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2.
BMC Plant Biol ; 21(1): 403, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488630

RESUMO

BACKGROUND: Winter freezing temperature impacts alfalfa (Medicago sativa L.) persistence and seasonal yield and can lead to the death of the plant. Understanding the genetic mechanisms of alfalfa freezing tolerance (FT) using high-throughput phenotyping and genotyping is crucial to select suitable germplasm and develop winter-hardy cultivars. Several clones of an alfalfa F1 mapping population (3010 x CW 1010) were tested for FT using a cold chamber. The population was genotyped with SNP markers identified using genotyping-by-sequencing (GBS) and the quantitative trait loci (QTL) associated with FT were mapped on the parent-specific linkage maps. The ultimate goal is to develop non-dormant and winter-hardy alfalfa cultivars that can produce extended growth in the areas where winters are often mild. RESULTS: Alfalfa FT screening method optimized in this experiment comprises three major steps: clone preparation, acclimation, and freezing test. Twenty clones of each genotype were tested, where 10 samples were treated with freezing temperature, and 10 were used as controls. A moderate positive correlation (r ~ 0.36, P < 0.01) was observed between indoor FT and field-based winter hardiness (WH), suggesting that the indoor FT test is a useful indirect selection method for winter hardiness of alfalfa germplasm. We detected a total of 20 QTL associated with four traits; nine for visual rating-based FT, five for percentage survival (PS), four for treated to control regrowth ratio (RR), and two for treated to control biomass ratio (BR). Some QTL positions overlapped with WH QTL reported previously, suggesting a genetic relationship between FT and WH. Some favorable QTL from the winter-hardy parent (3010) were from the potential genic region for a cold tolerance gene CBF. The BLAST alignment of a CBF sequence of M. truncatula, a close relative of alfalfa, against the alfalfa reference showed that the gene's ortholog resides around 75 Mb on chromosome 6. CONCLUSIONS: The indoor freezing tolerance selection method reported is useful for alfalfa breeders to accelerate breeding cycles through indirect selection. The QTL and associated markers add to the genomic resources for the research community and can be used in marker-assisted selection (MAS) for alfalfa cold tolerance improvement.


Assuntos
Mapeamento Cromossômico , Congelamento , Regulação da Expressão Gênica de Plantas/fisiologia , Medicago sativa/metabolismo , Locos de Características Quantitativas , Adaptação Fisiológica/genética , Cromossomos de Plantas/genética , Genótipo , Medicago sativa/genética , Fenótipo , Melhoramento Vegetal
3.
BMC Plant Biol ; 21(1): 377, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399687

RESUMO

BACKGROUND: C. panzhihuaensis is more tolerant to freezing than C. bifida but the mechanisms underlying the different freezing tolerance are unclear. Photosynthesis is one of the most temperature-sensitive processes. Lipids play important roles in membrane structure, signal transduction and energy storage, which are closely related to the stress responses of plants. In this study, the chlorophyll fluorescence parameters and lipid profiles of the two species were characterized to explore the changes in photosynthetic activity and lipid metabolism following low-temperature exposure and subsequent recovery. RESULTS: Photosynthetic activity significantly decreased in C. bifida with the decrease of temperatures and reached zero after recovery. Photosynthetic activity, however, was little affected in C. panzhihuaensis. The lipid composition of C. bifida was more affected by cold and freezing treatments than C. panzhihuaensis. Compared with the control, the proportions of all the lipid categories recovered to the original level in C. panzhihuaensis, but the proportions of most lipid categories changed significantly in C. bifida after 3 d of recovery. In particular, the glycerophospholipids and prenol lipids degraded severely during the recovery period of C. bifida. Changes in acyl chain length and double bond index (DBI) occurred in more lipid classes immediately after low-temperature exposure in C. panzhihuaensis compare with those in C. bifida. DBI of the total main membrane lipids of C. panzhihuaensis was significantly higher than that of C. bifida following all temperature treatments. CONCLUSIONS: The results of chlorophyll fluorescence parameters confirmed that the freezing tolerance of C. panzhihuaensis was greater than that of C. bifida. The lipid metabolism of the two species had differential responses to low temperatures. The homeostasis and plastic adjustment of lipid metabolism and the higher level of DBI of the main membrane lipids may contribute to the greater tolerance of C. panzhihuaensis to low temperatures.


Assuntos
Aclimatação , Cycas/fisiologia , Lipídeos de Membrana/metabolismo , China , Clorofila/metabolismo , Cycas/metabolismo , Congelamento , Glicerofosfatos/metabolismo , Homeostase , Especificidade da Espécie , Temperatura
4.
Theriogenology ; 173: 279-294, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34411905

RESUMO

In situations where an excessive generation of reactive oxygen species overwhelms antioxidant capacity, a harmful effect on sperm function is exerted. Antioxidants are molecules capable of minimizing this detrimental effect, which is important in pig sperm due to the high content of polyunsaturated fatty acids in their plasma membrane. The present systematic review aims at evaluating whether supplementing semen extenders (for liquid storage at 17 °C) or freezing and/or thawing media (for cryopreservation) with antioxidants influences sperm quality and functionality parameters, and in vitro/in vivo fertility outcomes. We defined inclusion and exclusion criteria in a PICOS table according to PRISMA guidelines, and conducted a literature search through MEDLINE-PubMed in November 2020. After systematic selection, 75 studies were included: 47 focused on cryopreservation and 28 on liquid storage at 17 °C. More than 70% of the studies included in this review showed that adding semen extenders for liquid storage and/or freezing/thawing media for cryopreservation with antioxidants enhances sperm quality and functionality parameters. In addition, this supplementation improves in vivo/in vitro fertility outcomes, supporting the hypothesis that the beneficial effect observed upon sperm quality has a positive impact on reproduction outcomes.


Assuntos
Antioxidantes , Preservação do Sêmen , Animais , Criopreservação/veterinária , Crioprotetores/farmacologia , Congelamento , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , Suínos
5.
BMC Plant Biol ; 21(1): 365, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380415

RESUMO

BACKGROUND: Kiwifruit (Actinidia Lindl.) is considered an important fruit species worldwide. Due to its temperate origin, this species is highly vulnerable to freezing injury while under low-temperature stress. To obtain further knowledge of the mechanism underlying freezing tolerance, we carried out a hybrid transcriptome analysis of two A. arguta (Actinidi arguta) genotypes, KL and RB, whose freezing tolerance is high and low, respectively. Both genotypes were subjected to - 25 °C for 0 h, 1 h, and 4 h. RESULTS: SMRT (single-molecule real-time) RNA-seq data were assembled using the de novo method, producing 24,306 unigenes with an N50 value of 1834 bp. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that they were involved in the 'starch and sucrose metabolism', the 'mitogen-activated protein kinase (MAPK) signaling pathway', the 'phosphatidylinositol signaling system', the 'inositol phosphate metabolism', and the 'plant hormone signal transduction'. In particular, for 'starch and sucrose metabolism', we identified 3 key genes involved in cellulose degradation, trehalose synthesis, and starch degradation processes. Moreover, the activities of beta-GC (beta-glucosidase), TPS (trehalose-6-phosphate synthase), and BAM (beta-amylase), encoded by the abovementioned 3 key genes, were enhanced by cold stress. Three transcription factors (TFs) belonging to the AP2/ERF, bHLH (basic helix-loop-helix), and MYB families were involved in the low-temperature response. Furthermore, weighted gene coexpression network analysis (WGCNA) indicated that beta-GC, TPS5, and BAM3.1 were the key genes involved in the cold response and were highly coexpressed together with the CBF3, MYC2, and MYB44 genes. CONCLUSIONS: Cold stress led various changes in kiwifruit, the 'phosphatidylinositol signaling system', 'inositol phosphate metabolism', 'MAPK signaling pathway', 'plant hormone signal transduction', and 'starch and sucrose metabolism' processes were significantly affected by low temperature. Moreover, starch and sucrose metabolism may be the key pathway for tolerant kiwifruit to resist low temperature damages. These results increase our understanding of the complex mechanisms involved in the freezing tolerance of kiwifruit under cold stress and reveal a series of candidate genes for use in breeding new cultivars with enhanced freezing tolerance.


Assuntos
Aclimatação/genética , Actinidia/genética , Actinidia/fisiologia , Congelamento , Regulação da Expressão Gênica de Plantas , Frutas/genética , Frutas/fisiologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Sistema de Sinalização das MAP Quinases , Anotação de Sequência Molecular , Fosfatidilinositóis/metabolismo , Melhoramento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Amido/metabolismo , Sacarose/metabolismo
6.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443445

RESUMO

Saponin is a biopesticide used to suppress the growth of the golden apple snail population. This study aims to determine the stabilized conditions for saponin storage. The maceration process was used for saponin extraction, and for saponin concentration, progressive freeze concentration (PFC) was used. Afterwards, stability analysis was performed by storing the sample for 21 days in two conditions: Room temperature (26 °C) and cold room (10 °C). The samples kept in a cold room were sterilized samples that undergo thermal treatment by placing the sample in the water bath. The non-sterilized samples were kept in room temperature condition for 21 days. The results showed that saponin stored in the cold room (sterilized sample) has low degradation with higher concentration than those stored at room temperature in stability analysis with the highest saponin concentration (0.730 mg/mL) at a concentration temperature of -6 °C and concentration time of 15 min. The lowest saponin concentration obtained by saponin stored at room temperature (non-sterilized sample) is 0.025 mg/mL at a concentration temperature of -6 °C and concentration time of 10 min. Thus, the finding concluded that saponin is sensitive to temperature. Hence, the best storage condition to store saponin after thermal treatment is to keep it in a cold room at 10 °C.


Assuntos
Congelamento , Saponinas/química , Esterilização , Estabilidade de Medicamentos , Fatores de Tempo
7.
Nat Biomed Eng ; 5(8): 793-804, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426675

RESUMO

The three classical core technologies for the preservation of live mammalian biospecimens-slow freezing, vitrification and hypothermic storage-limit the biomedical applications of biospecimens. In this Review, we summarize the principles and procedures of these three technologies, highlight how their limitations are being addressed via the combination of microfabrication and nanofabrication, materials science and thermal-fluid engineering and discuss the remaining challenges.


Assuntos
Manejo de Espécimes/métodos , Preservação de Tecido/métodos , Animais , Congelamento , Humanos , Hidrogéis/química , Magnetismo , Nanotecnologia , Temperatura , Vitrificação
8.
Sci Total Environ ; 795: 148894, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34252772

RESUMO

Understanding the migration of engineered nanoparticles (ENPs) in soil is of great significance for evaluating the potential risks of ENPs to ecosystem. So far, their migration under freeze-thaw cycles (FTCs) has not been investigated. This study explored the impacts of FTCs on the migration of three commonly used ENPs, copper oxide (CuO-NPs), cerium oxide (CeO2-NPs), and zinc oxide (ZnO-NPs), in three types of soil. After 32 FTC cycles, the highest migration rate of ENPs was found in black soil due to its higher clay particle content. CeO2-NPs with low surface charge exhibited the highest mobility among three ENPs, which migrated to 9-11 cm layer with the concentration of 42.1 mg/kg in the black soil column. ZnO-NPs were less influenced by FTCs as they were adsorbed onto sand grains due to electrostatic interaction, which migrated to 3-5 cm layer with the concentration of 25.2 mg/kg in the black soil. Higher moisture contents (50% and 100%) resulted in increased migration depth of the ENPs in all soils. Lower freezing temperature (-25 °C) caused fragmentation of large soil particles and produced more clay colloids. FTCs promoted the movement of moisture, which penetrated the soil and thus facilitated the movement of ENPs by increasing the contents and movement of clay colloids. This work reveals the migration behavior of ENPs in soils in freeze-thaw period and provides insights into the fate and environmental risk of nanomaterial at middle and high latitudes.


Assuntos
Nanopartículas Metálicas , Nanopartículas , Ecossistema , Congelamento , Óxidos , Solo
9.
Ultrason Sonochem ; 76: 105657, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34229120

RESUMO

This research evaluated the effects of multi-frequency ultrasound assisted freezing (UAF) on the freezing rate, structural characteristics, and quality properties of cultured large yellow croaker. The freezing effects with triple ultrasound-assisted freezing (TUF) at 20, 28 and 40 kHz under 175 W was more obvious than that of single ultrasound-assisted freezing (SUF) at 20 kHz and dual ultrasound-assisted freezing (DUF) at 20 and 28 kHz. The results showed that UAF significantly increased the freezing rate and better preserved the quality of frozen large yellow croaker samples. Specifically, the quality parameters of the TUF-treated samples were closer to those of the fresh samples, with greater texture characteristics, a larger water holding capacity (lower thawing loss and cooking loss), lower K values and lower thiobarbituric acid reactive substances values. Light microscopy observation images revealed that the ice crystals formed by TUF were fine and evenly distributed, resulting in less damage to the frozen large yellow croaker samples. Therefore, multi-frequency UAF could improve the quality properties of the large yellow croaker samples.


Assuntos
Qualidade dos Alimentos , Congelamento , Perciformes , Ondas Ultrassônicas , Animais
10.
Spectrochim Acta A Mol Biomol Spectrosc ; 263: 120203, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34325172

RESUMO

In this paper, CdSe/ZnS quantum dots (QDs) with particle size of 5.5 ~ 9.3 nm were synthesized, and the fluorescence emission ranged from 545 ~ 616 nm. When the volume fraction of ethanol was 30%, the water-soluble QD dispersion system remained liquid under -20 °C freezing conditions, the fluorescence intensity increased with a decrease in temperature, and the quantum yield reached 79% at -20 °C. The endothelial cell adhesion molecule CD31 antibody (anti-CD31) was used as the primary antibody, QDs were coupled with IgG as the secondary antibody (QD-Ab), and effective labeling of hepatic sinusoid endothelial cells was achieved at -20 °C. Fluorescence imaging and flow cytometry analysis showed that the labeling efficiency was as high as 97%, indicating that QDs have an important application prospect in microscopic section tomography of the liver.


Assuntos
Compostos de Cádmio , Pontos Quânticos , Compostos de Selênio , Células Endoteliais , Fluorescência , Corantes Fluorescentes , Congelamento , Fígado , Sulfetos , Compostos de Zinco
11.
Molecules ; 26(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207540

RESUMO

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen-thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen-thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing-thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen-thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.


Assuntos
Fígado Gorduroso/metabolismo , Produtos Avícolas/análise , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Patos , Fígado Gorduroso/classificação , Congelamento , Análise de Componente Principal , Proteoma/análise , Controle de Qualidade
12.
J Agric Food Chem ; 69(29): 8213-8226, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34264653

RESUMO

The study investigated the main characteristic micromolecular changes in tilapia fillets after partial freezing treatment with polyphenols by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. A total of 2121 metabolite ion features were identified. The result suggested that procyanidin treatment increased the sweet, salty, and thick peptides' contents and suppressed the formation of bitter peptides. The levels of cis-4-octenedioic acid, 2-amino-heptanoic acid, indoleacrylic acid, and 2-amino-3-methyl-1-butanol in polyphenol treatments were significantly lower compared to those in the control group (P < 0.05), which delayed the formation of micromolecule of acids and alcohols associated with spoilage and inhibited the protein and lipid oxidation and degradation. Polyphenol treatments suppressed the formation of biogenic amines (lower levels of spermidine and 1-naphthylacetylspermine) and reduced fillet quality deterioration. It provided critical novel insights into the understanding of the molecular mechanism for inhibiting the quality deterioration of fillets treated with polyphenols during storage.


Assuntos
Tilápia , Animais , Aminas Biogênicas , Congelamento , Oxirredução , Polifenóis
13.
Poult Sci ; 100(8): 101091, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34225205

RESUMO

The aim is to optimize the dimethylacetamide (DMA) straw freezing technology of Black silkies rooster semen through the handy patent equipment, screening the formula of freezing basic extender and optimizing the DMA addition method, and then by comparing the fertility of DMA straw frozen semen with the pellet frozen semen. After the DMA straw freezing technology is optimized, it is extended to the Youxian Partridge drake semen. The result showed that the frozen sperm motility of Lake and Ravie (LR) group is 64%, the fertility 49.57% and the hatchability 91.52%, all of which are superior to those of FEB, Beltsville Poultry Semen Extender (BPSE) and Lake (P < 0.05). The sperm motility of adding DMA stock solution is 59%, which is superior to adding DMA directly into diluted semen (P > 0.05). The fertility and hatchability of DMA straw group are 77.61% and 92.30%, respectively, and it is significantly higher than those in the pellet group (P < 0.01; P < 0.05). The fresh drake sperm motility of induction collection method is 71%, the massage collection method 61% and the frozen drake sperm motility of induction 33% while the massage 19%. The fertility of frozen drake semen group is 85.93%, while that of the fresh semen group is 88.17%. The frozen drake semen fertility of the highest batch is 93.8%. In conclusion, the world's advanced fertility of frozen semen can be obtained both in the chicken and drake through the optimized DMA straw freezing technology and the method of screening freeze-resistant individuals.


Assuntos
Preservação do Sêmen , Sêmen , Acetamidas , Animais , Galinhas , Criopreservação/veterinária , Crioprotetores , Fertilidade , Congelamento , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
14.
Sensors (Basel) ; 21(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208559

RESUMO

This study was performed to test bioimpedance as a tool to detect the effect of different thawing methods on meat quality to aid in the eventual creation of an electric impedance-based food quality monitoring system. The electric impedance was measured for fresh pork, thawed pork, and during quick and slow thawing. A clear difference was observed between fresh and thawed samples for both impedance parameters. Impedance was different between the fresh and the frozen-thawed samples, but there were no impedance differences between frozen-thawed samples and the ones that were frozen-thawed and then stored at +3 °C for an additional 16 h after thawing. The phase angle was also different between fresh and the frozen-thawed samples. At high frequency, there were small, but clear phase angle differences between frozen-thawed samples and the samples that were frozen-thawed and subsequently stored for more than 16 h at +3 °C. Furthermore, the deep learning model LSTM-RNN (long short-term memory recurrent neural network) was found to be a promising way to classify the different methods of thawing.


Assuntos
Espectroscopia Dielétrica , Carne , Estudos de Viabilidade , Qualidade dos Alimentos , Congelamento , Carne/análise
15.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206693

RESUMO

Plant overwintering may be affected in the future by climate change. Low-temperature waterlogging, associated with a predicted increase in rainfall during autumn and winter, can affect freezing tolerance, which is the main component of winter hardiness. The aim of this study was to elucidate the mechanism of change in freezing tolerance caused by low-temperature waterlogging in Lolium perenne, a cool-season grass that is well adapted to a cold climate. The work included: (i) a freezing tolerance test (plant regrowth after freezing); (ii) analysis of plant phytohormones production (abscisic acid [ABA] content and ethylene emission); (iii) measurement of leaf water content and stomatal conductance; (iv) carbohydrate analysis; and (v) analysis of Aco1, ABF2, and FT1 transcript accumulation. Freezing tolerance may be improved as a result of cold waterlogging. The mechanism of this change is reliant on multifaceted actions of phytohormones and carbohydrates, whereas ethylene may counteract ABA signaling. The regulation of senescence processes triggered by concerted action of phytohormones and glucose signaling may be an essential component of this mechanism.


Assuntos
Ácido Abscísico/metabolismo , Aclimatação , Etilenos/metabolismo , Congelamento , Estresse Fisiológico , Açúcares/metabolismo , Água , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/genética
16.
BMC Plant Biol ; 21(1): 323, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225663

RESUMO

BACKGROUND: Sufficient low temperature accumulation is the key strategy to break bud dormancy and promote subsequent flowering in tree peony anti-season culturing production. Exogenous gibberellins (GAs) could partially replace chilling to accelerate dormancy release, and different kinds of GAs showed inconsistent effects in various plants. To understand the effects of exogenous GA3 and GA4 on dormancy release and subsequent growth, the morphological changes were observed after exogenous GAs applications, the differentially expressed genes (DEGs) were identified, and the contents of endogenous phytohormones, starch and sugar were measured, respectively. RESULTS: Morphological observation and photosynthesis measurements indicated that both GA3 and GA4 applications accelerated bud dormancy release, but GA3 feeding induced faster bud burst, higher shoot and more flowers per plant. Full-length transcriptome of dormant bud was used as the reference genome. Totally 124 110 459, 124 015 148 and 126 239 836 reads by illumina transcriptome sequencing were obtained in mock, GA3 and GA4 groups, respectively. Compared with the mock, there were 879 DEGs and 2 595 DEGs in GA3 and GA4 group, 1 179 DEGs in GA3 vs GA4, and 849 DEGs were common in these comparison groups. The significant enrichment KEGG pathways of 849 DEGs highlighted plant hormone signal transduction, starch and sucrose metabolism, cell cycle, DNA replication, etc. Interestingly, the contents of endogenous GA1, GA3, GA4, GA7 and IAA significantly increased, ABA decreased after GA3 and GA4 treatments by LC-MS/MS. Additionally, the soluble glucose, fructose and trehalose increased after exogenous GAs applications. Compared to GA4 treatment, GA3 induced higher GA1, GA3 and IAA level, more starch degradation to generate more monosaccharide for use, and promoted cell cycle and photosynthesis. Higher expression levels of dormancy-related genes, TFL, FT, EBB1, EBB3 and CYCD, and lower of SVP by GA3 treatment implied more efficiency of GA3. CONCLUSIONS: Exogenous GA3 and GA4 significantly accelerated bud dormancy release and subsequent growth by increasing the contents of endogenous bioactive GAs, IAA, and soluble glucose such as fructose and trehalose, and accelerated cell cycle process, accompanied by decreasing ABA contents. GA3 was superior to GA4 in tree peony forcing culture, which might because tree peony was more sensitive to GA3 than GA4, and GA3 had a more effective ability to induce cell division and starch hydrolysis. These results provided the value data for understanding the mechanism of dormancy release in tree peony.


Assuntos
Flores/fisiologia , Giberelinas/metabolismo , Paeonia/fisiologia , Flores/efeitos dos fármacos , Congelamento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Giberelinas/farmacologia , Modelos Biológicos , Paeonia/efeitos dos fármacos , Paeonia/genética , Reguladores de Crescimento de Plantas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Amido/metabolismo , Sacarose/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
17.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199294

RESUMO

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.


Assuntos
Aclimatação/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Membrana Celular/enzimologia , Temperatura Baixa , Proteínas Quinases Ativadas por Mitógeno/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Congelamento , Cinética , Fluidez de Membrana , Biossíntese de Proteínas , Transcrição Genética
18.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206357

RESUMO

In the current work, a simple, economical, accurate, and precise HPLC method with UV detection was developed to quantify Favipiravir (FVIR) in spiked human plasma using acyclovir (ACVR) as an internal standard in the COVID-19 pandemic time. Both FVIR and ACVR were well separated and resolved on the C18 column using the mobile phase blend of methanol:acetonitrile:20 mM phosphate buffer (pH 3.1) in an isocratic mode flow rate of 1 mL/min with a proportion of 30:10:60 %, v/v/v. The detector wavelength was set at 242 nm. Maximum recovery of FVIR and ACVR from plasma was obtained with dichloromethane (DCM) as extracting solvent. The calibration curve was found to be linear in the range of 3.1-60.0 µg/mL with regression coefficient (r2) = 0.9976. However, with acceptable r2, the calibration data's heteroscedasticity was observed, which was further reduced using weighted linear regression with weighting factor 1/x. Finally, the method was validated concerning sensitivity, accuracy (Inter and Intraday's % RE and RSD were 0.28, 0.65 and 1.00, 0.12 respectively), precision, recovery (89.99%, 89.09%, and 90.81% for LQC, MQC, and HQC, respectively), stability (% RSD for 30-day were 3.04 and 1.71 for LQC and HQC, respectively at -20 °C), and carry-over US-FDA guidance for Bioanalytical Method Validation for researchers in the COVID-19 pandemic crisis. Furthermore, there was no significant difference for selectivity when evaluated at LLOQ concentration of 3 µg/mL of FVIR and relative to the blank.


Assuntos
Amidas/análise , Amidas/sangue , Antivirais/análise , Antivirais/sangue , Bioensaio/métodos , COVID-19/tratamento farmacológico , Cromatografia Líquida de Alta Pressão/métodos , Extração Líquido-Líquido/métodos , Pirazinas/análise , Pirazinas/sangue , Aciclovir/análise , Aciclovir/sangue , COVID-19/sangue , Calibragem , Estabilidade de Medicamentos , Congelamento , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Solventes/química
19.
Analyst ; 146(16): 5186-5197, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34297019

RESUMO

Lithium salts are commonly used as medication for Bipolar Disorder (BD) and depression. However, there are limited methods to quantify intracellular lithium. Most methods to analyze intracellular electrolytes require tedious sample processing, specialized and often expensive machinery, sometimes involving harmful chemicals, and a bulk amount of the sample. In this work, we report a novel method (FROZEN!) based on cell isolation (from the surrounding medium) through rapid de-ionized water cleaning, followed by flash freezing for preservation. SKOV3 cells were cultured in normal medium and a medium containing 1.0 mM lithium. Lithium and other intracellular electrolytes in the isolated and preserved cells were simultaneously analyzed with laser-induced breakdown spectroscopy (LIBS) and X-ray fluorescence spectroscopy (XRF). Key electrolytes such as sodium, potassium, and magnesium, along with lithium, were detectable at the single-cell level. We found that cells cultured in the lithium medium have an intracellular lithium concentration of 0.5 mM. Concurrently, the intracellular concentrations of other positively charged electrolytes (sodium, potassium, and magnesium) were reduced by the presence of lithium. FROZEN! will greatly facilitate research in intracellular electrolyte balance during drug treatment, or other physiological stresses. In particular, the cell isolation and preservation steps can be easily performed by many laboratories worldwide, after which the samples are sent to an analytical laboratory for electrolyte analysis.


Assuntos
Eletrólitos , Lítio , Animais , Congelamento , Potássio , Sódio
20.
Int J Pharm ; 606: 120932, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34310956

RESUMO

The use of inorganic nanoparticles (NPs) gains interest for pharmaceutical applications, e.g. as adjuvants or drug delivery vehicles. Colloidal stability of NPs in aqueous suspensions is a major development challenge. Both frozen and lyophilized liquids are alternative presentations to liquid dispersion. To improve the basic understanding, we investigated the freeze-thawing stability of model α-Al2O3 NPs. Freeze-thawing was conducted in three different buffer types at pH5 and 8 without and with additives to determine fundamental formulation principles. Before freeze-thawing, α-Al2O3 NPs could be stabilized in sodium citrate buffer at pH5 and 8, and in sodium or potassium phosphate at pH8. Particles revealed low zeta potential values in phosphate buffers at pH5 indicating insufficient electrostatic stabilization. After freeze-thawing, an increase in NP size was strongly reduced in potassium phosphate and sodium citrate buffers. Subsequent pH measurements upon freezing revealed a drastic acidic pH shift in sodium phosphate which was further demonstrated to destabilize NPs. The ionic stabilizers gelatin A/B, Na-CMC, and SDS, were suitable to improve colloidal stability in phosphate buffers at pH5 highlighting the importance of charge stabilization. Freeze-thawing stability was best in presence of gelatin A/B, followed by PVA, mannitol, or sucrose. Depletion and steric stabilization were insufficient using PEG and surfactants respectively. Thus, we could identify the fundamental formulation principles to preserve inorganic NPs upon freezing: i) sufficient charge stabilization, ii) a maintained pH during freezing, and iii) the addition of a suitable stabilizer, preferably gelatin, not necessarily surfactants. This forms the basis for future studies, e.g. on lyophilization.


Assuntos
Óxido de Alumínio , Nanopartículas , Estabilidade de Medicamentos , Excipientes , Liofilização , Congelamento
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