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Abstract Background Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy to increase the production of those metabolites. Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family which are derivatives of the trans-resveratrol structure and are produced by plants and cell cultures as a response to biotic and abiotic stresses. The potential benefits of resveratrol on human health have made it one of the most thoroughly studied phytochemical molecules. The aim of this study was to evaluate the elicitor effect of both cyclodextrin (CD) and methyljasmonate (MeJA) on grapevine cell cultures by carrying out a quantitative analysis of their role on resveratrol production and on the expression of stilbene biosynthetic genes in Vitis vinifera cv Monastrell albino cell suspension cultures. Findings MeJA and CD significantly but transiently induced the expression of stilbene biosynthetic genes when independently used to treat grapevine cells. This expression correlated with resveratrol production in CD-treated cells but not in MeJA-treated cells, which growth was drastically affected. In the combined treatment of CD and MeJA cell growth was similarly affected, however resveratrol production was almost one order of magnitude higher, in correlation with maximum expression values for stilbene biosynthetic genes. Conclusion The effect of MeJA on cell division combined with a true and strong elicitor like CD could be responsible for the observed synergistic effect of both compounds on resveratrol production and on the expression of genes in the stilbene pathway.

Sound vibrations (SV) are known to influence molecular and physiological processes that can improve crop performance and yield. In this study, the effects of three audible frequencies (100, 500 and 1000 Hz) at constant amplitude (90 dB) on tomato Micro-Tom physiological responses were evaluated 1 and 3 days post-treatment. Moreover, the potential use of SV treatment as priming agent for improved Micro-Tom resistance to Pseudomonas syringae pv. tomato DC3000 was tested by microarray. Results showed that the SV-induced physiological changes were frequency- and time-dependent, with the largest changes registered at 1000 Hz at day 3. SV treatments tended to alter the foliar content of photosynthetic pigments, soluble proteins, sugars, phenolic composition, and the enzymatic activity of polyphenol oxidase, peroxidase, superoxide dismutase and catalase. Microarray data revealed that 1000 Hz treatment is effective in eliciting transcriptional reprogramming in tomato plants grown under normal conditions, but particularly after the infection with Pst DC3000. Broadly, in plants challenged with Pst DC3000, the 1000 Hz pretreatment provoked the up-regulation of unique differentially expressed genes (DEGs) involved in cell wall reinforcement, phenylpropanoid pathway and defensive proteins. In addition, in those plants, DEGs associated with enhancing plant basal immunity, such as proteinase inhibitors, pathogenesis-related proteins, and carbonic anhydrase 3, were notably up-regulated in comparison with non-SV pretreated, infected plants. These findings provide new insights into the modulation of Pst DC3000-tomato interaction by sound and open up prospects for further development of strategies for plant disease management through the reinforcement of defense mechanisms in Micro-Tom plants., (© 2024 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Modern agriculture is being challenged by deteriorating edaphoclimatic conditions and increasing anthropogenic pressure. This necessitates the development of innovative crop production systems that can sustainably meet the demands of a growing world population while minimizing the environmental impact. The use of plant biostimulants is gaining ground as a safe and ecologically sound approach to improving crop yields. In this review, biostimulants obtained from different higher plant sources are presented under the term higher plant-derived biostimulants (hPDBs). Their mechanisms of action regulate physiological processes in plants from germination to fructification, conditioned by responses induced in plant mineral nutrition and primary metabolism, specialized metabolism, photosynthetic processes, oxidative metabolism, and signaling-related processes. The aim of this review is to collect and unify the abundant information dispersed in the literature on the effects of these biostimulants, focusing on crops subjected to abiotic stress conditions and the underlying mechanisms of action.

Objectives: This study aimed to determine the impact of acute stress on tear production in companion cats to provide a basis for minimizing stress-inducing stimuli during ophthalmic evaluations., Methods: A total of 24 healthy owned cats (12 males, 12 females) of mixed breed, aged 8 months to 7 years, with no history of ocular diseases, were selected for the study. The cats were housed in individual cages under controlled conditions for 6 days. The Schirmer tear test-1 (STT-1) was performed in the morning (between 9:00 am and 11:00 am) using test strips from the same batch. The first test (without stress) was conducted on the fifth day of acclimation, and the second test (with stress) on the sixth day. The stress stimulus consisted of recordings of barking dogs, cats fighting and the murmuring of people. For both tests, the heart rate was assessed with a stethoscope before, during and after the tests, and the environmental stress level was also evaluated. Results are presented as mean ± SD and 95% confidence interval (CI)., Results: The study found that STT-1 values were significantly higher ( P  = 0.009) with stress (22.2 ± 6.0 mm/min [95% CI 19.9-24.6]) than without stress (17.5 ± 6.9 mm/min [95% CI 14.8-20.2]). Similarly, the heart rate was significantly higher ( P  = 0.028) in stress vs non-stress conditions (213.4 ± 37.5 beats per minute [bpm] [95% CI 198.7-228.1] vs 171.5 ± 28.6 bpm [95% CI 160.3-182.7], respectively), and the environmental stress score was significantly higher ( P  <0.001) in stress vs non-stress conditions (3.3 ± 0.5 [95% CI 3.1-3.5] vs 1.2 ± 0.4 [95% CI 1.1-1.4], respectively)., Conclusions and Relevance: Stress increased tear production in cats. Although the mean STT-1 value obtained under stress conditions was within the normal range, stress can influence the test results. The use of cat friendly handling techniques facilitates execution of the STT-1., Competing Interests: Conflict of interestThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Multiple myeloma is the second most common hematological malignancy in adults and remains an incurable disease. B cell maturation antigen (BCMA)-directed immunotherapy, including T cells bearing chimeric antigen receptors (CARs) and systemically injected bispecific T cell engagers (TCEs), has shown remarkable clinical activity, and several products have received market approval. However, despite promising results, most patients eventually become refractory and relapse, highlighting the need for alternative strategies. Engineered T cells secreting TCE antibodies (STAb) represent a promising strategy that combines the advantages of adoptive cell therapies and bispecific antibodies. Here, we undertook a comprehensive preclinical study comparing the therapeutic potential of T cells either expressing second-generation anti-BCMA CARs (CAR-T) or secreting BCMAxCD3 TCEs (STAb-T) in a T cell-limiting experimental setting mimicking the conditions found in patients with relapsed/refractory multiple myeloma. STAb-T cells recruited T cell activity at extremely low effector-to-target ratios and were resistant to inhibition mediated by soluble BCMA released from the cell surface, resulting in enhanced cytotoxic responses and prevention of immune escape of multiple myeloma cells in vitro. These advantages led to robust expansion and persistence of STAb-T cells in vivo, generating long-lived memory BCMA-specific responses that could control multiple myeloma progression in xenograft models, outperforming traditional CAR-T cells. These promising preclinical results encourage clinical testing of the BCMA-STAb-T cell approach in relapsed/refractory multiple myeloma.

Background: During infectious diseases, proinflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung, the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG (erythroblast transformation-specific-related gene) as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury., Methods: Cytokine-dependent ubiquitination and proteasomal degradation of ERG were analyzed in cultured HUVECs (human umbilical vein ECs). Systemic administration of TNFα (tumor necrosis factor alpha) or the bacterial cell wall component lipopolysaccharide was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs ( Erg fl/fl ;Cdh5[PAC]-Cre ERT2 ), and multiple organs were analyzed by histology, immunostaining, and electron microscopy., Results: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or lipopolysaccharide resulted in a rapid and substantial degradation of ERG within lung ECs but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Erg fl/fl ;Cdh5(PAC)-Cre ERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek -a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation., Conclusions: Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases., Competing Interests: Disclosures None.

Morus alba L. is used for a range of therapeutic purposes in Asian traditional medicine, and its extracts are reported to be effective against lipidemia, diabetes, and obesity, as well as being hepatoprotective and tyrosinase-inhibitory. They are also included in cosmetic products as anti-aging and skin-whitening agents. Stilbenes, the major bioactive compounds found in M. alba , have received renewed attention recently because of their putative activity against COVID-19. In this study M. alba plants were established in vitro , and the effect of elicitation on plant growth and stilbene accumulation, specifically oxyresveratrol and trans -resveratrol, was investigated. Different concentrations of the elicitors including methyl jasmonate and cyclodextrins were applied, and stilbene levels were determined in leaves, roots, and the culture medium. Elicitation of the M. alba plants with 5 mM cyclodextrins, alone or in combination with 10 µM methyl jasmonate, significantly increased the total phenolic content in the culture medium and leaves after 7 days of treatment. The higher total phenolic content in the roots of control plants and those treated only with methyl jasmonate indicated that cyclodextrins promoted metabolite release to the culture medium. Notably, the cyclodextrin-treated plants with the highest levels of oxy- and trans -resveratrol also had the highest total phenolic content and antioxidant capacity. These results indicate that elicited M. alba in vitro plants constitute a promising alternative source of bioactive stilbenes to supply pharmaceutical and cosmeceutical industries.

Cyclopropanation reactions between C 60 and different malonates decorated with monosaccharides and steroids using the Bingel-Hirsch methodology have allowed the obtention of a new family of hybrid compounds in good yields. A complete set of instrumental techniques has allowed us to fully characterize the hybrid derivatives and to determine the chemical structure of monocycloadducts. Besides, the proposed structures were investigated by cyclic voltammetry, which evidenced the exclusive reductive pattern of fullerene Bingel-type monoadducts. Theoretical calculations at the DFT-D3(BJ)/PBE 6-311G(d,p) level of the synthesized conjugates predict the most stable conformation and determine the factors that control the hybrid molecules' geometry. Some parameters such as polarity, lipophilicity, polar surface area, hydrophilicity index, and solvent-accessible surface area were also estimated, predicting its potential permeability and capability as cell membrane penetrators. Additionally, a molecular docking simulation has been carried out using the main protease of SARS-CoV-2 (Mpro) as the receptor, thus paving the way to study the potential application of these hybrids in biomedicine., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. European Journal of Organic Chemistry published by Wiley-VCH GmbH.)

This article aims to provide in-depth insight into how consumers perceive recycled materials in comparison with natural raw materials at both the perceptual and attitudinal levels. To this end, we combined classic self-reported measures of sensory aspects, preferences, environmental attitudes, and consumption habits together with physiological measures of cognitive-emotional processing. Three different materials-two recycled materials, M2 and M3, and one raw material, M1-were chosen for inspection through three different sensory conditions, which we refer to as channels -visual, tactile, and visuo-tactile. The assignation of materials to sensory channels was counterbalanced so that each participant evaluated only one of the materials per channel. Although participants in general were not very accurate in discriminating between the materials, self-reported sensory evaluations showed that M3 (a recycled material that is made to look non-recycled), was clearly less liked. Meanwhile, the psychophysiological analyses revealed higher levels of electrodermal activity for the tactile evaluations of both recycled materials (M2 and M3). Finally, the results from the attitudes and habits evaluations indicate that the participants had positive environmental attitudes yet poor consumption habits. Altogether, these results suggest that some sensorial properties differ between recycled materials and natural raw materials and that there is a chance to improve and implement new consumption habits. The implications of these results are further discussed both in terms of suggestions for designers and methodological recommendations for researchers.

The impact of climate change entails a progressive and inexorable modification of the Earth's climate and events such as salinity, drought, extreme temperatures, high luminous intensity and ultraviolet radiation tend to be more numerous and prolonged in time. Plants face their exposure to these abiotic stresses or their combination through multiple physiological, metabolic and molecular mechanisms, to achieve the long-awaited acclimatization to these extreme conditions, and to thereby increase their survival rate. In recent decades, the increase in the intensity and duration of these climatological events have intensified research into the mechanisms behind plant tolerance to them, with great advances in this field. Among these mechanisms, the overproduction of molecular reactive species stands out, mainly reactive oxygen, nitrogen and sulfur species. These molecules have a dual activity, as they participate in signaling processes under physiological conditions, but, under stress conditions, their production increases, interacting with each other and modifying and-or damaging the main cellular components: lipids, carbohydrates, nucleic acids and proteins. The latter have amino acids in their sequence that are susceptible to post-translational modifications, both reversible and irreversible, through the different reactive species generated by abiotic stresses (redox-based PTMs). Some research suggests that this process does not occur randomly, but that the modification of critical residues in enzymes modulates their biological activity, being able to enhance or inhibit complete metabolic pathways in the process of acclimatization and tolerance to the exposure to the different abiotic stresses. Given the importance of these PTMs-based regulation mechanisms in the acclimatization processes of plants, the present review gathers the knowledge generated in recent years on this subject, delving into the PTMs of the redox-regulated enzymes of plant metabolism, and those that participate in the main stress-related pathways, such as oxidative metabolism, primary metabolism, cell signaling events, and photosynthetic metabolism. The aim is to unify the existing information thus far obtained to shed light on possible fields of future research in the search for the resilience of plants to climate change., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Martí-Guillén, Pardo-Hernández, Martínez-Lorente, Almagro and Rivero.)

In this work, Solanum lycopersicum cv. Micro-Tom suspension-cultured cells were used to analyze the effect of different elicitors including β-cyclodextrins (CD), methyl jasmonate (MJ), β-glucan (Glu) and 3-hexenol (Hex) separately and the combined treatments of CD + MJ, CD + glu and CD + Hex on triterpene compound production after 24, 72 and 96 h. Moreover, we studied the changes induced by elicitors in the expression of key biosynthetic genes to elucidate the regulation of the triterpene biosynthetic pathway. The relative abundance of the triterpene compounds identified in the extracellular medium after elicitation (squalene, fucosterol, avenasterol, β-sitosterol, cycloartenol and taraxasterol) was determined by gas chromatography coupled to mass spectrometry, and the expression level of genes in treated-cells was analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). Results showed that, in CD-treated cells (CD, CD + MJ, CD + Glu, CD + Hex), specialized metabolites were accumulated mainly in the extracellular medium after 72 h of elicitation. Moreover, qRT-PCR analysis revealed that the highest triterpene levels in CD-treated cells (CD, CD + MJ, CD + Glu, CD + Hex) were highly correlated with the expression of cycloartenol synthase , 3-hydroxy-3-methylglutaryl-CoA reductase and squalene epoxidase genes at 24 h of treatment, whereas the expression of sterol methyltransferase was increased at 72 h. According to our findings, CD acts as a true elicitor of triterpene biosynthesis and can promote the release of bioactive compounds from the tomato cells into the extracellular medium. The results obtained provide new insights into the regulation of the triterpene metabolic pathway, which might be useful for implementing metabolic engineering techniques in tomato.

The prevalence of type 2 diabetes (T2D) and impaired glucose tolerance (IGT) increases with ageing. T2D generally results from progressive impairment of the pancreatic islets to adapt β-cell mass and function in the setting of insulin resistance and increased insulin demand. Several studies have shown an age-related decline in peripheral insulin sensitivity. However, a precise understanding of the pancreatic β-cell response in ageing is still lacking. In this review, we summarize the age-related alterations, adaptations and/or failures of β-cells at the molecular, morphological and functional levels in mouse and human. Age-associated alterations include processes such as β-cell proliferation, apoptosis and cell identity that can influence β-cell mass. Age-related changes also affect β-cell function at distinct steps including electrical activity, Ca 2+ signaling and insulin secretion, among others. We will consider the potential impact of these alterations and those mediated by senescence pathways on β-cells and their implications in age-related T2D. Finally, given the great diversity of results in the field of β-cell ageing, we will discuss the sources of this heterogeneity. A better understanding of β-cell biology during ageing, particularly at older ages, will improve our insight into the contribution of β-cells to age-associated T2D and may boost new therapeutic strategies., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Main Conclusion: β-carotene is biologically active compound widely distributed in plants. The use of plant in vitro cultures and genetic engineering is a promising strategy for its sustainable production. β-carotene is an orange carotenoid often found in leaves as well as in fruits, flowers, and roots. A member of the tetraterpene family, this 40-carbon isoprenoid has a conjugated double-bond structure, which is responsible for some of its most remarkable properties. In plants, β-carotene functions as an antenna pigment and antioxidant, providing protection against photooxidative damage caused by strong UV-B light. In humans, β-carotene acts as a precursor of vitamin A, prevents skin damage by solar radiation, and protects against several types of cancer such as oral, colon and prostate. Due to its wide spectrum of applications, the global market for β-carotene is expanding, and the demand can no longer be met by extraction from plant raw materials. Considerable research has been dedicated to finding more efficient production alternatives based on biotechnological systems. This review provides a detailed overview of the strategies used to increase the production of β-carotene in plant in vitro cultures, with particular focus on culture conditions, precursor feeding and elicitation, and the application of metabolic engineering., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The glucose oligosaccharide-derived cyclodextrins (CDs) are used for improving bioactive compound production in plant cell cultures because, in addition to their elicitation activity, CDs promote product removal from cells. However, despite these advantages, the industrial application of CDs is hampered by their high market price. A strategy to overcome this constraint was recently tested, in which reusable CD polymers coated with magnetic Fe 3 O 4 nanoparticles were harnessed in Vitis vinifera cell cultures to produce t-resveratrol (t-R). In this study, we applied hydroxypropyl-β-CDs (HPCD) and HPCDs coated with magnetic nanoparticles (HPCD-EPI-MN) in methyl jasmonate (MJ)-treated transgenic Silybum marianum cultures ectopically expressing either a stilbene synthase gene (STS) or a chalcone synthase gene (CHS), and compared their effects on the yields of t-R and naringenin (Ng), respectively. HPCD-EPI-MN at 15 g/L stimulated the accumulation of metabolites in the culture medium of the corresponding transgenic cell lines, with up to 4 mg/L of t-R and 3 mg/L of Ng released after 3 days. Similar amounts were produced in cultures treated with HPCD. Concentrations higher than 15 g/L of HPCD-EPI-MN and prolonged incubation periods negatively affected cell growth and viability in both transgenic cell lines. Reutilization of HPCD-EPI-MN was possible in three elicitation cycles (72 h each), after which the polymer retained 25-30% of its initial efficiency, indicating good stability and reusability. Due to their capacity to adsorb metabolites and their recyclability, the application of magnetic CD polymers may reduce the costs of establishing efficient secondary metabolite production systems on a commercial scale. KEY POINTS: • Long-term transgenic S. marianum suspensions stably produce transgene products • t-R and Ng accumulated extracellularly in cultures elicited with HPCD and HPCD-EPI-MN • The recyclability of HPCD-EPI-MN for metabolite production was proven., (© 2022. The Author(s).)

Aging is associated with a decline in peripheral insulin sensitivity and an increased risk of impaired glucose tolerance and type 2 diabetes. During conditions of reduced insulin sensitivity, pancreatic β cells undergo adaptive responses to increase insulin secretion and maintain euglycemia. However, the existence and nature of β-cell adaptations and/or alterations during aging are still a matter of debate. In this study, we investigated the effects of aging on β-cell function from control (3-month-old) and aged (20-month-old) mice. Aged animals were further categorized into 2 groups: high insulin sensitive (aged-HIS) and low insulin sensitive (aged-LIS). Aged-LIS mice were hyperinsulinemic, glucose intolerant, and displayed impaired glucose-stimulated insulin and C-peptide secretion, whereas aged-HIS animals showed characteristics in glucose homeostasis similar to controls. In isolated β cells, we observed that glucose-induced inhibition of KATP channel activity was reduced with aging, particularly in the aged-LIS group. Glucose-induced islet NAD(P)H production was decreased in aged mice, suggesting impaired mitochondrial function. In contrast, voltage-gated Ca2+ currents were higher in aged-LIS β cells, and pancreatic islets of both aged groups displayed increased glucose-induced Ca2+ signaling and augmented insulin secretion compared with controls. Morphological analysis of pancreas sections also revealed augmented β-cell mass with aging, especially in the aged-LIS group, as well as ultrastructural β-cell changes. Altogether, these findings indicate that aged mouse β cells compensate for the aging-induced alterations in the stimulus-secretion coupling, particularly by adjusting their Ca2+ influx to ensure insulin secretion. These results also suggest that decreased peripheral insulin sensitivity exacerbates the effects of aging on β cells., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Highly productive trans -resveratrol ( t -R) grapevine suspension cultured cells (SCC) and two effective elicitors, methyl jasmonate (MJ) and randomly methylated β-cyclodextrins (CDs), were used to analyze the extent to which salt treatments alter the production of bioactive phenolic compounds. The expression/activity profile of the enzymes involved in phenol metabolism and antioxidant networks were also studied. A marked extracellular accumulation of phenolic compounds, especially t -R, was found in SCC elicited with CDs and/or MJ under saline conditions. However, the treatments with MJ alone and all those combined with salt favored the intracellular accumulation of catechin and ferulic acid. The salt-induced accumulation of phenolics was correlated with the higher total antioxidant capacity values found in cells, suggesting that cellular redox homeostasis under saline conditions was largely maintained by increasing phenolic compound production. These higher levels of phenolics found in elicited cells under saline conditions fit well with the highest activity of phenylalanine ammonia-lyase. Moreover, antioxidant enzyme activities were boosted in treatments with MJ and/or in those combined with salt and decreased in those treated with CDs. These results suggest a differential response of the antioxidative network to the presence of elicitors under saline conditions.

Materials are elements that configure our built environment and are key components in design and engineering education. This research aims to understand learners' sensorial perception of materials as stimuli and what constitutes the most appropriate communication channel for learning about their characteristics without losing information in accordance with their VAK learning styles-visual, auditory and kinaesthetic-. Seventy-five people participated in this workshop. Using evaluation tools in a test format, they evaluated all the sensory properties of the following materials: Alusion™, PolarMoss, and Silkworms. These three materials were presented in various interaction formats, called channels: a text plus image-C1-, video-C2-, and a physical sample plus audio-C3-. Two types of experiments were carried out: mixed per person-interaction with the three materials in a different channel each time in random order-and blocked per person-interaction with the same material in the three channels, in an order of C1-C3. The data obtained was analysed using mixed models with the channel as a fixed factor and the individuals and material as the random factors. The most relevant results indicate significant differences between channels in accordance with the sensory property, normally C2 and C3. The level of responses between the two experiments is similar, therefore showing that the order C1-C2-C3 does not affect perception. Although the three learning styles-visual, auditory, and kinaesthetic-coexist in the participants, the vast majority agree that they prefer C3 as easier to evaluate, more entertaining and the best way to learn., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

Honing processes are currently employed to obtain a cross-hatched pattern on the internal surfaces of cylinders that favors oil flow in combustion engines or hydraulic cylinders. The main aim of the present paper is to optimize the machining conditions in honing processes with respect to surface roughness, material removal rate and tool wear by means of the desirability function. Five process variables are considered: grain size, density, pressure, linear speed and tangential speed. Later, a sensitivity analysis is performed to determine the effect of the variation of the importance given to each response on the results of the optimization process. In the rough and semi-finish honing steps, variations of less than 5% of the importance value do not cause substantial changes in the optimization process. On the contrary, in the finish honing step, small changes in the importance values lead to modifications in the optimization process, mainly regarding pressure. Thus, the finish honing phase is more sensitive to changes in the optimization process than the rough and the semi-finish honing phases. The present paper will help users of honing machines to select proper values for the process variables.

We report the synthesis and characterization of a fullerene-steroid hybrid that contains H 2 @C 60 and a dehydroepiandrosterone moiety synthesized by a cyclopropanation reaction with 76 % yield. Theoretical calculations at the DFT-D3(BJ)/PBE 6-311G(d,p) level predict the most stable conformation and that the saturation of a double bond is the main factor causing the upfield shielding of the signal appearing at -3.13 ppm, which corresponds to the H 2 located inside the fullerene cage. Relevant stereoelectronic parameters were also investigated and reinforce the idea that electronic interactions must be considered to develop studies on chemical-biological interactions. A molecular docking simulation predicted that the binding energy values for the protease-hybrid complexes were -9.9 kcal/mol and -13.5 kcal/mol for PL pro and 3CL pro respectively, indicating the potential use of the synthesized steroid-H 2 @C 60 as anti-SARS-Cov-2 agent., (© 2021 Wiley-VCH GmbH.)

Paclitaxel (PTX), a widely used anticancer agent, is found in the inner bark of several Taxus species, although at such low levels that its extraction is ecologically unsustainable. Biotechnological platforms based on Taxus sp. cell cultures offer an eco-friendlier approach to PTX production, with yields that can be improved by elicitation. However, the also limited excretion of target compounds from the producer cells to the medium hampers their extraction and purification. In this context, we studied the effect of treating T. media cell cultures with the elicitor coronatine (COR) and calix[8]arenes (CAL), nanoparticles that can host lipophilic compounds within their macrocyclic scaffold. The highest taxane production (103.5 mg.L -1 ), achieved after treatment with COR (1 μM) and CAL (10 mg.L -1 ), was 15-fold greater than in the control, and PTX represented 82% of the total taxanes analyzed. Expression levels of the flux-limiting PTX biosynthetic genes, BAPT and DBTNBT, increased after the addition of COR, confirming its elicitor action, but not CAL. The CAL treatment significantly enhanced taxane excretion, especially when production levels were increased by COR; 98% of the total taxanes were found in the culture medium after COR + CAL treatment. By forming complexes with PTX, the nanoparticles facilitated its excretion to the medium, and by protecting cells from PTX toxicity, its intra-and extra-cellular degradation may have been avoided. The addition of COR and CAL to T. media cell cultures is therefore a bio-sustainable and economically viable system to improve the yield of this important anticancer compound., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Monitoring of hydrogen peroxide (H 2 O 2 ) in living cells has high significance for understanding its functions. We herein report an enzymeless H 2 O 2 sensor consisting of a previously activated screen-printed carbon electrode modified with Pt nanoparticles electrogenerated on a supporting conductive layer of polyazure A-dodecyl sulfate. This electrode was used to investigate the dynamic process of H 2 O 2 release from living grapevine cells under different (a)biotic stresses. The modified surfaces were characterized by FESEM/EDX, EIS and cyclic voltammetry. Sensor analytical performance was studied in a cell culture medium under aerobic conditions, as required for cell survival. In relation to the synergistic effect between the metal nanoparticles and the conjugated polymer, this electrode showed good stability, excellent analytical performance combined with a rapid response (<2s) and limit of detection of 24.9 nM in the culture medium. The modified electrodes could fulfill the real-time measurement requirement of H 2 O 2 release from living plant cells to the extracellular medium operating continuously, even in experiments lasting more than 12 h. Methyl jasmonate, L-methionine, clopyralid and the fungus Botrytis cinerea were the eliciting agents chosen to induce oxidative stress in the plant cells. This work demonstrates the huge potential of this sensor for the real-time tracking of the H 2 O 2 released from living cells under different physiological conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Engineered plant cell lines have the potential to achieve enhanced metabolite production rates, providing a high-yielding source of compounds of interest. Improving the production of taxanes, pharmacologically valuable secondary metabolites of Taxus spp., is hindered by an incomplete knowledge of the taxane biosynthetic pathway. Of the five unknown steps, three are thought to involve cytochrome P450-like hydroxylases. In the current work, after an in-depth in silico characterization of four candidate enzymes proposed in a previous cDNA-AFLP assay, TB506 was selected as a candidate for the hydroxylation of the taxane side chain. A docking assay indicated TB506 is active after the attachment of the side chain based on its affinity to the ligand 3' N -dehydroxydebenzoyltaxol. Finally, the involvement of TB506 in the last hydroxylation step of the paclitaxel biosynthetic pathway was confirmed by functional assays. The identification of this hydroxylase will contribute to the development of alternative sustainable paclitaxel production systems using synthetic biology techniques., (Copyright © 2020 Sanchez-Muñoz, Perez-Mata, Almagro, Cusido, Bonfill, Palazon and Moyano.)

Elicited broccoli suspension-cultured cells (SCC) provide a useful system for obtaining bioactive compounds, including glucosinolates (GS) and phenolic compounds (PCs). In this work, coronatine (Cor) and methyl jasmonate (MJ) were used to increase the bioactive compound production in broccoli SCC. Although the use of Cor and MJ in secondary metabolite production has already been described, information concerning how elicitors affect cell metabolism is scarce. It has been suggested that Cor and MJ trigger defence reactions affecting the antioxidative metabolism. In the current study, the concentration of 0.5 μM Cor was the most effective treatment for increasing both the total antioxidant capacity (measured as ferulic acid equivalents) and glucosinolate content in broccoli SCC. The elicited broccoli SCC also showed higher polyphenol oxidase activity than the control cells. Elicitation altered the antioxidative metabolism of broccoli SCC, which displayed biochemical changes in antioxidant enzymes, a decrease in the glutathione redox state and an increase in lipid peroxidation levels. Furthermore, we studied the effect of elicitation on the protein profile and observed an induction of defence-related proteins. All of these findings suggest that elicitation not only increases bioactive compound production, but it also leads to mild oxidative stress in broccoli SCC that could be an important factor triggering the production of these compounds., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

One of the most successfully procedures used to increase the production of t -resveratrol in Vitis vinifera suspension-cultured cells is the application of cyclodextrins (CDs) and methyl jasmonate (MJ) as elicitors. In particular, β-CDs are characterized by their chemical structure which makes them special, not only by acting as elicitors, but also because they are compounds capable of trapping high added-value hydrophobic molecules such as t -resveratrol. However, the use of β-CDs as elicitors increases the production costs of this compound, making their industrial exploitation economically unfeasible. Therefore, the development of β-CDs recovery strategies is necessary to provide a viable solution to their industrial use. In this work, carboxymethylated and hydroxypropylated β-CDs have been used to form polymers using epichlorohydrin (EPI) as a cross-linking agent. The polymers were coated to Fe 3 O 4 nanoparticles and were jointly used with MJ to elicit V. vinifera suspension-cultured cells. Once elicitation experiments were finished, a magnet easily allowed the recovery of polymers, and t -resveratrol was extracted from them by using ethyl acetate. The results indicated that the production of t -resveratrol in the presence of free carboxymethyl-β-CDs was much lower than that found in the presence of carboxymethyl-β-cyclodextrins-EPI polymer coated magnetic nanoparticles. In addition, the maximal levels of t -resveratrol were found at 168 h of elicitation in the presence of 15 g/L hydroxypropyl-β-CDs polymer coated magnetic nanoparticles and MJ, and non- t -resveratrol was found in the extracellular medium, indicating that all the t -resveratrol produced by the cells and secreted into the culture medium was trapped by the polymer and extracted from it. This work also showed that polymers can be regenerated and reused during three cycles of continuous elicitation since the induction and adsorption capacity of hydroxypropyl-β-CDs polymer-coated magnetic nanoparticles after these cycles of elicitation remained high, allowing high concentrations of t -resveratrol to be obtained.

Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatment of cancer. Taxus spp. biofactories have been used for taxane production, but the lack of knowledge about the taxane biosynthetic pathway and its molecular regulation hinders their optimal function. The difficulties in introducing foreign genes in Taxus spp. genomes hinder the study of the molecular mechanisms involved in taxane production, and a new approach is required to overcome them. In this study, a reliable, simple and fast method to obtain Taxus � media protoplasts was developed, allowing their manipulation in downstream assays for the study of physiological changes in Taxus spp. cells. Using this method, Taxus protoplasts were transiently transfected for the first time, corroborating their suitability for transfection assays and the study of specific physiological responses. The two assayed transcription factors (BIS2 and TSAR2) had a positive effect on the expression of several taxane-related genes, suggesting their potential use for the improvement of taxane yields. Furthermore, the results indicate that the developed method is suitable for obtaining T. � media protoplasts for transfection with the aim of unraveling regulatory mechanisms in taxane production., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The reaction of C 60 with pregnen-20-carboxaldehyde, a biologically active synthetic steroid, by using a 1,3-dipolar cycloaddition reaction (Prato's protocol) results in the formation of pyrrolidine rings bearing a new stereogenic center on the C2 of the five-membered ring. The formation of the fullerene-steroid hybrids proceeds with preference for the Re face of the 1,3-dipole, with formation of a diastereomeric mixture in 73:15 ratio. The investigation of the chiroptical properties of these conjugates allowed determining the absolute configuration of the new fulleropyrrolidines. In addition, a thorough spectroscopical study permitted to determine the structure of the two mono-cycloadducts. The electrochemical properties of the new hybrids were also evaluated by cyclic voltammetry, both systems exhibit three quasi-reversible reduction waves which are cathodically shifted in regard to the parent C 60 . Theoretical calculations help supporting the experimental data. A conformational study combining semiempirical methods and density functional theory has predicted the most stable diastereomer. On the basis of this agreement, a possible reaction mechanism is presented. Additionally, a molecular docking simulation has been carried out using the HIV-1 protease as receptor, thus paving the way to study the possible application of these stereoisomers in biomedicine.

The use of methyl jasmonate, alone or in combination with cyclic oligosaccharides such as cyclodextrins, has proved to be a successful strategy for increasing the production of trans-resveratrol in Vitis vinifera cell cultures. However, understanding the intracellular signalling pathways involved in its production would improve the management of grapevine cells as biofactories of this high-value natural product. The results obtained herein confirm the involvement of hydrogen peroxide and nitric oxide in cyclodextrins and methyl jasmonate-induced trans-resveratrol production in grapevine cell cultures. In fact, methyl jasmonate led to maximal intracellular levels of hydrogen peroxide and nitric oxide after 24 h of treatment, but extracellular hydrogen peroxide was only detected in the culture medium when grapevine cells were treated with cyclodextrins. The results derived from the cytochemical detection of H 2 O 2 in elicited grapevine cell cultures also suggested that the combined treatment with cyclodextrins and methyl jasmonate not only increased the production of H 2 O 2 but also released cell wall fragments with electron-dense deposits. Moreover, nitric oxide was localized in all the cellular compartments, particularly in the nucleus and cytoplasmic organelles, whereas hydrogen peroxide was mainly found in cytoplasmic areas close to the cell wall, and in the nucleoplasm., (Copyright © 2019. Published by Elsevier GmbH.)

Rationale: The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG's homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers)., Objective: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers., Methods and Results: Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms from genome-wide association studies lie within noncoding regions and perturb transcription factor recognition sequences in relevant cell types. Analysis of genome-wide association studies data shows significant enrichment of risk variants for cardiovascular disease and other diseases, at ERG endothelial enhancers and super-enhancers., Conclusions: The transcription factor ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of cardiovascular disease-associated single nucleotide polymorphisms at ERG super-enhancers suggests that ERG-dependent transcription modulates disease risk.

In this work, the effect of different elicitors and culture conditions on the production of glucosinolates in broccoli cell cultures was studied. The results showed that 0.5 μM coronatine was the best elicitor for increasing glucosinolate production (205-fold increase over untreated cells after 72 h of treatment). Furthermore, the expression levels of some genes related to the biosynthetic pathway of glucosinolates as well as three Myb transcription factors also have been studied. The highest glucosinolate levels found in coronatine-treated cells were closely correlated with the highest gene expression levels of Cyp79b2, Cyp83b1, St5a, Myb51, and Myb122 after 6 h of treatment. The data shown in this study provide new insight into the key metabolic steps involved in the biosynthesis of glucosinolates, which will be of use for future applications of metabolic engineering techniques in broccoli.

Main Conclusion: Diflufenican increased 493-fold the level of phytoene. Diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cells resulted in an increased production of phytoene. This work analyzes the effect of diflufenican, an inhibitor of phytoene desaturase, on the gene expression profiles of the biosynthetic pathway of carotenoids related with the production of these compounds in carrot cell cultures. The results showed that the presence of 10 µM diflufenican in the culture medium increased phytoene levels, which was 493-fold higher than in control cells after 7 days of treatment but did not alter cell growth in carrot cell cultures. The maximal production of phytoene was reached with 10 µM diflufenican after 7 days of incubation in the presence of light and with 30 g/L sucrose in the culture medium. Moreover, diflufenican decreased the expression of phytoene synthase and phytoene desaturase genes at all the times studied. This diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cell cultures resulted in an increased production of phytoene. Our results provide new insights into the action of diflufenican in carrot cell cultures, which could represent an alternative more sustainable and environmentally friendly system to produce phytoene than those currently used.

Two new functionalized steroids containing both chloro and formyl substituents in ring A, and intended as precursors for the synthesis of hybrid systems, have been synthesized from ketosteroid precursors. 3-Chloro-2-formyl-17,17-dimethyl-18-nor-5α-androstane-2,13-diene, (I), and methyl 3-chloro-4-formyl-12-oxo-5β-cholan-3-ene-24-oate, C 26 H 37 ClO 4 , (IV), have been synthesized using Vilsmeier reactions with 17β-hydroxy-17α-methyl-5α-androstan-3-one and methyl 3,13-dioxo-5β-cholan-24-oate, respectively. These products have been fully characterized using IR spectroscopy, 1 H and 13 C NMR spectroscopy, and high-resolution mass spectrometry, and in the case of (IV), a single-crystal X-ray diffraction study. Crystal structures have also been determined for the known analogues 3-chloro-2-formyl-17-oxo-5α-androst-2-ene, C 20 H 27 ClO 2 , (II), 3-chloro-2-formyl-5α-cholest-2-ene, C 28 H 45 ClO, (III), and the absolute and relative configurations are assigned for all four compounds (I)-(IV): when the fusion between rings A and B is trans, 3-chloro-2-formyl products are formed, but when this ring fusion is cis, a 3-chloro-4-formyl product results. The formation of (I) involves not only chloroformylation at ring A, but also dehydration and the 1,2 migration of a methyl group at ring D. In each of (II), (III) and (IV), rings B and C adopt almost perfect chair conformations, while ring A adopts a half-chair conformation. Ring D adopts an envelope conformation in each of (II) and (III), albeit differently folded in the two compounds, while in (IV), it adopts a half-chair conformation. A single C-H...O hydrogen bond links the molecules of (II) into C(6) chains which are linked into sheets by means of carbonyl-carbonyl interactions. The molecules of (IV) are linked into simple C(7) chains, again by a single C-H...O hydrogen bond, but there are no direction-specific interactions in (III) that are structurally significant.