The study of galactomannans with different molecular weights and their ability to form microparticles suitable for pulmonary delivery.

The influence of locust bean gum (LBG) galactomannans (GMs) molecular weight (Mw) to assemble microparticulate systems was evaluated, and carriers for deep lung delivery were developed. A commercial batch of LBG with a mannose/galactose (M/G) ratio of 2.4 (batch 1) was used to study the influence of different microwave partial acid hydrolysis conditions on carbohydrate composition, glycosidic linkages, and aqueous solutions viscosity. The microwave treatment did not affect the composition, presenting 4-Man (36-42 %), 4,6-Man (27-35 %), and T-Gal (24-25 %) as the main glycosidic linkages. Depolymerization led to a viscosity reduction (≤0.005 Pa·s) with no major impact on polysaccharide debranching. The structural composition of the LBG galactomannans were further elucidated with sequence-specific proteins using carbohydrate microarray technologies. A second batch of LBG (M/G 3.3) was used to study the impact of GMs with different Mw on microparticle assembling, characteristics, and insulin release kinetics. The low-Mw GMs microparticles led to a faster release (20 min) than the higher-Mw (40 min) ones, impacting the release kinetics. All microparticles exhibited a safety profile to cells of the respiratory tract. However, only the higher-Mw GMs allowed the assembly of microparticles with sizes suitable for this type of administration.

Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography.

DNA origami is an emerging technology that can be used as a nanoscale platform in numerous applications ranging from drug delivery systems to biosensors. The DNA nanostructures are assembled from large single-stranded DNA (ssDNA) scaffolds, ranging from hundreds to thousands of nucleotides and from short staple strands. Scaffolds are usually obtained by asymmetric PCR (aPCR) or Escherichia coli infection/transformation with phages or phagemids. Scaffold quantification is typically based on agarose gel electrophoresis densitometry for molecules obtained by aPCR, or by UV absorbance, in the case of scaffolds obtained by infection or transformation. Although these methods are well-established and easy-to-apply, the results obtained are often inaccurate due to the lack of selectivity and sensitivity in the presence of impurities. Herein, we present an HPLC method based on ion-pair reversed-phase (IP-RP) chromatography to quantify DNA scaffolds. Using IP-RP chromatography, ssDNA products (449 and 1000 nt) prepared by aPCR were separated from impurities and from the double stranded (ds) DNA byproduct. Additionally, both ss and dsDNA were quantified with high accuracy. The method was used to guide the optimization of the production of ssDNA by aPCR, which targeted the maximization of the ratio of ssDNA to dsDNA obtained. Moreover, ssDNA produced from phage infection of E. coli cells was also quantified by IP-RP using commercial ssDNA from the M13mp18 phage as a standard.

G-protein-coupled receptor 84 regulates acute inflammation in normal and diabetic skin wounds.

Lipids have emerged as potent regulators of immune cell function. In the skin, adipocyte lipolysis increases the local pool of free fatty acids and is essential for coordinating early macrophage inflammation following injury. Here, we investigate G-protein-coupled receptor 84 (GPR84), a medium-chain fatty acid (MCFA) receptor, for its potential to propagate pro-inflammatory signaling after skin injury. GPR84 signaling was identified as a key component of regulating myeloid cell numbers and subsequent tissue repair through in vivo administration of a pharmacological antagonist and the MCFA decanoic acid. We found that impaired injury-induced dermal adipocyte lipolysis is a hallmark of diabetes, and lipidomic analysis demonstrated that MCFAs are significantly reduced in diabetic murine wounds. Furthermore, local administration of decanoic acid rescued myeloid cell numbers and tissue repair during diabetic wound healing. Thus, GPR84 is a readily targetable lipid signaling pathway for manipulating injury-induced tissue inflammation with beneficial effects on acute diabetic healing.

In vivo cisplatin-resistant neuroblastoma metastatic model reveals tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as an independent prognostic factor of survival in neuroblastoma.

Neuroblastoma is the most common solid extracranial tumour in children. Despite major advances in available therapies, children with drug-resistant and/or recurrent neuroblastoma have a dismal outlook with 5-year survival rates of less than 20%. Therefore, tackling relapsed tumour biology by developing and characterising clinically relevant models is a priority in finding targetable vulnerability in neuroblastoma. Using matched cisplatin-sensitive KellyLuc and resistant KellyCis83Luc cell lines, we developed a cisplatin-resistant metastatic MYCN-amplified neuroblastoma model. The average number of metastases per mouse was significantly higher in the KellyCis83Luc group than in the KellyLuc group. The vast majority of sites were confirmed as having lymph node metastasis. Their stiffness characteristics of lymph node metastasis values were within the range reported for the patient samples. Targeted transcriptomic profiling of immuno-oncology genes identified tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as a significantly dysregulated MYCN-independent gene. Importantly, differential TNFRSF4 expression was identified in tumour cells rather than lymphocytes. Low TNFRSF4 expression correlated with poor prognostic indicators in neuroblastoma, such as age at diagnosis, stage, and risk stratification and significantly associated with reduced probability of both event-free and overall survival in neuroblastoma. Therefore, TNFRSF4 Low expression is an independent prognostic factor of survival in neuroblastoma.

From sensor fusion to knowledge distillation in collaborative LIBS and hyperspectral imaging for mineral identification.

Multimodal spectral imaging offers a unique approach to the enhancement of the analytical capabilities of standalone spectroscopy techniques by combining information gathered from distinct sources. In this manuscript, we explore such opportunities by focusing on two well-known spectral imaging techniques, namely laser-induced breakdown spectroscopy, and hyperspectral imaging, and explore the opportunities of collaborative sensing for a case study involving mineral identification. In specific, the work builds upon two distinct approaches: a traditional sensor fusion, where we strive to increase the information gathered by including information from the two modalities; and a knowledge distillation approach, where the Laser Induced Breakdown spectroscopy is used as an autonomous supervisor for hyperspectral imaging. Our results show the potential of both approaches in enhancing the performance over a single modality sensing system, highlighting, in particular, the advantages of the knowledge distillation framework in maximizing the potential benefits of using multiple techniques to build more interpretable models and paving for industrial applications.

Identification of Relevant Spectral Ranges in Laser-Induced Breakdown Spectroscopy Imaging Using the Fourier Space.

Laser-induced breakdown spectroscopy (LIBS) imaging has now a well-established position in the subject of spectral imaging, leveraging multi-element detection capabilities and fast acquisition rates to support applications both at academic and technological levels. In current applications, the standard processing pipeline to explore LIBS imaging data sets revolves around identifying an element that is suspected to exist within the sample and generating maps based on its characteristic emission lines. Such an approach requires some previous expert knowledge both on the technique and on the sample side, which hinders a wider and more transparent accessibility of the LIBS imaging technique by non-specialists. To address this issue, techniques based on visual analysis or peak finding algorithms are applied on the average or maximum spectrum, and may be employed for automatically identifying relevant spectral regions. Yet, maps containing relevant information may often be discarded due to low signal-to-noise ratios or interference with other elements. In this context, this work presents an agnostic processing pipeline based on a spatial information ratio metric that is computed in the Fourier space for each wavelength and that allows for the identification of relevant spectral ranges in LIBS. The results suggest a more robust and streamlined approach to feature extraction in LIBS imaging compared with traditional inspection of the spectra, which can introduce novel opportunities not only for spectral data analysis but also in the field of data compression.

TRIB3 silencing promotes the downregulation of Akt pathway and PAX3-FOXO1 in high-risk rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), such as other childhood tumors, has witnessed treatment advancements in recent years. However, high-risk patients continue to face poor survival rates, often attributed to the presence of the PAX3/7-FOXO1 fusion proteins, which has been associated with metastasis and treatment resistance. Despite efforts to directly target these chimeric proteins, clinical success remains elusive. In this study, the main aim was to address this challenge by investigating regulators of FOXO1. Specifically, we focused on TRIB3, a potential regulator of the fusion protein in RMS. Our findings revealed a prominent TRIB3 expression in RMS tumors, highlighting its correlation with the presence of fusion protein. By conducting TRIB3 genetic inhibition experiments, we observed an impairment on cell proliferation. Notably, the knockdown of TRIB3 led to a decrease in PAX3-FOXO1 and its target genes at protein level, accompanied by a reduction in the activity of the Akt signaling pathway. Additionally, inducible silencing of TRIB3 significantly delayed tumor growth and improved overall survival in vivo. Based on our analysis, we propose that TRIB3 holds therapeutic potential for treating the most aggressive subtype of RMS. The findings herein reported contribute to our understanding of the underlying molecular mechanisms driving RMS progression and provide novel insights into the potential use of TRIB3 as a therapeutic intervention for high-risk RMS patients.

Phase 1, first-in-human study of TYRP1-TCB (RO7293583), a novel TYRP1-targeting CD3 T-cell engager, in metastatic melanoma: active drug monitoring to assess the impact of immune response on drug exposure.

Introduction: Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy. Methods: TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352). Results: Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1-2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro. Discussion: This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed.

Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

Molecular Epidemiology of Mayaro Virus among Febrile Patients, Roraima State, Brazil, 2018-2021.

We detected Mayaro virus (MAYV) in 3.4% (28/822) of febrile patients tested during 2018-2021 from Roraima State, Brazil. We also isolated MAYV strains and confirmed that these cases were caused by genotype D. Improved surveillance is needed to better determine the burden of MAYV in the Amazon Region.

Revealing the microbiome diversity and biocontrol potential of field Aedes ssp.: Implications for disease vector management.

The mosquito Aedes spp. holds important relevance for human and animal health, as it serves as a vector for transmitting multiple diseases, including dengue and Zika virus. The microbiome's impact on its host's health and fitness is well known. However, most studies on mosquito microbiomes have been conducted in laboratory settings. We explored the mixed microbial communities within Aedes spp., utilizing the 16S rRNA gene for diversity analysis and shotgun metagenomics for functional genomics. Our samples, which included Ae. aegypti and Ae. albopictus, spanned various developmental stages-eggs, larvae, and adults-gathered from five semiurban areas in Mexico. Our findings revealed a substantial diversity of 8,346 operational taxonomic units (OTUs), representing 967 bacterial genera and 126,366 annotated proteins. The host developmental stage was identified as the primary factor associated with variations in the microbiome composition. Subsequently, we searched for genes and species involved in mosquito biocontrol. Wolbachia accounted for 9.6% of the 16S gene sequences. We observed a high diversity (203 OTUs) of Wolbachia strains commonly associated with mosquitoes, such as wAlb, with a noticeable increase in abundance during the adult stages. Notably, we detected the presence of the cifA and cifB genes, which are associated with Wolbachia's cytoplasmic incompatibility, a biocontrol mechanism. Additionally, we identified 221 OTUs related to Bacillus, including strains linked to B. thuringiensis. Furthermore, we discovered multiple genes encoding insecticidal toxins, such as Cry, Mcf, Vip, and Vpp. Overall, our study contributes to the understanding of mosquito microbiome biodiversity and metabolic capabilities, which are essential for developing effective biocontrol strategies against this disease vector.

Whole exome sequencing and machine learning germline analysis of individuals presenting with extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma.

BACKGROUND: Tobacco is the main risk factor for developing lung cancer. Yet, while some heavy smokers develop lung cancer at a young age, other heavy smokers never develop it, even at an advanced age, suggesting a remarkable variability in the individual susceptibility to the carcinogenic effects of tobacco. We characterized the germline profile of subjects presenting these extreme phenotypes with Whole Exome Sequencing (WES) and Machine Learning (ML). METHODS: We sequenced germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age (extreme cases) or who did not develop lung cancer at an advanced age (extreme controls), selected from databases including over 6600 subjects. We selected individual coding genetic variants and variant-rich genes showing a significantly different distribution between extreme cases and controls. We validated the results from our discovery cohort, in which we analysed by WES extreme cases and controls presenting similar phenotypes. We developed ML models using both cohorts. FINDINGS: Mean age for extreme cases and controls was 50.7 and 79.1 years respectively, and mean tobacco consumption was 34.6 and 62.3 pack-years. We validated 16 individual variants and 33 variant-rich genes. The gene harbouring the most validated variants was HLA-A in extreme controls (4 variants in the discovery cohort, p = 3.46E-07; and 4 in the validation cohort, p = 1.67E-06). We trained ML models using as input the 16 individual variants in the discovery cohort and tested them on the validation cohort, obtaining an accuracy of 76.5% and an AUC-ROC of 83.6%. Functions of validated genes included candidate oncogenes, tumour-suppressors, DNA repair, HLA-mediated antigen presentation and regulation of proliferation, apoptosis, inflammation and immune response. INTERPRETATION: Individuals presenting extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma show different germline profiles. Our strategy may allow the identification of high-risk subjects and the development of new therapeutic approaches. FUNDING: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

Perspectives and awareness of endoscopy healthcare professionals on sustainable practices in gastrointestinal endoscopy: results of the LEAFGREEN survey.

BACKGROUND: Gastrointestinal (GI) endoscopy is one of healthcare's main contributors to climate change. We aimed to assess healthcare professionals' attitudes and the perceived barriers to implementation of sustainable GI endoscopy. METHODS: The LEAFGREEN web-based survey was a cross-sectional study conducted by the European Society of Gastrointestinal Endoscopy (ESGE) Green Endoscopy Working Group. The questionnaire comprised 39 questions divided into five sections (respondent demographics; climate change and sustainability beliefs; waste and resource management; single-use endoscopes and accessories; education and research). The survey was available via email to all active members of the ESGE and the European Society of Gastroenterology and Endoscopy Nurses and Associates (ESGENA) in March 2023. RESULTS: 407 respondents participated in the survey (11% response rate). Most participants (86%) agreed climate change is real and anthropogenic, but one-third did not consider GI endoscopy to be a significant contributor to climate change. Improvement in the appropriateness of endoscopic procedures (41%) and reduction in single-use accessories (34%) were considered the most important strategies to reduce the environmental impact of GI endoscopy. Respondents deemed lack of institutional support and knowledge from staff to be the main barriers to sustainable endoscopy. Strategies to reduce unnecessary GI endoscopic procedures and comparative studies of single-use versus reusable accessories were identified as research priorities. CONCLUSIONS: In this survey, ESGE and ESGENA members acknowledge climate change as a major threat to humanity. Further improvement in sustainability beliefs and professional attitudes, reduction in inappropriate GI endoscopy, and rational use of single-use accessories and endoscopes are critically required.

Metabolic and Bariatric Surgery in Patients with Obesity Class V (BMI > 60 kg/m2): a Modified Delphi Study.

BACKGROUND: Metabolic and bariatric surgery (MBS) is the preferred method to achieve significant weight loss in patients with Obesity Class V (BMI > 60 kg/m2). However, there is no consensus regarding the best procedure(s) for this population. Additionally, these patients will likely have a higher risk of complications and mortality. The aim of this study was to achieve a consensus among a global panel of expert bariatric surgeons using a modified Delphi methodology. METHODS: A total of 36 recognized opinion-makers and highly experienced metabolic and bariatric surgeons participated in the present Delphi consensus. 81 statements on preoperative management, selection of the procedure, perioperative management, weight loss parameters, follow-up, and metabolic outcomes were voted on in two rounds. A consensus was considered reached when an agreement of ≥ 70% of experts' votes was achieved. RESULTS: A total of 54 out of 81 statements reached consensus. Remarkably, more than 90% of the experts agreed that patients should be notified of the greater risk of complications, the possibility of modifications to the surgical procedure, and the early start of chemical thromboprophylaxis. Regarding the choice of the procedure, SADI-S, RYGB, and OAGB were the top 3 preferred operations. However, no consensus was reached on the limb length in these operations. CONCLUSION: This study represents the first attempt to reach consensus on the choice of procedures as well as perioperative management in patients with obesity class V. Although overall consensus was reached in different areas, more research is needed to better serve this high-risk population.

A narrative on diversity, equity, and inclusion in surgery: insights from the European Society of Coloproctology and identification of points for action.

The focus of the 2022 European Society of Coloproctology (ESCP) annual campaign was diversity, equity, and inclusion (DEI) in surgery. The ESCP "Operation Equal Access" campaign sought to interview key-opinion leaders and trainees, to raise awareness on inequalities, inform the community of the status of the topic, and to identify future areas for improvement. The ESCP Social Media Working Group interviewed experts who have made significant contributions to DEI in colorectal surgery and were acknowledged opinion leaders in the field. The interviews focused on their career, professional life, experiences, and opportunities during their training, and their views on DEI in colorectal surgery. DEI principles, education, and values need further promotion to reduce and address bias within the profession and overall improve the experience of minority community including health professionals and patients. International Societies are working to facilitate training opportunities and overcome DEI, and networking have contributed to that. Collaborations between societies will be pivotal to contribute to offering research and leadership opportunities equally. Access to advanced workshops including cadaveric training and simulation can be consistently promoted and provided globally via societies through telemonitoring. Involving patients in research should be encouraged, as it brings the perspective of a living experience.

Thermodynamic Guidelines for the Mechanosynthesis or Solid-State Synthesis of MnFe2O4 at Relatively Low Temperatures.

Herein, thermodynamic assessment is proposed to screen suitable precursors for the solid-state synthesis of manganese ferrite, by mechanosynthesis at room temperature or by subsequent calcination at relatively low temperatures, and the main findings are validated by experimental results for the representative precursor mixtures MnO + FeO3, MnO2 + Fe2O3, and MnO2 +2FeCO3. Thermodynamic guidelines are provided for the synthesis of manganese ferrite from (i) oxide and/or metallic precursors; (ii) carbonate + carbonate or carbonate + oxide powder mixtures; (iii) other precursors. It is also shown that synthesis from metallic precursors (Mn + 2Fe) requires a controlled oxygen supply in limited redox conditions, which is hardly achieved by reducing gases H2/H2O or CO/CO2. Oxide mixtures with an overall oxygen balance, such as MnO + Fe2O3, act as self-redox buffers and offer prospects for mechanosynthesis for a sufficient time (>9 h) at room temperature. On the contrary, the fully oxidised oxide mixture MnO2 + Fe2O3 requires partial reduction, which prevents synthesis at room temperature and requires subsequent calcination at temperatures above 1100 °C in air or in nominally inert atmospheres above 750 °C. Oxide + carbonate mixtures, such as MnO2 +2FeCO3, also yield suitable oxygen balance by the decomposition of the carbonate precursor and offer prospects for mechanosynthesis at room temperature, and residual fractions of reactants could be converted by firing at relatively low temperatures (≥650 °C).

Sequential immunotherapy and targeted therapy for metastatic BRAF V600 mutated melanoma: 4-year survival and biomarkers evaluation from the phase II SECOMBIT trial.

No prospective data were available prior to 2021 to inform selection between combination BRAF and MEK inhibition versus dual blockade of programmed cell death protein-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) as first-line treatment options for BRAFV600-mutant melanoma. SECOMBIT (NCT02631447) was a randomized, three-arm, noncomparative phase II trial in which patients were randomized to one of two sequences with immunotherapy or targeted therapy first, with a third arm in which an 8-week induction course of targeted therapy followed by a planned switch to immunotherapy was the first treatment. BRAF/MEK inhibitors were encorafenib plus binimetinib and checkpoint inhibitors ipilimumab plus nivolumab. Primary outcome of overall survival was previously reported, demonstrating improved survival with immunotherapy administered until progression and followed by BRAF/MEK inhibition. Here we report 4-year survival outcomes, confirming long-term benefit with first-line immunotherapy. We also describe preliminary results of predefined biomarkers analyses that identify a trend toward improved 4-year overall survival and total progression-free survival in patients with loss-of-function mutations affecting JAK or low baseline levels of serum interferon gamma (IFNy). These long-term survival outcomes confirm immunotherapy as the preferred first-line treatment approach for most patients with BRAFV600-mutant metastatic melanoma, and the biomarker analyses are hypothesis-generating for future investigations of predictors of durable benefit with dual checkpoint blockade and targeted therapy.

Validation of the open biological negative image set for a Portuguese population: Comparing Japanese and Portuguese samples and an exploration of low-order visual properties of the stimuli.

Recently, Shirai and Watanabe Royal Society Open Science, 9(1), 211128 (2022) developed OBNIS (Open Biological Negative Image Set), a comprehensive database containing images (primarily animals but also fruits, mushrooms, and vegetables) that visually elicit disgust, fear, or neither. OBNIS was initially validated for a Japanese population. In this article, we validated the color version of OBNIS for a Portuguese population. In study 1, the methodology of the original article was used. This allowed direct comparisons between the Portuguese and Japanese populations. Aside from a few emotional classification mismatches between disgust, fear, or neither-related images, we found that arousal and valence relate distinctively in both populations. In contrast to the Japanese sample, the Portuguese reported increased arousal for more positive valenced stimuli, suggesting that OBNIS images elicit positive emotions in the Portuguese population. These results showed important cross-cultural differences regarding OBNIS. In study 2, a methodological change was introduced: instead of the three classification options used originally (fear, disgust, or neither), six basic emotions were used (fear, disgust, sadness, surprise, anger, happiness), and a "neither" option, to confirm whether some of the originally "neither-related" images are associated with positive emotions (happiness). Additionally, the low-order visual properties of images (luminosity, contrast, chromatic complexity, and spatial frequency distribution) were explored due to their important role in emotion-related research. A fourth image group associated with happiness was found in the Portuguese sample. Moreover, image groups present differences regarding the low-order visual characteristics, which are correlated with arousal and valence ratings, highlighting the importance of controlling such characteristics in emotion-related research.