The benefits of contributing to the citizen science platform iNaturalist as an identifier.

As the number of observations submitted to the citizen science platform iNaturalist continues to grow, it is increasingly important that these observations can be identified to the finest taxonomic level, maximizing their value for biodiversity research. Here, we explore the benefits of acting as an identifier on iNaturalist.

Transcriptomic profiling reveals histone acetylation-regulated genes involved in somatic embryogenesis in Arabidopsis thaliana.

BACKGROUND: Somatic embryogenesis (SE) exemplifies the unique developmental plasticity of plant cells. The regulatory processes, including epigenetic modifications controlling embryogenic reprogramming of cell transcriptome, have just started to be revealed. RESULTS: To identify the genes of histone acetylation-regulated expression in SE, we analyzed global transcriptomes of Arabidopsis explants undergoing embryogenic induction in response to treatment with histone deacetylase inhibitor, trichostatin A (TSA). The TSA-induced and auxin (2,4-dichlorophenoxyacetic acid; 2,4-D)-induced transcriptomes were compared. RNA-seq results revealed the similarities of the TSA- and auxin-induced transcriptomic responses that involve extensive deregulation, mostly repression, of the majority of genes. Within the differentially expressed genes (DEGs), we identified the master regulators (transcription factors - TFs) of SE, genes involved in biosynthesis, signaling, and polar transport of auxin and NITRILASE-encoding genes of the function in indole-3-acetic acid (IAA) biosynthesis. TSA-upregulated TF genes of essential functions in auxin-induced SE, included LEC1/LEC2, FUS3, AGL15, MYB118, PHB, PHV, PLTs, and WUS/WOXs. The TSA-induced transcriptome revealed also extensive upregulation of stress-related genes, including those related to stress hormone biosynthesis. In line with transcriptomic data, TSA-induced explants accumulated salicylic acid (SA) and abscisic acid (ABA), suggesting the role of histone acetylation (Hac) in regulating stress hormone-related responses during SE induction. Since mostly the adaxial side of cotyledon explant contributes to SE induction, we also identified organ polarity-related genes responding to TSA treatment, including AIL7/PLT7, RGE1, LBD18, 40, HB32, CBF1, and ULT2. Analysis of the relevant mutants supported the role of polarity-related genes in SE induction. CONCLUSION: The study results provide a step forward in deciphering the epigenetic network controlling embryogenic transition in somatic cells of plants.

DNA methylation analysis of floral parts revealed dynamic changes during the development of homostylous Fagopyrum tataricum and heterostylous F. esculentum flowers.

BACKGROUND: Proper flower development is essential for plant reproduction, a crucial aspect of the plant life cycle. This process involves precisely coordinating transcription factors, enzymes, and epigenetic modifications. DNA methylation, a ubiquitous and heritable epigenetic mechanism, is pivotal in regulating gene expression and shaping chromatin structure. Fagopyrum esculentum demonstrates anti-hypertensive, anti-diabetic, anti-inflammatory, cardio-protective, hepato-protective, and neuroprotective properties. However, the heteromorphic heterostyly observed in F. esculentum poses a significant challenge in breeding efforts. F. tataricum has better resistance to high altitudes and harsh weather conditions such as drought, frost, UV-B radiation damage, and pests. Moreover, F. tataricum contains significantly higher levels of rutin and other phenolics, more flavonoids, and a balanced amino acid profile compared to common buckwheat, being recognised as functional food, rendering it an excellent candidate for functional food applications. RESULTS: This study aimed to compare the DNA methylation profiles between the Pin and Thrum flower components of F. esculentum, with those of self-fertile species of F. tataricum, to understand the potential role of this epigenetic mechanism in Fagopyrum floral development. Notably, F. tataricum flowers are smaller than those of F. esculentum (Pin and Thrum morphs). The decline in DNA methylation levels in the developed open flower components, such as petals, stigmas and ovules, was consistent across both species, except for the ovule in the Thrum morph. Conversely, Pin and Tartary ovules exhibited a minor decrease in DNA methylation levels. The highest DNA methylation level was observed in Pin stigma from closed flowers, and the most significant decrease was in Pin stigma from open flowers. In opposition, the nectaries of open flowers exhibited higher levels of DNA methylation than those of closed flowers. The decrease in DNA methylation might correspond with the downregulation of genes encoding methyltransferases. CONCLUSIONS: Reduced overall DNA methylation and the expression of genes associated with these epigenetic markers in fully opened flowers of both species may indicate that demethylation is necessary to activate the expression of genes involved in floral development.

Vitamin D status of pregnant women with obesity in the UK and its association with pregnancy outcomes: a secondary analysis of the UK Pregnancies Better Eating and Activity Trial (UPBEAT) study.

Prenatal vitamin D deficiency is widely reported and may affect perinatal outcomes. In this secondary analysis of the UK Pregnancies Better Eating and Activity Trial, we examined vitamin D status and its relationship with selected pregnancy outcomes in women with obesity (BMI ≥ 30 kg/m2) from multi-ethnic inner-city settings in the UK. Determinants of vitamin D status at a mean of 17 ± 1 weeks' gestation were assessed using multivariable linear regression and reported as percent differences in serum 25-hydroxyvitamin D (25(OH)D). Associations between 25(OH)D and clinical outcomes were examined using logistic regression. Among 1089 participants, 67 % had 25(OH)D < 50 nmol/l and 26 % had concentrations < 25 nmol/l. In fully adjusted models accounting for socio-demographic and anthropometric characteristics, 25(OH)D was lower among women of Black (% difference = -33; 95 % CI: -39, -27), Asian (% difference = -43; 95 % CI: -51, -35) and other non-White (% difference = -26; 95 % CI: -35, -14) ethnicity compared with women of White ethnicity (n 1086; P < 0·001 for all). In unadjusted analysis, risk of gestational diabetes was greater in women with 25(OH)D < 25 nmol/l compared with ≥ 50 nmol/l (OR = 1·58; 95 % CI: 1·09, 2·31), but the magnitude of effect estimates was attenuated in the multivariable model (OR = 1·33; 95 % CI: 0·88, 2·00). There were no associations between 25(OH)D and risk of preeclampsia, preterm birth or small for gestational age or large-for-gestational-age delivery. These findings demonstrate low 25(OH)D among pregnant women with obesity and highlight ethnic disparities in vitamin D status in the UK. However, evidence for a greater risk of adverse perinatal outcomes among women with vitamin D deficiency was limited.

Threats to conservation from national security interests.

There is a growing trend of nation states invoking national security and emergency declarations to build state-sponsored infrastructure projects for border defense, energy production, and transportation. Established laws, regulations, and agreements for the protection of nature and cultural heritage within and between countries are becoming secondary to national security, compromising the function of protected areas, such as national parks, wilderness areas, and biosphere reserves that safeguard biodiversity, climate, and human health. We considered cases where decades-long multinational cross-border endangered species recovery programs have been jeopardized by waivers of environmental protection laws to facilitate rapid construction of border barriers that impede the movement and migration of animals, such as at the US-Mexico and Poland-Belarus borders. Renewable energy megaprojects, such as the Pinacate solar plant in Mexico, coupled with power transmission lines and road networks likewise cast a large footprint on the land and are being carried out with minimal to no environmental compliance under the guise of national security. National sovereignty likewise has been used as justification for bypassing laws to proceed with similar projects, such as Mexico's Dos Bocas refinery and Poland's Vistula Spit canal. Emphasis on security is also apparent in increasing military expenditure by the world's largest economies, which has created a mismatch with improvement in environmental policy stringency. Decisions to prioritize security can undermine democratic principles and environmental review protocols, trivialize humanity's dependence on functioning ecosystems, and contradict the United Nation's resolution on the human right to a healthy environment. Framing infrastructure projects as matters of national security also foments civil and political unrest by the labeling and casting of dissenters, including conservation scientists and environmental defenders, as antinational. World leaders must refrain from misusing extraordinary powers, adhere to laws and international agreements, and consult experts and local people before taking unilateral action on projects that affect ecological and human communities.

Amenazas para la conservación a partir de los intereses nacionales de seguridad Resumen En los países existe una tendencia creciente por invocar la seguridad nacional y las declaraciones de emergencia para construir infraestructuras financiadas por el estado para la defensa de las fronteras, producción de energía y transporte. Las leyes, regulaciones y acuerdos establecidos para la protección de la naturaleza y el patrimonio cultural dentro y entre los países se están relegando por la seguridad nacional, lo que compromete la función de las áreas protegidas (parques nacionales, áreas silvestres y reservas de la biósfera) que resguardan la biodiversidad, el clima y la salud humana. Consideramos los casos en donde se han puesto en peligro los programas longevos y multinacionales de recuperación de especies en peligro por las exenciones a las leyes de protección ambiental para facilitar la construcción rápida de barreras fronterizas que impiden el movimiento y la migración de animales, como es el caso de las fronteras entre EU y México y Polonia y Bielorrusia. Los megaproyectos de energía renovable, como la planta solar del Pinacate en México, en conjunto con las líneas de transmisión eléctrica y las redes de carreteras también dejan una gran huella sobre la tierra y se realizan con el mínimo o ningún cumplimiento bajo el aspecto de la seguridad nacional. La soberanía nacional también se ha usado para justificar la omisión de las leyes para proceder con proyectos similares, como la refinería de Dos Bocas en México y el canal Vistula Spit en Polonia. El énfasis sobre la seguridad también es evidente con el incremento del gasto militar de las mayores economías mundiales, lo que ha creado una desigualdad con las mejoras en la exigencia de la política ambiental. Las decisiones para priorizar la seguridad pueden debilitar los principios democráticos y los protocolos de revisión ambiental, banalizar la dependencia de la humanidad por los ecosistemas funcionales y contradecir la resolución de las Naciones Unidas sobre el derecho humano a un ambiente saludable. Cuando se denominan los proyectos de infraestructura como asuntos de seguridad nacional, también se fomenta el malestar civil y político al etiquetar como antinacionales a los disidentes, incluidos los defensores ambientales y los científicos de la conservación. Los líderes mundiales deben abstenerse de usar indebidamente los poderes extraordinarios, adherirse a las leyes y acuerdos internacionales y consultar con expertos y personas locales antes de actuar de forma unilateral en cuanto a proyectos que afectan las comunidades humanas y ecológicas.

The RdRp genotyping of SARS-CoV-2 isolated from patients with different clinical spectrum of COVID-19.

BACKGROUND: The evolution of SARS-CoV-2 has been observed from the very beginning of the fight against COVID-19, some mutations are indicators of potentially dangerous variants of the virus. However, there is no clear association between the genetic variants of SARS-CoV-2 and the severity of COVID-19. We aimed to analyze the genetic variability of RdRp in correlation with different courses of COVID-19. RESULTS: The prospective study included 77 samples of SARS-CoV-2 isolated from outpatients (1st degree of severity) and hospitalized patients (2nd, 3rd and 4th degree of severity). The retrospective analyses included 15,898,266 cases of SARS-CoV-2 genome sequences deposited in the GISAID repository. Single-nucleotide variants were identified based on the four sequenced amplified fragments of SARS-CoV-2. The analysis of the results was performed using appropriate statistical methods, with p < 0.05, considered statistically significant. Additionally, logistic regression analysis was performed to predict the strongest determinants of the observed relationships. The number of mutations was positively correlated with the severity of the COVID-19, and older male patients. We detected four mutations that significantly increased the risk of hospitalization of COVID-19 patients (14676C > T, 14697C > T, 15096 T > C, and 15279C > T), while the 15240C > T mutation was common among strains isolated from outpatients. The selected mutations were searched worldwide in the GISAID database, their presence was correlated with the severity of COVID-19. CONCLUSION: Identified mutations have the potential to be used to assess the increased risk of hospitalization in COVID-19 positive patients. Experimental studies and extensive epidemiological data are needed to investigate the association between individual mutations and the severity of COVID-19.

Steady-State and Time-Resolved Fluorescence Study of Selected Tryptophan-Containing Peptides in an AOT Reverse Micelle Environment.

The aim of this study was to demonstrate the utility of time-resolved fluorescence spectroscopy in the detection of subtle changes in the local microenvironment of a tryptophan chromophore in a confined and crowded medium of AOT reverse micelles, which mimic biological membranes and cell compartmentalization. For this purpose, fluorescence properties of L-tryptophan and several newly synthesized tryptophan-containing peptides in buffer and in an AOT reverse micelle medium were determined. It was shown that insertion of tryptophan and its short di- and tripeptides inside micelles led to evident changes in both the steady-state emission spectra and in fluorescence decay kinetics. The observed differences in spectral characteristics, such as a blue shift in the emission maxima, changes in the average fluorescence lifetime, and the appearance of environmental-dependent fluorescent species, showed the utility of time-resolved fluorescence spectroscopy as a sensitive tool for detecting subtle conformational modifications in tryptophan and its peptides induced by changes in polarity, viscosity, and specific interactions between chromophores and water molecules/polar groups/ions that occur inside reverse micelles.

A Brief Look at Hashimoto's Disease, Adrenal Incidentalomas, Obesity and Insulin Resistance-Could Endocrine Disruptors Be the Other Side of the Same Coin?

Hashimoto's disease (HD) is the most common cause of hypothyroidism in developed countries. The exact pathomechanism behind it has not been clearly established; however, an interplay of genetic susceptibility, environmental triggers (including diet) and epigenetic factors seems to be involved. Among the latter, increasingly more attention has been paid to some hormonally active substances, known as endocrine disruptors, which are commonly used worldwide. HD has become a condition widely reported in the media, acting as a culprit for inexplicable weight gain, chronic fatigue or weakness. Nevertheless, the recognition of HD is undeniably increasing and represents a major public health burden. At the same time, improving access to imaging tests has increased the number of incidentally diagnosed adrenal tumors. Above all, the widespread use of chest computed tomography (CT) due to the COVID-19 pandemic has contributed to frequent incidental detection of adrenal lesions. Fortunately, a vast majority of these findings are asymptomatic benign tumors with no excessive hormonal activity, and therefore, they are defined as adrenal incidentalomas (AIs). Interestingly, recent studies have indicated that patients with AIs are more prone to obesity and insulin resistance. Although mutual relationships between the thyroid and the adrenal glands have been studied widely, still, little is known about the possible pathophysiological associations between thyroid autoimmunity and the occurrence of adrenal incidentalomas. This article presents a brief review of the common endocrine disorders with a special focus on the frequently coexisting insulin resistance and/or obesity. Furthermore, in response to the recent growing interest in endocrine disruptors, with their transgenerational epigenetic effects that influence hormonal system function, a concise overview of the topic has also been included.

Request a woman scientist: A database for diversifying the public face of science.

A global online register of women scientists, ready to share their science, was established by a cohort of volunteer women from the grassroots organization 500 Women Scientists on January 17th, 2018. In less than one year, the database "Request a Woman Scientist" comprised over 7,500 women from 174 scientific disciplines and 133 countries. The database is built upon a voluntary questionnaire regarding career stage, degree, scientific discipline, geographic location, and other self-identifying dimensions of representation. The information was visualized using the software platform Tableau, with dropdown menus that help query the database and output a list of names, email addresses, and websites. The biological sciences and women scientists from the United States of America were best represented in the database. A survey of women in the database conducted in November 2018 showed that of 1,278 respondents, 11% had been contacted since signing up for a variety of engagements, including media, peer review, panel participation, educational outreach, and professional/research connections. These engagements resulted in consultations for articles, video chats with students, and speaking opportunities at conferences and events. With improved functionality and marketing, outreach in the global south, and future translation in other languages, this database will further promote the profile and participation of women scientists across society, which in turn will benefit the advancement of science.

Association between systemic sclerosis, palmar fasciitis with polyarthritis, Raynaud's phenomenon and erythromelalgia with underlying malignancy.

The symptoms of a rheumatic disease may also be a sign of a proliferative process. These include conditions that present with skin and vascular changes such as systemic sclerosis and Raynaud's phenomenon with peripheral ischaemia and ulceration. Furthermore, the less common conditions - erythromelalgia or palmar fasciitis with polyarthritis may also accompany cancer. In this article, we discuss the association of diffuse systemic sclerosis with anorectal tumor, palmar fasciitis and polyarthritis with ovarian cancer, erythromelalgia with underlying ovarian malignancy and Raynaud's phenomenon and digital ischemia associated with renal carcinoma. Based on the literature review on this topic we highlighted the importance of recognizing paraneoplastic syndromes at an early stage. This is a crucial point in the adequate management of a patient. Many of the paraneoplastic symptoms of rheumatic and other described conditions may regress with the management of the underlying malignancy.

Synthesis and antimicrobial evaluation of a pyrazoline-pyridine silver(I) complex: DNA-interaction and anti-biofilm activity.

The emergence of resistant bacterial strains mainly due to misuse of antibiotics has seriously affected our ability to treat bacterial illness, and the development of new classes of potent antimicrobial agents is desperately needed. In this study, we report the efficient synthesis of a new pyrazoline-pyridine containing ligand L1 which acts as an NN-donor for the formation of a novel silver (I) complex 2. The free ligand did not show antibacterial activity. High potency was exhibited by the complex against three Gram-negative bacteria, namely Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumanii with the minimum inhibitory concentration (MIC) ranging between 4 and 16 µg/mL (4.2-16.7 µM), and excellent activity against the fungi Candida albicans and Cryptococcus neoformans (MIC ≤ 0.25 µg/mL = 0.26 µM). Moreover, no hemolytic activity within the tested concentration range was observed. In addition to the planktonic growth inhibition, the biofilm formation of both Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was significantly reduced by the complex at MIC concentrations in a dose-dependent manner for Pseudomonas aeruginosa, whereas a biphasic response was obtained for MRSA showing that the sub-MIC doses enhanced biofilm formation before its reduction at higher concentration. Finally, complex 2 exhibited strong DNA binding with a large drop in DNA viscosity indicating the absence of classical intercalation and suggesting the participation of the silver ion in DNA binding which may be related to its antibacterial activity. Taken together, the current results reveal that the pyrazoline-pyridine silver complexes are of high interest as novel antibacterial agents, justifying further in vitro and in vivo investigation.

FoxO restricts growth and differentiation of cells with elevated TORC1 activity under nutrient restriction.

TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In Drosophila, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of PTEN mutant tissue under NR. Here we demonstrate that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of Rheb is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, Tsc1 mutant cells show decreased PKB activity, and the extent of Tsc1 mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from Tsc1 mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of Tsc1 mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1-in contrast to PTEN-rarely become malignant.

Molecular and Functional Characterization of MobK Protein-A Novel-Type Relaxase Involved in Mobilization for Conjugational Transfer of Klebsiella pneumoniae Plasmid pIGRK.

Conjugation, besides transformation and transduction, is one of the main mechanisms of horizontal transmission of genetic information among bacteria. Conjugational transfer, due to its essential role in shaping bacterial genomes and spreading of antibiotics resistance genes, has been widely studied for more than 70 years. However, new and intriguing facts concerning the molecular basis of this process are still being revealed. Most recently, a novel family of conjugative relaxases (Mob proteins) was distinguished. The characteristic feature of these proteins is that they are not related to any of Mobs described so far. Instead of this, they share significant similarity to tyrosine recombinases. In this study MobK-a tyrosine recombinase-like Mob protein, encoded by pIGRK cryptic plasmid from the Klebsiella pneumoniae clinical strain, was characterized. This study revealed that MobK is a site-specific nuclease and its relaxase activity is dependent on both a conserved catalytic tyrosine residue (Y179) that is characteristic of tyrosine recombinases and the presence of Mg2+ divalent cations. The pIGRK minimal origin of transfer sequence (oriT) was also characterized. This is one of the first reports presenting tyrosine recombinase-like conjugative relaxase protein. It also demonstrates that MobK is a convenient model for studying this new protein family.

Impact of curcumin on replicative and chronological aging in the Saccharomyces cerevisiae yeast.

Curcumin is a biologically active compound of vegetable origin which has a hormetic effect. Pro-health and anti-aging properties of curcumin have been known for years. The main benefit of curcumin is thought to be its anti-oxidative action. Despite vast amount of data confirming age-delaying activity of curcumin in various groups of organisms, so far little has been discovered about curcumin's impact on cell aging in the experimental model of the Saccharomyces cerevisiae budding yeast. We have been able to demonstrate that curcumin significantly increases oxidative stress and accelerates replicative and chronological aging of yeast cells devoid of anti-oxidative protection (with SOD1 and SOD2 gene deletion) and deprived of DNA repair mechanisms (RAD52). Interestingly, curcumin delays aging, probably through hormesis, of the wild-type strain BY4741.

Hypermethylation of Auxin-Responsive Motifs in the Promoters of the Transcription Factor Genes Accompanies the Somatic Embryogenesis Induction in Arabidopsis.

The auxin-induced embryogenic reprogramming of plant somatic cells is associated with extensive modulation of the gene expression in which epigenetic modifications, including DNA methylation, seem to play a crucial role. However, the function of DNA methylation, including the role of auxin in epigenetic regulation of the SE-controlling genes, remains poorly understood. Hence, in the present study, we analysed the expression and methylation of the TF genes that play a critical regulatory role during SE induction (LEC1, LEC2, BBM, WUS and AGL15) in auxin-treated explants of Arabidopsis. The results showed that auxin treatment substantially affected both the expression and methylation patterns of the SE-involved TF genes in a concentration-dependent manner. The auxin treatment differentially modulated the methylation of the promoter (P) and gene body (GB) sequences of the SE-involved genes. Relevantly, the SE-effective auxin treatment (5.0 µM of 2,4-D) was associated with the stable hypermethylation of the P regions of the SE-involved genes and a significantly higher methylation of the P than the GB fragments was a characteristic feature of the embryogenic culture. The presence of auxin-responsive (AuxRE) motifs in the hypermethylated P regions suggests that auxin might substantially contribute to the DNA methylation-mediated control of the SE-involved genes.

AGL15 Controls the Embryogenic Reprogramming of Somatic Cells in Arabidopsis through the Histone Acetylation-Mediated Repression of the miRNA Biogenesis Genes.

The embryogenic transition of somatic cells requires an extensive reprogramming of the cell transcriptome. Relevantly, the extensive modulation of the genes that have a regulatory function, in particular the genes encoding the transcription factors (TFs) and miRNAs, have been indicated as controlling somatic embryogenesis (SE) that is induced in vitro in the somatic cells of plants. Identifying the regulatory relationships between the TFs and miRNAs during SE induction is of central importance for understanding the complex regulatory interplay that fine-tunes a cell transcriptome during the embryogenic transition. Hence, here, we analysed the regulatory relationships between AGL15 (AGAMOUS-LIKE 15) TF and miR156 in an embryogenic culture of Arabidopsis. Both AGL15 and miR156 control SE induction and AGL15 has been reported to target the MIR156 genes in planta. The results showed that AGL15 contributes to the regulation of miR156 in an embryogenic culture at two levels that involve the activation of the MIR156 transcription and the containment of the abundance of mature miR156 by repressing the miRNA biogenesis genes DCL1 (DICER-LIKE1), SERRATE and HEN1 (HUA-ENHANCER1). To repress the miRNA biogenesis genes AGL15 seems to co-operate with the TOPLESS co-repressors (TPL and TPR1-4), which are components of the SIN3/HDAC silencing complex. The impact of TSA (trichostatin A), an inhibitor of the HDAC histone deacetylases, on the expression of the miRNA biogenesis genes together with the ChIP results implies that histone deacetylation is involved in the AGL15-mediated repression of miRNA processing. The results indicate that HDAC6 and HDAC19 histone deacetylases might co-operate with AGL15 in silencing the complex that controls the abundance of miR156 during embryogenic induction. This study provides new evidence about the histone acetylation-mediated control of the miRNA pathways during the embryogenic reprogramming of plant somatic cells and the essential role of AGL15 in this regulatory mechanism.

Mechanism of Binding of Antifungal Antibiotic Amphotericin B to Lipid Membranes: An Insight from Combined Single-Membrane Imaging, Microspectroscopy, and Molecular Dynamics.

Amphotericin B is a lifesaving polyene antibiotic used in the treatment of systemic mycoses. Unfortunately, the pharmacological applicability of this drug is limited because of its severe toxic side effects. At the same time, the lack of a well-defined mechanism of selectivity hampers the efforts to rationally design safer derivatives. As the drug primarily targets the biomembranes of both fungi and humans, new insights into the binding of amphotericin B to lipid membranes can be helpful in unveiling the molecular mechanisms underlying both its pharmacological activity and toxicity. We use fluorescence-lifetime-imaging microscopy combined with fluorescence-emission spectroscopy in the microscale to study the interaction of amphotericin B with single lipid bilayers, using model systems based on giant unilamellar liposomes formed with three lipids: dipalmitoylphosphatidylcholine (DPPC), dimirystoylphosphatidylcholine (DMPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). The results show that amphotericin B introduced into the water phase as a DMSO solution binds to the membrane as dimers and small-molecular aggregates that we identify as tetramers and trimers. Fluorescence-detected linear-dichroism measurements revealed high orientational freedom of all the molecular-organization forms with respect to the membrane plane, which suggests that the drug partially binds to the membrane surface. The presence of sterols in the lipid phase (cholesterol but particularly ergosterol at 30 mol %) promotes the penetration of drug molecules into the lipid membrane, as concluded on the basis of the decreased orientation angle of amphotericin B molecules with respect to the axis normal to the membrane plane. Moreover, ergosterol facilitates the association of amphotericin B dimers into aggregated structures that can play a role in membrane destabilization or permeabilization. The presence of cholesterol inhibits the formation of small aggregates in the lipid phase of liposomes, making this system a promising candidate for a low-toxicity antibiotic-delivery system. Our conclusions are supported with molecular simulations that reveal the conformational properties of AmB oligomers in both aqueous solution and lipid bilayers of different compositions.

5-Azacitidine Induces Cell Death in a Tissue Culture of Brachypodium distachyon.

Morphological and histological observations revealed that, at a concentration of 50 µM, 5-azacitidine (5-azaC) totally inhibited the induction of embryogenic masses (EM), while the cultivation of explants (zygotic embryos; ZEs) in the presence of 5 µM of 5-azaC led to the formation of a callus with EM in 10% of the cases. Transmission electron microscopy (TEM) analyzes revealed the presence of the morphological and ultrastructural features that are typical for the vacuolar type of cell death in the callus cells that were treated. A TUNEL assay confirmed the presence of DNA double-strand breaks for the callus cells that had been treated with both 5 and 50 µM 5-azaC concentrations. Analysis of the gene expression of selected cell death markers demonstrated a reduced expression of metacaspase, protein executer 1 (EX1), and thioredoxin (TRX) in the callus cells that had been treated compared to the control culture. The strongest increase in the gene activity was characteristic for glutathione S-transferase (GST). Our studies also included an analysis of the distribution of some arabinogalactan proteins (AGPs) and extensin epitopes, which can be used as markers of cells that are undergoing death in a Brachypodium distachyon tissue culture.

Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture.

Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.