Objective.To develop and optimise a dedicated low-energy proton beamline at the Centre for Micro-Analysis of Materials (CMAM, Madrid, Spain) for radiobiological applications.Approach.An automated irradiation system was implemented, integrating Gafchromic EBT3 radiochromic film dosimetry corrected for linear energy transfer dependent quenching and post-irradiation darkening. Dosimetric calibration was performed using multichannel analysis, and beam performance was systematically evaluated as a function of distance, raster scanning area, beam intensity and reproducibility.Main results.Optimal operating conditions were identified at moderate beam currents (⩽1 nA) and scanning areas of40×40to50×50 mm2, yielding homogeneous dose distributions with reproducibility better than 8%. The dosimetric protocol demonstrated linearity across clinically relevant dose ranges and allowed a reliable correlation between irradiation parameters and absorbed dose. Proof-of-concept experiments on U-87 MG glioblastoma cells confirmed the system's ability to deliver controlled and biologically effective proton exposures, as demonstrated by clonogenic survival assays.Significance.These results establish the CMAM implantation beamline as a robust and versatile platform for preclinical proton radiobiology, providing accurate dosimetric control and supporting investigations of relative biological efficacy. The system facilitates translational advances in proton radiobiology, bridging physical and biological studies in low-energy proton irradiation.

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurological disorder caused by mutations in the SACS gene, resulting in the loss of sacsin function. Sacsin is a multidomain protein that plays key roles in chaperone regulation, protein quality control, and neurofilament dynamics. Sacsin deficiency leads to disruption of intermediate filament and mitochondrial networks. S100B, a multifunctional brain-enriched protein, exhibits protective neuroprotective functions that include chaperone activity and interactions with filament proteins and mitochondria. In this study, we used an established astroglial C6 cell model of ARSACS to investigate the potential compensatory effects of S100B on sacsin loss with respect to neurofilament integrity and mitochondrial morphological and functional hallmarks. Our results demonstrate that sacsin deletion induces S100B upregulation at both mRNA and protein levels, with the S100B protein colocalizing with perinuclear nestin aggregates and filamentous mitochondria networks. Genetic silencing and pharmacological inhibition of S100B exacerbate filament protein aggregation and mitochondrial defects, while supplementation with exogenous recombinant S100B improves ARSACS hallmarks, including decreased nestin aggregates. These findings provide evidence for functional compensation of sacsin loss by S100B in glial cells, and suggests a potential role for glial cells in ARSACS.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a movement disorder caused by loss-offunction mutations in the sacsin gene. The most common hallmark of this disease is the disruption of intermediate filament networks in cells as diverse as neurons, kidney cells, fibroblasts, astroglia and microglia. Intermediate filaments are the main filaments responsible for the mechanical and viscoelastic properties of cells and tissues, but these have never been investigated in the context of ARSACS. Here, we analyzed the consequences of sacsin loss on the mechanical functions of astroglial-like C6 cells. The phenotype of C6Sacs- /- cells was analyzed by immunocytochemistry, electron microscopy, mass spectrometry, atomic force microscopy and motility/proliferation assays. C6Sacs- /- cells presented an abnormal cytoskeletal and organelle distribution, global proteome alterations linked to cell motility and mechanics, a significant decrease in cell elasticity in the cytoplasm, and a striking reduction in cell motility. These mechanical alterations in glial-like cells could be especially relevant for neuroinflammation and glial scar formation upon CNS injury. Our results support a possible role for alterations in glial functions in ARSACS and provide new tools for understanding the glial-specific mechanisms involved in this movement disorder.

center dot Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare disorder caused by loss-of-function mutations in the sacsin chaperone. center dot Sacsin was initially described as a neuronal protein but is also found in astroglia and microglia. center dot We developed and characterized a new microglial cell model of ARSACS based on human HMC3 cells.. center dot HMC3Sacs- /- cells show aberrant distribution of vimentin and mitochondrial networks, and other alterations with potential consequences on neuroinflammation. center dot Our results support a possible role for microglial cells in ARSACS and provide a new tool to understand glial-specific mechanisms involved in this pathology

ABSTRACT LAMA2 -congenital muscular dystrophy (LAMA2-CMD) is the most common congenital muscular dystrophy. This often-lethal disease is triggered by mutations in LAMA2 , coding for laminin-α2 chain, a key extracellular matrix (ECM) component, prevalent in the skeletal muscle. Several phenotypes have been associated with LAMA2-CMD, however, it is not yet known what mechanisms are faulty, right at disease onset in utero . Using the dy W mouse model of LAMA2-CMD we showed that the disease onset is characterized by a profound downregulation of gene expression, with a marked effect on cytoskeletal organization, myoblast differentiation and fusion and altered DNA repair and oxidative stress responses. Concordantly, we found that Lama2 -deficient myoblast cells displayed proliferation and differentiation defects, increased oxidative stress and DNA damage. Together, our findings provide unique insights into the processes dependent on laminin-α2 chain during muscle development, revealing its critical importance to maintain muscle cell homeostasis already at fetal stages.

The FLT3-ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1 alpha, one of the main proteins of the protein folding machinery at the ER. Overexpression of ERO1 alpha resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non-mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1 alpha depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.

LAMA2, coding for the laminin-α2 chain, is a crucial ECM component, particularly abundant in skeletal muscle. Mutations inLAMA2trigger the often-lethalLAMA2-congenital muscular dystrophy (LAMA2-CMD). Various phenotypes have been linked to LAMA2-CMD; nevertheless, the precise mechanisms that malfunction during disease onset in utero remain unknown. We generatedLama2-deficient C2C12 cells and found thatLama2-deficient myoblasts display proliferation, differentiation, and fusion defects, DNA damage, oxidative stress, and mitochondrial dysfunction. Moreover, fetal myoblasts isolated from thedyWmouse model of LAMA2-CMD display impaired differentiation and fusion in vitro. We also showed that disease onset during fetal development is characterized by a significant down-regulation of gene expression in muscle fibers, causing pronounced effects on cytoskeletal organization, muscle differentiation, and altered DNA repair and oxidative stress responses. Together, our findings provide unique insights into the critical importance of the laminin-α2 chain for muscle differentiation and muscle cell homeostasis.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a developmental and degenerative disorder caused by loss-of-function mutations in the gene that codifies for the sacsin chaperone. Sacsin was initially described as a neuronal protein but is found in various cell types, including astroglial, microglial, kidney, and skin cell lines. We and others have shown that virtually all cell and animal models of ARSACS show disruption of intermediate filament (IF) cytoskeleton and organelle distribution. This article extends previous observations from our lab on the C6 astroglial-like model and describes the development and characterization of a new human microglial cell model based on HMC3 cells. HMC3 cells knocked out for sacsin show similar alterations to C6 astroglial-like cells: aberrant distribution of IF network and organelles, downregulation of developmental transcription factors STAT3 and Smad1, and alterations in autophagy machinery and markers of intracellular stress, resulting in morphological changes. Our results extend previous observations suggesting a possible role for glial cells in ARSACS and provide new tools to understand the glial-specific mechanisms involved in this pathology. ### Competing Interest Statement The authors have declared no competing interest.

center dot Loss-of-function mutations in the NKX6-2 transcription factor are linked to autosomal recessive spastic ataxia 8 (SPAX8).center dot SPAX8-related mutations show lower levels of protein expression, potentially due to nonsense -mediated mRNA decay (NMD).center dot We developed new molecular tools to study SPAX8-related NKX6-2 mutants in living cells, which are not targeted by NMD.center dot SPAX8-causing mutations lose their exclusive nuclear localization, display lower protein levels, or aggregate.center dot Our results indicate that at least some forms of SPAX8 belong to the superfamily of protein misfolding disorders.

Glioblastoma (GBM) treatment is extremely challenging due to the high complexity of the tumor. It is one of the tumors in which a subpopulation of highly resistant glioma initiating cells (GICs) has been clearly identified. Thus, understanding the differences between GICs and tumor bulk cells is therefore essential to move to less conventional but more efficient approaches. It was found that, unlike their differentiated progeny, GICs survival and maintenance of stem cell properties depend on mitochondrial metabolism. GICs present higher glucose uptake and mitochondrial membrane potential and reduced lactate dehydrogenase activity, being more sensitive to mitochondrial inhibition than their differentiated counterparts. Calcium flux to the mitochondria appears to play an essential role in the maintenance of this distinct metabolic phenotype with a decrease in the expression of voltage‑dependent anionic channel (VDAC) and Grp75, two of the proteins of the IP3R‑Grp75‑VDAC complex that transfers calcium from the endoplasmic reticulum (ER) to the mitochondria. Disruption of ER homeostasis using ER stress inducers or inhibition of ER‑mitochondrial contact sites using the Grp75 inhibitor MKT‑077 resulted in cytotoxicity of GICs and loss of stemness. Moreover, MKT‑077 also potentiates the effect of temozolomide, current treatment for glioblastoma. In summary, the present data indicated that ER‑mitochondrial homeostasis is essential for regulation of GICs glucose metabolism and survival.

Platinum(II) complexes bearing N-heterocyclic carbenes based guanosine and caffeine have been synthesized by unassisted C-H oxidative addition, leading to the corresponding trans-hydride complexes. Platinum guanosine derivatives bearing triflate as counterion or bromide instead of hydride as co-ligand were also synthesized to facilitate correlation between structure and activity. The hydride compounds show high antiproliferative activity against all cell lines (TC-71, MV-4-11, U-937 and A-172). Methyl Guanosine complex 3, bearing a hydride ligand, is up to 30 times more active than compound 4, with a bromide in the same position. Changing the counterion has no significant effect in antiproliferative activity. Increasing bulkiness at N7, with an isopropyl group (compound 6), allows to maintain the antiproliferative activity while decreasing toxicity for non-cancer cells. Compound 6 leads to an increase in endoplasmic reticulum and autophagy markers on TC71 and MV-4-11 cancer cells, induces reductive stress and increases glutathione levels in cancer cells but not in non-cancer cell line HEK-293.

Background: Acute myeloid leukemia (AML) is the most common myeloid tumour in adults. ITD mutations in the FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This mutation prevents the correct processing of this receptor in the endoplasmic reticulum (ER) and it is retained in this organelle. In addition to being a cellular compartment for protein synthesis and processing, the ER plays an important role in the regulation of tumor metabolism, specifically mitochondrial metabolism, through the mitochondria-associated membranes (MAM). Interestingly, we and other have previously described differences in tumor glucose metabolism in FLT3 wild-type and ITD AML cell lines. Aims: The main objective of our study was to study if the accumulation of FLT3-ITD affects the functioning of the ER, and if it has any role in the regulation of glucose metabolism in AML. Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, ROS, GSH levels, mitochondrial membrane potential and calcium levels were determined by Thioflavin T, DCFH-DA, Thioltracker Violet, Rhodamine and Rhod-2 fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: The ER depends on GSH to neutralize reactive oxygen species (ROS) produced in the process of protein folding. We found FLT3-ITD cell lines to have lower levels of protein aggregates, ROS and GSH, additionally to greater levels of ERO1, an ER protein folding oxidorreductase. We verified that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol, compared to wild-type cell lines, which showed high levels of cell death after the treatment. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We found FLT3-ITD cell lines to have lower levels of UPR proteins (PERK and IRE), together with the main chaperone in the ER, BiP. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the mutated FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell line carrying the normal or mutated FLT3 receptor. In agreement with results in human cell lines, we found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cells. The IP3R and GRP75 are proteins involved in MAM formation and promote oxidative phosphorylation through the transfer of calcium from the ER to the mitochondria. We found these proteins to be overexpressed in AML cell lines, as well as the BaF3 cell model, carrying the wild-type FLT3 receptor together with greater mitochondrial activity, reflected by increased levels of mitochondrial calcium, mitochondria membrane potential and lower cytotoxic response to oxamate (LDH inhibitor). Summary/Conclusion: In this study we describe that AML cell lines carrying the ITD mutation have more efficient protein folding mechanisms in which ERO1 seems to play an important role. Additionally, we show that the ER could be involved in the regulation of mitochondria glucose metabolism by directly interacting with this organelle through MAM. Both processes could be directly regulated by the FLT3 receptor. More studies are needed to fully understand this process in view of describing new therapeutic targets. Keywords: Mitochondria, FLT3, Acute myeloid leukemia, Signaling

STAT3 is a pleiotropic transcription factor overactivated in 70% of solid tumours. We have recently reported that inactivating mutations on residues susceptible to post-translational modifications (PTMs) in only one of the monomers (i.e. asymmetric) caused changes in the cellular distribution of STAT3 homodimers. Here, we used more controlled experimental conditions, i.e. without the interference of endogenous STAT3 (STAT3-/- HeLa cells) and in the presence of a defined cytokine stimulus (Leukemia Inhibitory Factor, LIF), to provide further evidence that asymmetric PTMs affect the nuclear translocation of STAT3 homodimers. Time-lapse microscopy for 20 min after LIF stimulation showed that S727 dephosphorylation (S727A) and K685 inactivation (K685R) slightly enhanced the nuclear translocation of STAT3 homodimers, while K49 inactivation (K49R) delayed STAT3 nuclear translocation. Our findings suggest that asymmetrically modified STAT3 homodimers could be a new level of STAT3 regulation and, therefore, a potential target for cancer therapy.

Proteomic characterisation of cell lines expressing different variants of STAT3 dimers (WT-WT, WT-Y507F, Y507F-Y507F, with and without LIF stimulation).

The synthesis and base pairing properties of platinum complexes based on uridine and deoxyuridine nucleosides are described. The synthesis was performed by C–I oxidative addition with protected and unprotected nucleosides. The metallated compounds feature an agostic interaction at H6. Uridine complexes undergo self-base pairing and also establish base pairs with adenosine. The formation of an intermolecular N-H-Pt bond is also observed

Abstract Purpose: Glioblastoma (GBM) treatment is extremely challenging due to the high complexity of the tumor, being one of the tumors in which a subpopulation of highly resistant cancer initiating cells (GICs) has been clearly identified. Thus, understanding the differences between GICs and tumor bulk cells is therefore essential to move to less conventional but more efficient approaches. Methods: fluorimetry was used to measure glucose uptake, mitochondrial calcium, intracellular oxidants and mitochondrial membrane potential. Spectrophotometry was used to monitor lactate dehydrogenase activity. Self-renewal was determined by the limiting dilution assay and cell death by trypan blue exclusion assays. Protein expression was determined by western blot while gene expression was determined by real-time PCR. Results: We found that, unlike their differentiated progeny, GICs survival and stemness depend on mitochondrial metabolism. GICs present higher glucose uptake and mitochondrial membrane potential and less LDH activity, being more sensitive to mitochondrial inhibition than their differentiated counterparts. Calcium flux seems to play an essential role in the maintenance of this distinct metabolic phenotype with a decrease in the expression of VDAC and Grp75, two of the main proteins in the transfer of calcium from endoplasmic reticulum (ER) to the mitochondria. Disruption of ER homeostasis using ER stress inducers or inhibition of ER-mitochondrial contact sites using the Grp75 inhibitor MKT-707 resulted in GICs cytotoxicity and loss of stemness. Moreover, MKT-077 also potentiates the effect of temozolomide, current treatment for glioblastoma. Conclusions: our data indicates that ER-mitochondrial homeostasis is essential for regulation of GICs metabolism, survival and stemness.

OBJECTIVE The objective of this review is to examine the effectiveness of oral hygiene care in the management of oral symptoms of cancer patients under specialist palliative care and the patients' experience of such symptoms and care. INTRODUCTION Oral symptoms, such as xerostomia, mouth pain or dysgeusia, are highly prevalent in specialist palliative care cancer patients and have a negative effect on their quality of life. Oral hygiene care can manage oral symptoms and could be improved with a more systematized approach, adequate guidelines, and training to properly integrate oral hygiene care into the care provided in specialist palliative care. INCLUSION CRITERIA This review will consider quantitative, qualitative, and mixed methods studies on the effectiveness and experience of oral hygiene care intended to manage oral symptoms of adult cancer patients aged 18 years or older, diagnosed with any type of cancer, under specialist palliative care. METHODS The search will be conducted in MEDLINE (PubMed), CINAHL (EBSCO), Cochrane Central Register of Controlled Trials, Dentistry and Oral Sciences Source (EBSCO), and MedicLatina (EBSCO). Sources of unpublished studies and gray literature to be searched will include Networked Digital Library of Theses and Dissertations and Repositórios Científicos de Acesso Aberto de Portugal. Studies in English, Portuguese, and Spanish from 2000 to the present will be considered. Methodological quality will be assessed and data will be extracted. Synthesis and integration will follow the JBI segregated approach for mixed methods reviews. REVIEW REGISTRATION PROSPERO CRD42023400554.

The synthesis and base pairing properties of platinum complexes based on uridine and deoxyuridine nucleosides and preliminary studies of their antiproliferative activity are described. Platinum(II) uridine and deoxyuridine complexes were synthesized by C-I oxidative addition to Pt(0)(PPh3)4. First, the synthesis was performed with protected nucleosides to generate complexes 1 and 2, which were deprotected under basic conditions, affording complexes 3 and 4 in good yields. The synthesis with the unprotected nucleosides was also performed and provided complexes 3 and 4 effectively. Base pairing interactions were measured for complex 1, either for self-base pairing or for the Watson-Crick base pair. Complex 1 undergoes self-base pairing in CDCl3, and this aggregation was found not to be dependent on metalation. Contrastingly, for the Watson-Crick base pair with adenine, base pairing was also observed, but metalation was found to affect hydrogen bonding considerably. Complexes 3 and 4 and the corresponding ligand precursors were evaluated for their antiproliferative activity against human glioblastoma cell line U-251. The compounds showed IC50 values of 3.30 (3) and 1.84 (4) μM but are also toxic for nontumorous cell lines.