Amanitin determination in bile samples by UHPLC-MS: LR-MS and HR-MS analytical performance.

Consumption of misidentified foraged mushrooms containing bicyclic amanitin octapeptides is a worldwide public health and veterinary problem, being considered one of the deadliest accidental human and canine food ingestion due to acute liver failure (ALF). Reversal of advanced ALF and complete clinical recovery can be achieved following definitive removal of accumulated amatoxin laden bile from the gallbladder. An accurate means of quantifying amanitin content in aspirated bile is, therefore, urgently needed. Building on our prior work validating a method to detect and quantify amanitin in hepatic autopsy tissue, the development of an accurate method of measuring α- and ß-amanitin in aspirated gallbladder bile was performed to evaluate the efficiency of this emergency procedure applied as a clinical treatment for intoxicated patients. A solid-phase extraction (SPE) procedure was optimized followed by detection based on ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Low resolution mass spectrometry (LRMS) was compared with high resolution (HRMS) by the validation of UHPLC-MS/MS (triple quadrupole MS) and UHPLC-ToF-MS (time-of-flight MS). Both methods were able to detect amatoxins in bile with limits of detection and quantification ranging from 2.71 to 3.46 µg.kg-1, and 8.36-9.03 µg.kg-1 for α-amanitin and, 0.32-1.69 µg.kg-1 and 0.55-5.62 µg.kg-1 for ß-amanitin, respectively. Validation was completed with the evaluation of linearity, specificity, robustness, recovery, and precision following the ICH guidelines and CIR 808/2021. The validated methods were finally applied to bile samples obtained 48-96 hours + post-ingestion from 4 amatoxin poisoning patients who underwent gallbladder drainage procedures in Vietnam, Canada, and California. Gallbladder bile from patients with amatoxin mushroom poisoning contained significant amanitin content, even when aspirated several days post-ingestion, thus confirming the important role of enterohepatic circulation in amatoxin hepatotoxicity. This work represents a high and unique analytical throughput in amanitin poisoning allowing to efficiently respond to this fatal health problem.

Regulated and Emerging Mycotoxins in Bulk Raw Milk: What Is the Human Risk?

Mycotoxins are abiotic hazards whose contamination occurs at the pre- and post-harvest stages of the maize value chain, with animal exposure through contaminated feed leading to their excretion into milk. Currently, only aflatoxin M1 is regulated in milk products. Since feed materials and complete feed present a multi-mycotoxin composition and are the main mycotoxin source into milk, it is important to recognize the occurrence of multiple toxins and their co-occurrence in this highly consumed food product. The aim of this study was to determine the content of regulated and emerging mycotoxins in milk samples, which allowed for evaluating the occurrence and co-occurrence patterns of different mycotoxins known to contaminate feed materials and complete animal feed. Human exposure considering the occurrence patterns obtained was also estimated. Aflatoxins, fumonisins, zearalenone, and emerging mycotoxins were among the mycotoxins found to be present in the 100 samples analyzed. Concentrations ranged from 0.006 to 16.3 µg L-1, with no sample exceeding the AFM1 maximum level. Though several mycotoxins were detected, no exceeding values were observed considering the TDI or PMTDI. It can be concluded that the observed exposure does not pose a health risk to milk consumers, though it is important to recognize vulnerable age groups.

Social wellbeing, values, and identity among Caiçara small-scale fishers in southeastern Brazil.

Although much in the lives of members of the Caiçara small-scale fishing communities of Lázaro and Saco da Ribeira in Ubatuba, southeastern Brazil would suggest hardship, that population expresses a surprising degree of satisfaction with life. In this paper, we use a social wellbeing lens as applied through an ethnographic, mixed methods approach to reflect on this overall sense that lives rooted in small-scale fishing are well worth living despite their many challenges. We see the classic maritime anthropology theme of identity at the heart of meaning and life satisfaction. Identity provides core aspects of how people engage with their realities and anchors values that are reference points in work and social relations. With reference to the relational nuances revealed by the social wellbeing perspective, however, we show that Caiçara and small-scale fishing identities are not monolithic, but reflect gender and other social positions, and personal and familial experiences. These experiences include grappling with the complex effects of economic, social, political, and environmental changes. We conclude by arguing that fisheries policy that seeks to prioritize human wellbeing would benefit by adopting a social wellbeing perspective. Fisheries policy could thereby take into account identity, values, and relational elements of social life that give meaning and a sense of belonging to small-scale fishers, while also recognizing the cross-cutting and often contradictory variations in human experience that arise from social and economic differences. This social fabric of small-scale fishers' lives shapes their intentions and actions and is thus a necessary complication to the practice of fisheries management that its proponents need to consider.

The impact of heart failure therapy in patients with mildly reduced ejection fraction: a network meta-analysis.

BACKGROUND: Recent heart failure (HF) guidelines have re-classified HF patients with left ventricular ejection fraction (LVEF) between 41% and 49% as HF with mildly reduced ejection fraction (HFmrEF). HFmrEF treatment is often considered a grey zone as no randomized controlled trials (RCTs) were conducted exclusively on these patients. AIMS: A network meta-analysis (NMA) was performed to compare treatment effect of mineralocorticoid receptor antagonists (MRA), angiotensin receptor neprilysin inhibitor (ARNi), angiotensin receptor blockers (ARB), angiotensin-converting-enzyme inhibitors (ACEi), sodium-glucose cotransporter-2 inhibitors (SGLT2i), and beta-blockers (BB) in HFmrEF cardiovascular (CV) outcomes. METHODS AND RESULTS: RCTs sub-analyses evaluating the efficacy of pharmacological treatment in HFmrEF patients were searched. Hazard ratios (HRs) and their variance were extracted from each RCT for (i) composite of CV death or HF hospitalizations, (ii) CV death, and (iii) HF hospitalizations. A random-effects NMA was performed to compare and assess the treatment efficiency. Six RCTs with subgroup analysis according to participants' ejection fraction, a patient-level pooled meta-analysis of two RCTs, and an individual patient-level analysis of eleven BB RCTs were included, totalling 7966 patients. To our primary endpoint, SGLT2i vs. placebo was the only comparison with significant results, with a 19% risk reduction in the composite of CV death or HF hospitalizations [HR 0.81, 95% confidence interval (CI) 0.67-0.98]. In HF hospitalizations, the impact of the pharmacological therapies was more notorious, and ARNi reduced in 40% the risk of HF hospitalizations (HR 0.60, 95% CI 0.39-0.92), SGLT2i in 26% (HR 0.74, 95% CI 0.59-0.93) and renin-angiotensin system inhibition (RASi) with ARB and ACEi in 28% (HR 0.72, 95% CI 0.53-0.98). Although BBs were globally less beneficial, they were the only class that supported a reduced risk of CV death (HR vs. placebo: 0.48, 95% CI 0.24-0.95). We did not observe a statistically significant difference in any comparison between active treatments. There was a sound reduction with ARNi on the primary endpoint (HR vs. BB: 0.81, 95% CI 0.47-1.41; HR vs. MRA 0.94, 95% CI 0.53-1.66) and on HF hospitalizations (HR vs. RASi 0.83, 95% CI 0.62-1.11; HR vs. SGLT2i 0.80, 95% CI 0.50-1.30). CONCLUSIONS: In addition to SGLT2i, pharmacological treatment recommended for HF with reduced LVEF, namely, ARNi, MRA, and BB, can also be effective in HFmrEF. This NMA did not show significant superiority over any pharmacological class.

Mycotoxins in Raw Bovine Milk: UHPLC-QTrap-MS/MS Method as a Biosafety Control Tool.

Mycotoxins are compounds produced by several fungi that contaminate agricultural fields and, either directly or by carry-over, final food products. Animal exposure to these compounds through contaminated feed can lead to their excretion into milk, posing threats to public health. Currently, aflatoxin M1 is the sole mycotoxin with a maximum level set in milk by the European Union, as well as the most studied. Nonetheless, animal feed is known to be contaminated by several groups of mycotoxins with relevance from the food safety point of view that can be carried over into milk. To evaluate the multi-mycotoxin occurrence in this highly consumed food product it is crucial to develop precise and robust analytical methodologies towards their determination. In this sense, an analytical method for the simultaneous identification of 23 regulated, non-regulated, and emerging mycotoxins in raw bovine milk using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was validated. A modified QuEChERS protocol for extraction purposes was used, and further validation was performed by assessing the selectivity and specificity, limits of detection and quantification (LOD and LOQ), linearity, repeatability, reproducibility, and recovery. The performance criteria were compliant with mycotoxin-specific and general European regulations for regulated, non-regulated, and emerging mycotoxins. The LOD and LOQ ranged between 0.001 and 9.88 ng mL-1 and 0.005 and 13.54 ng mL-1, respectively. Recovery values were between 67.5 and 119.8%. The repeatability and reproducibility parameters were below 15 and 25%, respectively. The validated methodology was successfully applied to determine regulated, non-regulated, and emerging mycotoxins in raw bulk milk from Portuguese dairy farms, proving the importance of widening the monitoring scope of mycotoxins in dairy products. Additionality, this method presents itself as a new strategic and integrated biosafety control tool for dairy farms for the analysis of these natural and relevant human risks.

Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is pathologically defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neurons in the brain. Predominately pre-synaptic, aSyn has been reported in various subcellular compartments in experimental models. Indeed, nuclear alpha-synuclein (aSynNuc) is evident in many models, the dysregulation of which is associated with altered DNA integrity, transcription and nuclear homeostasis. However, the presence of aSynNuc in human brain cells remains controversial, yet the determination of human brain aSynNuc and its pathological modification is essential for understanding synucleinopathies. Here, using a multi-disciplinary approach employing immunohistochemistry, immunoblot, and mass-spectrometry (MS), we confirm aSynNuc in post-mortem brain tissue obtained from DLB and control cases. Highly dependent on antigen retrieval methods, in optimal conditions, intra-nuclear pan and phospho-S129 positive aSyn puncta were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations in all cases examined. Furthermore, an increase in nuclear phospho-S129 positive aSyn immunoreactivity was apparent in DLB cases compared to controls, in both neuronal and non-neuronal cell types. Our initial histological investigations identified that aSynNuc is affected by epitope unmasking methods but present under optimal conditions, and this presence was confirmed by isolation of nuclei and a combined approach of immunoblotting and mass spectrometry, where aSynNuc was approximately tenfold less abundant in the nucleus than cytoplasm. Notably, direct comparison of DLB cases to aged controls identified increased pS129 and higher molecular weight species in the nuclei of DLB cases, suggesting putative pathogenic modifications to aSynNuc in DLB. In summary, using multiple approaches we provide several lines of evidence supporting the presence of aSynNuc in autoptic human brain tissue and, notably, that it is subject to putative pathogenic modifications in DLB that may contribute to the disease phenotype.

RAB39B is redistributed in dementia with Lewy bodies and is sequestered within aß plaques and Lewy bodies.

Loss of function mutations within the vesicular trafficking protein Ras analogy in brain 39B (RAB39B) are associated with rare X-linked Parkinson's disease (PD). Physiologically, RAB39B is localized to Golgi vesicles and recycling endosomes and is required for glutamatergic receptor maturation but also for alpha-Synuclein (aSyn) homeostasis and the inhibition of its aggregation. Despite evidence linking RAB39B to neurodegeneration, the involvement of the protein in idiopathic neurodegenerative diseases remains undetermined. Here, analysis of the spatial distribution and expression of RAB39B was conducted in post-mortem human brain tissue from cases of dementia with Lewy bodies (DLB, n = 10), Alzheimer's disease (AD, n = 12) and controls (n = 12). Assessment of cortical RAB39B immunoreactivity using tissue microarrays revealed an overall reduction in the area of RAB39B positive gray matter in DLB cases when compared to controls and AD cases. Strikingly, RAB39B co-localized with beta-amyloid (Aß) plaques in all cases examined and was additionally present in a subpopulation of Lewy bodies (LBs) in DLB. Biochemical measures of total RAB39B levels within the temporal cortex were unchanged between DLB, AD and controls. However, upon subcellular fractionation, a reduction of RAB39B in the cytoplasmic pool was found in DLB cases, alongside an increase of phosphorylated aSyn and Aß in whole tissue lysates. The reduction of cytoplasmic RAB39B is consistent with an impaired reserve capacity for RAB39B-associated functions, which in turn may facilitate LB aggregation and synaptic impairment. Collectively, our data support the involvement of RAB39B in the pathogenesis of DLB and the co-aggregation of RAB39B with Aß in plaques suggests that age-associated cerebral Aß pathology may be contributory to the loss of RAB39B. Thus RAB39B, its associated functional pathways and its entrapment in aggregates may be considered as future targets for therapeutic interventions to impede the overall pathological burden and cellular dysfunction in Lewy body diseases.

Endoscopic stent in malignant colonic obstruction: the risk of tumor seeding / Prótese endoscópica no carcinoma do cólon em obstrução: risco de disseminação tumoral

Abstract Introduction: Malignant colonic occlusion is traditionally considered a surgical emergency. With the development of endoscopic techniques, metallic stents have emerged to ensure the colonic patency in nonsurgical candidates and, more recently, as a temporary measure until elective resection surgery is possible. Materials and methods: The research was conducted in PubMed and collected a total of 46 articles, including cross-references. Results: Ideally, intestinal occlusion should be resolved through tumor's primary resection with direct anastomosis. To avoid dehiscence of the anastomosis, tumor's resection may be performed with Hartmann's procedure. Metal stents are an alternative to emergency surgery and show excellent results in reliving colonic obstruction. However, they may have serious complications related to colonic perforation, migration and tumor dissemination. Discussion and conclusion: Observational studies and clinical trials show discrepant results. Metal stents are increasingly accepted in palliative care but are not yet recommended as a bridge to curative surgery. Treatment should be individualized, according to surgical risk and the probability of endoscopic complications.

Resumo Introdução: A oclusão intestinal aguda maligna é tradicionalmente considerada uma emergência cirúrgica. Com o desenvolvimento das técnicas endoscópicas surgiram os stents metálicos que asseguram a patência do cólon em doentes não candidatos a cirurgia e, mais recentemente, como uma medida temporária até ser possível realizar uma cirurgia de resseção eletiva. Materiais e métodos: A pesquisa decorreu na PubMed e reuniu um total de 46 artigos, incluindo referências cruzadas. Resultados: Idealmente, a oclusão intestinal deve ser abordada através da resseção primária do tumor com anastomose primária. Para evitar a deiscência da anastomose, a resseção tumoral pode ser realizada recorrendo à cirurgia Hartmann ou a um estoma derivativo sem resseção tumoral. Os stents metálicos são uma alternativa à cirurgia de emergência com resultados excelentes na resolução da obstrução cólica. Contudo, poderão levar a complicações como a perfuração cólica, a migração e a disseminação tumoral. Discussão e conclusão: Os estudos observacionais e ensaios clínicos mostram resultados discrepantes. O uso dos stents metálicos é cada vez mais aceite como tratamento paliativo, mas ainda não são inequivocamente recomendados como ponte para uma cirurgia curativa. O tratamento deve ser individualizado de acordo com o risco cirúrgico e a probabilidade de complicações endoscópicas.

The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood.

Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 µM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.

Development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry for determination of amanitins in urine and liver samples.

Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and ß-amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and ß-amanitin of 0.22 and 0.20 ng mL(-1) in urine and 10.9 and 9.7 ng g(-1) in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL(-1) in urine and 12.3-14.7 ng g(-1) in tissue, for both amanitins. Linearity, in the range of 10.0-200.0 ng mL(-1) or ng g(-1), shows that coefficients of correlation were greater than 0.997 for α-amanitin and 0.993 for ß-amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and ß-amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and mushrooms.

Histiocitose x (síndrome de hand-schuller-christian): relato de caso / Histiocytosis X (Hand-Schuller-Christian disease: a case report

Os autores relatam o caso de uma criança do sexo feminino, quatro anos de idade, que apresentava cefaléia, irritabilidade, otorréia e exoftalmia unilateral. No estudo tomográfico e na radiologia convencional encontraram-se grandes áreas líticas no crânio e na sua base. O restante do esqueleto não evidenciou anormalidades. O diagnóstico de histiocitose X (síndrome de Hand-Schuller-Christian) foi confirmado pelo mielograma. Este estudo mostra achados radiológicos e citológicos, além de apresentar uma revisão da literatura sobre o caso.

O processo de enfermagem no cotidiano dos acadêmicos de enfermagem e enfermeiros / The nursing process on the dailly lives of nursing students and nurses

O presente estudo tem como objetivo contribuir para a reflexäo do Processo de Enfermagem no cotidiano dos acadêmicos de enfermagem e enfermeiros, sendo utilizado como método de investigaçäo uma abordagem qualitativa, com aplicaçäo de entrevistas semi-estruturadas, cujos resultados obtidos foram confrontados com o referencial teórico e agrupados na forma de temas...

Carcinossarcoma de pulmäo: caracterizaçäo histopatológica e imuno-histoquímica dos componentes epiteliais e mesenquimais; relato de dois casos / Pulmonary carcinosarcoma: histopathological and immunohistochemical characterization of epithelial and mesenchimal components-report of two cases

Carcinosarcomas de pulmäo säo neoplasias raras que recorrem em posiçäo endobrônquica e perifericamente no parênquima pulmonar. Histologicamente diferenciam-se dos blastomas, podendo no entanto existir formas transicionais entre ambos. Dois novos casos dessa entidade säo relatados. É abordada com maior ênfase a caracterizaçäo histopatológica dos elementos epiteliais e mesenquimais e seus aspectos imuno-histoquímicos. Com esse objetivo, foram usados anticorpos policlonais, como citoqueratina, antígeno carcinoembrionário, vimentina, enolase neuro-específica, proteína S-100 e p anticorpo HHF35, dirigido para actina músculo-específica. No primeiro caso o componente epitelial se caracterizou pela diferenciaçäo epidermóide e de adenocarcinoma, incluindo áreas com padräo tipo carcinoma bronquíolo-alveolar. Este último aspecto esteve presente em um único caso daqueles descritos anteriormente. A imuno-histoquímica demonstrou positividade para citoqueratina nos diversos componentes epiteliais. O elemento mesenquimal expressou vimentina e actina músculo-específica. O segundo caso exibiu o componente epitelial com diferenciaçäo neuroenócrina, traduzida no estudo histológico convencional por carinoma anaplásico de pequenas células; a imuno-histoquímica mostrou positividade acentuada para a enolase neuro-especifica, um marcador neuroendócrino bem conhecido. Algumas células do componente sarcomatoso também mostraram-se positivas com esse marcador. A vimentina corou exclusivamente as células mesenquimais. A diferenciaçäo do tipo carcinoma de pequenas células correlaciona-se com a curta sobrevida do paciente, mesmo com todas as tentativas terapêuticas efetuadas. Esse tipo de diferenciaçäo do elemento epitelial foi poucas vezes observado nos casos relatados, e a maioria dos autores, classificando as lesöes como carcinomas anaplásicos, näo fazem distinçäo entre aqueles de grandes e pequenas células