Detection of Hypoglycin A and MCPrG Metabolites in the Milk and Urine of Pasture Dairy Cows after Intake of Sycamore Seedlings.

Hypoglycin A (HGA), methylenecyclopropylglycine (MCPrG), hypoglycin B (HGB), and γ-glutamyl-α-(methylenecyclopropyl) glycine (γ-glutamyl-MCPrG) are secondary plant metabolites occurring in sycamore maple (Acer pseudoplatanus) as well as several other Sapindaceae (e.g., Blighia sapida). By interfering with energy metabolism, they may cause severe intoxication in humans and other species. However, to date, there is not enough data available concerning the intake, metabolism, or excretion of sycamore maple toxins in dairy cows. In May 2022, five cows were observed over four days, when they had first access to a pasture with two sycamore maples. Grazing of their seedlings that grew numerously in between the pasture plants was monitored by direct observation. Milk samples were drawn both from individual cows and from the bulk tank. Spontaneous urine samples were collected from all cows on day 3 after access to the pasture. Seedlings (100 g) were sampled on the pasture and analyzed, together with milk and urine samples, for sycamore toxins and their metabolites using liquid chromatography-tandem mass spectrometry and liquid chromatography-high-resolution mass spectrometry. Cows ingested sycamore seedlings while grazing. Values of HGA in milk were below the limit of quantification. However, metabolites of HGA and MCPrG were detected in individual milk samples already at the end of the first day of grazing. Urine samples of all five cows showed higher concentrations of conjugated HGA and MCPrG metabolites than in milk. Observations suggest that dairy cows may have a low susceptibility toward sycamore maple toxins. However, whether this could be attributed to foregut fermenting species in general requires further elucidation.

Acute right insular ischaemic lesions and poststroke left ventricular dysfunction.

INTRODUCTION: Myocardial injury related to acute ischaemic stroke is common even without primary cardiac disease. We intended to determine associations between values of left ventricular ejection fraction (LVEF) and ischaemic stroke lesion sites. METHODS: Of a local database, patients with acute first-ever ischaemic stroke confirmed by brain imaging but without pre-existing heart disease were included. The cardiac morphology and LVEF were obtained from transthoracic or transesophageal echocardiography, and impaired LVEF was categorised as mild (35%-50%), moderate (34%-25%) and severe (<25%). Patient age, stroke severity, ischaemic lesion volume, prevalence of troponin I increase (>0.1 ng/mL), atrial fibrillation and cardiac wall motion abnormalities were assessed and compared between patients with and without impaired LVEF after stroke (significance: p<0.05). A multivariate voxelwise lesion analysis correlated LVEF after stroke with sites of ischaemic lesions. RESULTS: Of 1209 patients who had a stroke, 231 (mean age 66.3±14.0 years) met the inclusion criteria; 40 patients (17.3%) had an impaired LVEF after stroke. Patients with impaired LVEF had higher infarct volumes (53.8 mL vs 30.0 mL, p=0.042), a higher prevalence of troponin increase (17.5% vs 4.2%, p=0.006), cardiac wall motion abnormalities (42.5% vs 5.2%, p<0.001) and atrial fibrillation (60.0% vs 26.2%, p<0.001) than patients with LVEF of >50%. The multivariate voxelwise lesion analysis yielded associations between decreased LVEF and damaged voxels in the insula, amygdala and operculum of the right hemisphere. CONCLUSION: Our imaging analysis unveils a prominent role of the right hemispheric central autonomic network, especially of the insular cortex, in the brain-heart axis. Our results support preliminary evidence that acute ischaemic stroke in distinct brain regions of the central autonomic network may directly impair cardiac function and thus further supports the concept of a distinct stroke-heart syndrome.

Integration of ICTs in teaching practices: propositions to the SAMR model.

This article aims to analyze the experiences of teachers on the uses of ICTs in the development of teaching practices within the framework of a new learning ecology. We use the SAMR model to scale the levels of ICTs contributions in each practice. 116 teachers from a public educational institution in Brazil answered a questionnaire during the pandemic scenario, while conducting emergency remote teaching. Teachers declared to develop the teaching practices with the integration of ICTs at the levels of Augmentation and Modification by correspondence to the SAMR model. Based on the analyzes carried out, we propose some reflections that help to rethink this model and to understand the second-order barriers that prevent the effective integration of ICT in teachers' practices.

Investigations on the Transfer of Quinolizidine Alkaloids from Lupinus angustifolius into the Milk of Dairy Cows.

Lupin varieties with a low content of quinolizidine alkaloids (QAs) like blue sweet lupin (BSL) have long been used as a protein source for dairy cows. A health concern for humans may arise from the transfer of acute toxic QAs from feed into cow's milk. This study is the first to quantify the transfer of QAs from BSL into cow's milk with experimental and modeling methods. Four lactating dairy cows were subjected to two 7 day feeding periods with 1 and 2 kg/d BSL, respectively, each followed by a depuration period. BSL contained 1774 mg/kg dry matter total QAs. Individual milk samples were taken twice daily and QA contents in feed and milk determined with liquid chromatography-tandem mass spectrometry. Transfer of QAs into the milk was already seen with the administration of 1 kg/d BSL, with differences in transfer rates (TRs) between individual QAs. A toxicokinetic model was derived to quantify and predict QA feed-to-food transfer. For the four most prominent QAs, our model shows an α-half-life of around 0.27 d. TRs were obtained for six QAs and were between 0.13 (sparteine) and 3.74% (multiflorine). A toxicological assessment of milk containing QAs as measured in this study indicated a potential health concern.

Co-Occurrence of Hypoglycin A and Hypoglycin B in Sycamore and Box Elder Maple Proved by LC-MS/MS and LC-HR-MS.

Hypoglycin A (HGA) and methylenecyclpropylglycine (MCPrG) are formed by some maple trees (Acer species) and have been associated with incidences of atypical myopathy among horses in pastures. In this work, a simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method without derivatization was developed for the quantification of HGA and MCPrG in maple samples and validated according to EU guidelines. The LOQ presented here for HGA (16.4 µg/kg) is considerably lower than the lowest published LOQ (500 µg/kg). This method confirms that sycamore and box elder maple contain considerable amounts of HGA and MCPrG. In addition, the presence of the dipeptides hypoglycin B and γ-glutamyl-MCPrG in these two maple species is shown using high-resolution MS. This is the first report on the presence of these dipeptides in maple since 1973. The presence of HGB and γ-glutamyl-MCPrG could change the way we understand animal intoxication following the ingestion of maple.

Efficient DNA Repair Mitigates Replication Stress Resulting in Less Immunogenic Cytosolic DNA in Radioresistant Breast Cancer Stem Cells.

Cancer stem cells (CSCs) are a major cause of tumor therapy failure. This is mainly attributed to increased DNA repair capacity and immune escape. Recent studies have shown that functional DNA repair via homologous recombination (HR) prevents radiation-induced accumulation of DNA in the cytoplasm, thereby inhibiting the intracellular immune response. However, it is unclear whether CSCs can suppress radiation-induced cytoplasmic dsDNA formation. Here, we show that the increased radioresistance of ALDH1-positive breast cancer stem cells (BCSCs) in S phase is mediated by both enhanced DNA double-strand break repair and improved replication fork protection due to HR. Both HR-mediated processes lead to suppression of radiation-induced replication stress and consequently reduction of cytoplasmic dsDNA. The amount of cytoplasmic dsDNA correlated significantly with BCSC content (p=0.0002). This clearly indicates that HR-dependent avoidance of radiation-induced replication stress mediates radioresistance and contributes to its immune evasion. Consistent with this, enhancement of replication stress by inhibition of ataxia telangiectasia and RAD3 related (ATR) resulted in significant radiosensitization (SER37 increase 1.7-2.8 Gy, p<0.0001). Therefore, disruption of HR-mediated processes, particularly in replication, opens a CSC-specific radiosensitization option by enhancing their intracellular immune response.

[German national survey of lecturers in ophthalmology on student teaching in the 2020/21 corona pandemic]. / Deutschlandweite Befragung der Lehrbeauftragten in der Augenheilkunde zur studentischen Lehre in der Corona-Pandemie 2020/21.

BACKGROUND: The corona pandemic has had a significant impact on the conditions of university student teaching. Due to the pandemic-related contact restrictions, digital teaching formats were widely used instead of the previous face-to-face teaching. In the summer semester of 2020 students received this well and evaluated it positively in recent publications. In this work, the main focus was on the experiences and assessments of teachers in ophthalmology during the winter semester 2020/2021. METHODS: By means of two anonymous surveys via online questionnaires, the lecturers in ophthalmology of German university hospitals as well as internal and external lecturers and staff members of the student teaching of the Department of Ophthalmology of the University Medical Center Mainz were asked about their experiences with the implementation of digital teaching. RESULTS: In this context 95% of the teaching staff of ophthalmology departments of university hospitals in Germany stated that they had established digital teaching concepts at the latest since the corona pandemic. Hybrid formats with a proportion of face-to-face teaching were used by 68%. A wide variety of teaching formats were used. Difficulties were also encountered, particularly in interaction with students. Despite predominantly digital teaching, examinations continued to be held in face-to-face settings; only 18% of respondents stated that they had conducted online examinations. In the future, 86% of respondents want to integrate digital formats into their teaching concepts and establish them as a supplement to existing face-to-face teaching. CONCLUSION: The development of student teaching during the corona pandemic can serve as an opportunity for shaping the future education of medical students in ophthalmology.

US EPA's TSCA risk assessment approach: a case study of asbestos in automotive brakes.

The United States Environmental Protection Agency (US EPA) is currently refining its approach for risk assessments conducted under the amended Toxic Substances Control Act (TSCA), largely based on recommendations from the National Academies of Sciences, Engineering, and Medicine (NASEM). We identified several issues with the current TSCA risk assessment approach that were not addressed by NASEM in its recommendations. Here, we demonstrate these issues with a case study of the 'Risk Evaluation for Asbestos, Part 1: Chrysotile Asbestos,' which US EPA released in December 2020. In this evaluation, US EPA found that occupational and some consumer uses of automotive brakes and clutches that contain asbestos result in unreasonable risks. These risks were calculated from estimated exposures during brake work and an inhalation unit risk (IUR) developed for chrysotile asbestos. We found that US EPA overestimated risk as a result of unrealistic inputs to both the exposure and toxicity components of the risk equation, and because the Agency did not fully consider relevant epidemiology and toxicity evidence in its systematic review. Our evaluation demonstrates areas in which the TSCA risk assessment approach could be improved to result in risk evaluations that are supported by the available scientific evidence.

Activating transcription factor 4 mediates adaptation of human glioblastoma cells to hypoxia and temozolomide.

The integrated stress response (ISR) is a central cellular adaptive program that is activated by diverse stressors including ER stress, hypoxia and nutrient deprivation to orchestrate responses via activating transcription factor 4 (ATF4). We hypothesized that ATF4 is essential for the adaptation of human glioblastoma (GB) cells to the conditions of the tumor microenvironment and is contributing to therapy resistance against chemotherapy. ATF4 induction in GB cells was modulated pharmacologically and genetically and investigated in the context of temozolomide treatment as well as glucose and oxygen deprivation. The relevance of the ISR was analyzed by cell death and metabolic measurements under conditions to approximate aspects of the GB microenvironment. ATF4 protein levels were induced by temozolomide treatment. In line, ATF4 gene suppressed GB cells (ATF4sh) displayed increased cell death and decreased survival after temozolomide treatment. Similar results were observed after treatment with the ISR inhibitor ISRIB. ATF4sh and ISRIB treated GB cells were sensitized to hypoxia-induced cell death. Our experimental study provides evidence for an important role of ATF4 for the adaptation of human GB cells to conditions of the tumor microenvironment characterized by low oxygen and nutrient availability and for the development of temozolomide resistance. Inhibiting the ISR in GB cells could therefore be a promising therapeutic approach.

Activation of Epidermal Growth Factor Receptor Sensitizes Glioblastoma Cells to Hypoxia-Induced Cell Death.

BACKGROUND: The epidermal growth factor receptor (EGFR) signaling pathway is genetically activated in approximately 50% of glioblastomas (GBs). Its inhibition has been explored clinically but produced disappointing results, potentially due to metabolic effects that protect GB cells against nutrient deprivation and hypoxia. Here, we hypothesized that EGFR activation could disable metabolic adaptation and define a GB cell population sensitive to starvation. METHODS: Using genetically engineered GB cells to model different types of EGFR activation, we analyzed changes in metabolism and cell survival under conditions of the tumor microenvironment. RESULTS: We found that expression of mutant EGFRvIII as well as EGF stimulation of EGFR-overexpressing cells impaired physiological adaptation to starvation and rendered cells sensitive to hypoxia-induced cell death. This was preceded by adenosine triphosphate (ATP) depletion and an increase in glycolysis. Furthermore, EGFRvIII mutant cells had higher levels of mitochondrial superoxides potentially due to decreased metabolic flux into the serine synthesis pathway which was associated with a decrease in the NADPH/NADP+ ratio. CONCLUSIONS: The finding that EGFR activation renders GB cells susceptible to starvation could help to identify a subgroup of patients more likely to benefit from starvation-inducing therapies.

Chronic inflammation, Adverse Outcome Pathways, and risk assessment: A diagrammatic exposition.

Inflammasomes are a family of pro-inflammatory signaling complexes that orchestrate inflammatory responses in many tissues. The NLRP3 inflammasome has been implicated in several diseases associated with chronic inflammation. In this paper, we present an Adverse Outcome Pathway (AOP) for NLRP3-induced chronic inflammatory diseases that demonstrates how NLRP3 can cause a transition from acute to chronic inflammation, and ultimately the onset of disease. We present a simple graphical description of the main features of internal dose time courses that are important when pharmacodynamics are governed by an activation threshold. Similar considerations hold for other AOPs that are rate-limited by processes with activation thresholds. The risk analysis implications of AOPs with threshold or threshold-like pharmacodynamic responses include the need to consider how cumulative dose per unit time is distributed over time and the possibility that safe, or virtually safe, exposure concentrations can be defined for such processes.

Serine-dependent redox homeostasis regulates glioblastoma cell survival.

BACKGROUND: The amino acid serine is an important substrate for biosynthesis and redox homeostasis. We investigated whether glioblastoma (GBM) cells are dependent on serine for survival under conditions of the tumour microenvironment. METHODS: Serine availability in GBM cells was modulated pharmacologically, genetically and by adjusting serine and glycine concentrations in the culture medium. Cells were investigated for regulation of serine metabolism, proliferation, sensitivity to hypoxia-induced cell death and redox homeostasis. RESULTS: Hypoxia-induced expression of phosphoglycerate dehydrogenase (PHGDH) and the mitochondrial serine hydroxymethyltransferase (SHMT2) was observed in three of five tested glioma cell lines. Nuclear factor erythroid 2-related factor (Nrf) 2 activation also induced PHGDH and SHMT2 expression in GBM cells. Low levels of endogenous PHGDH as well as PHGDH gene suppression resulted in serine dependency for cell growth. Pharmacological inhibition of PHGDH with CBR-5884 reduced proliferation and sensitised cells profoundly to hypoxia-induced cell death. This effect was accompanied by an increase in reactive oxygen species and a decrease in the NADPH/NADP+ ratio. Similarly, hypoxia-induced cell death was enhanced by PHGDH gene suppression and reduced by PHGDH overexpression. CONCLUSIONS: Serine facilitates adaptation of GBM cells to conditions of the tumour microenvironment and its metabolism could be a plausible therapeutic target.

Alien limb phenomenon following posterior cerebral artery stroke: a distinct clinical entity.

Alien limb syndrome following stroke within the territory of the posterior cerebral artery is exceedingly rare. A right-handed female experienced left homonymous hemianopia, visuospatial neglect, and proprioceptive loss of her left hemi-body. She experienced unintended, involuntary movements of her left arm and hand, which interfered with and disturbed motor actions of daily life performed with her right upper limb. There was no denial of ownership, but she interpreted movements of her left upper limb to be annoying, out of her will and unwanted. The alien limb phenomenon improved in parallel with improvement of proprioceptive loss over a 12-week of in-patient rehabilitation. A recently proposed theoretical concept of the alien limb phenomenon after posterior artery stroke is discussed.

Critical review of the evidence for a causal association between exposure to asbestos and esophageal cancer.

Esophageal cancers comprise about 1% of all cancers diagnosed in the US but are more prevalent in other regions of the world. Several regulatory agencies have classified asbestos as a known human carcinogen, and it is linked to multiple diseases and malignancies, including lung cancer and mesothelioma. In a 2006 review of the epidemiological literature, the Institute of Medicine (IOM) did not find sufficient evidence to demonstrate a causal relationship between asbestos exposure and esophageal cancer. To reevaluate this conclusion, we performed a critical review of the animal toxicological, epidemiological, and mechanism of action literature on esophageal cancer and asbestos, incorporating studies published since 2006. Although there is some evidence in the epidemiological literature for an increased risk of esophageal cancer in asbestos-exposed occupational cohorts, these studies generally did not control for critical esophageal cancer risk factors (e.g. smoking, alcohol consumption). Furthermore, data from animal toxicological studies do not indicate that asbestos exposure increases esophageal cancer risk. Based on our evaluation of the literature, and reaffirming the IOM's findings, we conclude that there is insufficient evidence to demonstrate a causal link between asbestos exposure and esophageal cancer.

Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems.

Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01⁻1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.

Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

Glioblastomas are characterized by fast uncontrolled growth leading to hypoxic areas and necrosis. Signalling from EGFR via mammalian target of rapamycin complex 1 (mTORC1) is a major driver of cell growth and proliferation and one of the most commonly altered signalling pathways in glioblastomas. Therefore, epidermal growth factor receptor and mTORC1 signalling are plausible therapeutic targets and clinical trials with inhibitors are in progress. However, we have previously shown that epidermal growth factor receptor and mTORC1 inhibition triggers metabolic changes leading to adverse effects under the conditions of the tumour microenvironment by protecting from hypoxia-induced cell death. We hypothesized that conversely mTORC1 activation sensitizes glioma cells to hypoxia-induced cell death. As a model for mTORC1 activation we used gene suppression of its physiological inhibitor TSC2 (TSC2sh). TSC2sh glioma cells showed increased sensitivity to hypoxia-induced cell death that was accompanied by an earlier ATP depletion and an increase in reactive oxygen species. There was no difference in extracellular glucose consumption but an altered intracellular metabolic profile with an increase of intermediates of the pentose phosphate pathway. Mechanistically, mTORC1 upregulated the first and rate limiting enzyme of the pentose phosphate pathway, G6PD. Furthermore, an increase in oxygen consumption in TSC2sh cells was detected. This appeared to be due to higher transcription rates of genes involved in mitochondrial respiratory function including PPARGC1A and PPARGC1B (also known as PGC-1α and -ß). The finding that mTORC1 activation causes an increase in oxygen consumption and renders malignant glioma cells susceptible to hypoxia and nutrient deprivation could help identify glioblastoma patient cohorts more likely to benefit from hypoxia-inducing therapies such as the VEGFA-targeting antibody bevacizumab in future clinical evaluations.

[Some aspects of energy-saving pacing in the light of the author's studies]. / Niektóre aspekty kardiostymulacji energooszczednej w swietle badan wlasnych.

UNLABELLED: Pacemaker working time, which was in the beginning not more than one year, reached the maximum in the first half of the 70s, then shortened to between a few to several years. Aim of the study was investigated the electrical properties of the endocavitary pacemaker leads, considered the possibility of manufacturing a longlasting pacemaker ("lifetime pacemaker") and examined the preference of patients in relation to dimensions of the implanted device. MATERIALS AND METHODS: The investigation included 190 electrodes with cathodes coated with titanium nitride (TIJ and TIR), 244--coated with iridium (SXA and SXV) and 90--coated with black platinum (DXA and DXV). A formula was developed to calculate the estimated pacemaker longevity: Longevity (years)=Qog (Ah)x10(6)/8760x[Isp (µA)+Ist (µA)], where Qog is the capacity of the electric cell, Ist--current stimulation, Isp--quiescent current of the generator. The survey was performed in a group of 145 patients with pacemakers, using an original questionnaire. RESULTS: All the tested electrodes manifested a good acute and distant pacing threshold and the small intra-electrode differences were not clinically significant. In distant measurement, the average rheobasis of the steroid platinum electrode was 0.59 V and the chronaxie was 0.23 ms. These parameters have direct impact on modern pacemaker programming methods. It was proven that increasing the electric cell capacity in the DDD pacemaker by every one-tenth of ampere-hours caused a significant increase in the working time (p=0.000). Thus, the increase of the electric cell capacity allows for returning to the concept of a "lifetime pacemaker". The results of the survey showed that patients were willing to accept larger, but longlasting pacemakers (p=0.000). CONCLUSIONS: The tested passive and active electrode with a fractalcoated cathode ensured good electrical parameters within several months of observation. The electrode equipped with dexamethasone deposit showed no advantage in terms of the stimulation threshold when compared to the passive electrode coated with titanium nitride. Significant differences in the pacing threshold, which were detected between the leads with different cathode coatings, had little effect on pacemaker longevity. The most extensive impact on the generator longevity was exerted by the capacity of the electrical cell and pacemaker quiescent current. Patients were able to accept a much larger device than currently produced providing a long working time was ensured.

[Progress in the diagnosis and treatment of cardiac amyloidosis]. / Najnowsze postepy w rozpoznawaniu i leczeniu amyloidozy serca.

The heart is an organ often occupied by various forms of amyloidosis; cardiomyopathies are the leading cause of mortality in patients with amyloidosis. Cardiac amyloidosis is often diagnosed late because of nonspecific symptoms and missed early signs in the imaging routine. A method for treating cardiac amyloidosis depends on the type of amyloid protein. In the treatment of amyloidosis associated with immunoglobulins systemic chemotherapy is used without transplant or stem cell transplantation and in the treatment of familial transthyretin amyloidosis liver transplantation is used. There are still clinical studies on the use of siRNA for the treatment of cardiomyopathy associated with transthy retin amyloidosis, and on the use of amyloid protein stabilizers. The prognosis depends on the type of amyloid protein; worse results observed in the case of light chain amyloidosis. Care support is the cornerstone of treatment; it is expected that advances in cardiac imaging and proteomics positive impact on our ability to diagnosis, prognosis and treatment outcomes of amyloidosis of the heart.