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Neuroinflammation is one of the core pathological features of Alzheimer’s disease (AD) as both amyloid β (Aβ) and tau monomers and oligomers can trigger the long-term pro-inflammatory phenotype of microglial cells with consequent overactivation of the inflammasomes. To investigate the NLRP1 inflammasome activation in AD, we analyzed the expression of NLRP1, ASC, cleaved gasdermin (cGSDMD), and active caspase-6 (CASP-6) proteins in each hippocampal subdivision (hilar part of CA3, CA2/3, CA1, subiculum) of postmortem tissue of 9 cognitively healthy controls (HC) and 11 AD patients whose disease duration varied from 3 to 7 years after the clinical diagnosis. The total number of neurons, along with the total number of neurofibrillary tangles (NFTs), were estimated in Nissl- and adjacent modified Bielschowsky-stained sections, respectively, using the optical disector method. The same 9 HC and 11 AD cases were additionally semiquantitatively analyzed for expression of IBA1, HLA-DR, and CD68 microglial markers. Our results show that the expression of NLRP1, ASC, and CASP-6 is present in a significantly greater number of hippocampal formation neurons in AD brains compared to controls, suggesting that the NLRP1 inflammasome is more active in the AD brain. None of the investigated inflammasome and microglial markers were found to correlate with the age of the subjects or the duration of AD. However, besides positive correlations with microglial IBA1 expression in the subiculum and with microglial CD68 expression in the CA1 field and subiculum in the AD group, the overall NLRP1 expression in the hippocampal formation was positively correlated with the number of NFTs, thus providing a causal link between neuroinflammation and neurofibrillary degeneration. The accumulation of AT8-immunoreactive phosphorylated tau proteins that we observed at nuclear pores of large pyramidal neurons of the Ammon’s horn further supports their role in the extent of neuronal dysfunction and degeneration in AD. This is important because unlike fibrillar amyloid-β deposits that are not related to dementia severity, total NFTs and neuron numbers in the hippocampal formation, especially in the CA1 field, are the best correlates of cognitive deterioration in both human brain aging and AD. Our findings also support the notion that the CA2 field vulnerability is strongly linked to specific susceptibilities to different tauopathies, including primary age-related tauopathy. Altogether, these findings contrast with reports of nonsignificant microglial activation in aged nonhuman primates and indicate that susceptibility to inflammasome activation may render the human brain comparatively more vulnerable to neurodegenerative changes and AD. In conclusion, our results confirm a key role of NLRP1 inflammasome in AD pathogenesis and suggest NLRP1 as a potential diagnostic marker and therapeutic target to slow or prevent AD progression.

Summary: Magnetic nanoparticles can be functionalized in many ways for biomedical applications. Here, we combine four advantageous features in a novel Fe-Pt-Yb2O3 core-shell nanoparticle. (a) The nanoparticles have a size of 10 nm allowing them to diffuse through neuronal tissue. (b) The particles are superparamagnetic after synthesis and ferromagnetic after annealing, enabling directional control by magnetic fields, enhance NMRI contrast, and hyperthermia treatment. (c) After neutron-activation of the shell, they carry low-energetic, short half-life β-radiation from 175Yb, 177Yb, and 177Lu. (d) Additionally, the particles can be optically visualized by plasmonic excitation and luminescence. To demonstrate the potential of the particles for cancer treatment, we exposed cultured human glioblastoma cells (LN-18) to non-activated and activated particles to confirm that the particles are internalized, and that the β-radiation of the radioisotopes incorporated in the neutron-activated shell of the nanoparticles kills more than 98% of the LN-18 cancer cells, promising for future anti-cancer applications.

BackgroundPast research indicates a higher prevalence, incidence, and severe clinical manifestations of alpha-synucleinopathies in men, leading to a suggestion of neuroprotective properties of female sex hormones (especially estrogen). The potential pathomechanisms of any such effect on alpha-synucleinopathies, however, are far from understood. With that aim, we undertook to systematically review, and to critically assess, contemporary evidence on sex and gender differences in alpha-synucleinopathies using a bench-to-bedside approach.MethodsIn this systematic review, studies investigating sex and gender differences in alpha-synucleinopathies (Rapid Eye Movement (REM) Behavior Disorder (RBD), Parkinson’s Disease (PD), Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA)) from 2012 to 2022 were identified using electronic database searches of PubMed, Embase and Ovid.ResultsOne hundred sixty-two studies were included; 5 RBD, 6 MSA, 20 DLB and 131 PD studies. Overall, there is conclusive evidence to suggest sex-and gender-specific manifestation in demographics, biomarkers, genetics, clinical features, interventions, and quality of life in alpha-synucleinopathies. Only limited data exists on the effects of distinct sex hormones, with majority of studies concentrating on estrogen and its speculated neuroprotective effects.ConclusionFuture studies disentangling the underlying sex-specific mechanisms of alpha-synucleinopathies are urgently needed in order to enable novel sex-specific therapeutics.

BackgroundThe presence of visual imagery in dreams of congenitally blind people has long been a matter of substantial controversy. We set to systematically review body of published work on the presence and nature of oneiric visuo-spatial impressions in congenitally and early blind subjects across different areas of research, from experimental psychology, functional neuroimaging, sensory substitution, and sleep research.MethodsRelevant studies were identified using the following databases: EMBASE, MEDLINE and PsychINFO.ResultsStudies using diverse imaging techniques and sensory substitution devices broadly suggest that the “blind” occipital cortex may be able to integrate non-visual sensory inputs, and thus possibly also generate visuo-spatial impressions. Visual impressions have also been reported by blind subjects who had near-death or out-of-body experiences.ConclusionDeciphering the mechanistic nature of these visual impression could open new possibility in utilization of neuroplasticity and its potential role for treatment of neurodisability.

Abstract Rapid eye movement (REM) sleep behaviour disorder (RBD) is a REM parasomnia that often predicts the later occurrence of alpha-synucleinopathies. Variants in the gene encoding for the lysosomal enzyme glucocerebrosidase, GBA, strongly increase the risk of RBD. In a GBA1-mouse model recently shown to mimic prodromal stages of α-synucleinopathy, we now demonstrate striking REM and NREM electroencephalographic sleep abnormalities accompanied by distinct structural changes in the more widespread sleep neurocircuitry.

It is unclear to what extent the absence of vision affects the sensory sensitivity for oneiric construction. Similarly, the presence of visual imagery in the mentation of dreams of congenitally blind people has been largely disputed. We investigate the presence and nature of oneiric visuo-spatial impressions by analysing 180 dreams of seven congenitally blind people identified from the online database DreamBank. A higher presence of auditory, haptic, olfactory, and gustatory sensation in dreams of congenitally blind people was demonstrated, when compared to normally sighted individuals. Nonetheless, oneiric visual imagery in reports of congenitally blind subjects was also noted, in opposition to some previous studies, and raising questions about the possible underlying neuro-mechanisms.

Background Inflammatory skin reactions and skin alterations are still a potential side effect in radiation therapy (RT), which also need attention for patients’ health care. Method In a pre-clinical study we consider alterations in irradiated in-vitro skin models of epidermal and dermal layers. Typical dose regimes in radiation therapy are applied for irradiation. For non-invasive imaging and characterization optical coherence tomography (OCT) is used. Histological staining method is additionally applied for comparison and discussion. Results Structural features, such as keratinization, modifications in epidermal cell layer thickness and disorder in the layering—as indications for reactions to ionizing radiation and aging—could be observed by means of OCT and confirmed by histology. We were able to recognize known RT induced changes such as hyper-keratosis, acantholysis, and epidermal hyperplasia as well as disruption and/or demarcation of the dermo-epidermal junction. Conclusion The results may pave the way for OCT to be considered as a possible adjunctive tool to detect and monitor early skin inflammation and side effects of radiotherapy, thus supporting patient healthcare in the future.

Abstract Synaptic glycoprotein neuroplastin is involved in synaptic plasticity and complex molecular events underlying learning and memory. Studies in mice and rats suggest that neuroplastin is essential for cognition, as it is needed for long-term potentiation and associative memory formation. Recently, it was found that some of the effects of neuroplastin are related to regulation of calcium homeostasis through interactions with plasma membrane calcium ATPases. Neuroplastin is increasingly seen as a key factor in complex brain functions, but studies in humans remain scarce. Here we summarize present knowledge about neuroplastin in human tissues and argue its genetic association with cortical thickness, intelligence, schizophrenia, and autism; specific immunolocalization depicting hippocampal trisynaptic pathway; potential role in tissue compensatory response in neurodegeneration; and high, almost housekeeping, level of spatio-temporal gene expression in the human brain. We also propose that neuroplastin acts as a housekeeper of neuroplasticity, and that it may be considered as an important novel cognition-related molecule in humans. Several promising directions for future investigations are suggested, which may complete our understanding of neuroplastin actions in molecular basis of human cognition.

This study was designed in a rat model to determine the hallmarks of possible permanent behavioral and structural brain alterations after a single moderate hypoxic insult. Eighty-two Wistar Han (RccHan: WIST) rats were randomly subjected to hypoxia (pO2 73 mmHg/2 h) or normoxia at the first postnatal day. The substantially increased blood lactate, a significantly decreased cytochrome-C-oxygenase expression in the brain, and depleted subventricular zone suggested a high vulnerability of subset of cell populations to oxidative stress and consequent tissue response even after a single, moderate, hypoxic event. The results of behavioral tests (open-field, hole-board, social-choice, and T-maze) applied at the 30–45th and 70–85th postnatal days revealed significant hyperactivity and a slower pace of learning in rats subjected to perinatal hypoxia. At 3.5 months after hypoxic insult, the histochemical examination demonstrated a significantly increased number of specific extracellular matrix—perineuronal nets and increased parvalbumin expression in a subpopulation of interneurons in the medial and retrosplenial cingulate cortex of these animals. Conclusively, moderate perinatal hypoxia in rats causes a long-lasting reorganization of the connectivity in the cingulate cortex and consequent alterations of related behavioral and cognitive abilities. This non-invasive hypoxia model in the rat successfully and complementarily models the moderate perinatal hypoxic injury in fetuses and prematurely born human babies and may enhance future research into new diagnostic and therapeutic strategies for perinatal medicine.

Gangliosides, amphiphilic glycosphingolipids, tend to associate laterally with other membrane constituents and undergo extensive interactions with membrane proteins in cis or trans configurations. Studies of human diseases resulting from mutations in the ganglioside biosynthesis pathway and research on transgenic mice with the same mutations implicate gangliosides in the pathogenesis of epilepsy. Gangliosides are reported to affect the activity of the Na+/K+-ATPase, the ubiquitously expressed plasma membrane pump responsible for the stabilization of the resting membrane potential by hyperpolarization, firing up the action potential and ion homeostasis. Impaired Na+/K+-ATPase activity has also been hypothesized to cause seizures by several mechanisms. In this review we present different epileptic phenotypes that are caused by impaired activity of Na+/K+-ATPase or changed membrane ganglioside composition. We further discuss how gangliosides may influence Na+/K+-ATPase activity by acting as lipid sorting machinery providing the optimal stage for Na+/K+-ATPase function. By establishing a distinct lipid environment, together with other membrane lipids, gangliosides possibly modulate Na+/K+-ATPase activity and aid in “starting up” and “turning off” this vital pump. Therefore, structural changes of neuronal membranes caused by altered ganglioside composition can be a contributing factor leading to aberrant Na+/K+-ATPase activity and ion imbalance priming neurons for pathological firing.

In this study, we developed a high-resolution tandem mass spectrometry (HR MS) approach to assess presumed changes in gangliosidome of a human hippocampus affected by temporal lobe epilepsy (TLE) in comparison with a normal hippocampus. Gangliosides, membrane glycolipids, are particularly diverse and abundant in the human brain, and participate in ion transport and modulation of neuronal excitability. Changes in structural ganglioside pattern potentially linked to TLE molecular pathogenesis have not been explored in detail. Aiming to characterize TLE-specific gangliosidome, we analyzed the native gangliosides purified from a human hippocampal tissue sample affected by TLE and a control hippocampus using HR MS. Marked differences of ganglioside expression were shown in TLE vs. control, particularly with respect to the sialylation degree of components, discovered as a characteristic feature of TLE. Another major finding is the occurrence of tetrasialofucogangliosides in TLE and species modified by either O-acetylation or CH3COO−. Structural analysis by higher-energy collisional dissociation (HCD) MS/MS gave rise to fragmentation patterns implying that the GQ1b (d18:1/18:0) isomer is specifically associated with TLE. Further investigation in a larger sample is needed in order to confirm the discovery of ganglioside structures specifically expressed in human TLE and to provide information on the probable role of gangliosides in the molecular events underlying seizures.

This study was a phase III, multicenter, double-blind, randomized, placebo-controlled trial to evaluate the safety and immunogenicity of a seasonal trivalent split, inactivated influenza vaccine (TIV) in healthy Serbian adults between the ages of 18 and 65 years. This egg-based vaccine was manufactured by the Institute of Virology, Vaccines and Sera, Torlak, Belgrade, Serbia. A total of 480 participants were assigned randomly in a ratio of 2:1 to receive a single intramuscular dose (0.5 ml) of the vaccine (15 µg of hemagglutinin per strain) or placebo (phosphate-buffered saline). Participants were monitored for safety, including solicited and unsolicited adverse events (AEs) and serious adverse events (SAEs). No SAEs related to vaccination were reported. Injection site pain (51.3%), injection site tenderness (40.4%), tiredness (17.0%), and headache (15.1%) were the most commonly reported solicited events in the vaccine group. Incidence of related unsolicited AEs was low (1.3%) among vaccinees. Hemagglutinin inhibition (HAI) titers were measured before and 21 days after vaccination in 151 participants. Overall, HAI seroconversion rates to H1 and H3 were observed in 90.1% and 76.2% of vaccinees, respectively. For B antigen, it was 51.5%, likely due to high pre-vaccination titers. Post-vaccination seroprotection rates were in the range of 78.2–95.0% for the three antigens. Post-vaccination geometric mean titers (GMT) were at least 3.8 times higher than baseline levels for all the three strains among vaccinees. Overall, the study showed that the vaccine was safe and well tolerated, and induced a robust immune response against all three vaccine strains. ClinicalTrials.gov identifier: NCT02935192, October 17, 2016

This study was a phase I double-blind, randomized, placebo-controlled trial to evaluate the safety and immunogenicity of a Serbian-produced seasonal trivalent split, inactivated influenza vaccine in healthy adults. The vaccine was manufactured in eggs by the Torlak Institute of Virology, Vaccines and Sera, Belgrade, Serbia and contained A/H1N1, A/H3N2 and B viruses. The clinical trial took place at the Clinical Center of Serbia in Belgrade. Sixty healthy volunteers, aged 18–45 years, were enrolled in the trial. On the day of immunization, volunteers were randomly assigned to receive either a single dose of the trivalent seasonal influenza vaccine (15 μg of hemagglutinin per strain) or placebo (phosphate-buffered saline). Subjects were monitored for adverse events through a clinical history and physical examination, and blood was taken for testing at screening and on day 8 to assess vaccine safety. Serum samples obtained before and 21 days after immunization were tested for influenza antibody titers using hemagglutination-inhibition (HAI) and microneutralization (MN) tests. No serious adverse events were reported. Pain and tenderness at the injection site were the most commonly reported symptoms in both vaccine and placebo groups. Overall, serum HAI responses of fourfold or greater magnitude were observed to H1, H3, and B antigen in 80%, 75%, and 70% of subjects, respectively. Seroprotection rates as measured by HAI were also high (100%, 100% and 86.67%, respectively, for H1, H3 and B). Thus, Torlak's seasonal trivalent influenza vaccine was not associated with adverse events, was well-tolerated and immunogenic. It should be further evaluated in clinical trials to provide sufficient safety and immunogenicity data for licensing in Serbia.

The recent identification of plasma membrane (Ca2+)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis.

Abstract The cell adhesion molecule neuroplastin (Np) is a novel candidate to influence human intelligence. Np-deficient mice display complex cognitive deficits and reduced levels of Plasma Membrane Ca2+ ATPases (PMCAs), an essential regulator of the intracellular Ca2+ concentration ([iCa2+]) and neuronal activity. We show abundant expression and conserved cellular and molecular features of Np in glutamatergic neurons in human hippocampal-cortical pathways as characterized for the rodent brain. In Nptn lox/loxEmx1Cre mice, glutamatergic neuron-selective Np ablation resulted in behavioral deficits indicating hippocampal, striatal, and sensorimotor dysfunction paralleled by highly altered activities in hippocampal CA1 area, sensorimotor cortex layers I-III/IV, and the striatal sensorimotor domain detected by single-photon emission computed tomography. Altered hippocampal and cortical activities correlated with reduction of distinct PMCA paralogs in Nptn lox/loxEmx1Cre mice and increased [iCa2+] in cultured mutant neurons. Human and rodent Np enhanced the post-transcriptional expression of and co-localized with PMCA paralogs in the plasma membrane of transfected cells. Our results indicate Np as essential for PMCA expression in glutamatergic neurons allowing proper [iCa2+] regulation and normal circuit activity. Neuron-type-specific Np ablation empowers the investigation of circuit-coded learning and memory and identification of causal mechanisms leading to cognitive deterioration.

Thy-1 is a small membrane glycoprotein and member of the immunoglobulin superfamily of cell adhesion molecules. It is abundantly expressed in many cell types including neurons and is anchored to the outer membrane leaflet via a glycosyl phosphatidylinositol tail. Thy-1 displays a number of interesting properties such as fast lateral diffusion, which allows it to get in and out of membrane nanodomains with different lipid composition. Thy-1 displays a broad expression in different cell types and plays confirmed roles in cell development, adhesion and differentiation. Here, we explored the functions of Thy-1 in neuronal signaling, initiated by extracellular binding of αVβ3 integrin, may strongly dependent on the lipid content of the cell membrane. Also, we assort literature suggesting the association of Thy-1 with specific components of lipid rafts such as sialic acid containing glycosphingolipids, called gangliosides. Furthermore, we argue that Thy-1 positioning in nanodomains may be influenced by gangliosides. We propose that the traditional conception of Thy-1 localization in rafts should be reconsidered and evaluated in detail based on the potential diversity of neuronal nanodomains.

Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams ≥ 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Submillimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation.

Proton radiotherapy using minibeams of sub-millimeter dimensions reduces side effects in comparison to conventional proton therapy due to spatial fractionation. Since the proton minibeams widen with depth, the homogeneous irradiation of a tumor can be ensured by adjusting the beam distances to tumor size and depth to maintain tumor control as in conventional proton therapy. The inherent advantages of protons in comparison to photons like a limited range that prevents a dosage of distal tissues are maintained by proton minibeams and can even be exploited for interlacing from different beam directions. A first animal study was conducted to systematically investigate and quantify the tissue-sparing effects of proton pencil minibeams as a function of beam size and dose distributions, using beam widths between σ = 95, 199, 306, 411, 561 and 883 μm (standard deviation) at a defined center-to-center beam distance (ctc) of 1.8 mm. The average dose of 60 Gy was distributed in 4x4 minibeams using 20 MeV protons (LET ~ 2.7 keV/μm). The induced radiation toxicities were measured by visible skin reactions and ear swelling for 90 days after irradiation. The largest applied beam size to ctc ratio (σ/ctc = 0.49) is similar to a homogeneous irradiation and leads to a significant 3-fold ear thickness increase compared to the control group. Erythema and desquamation was also increased significantly 3-4 weeks after irradiation. With decreasing beam sizes and thus decreasing σ/ctc, the maximum skin reactions are strongly reduced until no ear swelling or other visible skin reactions should occur for σ/ctc < 0.032 (extrapolated from data). These results demonstrate that proton pencil minibeam radiotherapy has better tissue-sparing for smaller σ/ctc, corresponding to larger peak-to-valley dose ratios PVDR, with the best effect for σ/ctc < 0.032. However, even quite large σ/ctc (e.g. σ/ctc = 0.23 or 0.31, i.e. PVDR = 10 or 2.7) show less acute side effects than a homogeneous dose distribution. This suggests that proton minibeam therapy spares healthy tissue not only in the skin but even for dose distributions appearing in deeper layers close to the tumor enhancing its benefits for clinical proton therapy.

X-ray microbeam radiotherapy can potentially widen the therapeutic window due to a geometrical redistribution of the dose. However, high requirements on photon flux, beam collimation, and system stability restrict its application mainly to large-scale, cost-intensive synchrotron facilities. With a unique laser-based Compact Light Source using inverse Compton scattering, we investigated the translation of this promising radiotherapy technique to a machine of future clinical relevance. We performed in vitro colony-forming assays and chromosome aberration tests in normal tissue cells after microbeam irradiation compared to homogeneous irradiation at the same mean dose using 25 keV X-rays. The microplanar pattern was achieved with a tungsten slit array of 50 μm slit size and a spacing of 350 μm. Applying microbeams significantly increased cell survival for a mean dose above 2 Gy, which indicates fewer normal tissue complications. The observation of significantly less chromosome aberrations suggests a lower risk of second cancer development. Our findings provide valuable insight into the mechanisms of microbeam radiotherapy and prove its applicability at a compact synchrotron, which contributes to its future clinical translation.

BACKGROUND: Aim of this study was to evaluate the relative biological effectiveness (RBE) of carbon (12C) and oxygen ion (16O)-irradiation applied in the raster-scanning technique at the Heidelberg Ion beam Therapy center (HIT) based on clonogenic survival in hepatocellular carcinoma cell lines compared to photon irradiation. METHODS: Four human HCC lines Hep3B, PLC, HepG2 and HUH7 were irradiated with photons, 12C and 16O using a customized experimental setting at HIT for in-vitro trials. Cells were irradiated with increasing physical photon single doses of 0, 2, 4 and 6 Gy and heavy ion-single doses of 0, 0.125, 0.5, 1, 2, 3 Gy (12C and 16O). SOBP-penetration depth and extension was 35 mm +/-4 mm and 36 mm +/-5 mm for carbon ions and oxygen ions respectively. Mean energy level and mean linear energy transfer (LET) were 130 MeV/u and 112 keV/um for 12C, and 154 MeV/u and 146 keV/um for 16O. Clonogenic survival was computated and relative biological effectiveness (RBE) values were defined. RESULTS: For all cell lines and both particle modalities α- and β-values were determined. As expected, α-values were significantly higher for 12C and 16O than for photons, reflecting a steeper decline of the initial slope of the survival curves for high-LET beams. RBE-values were in the range of 2.1-3.3 and 1.9-3.1 for 12C and 16O, respectively. CONCLUSION: Both irradiation with 12C and 16O using the raster-scanning technique leads to an enhanced RBE in HCC cell lines. No relevant differences between achieved RBE-values for 12C and 16O were found. Results of this work will further influence biological-adapted treatment planning for HCC patients that will undergo particle therapy with 12C or 16O.