Anti-Glycation Properties of Zinc-Enriched Arthrospira platensis (Spirulina) Contribute to Prevention of Metaflammation in a Diet-Induced Obese Mouse Model.

Advanced glycation end products (AGEs) exert a key pathogenic role in the development of obesity and insulin resistance. Thanks to its abundance in bioactive compounds, the microalga Arthrospira platensis (spirulina, SP) is proposed as a nutritional supplement. Here, we investigated the potential anti-glycating properties of SP enriched with zinc (Zn-SP) and the following impact on diet-induced metabolic derangements. Thirty male C57Bl6 mice were fed a standard diet (SD) or a high-fat high-sugar diet (HFHS) for 12 weeks, and a subgroup of HFHS mice received 350 mg/kg Zn-SP three times a week. A HFHS diet induced obesity and glucose intolerance and increased plasma levels of pro-inflammatory cytokines and transaminases. Zn-SP administration restored glucose homeostasis and reduced hepatic dysfunction and systemic inflammation. In the liver of HFHS mice, a robust accumulation of AGEs was detected, paralleled by increased expression of the main AGE receptor (RAGE) and depletion of glyoxalase-1, whereas Zn-SP administration efficiently prevented these alterations reducing local pro-inflammatory responses. 16S rRNA gene profiling of feces and ileum content revealed altered bacterial community structure in HFHS mice compared to both SD and HFHS + Zn-SP groups. Overall, our study demonstrates relevant anti-glycation properties of Zn-SP which contribute to preventing AGE production and/or stimulate AGE detoxification, leading to the improvement of diet-related dysbiosis and metabolic derangements.

Effectiveness and safety of IL1 inhibition with anakinra in chronic refractory idiopathic myocarditis.

AIM: The aim of this study was to evaluate the effectiveness and safety of the anti-IL-1 receptor anakinra in patients with chronic active myocarditis refractory to standard therapy. METHODS AND RESULTS: In this retrospective, observational study, we enrolled 6 patients with chronically active myocarditis treated with anakinra on-top-of standard treatment. Response to treatment was evaluated at different time points [disease onset (T0), anakinra beginning (T1), three months from anakinra beginning (T2), last available follow-up (T3)], and was assessed by variations in New York Heart Association (NYHA) functional class, laboratory tests [C-reactive protein (CRP), a high-sensitivity cardiac troponin T (cTnT), and Nt-proBNP], left ventricular ejection fraction (LVEF), and cardiac magnetic resonance (CMR) edema or late gadolinium enhancement. The number of premature ventricular complexes (PVCs) at 24-h EKG-recordings was considered in patients with arrhythmic manifestations. No differences were found between T0 and T1 in terms of CRP, Nt-ProBNP, and LVEF. Before anakinra beginning, all patients were still symptomatic. At T2, all patients were symptom-free, in NYHA class I. A significant decrease in CRP (p = 0.03) and a significant improvement in LVEF (p = 0.03) were observed. Sustained arrhythmic manifestations were found in 4 out of 6 patients. In this subgroup, anakinra showed effectiveness in reducing the arrhythmic burden. At T3, the improvement in laboratory values and cardiac function persisted. The arrhythmic burden remained abated. CONCLUSIONS: All patients had a rapid improvement in systemic inflammation, cardiac function, and arrhythmic burden with anti-IL1 therapy, indicating that anakinra may be an effective treatment in chronic active idiopathic myocarditis, refractory to standard treatment.

Dietary Intake of Fructooligosaccharides Protects against Metabolic Derangements Evoked by Chronic Exposure to Fructose or Galactose in Rats.

SCOPE: Diets rich in fat and sugars evoke chronic low-grade inflammation, leading to metabolic derangements. This study investigates the impact of fructose and galactose, two commonly consumed simple sugars, on exacerbation of the harmful effects caused by high fat intake. Additionally, the potential efficacy of fructooligosaccharides (FOS), a fermentable dietary fiber, in counteracting these effects is examined. METHODS AND RESULTS: Male Sprague-Dawley rats (six/group) are fed 8 weeks as follows: control 5% fat diet (CNT), 20% fat diet (FAT), FAT+10% FOS diet (FAT+FOS), FAT+25% galactose diet (FAT+GAL), FAT+GAL+10% FOS diet (FAT+GAL+FOS), FAT+25% fructose diet (FAT+FRU), FAT+FRU+10% FOS diet (FAT+FRU+FOS). The dietary manipulations tested do not affect body weight gain, blood glucose, or markers of systemic inflammation whereas significant increases in plasma concentrations of triacylglycerols, cholesterol, aspartate aminotransferase, and alanine aminotrasferase are detected in both FAT+FRU and FAT+GAL compared to CNT. In the liver and skeletal muscle, both sugars induce significant accumulation of lipids and advanced glycation end-products (AGEs). FOS supplementation prevents these impairments. CONCLUSION: This study extends the understanding of the deleterious effects of a chronic intake of simple sugars and demonstrates the beneficial role of the prebiotic FOS in dampening the sugar-induced metabolic impairments by prevention of lipid and AGEs accumulation.

Different Effects of High-Fat/High-Sucrose and High-Fructose Diets on Advanced Glycation End-Product Accumulation and on Mitochondrial Involvement in Heart and Skeletal Muscle in Mice.

Diets with an elevated content of fat, sucrose, or fructose are recognized models of diet-induced metabolic alterations, since they induce metabolic derangements, oxidative stress, and chronic low-grade inflammation associated with local and systemic accumulation of advanced glycation end-products (AGEs). This study used four-week-old C57BL/6 male mice, randomly assigned to three experimental dietary regimens: standard diet (SD), high-fat high-sucrose diet (HFHS), or high fructose diet (HFr), administered for 12 weeks. Plasma, heart, and tibialis anterior (TA) skeletal muscle were assayed for markers of metabolic conditions, inflammation, presence of AGEs, and mitochondrial involvement. The HFHS diet induced a tissue-specific differential response featuring (1) a remarkable adaptation of the heart to HFHS-induced heavy oxidative stress, demonstrated by an increased presence of AGEs and reduced mitochondrial biogenesis, and efficaciously counteracted by a conspicuous increase in mitochondrial fission and PRXIII expression; (2) the absence of TA adaptation to HFHS, revealed by a heavy reduction in mitochondrial biogenesis, not counteracted by an increase in fission and PRXIII expression. HFr-induced mild oxidative stress elicited tissue-specific responses, featuring (1) a decrease in mitochondrial biogenesis in the heart, likely counteracted by a tendency for increased fission and (2) a mild reduction in mitochondrial biogenesis in TA, likely counteracted by a tendency for increased fusion, showing the adaptability of both tissues to the diet.

Cluster Analysis to Explore Clinical Subphenotypes of Eosinophilic Granulomatosis With Polyangiitis.

OBJECTIVE: Previous studies suggested that distinct phenotypes of eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg-Strauss syndrome) could be determined by the presence or absence of antineutrophil cytoplasmic antibodies (ANCA), reflecting predominant vasculitic or eosinophilic processes, respectively. This study explored whether ANCA-based clusters or other clusters can be identified in EGPA. METHODS: This study used standardized data of 15 European centers for patients with EGPA fulfilling widely accepted classification criteria. We used multiple correspondence analysis, hierarchical cluster analysis, and a decision tree model. The main model included 10 clinical variables (musculoskeletal [MSK], mucocutaneous, ophthalmological, ENT, cardiovascular, pulmonary, gastrointestinal, renal, central, or peripheral neurological involvement); a second model also included ANCA results. RESULTS: The analyses included 489 patients diagnosed between 1984 and 2015. ANCA were detected in 37.2% of patients, mostly perinuclear ANCA (85.4%) and/or antimyeloperoxidase (87%). Compared with ANCA-negative patients, those with ANCA had more renal (P < 0.001) and peripheral neurological involvement (P = 0.04), fewer cardiovascular signs (P < 0.001), and fewer biopsies with eosinophilic tissue infiltrates (P = 0.001). The cluster analyses generated 4 (model without ANCA) and 5 clusters (model with ANCA). Both models identified 3 identical clusters of 34, 39, and 40 patients according to the presence or absence of ENT, central nervous system, and ophthalmological involvement. Peripheral neurological and cardiovascular involvement were not predictive characteristics. CONCLUSION: Although reinforcing the known association of ANCA status with clinical manifestations, cluster analysis does not support a complete separation of EGPA in ANCA-positive and -negative subsets. Collectively, these data indicate that EGPA should be regarded as a phenotypic spectrum rather than a dichotomous disease.

ROS-driven structural and functional fibrinogen modifications are reverted by interleukin-6 inhibition in Giant Cell Arteritis.

BACKGROUND: Cranial and extra-cranial vascular events are among the major determinants of morbidity and mortality in Giant Cell Arteritis (GCA). Vascular events seem mostly of inflammatory nature, although the precise pathogenetic mechanisms are still unclear. We investigated the role of oxidation-induced structural and functional fibrinogen modifications in GCA. The effects of the anti-IL6R tocilizumab in counteracting these mechanisms were also assessed. MATERIALS AND METHODS: A cross-sectional study was conducted on 65 GCA patients and 65 matched controls. Leucocyte reactive oxygen species (ROS) production, redox state, and fibrinogen structural and functional features were compared between patients and controls. In 19 patients receiving tocilizumab, pre vs post treatment variations were assessed. RESULTS: GCA patients displayed enhanced blood lymphocyte, monocyte and neutrophil ROS production compared to controls, with an increased plasma lipid peroxidation and a reduced total antioxidant capacity. This oxidative impairment resulted in a sustained fibrinogen oxidation (i.e. dityrosine content 320 (204-410) vs 136 (120-176) Relative Fluorescence Units (RFU), p < 0.0001), with marked alterations in fibrinogen secondary and tertiary structure [intrinsic fluorescence: 134 (101-227) vs 400 (366-433) RFU, p < 0.001]. Structural alterations paralleled a remarkable fibrinogen functional impairment, with a reduced ability to polymerize into fibrin and a lower fibrin susceptibility to plasmin-induced lysis. In patients receiving tocilizumab, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features (p < 0.001). CONCLUSIONS: An impaired redox status accounts for structural and functional fibrinogen modifications in GCA, suggesting a potential role of tocilizumab for cardiovascular prevention in GCA.

High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Prevalence and clinical associations of ultrasound-confirmed enthesitis in systemic lupus erythematosus.

OBJECTIVES: To assess the prevalence of US-confirmed enthesitis in a cohort of patients with SLE and to analyse the clinical associations to enthesitis during the course of SLE. METHODS: In a retrospective analysis of the SLE cohort of the Lupus Unit of the Careggi University Hospital, US examinations of SLE patients presenting with tender and/or swollen joints were retrieved to assess the presence of enthesitis. Patients with US-proven enthesitis were compared with SLE controls with tender and/or swollen joints who showed no US evidence of enthesitis. Clinical and laboratory features were compared at disease onset and during follow-up. RESULTS: A total of 400 patients fulfilling EULAR/ACR classification criteria for SLE were assessed. Of these, 106 underwent articular US examination. Evidence of enthesitis was found in 31/106 (29.2%) patients. Seventy-one patients without US-enthesitis were included as controls; four were excluded due to lack of follow-up data. Laboratory and clinical features were comparable between cases and controls at disease onset. Throughout a median follow-up of 10.0 (interquartile range [IQR] 8.3-23.3) years for cases and 12.4 (IQR 7.2-13.3) years for controls, patients with enthesitis were less likely to develop renal involvement (22.6% vs 46.5%, P = 0.028) and failed B cell depletion more frequently (75.0% vs 0%). CONCLUSION: In SLE patients with clinically active joints, US-proven enthesitis is a fairly common finding. Enthesitis in SLE could be the hallmark of a distinct disease phenotype with less renal involvement, more arthritis and low response to anti-CD 20 therapy, potentially requiring a tailored treatment.

Hypothalamic neuroglial plasticity is regulated by anti-Müllerian hormone and disrupted in polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common reproductive-endocrine disorder affecting between 5 and 18% of women worldwide. An elevated frequency of pulsatile luteinizing hormone (LH) secretion and higher serum levels of anti-Müllerian hormone (AMH) are frequently observed in women with PCOS. The origin of these abnormalities is, however, not well understood. METHODS: We studied brain structure and function in women with and without PCOS using proton magnetic resonance spectroscopy (MRS) and diffusion tensor imaging combined with fiber tractography. Then, using a mouse model of PCOS, we investigated by electron microscopy whether AMH played a role on the regulation of hypothalamic structural plasticity. FINDINGS: Increased AMH serum levels are associated with increased hypothalamic activity/axonal-glial signalling in PCOS patients. Furthermore, we demonstrate that AMH promotes profound micro-structural changes in the murine hypothalamic median eminence (ME), creating a permissive environment for GnRH secretion. These include the retraction of the processes of specialized AMH-sensitive ependymo-glial cells called tanycytes, allowing more GnRH neuron terminals to approach ME blood capillaries both during the run-up to ovulation and in a mouse model of PCOS. INTERPRETATION: We uncovered a central function for AMH in the regulation of fertility by remodeling GnRH terminals and their tanycytic sheaths, and provided insights into the pivotal role of the brain in the establishment and maintenance of neuroendocrine dysfunction in PCOS. FUNDING: INSERM (U1172), European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement n° 725149), CHU de Lille, France (Bonus H).

Evidence of subclinical atherosclerosis in eosinophilic granulomatosis with polyangiitis.

OBJECTIVES: Patients affected by eosinophilic granulomatosis with polyangiitis (EGPA) display an increased risk of atherothrombotic events compared with the general population. An increased frequency of subclinical markers of atherosclerosis has been observed in other ANCA-associated vasculitis, but no specific study focused on EGPA. We therefore evaluated subclinical atherosclerosis in EGPA patients and in a control population. METHODS: Forty EGPA patients and 80 controls matched by age, sex and traditional cardiovascular risk factors underwent sonographic assessment of common carotid artery (CCA) intima-media thickness (IMT). The presence of plaques of the CCA was also investigated. The correlation between CCA-IMT and clinical and laboratory features was also assessed. RESULTS: Median CCA-IMT was significantly higher in EGPA patients compared with controls (P = 0.002). Also, the proportion of subjects with increased CCA-IMT and with presence of plaques was significantly higher among EGPA patients (P < 0.001 for both). Moreover, within the EGPA cohort, CCA-IMT tended to increase with disease duration (P = 0.034) and corticosteroid cumulative dose (P = 0.004). No significant associations were found between CCA-IMT, ANCA status, other clinical features and therapeutic regimens. Notably, the prevalence of traditional cardiovascular risk factors was comparable in patients with vs without an increased CCA-IMT. CONCLUSION: Ultrasound markers of subclinical atherosclerosis are increased in EGPA patients as compared with controls, independently of traditional cardiovascular risk factors.

Solar photodegradation of irinotecan in water: optimization and robustness studies by experimental design.

Irinotecan, a widely prescribed anticancer drug, is an emerging contaminant of concern that has been detected in various aquatic environments due to ineffective removal by traditional wastewater treatment systems. Solar photodegradation is a viable approach that can effectively eradicate the drug from aqueous systems. In this study, we used the design of experiment (DOE) approach to explore the robustness of irinotecan photodegradation under simulated solar irradiation. A full factorial design, including a star design, was applied to study the effects of three parameters: initial concentration of irinotecan (1.0-9.0 mg/L), pH (5.0-9.0), and irradiance (450-750 W/m2). A high-performance liquid chromatography coupled with a high-resolution mass spectrometry (HPLC-HRMS) system was used to determine irinotecan and identify transformation products. The photodegradation of irinotecan followed a pseudo-first order kinetics. In the best-fitted linear model determined by the stepwise model fitting approach, pH was found to have about 100-fold greater effect than either irinotecan concentration or solar irradiance. Under optimal conditions (irradiance of 750 W/m2, 1.0 mg/L irinotecan concentration, and pH 9.0), more than 98% of irinotecan was degraded in 60 min. With respect to irradiance and irinotecan concentration, the degradation process was robust in the studied range, implying that it may be effectively applied in locations and/or seasons with solar irradiance as low as 450 W/m2. However, pH needs to be strictly controlled and kept between 7.0 and 9.0 to maintain the degradation process robust. Considerations about the behavior of degradation products were also drawn.

Biologics for eosinophilic granulomatosis with polyangiitis.

PURPOSE OF REVIEW: The link between severe asthma and eosinophilic granulomatosis with polyangiitis (EGPA) in terms of pathophysiological background, clinical manifestations and disease evolution has leaded to investigate the relevance of anti T2 monoclonal antibodies licensed for severe asthma patients as a treatment option for EGPA. The present review aimed to provide un update on EGPA pathophysiology and to critically summarize the most robust evidence coming from trials and real-life setting on the use of anti T2 biologics in EGPA patients. RECENT FINDINGS: Mepolizumab, an anti-interleukin-5 monoclonal antibody, is the only biologic drug targeting eosinophilic inflammation currently approved for EGPA treatment at the dose of 300 mg/4 weeks. Its use is restricted by the American College of Rheumatology guidelines to specific diseases phases and severity grades. However the most appropriate mepolizumab positioning and dose is still under investigation in the real life practice, which is providing an increasing amount of evidence confirming its efficacy, alone or in combination with other options in different disease stages. The relevance of other monoclonal antibodies interfering with T2 inflammation, including omalizumab and benralizumab, is under investigation but the evidence is still scarce. SUMMARY: Taking into account the suboptimal medium-long term safety profile of conventional EGPA treatments, the opportunity of selectively targeting eosinophilic inflammation certainly represents a revolutionary approach. However, further real-word evidence is required to effectively position the new treatments in the light of the disease complexity, including different immunological drivers, and individual variability.

Benralizumab for eosinophilic granulomatosis with polyangiitis: a retrospective, multicentre, cohort study.

BACKGROUND: Interleukin-5 (IL-5) inhibitors represent novel therapies for eosinophilic granulomatosis with polyangiitis (EGPA). This study assessed the effectiveness and safety of the IL-5 receptor inhibitor benralizumab in a European cohort of patients with EGPA. METHODS: This retrospective cohort study included patients with EGPA from 28 European referral centres of the European EGPA Study Group across six countries (Italy, France, UK, Russia, Spain, and Switzerland) who received benralizumab as any line of treatment between Jan 1, 2019, and Sep 30, 2022. We assessed the rates of complete response, defined as no disease activity (Birmingham Vasculitis Activity Score [BVAS] of 0) and a prednisone dose of up to 4 mg/day, in contrast to partial response, defined as a BVAS of 0 and a prednisone dose greater than 4 mg/day. Active disease manifestations, pulmonary function, variation in glucocorticoid dose, and safety outcomes were also assessed over a 12-month follow-up. FINDINGS: 121 patients with relapsing-refractory EGPA treated with benralizumab at the dose approved for eosinophilic asthma were included (64 [53%] women and 57 [47%] men; median age at the time of beginning benralizumab treatment 54·1 years [IQR 44·2-62·2]). Complete response was reported in 15 (12·4%, 95% CI 7·1-19·6) of 121 patients at month 3, 25 (28·7%, 19·5-39·4) of 87 patients at month 6, and 32 (46·4%, 34·3-58·8) of 69 patients at month 12; partial response was observed in an additional 43 (35·5%, 27·0-44·8) patients at month 3, 23 (26·4%, 17·6-37·0) at month 6, and 13 (18·8%, 10·4-30·1) at month 12. BVAS dropped from 3·0 (IQR 2·0-8·0) at baseline to 0·0 (0·0-2·0) at months 3 and 6, and to 0·0 (0·0-1·0) at month 12. The proportion of patients with systemic manifestations, active peripheral neurological disease, ear, nose, and throat involvement, and pulmonary involvement decreased, with an improvement in lung function tests. Six patients relapsed after having a complete response. The oral prednisone (or equivalent) dose decreased from 10·0 mg/day (5·0-12·5) at baseline to 5·0 mg/day (3·6-8·5) at month 3 (p<0·01), to 5·0 mg/day (2·5-6·3) at month 6, and to 2·5 mg/day (0·0-5·0) at month 12 (p<0·0001). 19 (16%) of 121 patients had adverse events and 16 (13%) discontinued benralizumab. INTERPRETATION: These data suggest that benralizumab could be an effective treatment for EGPA in real-life clinical practice. Further clinical trials are required to confirm the efficacy of benralizumab in patients with a higher baseline disease activity. FUNDING: None.

At the Heart of Eosinophilic Granulomatosis with Polyangiitis: into Cardiac and Vascular Involvement.

PURPOSE OF REVIEW: To provide an overview of existing literature on pathogenetic and clinical aspects of cardiac and vascular involvement in eosinophilic granulomatosis with polyangiitis (EGPA). RECENT FINDINGS: In EGPA, cardiac and vascular involvement are more common than previously thought. However, no international recommendations on the topic are available yet. Herein, we summarize the existing evidence on the topic and propose a diagnostic approach for cardiac involvement in EGPA. The prevalence of cardiovascular involvement in patients with EGPA varies greatly among published studies, ranging between 3.1-18.7% for occlusive arterial disease, 5.8-30% for venous thrombosis and 17-92% for heart involvement. Cardiac involvement in EGPA is associated with high mortality even though manifestations are heterogeneous. In principle, every anatomical structure of the heart can be involved, and EGPA-related heart disease may be completely asymptomatic at first. A careful diagnostic work-up for early detection and prompt treatment initiation is therefore required. While cardiac manifestations are more common in anti-neutrophil cytoplasmic antibodies (ANCA)-negative patients, arterial and venous thrombotic events are not linked to ANCA status but correlate closely with disease activity and accumulate at disease onset. Thrombotic events (mainly venous) are considerably more frequent in EGPA than in the general population contributing substantially to morbidity and highlighting the importance of developing specific prevention strategies for patients who are diagnosed with EGPA.

Heterozygous Loss of KRIT1 in Mice Affects Metabolic Functions of the Liver, Promoting Hepatic Oxidative and Glycative Stress.

KRIT1 loss-of-function mutations underlie the pathogenesis of Cerebral Cavernous Malformation (CCM), a major vascular disease affecting the central nervous system (CNS). However, KRIT1 is also expressed outside the CNS and modulates key regulators of metabolic and oxy-inflammatory pathways, including the master transcription factor FoxO1, suggesting a widespread functional significance. Herein, we show that the KRIT1/FoxO1 axis is implicated in liver metabolic functions and antioxidative/antiglycative defenses. Indeed, by performing comparative studies in KRIT1 heterozygous (KRIT1+/-) and wild-type mice, we found that KRIT1 haploinsufficiency resulted in FoxO1 expression/activity downregulation in the liver, and affected hepatic FoxO1-dependent signaling pathways, which are markers of major metabolic processes, including gluconeogenesis, glycolysis, mitochondrial respiration, and glycogen synthesis. Moreover, it caused sustained activation of the master antioxidant transcription factor Nrf2, hepatic accumulation of advanced glycation end-products (AGEs), and abnormal expression/activity of AGE receptors and detoxifying systems. Furthermore, it was associated with an impairment of food intake, systemic glucose disposal, and plasma levels of insulin. Specific molecular alterations detected in the liver of KRIT1+/- mice were also confirmed in KRIT1 knockout cells. Overall, our findings demonstrated, for the first time, that KRIT1 haploinsufficiency affects glucose homeostasis and liver metabolic and antioxidative/antiglycative functions, thus inspiring future basic and translational studies.

Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma.

Gonadotropin-releasing hormone isoform I (GnRH), a neuro-deca-peptide, plays a fundamental role in development and maintenance of the reproductive system in vertebrates. The anomalous release of GnRH is observed in reproductive disorder such as hypogonadotropic hypogonadism, polycystic ovary syndrome (PCOS), or following prenatal exposure to elevated androgen levels. Quantitation of GnRH plasma levels could help to diagnose and better understand these pathologies. Here, a validated nano-high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) method to quantify GnRH in ewe plasma samples is presented. Protein precipitation and solid-phase extraction (SPE) pre-treatment steps were required to purify and enrich GnRH and internal standard (lamprey-luteinizing hormone-releasing hormone-III, l-LHRH-III). For the validation process, a surrogate matrix approach was chosen following the International Council for Harmonisation (ICH) and FDA guidelines. Before the validation study, the validation model using the surrogate matrix was compared with those using a real matrix such as human plasma. All the tested parameters were analogous confirming the use of the surrogate matrix as a standard calibration medium. From the validation study, limit of detection (LOD) and limit of quantitation (LOQ) values of 0.008 and 0.024 ng/mL were obtained, respectively. Selectivity, accuracy, precision, recovery, and matrix effect were assessed with quality control samples in human plasma and all values were acceptable. Sixteen samples belonging to healthy and prenatal androgen (PNA) exposed ewes were collected and analyzed, and the GnRH levels ranged between 0.05 and 3.26 ng/mL. The nano-HPLC-HRMS developed here was successful in measuring GnRH, representing therefore a suitable technique to quantify GnRH in ewe plasma and to detect it in other matrices and species.

Great Abilities of Shinella zoogloeoides Strain from a Landfarming Soil for Crude Oil Degradation and a Synergy Model for Alginate-Bead-Entrapped Consortium Efficiency.

Oil contamination is of great concern worldwide and needs to be properly addressed. The present work aimed to contribute to the development of bacterial consortia for oil recovery. We investigated the community structure of a landfarming-treated soil (LF2) by metagenomics to unravel the presence of hydrocarbon degraders. Moreover, we isolated Shinella zoogloeoides LFG9 and Bacillus swezeyi LFS15 from LF2 and combined them with Pseudomonas guguanensis SGPP2 isolated from an auto mechanic workshop soil to form the mixed consortium COG1. Bacterial isolates were tested for biosurfactant production. Additionally, the bioremediation potential of COG1 was studied as free and entrapped consortia by gas chromatography-mass spectrometry, in comparison to the single strains. Results revealed the presence of Actinobacteria (66.11%), Proteobacteria (32.21%), Gammaproteobacteria (5.39%), Actinomycetales (65.15%), Burkholderiales (13.92%), and Mycobacterium (32.22%) taxa, indicating the presence of hydrocarbon degraders in soil LF2. All three isolated strains were biosurfactant producers capable of degrading crude oil components within 14 days. However, Shinella zoogloeoides LFG9 performed best and was retained as candidate for further bioremediation investigation. In addition, COG1 performed better when immobilized, with entrapment effectiveness manifested by increased fatty acids and aromatic compound degradation. Attempt to improve crude oil biodegradation by adding surfactants failed as sodium dodecyl sulfate restrained the immobilized consortium performance.