Effects of long-term individual housing of middle-aged female Octodon degus on spatial learning and memory in the Barnes maze task.

Introduction: Prolonged social isolation is a form of passive chronic stress that has consequences on human and animal behavior. The present study was undertaken to elucidate whether the long-term isolation would precipitate age-related changes in anxiety and spatial learning and memory in degus. Methods: We investigated the effects of long-term social isolation on anxiety levels in the light-dark test, and spatial orientation abilities in the Barnes maze. Middle-aged female Octodon degus were allocated to either group-housed (3 animals per cage) or individually-housed for 5 months. Results: Under this experimental condition, there were no significant group differences in the anxiety level tested in the light-dark test and in the motivation to escape from the Barnes maze. There were no significant differences in cortisol levels between individually- and group-housed animals. On the last acquisition training day of spatial learning, individually- housed animals had a significantly higher number of correct responses and a smaller number of reference and working memory errors than the group-housed animals. In addition, isolated animals showed a tendency for reference and working memory impairment on the retention trial, while group-housed degus showed improvement in these parameters. Discussion and conclusion: The present study indicates that prolonged social isolation during adulthood in female degus has a dual effect on spatial orientation. Specifically, it results in a significant improvement in acquisition skills but a slight impairment in memory retention. The obtained cognitive changes were not accompanied by modification in anxiety and cortisol levels.

Incomplete penetrance and variable expressivity in monogenic diabetes; a challenge but also an opportunity.

Monogenic Forms of Diabetes (MFD) account for about 3% of all diabetes, and their accurate diagnosis often results in life-changing therapeutic reassignment for the patients. Like other Mendelian diseases, reduced penetrance and variable expressivity are often seen in several different types of MFD, where symptoms develop only in a portion of the persons who carry the pathogenic variant or vary widely in symptom severity and age of onset. This complicates diagnosis and disease management in MFD. In addition to its clinical importance, knowledge of genetic modifiers that confer penetrance and expressivity variability opens possibilities to identify protective genetic variants which may help probe the mechanisms of more common forms of diabetes and shed light in new therapeutic strategies. In this review, we will mainly address penetrance and expressivity variation in different types of MFD, factors that confer such variations and opportunities that come with such knowledge. Related literature was searched in PubMed, Medline and Embase. Papers with publication year from 1974 to 2023 are included. Data are either sourced from literatures or from OMIM, Clinvar and 1000 genome browser.

Rats conserve passive avoidance retention level throughout the light phase of diurnal cycle.

Emotions and memory formation are sensible to circadian rhythm. Here we study whether the time of day during the light phase of the diurnal cycle affects emotional memory in male Wistar rats using the passive avoidance (PA) test. Experiments were conducted at the beginning of Zeitgeber time (ZT) (ZT0.5-2), mid-time (ZT5-6.5), and end (ZT10.5-12) of the light period. Our results suggest that time of day has no impact on emotional response during acquisition trials, but slightly influences cognitive response during the 24-hour retention trial. Retention response was highest for ZT5-6.5, followed by ZT0.5-2, and lowest for ZT10.5-12.

Effect of seasonal temperature shifts on body homeostasis, biochemical parameters and chemical composition of green ormer (Haliotis tuberculata) in the Northern Adriatic.

Seasonal responses of green ormer in terms of antioxidant capacity and lipid peroxidation, proximate and fatty acid tissue composition, trace and macro elements concentrations over the seasons were studied in relation to temperature shifts in the Northern Adriatic Sea. Overall antioxidative defenses (SOD, TBARS, TAS, LDH) varied significantly (p < 0.001) according to seasons (primarily spring and summer). The proportions of overall SFA were highest in summer. The proportions of MUFA increased in autumn, with significant differences between genders in spring and summer, and spring, summer and autumn for C18:1n7 and C20:4n6. The only fatty acid lacking significant variation between seasons was C22:5n3. Significant overall differences were observed in summer vs. winter samples for As, Ba, Co, Ni, Mn, Pb, Sb, and Se content in soft tissues, however, gender variations were not significant. The data obtained in the study are of utmost importance for the management of this under-investigated species.

Fish liver damage related to the wastewater treatment plant effluents.

Wastewater treatment plants (WWTPs) continuously release a complex mixture of municipal, hospital, industrial, and runoff chemicals into the aquatic environment. These contaminants are both legacy contaminants and emerging-concern contaminants, affecting all tissues in a fish body, particularly the liver. The fish liver is the principal detoxifying organ and effects of consistent pollutant exposure can be evident on its cellular and tissue level. The objective of this paper is thus to provide an in-depth analysis of the WWTP contaminants' impact on the fish liver structure, physiology, and metabolism. The paper also gives an overview of the fish liver biotransformation enzymes, antioxidant enzymes, and non-enzymatic antioxidants, their role in metabolizing xenobiotic compounds and coping with oxidative damage. Emphasis has been placed on highlighting the vulnerability of fish to xenobiotic compounds, and on biomonitoring of exposed fish, generally involving observation of biomarkers in caged or native fish. Furthermore, the paper systematically assesses the most common contaminants with the potential to affect fish liver tissue.

A Novel Somatic Mutation Implicates ATP6V0D1 in Proinsulin Processing.

Context: Prohormone convertase 1/3 (PC1/3), encoded by protein convertase subtilisin kexin type 1 (PCSK1), converts inactive prohormones into biologically active peptides. Somatic mutations of insulinomas are associated with genetic defects interfering with control of insulin secretion from pancreatic beta cells. However, somatic mutations in proinsulinomas have not been described. Objective: We report a case of a proinsulinoma, with suppressed insulin and C-peptide levels. Methods: A 70-year-old woman presented with a 20-year history of "blackouts." During a 72-hour fast, blood glucose level dropped to 1.9 mmol/L with suppressed plasma insulin and C-peptide levels, but proinsulin levels were raised at 37 pmol/L (<10 pmol/L). Results: Imaging revealed 3 distinct DOTATATE-avid pancreatic lesions. Laparoscopic spleen-preserving distal pancreatomy was performed. In view of discordant insulin, C-peptide, and proinsulin levels, whole exome sequencing analysis was performed on the tumor. In the somatic exome of the tumor, we found mutations in PCSK expression regulators, as well as a novel truncating somatic mutation in ATP6V0D1, a subunit of the ion pump that acidifies the ß-cell compartments where the PCSKs act. Conclusion: Appropriately suppressed insulin levels in the context of hypoglycemia do not always indicate the absence of a neuroendocrine islet cell tumor and proinsulin levels may be indicated to solidify the diagnosis. In the context of elevated proinsulin levels, low insulin and C-peptide levels might be explained by somatic mutations that likely implicate proinsulin processing within the tumor. Furthermore, we propose several mechanistic candidates, including ATP6V0D1. Experimental validation using cellular approaches may in future confirm pathomechanisms involved in this rare condition.

Sample preparation and culture condition effects on MALDI-TOF MS identification of bacteria: A review.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI-TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI-TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements.

Identification of environmental aquatic bacteria by mass spectrometry supported by biochemical differentiation.

In this study, the purposefulness of using the API20E biochemical identification system as a supportive tool for enhancing the discrimination of environmental bacteria by MALDI-TOF MS method was evaluated. The identification results of MALDI-TOF MS and API20E for 321 Gram-negative strains isolated from the riverine freshwater and its sediment, and from the tissues of fish from the same water body were compared. Of 190 isolates identified with probable to highly probable species-level identification, and secure genus to probable species identification, 14 isolates (7.37%) had identification score over 2.300, and from the same group 19 isolates (10%) had excellent or very good identification to the genus by API20E system. With regard to agreement at genus level, out of 231 strains with genus designation available by API20E at any level of identification reliability, MALDI-TOF MS genus identification agreed in 163 (70.6%) strains. Of these, 135 (82.8%) were Aeromonas species and the remaining isolates belonged to 7 different genera. Although API20E resulted in frequent misidentification due to a limited profile index, its individual biochemical reactions might contribute to overall characterization of isolates. For example, for all reliable A. hydrophila strain identifications with MALDI-TOF MS, ONPG, GLU and OX reactions were unarguably positive for all fish and water/sediment isolates, whereas only fish isolates yielded additional 100% positive TDA and VP reactions. Thus, after initial identification with MALDI-TOF MS, environmental isolates with lower identification scores should be further analyzed. Before commencing confirmatory testing with nucleic acid-based methods, biochemical API20E tests could be applied as a purposeful and inexpensive identification support in targeting better identification accuracy. In this study, this was particularly evident with A. hydrophila, Chryseobacterium sp. and Pseudomonas sp. This identification strategy could significantly resolve methodological and cost-related shortcomings frequently occurring with large number of environmental isolates.

Shewanella spp. from wastewater treatment plant-affected environment: isolation and characterization.

Bacteria from the genus Shewanella are inhabitants of marine and freshwater ecosystems, recognized fish spoilage bacteria, but less known as fish disease agents. Shewanella spp. isolated from fish living in waters close to effluents of a wastewater treatment plant (WWTP) were not previously characterized. We have tested Shewanella isolates from WWTP-affected waters and related fish. Genotypic characterization identified most strains as S. baltica and S. oneidensis. In order to investigate the sensibility and accuracy of their MALDI-TOF MS identification, they were grown on two culture media enriched by various NaCl concentrations, incubated at different temperatures and duration. We analyzed their antimicrobial susceptibility on a panel of antimicrobial drugs and capacity for biofilm production. With a view to demonstrate their capacity to produce fatty acids, we assessed the impact of different culture media on their lipid profile. We performed zebrafish embryotoxicity tests to simulate the environmental infection of the earliest life stages in S. baltica-contaminated waters. The best MALDI-TOF MS identification scores were for strains cultivated on TSA for 24 h at 22 °C and with supplementation of 1.5% NaCl. Less than 17% of isolates demonstrated antimicrobial resistance. Most isolates were weak biofilm producers. Strain-to-strain variation of MIC and MBC was low. The major fatty acids were C15:0, C16:0, C16:1, C17:1, and iC15:0. Exposure of Danio rerio to different S. baltica concentrations induced severe effects on zebrafish development: decreased heartbeat rate, locomotor activity, and melanin pigmentation. S. baltica passed through chorionic pores of zebrafish.

Selenite as a Lipid Inductor in Marine Microalga Dunaliella tertiolecta: Comparison of One-Stage and Two-Stage Cultivation Strategies.

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00-40.00 mg L-1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00 mg L-1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00 mg L-1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta.

Chemical Evaluation, Antioxidant, Antiproliferative, Anti-Inflammatory and Antibacterial Activities of Organic Extract and Semi-Purified Fractions of the Adriatic Sea Fan, Eunicella cavolini.

Due to sedentary lifestyle and harsh environmental conditions, gorgonian coral extracts are recognized as a rich source of novel compounds with various biological activities, of interest to the pharmaceutical and cosmetic industries. The presented study aimed to perform chemical screening of organic extracts and semi-purified fractions obtained from the common Adriatic gorgonian, sea fan, Eunicella cavolini (Koch, 1887) and explore its abilities to exert different biological effects in vitro. Qualitative chemical evaluation revealed the presence of several classes of secondary metabolites extended with mass spectrometry analysis and tentative dereplication by using Global Natural Product Social Molecular Networking online platform (GNPS). Furthermore, fractions F4 and F3 showed the highest phenolic (3.28 ± 0.04 mg GAE/g sample) and carotene (23.11 ± 2.48 mg ß-CA/g sample) content, respectively. The fraction F3 inhibited 50% of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ABTS (2,2'-azino-bis (3-ethylbenzthiazolin-6-yl) sulfonic acid) radicals at the concentrations of 767.09 ± 11.57 and 157.16 ± 10.83 µg/mL, respectively. The highest anti-inflammatory potential was exhibited by F2 (IC50 = 198.70 ± 28.77 µg/mL) regarding the inhibition of albumin denaturation and F1 (IC50 = 254.49 ± 49.17 µg/mL) in terms of soybean lipoxygenase inhibition. In addition, the most pronounced antiproliferative effects were observed for all samples (IC50 ranging from 0.82 ± 0.14-231.18 ± 46.13 µg/mL) against several carcinoma cell lines, but also towards non-transformed human fibroblasts pointing to a generally cytotoxic effect. In addition, the antibacterial activity was tested by broth microdilution assay against three human pathogenic bacteria: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The latter was the most affected by fractions F2 and F3. Finally, further purification, isolation and characterization of pure compounds from the most active fractions are under investigation.

Widespread Doublecortin Expression in the Cerebral Cortex of the Octodon degus.

It has been demonstrated that in adulthood rodents show newly born neurons in the subgranular layer (SGL) of the dentate gyrus (DG), and in the subventricular zone (SVZ). The neurons generated in the SVZ migrate through the rostral migratory stream (RMS) to the olfactory bulb. One of the markers of newly generated neurons is doublecortin (DCX). The degu similarly shows significant numbers of DCX-labeled neurons in the SGL, SVZ, and RMS. Further, most of the nuclei of these DCX-expressing neurons are also labeled by proliferating nuclear antigen (PCNA) and Ki67. Finally, whereas in rats and mice DCX-labeled neurons are predominantly present in the SGL and SVZ, with only a few DCX neurons present in piriform cortex, the degu also shows significant numbers of DCX expressing neurons in areas outside of SVZ, DG, and PC. Many areas of neocortex in degu demonstrate DCX-labeled neurons in layer II, and most of these neurons are found in the limbic cortices. The DCX-labeled cells do not stain with NeuN, indicating they are immature neurons.

COCO/DAND5 inhibits developmental and pathological ocular angiogenesis.

Neovascularization contributes to multiple visual disorders including age-related macular degeneration (AMD) and retinopathy of prematurity. Current therapies for treating ocular angiogenesis are centered on the inhibition of vascular endothelial growth factor (VEGF). While clinically effective, some AMD patients are refractory or develop resistance to anti-VEGF therapies and concerns of increased risks of developing geographic atrophy following long-term treatment have been raised. Identification of alternative pathways to inhibit pathological angiogenesis is thus important. We have identified a novel inhibitor of angiogenesis, COCO, a member of the Cerberus-related DAN protein family. We demonstrate that COCO inhibits sprouting, migration and cellular proliferation of cultured endothelial cells. Intravitreal injections of COCO inhibited retinal vascularization during development and in models of retinopathy of prematurity. COCO equally abrogated angiogenesis in models of choroidal neovascularization. Mechanistically, COCO inhibited TGFß and BMP pathways and altered energy metabolism and redox balance of endothelial cells. Together, these data show that COCO is an inhibitor of retinal and choroidal angiogenesis, possibly representing a therapeutic option for the treatment of neovascular ocular diseases.

Utilization of the zebrafish model to unravel the harmful effects of biomass burning during Amazonian wildfires.

Amazonian wildfires in 2019 have raised awareness about rainforest burning due to increased emissions of particulate matter and carbon. In the context of these emissions, by-products of lignin thermal degradation (i.e. methoxyphenols) are often neglected. Methoxyphenols entering the atmosphere may form intermediates with currently unknown reaction mechanisms and toxicity. This study for the first time provides a comprehensive insight into the impact of lignin degradation products [guaiacol, catechol], and their nitrated intermediates [4-nitrocatechol, 4,6-dinitroguaiacol, 5-nitroguaiacol] on zebrafish Danio rerio. Results revealed 4-nitrocatechol and catechol as the most toxic, followed by 4,6DNG > 5NG > GUA. The whole-organism bioassay integrated with molecular modeling emphasized the potential of methoxyphenols to inhibit tyrosinase, lipoxygenase, and carbonic anhydrase, consequently altering embryonic development (i.e. affected sensorial, skeletal, and physiological parameters, pigmentation formation failure, and non-hatching of larvae). The whole-organism bioassay integrated with in silico approach confirmed the harmful effects of lignin degradation products and their intermediates on aquatic organisms, emphasizing the need for their evaluation within ecotoxicity studies focused on aquatic compartments.

The diurnal variation of open-field habituation in rats.

The present study aimed to establish the effect of time of the day on habituation in the open-field test, one of the most elementary forms of non-associative hippocampal-dependent learning. Open-field test was performed in young adult male Wistar rats at the beginning (08:30-10:00 h; defined as Zeitgeber time (ZT) ZT0.5-2), mid-time (13:00-14:30 h, ZT5-6.5) and at the end (18:30-20:00 h, ZT10.5-12) of the light period. Our results revealed that in the acquisition trial there were no significant differences among the six parameters recorded through tested periods. In contrast, the level of habituation in the ambulation and rearing rose as followed: ZT0.5-2 < ZT10.5-12 < ZT5-6.5. In both trials, the principal component analysis highlights two components: component 1 was mainly loaded by ambulation in the outer and inner area, rearing and freezing behaviors, whereas component 2 was mostly loaded on grooming activity and defecation. The correlation between parameters varied across the period of day and trial. Animals that expressed a higher level of grooming, defecation (ZT5-6.5) and freezing behavior (ZT0.5-2 and ZT5-6.5) at acquisition trial habituated better on those parameters on the retention trial. In conclusion, habituation outcomes to the open-field test and correlation between tested parameters highly depend on daytime.

Sex and Time-of-Day Impact on Anxiety and Passive Avoidance Memory Strategies in Mice.

In humans, anxiety and cognitive processes are age, gender, and time of day dependent. The purpose of the present study was to assess whether the time of day and sex have an influence on anxiety and emotional memory in adult mice. Light-dark and passive avoidance (PA) tests were performed at the beginning and at the end of the light cycle, defined as Zeitgeber time (ZT) ZT0-2.5 and ZT9.5-12, respectively. A baseline difference in anxiety was not found, but on the 24 h retention trial of the PA test, females presented longer latencies to enter into the dark compartment at the ZT0-2.5 time point of the day. The data from the second test day (PA reversal trial) indicated that some animals associated the dark compartment with an aversive stimulus (shock), while others associated the aversive stimulus with crossing from one compartment to another. At the ZT9.5-12, female mice mainly related the aversive stimulus to transferring from one compartment to another, while male mice associated darkness with the aversive stimulus. There was a negative correlation between the frequency of light-dark transitions in the light-dark test and the PA latency on the 24 h retention trial in males tested at ZT0-2.5. The PA latency on the reversal and 24 h retention trials negatively correlated with a risk assessment behavior in male mice tested on ZT0-2.5 and ZT9.5-12, respectively. In conclusion, our data reveal that the impact of motor activity and risk assessment behavior on PA memory formation and applied behavioral strategies are time of day and sex dependent.

Verapamil and Alzheimer's Disease: Past, Present, and Future.

Verapamil is a phenylalkylamine class calcium channel blocker that for half a century has been used for the treatment of cardiovascular diseases. Nowadays, verapamil is also considered as a drug option for the treatment of several neurological and psychiatric disorders, such as cluster headache, bipolar disorders, epilepsy, and neurodegenerative diseases. Here, we review insights into the potential preventive and therapeutic role of verapamil on Alzheimer's disease (AD) based on limited experimental and clinical data. Pharmacological studies have shown that verapamil has a wide therapeutic spectrum, including antihypertensive, anti-inflammatory, and antioxidative effects, regulation of the blood-brain barrier function, due to its effect on P-glycoprotein, as well as adjustment of cellular calcium homeostasis, which may result in the delay of AD onset or ameliorate the symptoms of patients. However, the majority of the AD individuals are on polypharmacotherapy, and the interactions between verapamil and other drugs need to be considered. Therefore, for an appropriate and successful AD treatment, a personalized approach is more than necessary. A well-known narrow pharmacological window of verapamil efficacy may hinder this approach. It is therefore important to note that the verapamil efficacy may be conditioned by different factors. The onset, grade, and brain distribution of AD pathological hallmarks, the time-sequential appearances of AD-related cognitive and behavioral dysfunction, the chronobiologic and gender impact on calcium homeostasis and AD pathogenesis may somehow be influencing that success. In the future, such insights will be crucial for testing the validity of verapamil treatment on animal models of AD and clinical approaches.

Seasonal antioxidant and biochemical properties of the Northern Adriatic Pecten jacobaeus.

The present work is the first study of Mediterranean scallop (Pecten jacobaeus) biochemical properties, antioxidant defenses, and free radical scavengers during the yearly seasons in the Northern Adriatic, off Istria. Scallop nutrient reserves (glucose, triglyceride, and cholesterol) in four tissues under examination were positively correlated and were predominant in digestive gland and gonad. The muscle energy maxima were in correlation with the maximum fall gonosomatic index (GSI), when diatoms and coccolithophorids thrive. The decrease of GSI in summer might be related to the spawning or resorption of gametes. Summer also revealed elevated levels of glucose in gonad and digestive gland, while muscle glucose and cholesterol significantly varied in spring vs. winter samples. In relation to the diatom seasonal abundance, carotenoids, namely astaxanthin peaks were found in digestive gland, which, being stimulators of calcium transport over cell membranes, could have contributed to the high digestive gland levels of calcium in winter. In winter, total antioxidative status (TAS) of scallop tissues was 3-fold higher than in other seasons, particularly in digestive gland, having a significant correlation with magnesium, a regulatory tool in oxidative processes. The winter maxima of TAS and thiobarbituric acid reactive substances TBARS in relation to summer maxima of glutathione peroxidase and superoxide dismutase in digestive glands indicate to a decrease in antioxidant defense during cold months, and are related to the accumulation of lipid peroxidation products (such as malondialdehyde) in digestive gland of scallops. Although the increased susceptibility to oxidative stress could be attributed to winter temperature, other factors such as the gonad maturation, availability of food supply, and salinity might counteract that effect. The seawater alterations of salinity, temperature and water quality are in relation to the river Po influx, which is very likely to influence the physiological and biochemical responses of scallops in the Northern Adriatic.

BMP9 signaling promotes the normalization of tumor blood vessels.

The presence of an immature tumor vascular network contributes to cancer dissemination and the development of resistance to therapies. Strategies to normalize the tumor vasculature are therefore of significant therapeutic interest for cancer treatments. VEGF inhibitors are used clinically to normalize tumor blood vessels. However, the time frame and dosage of these inhibitors required to achieve normalization is rather narrow, and there is a need to identify additional signaling targets to attain vascular normalization. In addition to VEGF, the endothelial-specific receptor Alk1 plays a critical role in vascular development and promotes vascular remodeling and maturation. Therefore, we sought to evaluate the effects of the Alk1 ligand BMP9 on tumor vascular formation. BMP9 overexpression in Lewis Lung Carcinoma (LLC) tumors significantly delayed tumor growth. Blood vessels in BMP9-overexpressing LLC tumors displayed markers of vascular maturation and were characterized by increased perivascular cell coverage. Tumor vasculature normalization was associated with decreased permeability and increased perfusion. These changes in vascular function in BMP9-overexpressing LLC tumors resulted in significant alterations of the tumor microenvironment, characterized by a decrease in hypoxia and an increase in immune infiltration. In conclusion, we show that BMP9 promotes vascular normalization in LLC tumors that leads to changes in the microenvironment.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream.

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48-72 hours.