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Gonad maturation is critical for the reproductive success of any organism, and in fish, captivity can significantly affect their reproductive performance, leading to maturation incompetence and spawning failure. The greater amberjack (Seriola dumerili), a fish species recently introduced to aquaculture fails to undergo oocyte maturation, ovulation, and spawning when reared in aquaculture facilities. Since confinement has been shown to influence gonad maturation and completion of the reproductive cycle, investigations into epigenetic mechanisms may shed light on the reasoning behind the reproductive dysfunctions of fish under captivity. Among the known important epigenetic regulators are small non-coding RNAs (sncRNAs), and in particular microRNAs (miRNAs). In this study, immature, maturing (late vitellogenesis), and spent ovaries of captive greater amberjack were collected, and the differential expression of miRNAs in the three different ovarian development stages was examined. Expression patterns of conserved and novel miRNAs were identified, and potential targets of highly differentially expressed miRNAs were detected. Additionally, read length distribution showed two prominent peaks in the three different ovarian maturation stages, corresponding to miRNAs and putative piwi-interacting RNAs (piRNAs), another type of ncRNAs with a germ-cell specific role. Furthermore, miRNA expression patterns and their putative target mRNAs are discussed, in relevance with the different ovarian maturation stages of captive greater amberjack. Overall, this study provides insights into the role of miRNAs in the reproductive dysfunctions observed in fish under captivity and highlights the importance of epigenetic mechanisms in understanding and managing the reproductive performance of economically important fish species., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Fas/Fas ligand (FasL) system is a major apoptosis-regulating pathway with a key role in tumor immune surveillance and metastasis. The expression of Fas/FasL on mammary tumor tissues holds prognostic value for breast cancer (BC) patients. We herein assessed Fas/FasL expression on circulating tumor cells (CTCs) and matched peripheral blood mononuclear cells (PBMCs) from 98 patients with metastatic BC receiving first-line treatment. Fas+, FasL+, and Fas+/FasL+ CTCs were identified in 88.5%, 92.3%, and 84.6% of CTC-positive patients, respectively. In addition, Fas+/FasL+, Fas-/FasL+, and Fas-/FasL- PBMCs were identified in 70.3%, 24.2%, and 5.5% of patients, respectively. A reduced progression-free survival (PFS) was revealed among CTC-positive patients (median PFS: 9.5 versus 13.4 months; p = 0.004), and specifically among those harboring Fas+/FasL+ CTCs (median PFS: 9.5 vs. 13.4 months; p = 0.009). On the other hand, an increased overall survival (OS) was demonstrated among patients with Fas+/FasL+ PBMCs rather than those with Fas-/FasL+ and Fas-/FasL- PBMCs (median OS: 35.7 vs. 25.9 vs. 14.4 months, respectively; p = 0.008). These data provide for the first time evidence on Fas/FasL expression on CTCs and PBMCs with significant prognostic value for patients with metastatic BC, thus highlighting the role of the Fas/FasL system in the peripheral immune response and metastatic progression of BC.

Purpose: To compare the proportion of epiretinal membrane (ERM) between individuals with diabetes mellitus (DM) and without DM, who live in Brussels, to investigate possible risk factors for ERM formation and to compare the results with the ones of large population studies., Methods: Participants were divided into two groups; 99 patients with DM (group A) and 103 individuals without DM (group B). All participants underwent an undilated 7-field color fundus photography and a spectral domain optical coherence tomography (SD-OCT). Age, gender, race, type of diabetes, duration of medical treatment of diabetes, HbA1C rate, smoking, previous cataract surgery and educational level were investigated as possible risk factors., Results: Epiretinal membrane was detected in 17.2% of group A and in 11.7% of group B participants. The difference is not statistically significant (χ2 (1) = 1.252, p = 0.263). The proportion of ERM was significantly associated with age in both groups (p = .009 and p < .001 respectively), as well as with smoking (p = .023) and previous cataract surgery (p = .028) in patients with DM., Conclusion: There is no statistically significant difference of ERM proportion between the two groups of the study. Age was recognized as a risk factor for both groups, while smoking and previous cataract surgery were identified as predictors only for diabetics., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

We compared the endocrine status of the pituitary-gonad axis of wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle (April - July), reporting on the expression and release of the two gonadotropins for the first time in the Mediterranean Sea. Ovaries from wild females were characterized histologically as DEVELOPING in early May and SPAWNING capable in late May-July, the latter having a 3 to 4-fold higher gonadosomatic index (GSI). SPAWNING capable wild females exhibited an increase in pituitary follicle stimulating hormone (Fsh) content, plasma testosterone (T) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), while almost a 10-fold increase was observed in pituitary luteinizing hormone (Lh) content. An increasing trend of plasma 17β-estradiol (E 2 ) was also recorded between the two reproductive stages in wild females. Captive-reared females sampled during the reproductive cycle exhibited two additional reproductive categories, with REGRESSED females having extensive follicular atresia and fish in the REGENERATING stage having only primary oocytes in their ovaries. Pituitary content of Fsh and Lh, fshb and lhb expression and plasma levels of Fsh and Lh remained unchanged among the four reproductive stages in captive females, in contrast with plasma E 2 and T that decreased in the REGENERATING stage, and 17,20β-P which increased after the DEVELOPING stage. In general, no significant hormonal differences were recorded between captive-reared and wild DEVELOPING females, in contrast to SPAWNING capable females, where pituitary Lh content, plasma Fsh and T were found to be lower in females in captivity. Overall, the captive females lagged behind in reproductive development compared to the wild ones and this was perhaps related to the multiple handling of the sea cages where all the sampled fish were maintained. Between wild males in the DEVELOPING and SPAWNING capable stages, pituitary Lh content, plasma T and 17,20β-P, and GSI exhibited 3 to 4-fold increases, while an increasing trend of pituitary Fsh content, lhb expression levels and plasma 11-ketotestosterone (11-KT) was also observed, and an opposite trend was observed in plasma Lh. Captive males were allocated to one more category, with REGRESSED individuals having no spermatogenic capacity. During the SPAWNING capable phase, almost all measured parameters were lower in captive males compared to wild ones. More importantly, captive males showed significant differences from their wild counterparts throughout the reproductive season, starting already from the DEVELOPING stage. Therefore, it appears that captivity already exerted negative effects in males prior to the onset of the study and the multiple handling of the cage where sampled fish were reared. Overall, the present study demonstrated that female greater amberjack do undergo full vitellogenesis in captivity, albeit with some dysfunctions that may be related to the husbandry of the experiment, while males, on the other hand, may be more seriously affected by captivity even before the onset of the study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Giant cell arteritis (GCA) is an autoimmune disease affecting large vessels in patients over 50 years old. It is an exemplary model of a classic inflammatory disorder with IL-6 playing the leading role. The main comorbidities that may appear acutely or chronically are vascular occlusion leading to blindness and thoracic aorta aneurysm formation, respectively. The tissue inflammatory bulk is expressed as acute or chronic delayed-type hypersensitivity reactions, the latter being apparent by giant cell formation. The activated monocytes/macrophages are associated with pronounced Th1 and Th17 responses. B-cells and neutrophils also participate in the inflammatory lesion. However, the exact order of appearance and mechanistic interactions between cells are hindered by the lack of cellular and molecular information from early disease stages and accurate experimental models. Recently, senescent cells and neutrophil extracellular traps have been described in tissue lesions. These structures can remain in tissues for a prolonged period, potentially favoring inflammatory responses and tissue remodeling. In this review, current advances in GCA pathogenesis are discussed in different inflammatory phases. Through the description of these-often overlapping-phases, cells, molecules, and small lipid mediators with pathogenetic potential are described.

Zingiber officinale Roscoe (ginger) is a plant from the Zingiberaceae family, and its extracts have been found to contain several compounds with beneficial bioactivities. Nowadays, the use of environmentally friendly and sustainable extraction methods has attracted considerable interest. The main objective of this study was to evaluate subcritical propane (scPropane), supercritical CO 2 (scCO 2 ), and supercritical CO 2 with ethanol (scCO 2 + EtOH) as co-solvent methods for the extraction of high value products from ginger. In addition, the reuse/recycling of the secondary biomass in a second extraction as a part of the circular economy was evaluated. Both the primary and the secondary biomass led to high yield percentages, ranging from 1.23% to 6.42%. The highest yield was observed in the scCO 2 + EtOH, with biomass prior used to scCO 2 extraction. All extracts presented with high similarities as far as their total phenolic contents, antioxidant capacity, and chemical composition. The most abundant compounds, identified by the two different gas chromatography-mass spectrometry (GC-MS) systems present, were a-zingiberene, β- sesquiphellandrene, a-farnesene, β-bisabolene, zingerone, gingerol, a-curcumene, and γ-muurolene. Interestingly, the reuse/recycling of the secondary biomass was found to be promising, as the extracts showed high antioxidant capacity and consisted of significant amounts of compounds with beneficial properties.

We evaluated the onset of puberty of first-generation (F1) hatchery-produced greater amberjack (Seriola dumerili) reared in sea cages for 5 years. Fish were sampled every year in June, at the expected peak of the spawning period in the Mediterranean Sea. No sexual dimorphism in body weight was observed in the study. The ovaries of 1 and 2-year-old (yo) females consisted of primary oocytes only, while at the age of 3-yo early vitellogenic (Vg) oocytes were also identified, but with extensive follicular atresia. At the age of 4-yo, late Vg oocytes were observed, but again extensive follicular atresia characterized the ovaries of 50 % of females. At the age of 5-yo, follicular atresia of Vg oocytes was very limited. In males, gametogenesis was evident already in 1- and 2-yo fish, and 100 % of sampled 3-yo males produced collectable viable sperm. Plasma testosterone (T), 17β-estradiol (E2), and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) remained similar in 3 - 5-yo females, with T and E2 levels being highest in females in advanced vitellogenesis or with significant follicular atresia, compared to immature females. In males, plasma T declined over the years, while 11-ketotestosterone (11-KT) and 17,20β-P were highest in 4 and 5-yo males, with spermatozoa motility characteristics being improved from the 4th year onwards. The administration of GnRHa implants to 5-yo fish induced only two spawns, albeit no fertilized eggs were obtained. The results indicate that hatchery-produced greater amberjack males mature well and within the same age observed in the wild, however with smaller gonad size. On the contrary, females mature later than in the wild, also with a smaller gonad size. Spawning in response to GnRHa treatment was not effective, suggesting that Mediterranean hatchery-produced broodstocks may be dysfunctional, and further research is needed to document any improvement as the fish get older, or to determine if the results may be related to the specific stock of fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Limited information exists on epidermal growth factor receptor (EGFR) molecular epidemiology in Greece. Next-generation sequencing (NGS) is the recommended method for EGFR genotyping in NSCLC. The Idylla Biocartis platform is a fully automated system for actionable EGFR mutation detection., Research Design and Methods: We describe the prevalence of EGFR mutations in NSCLC patients in two high-volume clinical centers in Greece and compare key methods used for their determination. Eight hundred and fifty-seven FFPE samples from NSCLC patients were tested for EGFR mutations at University of Crete (UoC; n  = 324) and at Evangelismos Hospital, Athens (Evangelismos; n  = 503)., Results: The prevalence of EGFR mutations was 11.1% in the whole cohort (11.5% in non-squamous). The detection rate was 11.0% by NGS, 9.8% by Sanger and 11.3% by Idylla for the whole cohort (12.0% in non-squamous). The agreement between Idylla and Sanger was 93.2%. A targetable EGFR mutation was detected in 10.0% using tissue NGS alone, and in 16.0% using concurrent Idylla ct EGFR testing., Conclusion: The frequency of EGFR mutations was as expected for a Caucasian population. The Idylla EGFR test performance is comparable to reference methods and with a shorter TAT. Adding a concurrent plasma Idylla test to tissue NGS testing increases the detection rate of EGFR mutations in NSCLC.

Decellularized porcine myocardium is commonly used as scaffolding for engineered heart tissues (EHTs). However, structural and mechanical heterogeneity in the myocardium complicate production of mechanically consistent tissues. In this study, we evaluate the porcine psoas major muscle (tenderloin) as an alternative scaffold material. Head-to-head comparison of decellularized tenderloin and ventricular scaffolds showed only minor differences in mean biomechanical characteristics, but tenderloin scaffolds were less variable and less dependent on the region of origin than ventricular samples. The active contractile behavior of EHTs made by seeding tenderloin versus ventricular scaffolds with human-induced pluripotent stem cell-derived cardiomyocytes was also comparable, with only minor differences observed. Collectively, the data reveal that the behavior of EHTs produced from decellularized porcine psoas muscle is almost identical to those made from porcine left ventricular myocardium, with the advantages of being more homogeneous, biomechanically consistent, and readily obtainable.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca 2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca 2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca 2+ activation. Additionally, in the low Ca 2+ -relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca 2+ , and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Bacteriophages represent the most extensive group of viruses within the human virome and have a significant impact on general health and well-being by regulating bacterial population dynamics. Staphylococcus aureus , found in the anterior nostrils, throat and skin, is an opportunistic pathobiont that can cause a wide range of diseases, from chronic inflammation to severe and acute infections. In this study, we developed a human cell-based homeostasis model between a clinically isolated strain of S. aureus 141 and active phages for this strain (PYO Sa141 ) isolated from the commercial Pyophage cocktail (PYO). The cocktail is produced by Eliava BioPreparations Ltd. (Tbilisi, Georgia) and is used as an add-on therapy for bacterial infections, mainly in Georgia. The triptych interaction model was evaluated by time-dependent analysis of cell death and inflammatory response of the nasal and bronchial epithelial cells. Inflammatory mediators (IL-8, CCL5/RANTES, IL-6 and IL-1β) in the culture supernatants were measured by enzyme-linked immunosorbent assay and cell viability was determined by crystal violet staining. By measuring trans-epithelial electrical resistance, we assessed the epithelial integrity of nasal cells that had differentiated under air-liquid interface conditions. PYO Sa141 was found to have a prophylactic effect on airway epithelial cells exposed to S. aureus 141 by effectively down-regulating bacterial-induced inflammation, cell death and epithelial barrier disruption in a time-dependent manner. Overall, the proposed model represents an advance in the way multi-component biological systems can be simulated in vitro .

Since circulating microRNAs (miRNAs) are involved in the modulation of the immune response, they are tested as liquid biopsy-based biomarkers in patients with NSCLC treated with immunotherapy. We analyzed the expression levels and examined the clinical significance of immunoregulatory miRNAs involved in immune checkpoint regulation (miR-34a, miR-200b, miR-200c), T-cell activity (miR-155), and the function of myeloid-derived suppressive cells (MDSCs) (miR-223) or regulatory T lymphocytes (Tregs) (miR-146a), in patients with advanced NSCLC (N = 69) treated with anti-PD-1 (Nivolumab) immunotherapy as 2nd or 3rd line of treatment therapy. Plasma levels of circulating miRNAs were analyzed by RT-qPCR before the initiation of immunotherapy. Expression of miR-34a, miR-146a, mir-200c, and miR-223 was found to be associated with response to immunotherapy. High miR-200c expression emerged as an independent prognostic factor for inferior overall survival in all patients with NSCLC (OS, HR: 2.243, 95% CI: 1.208-4.163; p = 0.010) and in patients with non-Squamous (non-SqCC) subtype (N = 38) (HR: 2.809, 95% CI: 1.116-7.074; p = 0.028). Low miR-34a expression independently predicted for shorter OS (HR: 3.189, 95% CI: 1.193-8.527; p = 0.021) in the non-SqCC subgroup. Our findings suggest that alterations in circulating miR-200c and miR-34a expression levels are associated with the response and outcome in patients with advanced NSCLC treated with anti-PD1 immunotherapy.

Environmental effects and, particularly, temperature changes have been demonstrated to influence the activity, function, and well-being of teleosts. Temperature may change seasonally in the wild, and in captivity under aquaculture operations. Moreover, climate change is expected to shift temperature profiles worldwide. MicroRNAs (miRNA) are important temperature-sensitive gene-expression regulators acting at the post-transcriptional level. They are known to be key regulators in development, reproduction, and immune responses. Therefore, early larval development of the European sea bass ( Dicentrarchus labrax ), one of the most extensively cultured species in Mediterranean aquaculture, was investigated at early rearing temperatures, i.e., 15, 17.5, and 20 °C, in regard to the impact of temperatures on miRNAs through sncRNA high-throughput sequencing but also at the phenotypic level in terms of growth, sex, vision, and skeletal deformities. Expression profiling revealed stage- and temperature-specific miRNA expression targeting genes with roles in reproduction and immune response mainly at the flexion and all-fins stages. Similar stage- and temperature-specific results were also observed concerning the number of rod cells and lower jaw elongation. The present work presents for the first time highly promising results on the influence of early rearing temperature at the post-transcriptional level during European sea bass development, with a putative impact on reproduction and immune response, as well as regarding teleost vision and larval development.

Background: Gastropod snails remain strongly understudied, despite their important role in transmitting parasitic diseases. Knowledge of their distribution and population dynamics increases our understanding of the processes driving disease transmission. We report the first study to use high-throughput sequencing (HTS) to elucidate the population genetic structure of the hermaphroditic snail Bulinus truncatus (Gastropoda, Heterobranchia) on a regional (17-150 km) and inter-regional (1000-5400 km) scale. This snail species acts as an intermediate host of Schistosoma haematobium and Schistosoma bovis, which cause human and animal schistosomiasis respectively., Methods: Bulinus truncatus snails were collected in Senegal, Cameroon, Egypt and France and identified through DNA barcoding. A single-end genotyping-by-sequencing (GBS) library, comprising 87 snail specimens from the respective countries, was built and sequenced on an Illumina HiSeq 2000 platform. Reads were mapped against S. bovis and S. haematobium reference genomes to identify schistosome infections, and single nucleotide polymorphisms (SNPs) were scored using the Stacks pipeline. These SNPs were used to estimate genetic diversity, assess population structure and construct phylogenetic trees of B. truncatus., Results: A total of 10,750 SNPs were scored and used in downstream analyses. The phylogenetic analysis identified five clades, each consisting of snails from a single country but with two distinct clades within Senegal. Genetic diversity was low in all populations, reflecting high selfing rates, but varied between locations due to habitat variability. Significant genetic differentiation and isolation by distance patterns were observed at both spatial scales, indicating that gene flow is not strong enough to counteract the effects of population bottlenecks, high selfing rates and genetic drift. Remarkably, the population genetic differentiation on a regional scale (i.e. within Senegal) was as large as that between populations on an inter-regional scale. The blind GBS technique was able to pick up parasite DNA in snail tissue, demonstrating the potential of HTS techniques to further elucidate the role of snail species in parasite transmission., Conclusions: HTS techniques offer a valuable toolbox to further investigate the population genetic patterns of intermediate schistosome host snails and the role of snail species in parasite transmission., (© 2022. The Author(s).)

Pharmaceutical Active Compounds (PhACs) are of particular interest among the emerging contaminants detected in the aquatic environment. Commonly, PhACs exist as complex mixtures in aquatic systems, causing potential adverse effects to the environment and human health than those of individual compounds. Based on the increasing interest in the contamination of water resources by PhACs, the photocatalytic degradation of Cimetidine and Amisulpride as a mixture in combination with their toxic and genotoxic effects before and after the treatment were evaluated for the first time. The toxic, genotoxic and cytotoxic effects were investigated using the Trypan Blue Exclusion Test and the Cytokinesis Block MicroNucleus (CBMN) assay in cultured human lymphocytes. The photocatalytic degradation of the PhACs was studied in ultrapure water and environmentally relevant matrices using UV-A and visible (Vis) irradiation and C-TiO 2 (TiO 2 Kronos vlp 7000) as photocatalyst. High removal percentages were observed for both compounds under UV-A and Vis irradiation in ultrapure water. In lake and drinking water a slower degradation rate was shown that could be attributed to the complex composition of these matrices. Scavenging experiments highlighted the significant role of h + and O 2 ●- in the degradation mechanisms under both irradiation sources. Oxidation, dealkylation and deamination were the main degradation pathways. Regarding the individual compounds, Amisulpride was found to be more cytotoxic than Cimetidine. No significant differences of the genotoxic effects during the treatment were observed. However, a slight increase in cytotoxicity was observed at the first stages of the process. At the end of the process under both UV-A and Vis light, non-significant cytotoxic/toxic effects were observed. Based on the results, heterogeneous photocatalysis can be considered as an effective process for the treatment of complex mixtures without the formation of harmful transformation products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We herein aimed to assess the effect of eribulin mesylate on the cancer stem cell (CSC)/EMT-like phenotype of CTCs, and to investigate the prognostic role of CTC detection and monitoring for eribulin-treated BC patients. Peripheral blood was obtained at baseline ( n = 42 patients) and 8 days after treatment initiation (C1D8: n = 22), and on disease progression (PD: n = 26). PBMCs cytospins were immunofluorescently stained for Cytokeratins/ALDH1/TWIST1/DAPI and analyzed via Ariol microscopy. CTCs were detected in 33.3%, 27.3%, and 23.1% of patients at baseline, C1D8, and PD, respectively. Accordingly, partial-EMT+ CTCs represented 61.3%, 0%, and 37.5% of total CTCs, whereas the CSC-like phenotype was consistently expressed by 87.5%, 75%, and 91.7% of CTCs at the respective time points. Interestingly, the CSC+/partial-EMT+ subset prevailed at baseline, but it was eradicated on C1D8 and resurged again during PD. CTC detection at baseline was associated with reduced PFS ( p = 0.007) and OS ( p = 0.005), and was an independent risk factor for death (HR: 3.779, p = 0.001; multivariate analysis). The CSC+/partial-EMT+ CTCs emerged as the only subset with adverse prognostic significance, while CTC monitoring during eribulin therapy improved the prediction of disease progression. These results indicate that resistant CTC subsets persevere eribulin treatment and highlight the prognostic implications of CTC analyses for eribulin-treated BC patients.

The present study investigated the cyto-genotoxic and antigenotoxic effects of four different extracts of Equisetum arvense L. (common name: field horsetail) on human lymphocytes. Specifically, Soxhlet's prepared extracts from E. arvense L., using different solvents (S1: methanol (MeOH)-, S2: ethanol (EtOH)-, S3: water-, and S4: ethanol/water (EtOH-W)-) were analyzed for (a) their total phenolic and flavonoid content (TPC and TFC, respectively), (b) their antioxidant activity (AA), via the DPPH, FRAP and ABTS assays, and (c) their cyto-genotoxic and/or protective efficiency against the mutagenic agent mitomycin C, via the Cytokinesis Block MicroNucleus assay. All extracts showed increased TPC, TFC, and AA values in almost all cases. S1, S3 and S4 demonstrated no cytotoxic potential, whereas S2 was cytotoxic only at the highest concentrations. Genotoxicity was not observed in the tested extracts. The highest antigenotoxic activity was observed for EtOH-W (S4) extract, which was found to be rich in flavonoids, flavonoid- O -glycosides, phytosterols, phenolic and fatty acids as well as in minerals and mainly in K, Ca, Mg, Si and P, as assessed by using various mass spectrometry techniques. Those findings confirm that E. arvense L. extracts could be valuable candidates for medicinal applications and pharmaceutical products, thus alleviating the effects of more conventional drugs.

This study provides a high-resolution reconstruction of the vegetation of the Argive Plain (Peloponnese, Greece) covering 5000 years from the Early Bronze Age onwards. The well dated pollen record from ancient Lake Lerna has been interpreted in the light of archaeological and historical sources, climatic data from the same core and other regional proxies. Our results demonstrate a significant degree of human impact on the environments of the Argive Plain throughout the study period. During the Early Bronze Age evidence of a thermophilous vegetation is seen in the pollen record, representing the mixed deciduous oak woodland of the Peloponnesian uplands. The plain was mainly used for the cultivation of cereals, whereas local fen conditions prevailed at the coring site. Towards the end of this period an increasing water table is recorded and the fen turns into a lake, despite more arid conditions. In the Late Bronze Age, the presence of important palatial centres modified the landscape resulting in decrease of mixed deciduous oak woodland and increase in open land, partly used for grazing. Possibly, the human management produced a permanent hydrological change at Lake Lerna. From the Archaic period onwards the increasing human pressure in association with local drier conditions caused landscape instability, as attested by a dramatic alluvial event recorded in the Pinus curve at the end of the Hellenistic Age. Wet conditions coincided with Roman times and favoured a forest regeneration pattern in the area, at the same time as we see the most intensive olive cultivation in the pollen record. The establishment of an economic landscape primarily based on pastures is recorded in the Byzantine period and continues until modern times. Overgrazing and fires in combination with arid conditions likely caused degradation of the vegetation into garrigue, as seen in the area of the Argive Plain today., Competing Interests: The authors have declared that no competing interests exist.

Pediatric chronic kidney disease (CKD) patients, as well as kidney transplant patients, are at an increased risk of developing cardiovascular disease. BNP measurement, as a biomarker of cardiovascular risk, has been recommended to this high-risk population. Plasma BNP levels were measured in 56 CKD children in either pre-dialysis stage, hemodialysis (HD) or renal transplant recipients (RTRs) and in 76 sex- and age-matched healthy controls. BNP levels were investigated in HD children, before and after the completion of their HD session. BNP levels in total CKD population, in pre-dialysis stage patients and on HD were significantly higher, compared to the respective controls. HD children had higher BNP levels compared to CKD patients in the pre-dialysis stage. Moreover, post-HD BNP concentration was slightly higher than pre-HD, with the difference being marginally statistically significant. BNP was positively correlated with eGFR, creatinine, cystatin-C and parathormone and negatively with albumin and 25-hydroxyvitamin D. A positive correlation between BNP concentration and the ratio of E/A in pulse-wave Doppler echocardiography was also observed. In conclusion, CKD pediatric patients, mainly those undergoing HD, have high plasma BNP levels which do not decrease after the HD session. This is indicative of a greater risk for future cardiovascular disease.

Background: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance., Methods: Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied., Results: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA., Conclusions: We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

TLR4 and pSTAT3 are key players in cancer inflammation and immune evasion; however, their role in the peripheral blood (PB) is largely unexplored. Herein we evaluated their expression in the circulating tumor cells (CTCs) and peripheral-blood mononuclear cells (PBMCs) of patients with early ( n = 99) and metastatic ( n = 100) breast cancer (BC). PB samples obtained prior to adjuvant and first-line therapy, were immunofluorescently stained for Cytokeratins/TLR4/pSTAT3/DAPI and analyzed via Ariol microscopy. TLR4+ CTCs were detected in 50% and 68% of early and metastatic CTC-positive patients, respectively, and pSTAT3+ CTCs in 83% and 68%, respectively. In metastatic patients, CTC detection was associated with a high risk of death (HR: 1.764, p = 0.038), while TLR4+ CTCs correlated with a high risk of disease progression (HR: 1.964, p = 0.030). Regarding PBMCs, TLR4 expression prevailed in metastatic disease ( p = 0.029), while pSTAT3 expression was more frequent in early disease ( p = 0.014). In early BC, TLR4 expression on PBMCs independently predicted for high risk of relapse (HR: 3.549; p = 0.009), whereas in metastatic BC, TLR4+/pSTAT3- PBMCs independently predicted for high risk of death (HR: 2.925; p = 0.012). These results suggest that TLR4/pSTAT3 signaling on tumor- and immune-cell compartments in the PB could play a role in BC progression, and may hold independent prognostic implications for BC patients.

Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been because of a paucity of methods for objective and quantitative analysis. Here, we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near-physiological conditions and demonstrate its superiority as compared with conventional histological assessments. Using small-angle x-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wild-type mouse myocardium from the left ventricle by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length of 1.9 μm, the angle σ was 0.23 ± 0.01 rad, decreased to 0.19 ± 0.01 rad at a sarcomere length of 2.1 μm, and further decreased to 0.15 ± 0.01 rad at a sarcomere length of 2.3 μm (p < 0.0001). Angle σ was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy model, porcine myocardium (0.24 ± 0.01 rad) compared with wild-type myocardium (0.14 ± 0.005 rad; p < 0.0001), as well as in human heart failure tissue (0.19 ± 0.006 rad) when compared with nonfailing samples (0.17 ± 0.007 rad; p = 0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared with healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological approaches that assess myocyte orientation and only indirectly evaluate myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent x-ray diffraction patterns from frozen human myocardium provides a new tool for investigating structural anomalies associated with cardiac diseases., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Modern information systems have to support the user in managing, understanding and interacting with, more and more data. Visualization could help users comprehend information more easily and reach conclusions in relative shorter time. However, the bigger the data is, the harder the problem of visualizing it becomes. In this paper we focus on the problem of placing a set of values in the 2D (or 3D) space. We present a novel family of algorithms that produces spiral-like layouts where the biggest values are placed in the centre of the spiral and the smaller ones in the peripheral area, while respecting the relative sizes. The derived layout is suitable not only for the visualization of medium-sized collections of values, but also for collections of values whose sizes follow power-law distribution because it makes evident the bigger values (and their relative size) and it does not leave empty spaces in the peripheral area which is occupied by the majority of the values which are small. Therefore, the produced drawings are both informative and compact. The algorithm has linear time complexity (assuming the values are sorted), very limited main memory requirements, and produces drawings of bounded space, making it appropriate for interactive visualizations, and visual interfaces in general. We showcase the application of the algorithms in various domains and interactive interfaces., (© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021.)

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 μM MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MG's functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosin's blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosin's position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Although several studies have been launched towards the prediction of risk factors for mortality and admission in the intensive care unit (ICU) in COVID-19, none of them focuses on the development of explainable AI models to define an ICU scoring index using dynamically associated biological markers., Methods: We propose a multimodal approach which combines explainable AI models with dynamic modeling methods to shed light into the clinical features of COVID-19. Dynamic Bayesian networks were used to seek associations among cytokines across four time intervals after hospitalization. Explainable gradient boosting trees were trained to predict the risk for ICU admission and mortality towards the development of an ICU scoring index., Results: Our results highlight LDH, IL-6, IL-8, Cr, number of monocytes, lymphocyte count, TNF as risk predictors for ICU admission and survival along with LDH, age, CRP, Cr, WBC, lymphocyte count for mortality in the ICU, with prediction accuracy 0.79 and 0.81, respectively. These risk factors were combined with dynamically associated biological markers to develop an ICU scoring index with accuracy 0.9., Conclusions: to our knowledge, this is the first multimodal and explainable AI model which quantifies the risk of intensive care with accuracy up to 0.9 across multiple timepoints.

Polycystic ovary syndrome (PCOS) is the most common endocrine condition affecting 6-18% of adolescents and is strongly associated with obesity and cardiovascular risk factors, enhancing the risk of atherosclerosis. Thirty-two adolescents with newly diagnosed PCOS were evaluated for lipid profile disorders, insulin resistance, inflammation, non-alcoholic fatty liver disease (NAFLD), and subclinical atherosclerosis through measurements of carotid intima-media thickness (cIMT). The relationships of the above markers with increased body mass index and abdominal obesity were investigated. Twenty-three adolescents (72%) were overweight (OW) or obese (OB). The OW/OB group had significantly higher insulin, HOMA-IR, high-sensitive C-reactive protein (hsCRP), visceral adiposity index (VAI), and lipid accumulation product (LAP) levels; and lower glucose-per-insulin ratios and HDL-C levels compared to the healthy weight group. The cIMT and small dense low-density lipoprotein cholesterol (sdLDL-C) levels did not differ between the two groups. Similarly, cIMT and sdLDL-C levels did not differ between PCOS-adolescents and healthy controls. CIMT was positively correlated with systolic blood pressure and waist circumference per height ratio. In conclusion, OW/OB PCOS-adolescents have a cluster of adverse factors predisposing them to atherosclerotic cardiovascular disease. Therefore, early cardiovascular risk assessment, as well as timely and targeted interventions, are necessary for prevention.

Disturbances in the primary colonization of the infant gut can result in lifelong consequences and have been associated with a range of host conditions. Although early-life factors have been shown to affect infant gut microbiota development, our current understanding of human gut colonization in early life remains limited. To gain more insights into the unique dynamics of this rapidly evolving ecosystem, we investigated the microbiota over the first year of life in eight densely sampled infants ( n  = 303 total samples). To evaluate the gut microbiota maturation transition toward an adult configuration, we compared the microbiome composition of the infants to that of the Flemish Gut Flora Project (FGFP) population ( n  = 1,106). We observed the infant gut microbiota to mature through three distinct, conserved stages of ecosystem development. Across these successional gut microbiota maturation stages, the genus predominance was observed to shift from Escherichia over Bifidobacterium to Bacteroides . Both disease and antibiotic treatment were observed to be associated occasionally with gut microbiota maturation stage regression, a transient setback in microbiota maturation dynamics. Although the studied microbiota trajectories evolved to more adult-like constellations, microbiome community typing against the background of the FGFP cohort clustered all infant samples within the (in adults) potentially dysbiotic Bacteroides 2 (Bact2) enterotype. We confirmed the similarities between infant gut microbial colonization and adult dysbiosis. Profound knowledge about the primary gut colonization process in infants might provide crucial insights into how the secondary colonization of a dysbiotic adult gut can be redirected. IMPORTANCE After birth, microbial colonization of the infant intestinal tract is important for health later in life. However, this initial process is highly dynamic and influenced by many factors. Studying this process in detail requires a dense longitudinal sampling effort. In the current study, the bacterial microbiota of >300 stool samples was analyzed from 8 healthy infants, suggesting that the infant gut microbial population matures along a path involving distinct microbial constellations and that the timing of these transitions is infant specific and can temporarily retrace upon external events. We also showed that the infant microbial populations show similarities to suboptimal bacterial populations in the guts of adults. These insights are crucial for a better understanding of the dynamics and characteristics of a "healthy gut microbial population" in both infants and adults and might allow the identification of intervention targets in cases of microbial disturbances or disease.

The histological process of gonadal differentiation, together with the endocrine changes of sex steroid hormones and some of their precursors, was studied in hatchery-produced greater amberjack Seriola dumerili from 101 until 408 days post-hatching (dph), with samplings conducted every 50 days. Histological processing showed that sex differentiation began at 101 dph with the formation of the ovarian cavity in females, while the presumptive males did not yet contain any germ cells in their gonad. At 150 dph, we observed the first germ cells in the developing testes. Sex differentiation in almost all sampled individuals was complete at 408 dph. No size dimorphism was observed between the sexes, and the sex ratio was 1:1, suggesting that there was no influence of early rearing in captivity on sex differentiation. Plasma concentrations of adrenosterone (Ad), androstenedione (Δ4), 11-ketotestosterone (11ΚΤ), testosterone (Τ), estradiol (Ε 2 ), progesterone (P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) were measured in males and females with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine their role in the sex differentiation process. From the seven hormones, the only one that exhibited differences between the sexes was 11-KT and the plasma 11-KT concentration was found to be a useful indication of greater amberjack sex. Variations were observed in the mean values of Ad, Δ4, 11-KT, T, P4 and 17,20βP over time in one or both sexes, indicating their involvement in the sex differentiation process., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The effect of fasting on spawning performance, maternal, and egg nutrient composition, and on embryo/larval development was monitored in gilthead seabream (Sparus aurata). Two broodstocks were fasted during two consecutive years, for a period of 43 and 54 days within the spawning season, in a preliminary (year 1, 5-year-old breeders) and the main study (year 2, 6-year-old breeders), respectively. Mean daily fecundity showed a declining trend during fasting in the main study only, while fertilization success was high in both years and it was not affected by fasting, as was hatching and 5-day larval survival. There was a loss of 23.5% of maternal body mass due to fasting, and a reduction in gonadosomatic and hepatosomatic indexes, as well as crude protein in maternal muscle and gonads, but not in the liver. After fasting, muscle Σω-6 PUFA and C18:3ω-3 were reduced while C20:4ω-6, 20:5ω-3/20:4ω-6, and C22:6ω-3/20:4ω-6 increased; in the liver, significant reductions were observed in C16:0, C18:3 ω-3, 20:5ω-3/C22:6ω-3 and increases in C18:0, C20:5ω-3, Σω-6 PUFA, and 20:5ω-3/20:4ω-6; in gonads, C15:0, ΣMUFA, 20:5ω-3/C22:6ω-3, 20:5ω-3/20:4ω-6 were increased, while C18:1ω-9 and C20:5ω-3 decreased. Contrary to maternal tissues, the energy density and proximate composition of the eggs did not change due to fasting. The study suggests that fasting of gilthead seabream breeders for 6-8 weeks during the spawning period does not affect spawning performance, egg proximate composition, or embryo and early larval development since maternal nutrient reserves are mobilized to maintain optimal egg nutrient composition., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: To investigate the association of serum vitamin D and nasal secretion antimicrobial peptides (AMPs) levels with the severity of acute bronchiolitis., Study Design: We conducted a prospective single pediatric tertiary care center cohort study of inpatients aged 0-18 months with a first episode of acute bronchiolitis from November 1st 2014 to April 30th 2017. Disease severity was determined by the length of hospitalization and supplemental hospital data. Qualitative measurements included serum 25(OH)D and nasal secretion LL-37 and β-defensin-2 levels. Correlations were examined with the Mann-Whitney and Kruskal-Wallis criteria for qualitative and the correlation coefficient Spearman's rho for quantitative factors. Multiple linear and logarithmic regression were performed to adjust for confounding factors., Results: The study population consisted of 153 infants and toddlers with median age 3.1 months (interquartile range:1.6-4.9). No association was found between serum 25(OH)D and AMPs nasal secretions levels. Serum 25(OH)D and nasal secretion β-defensin-2 levels were not associated with the severity of bronchiolitis. In contrast, LL-37 levels were inversely associated with the length of hospitalization (rho = -0.340, p = .001), the need for medication use (p = .001), as well as the duration of oxygen supplementation (rho = -0.339, p = .001), and intravenous fluid administration (rho = -0.323, p = .001). This association remained significant after adjustment for potential confounders., Conclusion: A significant association between LL-37 nasal secretions levels with the severity of acute bronchiolitis was found in hospitalized infants and toddlers. The role of LL-37 in the pathogenesis of bronchiolitis merits further investigation., (© 2021 Wiley Periodicals LLC.)

Sturgeon sperm maturation occurs outside the testes during the transit of testicular spermatozoa (TS) through the kidneys and the Wolffian ducts. A method of in vitro TS maturation in sterlet Acipenser ruthenus was used to investigate the effects of temperature and hormonal stimulation of spermiation on the ability of TS to complete this process. Spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology were studied in three groups of sterlet: (1) after overwintering in ponds (OW), (2) adapted to spawning temperature (ST), and (3) adapted to spawning temperature with hormonal induction of spermiation (ST-HI). Blood plasma concentrations of testosterone, 11-ketotestosterone and 17,20β-dihydroxy-pregnenolone increased significantly after hormonal induction of spermiation (group ST-HI). In all groups, TS were not motile. After in vitro sperm maturation, motility was up to 60% only in group ST-HI. The data suggest that the ability of TS to be matured in vitro was not related to the environmental temperature, while hormonal stimulation of spermiation during the spawning season was an absolute requirement for optimal in vitro maturation.

MicroRNAs (miRNAs) are involved in the regulation of immune response and hold an important role in tumor immune escape. We investigated the differential expression of the immunomodulatory miR-10b, miR-19a, miR-20a, miR-126, and miR-155 in the plasma of healthy women and patients with early stage breast cancer and interrogated their role in the prediction of patients' relapse. Blood samples were obtained from healthy women ( n = 20) and patients with early stage breast cancer ( n = 140) before adjuvant chemotherapy. Plasma miRNA expression levels were assessed by RT-qPCR. Relapse predicting models were developed using binary logistic regression and receiver operating curves (ROC) were constructed to determine miRNA sensitivity and specificity. Only miR-155 expression was lower in patients compared with healthy women ( p = 0.023), whereas miR-155 and miR-10b were lower in patients who relapsed compared with healthy women ( p = 0.039 and p = 0.002, respectively). MiR-155 expression combined with axillary lymph node infiltration and tumor grade demonstrated increased capability in distinguishing relapsed from non-relapsed patients [(area under the curve, (AUC = 0.861; p < 0.001)]. Combined miR-19a and miR-20a expression had the highest performance in discriminating patients with early relapse (AUC = 0.816; p < 0.001). Finally, miR-10b in combination with lymph node status and grade had the highest accuracy to discriminate patients with late relapse (AUC = 0.971; p < 0.001). The robustness of the relapse predicting models was further confirmed in a 10-fold cross validation. Deregulation of circulating miRNAs involved in tumor-immune interactions may predict relapse in early stage breast cancer. Their successful clinical integration could potentially address the significance challenge of treatment escalation or de-escalation according to the risk of recurrence.

Carotid intima-media thickness (cIMT) has been proposed as an early marker of subclinical atherosclerosis in high risk children. Children with heterozygous familial hypercholesterolemia have greater cIMT than matched healthy controls or their unaffected siblings. Statin therapy may delay the progression of cIMT, although long-term studies in children are scarce. We evaluated the effect of atorvastatin treatment on cIMT in children with dyslipidemia. We studied 81 children/adolescents, 27 with severe dyslipidemia (low-density lipoprotein cholesterol [LDL-C] ≥190 mg/dL) and 54 sex- and age-matched healthy controls; LDL-C ≤ 130 mg/dL and lipoprotein (a), Lp(a), ≤30 mg/dL. In the children with dyslipidemia, cIMT was measured twice, before and on treatment (18.2 ± 7.7 months). Anthropometric data, a full lipid profile, liver, kidney, and thyroid function were evaluated. Males with dyslipidemia had a greater cIMT than male controls after adjustment for other factors ( P = .049). In addition, a nonstatistically significant decrease in cIMT was observed after treatment ( P = .261). Treatment with atorvastatin resulted in a significantly improved lipid profile. Females with dyslipidemia had a significantly thinner cIMT than males. Children with normal and high Lp(a) levels had similar cIMT values. In conclusion, treatment with atorvastatin had a beneficial effect on the lipid profile and cIMT progression in children with severe dyslipidemia.

Background: To investigate the correlation between circulating tumor cells (CTCs) bearing cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) phenotypes and the different immunosuppressive cells in peripheral blood of patients with metastatic breast cancer (mBC)., Materials and Methods: Blood was obtained from 38 pre-treated patients with mBC before a new line of treatment. CTC detection and characterization was performed by triple immunofluorescent staining, while Myeloid-derived Suppressor Cells (MDSCs) and T regulatory cells (Tregs) were analyzed by multi-flow cytometry., Results: CTCs were detected in 16 (42.1%) of patients. Based on the co-expression of ALDH1, TWIST and CK, CTCs revealed an important heterogeneity: CTCs with a CSC/partial-EMT, CSC/Epithelial-like, non-CSC/partial-EMT and non-CSC/Epithelial-like phenotype were detected in 7 (18.4%), 7 (18.4%), 1 (1.4%) and 9 (23.7%) of patients, respectively. Immunophenotyping of MDSCs identified 2 monocytic [M-MDSCs; CD14 + CD15 + CD11b + CD33 + HLA-DR - Lin - (CD14 + CD15 + ) and CD14 + CD15 - CD11b + CD33 + HLA-DR - Lin - (CD14 + CD15 - )] and one granulocytic [G-MDSCs; CD14 - CD15 + CD11b + CD33 + HLA-DR - Lin - (CD14 - CD15 + )] subpopulations, expressing inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS), respectively. Patients with detectable CTCs had a higher frequency of Tregs (CD3 + CD4 + CD25 high ; p=0.022) whereas a positive correlation was found between CTC counts and the percentage of Tregs (p=0.005) and CD14 + CD15 + M-MDSCs (p=0.024). Patients with a partial-EMT phenotype had a higher frequency of CD14 + CD15 + M-MDSCs (p=0.023). Patients harboring the non-CSC/epithelial-like CTC subpopulation had an increased frequency of CD14-CD15 + G-MDSCs (p=0.020), along with decreased levels of CD3 + CD4 + CD25 high FoxP3+ Tregs (p=0.020)., Conclusion: These findings provide evidence that CTCs in ER + /HER2 - mBC patients may be under the control of the immune system and various immune escape mechanisms might be involved during the different stages of their biological evolution., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

We herein investigated the detection frequency and clinical relevance of circulating tumor cells (CTCs) in chemotherapy-naïve stage IIIB/IV non-small cell lung cancer (NSCLC), by using the CellSearch and real-time CEACAM5mRNA assays. Blood samples from 43 patients were obtained at different time points during first-line chemotherapy. CellSearch revealed the detection of ≥1 CTCs in 41.9%, 40.9%, and 16.7% of patients at baseline, post-1st, and post-2nd treatment cycle, respectively, and of ≥5 CTCs in 11.6%, 9.1%, and 5.6%, respectively. CEACAM5mRNA+ CTCs were detected in 29.3% and 16% of patients pre- and post-treatment, respectively. The positivity concordance between the two assays was 2.2%. CTC-detection by CellSearch (≥5 CTCs: p = 0.004), CEACAM5mRNA ( p = 0.010), or by any assay ( p = 0.000) was associated with disease progression. Reduced survival was demonstrated for patients harboring ≥5 CTCs (progression-free survival; PFS: p = 0.000; overall survival; OS: p = 0.009), CEACAM5mRNA+ CTCs (PFS: p = 0.043; OS: p = 0.039), and CTCs by any assay (PFS: p = 0.005; OS: p = 0.006, respectively). CTC-detection by any assay independently predicted for increased risk of relapse (hazard ratio; HR: 3.496; p = 0.001) and death (HR: 2.866; p = 0.008). CellSearch-positivity either pre-, post-1st, or post-2nd cycle, was predictive for shorter PFS ( p = 0.036) compared to negativity in all time points. Persistent CEACAM5mRNA-positivity pre- and post-treatment was associated with reduced PFS ( p = 0.036) and OS ( p = 0.026). In conclusion, CTC detection and monitoring using the CellSearch and CEACAM5mRNA assays provides valuable and complementary clinical information for chemo-naïve advanced or metastatic NSCLC.

We aimed to evaluate the co-expression of PD-L1 and epithelial-mesenchymal markers in CTCs from metastatic breast cancer (MBC) patients and to determine if there is any relationship with patients' outcome after eribulin treatment. Using cytospin preparations of peripheral blood mononuclear cells (PBMCs) from MBC patients treated with eribulin and a combination of immunocytochemistry and immunofluorescence, we quantified PD-L1, keratins and vimentin in single and cluster CTCs on days 1 and 8 of the first-treatment cycle. CTCs ( n = 173) were found in 31 out of 38 patients. At baseline, the presence of cluster CTCs ( p = 0.048), cluster mesenchymal CTCs (mCTCs) ( p = 0.0003) or cluster PD-L1 + mCTCs ( p = 0.006) was associated with shorter overall survival (OS). In multivariate cox regression analysis, the detection of cluster mCTCs was the only parameter associated with increased risk of death ( p = 0.024). On day 8 post-eribulin administration, PD-L1 + mCTCs and especially single PD-L1 + mCTCs decreased in 75% and 89% of patients, respectively. The detection of single PD-L1 + mCTCs after eribulin treatment was correlated with shorter PFS ( p = 0.047) and OS ( p = 0.020). In conclusion, our study identified for the first time that cluster and single PD-L1 + mCTCs subpopulations are of clinical significance in patients with MBC and highlighted the importance of CTC phenotyping during treatment with eribulin.

KRAS mutations are found in approximately one third of non-small cell lung cancer (NSCLC) patients. In this study, we aim to investigate whether KRAS G12/G13 mutant allele fraction (MAF) in cell-free DNA (cfDNA) can provide meaningful prognostic information in NSCLC. Multiplex droplet-digital PCR was used to quantitatively assess KRAS G12/G13 MAF in cfDNA from 114 pre-treated advanced disease NSCLC patients. In 14 patients, changes in KRAS G12/G13 MAF were longitudinally monitored during treatment. Plasma KRAS G12/G13 status was associated with poor patients' outcome in terms of progression-free survival (PFS) ( p < 0.001) and overall survival (OS) ( p < 0.001). In multivariate analysis, the detection of plasma KRAS mutations was an independent predictor of adverse PFS (HR = 3.12; p < 0.001) and OS (HR = 2.53; p = 0.002). KRAS G12/G13 MAF at first treatment evaluation (T1) was higher ( p = 0.013) among patients experiencing progressive disease compared to those with disease control, and increased KRAS MAF at T1 was associated ( p = 0.005) with shorter PFS. On the contrary, no association was observed between tissue KRAS mutation status and patients' prognosis. Our results show that ddPCR-based detection of KRAS G12/G13 mutations in plasma could serve as an independent biomarker of unfavorable prognosis in NSCLC patients. Changes in KRAS MAF can provide valuable information for monitoring patient outcome during treatment.

Meagre (Argyrosomus regius) undergo spermatogenesis and spermiation when reared in captivity, but often produce low milt volumes, sometimes with reduced quality and for a limited time period. In the present study we a) compared the efficiency of gonadotropin releasing hormone agonist (GnRHa) implants versus injections on testicular stimulation and spermiation enhancement, b) investigated the effect of GnRHa on the endocrine spermiation regulation (sex steroid hormones), and c) evaluated a commercial induced spawning simulation scenario. Firstly, males (n = 5) were injected with 15.0 ± 0.2 μg GnRHa kg -1 (Injections) or implanted with 51.0 ± 5.1 μg GnRHa kg -1 (Implant) and compared their sperm production response. Secondly, the best hormonal treatment (Implant) was tested treating males (n = 8) with 57.5 ± 7.5 μg GnRHa kg -1 every 3 weeks for a period of 70 days. Milt production was improved by the GnRHa implants with only minor sperm quality alterations (improved sperm motility percentage). Elevated plasma testosterone (T) and 11-ketotestosterone (11-KT) levels were recorded in response to GnRHa implants, while no significant difference for 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was observed. In the commercial induced spawning simulation, it was shown that meagre females are capable of on-demand induction of spawning at random intervals (5-21 days) using GnRHa injections, over a period of at least 2.5 months. During this period, spermiation enhancement was achieved with GnRHa implants every 3 weeks, producing sperm with stable, in general, quality and motility parameters. Percentage of motile cells, motility duration and density fluctuated significantly, but remained within levels that are considered appropriate for high fertilization success in this species., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Non-coding RNAs (ncRNAs) are involved in several different regulatory pathways including reproduction. In teleost fish, efficacious reproduction is heavily dependent on the completion of the reproductive cycle. The presence of ncRNA, however, and their expression dynamics and putative regulatory role in mature and immature gonads have not yet been extensively explored. Therefore, the abundance of ncRNAs in mature and immature female sharpsnout seabream (Diplodus puntazzo) was investigated. The sharpsnout seabream is a rudimentary hermaphrodite which, in captivity, displays dysfunctions in the gonad maturation process. Our analyses revealed a gonad specific read length distribution with two main peaks representing miRNAs (21-26 nt) and PIWI RNA (27-34 nt). Besides, distinct expression patterns for several ncRNA biotypes including microRNAs (miRNAs), PIWI RNAs (piRNAs), and ribosomal RNAs (rRNAs) were detected. Identified miRNA accounted to 938, corresponding to ~ 13% of obtained transcripts. Among the differential expressed ncRNAs, 10 (~ 7%) were annotated as miRNA, out of which 2 were found in higher abundance in immature gonads (miR-125c and miR-24) and 8 (miR-451, miR-7a, miR-122-1, miR190a, miR129, ENSGACT00000029608, ENSGACT00000029489, and ENSGACT00000029667) were found to be higher expressed in mature gonads. Putative miRNA targets, including long non-coding RNAs (lncRNAs) and genes, are proposed. Target genes are involved in several processes of fish oocyte development, such as steroidogenesis, proteolysis, and apoptosis, and may explain hormone regulation. This study demonstrates a gonad maturation biased ncRNA profile which in turn may support the role of ncRNAs in ovarian physiology and reproductive performance of fish, stressing the specific function of each RNA biotype in oocyte development.

Human heart samples from the Sydney Heart Bank have become a de facto standard against which others can be measured. Crucially, the heart bank contains a lot of donor heart material: for most researchers this is the hardest to obtain and yet is necessary since we can only study the pathological human heart in comparison with a control, preferably a normal heart sample. It is not generally realised how important the control is for human heart studies. We review our studies on donor heart samples. We report the results obtained with 17 different donor samples collected from 1994 to 2011 and measured from 2005 to 2015 by our standard methodology for in vitro motility and troponin I phosphorylation measurements. The donor heart sample parameters are consistent between the hearts, over time and with different operators indicating that Sydney Heart Bank donor hearts are a valid baseline control for comparison with pathological heart samples. We also discuss to what extent donor heart samples are representative of the normal heart.

The current study aimed at the optimization of circulating tumor cell (CTC) enrichment for downstream protein expression analyses in non-small cell lung cancer (NSCLC) to serve as a tool for the investigation of immune checkpoints in real time. Different enrichment approaches-ficoll density, erythrolysis, their combination with magnetic separation, ISET, and Parsortix-were compared in spiking experiments using the A549, H1975, and SKMES-1 NSCLC cell lines. The most efficient methods were tested in patients ( n = 15) receiving immunotherapy targeting programmed cell death-1 (PD-1). Samples were immunofluorescently stained for a) cytokeratins (CK)/epithelial cell adhesion molecule (EpCAM)/leukocyte common antigen (CD45), and b) CK/programmed cell death ligand-1 (PD-L1)/ indoleamine-2,3-dioxygenase (IDO). Ficoll, ISET, and Parsortix presented the highest yields and compatibility with phenotypic analysis; however, at the patient level, they provided discordant CTC positivity (13%, 33%, and 60% of patients, respectively) and enriched for distinct CTC populations. IDO and PD-L1 were expressed in 44% and 33% and co-expressed in 19% of CTCs. CTC detection was associated with progressive disease (PD) ( p = 0.006), reduced progression-free survival PFS ( p = 0.007), and increased risk of relapse (hazard ratio; HR: 10.733; p = 0.026). IDO-positive CTCs were associated with shorter PFS ( p = 0.039) and overall survival OS ( p = 0.021) and increased risk of death (HR: 5.462; p = 0.039). The current study indicates that CTC analysis according to distinct immune checkpoints is feasible and may provide valuable biomarkers to monitor NSCLC patients treated with anti-PD-1 agents.

The role of CD47 and PD-L1 expression on circulating tumor cells (CTCs) remains unclear, and it is currently unknown whether their distribution varies between the blood and tumor tissue in breast cancer (BC). In this study, CD47 and PD-L1 expression was investigated a) on peripheral blood mononuclear cell (PBMC) cytospins from early ( n = 100) and metastatic ( n = 98) BC patients, by triple immunofluorescence for CD47/PD-L1/Cytokeratins, and b) on matched primary and/or metastatic tumor tissue from CTC-positive patients using immunohistochemistry. CD47+and/orPD-L1+ CTCs were detected in 11%, 16.9%, and 29.6% of early, recurrent, and de novo metastatic patients ( p = 0.016). In metastatic disease, CD47 high and/orPD-L1 high CTCs were associated with disease progression ( p = 0.005) and shorter progression-free survival (PFS) ( p = 0.010), and independently predicted for an increased risk of relapse (HR: 2.719; p = 0.008) and death (HR: 2.398; p = 0.034). PD-L1 expression rates differed between CTCs and tissue tumor cells and between peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) (positive concordance of 3.8% and 4%, respectively). CD47 expression also differed between CTCs and tumor cells (positive concordance of 11.5%). In conclusion, CTCs expressing CD47 and PD-L1 have independent poor prognostic implications in metastatic BC, indicating a potential role of innate and adaptive immune evasion mechanisms in their metastatic potential. The clinical value of the parallel assessment of the peripheral and local immune response merits further evaluation in BC., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

The greater amberjack (Seriola dumerili) is one of the most promising finfish species for aquaculture, due to its cosmopolitan distribution and acceptability, high growth rates and large size. However, lack of reproduction control has been one of the main bottlenecks for its commercialization. The study examined two endocrine methods for the induction of oocyte maturation/ovulation and spawning, which are based on the induction of endogenous Luteinizing Hormone (LH) release from the pituitary, through the use of Gonadotropin Releasing Hormone agonist (GnRHa) either in the form of sustained-release delivery systems (implants) or injections (acute release). The stock (n = 28) consisted of wild fish captured in 2011 and each GnRHa administration method was conducted in two replicates, with 6 and 8 fish per tank, respectively, at a 1:1 sex ratio. Fish were given a GnRHa injection once a week (three administrations), or a GnRHa implant every 2 weeks (two administrations). Mean daily relative fecundity was significantly higher (t 1.3  = -5.24, P = 0.012) in the implanted fish (15,170 ± 2,738 eggs kg -1 female day -1 ) compared to the injected fish (6,119 ± 2,790 eggs kg -1 female day -1 ). Total relative fecundity was also significantly higher (t 1.3  = -9.93, P = 0.003) in the implanted fish (102,402 ± 20,337 eggs kg -1 female) compared to the injected ones (26,517 ± 9,938 eggs kg -1 female), but there were no differences in the quality of eggs in terms of fertilization, 24-h embryo survival, hatching and 5-d larval survival. The number of females with fully vitellogenic oocytes eligible for induction of spawning decreased from the initial to the final sampling, from 7 to 6 females for the GnRHa implant treatment and from 7 to 3 females for the GnRHa injection treatment. In addition to apparently promoting the proper endocrine changes leading to multiple cycles of oocyte maturation, ovulation and spawning, and thus producing larger numbers of eggs; the use of GnRHa implants may be more appropriate in greater amberjack than multiple injections because (a) it also stimulates vitellogenesis and (b) involves less handling of the fish., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Polymerization reactions have caused a number of serious incidents in the past; they are prone to reaction runaways because of their exothermic and autoaccelerating nature. To minimalize the risk, the reaction is commonly performed in a solvent as empirical industrial practice. In this work, the thermal runaway hazards of the ethylbenzene-styrene system with different monomer mass fractions were calorimetrically investigated up to temperatures where decomposition products are unlikely to be produced. Experiments showed that the polymerization runaway "onset" temperature inversely increased with the monomer mass fraction. Experiment and thermodynamic calculations showed that volatile diluent increased system vapor pressure even at a lower adiabatic temperature rise and verified that moderation of the risks could be achieved if the monomer mass fraction is below ca. 85%. A lumped kinetic model developed by Hui and Hamielec was used to predict the runaway profile of this reaction under different dilutions, and the agreement was excellent., Competing Interests: The authors declare no competing financial interest.

Diisononyl phthalate (DiNP) is a plasticizer used to improve plastic performance in a large variety of items which has been reported as an endocrine-disrupting chemical (EDC) in several organisms. The endocannabinoid system (ECS) is a cellular signaling system, whose functionality is tightly involved with reproductive function. The aim of the present study was the assessment of the effects of DiNP on the gonadal ECS and on the reproductive function of male gilthead sea bream Sparus aurata, an important marine aquacultured species in Europe, during the reproductive season. Fish were fed for 21 days with two diets contaminated with different nominal concentrations of DiNP (DiNP LOW at 15 µg DiNP kg -1 bw day -1 and DiNP HIGH at 1500 µg DiNP kg -1 bw day -1 ), based on the tolerable daily intake (TDI) ruled by the European Food Safety Authority for humans. The transcription of several genes related to the ECS was affected by the DiNP. Specifically, DiNP reduced the levels of endocannabinoids and endocannabinoid-like mediators, concomitant with the increase of fatty acid amide hydrolase (FAAH) activity. At the histological level, DiNP LOW induced the highest occurrence of individuals with regressed testes. Steroidogenesis was affected significantly, since plasma 11-ketotestosterone (11-KT), the main active androgen in fish, was significantly decreased by the DiNP HIGH treatment, while plasma 17β-estradiol (E 2 ) levels were raised, associated with an increase of the gonadosomatic index (GSI). Additionally, the level of testosterone (T) was significantly increased in the DiNP LOW group, however, the same DiNP concentration reduced the levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P). The production of sperm was in general not affected, since spermiation index, sperm density, survival and the duration of forward motility did not exhibit any changes compared to controls. However, computer-assisted sperm analysis (CASA) showed that DiNP reduced the percentage of motile cells. The results clearly suggest a negative effect of DiNP via the diet on the male endocrine system of gilthead sea bream during the reproductive season.

Receptor activator of nuclear factor-κB ligand (RANKL), a member of the Tumor Necrosis Factor (TNF) superfamily, constitutes the master regulator of osteoclast formation and bone resorption, whereas its involvement in inflammatory diseases remains unclear. Here, we used the human TNF transgenic mouse model of erosive inflammatory arthritis to determine if the progression of inflammation is affected by either genetic inactivation or overexpression of RANKL in transgenic mouse models. TNF-mediated inflammatory arthritis was significantly attenuated in the absence of functional RANKL. Notably, TNF overexpression could not compensate for RANKL-mediated osteopetrosis, but promoted osteoclastogenesis between the pannus and bone interface, suggesting RANKL-independent mechanisms of osteoclastogenesis in inflamed joints. On the other hand, simultaneous overexpression of RANKL and TNF in double transgenic mice accelerated disease onset and led to severe arthritis characterized by significantly elevated clinical and histological scores as shown by aggressive pannus formation, extended bone resorption, and massive accumulation of inflammatory cells, mainly of myeloid origin. RANKL and TNF cooperated not only in local bone loss identified in the inflamed calcaneous bone, but also systemically in distal femurs as shown by microCT analysis. Proteomic analysis in inflamed ankles from double transgenic mice overexpressing human TNF and RANKL showed an abundance of proteins involved in osteoclastogenesis, pro-inflammatory processes, gene expression regulation, and cell proliferation, while proteins participating in basic metabolic processes were downregulated compared to TNF and RANKL single transgenic mice. Collectively, these results suggest that RANKL modulates modeled inflammatory arthritis not only as a mediator of osteoclastogenesis and bone resorption but also as a disease modifier affecting inflammation and immune activation.

Circulating tumor cells (CTCs) bearing phenotypes related to cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) have been identified in breast cancer; however, their clinical significance is not clear. In the current study, we investigated the prognostic relevance of single CSC + /partial-EMT + CTCs in patients with metastatic breast cancer and the effect of first-line chemotherapy on their incidence. For this purpose, triple immunofluorescence against cytokeratin, ALDH1, and TWIST1 was performed in peripheral blood mononuclear cell (PBMC) cytospins from 130 patients before and after first-line chemotherapy. CSC + /partial-EMT + CTCs were characterized as cells co-expressing cytokeratin, high levels of ALDH1, and nuclear TWIST1. CSC + /partial-EMT + CTCs were evident in 27.7% of patients at baseline and were correlated to lung metastases ( P = 0.010) and decreased progression-free survival [PFS; median 10.2 (8.9-11.6) vs. 13.5 (11.3-15.7) months; P = 0.024]. Their detection was an independent factor predicting for increased risk of relapse [multivariate analysis; HR (95% confidence interval (CI)): 1.785 (1.171-2.720); P = 0.007]. In HER-2-negative patients, CSC + /partial-EMT + CTCs were additionally associated with reduced overall survival (OS) [median 39 (26.2-51.9) vs. 51 (15.7-86.4) months; P = 0.020] and increased risk of death [multivariate analysis; HR (95% CI): 2.228 (1.066-4.655); P = 0.033]. Chemotherapy resulted in a significant increase in the incidence of CSC + /partial-EMT + CTCs (mean CTC% per patient: 59.4% post vs. 39.5% pre; P = 0.018), which was subsequently confirmed only in HER2-negative patients ( P = 0.040) and in non-responders at the end of treatment ( P = 0.020). In conclusion, CSC + /partial-EMT + CTCs represent a chemoresistant subpopulation, which independently predicts for unfavorable outcome in metastatic breast cancer. Efficient targeting of these CTCs could potentially increase patient survival., (©2018 American Association for Cancer Research.)