Elucidating the phytoremediation potentials and ecophysiological mechanisms of indicator plants in the industrial polluted region.

The integrity of natural ecosystems, particularly in the Global South, is increasingly compromised by industrial contaminants. Our study examines the growth of plant species adapted to ecosystems impacted by heavy metal pollution, specifically focusing on their phytoremediation capabilities and tolerance to contaminants. The potential of pollution-tolerant species was evaluated in the industrial subtropical wetland of Sialkot, Pakistan. Employing quantitative ecological methods, data on vegetation, phytosociological attributes, and soil properties were gathered from 450 plots across different pollution gradients. The study pinpointed 17 key indicator species tolerating high heavy metal pollution out of 182 surveyed, using a combination of Indicator Species Analysis (ISA) and the Importance Value Index (IVI). These species demonstrated diverse capacities to extract, stabilize, and accumulate heavy metals (Cr, Zn, Cu, As, Cd, Ni, Hg, and Pb) across varying pollution zones. Notably, Cannabis sativa demonstrated substantial phytoextraction of Zn and Cd, with concentrations reaching 1977.25 µg/g and 1362.78 µg/g, respectively. Arundo donax showed marked hyperaccumulation of Cd, peaking at 410.531 µg/g. Achyranthes aspera was remarkable for its extraction and accumulation of Ni and Cu, with concentrations of 242.412 µg/g and 77.2997 µg/g, respectively. Physiological changes, such as increased proline levels in Cannabis sativa and Achyranthes aspera reaching 39.041 µg/g and 27.523 µg/g under high metal concentrations, indicated adaptation to metal stress. Declines in chlorophyll and carotenoid levels were also observed as metal contamination increased, with up to 35% reductions in some species. These findings underscore the potential efficacy of selected plant species in phytoremediation and highlight the importance of physiological responses in their tolerance to metals, providing valuable information for targeted remediation strategies in polluted ecosystems and improving environmental management and sustainable practices.

Revisiting the dipeptidyl carboxypeptidase inhibitor captopril as a source of pan anti-trypanosomatid agents.

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for continued propagation of neglected tropical diseases such as African sleeping sickness, Chagas disease and leishmaniasis respectively. Following a report that captopril targets Leishmania donovani dipeptidyl carboxypeptidase, a series of simple proline amides and captopril analogues were synthesized and found to exhibit 1-2 µM in vitro inhibition and selectivity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. The results were corroborated with computational docking studies. Arguably, the synthetic proline amides represent the structurally simplest examples of in vitro pan antiprotozoal compounds.

The Antioxidant Defense System of Tomato (Solanum lycopersicum L.) Varieties under Drought Stress and upon Post-Drought Rewatering.

Water shortage induces physiological, biochemical, and molecular alterations in plant leaves that play an essential role in plant adaptive response. The effects of drought and post-drought rewatering on the activity of antioxidant enzymes and levels of H2O2, phenolic compounds, ascorbic acid, and proline were studied in six local tomato (Solanum lycopersicum L.) varieties. The contents of H2O2 and ascorbic acid increased in all drought-exposed tomato plants and then decreased upon rewatering. The level of phenolic compounds also decreased in response to water shortage and then recovered upon rehydration, although the extent of this response was different in different varieties. The activities of ascorbate peroxidase (APX) and guaiacol peroxidase (POX) and the content of proline significantly increased in the drought-stressed plants and then decreased when the plants were rewatered. The activities of 8 constitutive APX isoforms and 2 constitutive POX isoforms varied upon exposure to drought and were observed after rewatering in all studied varieties. The information on the response of tomato plants to drought and subsequent rewatering is of great importance for screening and selection of drought-tolerant varieties, as well as for development of strategies for increasing plant productivity under adverse environmental conditions.

Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.

L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.

Targeting prolidase. A survey of the literature data to depict a structure-activity relationship frame and to address future studies for drug development.

Prolidase (EC.3.4.13.9) is a Mn+2-dependent dipeptidase that is well known to play a crucial role in several physiological and pathological processes affecting humans. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), providing a fine regulation of the homeostasis of these two amino acids. Hyperactivity or deficiency of prolidase have been clearly associated to the development and progress of several acute and chronic syndromes (e.g. chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection). Thus, targeting prolidase and modulating its activity is an intriguing field of research with a great therapeutic potential for the next future and for the design of specific and selective drugs. Prolidase can be exploited in two essential ways: as an activator of proline containing prodrugs and by direct interaction. In this latter case, few specific ligands for the title enzyme have been described, but with no reports about their structure-activity relationship. The aim of this comprehensive review is to gather all available information on prolidase targeting so far reported in the literature, to rationalize the observed data and effect into a preliminary structure-relationship picture, to comment about the effectiveness of each reported ligands, and to address future research activities providing new potential and putative natural, semisynthetic, and purely synthetic molecules able to trigger prolidase as the main biological target.

Biochemical insights into proline metabolism and its contribution to the endurant cell wall structure under metal stress.

The cell wall serves as the primary barrier against the entry of heavy metal ions into cells. However, excessive accumulation of heavy metals within plants can lead to alterations in the spatial structure and physical properties of the cell wall, thereby affecting the capacity of plants to capture heavy metals. Proline (Pro) is involved in the synthesis of the cell wall, modulating the stability and integrity of its structure. Extensins, core proteins that maintain the cell wall structure, are proline/hydroxyproline-rich glycoproteins that contain the characteristic sequence Ser-[Pro]3-5. They act as intermediates in the regulation of biological processes such as cell wall synthesis, assembly, and signal transduction, typically forming a network structure of cell wall proteins through cross-linking with pectin. This network is essential for the self-assembly expansion of the plant cell wall and plays an indispensable role in cell wall stress signal transduction through its interaction with intracellular signalling molecules. However, the mechanisms by which Pro affects the synthesis of cell wall structural proteins, cell wall assembly, and the sensing of cell wall stress under heavy metal stress remain unclear. This review, from the perspectives of biochemistry and molecular biology, comprehensively elaborates on the impact of Pro and Pro-rich proteins on the structure and function of the cell wall. These findings emphasize the mechanism by which Pro enhances the ability of the cell wall to capture heavy metals, providing new research ideas for the use of genetic engineering to manipulate cell wall synthesis and repair, thereby reducing the phytotoxicity of heavy metals.

Inhibition of ACE2-S Protein Interaction by a Short Functional Peptide with a Boomerang Structure.

Considering the high evolutionary rate and great harmfulness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is imperative to develop new pharmacological antagonists. Human angiotensin-converting enzyme-2 (ACE2) functions as a primary receptor for the spike protein (S protein) of SARS-CoV-2. Thus, a novel functional peptide, KYPAY (K5), with a boomerang structure, was developed to inhibit the interaction between ACE2 and the S protein by attaching to the ACE2 ligand-binding domain (LBD). The inhibition property of K5 was evaluated via molecular simulations, cell experiments, and adsorption kinetics analysis. The molecular simulations showed that K5 had a high affinity for ACE2 but a low affinity for the cell membrane. The umbrella sampling (US) simulations revealed a significant enhancement in the binding potential of this functional peptide to ACE2. The fluorescence microscopy and cytotoxicity experiments showed that K5 effectively prevented the interaction between ACE2 and the S protein without causing any noticeable harm to cells. Further flow cytometry research indicated that K5 successfully hindered the interaction between ACE2 and the S protein, resulting in 78% inhibition at a concentration of 100 µM. This work offers an innovative perspective on the development of functional peptides for the prevention and therapy of SARS-CoV-2.

Synergistic Catalytic Effects on Nitrogen Transformation during Biomass Pyrolysis: A Focus on Proline as a Model Compound.

To investigate the control mechanisms of NOx precursors and the synergistic effects of composite catalysts during proline pyrolysis, a systematic series of experiments was conducted utilizing composite catalysts with varying Fe-Ca ratios. Product distribution analysis was employed to elucidate the catalysts' mechanisms in reducing NOx precursor emissions. The synergistic interactions between Fe and Ca were quantitatively assessed through comparative theoretical and experimental release calculations. The results indicate that an increase in the Fe content in the catalyst led to a rise in amine concentrations from 0.9% to 2.95%, implying that Fe facilitates the generation of amine-N through ring-opening and substitution reactions. When the Fe to Ca ratio was balanced at 1:1, nitrogen predominantly participated in the formation of purines via cyclization and substitution reactions. Additionally, all composite catalysts exhibited a suppressive effect on the release of NOx precursors, attributed to their significant enhancement of solid product retention. Fe-Ca composite catalyst synergistically inhibits the release of gaseous nitrogen. Notably, the strongest synergistic effect was observed with a 1:3 Fe to Ca ratio, which reduced the release of NH3 by 38.7% and HCN by 53.6% during proline pyrolysis. This study offers valuable insights into the control of NOx precursors and the optimization of nitrogen-rich biomass pyrolysis processes.

Design, Synthesis, Nematicidal Evaluation, and Molecular Docking Study of Pyrano[3,2-c]pyridones against Meloidogyne incognita.

Root-knot nematodes pose a serious threat to crops by affecting production and quality. Over a period of time, substantial work has been done toward the development of effective and environmentally benign nematicidal compounds. However, due to the inefficiencies of previously reported synthetics in achieving the target of safe, selective, and effective treatment, it is necessary to develop new efficacious and safer nematicidal agents considering human health and environment on top priority. This work aims to highlight the efficient and convenient l-proline catalyzed synthesis of pyrano[3,2-c]pyridone and their use as potential nematicidal agents. In vitro results of larval mortality and egg hatching inhibition revealed maximum nematicidal activity against Meloidogyne incognita from compounds 15b, 15m, and 15w with LC50 values of 28.8, 46.8, and 49.18 µg/mL at 48 h, respectively. Under similar conditions, pyrano[3,2-c]pyridones derivatives 15b (LC50 = 28.8 µg/mL) was found at par with LC50 (26.92 µg/mL) of commercial nematicide carbofuran. The in vitro results were further validated with in silico studies with the most active compound 15b nematicidal within the binding to the pocket of acetylcholine esterase (AChE). In docking, binding free energy values for compound 15b were found to be -6.90 kcal/mol. Results indicated that pyrano[3,2-c]pyridone derivatives have the potential to control M. incognita.

The C-terminal proline-rich repeats of Enteropathogenic E. coli effector EspF are sufficient for the depletion of tight junction membrane proteins and interactions with early and recycling endosomes.

BACKGROUND: Enteropathogenic E. coli (EPEC) causes acute infantile diarrhea accounting for significant morbidity and mortality in developing countries. EPEC uses a type three secretion system to translocate more than twenty effectors into the host intestinal cells. At least four of these effectors, namely EspF, Map, EspG1/G2 and NleA, are reported to disrupt the intestinal tight junction barrier. We have reported earlier that the expression of EspF and Map in MDCK cells causes the depletion of the TJ membrane proteins and compromises the integrity of the intestinal barrier. In the present study, we have examined the role of the proline-rich repeats (PRRs) within the C-terminus of EspF in the depletion of the tight junction membrane proteins and identified key endocytosis markers that interact with EspF via these repeats. RESULTS: We generated mutant EspF proteins which lacked one or more proline-rich repeats (PRRs) from the N-terminus of EspF and examined the effect of their expression on the cellular localization of tight junction membrane proteins. In lysates derived from cells expressing the mutant EspF proteins, we found that the C-terminal PRRs of EspF are sufficient to cause the depletion of TJ membrane proteins. Pull-down assays revealed that the PRRs mediate interactions with the TJ adaptor proteins ZO-1 and ZO-2 as well as with the proteins involved in endocytosis such as caveolin-1, Rab5A and Rab11. CONCLUSIONS: Our study demonstrates the direct role of the proline-rich repeats of EspF in the depletion of the TJ membrane proteins and a possible involvement of the PRRs in the endocytosis of host proteins. New therapeutic strategies can target these PRR domains to prevent intestinal barrier dysfunction in EPEC infections.

Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH.

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

Expression of an engineered salt-inducible proline biosynthetic operon in a Glutamic acid Over-Producing mutant, Halomonas elongata GOP, confers increased proline yield due to enhanced growth under high-salinity conditions.

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata GOP, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.

Unraveling the metabolomic architecture of autism in a large Danish population-based cohort.

BACKGROUND: The prevalence of autism in Denmark has been increasing, reaching 1.65% among 10-year-old children, and similar trends are seen elsewhere. Although there are several factors associated with autism, including genetic, environmental, and prenatal factors, the molecular etiology of autism is largely unknown. Here, we use untargeted metabolomics to characterize the neonatal metabolome from dried blood spots collected shortly after birth. METHODS: We analyze the metabolomic profiles of a subset of a large Danish population-based cohort (iPSYCH2015) consisting of over 1400 newborns, who later are diagnosed with autism and matching controls and in two Swedish population-based cohorts comprising over 7000 adult participants. Mass spectrometry analysis was performed by a timsTOF Pro operated in QTOF mode, using data-dependent acquisition. By applying an untargeted metabolomics approach, we could reproducibly measure over 800 metabolite features. RESULTS: We detected underlying molecular perturbations across several metabolite classes that precede autism. In particular, the cyclic dipeptide cyclo-leucine-proline (FDR-adjusted p = 0.003) and the carnitine-related 5-aminovaleric acid betaine (5-AVAB) (FDR-adjusted p = 0.03), were associated with an increased probability for autism, independently of known prenatal and genetic risk factors. Analysis of genetic and dietary data in adults revealed that 5-AVAB was associated with increased habitual dietary intake of dairy (FDR-adjusted p < 0.05) and with variants near SLC22A4 and SLC22A5 (p < 5.0e - 8), coding for a transmembrane carnitine transporter protein involved in controlling intracellular carnitine levels. CONCLUSIONS: Cyclo-leucine-proline and 5-AVAB are associated with future diagnosis of autism in Danish neonates, both representing novel early biomarkers for autism. 5-AVAB is potentially modifiable and may influence carnitine homeostasis.

The Spatial Extracellular Proteomic Tumor Microenvironment Distinguishes Molecular Subtypes of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) mortality rates continue to increase faster than those of other cancer types due to high heterogeneity, which limits diagnosis and treatment. Pathological and molecular subtyping have identified that HCC tumors with poor outcomes are characterized by intratumoral collagenous accumulation. However, the translational and post-translational regulation of tumor collagen, which is critical to the outcome, remains largely unknown. Here, we investigate the spatial extracellular proteome to understand the differences associated with HCC tumors defined by Hoshida transcriptomic subtypes of poor outcome (Subtype 1; S1; n = 12) and better outcome (Subtype 3; S3; n = 24) that show differential stroma-regulated pathways. Collagen-targeted mass spectrometry imaging (MSI) with the same-tissue reference libraries, built from untargeted and targeted LC-MS/MS was used to spatially define the extracellular microenvironment from clinically-characterized, formalin-fixed, paraffin-embedded tissue sections. Collagen α-1(I) chain domains for discoidin-domain receptor and integrin binding showed distinctive spatial distribution within the tumor microenvironment. Hydroxylated proline (HYP)-containing peptides from the triple helical regions of fibrillar collagens distinguished S1 from S3 tumors. Exploratory machine learning on multiple peptides extracted from the tumor regions could distinguish S1 and S3 tumors (with an area under the receiver operating curve of ≥0.98; 95% confidence intervals between 0.976 and 1.00; and accuracies above 94%). An overall finding was that the extracellular microenvironment has a high potential to predict clinically relevant outcomes in HCC.

Phytoremediation ability of three succulent ornamental plants; cactus (Opuntia humifusa), kalanchoe (Kalanchoe blossfeldiana) and bryophyllum (Bryophyllum delagoensis) under heavy metals pollution.

The current status of environmental pollution by heavy metals (HMs) will affect the entire ecosystem components. The results obtained so far indicate that some plants can be effective in removing toxic metals from the soil. For this purpose, the phytoremediation ability of three fleshy ornamental plants; cactus (Opuntia humifusa), kalanchoe (Kalanchoe blossfeldiana) and bryophyllum (Bryophyllum delagoensis), was evaluated under the stress of HMs. These succulents are known for their remarkable adaptive capabilities, allowing them to thrive in harsh environmental conditions, including those with high levels of contaminants. Their robust nature, efficient water-use strategies, and proven potential for heavy metal accumulation made them viable candidates for investigating their phytoremediation potential. This experiment was performed as factorial based on completely randomized block design with two factors; the first factor included the type of plant in 3 levels (cactus, kalanchoe and bryophyllum) and the second one included the type of metal in 5 levels (control, silver, cadmium, lead and nickel) in 3 repetitions. The concentration of each salt used was 100 ppm. The measured parameters included stem height, relative growth, diameter, dry matter percentage of root and shoot, chlorophyll a, b and total chlorophyll, carotenoid, anthocyanin, proline, and elements of nickel, silver, lead and cadmium, as well biological concentration factor. The results showed that the highest amount of final stem height, relative growth, dry matter percentage of shoot and the highest amount of chlorophyll a and b, carotenoid and anthocyanin were obtained in bryophyllum. Also, the results of mean comparison of the data related to the effect of metal type on the plants showed that the highest amount of carotenoid, anthocyanin and biological concentration factor were induced by cadmium. On the other hand, the highest and lowest amount of proline as well anthocyanin and proline were induced by silver and lead, respectively. Totally, bryophyllum had a high resistance to HMs and the examined HMs had less effect on the growth of this plant. Cactus, among trial species, exhibited superior potential for HM absorption compared to kalanchoe and bryophyllum. The study underscores cactus as an excellent phytoremediator.

Effects of resistance exercise, collagen ingestion and circulating oestrogen concentration on collagen synthesis in a female athlete: A case report.

We investigated the effects of resistance exercise (RE), hydrolysed collagen (HC) ingestion and circulating oestrogen concentration on collagen synthesis in a naturally menstruating female CrossFit athlete. In a double-blind, randomised cross-over design, the participant (36 years; height 1.61 m; mass 82.6 kg) consumed 0 or 30 g HC prior to performing back-squat RE when endogenous circulating oestrogen concentration was low (onset of menses, OM) and high (late follicular phase, LF) during two consecutive menstrual cycles. Ten 5-mL blood samples were collected during each of the four interventions to analyse concentrations of serum 17ß-oestradiol, and biomarkers of type I collagen turnover, that is serum procollagen type I N-terminal propeptide (PINP, a biomarker of collagen synthesis) and plasma ß-isomerised C-terminal telopeptide of type I collagen (ß-CTX, a biomarker of collagen breakdown), as well as the serum concentration of 18 collagen amino acids. 17ß-Oestradiol concentration was 5-fold higher at LF (891 ± 116 pmol L-1) than OM (180 ± 13 pmol L-1). The PINP concentration × time area under the curve (AUC) was higher in the 30 g HC OM intervention (201 µg L-1 h) than the 30 g HC LF (144 µg L-1 h), 0 g HC OM (151 µg L-1 h) and 0 g HC LF (122 µg L-1 h) interventions. ß-CTX concentration decreased 1.4-fold from pre-RE to 6 h post-RE in all interventions. Thus, high circulating oestrogen concentration was associated with lower collagen synthesis following RE in this female athlete. Ingesting 30 g HC, however, augmented the collagen synthesis response at LF and particularly at OM. HIGHLIGHTS: What is the central question of this study? Does resistance exercise-induced collagen synthesis vary according to circulating oestrogen concentration in a naturally menstruating female athlete, and if so, does hydrolysed collagen ingestion have any impact? What is the main finding and its importance? Exercise-induced collagen synthesis was low when circulating oestrogen concentration was high and vice versa. However, ingesting 30 g hydrolysed collagen prior to exercise reduced the negative effect of oestrogen on collagen synthesis. As high circulating oestrogen has been associated with greater injury risk in females, supplementing exercise with hydrolysed collagen may help protect these tissues from injury.

Elicitors directed in vitro growth and production of stevioside and other secondary metabolites in Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA3) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA3 resulted in the highest accumulation of proline (0.99 µg/mL) as compared to controls (0.37 µg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.

Hepatitis C Virus Dysregulates Polyamine and Proline Metabolism and Perturbs the Urea Cycle.

Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV's imprint on cell metabolism.

Melatonin Combined with Wax Treatment Enhances Tolerance to Chilling Injury in Red Bell Pepper.

Bell peppers (Capsicum annuum L.) are prone to chilling injury (CI) when stored at temperatures below 7 °C. Melatonin, a natural plant regulator, plays a critical role in defending against different pre- and post-harvest abiotic stresses, including those associated with cold storage. This study aimed to assess the effects of applying exogenous melatonin alone and in combination with a commercial wax on the CI tolerance, postharvest life, and potential biomarker search of red bell peppers. In the initial experiment, the effective melatonin concentration to reduce CI effects was determined. Peppers were sprayed with either distilled water (control) or a melatonin aqueous solution (M100 = 100 µM or M500 = 500 µM) and then stored for 33 d at 4 °C, followed by 2 d at 20 °C. The M500 treatment proved to be more effective in reducing fruit CI incidence (superficial scalds) and metabolic rate, while weight loss, softening, and color were comparable to the control. A second experiment assessed the potential synergistic effects of a combined melatonin and commercial wax treatment on pepper CI and quality. Fruits were sprayed with distilled water (control), melatonin (M500), commercial wax (Wax), or the combined treatment (Wax + M500) and stored for 28 d at 4 °C, followed by 2 d at 20 °C. The Wax + M500 was the most effective in significantly reducing the incidence of fruit CI symptoms and calyx fungal infection. Furthermore, this combined treatment enhanced fruit weight loss prevention compared with individual melatonin or wax treatment. Also, Wax + M500-treated peppers exhibited notable proline accumulation, indicative of a metabolic response counteracting the cold effects, resulting in better fruit stress acclimation. This treatment also preserved the peppers' color and antioxidant capacity. In summary, these findings highlight the suitability of applying a combined Wax + M500 treatment as a highly effective strategy to enhance the CI tolerance of peppers and extend their postharvest life.

L-proline determination by molecularly imprinted nanoparticles: A potential nanoscale tool for the diagnosis of metabolic disorders.

Detecting and quantifying amino acids is vital in biochemical analyses, especially for diagnosing metabolic disorders. L-proline, among these amino acids, holds significant relevance for various metabolic disorders in living organisms, particularly in humans. hyperprolinemia arises when ineffective breakdown of L-proline occurs due to enzyme deficiencies, leading to its accumulation in the body and underscoring the need for precise monitoring. To address this challenge, molecular imprinting offers a reliable single-step technique for detecting target molecules like proteins, peptides, amino acids, or ions with high selectivity. Moreover, nanoparticles, with significant surface area-to-volume ratios, enable high-level mass transfer and binding kinetics, making them ideal for nano-scale sensitive applications. In this study, 2-hydroxyethyl methacrylate-based molecularly imprinted nanoparticles were synthesized via mini-emulsion polymerization, combining the advantages of molecular imprinting technique and nanoparticles for the specific recognition of L-proline, and were well-characterized by Scanning Electron Microscopy, zeta-sizer particle size analysis, and Fourier Transform Infrared Spectroscopy. Based on zeta-sizer analysis, the estimated diameters of L-proline-imprinted and non-imprinted nanoparticles (Pro-MIPs and NIPs) were determined to be approximately 27.51 nm and 20.66 nm, respectively. The adsorption of L-proline onto nanoparticles from aqueous solutions was investigated in a batch system, and the maximum L-proline adsorption capacity was determined to be 26.58 mg/g for Pro-MIPs and 4.65 mg/g for and NIPs. The selectivity of Pro-MIPs was assessed using Liquid Chromatography-Tandem Mass Spectrometry, even in human serum and in the presence of competing molecules (L-histidine and L-phenylalanine). Additionally, Pro-MIPs maintained their adsorption capacity through up to 10 adsorption-desorption cycles without significant decrease.