. One such drug is sulfadiazine, which is an analog of p-aminobenzoic acid (pABA), and acts as a competitive inhibitor of dihydropteroate synthase, which is an essential enzyme in the parasite's folate synthesis pathway that is required for DNA synthesis. Sulfadiazine treatment of mice infected with P. yoelii and P. berghei is routinely used to enrich for gametocytes by killing asexual blood stage parasites, but it is not well known if the exposed gametocytes are perturbed or if there is a detrimental effect on transmission. To determine if there was a significant effect of sulfadiazine exposure upon host-to-vector transmission, we transmitted Plasmodium yoelii (17XNL strain) parasites to Anopheles stephensi mosquitoes and evaluated the prevalence of infection (percent of mosquitoes infected) and intensity of infection (number of oocysts per infected mosquito) under different sulfadiazine treatment conditions of the mouse or of the mosquitoes. We observed that parasites exposed to sulfadiazine either in the mouse host or in the mosquito vector had a reduction in both the number of mosquitoes that became infected and in the intensity of infection compared to untreated controls. We also observed that provision of freshly prepared pABA in the mosquito sugar water could only marginally overcome the defects caused by sulfadiazine treatment. In contrast, we determined that gametocytes exposed to sulfadiazine were able to be fertilized and develop into morphologically mature ookinetes in vitro, and thus that sulfadiazine exposure in the host may be reversible if the drug is washed out and the parasites are supplemented with pABA in the culture media. Overall, this indicates that sulfadiazine dampens host-to-vector transmission, and that this inhibition can only be partially overcome by exposure to fresh pABA in vivo and in vitro. Because gametocytes are of great interest for developing transmission blocking interventions, we recommend that less disruptive approaches for gametocyte enrichment be used in order to study minimally perturbed parasites.

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