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The extremotolerant red yeast Rhodotorula mucilaginosa displays resilience to diverse environmental stressors, including cold, osmolarity, salinity, and oligotrophic conditions. Particularly, this yeast exhibits a remarkable ability to accumulate lipids and carotenoids in response to stress conditions. However, research into lipid biosynthesis has been hampered by limited genetic tools and a scarcity of studies on adaptive responses to nutrient stressors stimulating lipogenesis. This study investigated the impact of nitrogen stress on the adaptive response in Antarctic yeast R. mucilaginosa M94C9. Varied nitrogen availability reveals a nitrogen-dependent modulation of biomass and lipid droplet production, accompanied by significant ultrastructural changes to withstand nitrogen starvation. In silico analysis identifies open reading frames of genes encoding key lipogenesis enzymes, including acetyl-CoA carboxylase (Acc1), fatty acid synthases 1 and 2 (Fas1/Fas2), and acyl-CoA diacylglycerol O-acyltransferase 1 (Dga1). Further investigation into the expression profiles of RmACC1, RmFAS1, RmFAS2, and RmDGA1 genes under nitrogen stress revealed that the prolonged up-regulation of the RmDGA1 gene is a molecular indicator of lipogenesis. Subsequent fatty acid profiling unveiled an accumulation of oleic and palmitic acids under nitrogen limitation during the stationary phase. This investigation enhances our understanding of nitrogen stress adaptation and lipid biosynthesis, offering valuable insights into R. mucilaginosa M94C9 for potential industrial applications in the future.
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Population expansion is a global issue, especially for food production. Meanwhile, global climate change is damaging our soils, making it difficult for crops to thrive and lowering both production and quality. Poor nutrition and salinity stress affect plant growth and development. Although the impact of individual plant stresses has been studied for decades, the real stress scenario is more complex due to the exposure to multiple stresses at the same time. Here we investigate using existing evidence and a meta-analysis approach to determine molecular linkages between two contemporaneous abiotic stimuli, phosphate (Pi) deficiency and salinity, on a single plant cell model, the root hairs (RHs), which is the first plant cell exposed to them. Understanding how these two stresses work molecularly in RHs may help us build super-adaptable crops and sustainable agriculture in the face of global climate change.
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Chondrosarcoma (CS) is a malignant bone tumor arising from cartilage-producing cells. The conventional subtype of CS typically develops within a dense cartilaginous matrix, creating an environment deficient in oxygen and nutrients, necessitating metabolic adaptation to ensure proliferation under stress conditions. Although ketone bodies (KBs) are oxidized by extrahepatic tissue cells such as the heart and brain, specific cancer cells, including CS cells, can undergo ketolysis. In this study, we found that KBs catabolism is activated in CS cells under nutrition-deprivation conditions. Interestingly, cytosolic ß-hydroxybutyrate dehydrogenase 2 (BDH2), rather than mitochondrial BDH1, is expressed in these cells, indicating a specific metabolic adaptation for ketolysis in this bone tumor. The addition of the KB, ß-Hydroxybutyrate (ß-HB) in serum-starved CS cells re-induced the expression of BDH2, along with the key ketolytic enzyme 3-oxoacid CoA-transferase 1 (OXCT1) and monocarboxylate transporter-1 (MCT1). Additionally, internal ß-HB production was quantified in supplied and starved cells, suggesting that CS cells are also capable of ketogenesis alongside ketolysis. These findings unveil a novel metabolic adaptation wherein nutrition-deprived CS cells utilize KBs for energy supply and proliferation.
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Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, we elaborated the characterization of Mt-PhoH2 through biophysical, biochemical, and computational methods. In addition to its intrinsic ATPase activity, the biochemical experiments revealed its GTPase activity and both activities are metal ion dependent. Magnesium, manganese, copper, iron, nickel, zinc, cesium, calcium, and lithium were examined for their effect on activity, and the optimum activity was found with 10 mM of Mg2+ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as Km 4.873 ± 0.44 µM, Vmax 12.3817 ± 0.084 µM/min/mg, Kcat 0.0075 ± 0.00005 s-1, and Kcat/Km 0.0015 ± 0.000001 µM-1 s-1 with GTP. In the case of GTP as a substrate, a 20% decrease in enzymatic activity and a 50% increase in binding affinity of Mt-PhoH2 were observed. The substrates ADP and GDP inhibit the ATPase and GTPase activity of Mt-PhoH2. CD spectroscopy showed the dominance of alpha helix in the secondary structure of Mt-PhoH2, and this structural pattern was altered upon addition of ATP and GTP. In silico inhibitor screening revealed ML141 and NAV_2729 as two potential inhibitors of the catalytic activity of Mt-PhoH2. Mt-PhoH2 is essential for mycobacterial growth as its knockdown strain showed a decreased growth effect. Overall, the present article emphasizes the factors essential for the proper functioning of Mt-PhoH2 which is a participant in the toxin-antitoxin machinery and may also play an important role in phosphate starvation.
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Proteínas de Bactérias , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cinética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/químicaRESUMO
Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.
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Proteínas de Arabidopsis , Arabidopsis , Citocromos c/genética , Citocromos c/metabolismo , Sirolimo/farmacologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Trifosfato de Adenosina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Extreme environmental conditions have profound impacts on shaping the evolutionary trajectory of organisms. Exposure to these conditions elicits stress responses, that can trigger phenotypic changes in novel directions. The Mexican Tetra, Astyanax mexicanus, is an excellent model for understanding evolutionary mechanisms in response to extreme or new environments. This fish species consists of two morphs; the classical surface-dwelling fish and the blind cave-dwellers that inhabit dark and biodiversity-reduced ecosystems. In this review, we explore the specific stressors present in cave environments and examine the diverse adaptive strategies employed by cave populations to not only survive but thrive as successful colonizers. By analyzing the evolutionary responses of A. mexicanus, we gain valuable insights into the genetic, physiological, and behavioral adaptations that enable organisms to flourish under challenging environmental conditions.
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Adaptação Fisiológica , Evolução Biológica , Cavernas , Characidae , Ambientes Extremos , Estresse Fisiológico , Animais , Characidae/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MSE proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, ß-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection.
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In cellular circumstances where carbohydrates are scarce, plants can use alternative substrates for cellular energetic maintenance. In plants, the main protein reserve is present in the chloroplast, which contains most of the total leaf proteins and represents a rich source of nitrogen and amino acids. Autophagy plays a key role in chloroplast breakdown, a well-recognised symptom of both natural and stress-induced plant senescence. Remarkably, an autophagic-independent route of chloroplast degradation associated with chloroplast vesiculation (CV) gene was previously demonstrated. During extended darkness, CV is highly induced in the absence of autophagy, contributing to the early senescence phenotype of atg mutants. To further investigate the role of CV under dark-induced senescence conditions, mutants with low expression of CV (amircv) and double mutants amircv1xatg5 were characterised. Following darkness treatment, no aberrant phenotypes were observed in amircv single mutants; however, amircv1xatg5 double mutants displayed early senescence and altered dismantling of chloroplast and membrane structures under these conditions. Metabolic characterisation revealed that the functional lack of both CV and autophagy leads to higher impairment of amino acid release and differential organic acid accumulation during starvation conditions. The data obtained are discussed in the context of the role of CV and autophagy, both in terms of cellular metabolism and the regulation of chloroplast degradation.
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Arabidopsis , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Carboidratos , Aminoácidos/metabolismo , Autofagia/fisiologia , Folhas de Planta/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The energy stored in fatty acids is essential for several critical activities of insects, such as embryogenesis, oviposition, and flight. Rhodnius prolixus is an obligatory hematophagous hemipteran and vector of Chagas disease, and it feeds infrequently on very large blood meals. As digestion slowly occurs, lipids are synthesized and accumulate in the fat body, mainly as triacylglycerol, in lipid droplets. Between feeding bouts, proper mobilization and oxidation of stored lipids are crucial for survival, and released fatty acids are oxidized by mitochondrial ß-oxidation. Carnitine palmitoyl transferase I (CPT1) is the enzyme that catalyzes the first reaction of the carnitine shuttle, where the activated fatty acid, acyl-CoA, is converted to acyl-carnitine to be transported into the mitochondria. Here, we investigated the role of CPT1 in lipid metabolism and in resistance to starvation in Rhodnius prolixus. The expression of the CPT1 gene (RhoprCpt1) was determined in the organs of adult females on the fourth day after a blood meal, and the flight muscle showed higher expression levels than the ovary, fat body, and anterior and posterior midgut. RhoprCpt1 expression in the fat body dramatically decreased after feeding, and started to increase again 10 days later, but no changes were observed in the flight muscle. ß-oxidation rates were determined in flight muscle and fat body homogenates with the use of 3H-palmitate, and in unfed females, they were higher in the flight muscle. In the fat body, lipid oxidation activity did not show any variation before or at different days after feeding, and was not affected by the presence of etomoxir or malonyl-CoA. We used RNAi and generated RhoprCPT1-deficient insects, which surprisingly did not show a decrease in measured 3H-palmitate oxidation rates. However, the RNAi-knockdown females presented increased amounts of triacylglycerol and larger lipid droplets in the fat body, but not in the flight muscle. When subjected to starvation, these insects had a shorter lifespan. These results indicated that the inhibition of RhoprCpt1 expression compromised lipid mobilization and affected resistance to starvation.
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Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.
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Adaptação Fisiológica , Doença de Chagas , Insetos , Estágios do Ciclo de Vida , Inanição , Trypanosoma cruzi , Animais , Diferenciação Celular , Doença de Chagas/genética , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Insetos/metabolismo , Insetos/parasitologia , Insetos/fisiologia , Mamíferos/parasitologia , Transcriptoma/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiologia , Inanição/genética , Inanição/parasitologia , Inanição/fisiopatologia , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/fisiologiaRESUMO
In triatomines, blood-feeding triggers many physiological processes including post embryonic development and reproduction. Different feeding habits, such as hematophagy, can shape gene functions to meet the challenges of each type of diet. The gut of blood-sucking insects faces particular challenges after feeding due to the quantity and the quality of the food ingested. A comparison of transcriptomic and proteomic data indicates that post transcriptional regulation of gene expression is crucial in the triatomine gut. It was proposed that eukaryotic translation initiation factor 3 subunit m (eIF3m) and eIF3e define 2 different eIF3 complexes with a distinct affinity for the different mRNAs, thus selecting the set of mRNAs to be translated and constituting a post transcriptional mode of regulation of gene expression. Because the eIF3m is mainly expressed in the gut, we evaluated its relevance in Rhodnius prolixus physiology through RNA interference-mediated gene silencing. The knockdown of eIF3m reduced the digestion rate, affecting the processes triggered by a blood meal. Its silencing inhibited molting and caused premature death in nymphs while impaired ovary development, oviposition and increased resistance to starvation in adult females. The survival of males after feeding (resistance to starvation) was not affected by eIF3m knockdown. The information regarding the eIF3m function in insects is scarce and the phenotypes observed in R. prolixus upon eIF3m silencing are different and more severe than those previously described in Drosophila melanogaster, indicating a pleiotropic role of this gene in triatomines.
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Titanium is a ubiquitous element with a wide variety of beneficial effects in plants, including enhanced nutrient uptake and resistance to pathogens and abiotic stresses. While there is numerous evidence supporting the beneficial effects that Ti fertilization give to plants, there is little information on which genetic signaling pathways the Ti application activate in plant tissues. In this study, we utilize RNA-seq and ionomics technologies to unravel the molecular signals that Arabidopsis plants unleash when treated with Ti. RNA-seq analysis showed that Ti activates abscisic acid and salicylic acid signaling pathways and the expression of NUCLEOTIDE BINDING SITE-LEUCINE RICH REPEAT receptors likely by acting as a chemical priming molecule. This activation results in enhanced resistance to drought, high salinity, and infection with Botrytis cinerea in Arabidopsis. Ti also grants an enhanced nutritional state, even at suboptimal phosphate concentrations by upregulating the expression of multiple nutrient and membrane transporters and by modifying or increasing the production root exudates. Our results suggest that Ti might act similarly to the beneficial element Silicon in other plant species.
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Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli, the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes (iscRSUAX-hscBA-fdx), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe2(SO4)3 for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.
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Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Extratos Celulares , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Óperon , Enxofre/metabolismoRESUMO
We demonstrated that serpinA3c/k relocates from the cytoplasm to the apical tubular membrane (ATM) in chronic kidney disease (CKD), suggesting its secretion in luminal space in pathophysiological contexts. Here, we studied serpinA3c/k expression and secretion under different stressful conditions in vitro and in vivo. HEK-293 cells were transfected with a FLAG-tagged serpinA3c/k clone and exposed to H2 O2 or starvation. Both stressors induced serpinA3c/k secretion but with a higher molecular weight. Glycanase treatment established that serpinA3c/k is glycosylated. Site-directed mutagenesis for each of the four glycosylation sites was performed. During cellular stress, serpinA3c/k secretion increased with each mutant except in the quadruple mutant. In rats and patients suffering acute kidney injury (AKI), an atypical urinary serpinA3c/k excretion (uSerpinA3c/k) was observed. In rats with AKI, the greater the induced kidney damage, the greater the uSerpinA3 c/k, together with relocation toward ATM. Our findings show that: (1) serpinA3c/k is glycosylated and secreted, (2) serpinA3c/k secretion increases during cellular stress, (3) its appearance in urine reveals a pathophysiological state, and (4) urinary serpinA3 excretion could become a potential biomarker for AKI.
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Injúria Renal Aguda/metabolismo , Estresse Fisiológico , alfa 1-Antiquimotripsina/metabolismo , Injúria Renal Aguda/urina , Animais , Glicosilação , Células HEK293 , Humanos , Masculino , Mutação , Ratos , alfa 1-Antiquimotripsina/genética , alfa 1-Antiquimotripsina/urinaRESUMO
KEY MESSAGE: The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.
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Proteínas de Arabidopsis , Arabidopsis , Flavoproteínas Transferidoras de Elétrons , Aminoácidos de Cadeia Ramificada/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Técnicas de Cultura de Células , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Regulação da Expressão Gênica de Plantas , Isovaleril-CoA Desidrogenase/genética , Isovaleril-CoA Desidrogenase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , MutaçãoRESUMO
Physcomitrium patens apical growing protonemal cells have the singularity that they continue to undergo cell divisions as the plant develops. This feature provides a valuable tool to study autophagy in the context of a multicellular apical growing tissue coupled to development. Herein, we showed that the core autophagy machinery is present in the moss P. patens, and characterized the 2D and 3D growth and development of atg5 and atg7 loss-of-function mutants under optimal and nutrient-deprived conditions. Our results showed that 2D growth of the different morphological and functional protonemata apical growing cells, chloronema and caulonema, is differentially modulated by this process. These differences depend on the protonema cell type and position along the protonemal filament, and growth condition. As a global plant response, the absence of autophagy favors the spread of the colony through protonemata growth at the expense of a reduction of the 3D growth, such as the buds and gametophore development, and thus the adult gametophytic and reproductive phases. Altogether this study provides valuable information suggesting that autophagy has roles during apical growth with differential responses within the cell types of the same tissue and contributes to life cycle progression and thus the growth and development of the 2D and 3D tissues of P. patens.
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An in vitro model for the formation of an Enterococcus faecalis endodontic biofilm under nutritional restriction was established, simulating clinical conditions for the evaluation of antimicrobial substances. Biofilm formation in dentin was standardized using root quarters incubated with E. faecalis ATCC 29212 at 37°C without nutritional changes. Biofilms were evaluated at 7, 14, and 30 days, counting bacterial colony-forming units using conventional culture and verified scanning electron microscopy. Bacterial viability and biovolume were determined with confocal laser microscopy. Colonization of E. faecalis and biofilm formation on the dentinal surface was confirmed after 7 and 14 days, respectively. Microorganism colonization was homogeneous over the entire root surface at each time point, without significant differences in the viability percentage and biovolume. On the contrary, a decrease in viability and an increase in biovolume were observed when the time was increased. Compared with other incubation times, 14 days was found to be the best time for the establishment of the biofilm in terms of biovolume and bacterial viability. This in vitro model for the formation of endodontic biofilm will allow future evaluation of the efficacy of antimicrobial substances with a more adequate clinical approach.
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Biofilmes , Enterococcus faecalis , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Viabilidade Microbiana , Microscopia Confocal , Microscopia Eletrônica de VarreduraRESUMO
Macroautophagy/autophagy, a mechanism of degradation of intracellular material required to sustain cellular homeostasis, is exacerbated under stress conditions like nutrient deprivation, protein aggregation, organelle senescence, pathogen invasion, and hypoxia, among others. Detailed in vivo description of autophagic responses triggered by hypoxia is limited. We have characterized the autophagic response induced by hypoxia in Drosophila melanogaster. We found that this process is essential for Drosophila adaptation and survival because larvae with impaired autophagy are hypersensitive to low oxygen levels. Hypoxia triggers a bona fide autophagic response, as evaluated by several autophagy markers including Atg8, LysoTracker, Lamp1, Pi3K59F/Vps34 activity, transcriptional induction of Atg genes, as well as by transmission electron microscopy. Autophagy occurs in waves of autophagosome formation and maturation as hypoxia exposure is prolonged. Hypoxia-triggered autophagy is induced cell autonomously, and different tissues are sensitive to hypoxic treatments. We found that hypoxia-induced autophagy depends on the basic autophagy machinery but not on the hypoxia master regulator sima/HIF1A. Overall, our studies lay the foundation for using D. melanogaster as a model system for studying autophagy under hypoxic conditions, which, in combination with the potency of genetic manipulations available in this organism, provides a platform for studying the involvement of autophagy in hypoxia-associated pathologies and developmentally regulated processes.Abbreviations: Atg: autophagy-related; FYVE: zinc finger domain from Fab1 (yeast ortholog of PIKfyve); GFP: green fluorescent protein; HIF: hypoxia-inducible factor; hsf: heat shock factor; Hx: hypoxia; mCh: mCherry; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; Rheb: Ras homolog enriched in brain; sima: similar; Stv: Starvation; TEM: transmission electron microscopy; Tor: target of rapamycin; UAS: upstream activating sequence; Vps: vacuolar protein sorting.
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Proteínas de Drosophila , Proteínas de Saccharomyces cerevisiae , Animais , Autofagia/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Hipóxia , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
We investigated a time-course larval transcriptional analysis (RNA-seq) in the longfin yellowtail Seriola rivoliana, from hatching to day four at 22 °C, without providing zooplankton as food. Larval starvation is a critical physiological stage that must be prevented to ensure survival. However, the transcriptional mechanisms to endure starvation have not been investigated in marine fish. Differential gene expression showed newly day-specific transcriptome events during larval development. On day 1 (yolk sac absorption), the predominant upregulated developmental processes were larval growth, muscle and vision development, cytoskeletal structure, protein synthesis, protein and fat digestion-absorption, and hormone biosynthesis, whereas the cell cycle was suppressed. On day 2 (yolk sac exhaustion), a new stage of energy regeneration (ATP) was supplied by the oil drop reserve, whereas protein digestion-absorption and growth were suppressed. On day 3 (mouth opening and starvation), stress signals and nutrition deprivation upregulated the p53 signal and triggered autophagy and the AMP-activated protein kinase (AMPK) pathways as an alternative catabolic pathway to enduring starvation, and the circadian rhythm was established. On day 4 (starving and weakened larvae condition), autophagy supported subsequent protein synthesis, activated the immune system, and promoted estrogen signaling and skeleton renovation. However, larvae suppressed muscle development, vision and carbohydrate, and fat digestion-absorption and became lethargic, evidencing limited physiological support by autophagy to maintain survival without exogenous nutrition in this species.
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Privação de Alimentos , Perciformes/crescimento & desenvolvimento , Perciformes/metabolismo , Animais , Autofagia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Perciformes/genética , TranscriptomaRESUMO
Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.