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ABSTRACT Plant phenotyping on morpho‐anatomical traits through image analysis, from microscope images to large‐scale acquisitions through remote sensing, represents a low‐invasive tool providing insight into physiological and structural trait variation, as well as plant–environment interactions. High phenotype diversity in the genus Amaranthus includes annual weed species with high invasiveness and impact on important summer crops, and nutritive grain or vegetable crops. Identification of morpho‐anatomical leaf characters at very young stages across weedy amaranths could be useful for better understanding their performance in agroecosystems. We used an innovative multi‐scale approach with phenotype analyses of about 20 single‐leaf morphometric traits of four Amaranthus species through processing confocal microscopy and camera acquisitions. The results highlight that determination of leaf traits at different investigation levels highlight species‐specific traits at a juvenile stage, which are crucial for plant development, competition and establishment. Specifically, leaf circularity and hairiness Aspect Ratio better discriminated A. tuberculatus from other species. Also, leaf DW, hairiness area and perimeter variables allowed identification of dioecious amaranth species as distinct from monoecious species. The methodology used here provides a promising, reliable and low‐impact approach for the functional characterization of phylogenetically related species and for statistical quantification of traits involved in taxonomy and biodiversity studies. [ABSTRACT FROM AUTHOR]

Placental insufficiency often correlates with fetal growth restriction (FGR), a condition that has both short- and long-term effects on the health of the newborn. In our study, we analyzed placental tissue from infants with FGR and from infants classified as small for gestational age (SGA) or appropriate for gestational age (AGA), performing comprehensive analyses that included transcriptomics and metabolomics. By examining villus tissue biopsies and 3D trophoblast organoids, we identified significant metabolic changes in placentas associated with FGR. These changes include adaptations to reduced oxygen levels and modifications in arginine metabolism, particularly within the polyamine and creatine phosphate synthesis pathways. Specifically, we found that placentas with FGR utilize arginine to produce phosphocreatine, a crucial energy reservoir for ATP production that is essential for maintaining trophoblast function. In addition, we found polyamine insufficiency in FGR placentas due to increased SAT1 expression. SAT1 facilitates the acetylation and subsequent elimination of spermine and spermidine from trophoblasts, resulting in a deficit of polyamines that cannot be compensated by arginine or polyamine supplementation alone, unless SAT1 expression is suppressed. Our study contributes significantly to the understanding of metabolic adaptations associated with placental dysfunction and provides valuable insights into potential therapeutic opportunities for the future., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Access to DNA is the first level of control in regulating gene transcription, a control that is also critical for maintaining DNA integrity. Cellular senescence is characterized by profound transcriptional rearrangements and accumulation of DNA lesions. Here, we discovered an epigenetic complex between HDAC4 and HDAC1/HDAC2 that is involved in the erase of H2BK120 acetylation. The HDAC4/HDAC1/HDAC2 complex modulates the efficiency of DNA repair by homologous recombination, through dynamic deacetylation of H2BK120. Deficiency of HDAC4 leads to accumulation of H2BK120ac, impaired recruitment of BRCA1 and CtIP to the site of lesions, accumulation of damaged DNA and senescence. In senescent cells this complex is disassembled because of increased proteasomal degradation of HDAC4. Forced expression of HDAC4 during RAS-induced senescence reduces the genomic spread of γH2AX. It also affects H2BK120ac levels, which are increased in DNA-damaged regions that accumulate during RAS-induced senescence. In summary, degradation of HDAC4 during senescence causes the accumulation of damaged DNA and contributes to the activation of the transcriptional program controlled by super-enhancers that maintains senescence., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The need to minimise the impact of phytosanitary treatments for disease control boosted researchers to implement techniques with less environmental impact. The development of technologies using molecular mechanisms based on the modulation of metabolism by short dsRNA sequences appears promising. The intrinsic fragility of polynucleotides and the high cost of these techniques can be circumvented by nanocarriers that protect the bioactive molecule enabling high efficiency delivery to the leaf surface and extending its half-life. In this work, a specific protocol was developed aiming to assess the best methodological conditions for the synthesis of low-size chitosan nanoparticles (NPs) to be loaded with nucleotides. In particular, NPs have been functionalised with partially purified Green Fluorescent Protein dsRNAs (GFP dsRNA) and their size, surface charge and nucleotide retention capacity were analysed. Final NPs were also stained with FITC and sprayed on Nicotiana benthamiana leaves to assess, by confocal microscopy, both a distribution protocol and the fate of NPs up to 6 days after application. Finally, to confirm the ability of NPs to increase the efficacy of dsRNA interference, specific tests were performed: by means of GFP dsRNA-functionalised NPs, the nucleotide permanence during time was assessed both in vitro on detached wild-type N. benthamiana leaves and in planta; lastly, the inhibition of Botrytis cinerea on single leaves was also evaluated, using a specific fungal sequence (Bc dsRNA) as the NPs' functionalising agent. The encouraging results obtained are promising in the perspective of long-lasting application of innovative treatments based on gene silencing., (© 2023 The Author(s).)

In pancreatic ductal adenocarcinomas (PDAC), the KRAS G12D -NRF2 axis controls cellular functions such as redox homeostasis and metabolism. Disruption of this axis through suppression of NRF2 leads to profound reprogramming of metabolism. Unbiased transcriptome and metabolome analyses showed that PDAC cells with disrupted KRAS G12D -NRF2 signaling ( NRF2 -/- cells) shift from aerobic glycolysis to metabolic pathways fed by amino acids. Metabolome, RNA-seq and qRT-PCR analyses revealed a blockade of the urea cycle, making NRF2 -/- cells dependent on exogenous arginine for survival. Arginine is channeled into anabolic pathways, including the synthesis of phosphocreatine, which generates an energy buffer essential for cell growth. A similar switch was observed in tumor clones that had survived FOLFIRINOX therapy or blockade of KRAS signaling. Inhibition of the creatine pathway with cyclocreatine reduced both ATP and invasion rate in 3D spheroids from NRF2 -deficient PDAC cells. Our study provides basis for the rational development of combination therapies for pancreatic cancer., Competing Interests: The authors declare that they have no conflicts of interest with the content of this article., (© 2023 The Author(s).)

Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC 50  ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1-18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4-5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1-18) on osteoblast cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Leiomyosarcomas are rare and aggressive tumors characterized by a complex karyotype. Surgical resection with or without radiotherapy and chemotherapy is the standard curative treatment. Unfortunately, a high percentage of leiomyosarcomas recurs and metastasizes. In these cases, doxorubicin and ifosfamide represent the standard treatment but with low response rates. Here, we evaluated the induction of proteotoxic stress as a possible strategy to kill leiomyosarcoma cells in a therapeutic perspective. We show that aggressive leiomyosarcomas coexist with high levels of proteotoxic stress. As a consequence, we hypothesized that leiomyosarcoma cells are vulnerable to further increases of proteotoxic stress. The small compound 2c is a strong inducer of proteotoxic stress. In leiomyosarcoma cells, it triggers cell death coupled to a profound reorganization of the mitochondrial network. By using stimulated emission depletion microscopy, we have unveiled the existence of DIABLO/SMAC clusters that are modulated by 2c. Finally, we have engineered a new version of 2c linked to polyethylene glycol though a short peptide, named 2cPP. This new prodrug is specifically activated by proteases present in the tumor microenvironment. 2cPP shows a strong antitumor activity in vivo against leiomyosarcomas and no toxicity against normal cells., (©2021 American Association for Cancer Research.)

This research was conducted to evaluate the potential use of saturated monoglyceride (MG)-based gels in the protection of probiotics upon in vitro digestion. For this purpose, a Lactobacillus rhamnosus strain was inoculated into binary and ternary systems, containing MGs, a water phase composed of an aqueous solution at controlled pH or UHT skimmed milk, and in ternary gels, sunflower oil. Gel structure characterization was initially performed just after preparation and after 14 days of storage at 4 °C by rheological, mechanical, thermal, and microscopy analyses. Afterwards, probiotic viability upon in vitro digestion was evaluated. The results highlighted that all freshly prepared samples showed good capability to protect L. rhamnosus with the exception of the binary system containing milk. However, the digestion of samples after 14 days of storage showed that the ternary system containing skimmed milk exhibited the best protection performance ensuring a L. rhamnosus viability of almost 10 6 CFU g -1 at the end of the gastrointestinal passage. Confocal microscopy results demonstrated that bacterial cells were located prevalently within the aqueous domain near the monoglycerides and protein aggregates. Under these conditions, they can simultaneously achieve physical protection and find nutrients to survive environmental stresses. These findings suggest that MG-based gels can be proposed as efficient carriers of probiotic bacteria not only during food processing and storage but also upon digestion.

A continuous state of oxidative stress during inflammation contributes to the development of 25% of human cancers. Epithelial and inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can damage DNA. ROS/RNS have biological implications in both chemoresistance and tumor recurrence. As several clinically employed anticancer drugs can generate ROS/RNS, we have addressed herein how inducible nitric oxide synthase and nitric oxide (iNOS/ • NO) affect the molecular pathways implicated in the tumor response to oxidative stress. To mimic the oxidative stress associated with chemotherapy, we used a photosensitizer (pheophorbide a) that can generate ROS/RNS in a controlled manner. We investigated how iNOS/ • NO modulates the tumor response to oxidative stress by involving the NF-κB and Nrf2 molecular pathways. We found that low levels of iNOS induce the development of a more aggressive tumor population, leading to survival, recurrence and resistance. By contrast, high levels of iNOS/ • NO sensitize tumor cells to oxidative treatment, causing cell growth arrest. Our analysis showed that NF-κB and Nrf2, which are activated in response to oxidative stress, communicate with each other through RKIP. For this critical role, RKIP could be an interesting target for anticancer drugs. Our study provides insight into the complex signaling response of cancer cells to oxidative treatments as well as new possibilities for the rational design of new therapeutic strategies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras -driven cancers.

Bacterial infection of orthopaedic implants, often caused by Staphylococcus species, may ultimately lead to implant failure. The development of infection-resistant, osteoblast-compatible biomaterials could represent an effective strategy to prevent bacterial colonization of implants, reducing the need for antibiotics. In this study, the widely used biomaterial titanium was functionalized with BMAP27(1-18), an α-helical cathelicidin antimicrobial peptide that retains potent staphylocidal activity when immobilized on agarose beads. A derivative bearing a short spacer with a free thiol at the N-terminus was coupled to silanized titanium disks via thiol-maleimide chemistry. Tethering was successful, as assessed by Contact angle, Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoelectron Spectroscopy (XPS), with an average surface mass density of 456 ng/cm 2 and a layer thickness of 3 nm. The functionalized titanium displayed antimicrobial properties against a reference strain of Staphylococcus epidermidis with well-known biofilm forming capability. Reduction of bacterial counts and morphological alterations of adhering bacteria, upon 2 h incubation, indicate a rapid contact-killing effect. The immobilized peptide was not toxic to osteoblasts, which adhered and spread better on functionalized titanium when co-cultured with bacteria, compared to non-coated surfaces. Results suggest that functionalization of titanium with BMAP27(1-18) could be promising for prevention of bacterial colonization in bone graft applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The prevention of implant-associated infection, one the most feared complications in orthopaedic surgery, remains a major clinical challenge and urges development of effective methods to prevent bacterial colonization of implanted devices. Alpha-helical antimicrobial peptides (AMPs) may be promising candidates in this respect due to their potent and broad-spectrum antimicrobial activity, their low tendency to elicit resistance and possible retention of efficacy in the immobilized state. The aim of this study was to evaluate the potential of five different helical AMPs, the cathelicidins BMAP-27 and BMAP-28, their (1-18) fragments and the rationally designed, artificial P19(9/G7) peptide, for the prevention of orthopaedic implant infections. Peptides were effective at micromolar concentrations against 22 Staphylococcus and Streptococcus isolates from orthopaedic infections, while only BMAP-28 and to a lesser extent BMAP-27 were active against Enterococcus faecalis. Peptides in solution showed activities comparable to those of cefazolin and linezolid, on a molar basis, and also a variable capacity to neutralize bacterial lipopolysaccharide, while devoid of adverse effects on MG-63 osteoblast cells at concentrations corresponding to the MIC. The (1-18) BMAP fragments and P19(9/G7) were selected for further examination, based on better selectivity indices, and showed effectiveness in the presence of hyaluronic acid and in synovial fluid, while human serum affected their activity to variable extents, with BMAP-27(1-18) best retaining activity. This peptide was immobilized on streptavidin-resin beads and retained activity against reference Staphylococcus epidermidis and Staphylococcus aureus strains, with negligible toxicity towards osteoblasts, underlining its potential for the development of infection-resistant biomaterials for orthopaedic application. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.)

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24-40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant β-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture., (Copyright © 2016 Elsevier Ltd. All rights reserved.)