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Arsenic compounds have been used as therapeutic alternatives for several diseases including cancer. In the following work, we obtained arsenic nanoparticles (AsNPs) produced by an anaerobic bacterium from the Salar de Ascotán, in northern Chile, and evaluated their effects on the human oral squamous carcinoma cell line OECM-1. Resazurin reduction assays were carried out on these cells using 1-100 µM of AsNPs, finding a concentration-dependent reduction in cell viability that was not observed for the non-tumoral gastric mucosa-derived cell line GES-1. To establish if these effects were associated with apoptosis induction, markers like Bcl2, Bax, and cleaved caspase 3 were analyzed via Western blot, executor caspases 3/7 via luminometry, and DNA fragmentation was analyzed by TUNEL assay, using 100 µM cisplatin as a positive control. OECM-1 cells treated with AsNPs showed an induction of both extrinsic and intrinsic apoptotic pathways, which can be explained by a significant decrease in P-Akt/Akt and P-ERK/ERK relative protein ratios, and an increase in both PTEN and p53 mRNA levels and Bit-1 relative protein levels. These results suggest a prospective mechanism of action for AsNPs that involves a potential interaction with extracellular matrix (ECM) components that reduces cell attachment and subsequently triggers anoikis, an anchorage-dependent type of apoptosis.

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Gastric diseases represent a significant global public health challenge, characterized by molecular dysregulation in redox homeostasis and heightened oxidative stress. Although prior preclinical studies have demonstrated the cytoprotective antioxidant effects of alginate oligosaccharides (AOSs) through the Nrf2 pathway, whether such mechanisms apply to gastric diseases remains unclear. In this study, we used the GES-1 gastric cell line exposed to hydrogen peroxide (H2O2) as a damage model to investigate the impact of AOS on cell viability and its associated mechanisms. Our results revealed that pre-incubation with AOS for either 4 h or 24 h significantly improved the viability of GES-1 cells exposed to H2O2. In addition, AOS reduced the intracellular ROS levels, activating the Nrf2 signaling pathway, with increased Nrf2 protein and mRNA expression and a significant upregulation of the target genes HO-1 and NQO1. The activation of Nrf2 was correlated with decreased Keap1 protein expression and an increased level of the autophagy protein p62/SQSTM1, suggesting the activation of Nrf2 through a noncanonical pathway. This study suggests that AOS is a potential treatment for protecting gastric epithelial cells from oxidative stress by activating the p62/SQSTM1-Keap1-Nrf2 axis and laying the foundation for future investigations about its specific therapeutic mechanisms.

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pH regulation is essential to allow normal cell function, and their imbalance is associated with different pathologic situations, including cancer. In this study, we present the synthesis of 2-(((2-aminoethyl)imino)methyl)phenol (HL1) and the iron (III) complex (Fe(L1)2Br, (C1)), confirmed by X-ray diffraction analysis. The absorption and emission properties of complex C1 were assessed in the presence and absence of different physiologically relevant analytes, finding a fluorescent turn-on when OH- was added. So, we determined the limit of detection (LOD = 3.97 × 10-9 M), stoichiometry (1:1), and association constant (Kas = 5.86 × 103 M-1). Using DFT calculations, we proposed a spontaneous decomposition mechanism for C1. After characterization, complex C1 was evaluated as an intracellular pH chemosensor on the human primary gastric adenocarcinoma (AGS) and non-tumoral gastric epithelia (GES-1) cell lines, finding fluorescent signal activation in the latter when compared to AGS cells due to the lower intracellular pH of AGS cells caused by the increased metabolic rate. However, when complex C1 was used on metastatic cancer cell lines (MKN-45 and MKN-74), a fluorescent turn-on was observed in both cell lines because the intracellular lactate amount increased. Our results could provide insights about the application of complex C1 as a metabolic probe to be used in cancer cell imaging.

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Plants colonized the land approximately 470 million years ago, coinciding with the development of apical cells that divide in three planes. The molecular mechanisms that underly the development of the 3D growth pattern are poorly understood, mainly because 3D growth in seed plants starts during embryo development. In contrast, the transition from 2D to 3D growth in the moss Physcomitrium patens has been widely studied, and it involves a large turnover of the transcriptome to allow the establishment of stage-specific transcripts that facilitate this developmental transition. N6 -Methyladenosine (m6 A) is the most abundant, dynamic and conserved internal nucleotide modification present on eukaryotic mRNA and serves as a layer of post-transcriptional regulation directly affecting several cellular processes and developmental pathways in many organisms. In Arabidopsis, m6 A has been reported to be essential for organ growth and determination, embryo development and responses to environmental signals. In this study, we identified the main genes of the m6 A methyltransferase complex (MTC), MTA, MTB and FIP37, in P. patens and demonstrate that their inactivation leads to the loss of m6 A in mRNA, a delay in the formation of gametophore buds and defects in spore development. Genome-wide analysis revealed several transcripts affected in the Ppmta background. We demonstrate that the PpAPB1-PpAPB4 transcripts, encoding central factors orchestrating the transition from 2D to 3D growth in P. patens, are modified by m6 A, whereas in the Ppmta mutant the lack of the m6 A marker is associated with a corresponding decrease in transcript accumulation. Overall, we suggest that m6 A is essential to enable the proper accumulation of these and other bud-specific transcripts directing the turnover of stage-specific transcriptomes, and thus promoting the transition from protonema to gametophore buds in P. patens.

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Vagus nerve innervates several organs including the heart, stomach, and pancreas among others. Somas of sensory neurons that project through the vagal nerve are located in the nodose ganglion. The presence of purinergic receptors has been reported in neurons and satellite glial cells in several sensory ganglia. In the nodose ganglion, calcium depletion-induced increases in neuron activity can be partly reversed by P2X7 blockers applied directly into the ganglion. The later suggest a possible role of P2X7 receptors in the modulation of neuronal activity within this sensory ganglion. We aimed to characterize the response to P2X7 activation in nodose ganglion neurons under physiological conditions. Using an ex vivo preparation for electrophysiological recordings of the neural discharges of nodose ganglion neurons, we found that treatments with ATP induce transient neuronal activity increases. Also, we found a concentration-dependent increase in neural activity in response to Bz-ATP (ED50 = 0.62 mM, a selective P2X7 receptor agonist), with a clear desensitization pattern when applied every ~ 30 s. Electrophysiological recordings from isolated nodose ganglion neurons reveal no differences in the responses to Bz-ATP and ATP. Finally, we showed that the P2X7 receptor was expressed in the rat nodose ganglion, both in neurons and satellite glial cells. Additionally, a P2X7 receptor negative allosteric modulator decreased the duration of Bz-ATP-induced maximal responses without affecting their amplitude. Our results show the presence of functional P2X7 receptors under physiological conditions within the nodose ganglion of the rat, and suggest that ATP modulation of nodose ganglion activity may be in part mediated by the activation of P2X7 receptors.

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Common bean (Phaseolus vulgaris L.) is one of the most consumed legumes in the human diet and a substantial source of dietary protein. A major problem for this rainfed crop is the decrease in grain yield caused by prolonged drought periods during the reproductive stage of plant development (terminal drought). Terminal drought remains a prevailing threat to the farming of this staple, with losses reaching >80%. Based on the high correlation between the resistance of common bean to terminal drought and efficient photoassimilate mobilization and biomass accumulation in seeds, we aimed to identify mechanisms implicated in its resistance to this stress. We used two representative Durango race common bean cultivars with contrasting yields under terminal drought, grown under well-watered or terminal drought conditions. Using comparative transcriptomic analysis focused on source leaves, pods, and seeds from both cultivars, we provide evidence indicating that under terminal drought the resistant cultivar promotes the build-up of transcripts involved in recycling carbon through photosynthesis, photorespiration, and CO2-concentrating mechanisms in pod walls, while in seeds, the induced transcripts participate in sink strength and respiration. Physiological data support this conclusion, implicating their relevance as key processes in the plant response to terminal drought.

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Acylpeptide hydrolase (APEH) is a serine protease involved in amino acid recycling from acylated peptides (exopeptidase activity) and degradation of oxidized proteins (endoproteinase activity). This enzyme is inhibited by dichlorvos (DDVP), an organophosphate compound used as an insecticide. The role of APEH in spermatogenesis has not been established; therefore, the aim of this study was to characterize the distribution and activity profile of APEH during this process. For this purpose, cryosections of male reproductive tissues (testis and epididymis) and isolated cells (Sertoli cells, germ cells, and spermatozoa) were obtained from adult rats in order to analyze the intracellular localization of APEH by indirect immunofluorescence. In addition, the catalytic activity profiles of APEH in the different male reproductive tissues and isolated cells were quantified. Our results show that APEH is homogeneously distributed in Sertoli cells and early germ cells (spermatocytes and round spermatids), but this pattern changes during spermiogenesis. Specifically, in elongated spermatids and spermatozoa, APEH was localized in the acrosome and the principal piece. The exopeptidase activity was higher in the germ cell pool, compared to sperm and Sertoli cells, while the endoproteinase activity in epididymal homogenates was higher compared to testis homogenates at 24 h of incubation. In isolated cells, this activity was increased in Sertoli and germ cell pools, compared to spermatozoa. Taken together, these results indicate that APEH is differentially distributed in the testicular epithelium and undergoes re-localization during spermiogenesis. A possible role of APEH as a component of a protection system against oxidative stress and during sperm capacitation is discussed.

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Terminal drought stress affects more than half of the areas planted with common bean (Phaseolus vulgaris), the main food legume globally, generating severe yield losses. Phenotyping water deficit responses and water use are central strategies to develop improved terminal drought resilience. The exploration and exploitation of genetic diversity in breeding programs are gaining importance, with a particular interest in related species with great adaptation to biotic and abiotic factors. This is the case with tepary beans (Phaseolus acutifolius), a bean that evolved and was domesticated in arid conditions and is considered well adapted to drought and heat stress. Under greenhouse conditions, using one genotype of tepary beans (resistant to drought) and two of common beans (one resistant and one susceptible to terminal drought), we evaluated phenotypic differences in traits such as water use efficiency (WUE), transpiration efficiency, rate of photosynthesis, photosynthetic efficiency, stomatal density, stomatal index, stomatal size, and the threshold for transpiration decline under well-watered and terminal drought conditions. Our results indicate two different water use strategies in drought-resistant genotypes: one observed in common bean aimed at conserving soil water by closing stomata early, inhibiting stomatal development, and limiting growth; and the other observed in tepary bean, where prolonged stomatal opening and higher carbon fixation, combined with no changes in stomata distribution, lead to higher biomass accumulation. Strategies that contribute to drought adaptation combined with other traits, such as greater mobilization of photoassimilates to the formation of reproductive structures, confer bean drought resistance and are useful targets in breeding programs.

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Neuroblastoma is one of the most frequent types of cancer found in infants, and traditional chemotherapy has limited efficacy against this pathology. Thus, the development of new compounds with higher activity and selectivity than traditional drugs is a current challenge in medicinal chemistry research. In this study, we report the synthesis of 21 chalcones with antiproliferative activity and selectivity against the neuroblastoma cell line SH-SY5Y. Then, we developed three-dimensional quantitative structure-activity relationship models (comparative molecular field analysis and comparative molecular similarity index analysis) with high-quality statistical values (q2 > 0.7; r2 > 0.8; r2 pred > 0.7), using IC50 and selectivity index (SI) data as dependent variables. With the information derived from these theoretical models, we designed and synthesized 16 new molecules to prove their consistency, finding good antiproliferative activity against SH-SY5Y cells on these derivatives, with three of them showing higher SI than the referential drugs 5-fluorouracil and cisplatin, displaying also a proapoptotic effect comparable to these drugs, as proven by measuring their effects on executor caspases 3/7 activity induction, Bcl-2/Bax messenger RNA levels alteration, and DNA fragmentation promotion.

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Chile is a mining country, where waste mining is frequently found in the vicinity of inhabited areas. To explore the association between metal exposure and alterations in glucose metabolism, inflammatory status, and oxidative stress in individuals with chronic exposure to metals, a cross-sectional study was performed with 25 volunteers, between 45-65 years old. Inductive coupled plasma mass spectrometry (ICP-MS) was used to measure urinary levels of total arsenic (As) and its metabolites, cooper, nickel, chromium, and lead. Lipid profile, glucose, and insulin were measured in blood, as well as inflammation (interleukin-6, IL-6) and oxidative stress (8-hydroxy-2'deoxyguanosine, 8-OHdG) markers. Increased levels of Low-density lipoprotein, high-density lipoproteins, cholesterol and 8-OHdG, and the index for homeostasis model assessment-insulin resistance (HOMA-IR) were observed in 72%, 60%, and 56% of the volunteers, respectively. Blood-glucose levels were correlated with dimethylarsinic acid (DMA) (R2 = 0.47, p = 0.019), inorganic As (Asi) (R2 = 0.40, p = 0.012), and Ni (R2 = 0.56; p = 0.044). The models with these compounds explained 72% of the glycemia variability (ßDMA = -6.47; ßAsi = 6.68; ßNi = 6.87). Ni showed a significantly influence on IL-6 variability (ß = 0.85: R2 = 0.36). Changes in glycemia could be related to exposure to low levels of Asi and Ni, representing risk factors for metabolic diseases. Body mass index would confuse the relation between IL-6 and Ni levels, probably due to known chronic inflammation present in obese people.

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Plants respond to adverse environmental cues by adjusting a wide variety of processes through highly regulated mechanisms to maintain plant homeostasis for survival. As a result of the sessile nature of plants, their response, adjustment and adaptation to the changing environment is intimately coordinated with their developmental programs through the crosstalk of regulatory networks. Germination is a critical process in the plant life cycle, and thus plants have evolved various strategies to control the timing of germination according to their local environment. The mechanisms involved in these adjustment responses are largely unknown, however. Here, we report that mutations in core elements of canonical RNA-directed DNA methylation (RdDM) affect the germination and post-germination growth of Arabidopsis seeds grown under salinity stress. Transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses support the involvement of this pathway in the control of germination timing and post-germination growth under salinity stress by preventing the transcriptional activation of genes implicated in these processes. Subsequent transcriptional effects on genes that function in relation to these developmental events support this conclusion.

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MicroRNAs are important regulators of gene expression in eukaryotes. Previously, we reported that in Phaseolus vulgaris, the precursor for miR2119 is located in the same gene as miR398a, conceiving a dicistronic MIR gene. Both miRNA precursors are transcribed and processed from a single transcript resulting in two mature microRNAs that regulate the mRNAs encoding ALCOHOL DEHYDROGENASE 1 (ADH1) and COPPER-ZINC SUPEROXIDE DISMUTASE 1 (CSD1). Genes for miR398 are distributed throughout the spermatophytes; however, miR2119 is only found in Leguminosae species, indicating its recent emergence. Here, we used public databases to explore the presence of the miR2119 sequence in several plant species. We found that miR2119 is present only in specific clades within the Papilionoideae subfamily, including important crops used for human consumption and forage. Within this subfamily, MIR2119 and MIR398a are found together as a single gene in the genomes of the Millettioids and Hologalegina. In contrast, in the Dalbergioids MIR2119 is located in a different locus from MIR398a, suggesting this as the ancestral genomic organization. To our knowledge, this is a unique example where two separate MIRNA genes have merged to generate a single polycistronic gene. Phylogenetic analysis of ADH1 gene sequences in the Papilionoideae subfamily revealed duplication events resulting in up to four ADH1 genes in certain species. Notably, the presence of MIR2119 correlates with the conservation of target sites in particular ADH1 genes in each clade. Our results suggest that post-transcriptional regulation of ADH1 genes by miR2119 has contributed to shaping the expansion and divergence of this gene family in the Papilionoideae. Future experimental work on ADH1 regulation by miR2119 in more legume species will help to further understand the evolutionary history of the ADH1 gene family and the relevance of miRNA regulation in this process.

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Unlike for structured proteins, the study of intrinsically disordered proteins (IDPs) requires selection of ad hoc assays and strategies to characterize their dynamic structure and function. Late embryogenesis abundant (LEA) proteins are important plant IDPs closely related to water-deficit stress response. Diverse hypothetical functions have been proposed for LEA proteins, such as membrane stabilizers during cold stress, oxidative regulators acting as ion metal binding molecules, and protein protectants during dehydration and cold/freezing conditions. Here we present two detailed protocols to characterize IDPs with potential protein/enzyme protection activity under partial dehydration and freeze-thaw treatments.

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Plant microRNAs are commonly encoded in transcripts containing a single microRNA precursor. Processing by DICER-LIKE 1 and associated factors results in the production of a small RNA, followed by its incorporation into an AGO-containing protein complex to guide silencing of an mRNA possessing a complementary target sequence. Certain microRNA loci contain more than one precursor stem-loop structure, thus encoding more than one microRNA in the same transcript. Here, we describe a unique case where the evolutionary conserved miR398a is encoded in the same transcript as the legume-specific miR2119. The dicistronic arrangement found in common bean was also observed in other legumes. In Phaseolus vulgaris, mature miR398 and miR2119 are repressed in response to water deficit, and we demonstrate that both are functional as they target the mRNAs for CSD1 and ADH1, respectively. Our results indicate that the repression of miR398 and miR2119 leads to coordinated up-regulation of CSD1 and ADH1 mRNAs in response to water deficit in common bean and possibly in other legumes. Furthermore, we show that miRNA directed CSD1 and ADH1 mRNAs up-regulation also occurs when common bean plants are exposed to flooding, suggesting that plant redox status and fermentation metabolism must be closely coordinated under different adverse conditions.

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Late embryogenesis abundant (LEA) proteins accumulate in plants during adverse conditions and their main attributed function is to confer tolerance to stress. One of the deleterious effects of the adverse environment is the accumulation of metal ions to levels that generate reactive oxygen species, compromising the survival of cells. AtLEA4-5, a member of group 4 of LEAs in Arabidopsis, is an intrinsically disordered protein. It has been shown that their N-terminal region is able to undergo transitions to partially folded states and prevent the inactivation of enzymes. We have characterized metal ion binding to AtLEA4-5 by circular dichroism, electronic absorbance spectroscopy (UV-vis), electron paramagnetic resonance, dynamic light scattering, and isothermal titration calorimetry. The data shows that AtLEA4-5 contains a single binding site for Ni(II), while Zn(II) and Cu(II) have multiple binding sites and promote oligomerization. The Cu(II) interacts preferentially with histidine residues mostly located in the C-terminal region with moderate affinity and different coordination modes. These results and the lack of a stable secondary structure formation indicate that an ensemble of conformations remains accessible to the metal for binding, suggesting the formation of a fuzzy complex. Our results support the multifunctionality of LEA proteins and suggest that the C-terminal region of AtLEA4-5 could be responsible for antioxidant activity, scavenging metal ions under stress conditions while the N-terminal could function as a chaperone.

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The establishment of the symbiosis between legumes and nitrogen-fixing rhizobia is finely regulated at the transcriptional, posttranscriptional and posttranslational levels. Argonaute5 (AGO5), a protein involved in RNA silencing, can bind both viral RNAs and microRNAs to control plant-microbe interactions and plant physiology. For instance, AGO5 regulates the systemic resistance of Arabidopsis against Potato Virus X as well as the pigmentation of soybean (Glycine max) seeds. Here, we show that AGO5 is also playing a central role in legume nodulation based on its preferential expression in common bean (Phaseolus vulgaris) and soybean roots and nodules. We also report that the expression of AGO5 is induced after 1 h of inoculation with rhizobia. Down-regulation of AGO5 gene in P. vulgaris and G. max causes diminished root hair curling, reduces nodule formation and interferes with the induction of three critical symbiotic genes: Nuclear Factor Y-B (NF-YB), Nodule Inception (NIN) and Flotillin2 (FLOT2). Our findings provide evidence that the common bean and soybean AGO5 genes play an essential role in the establishment of the symbiosis with rhizobia.

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Plants are sessile organisms. This intriguing nature provokes the question of how they survive despite the continual perturbations caused by their constantly changing environment. The large amount of knowledge accumulated to date demonstrates the fascinating dynamic and plastic mechanisms, which underpin the diverse strategies selected in plants in response to the fluctuating environment. This phenotypic plasticity requires an efficient integration of external cues to their growth and developmental programs that can only be achieved through the dynamic and interactive coordination of various signaling networks. Given the versatility of intrinsic structural disorder within proteins, this feature appears as one of the leading characters of such complex functional circuits, critical for plant adaptation and survival in their wild habitats. In this review, we present information of those intrinsically disordered proteins (IDPs) from plants for which their high level of predicted structural disorder has been correlated with a particular function, or where there is experimental evidence linking this structural feature with its protein function. Using examples of plant IDPs involved in the control of cell cycle, metabolism, hormonal signaling and regulation of gene expression, development and responses to stress, we demonstrate the critical importance of IDPs throughout the life of the plant.

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Recent studies have identified microRNAs as post-transcriptional regulators involved in stress responses. miR1514a is a legume microRNA that is induced in response to drought stress in Phaseolus vulgaris (common bean) and shows differential accumulation levels in roots during water deficit in two cultivars with different drought tolerance phenotypes. A recent degradome analysis revealed that miR1514a targets the transcripts of two NAC transcription factors (TFs), Phvul.010g121000 and Phvul.010g120700. Furthermore, expression studies and small RNA-seq data indicate that only Phvul.010g120700 generates phasiRNAs, which also accumulate under water deficit conditions. To confirm these results, we over-expressed miR1514a in transgenic hairy roots, and observed a reduced accumulation of Phvul.010g120700 and an increase in NAC-derived phasiRNAs; inhibition of miR1514a activity resulted in the opposite effect. Moreover, we determined that a NAC-derived phasiRNA associates with ARGONAUTE 1 (AGO1), suggesting that it is functional. In addition, a transcriptome analysis of transgenic hairy roots with reduced miR1514a levels revealed several differentially expressed transcripts, mainly involved in metabolism and stress responses, suggesting they are regulated by the NAC TF and/or by phasiRNAs. This work therefore demonstrates the participation of miR1514 in the regulation of a NAC transcription factor transcript through phasiRNA production during the plant response to water deficit.

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High-throughput sequencing techniques have been applied to the discovery of several different regulatory small RNAs, including siRNAs and microRNAs in different plant species. Their subsequent characterization demands the use of sensitive and quantitative methods for their detection. Here we describe the use of northern blot and quantitative PCR for these purposes. We highlight the advantages and shortcomings of each method and offer a detailed description of those techniques that best work in our hands, in particular having in mind their use for the study of several small RNAs in a given sample. We enumerate relevant alternatives as well as cautionary comments in cases where we have detected potential difficulties.

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Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions.