The Effect of Honey, Aloe Vera, and Hydrocolloid Dressing on the Healing Process of Murine Excisional Wounds.

Chronic ulcers are a major health problem associated with high costs and a loss of quality of life. Because of this, the search for products that accelerate wound healing is a constant, given the need for alternatives that help to alleviate this serious health problem. We analyzed the efficacy of 2 natural products-honey and aloe vera-versus hydrocolloid (HC) dressings as a control group in healing full-thickness wounds. For this purpose, we performed full-thickness excisions of the skin, including the panniculus carnosus, in mice. We inserted a nitrile ring into the subcutaneous cellular tissue simulating the second-intention wound healing course. We found that aloe vera reduced the diameter of the wounds compared to honey (p < .001) and the control group (p < .001).

Topographic distribution of inflammation factors in a healing aneurysm.

BACKGROUND: Healing of intracranial aneurysms following endovascular treatment relies on the organization of early thrombus into mature scar tissue and neointima formation. Activation and deactivation of the inflammation cascade plays an important role in this process. In addition to timely evolution, its topographic distribution is hypothesized to be crucial for successful aneurysm healing. METHODS: Decellularized saccular sidewall aneurysms were created in Lewis rats and coiled. At follow-up (after 3 days (n = 16); 7 days (n = 19); 21 days (n = 8)), aneurysms were harvested and assessed for healing status. In situ hybridization was performed for soluble inflammatory markers (IL6, MMP2, MMP9, TNF-α, FGF23, VEGF), and immunohistochemical analysis to visualize inflammatory cells (CD45, CD3, CD20, CD31, CD163, HLA-DR). These markers were specifically documented for five regions of interest: aneurysm neck, dome, neointima, thrombus, and adjacent vessel wall. RESULTS: Coiled aneurysms showed enhanced patterns of thrombus organization and neointima formation, whereas those without treatment demonstrated heterogeneous patterns of thrombosis, thrombus recanalization, and aneurysm growth (p = 0.02). In coiled aneurysms, inflammation markers tended to accumulate inside the thrombus and in the neointima (p < 0.001). Endothelial cells accumulated directly in the neointima (p < 0.0001), and their presence was associated with complete aneurysm healing. CONCLUSION: The presence of proinflammatory cells plays a crucial role in aneurysm remodeling after coiling. Whereas thrombus organization is hallmarked by a pronounced intra-thrombotic inflammatory reaction, neointima maturation is characterized by direct invasion of endothelial cells. Knowledge concerning topographic distribution of regenerative inflammatory processes may pave the way for future treatment modalities which enhance aneurysm healing after endovascular therapy.

Targeted removal of macrophage-secreted interleukin-1 receptor antagonist protects against lethal Candida albicans sepsis.

Invasive fungal infections are associated with high mortality rates, and the lack of efficient treatment options emphasizes an urgency to identify underlying disease mechanisms. We report that disseminated Candida albicans infection is facilitated by interleukin-1 receptor antagonist (IL-1Ra) secreted from macrophages in two temporally and spatially distinct waves. Splenic CD169+ macrophages release IL-1Ra into the bloodstream, impeding early neutrophil recruitment. IL-1Ra secreted by monocyte-derived tissue macrophages further impairs pathogen containment. Therapeutic IL-1Ra neutralization restored the functional competence of neutrophils, corrected maladapted hyper-inflammation, and eradicated the otherwise lethal infection. Conversely, augmentation of macrophage-secreted IL-1Ra by type I interferon severely aggravated disease mortality. Our study uncovers how a fundamental immunoregulatory mechanism mediates the high disease susceptibility to invasive candidiasis. Furthermore, interferon-stimulated IL-1Ra secretion may exacerbate fungal dissemination in human patients with secondary candidemia. Macrophage-secreted IL-1Ra should be considered as an additional biomarker and potential therapeutic target in severe systemic candidiasis.

Minor intron splicing is critical for survival of lethal prostate cancer.

The evolutionarily conserved minor spliceosome (MiS) is required for protein expression of ∼714 minor intron-containing genes (MIGs) crucial for cell-cycle regulation, DNA repair, and MAP-kinase signaling. We explored the role of MIGs and MiS in cancer, taking prostate cancer (PCa) as an exemplar. Both androgen receptor signaling and elevated levels of U6atac, a MiS small nuclear RNA, regulate MiS activity, which is highest in advanced metastatic PCa. siU6atac-mediated MiS inhibition in PCa in vitro model systems resulted in aberrant minor intron splicing leading to cell-cycle G1 arrest. Small interfering RNA knocking down U6atac was ∼50% more efficient in lowering tumor burden in models of advanced therapy-resistant PCa compared with standard antiandrogen therapy. In lethal PCa, siU6atac disrupted the splicing of a crucial lineage dependency factor, the RE1-silencing factor (REST). Taken together, we have nominated MiS as a vulnerability for lethal PCa and potentially other cancers.

Spatial distribution of CD3- and CD8-positive lymphocytes as pretest for POLE wild-type in molecular subgroups of endometrial carcinoma.

Introduction: Over the years, the molecular classification of endometrial carcinoma has evolved significantly. Both POLEmut and MMRdef cases share tumor biological similarities like high tumor mutational burden and induce strong lymphatic reactions. While therefore use case scenarios for pretesting with tumor-infiltrating lymphocytes to replace molecular analysis did not show promising results, such testing may be warranted in cases where an inverse prediction, such as that of POLEwt, is being considered. For that reason we used a spatial digital pathology method to quantitatively examine CD3+ and CD8+ immune infiltrates in comparison to conventional histopathological parameters, prognostics and as potential pretest before molecular analysis. Methods: We applied a four-color multiplex immunofluorescence assay for pan-cytokeratin, CD3, CD8, and DAPI on 252 endometrial carcinomas as testing and compared it to further 213 cases as validation cohort from a similar multiplexing assay. We quantitatively assessed immune infiltrates in microscopic distances within the carcinoma, in a close distance of 50 microns, and in more distant areas. Results: Regarding prognostics, high CD3+ and CD8+ densities in intra-tumoral and close subregions pointed toward a favorable outcome. However, TCGA subtyping outperforms prognostication of CD3 and CD8 based parameters. Different CD3+ and CD8+ densities were significantly associated with the TCGA subgroups, but not consistently for histopathological parameter. In the testing cohort, intra-tumoral densities of less than 50 intra-tumoral CD8+ cells/mm2 were the most suitable parameter to assume a POLEwt, irrespective of an MMRdef, NSMP or p53abn background. An application to the validation cohort corroborates these findings with an overall sensitivity of 95.5%. Discussion: Molecular confirmation of POLEmut cases remains the gold standard. Even if CD3+ and CD8+ cell densities appeared less prognostic than TCGA, low intra-tumoral CD8+ values predict a POLE wild-type at substantial percentage rates, but not vice versa. This inverse correlation might be useful to increase pretest probabilities in consecutive POLE testing. Molecular subtyping is currently not conducted in one-third of cases deemed low-risk based on conventional clinical and histopathological parameters. However, this percentage could potentially be increased to two-thirds by excluding sequencing of predicted POLE wild-type cases, which could be determined through precise quantification of intra-tumoral CD8+ cells.

Smarcd3 is an epigenetic modulator of the metabolic landscape in pancreatic ductal adenocarcinoma.

Pancreatic cancer is characterized by extensive resistance to conventional therapies, making clinical management a challenge. Here we map the epigenetic dependencies of cancer stem cells, cells that preferentially evade therapy and drive progression, and identify SWI/SNF complex member SMARCD3 as a regulator of pancreatic cancer cells. Although SWI/SNF subunits often act as tumor suppressors, we show that SMARCD3 is amplified in cancer, enriched in pancreatic cancer stem cells and upregulated in the human disease. Diverse genetic mouse models of pancreatic cancer and stage-specific Smarcd3 deletion reveal that Smarcd3 loss preferentially impacts established tumors, improving survival especially in context of chemotherapy. Mechanistically, SMARCD3 acts with FOXA1 to control lipid and fatty acid metabolism, programs associated with therapy resistance and poor prognosis in cancer. These data identify SMARCD3 as an epigenetic modulator responsible for establishing the metabolic landscape in aggressive pancreatic cancer cells and a potential target for new therapies.

Immunohistochemical Detection of the Autophagy Markers LC3 and p62/SQSTM1 in Formalin-Fixed and Paraffin-Embedded Tissue.

Autophagy is a highly conserved cellular mechanism of "self-digestion," ensuring cellular homeostasis and playing a role in many diseases including cancer. As a stress response mechanism, it may also be involved in cellular response to therapy. LC3 and Sequestosome 1 (p62/SQSTM1) are among the most widely used markers to monitor autophagy and can be visualized in formalin-fixed and paraffin-embedded tissue by immunohistochemistry. Here we describe a validated staining protocol using an automated staining system available in many routine pathology laboratories, enabling high-throughput staining under standardized conditions.

Immunohistochemical Detection of the Chaperone-Mediated Autophagy Markers LAMP2A and HSPA8 in Formalin-Fixed and Paraffin-Embedded Tissues.

Autophagy is crucial for maintaining cellular homeostasis and its deregulation is involved in disease development, including cancer. The key players of chaperone-mediated autophagy (CMA), a particular selective subtype of autophagy, are HSPA8 and LAMP2A. Both proteins can be immunohistochemically detected in formalin-fixed paraffin-embedded (FFPE) tissue. LAMP2A is frequently overexpressed in a variety of cancers where it likely supports cancer cell survival and resistance to anti-cancer therapies in a context-dependent manner. Here we present the immunohistochemical staining protocol of antibodies against LAMP2A and HSPA8, using an automated staining system, suitable for routine diagnostics. Additionally, we also suggest a staining evaluation method.

Loss of the volume-regulated anion channel components LRRC8A and LRRC8D limits platinum drug efficacy.

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.

Aberrant hyperexpression of the RNA binding protein FMRP in tumors mediates immune evasion.

Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP's immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators-interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP's cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.

A combined spatial score of granzyme B and CD68 surpasses CD8 as an independent prognostic factor in TNM stage II colorectal cancer.

BACKGROUND: Previous assessments of peritumoral inflammatory infiltrate in colorectal cancer (CRC) have focused on the role of CD8+ T lymphocytes. We sought to compare the prognostic value of CD8 with downstream indicators of active immune cell function, specifically granzyme B (GZMB) and CD68 in the tumour microenvironment. METHODS: Immunohistochemical (IHC) staining was performed for CD8, GZMB, CD68 and CD163 on next-generation tissue microarrays (ngTMAs) in a primary cohort (n = 107) and a TNM stage II validation cohort (n = 151). Using digital image analysis, frequency of distinct immune cell types was calculated for tumour proximity (TP) zones with varying radii (10 µm-100 µm) around tumour cells. RESULTS: Associations notably of advanced TNM stage were observed for low density of CD8 (p = 0.002), GZMB (p < 0.001), CD68 (p = 0.034) and CD163 (p = 0.011) in the primary cohort. In the validation cohort only low GZMB (p = 0.036) was associated with pT4 stage. Survival analysis showed strongest prognostic effects in the TP25µm zone at the tumour centre for CD8, GZMB and CD68 (all p < 0.001) in the primary cohort and for CD8 (p = 0.072), GZMB (p = 0.035) and CD68 (p = 0.004) in the validation cohort with inferior prognostic effects observed at the tumour invasive margin. In a multivariate survival analysis, joint analysis of GZMB and CD68 was similarly prognostic to CD8 in the primary cohort (p = 0.007 vs. p = 0.002) and superior to CD8 in the validation cohort (p = 0.005 vs. p = 0.142). CONCLUSION: Combined high expression of GZMB and CD68 within 25 µm to tumour cells is an independent prognostic factor in CRC and of superior prognostic value to the well-established CD8 in TNM stage II cancers. Thus, assessment of antitumoral effect should consider the quality of immune activation in peritumoral inflammatory cells and their actual proximity to tumour cells.

Maximum SNP FST Outperforms Full-Window Statistics for Detecting Soft Sweeps in Local Adaptation.

Local adaptation can lead to elevated genetic differentiation at the targeted genetic variant and nearby sites. Selective sweeps come in different forms, and depending on the initial and final frequencies of a favored variant, very different patterns of genetic variation may be produced. If local selection favors an existing variant that had already recombined onto multiple genetic backgrounds, then the width of elevated genetic differentiation (high FST) may be too narrow to detect using a typical windowed genome scan, even if the targeted variant becomes highly differentiated. We, therefore, used a simulation approach to investigate the power of SNP-level FST (specifically, the maximum SNP FST value within a window, or FST_MaxSNP) to detect diverse scenarios of local adaptation, and compared it against whole-window FST and the Comparative Haplotype Identity statistic. We found that FST_MaxSNP had superior power to detect complete or mostly complete soft sweeps, but lesser power than full-window statistics to detect partial hard sweeps. Nonetheless, the power of FST_MaxSNP depended highly on sample size, and confident outliers depend on robust precautions and quality control. To investigate the relative enrichment of FST_MaxSNP outliers from real data, we applied the two FST statistics to a panel of Drosophila melanogaster populations. We found that FST_MaxSNP had a genome-wide enrichment of outliers compared with demographic expectations, and though it yielded a lesser enrichment than window FST, it detected mostly unique outlier genes and functional categories. Our results suggest that FST_MaxSNP is highly complementary to typical window-based approaches for detecting local adaptation, and merits inclusion in future genome scans and methodologies.

Systematic Investigation of SARS-CoV-2 Receptor Protein Distribution along Viral Entry Routes in Humans.

BACKGROUND: The novel beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), enters the human body via mucosal surfaces of the upper and/or lower respiratory tract. Viral entry into epithelial cells is mediated via angiotensin-converting enzyme 2 (ACE2) and auxiliary molecules, but the precise anatomic site of infection still remains unclear. METHODS: Here, we systematically investigated the main SARS-CoV-2 receptor proteins ACE2 and transmembrane serine protease 2 (TMPRSS2), as well as 2 molecules potentially involved in viral entry, furin and CD147, in formalin-fixed, paraffin-embedded human tissues. Tissue microarrays incorporating a total of 879 tissue cores from conjunctival (n = 84), sinonasal (n = 95), and lung (bronchiolar/alveolar; n = 96) specimens were investigated for protein expression by immunohistochemistry. RESULTS: ACE2 and TMPRSS2 were expressed in ciliated epithelial cells of the conjunctivae and sinonasal tissues, with highest expression levels observed in the apical cilia. In contrast, in the lung, the expression of those molecules in bronchiolar and alveolar epithelial cells was much rarer and only very focal when present. Furin and CD147 were more uniformly expressed in all tissues analyzed, including the lung. Interestingly, alveolar macrophages consistently expressed high levels of all 4 molecules investigated. CONCLUSIONS: Our study confirms and extends previous findings and contributes to a better understanding of potential SARS-CoV-2 infection sites along the human respiratory tract.

Rhesus Macaque CODEX Multiplexed Immunohistochemistry Panel for Studying Immune Responses During Ebola Infection.

Non-human primate (NHP) animal models are an integral part of the drug research and development process. For some biothreat pathogens, animal model challenge studies may offer the only possibility to evaluate medical countermeasure efficacy. A thorough understanding of host immune responses in such NHP models is therefore vital. However, applying antibody-based immune characterization techniques to NHP models requires extensive reagent development for species compatibility. In the case of studies involving high consequence pathogens, further optimization for use of inactivated samples may be required. Here, we describe the first optimized CO-Detection by indEXing (CODEX) multiplexed tissue imaging antibody panel for deep profiling of spatially resolved single-cell immune responses in rhesus macaques. This 21-marker panel is composed of a set of 18 antibodies that stratify major immune cell types along with a set three Ebola virus (EBOV)-specific antibodies. We validated these two sets of markers using immunohistochemistry and CODEX in fully inactivated Formalin-Fixed Paraffin-Embedded (FFPE) tissues from mock and EBOV challenged macaques respectively and provide an efficient framework for orthogonal validation of multiple antibody clones using CODEX multiplexed tissue imaging. We also provide the antibody clones and oligonucleotide tag sequences as a valuable resource for other researchers to recreate this reagent set for future studies of tissue immune responses to EBOV infection and other diseases.

LAG-3 Expression Predicts Outcome in Stage II Colon Cancer.

INTRODUCTION: LAG-3 is an inhibitory immune checkpoint molecule that suppresses T cell activation and inflammatory cytokine secretion. T cell density in the tumor microenvironment of colon cancer plays an important role in the host's immunosurveillance. We therefore hypothesized that LAG-3 expression on tumor-infiltrating lymphocytes (TILs) predicts outcome in patients with stage II colon cancer. PATIENTS AND METHODS: Immunohistochemical staining for LAG-3 was performed on tissue microarrays (TMAs) of formalin-fixed paraffin-embedded tissue from 142 stage II colon cancer patients. LAG-3 expression was assessed in TILs within both the tumor front and tumor center and scored as either positive or negative. The primary endpoint was disease-free survival (DFS). RESULTS: In patients diagnosed with stage II colon cancer, the presence of LAG-3 expression on TILs was significantly associated with better 5-year DFS (HR 0.34, 95% CI 0.14-0.80, p = 0.009). The effect on DFS was mainly due to LAG-3-positive TILs in the tumor front (HR 0.33, 95% CI 0.13-0.82, p = 0.012). CONCLUSION: Assessment of LAG-3 might help to predict outcomes in patients with stage II colon cancer and potentially identify those patients who might benefit from adjuvant chemotherapy. Therefore, LAG-3 may serve as a prognostic biomarker in stage II colon cancer.

Liver proteome alterations in psychologically distressed rats and a nootropic drug.

BACKGROUND: Chronic psychological distress is considered today a pandemic due to the modern lifestyle and has been associated with various neurodegenerative, autoimmune, or systemic inflammation-related diseases. Stress is closely related to liver disease exacerbation through the high activity of the endocrine and autonomic nervous systems, and the connection between the development of these pathologies and the physiological effects induced by oxidative stress is not yet completely understood. The use of nootropics, as the cognitive enhancer and antioxidant piracetam, is attractive to repair the oxidative damage. A proteomic approach provides the possibility to obtain an in-depth comprehension of the affected cellular processes and the possible consequences for the body. Therefore, we considered to describe the effect of distress and piracetam on the liver proteome. METHODS: We used a murine model of psychological stress by predatory odor as a distress paradigm. Female Sprague-Dawley rats were distributed into four experimental groups (n = 6 - 7/group) and were exposed or not to the stressor for five days and treated or not with piracetam (600 mg/kg) for six days. We evaluated the liver proteome by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE) followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Besides, we analyzed the activity of liver antioxidant enzymes, the biochemical parameters in plasma and rat behavior. RESULTS: Our results showed that distress altered a wide range of proteins involved in amino acids metabolism, glucose, and fatty acid mobilization and degradation on the way to produce energy, protein folding, trafficking and degradation, redox metabolism, and its implications in the development of the non-alcoholic fatty liver disease (NAFLD). Piracetam reverted the changes in metabolism caused by distress exposure, and, under physiological conditions, it increased catabolism rate directed towards energy production. These results confirm the possible relationship between chronic psychological stress and the progression of NAFLD, as well as we newly evidenced the controversial beneficial effects of piracetam. Finally, we propose new distress biomarkers in the liver as the protein DJ-1 (PARK7), glutathione peroxidase 1 (GPX), peroxiredoxin-5 (PRDX5), glutaredoxin 5 (GLRX5), and thioredoxin reductase 1 (TXNDR1), and in plasma as biochemical parameters related to kidney function such as urea and blood urea nitrogen (BUN) levels.

RHAMM in liver metastases of stage IV colorectal cancer with mismatch-repair proficient status correlates with tumor budding, cytotoxic T-cells and PD-1/PD-L1.

BACKGROUND: During the last decades, the management for metastatic colorectal cancer patients has improved due to novel therapeutic approaches. A mismatch-repair deficient status seems to favour a better response to checkpoint inhibitor therapy, but the question arises whether a specific subgroup of stage IV patients with mismatch-repair (MMR) proficient status should also be considered. RHAMM (Receptor for Hyaluronic Acid Mediated Motility/HAMMR/CD168) is characterized by tumor progression and immunogenicity. Therefore, the aim of this study is to determine whether RHAMM within the CRLM of MMR-proficient patients correlate with a more immunological microenvironment, represented by cytotoxic T-cells, PD-1 and PD-1. METHODS: Two patient cohorts of liver metastases from MMR colorectal cancers were included into the study (n = 81 and 76) using ngTMA® technology and immunohistochemically analyzed for RHAMM, cytotoxic T-cells (CD8+), PD-1/PD-L1, intrametastatic budding (IMB) and perimetastatic budding (PMB). RESULTS: RHAMM-positive IMB was linked to a higher PD-L1 expression (r = 0.32; p = 0.233 and r = 0.28; p = 0.044) in the center and periphery of the metastasis and RHAMM-positive PMB was associated with a higher expression of PD-1 (r = 0.33; p = 0.0297), and especially PD-L1 (r = 0.604; p < 0.0001 and r = 0.43; p = 0.003) in the center and periphery of the metastasis. IMB and PMB were additionally associated with a higher count of CD8+ T-cells (p < 0.0001; r = 0.58; p < 0.0001; r = 0.53). CONCLUSIONS: The RHAMM status can be assessed in IMB/PMB either in biopsies or in resections of colorectal cancer liver metastases. A positive RHAMM status in IMB and/or PMB may be a potential indicator for a checkpoint inhibitor therapy for stage IV colorectal cancer patients with MMR proficient status.

Interspecific Variation between the American Quercus virginiana and Mediterranean Quercus Species in Terms of Seed Nutritional Composition, Phytochemical Content, and Antioxidant Activity.

This study aimed to evaluate a complete nutritional composition in the seeds Quercus virginiana to compare this nutritional composition with three Mediterranean Quercus species. We analyzed the seed morphometry, proximate composition, phytochemicals, and antioxidant capacity. The seed of Q. virginiana presented the smaller seed size and weight, while Q. suber presented the highest values. Moreover, Q. virginiana seeds showed the highest amounts of sugar and total lipids, digestibility, energy, palmitic acid, and stearic acid. On the other hand, Q. virginiana seeds showed the lowest values of linoleic acid. Moreover, Q. coccifera seeds presented the highest total phenolics and flavonoids contents and antioxidant activity. The clustering analysis revealed a significant similarity in seed morphometry and nutritional composition between the Mediterranean Q. ilex and Q. suber, grouping with the American Q. virginiana, but to a considerable distance; by contrast, the Mediterranean Q. coccifera was the most distant in the clustering analysis. The content of phenolics and flavonoids and digestibility value were the variables that contributed to the separation to a greater extent in the clustering of the four species. The nutritional and biological activity assessment of plant seed may be considered as an essential mission to find new sustainable sources and novel chemical agents. In this sense, Quercus seeds may be an alternative and a competitive food source for the agri-food industry.

Sensory attributes, physicochemical and antioxidant characteristics, and protein profile of wild prickly pear fruits (O. macrocentra Engelm., O. phaeacantha Engelm., and O. engelmannii Salm-Dyck ex Engelmann.) and commercial prickly pear fruits (O. ficus-indica (L.) Mill.).

Mexico presents the highest richness of Opuntia Mill. species. These species are an important economic factor for the country, and source of nutrients, bioactive compounds, pigments, and nutraceuticals which can be of interest for the food and pharmaceutical industry. However, there are some wild Opuntia species in the Chihuahua desert, that have not been analyzed to establish their properties and potential use. The aim of study was to evaluate the sensory, physicochemical and protein profile in wild prickly pear fruits (O. macrocentra Engelm. (OM), O. phaeacantha Engelm. (OP), and O. engelmannii Salm-Dyck ex Engelmann. (OE)) from Samalayuca, Chihuahua and compare them with two commercial prickly pear fruits (O. ficus-indica (L.) Mill. (green-OFG, red-OFR). The sensory profile of wild species was characterized by highest color, odor, and sour taste compared to the commercial fruits. Pulp, peel, and seeds from wild prickly pear fruits showed lower pH, and higher titratable total acidity, total phenolic compounds, total flavonoids, antioxidant capacity, protein, lipids, ash, carbohydrates (only peel), and crude fiber content than commercial Opuntia species. Furthermore, O. engelmannii showed a tendency to present the highest betacyanins, betaxanthins, and betalains contents. A total of 181, 122, 113, 183 and 140 different proteins were identified in OM, OP, OE, OFG, OFR species, respectively. All species showed the highest enrichment in three main pathways such as amino acids biosynthesis, glycolysis (dark)/gluconeogenesis (light), and the citric acid cycle. The wild prickly pear fruits of this study showed important nutritional, protein, and antioxidant properties with biological interest, and can be a potential source of functional ingredients and nutraceuticals.

Tumour budding and CD8+ T cells: 'attackers' and 'defenders' in rectal cancer with and without neoadjuvant chemoradiotherapy.

AIM: Tumour budding ('attacker') and CD8+ T cells ('defender') are recognised as important parameters for risk stratification in colon cancers and, combined, may have an even stronger clinical impact. Here, we determine the value of tumour budding and CD8+ in rectal cancer patients treated with/without neoadjuvant therapy. METHODS AND RESULTS: Using digital scans of all tumour slides/case, we analysed CD8+ T cell counts in two patient cohorts: 45 neoadjuvantly treated and 47 primarily surgically treated (totalling n = 543 slides) after double-staining of the surgical resection specimen for pan-cytokeratin and CD8+ . Tumour buds in hot-spots were manually counted (area = 0.785 mm2 ) and CD8+ T cell counts were analysed separately both in tumour budding hot-spots and the densest CD8+ regions throughout the tumour. In neoadjuvantly treated patients, only tumour budding and not CD8+ T cells was associated with tumour features, including more advanced ypT (P = 0.0062), venous invasion (P = 0.002), lymphatic invasion (P = 0.0003) and perineural invasion (P = 0.0017), as well as higher American Joint Committee on Cancer (AJCC) tumour regression score (P = 0.0035), indicating less tumour response. Overall survival was also worse in patients with high-grade budding in univariate analysis only. In contrast, all three variables, namely tumour budding (P = 0.0347), CD8+ T cells in budding hot-spots (P = 0.0382) and CD8+ T cells in the densest areas (P = 0.0117) were also associated with worse (budding) and better (CD8) survival time in the multivariate setting. CONCLUSION: In rectal cancer, tumour budding has clinical relevance in both primarily surgically treated patients and in those with neoadjuvantly treated patients, where it characterises highly aggressive residual disease. CD8+ T cell counts appear not to have prognostic relevance in the neoadjuvant context.