Lysins as a powerful alternative to combat Bacillus anthracis.

This review gathers all, to the best of our current knowledge, known lysins, mainly bacteriophage-derived, that have demonstrated activity against Bacillus anthracis strains. B. anthracis is a spore-forming, toxin-producing bacteria, naturally dwelling in soil. It is best known as a potential biowarfare threat, an etiological agent of anthrax, and a severe zoonotic disease. Anthrax can be treated with antibiotics (ciprofloxacin, penicillin, doxycycline); however, their administration may take up even to 60 days, and different factors can compromise their effectiveness. Bacterial viruses, bacteriophages (phages), are natural enemies of bacteria and use their lytic enzymes, endolysins (lysins), to specifically kill bacterial cells. Harnessing the potential of lysins to combat bacterial infections holds promise for diminishing antibiotic usage and, consequently, addressing the escalating antibiotic resistance in bacteria. In this context, we list the lysins with the activity against B. anthracis, providing a summary of their lytic properties in vitro and the outcomes observed in animal models. Bacillus cereus strain ATCC 4342/RSVF1, a surrogate for B. anthracis, was also included as a target bacteria. KEY POINTS: • More than a dozen different B. anthracis lysins have been identified and studied. • They fall into three blocks regarding their amino acid sequence similarity and most of them are amidases. • Lysins could be used in treating B. anthracis infections.

Development, in-house validation and application of a method using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the quantification of 12 ergot alkaloids in compound feeds.

Introduction: Ergot alkaloids (EAs) are toxic substances naturally produced by Claviceps fungi. These fungi infest a wide range of cereals and grasses. When domestic animals are exposed to EAs through contaminated feeds, it is detrimental to them and leads to significant economic losses. For that reason, it is important to monitor feed for the presence of EAs, especially with methods enabling their determination in processed materials. Material and Methods: Ergot alkaloids were extracted with acetonitrile, and dispersive solid phase extraction (d-SPE) was used for clean-up of the extracts. After evaporation, the extracts were reconstituted in ammonium carbonate and acetonitrile and subjected to instrumental analysis using high-performance liquid chromatography with fluorescence detection. The developed method was validated in terms of linearity, selectivity, repeatability, reproducibility, robustness, matrix effect, limits of quantification and detection and uncertainty. The EA content of 40 compound feeds was determined. Results: All the assessed validation parameters fulfilled the requirements of Regulation (EU) 2021/808. At least one of the monitored alkaloids was determined in 40% of the samples. The EAs with the highest incidence rate were ergocryptine, ergometrinine and ergocornine. The total concentrations of EAs ranged from under the limit of quantification to 62.3 µg kg-1. Conclusion: The results demonstrated that the developed method was suitable for simultaneously determining twelve EAs in compound feed and could be used for routine analysis.

Simultaneous Determination of Pyrrolizidine and Tropane Alkaloids in Honey by Liquid Chromatography-mass Spectrometry.

Introduction: Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography- mass spectrometry was developed. Material and Methods: The analytical protocol used sulphuric acid extraction and solid-phase extraction purification. The developed procedure was subjected to validation in terms of linearity, selectivity, repeatability, reproducibility, limits of quantification and determination, matrix effect and uncertainty. A total of 29 honey samples were analysed for the determination of PAs and TAs. Results: All the evaluated validation parameters fulfilled the requirements of European Commission Decision 2002/657/EC. At least one of the monitored alkaloids was determined in 52% of the samples. Among the most abundant alkaloids were echimidine, intermedine and lycopsamine. The total PA concentrations ranged from 2.2 to 147.0 µg kg-1. Contrastingly, none of the monitored TAs was detected in the analysed samples. An assessment of the dietary exposure to PAs from the consumption of the contaminated honeys showed that three of them would pose a risk to consumers, especially if they were children. Conclusion: A sensitive method suitable for simultaneous determination of PAs and TAs in honey was developed and validated. The analysis of 29 honey samples for PAs and TAs revealed that honey destined for retail could pose a risk to consumers.

Risk assessment of rare earth elements, antimony, barium, boron, lithium, tellurium, thallium and vanadium in teas.

In recent years, a great intensification in the use of various elements especially in modern technology can be observed. However, the anthropogenic activities, including industrialisation, urbanisation or intensive agriculture, have led to the release of many of the elements into the environment. The consequence of the accumulation of the elements both in soil and water systems is their presence in the food chain. Inhalation and consumption of the contaminated food and beverages have been indicated as the main pathways of the exposure to many elements. Due to the fact, that tea is considered the second most popular beverage worldwide and its consumption is constantly increasing, it is crucial to evaluate the safety of the product, especially for toxic elements contamination. Thus, the aim of the project was to evaluate the contamination levels of rare earth elements (REEs) including lanthanides, scandium (Sc) and yttrium (Y) and also antimony (Sb), barium (Ba), boron (B), lithium (Li), tellurium (Te), thallium (Tl) and vanadium (V) in teas. Subsequently, the risk assessment was carried out. Additionally, the Fellowship provided hands-on training on the evaluation of applications of new biocides and participation in the science-based advises given to the Danish Food and Veterinary Administration, Danish Environment Protection Agency and Danish Medical Agency.

Scopolamine and atropine in feeds - determination with liquid chromatography mass spectrometry.

Tropane alkaloids (TAs) are naturally occurring plant toxins. Due to the fact that TA-producing plants can enter the food chain, they pose a risk for animals and human health. Therefore, sensitive analytical methods need to be developed to provide an adequate safety of feed and food. The presented method is based on liquid chromatography-mass spectrometry detection and enables the determination of scopolamine and atropine in compound feeds at a low level of contamination. Limits of quantification for scopolamine and atropine were 0.92 and 0.93 µg kg-1, respectively. Scopolamine-D3 and atropine-D3 were used for quantification. The method was successfully validated and applied to the analysis of 42 feed samples. Among investigated feeds, 67% contained at least one of the monitored alkaloids. Soybean meals were the feed materials contaminated most often, also with the highest determined concentrations of TAs, which reached 147.9 µg kg-1.

Application of the sum parameter method for the determination of pyrrolizidine alkaloids in teas.

Tea samples from the Polish market were analysed for the determination of pyrrolizidine alkaloids (PAs). The method applied in the study is based on the GC-MS technique and uses the sum parameter approach for quantification. The analytical protocol was subjected to validation in terms of linearity, repeatability, reproducibility, the limit of quantification and determination, matrix effect, selectivity, and uncertainty, and all evaluated parameters fulfilled the requirements of Commission Decision 2002/657. A total of 78% of the analysed samples contained 1,2-unsaturated PAs, and the concentrations were determined to fall in the range of 2-6498 µg kg-1. The highly contaminated samples were additionally subjected to LC-MS analysis. Much higher PA concentrations were determined with the sum parameter method, proving its superior quantification capability. The high contamination found prompted an assessment of the dietary exposure from the consumption of infusions prepared from the analysed teas. Two approaches were adopted: one assuming 100% transfer of PAs to the infusions and the second assuming only partial transfer. Regardless of the adopted approach, consumption of several infusions was shown to greatly exceed the maximum acceptable daily intake of PAs.

Diagnostic utility of two-dimensional speckle tracking echocardiography to identify ischemic etiology of left ventricular systolic dysfunction.

BACKGROUND: The differentiation between dilated cardiomyopathy (DCM) and systolic dysfunction caused by coronary artery disease (CAD) based on clinical and echocardiographic presentation can be difficult in some cases. AIM: Our study aimed to define differences in myocardial function between patients with ischemic and nonischemic etiology of reduced left ventricular ejection fraction using 2D speckle tracking echocardiography (STE). MATERIAL AND METHODS: We retrospectively analyzed 90 patients (mean age 65 ± 11 years, 70% male) with de novo diagnosed reduced left ventricular ejection fraction (≤45% as determined by the transthoracic echocardiography), who were referred for coronary angiography to determine the presence of significant CAD. On the basis of coronary angiography results, patients were divided into two subgroups as follows: 45 subjects with significant CAD (group A) and 45 subjects without CAD (group B). Acquired transthoracic echocardiographic images were assessed off-line using 2DSTE. In each patient, we calculated arithmetic mean, median value, and standard deviation of regional longitudinal strain and strain rate of 18 left ventricular segments. RESULTS: Standard deviation of the regional peak early diastolic strain rate was significantly higher in group A than in group B (P = 0.01). The cutoff value with the highest diagnostic value was >0.37/s-its sensitivity and specificity for the diagnosis of CAD were 73% and 53%, respectively. The remaining parameters did not differ significantly between the study groups. CONCLUSIONS: Patients with systolic dysfunction resulting from CAD have significantly more heterogeneous regional longitudinal early diastolic strain rate assessed by 2DSTE than patients with DCM.

Use of a New LC-MS Method for The Determination of Pyrrolizidine Alkaloids in Feeds.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed. MATERIAL AND METHODS: PAs were extracted with sulphuric acid and purified with cation exchange cartridges. A newly developed solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis. RESULTS: The developed method was validated according to SANTE/11945/2015 guidelines. The recovery was from 84.1% to 112.9%, the repeatability ranged from 3.0% to 13.6%, and the reproducibility was from 4.8% to 18.9%. CONCLUSIONS: A sensitive and selective method for determination of PAs in feed materials has been developed and validated. All evaluated validation parameters were in accordance with EU Reference Laboratories document no. SANTE/11945/2015. Almost 41% of the analysed feed samples were positive for the presence of at least one PA.

Pyrrolizidine Alkaloids in Honey: Determination with Liquid Chromatography-mass Spectrometry Method.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) are probably the most widespread toxins of natural origin. More than 6,000 plant species produce these toxic compounds. Bees can forage on flowers of plants producing PAs, which leads to contamination of honey with the toxic compounds. To determine the contamination of honey with PAs, a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed. MATERIAL AND METHODS: PAs were extracted with 0.05 M sulphuric acid and purified with MCX cartridges. A solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine (8:1:1:0.1:0.1, v/v) was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis. RESULTS: The developed method was validated according to SANTE/11945/2015 requirements. The recovery was from 80.6% to 114.5%. The repeatability ranged from 2.3% to 14.6%, and the reproducibility was from 4.9% to 17.7%. CONCLUSIONS: A new method for the determination of PAs in honey has been developed and validated. All evaluated parameters were in accordance with the SANTE/11945/2015 guidance document. Out of 50 analysed honey samples, 16 (32%) were positive for the content of at least one PA.

Evaluation of EX-PRESS glaucoma implant in elderly diabetic patients after 23G vitrectomy.

PURPOSE: The most frequent qualifications for pars plana vitrectomy (PPV) in diabetic patients include recurrent hemorrhage into the vitreous body chamber and vitreoretinal proliferation, also with traction retinal detachment. The aim of this study was to evaluate the effectiveness of EX-PRESS implant for the treatment of secondary glaucoma in elderly diabetic patients following PPV 23G with silicon oil or SF6 gas endotamponade. MATERIALS AND METHODS: This retrospective analysis comprised 18 patients (19 eyes). We applied EX-PRESS implants in 9 patients with neovascular glaucoma (NVG) and in 10 patients with non-NVG. All patients had earlier history of diabetes and vitrectomy 23G for diabetic complications. Intraocular pressure (IOP) was measured and compared before; 7 days; 1, 3, 6, and 12 months; and a year after the surgery. RESULTS: Application of the implant exhibited a lowering effect on IOP. After 1 month, 53% of patients had IOP values beyond 22 mmHg, while 86% after 1 year. CONCLUSION: The implant can be used to treat both NVG and non-NVG in diabetic patients following PPV.

Determination of pyrrolizidine alkaloids in selected feed materials with gas chromatography-mass spectrometry.

1,2-Dehydropyrrolizidine alkaloids are known to be toxic to many animals and humans. To provide safety of feeds a method based on gas chromatography-mass spectrometry enabling the determination of a content of 1,2-unsaturated PAs in feed materials was developed. After extraction with aqueous solution of HCl and purification of the extract, 1,2-unsaturated alkaloids are reduced to their common backbone structures and subsequently derivatised with heptafluorobutyric anhydride (HFBA). The method was validated according to SANTE/11945/2015. All received parameters are consistent with the document requirements as recovery of a final compound retronecine derivative was from 81.8% to 94.4% when retrosine was used for spiking and from 72.7% to 85.5% when retrorsine N-oxide was spiked. The repeatability was calculated as relative standards deviation and ranged from 7.5% to 14.4%, for N-oxide was from 7.9% to 15.4%. The reproducibility was in the range from 14.2% to 16.3% and from 17.0% to 18.1% for free base and N-oxide respectively. The limit of quantification was determined as 10 µg kg-1. Good linearity of the method was obtained with coefficient of determination R2 > 0.99. The method was applied to 35 silage and two hay samples analysis.

Heart failure as an independent predictor of thrombus persistence in nonvalvular atrial fibrillation: a transesophageal echocardiography-based study.

INTRODUCTION: Formation of left atrial (LA) thrombus is one of the most serious complications in patients with atrial fibrillation (AF). OBJECTIVES: The aim of our study was to determine the predictors of LA thrombus resolution among patients with AF receiving oral anticoagulation. PATIENTS AND METHODS: After a retrospective analysis of 1877 transesophageal echocardiographic examinations (TEEs) performed in our department between January 2009 and June 2013, we included 64 patients (women, 36%; mean age at diagnosis, 64 ±8.8 years) with nonvalvular AF and LA thrombi on TEE into the study. All patients received oral anticoagulation and underwent follow-up TEE within a few months since diagnosis. RESULTS: After a mean follow-up period of 88 ±107 days, thrombus resolution was observed in 30 patients (47%). The univariate Cox proportional regression model showed that heart failure and reduced left ventricular ejection fraction were associated with the persistence of LA thrombus (hazard ratio [HR], 2.72; 95% confidence interval [CI], 1.32-5.61; P = 0.007 and HR, 0.97; 95% CI, 0.94-0.99; P = 0.04; respectively). The international normalized ratio and CHA2DS2-VASc score were not prognostic for thrombus resolution (HR, 0.64; 95% CI, 0.37-1.1; P = 0.1 and HR 1.10; 95% CI, 0.91-1.33; P = 0.3; respectively). In a multivariate analysis, heart failure was the only independent factor predicting unsuccessful resolution of LA thrombus (P = 0.04). CONCLUSIONS: Heart failure is an independent negative predictor of LA thrombus resolution in patients with AF receiving oral anticoagulation.

Prognostic Implications of Discordant Results of Myocardial Perfusion Single-Photon Emission Computed Tomography and Exercise ECG Test in Patients with Stable Angina.

BACKGROUND: ECG exercise treadmill test (ExT) and myocardial perfusion SPECT (single photon emission computed tomography) study are widely used for the non-invasive evaluation of patients with coronary artery disease (CAD). OBJECTIVES: To assess long-term prognosis in patients with suspected or known coronary artery disease (CAD), in whom ECG exercise treadmill test (ExT) and myocardial perfusion single photon emission computed tomography (SPECT) provided discordant results are lacking. MATERIAL AND METHODS: Four hundred eighty three patients with suspected or known stable CAD underwent 99mTc-methoxyisobutylisonitrile SPECT and ExT. SPECT was considered positive (+) if inducible or mixed perfusion defects were detected. ExT was evaluated using widely accepted criteria. Based on the results of both examinations the patients were divided into 4 subgroups: group 1 - SPECT (+) and ExT (+), group 2 - SPECT (+) and ExT (-), group 3 - SPECT (-) and ExT (+), group 4 - SPECT (-) and ExT (-). RESULTS: After a mean follow-up of 59 ± 7 months, major cardiac events (cardiac death and nonfatal myocardial infarction combined) and revascularizations were more prevalent in groups 1 and 2 than in groups 3 and 4. However, the statistical significance (p ≤ 0.01) was reached only for the following differences: in major cardiac events - group 1 vs group 3 and group 1 vs group 4; in revascularizations - group 1 vs. group 3, group 1 vs. group 4 and group 2 vs group 4 and in cardiac hospitalizations - group 1 vs. group 4 and group 2 vs. group 4. CONCLUSIONS: Positive myocardial perfusion SPECT result is associated with similar clinical outcome irrespectively of ExT result in long-term follow-up.

Giant J-wave and Brugada-like pattern in a patient with severe hypothermia.

We describe a patient with severe hypothermia, in whom the electrocardiogram showed giant J-waves, also known as Osborn waves, distinguishable in the inferior and anterolateral leads. Moreover, a Brugada-like pattern was also visible in leads V2 and V3. The presented case and ECG findings may contribute to the discussion about the pathophysiologic mechanism underlying Brugada syndrome and giant J-wave.

Development and validation method for the determination of selected tetracyclines in animal medicated feedingstuffs with the use of micellar liquid chromatography.

A chromatographic procedure for the determination of oxytetracycline (OTC), tetracycline (TC), chlorotetracycline (CTC), and doxycycline (DC) in medicated feedingstuffs was developed. Samples were extracted with 0.01 M citric buffer/acetonitrile (pH 3.0) and further purified with 0.45 µm syringe filters. The purified extract was separated on Thermo column C18, 150 × 4 mm, 5 µm and detection was carried out at 360 nm for OTC, and TC, 370 nm for CTC, and 350 nm for DC. TCs were eluted with a mobile phase of 0.03 M SDS/7% 1-butanol/0.02 M oxalic acid/NaOH at pH 2.5. This method provided average recoveries of 80.4% to 100.2%, with CVs of 0.5% to 6.6% in the range of 50 to 1500 mg/kg OTC, TC, CTC, and DC in feeds. The linearity for the four TCs was determined by high-performance liquid chromatography-diode array detector (HPLC-DAD) in the range 10-300 µg/mL (50-1500 mg/kg), with a linear correlation coefficient (R) > 0.99. The LOD and LOQ for TCs in pig and poultry feeds ranged from 4.0 to 10.7 and 4.7 to 12.6 mg/kg, respectively. The methodology was applied to the analysis of animal feedingstuffs collected from poultry and pig farms.

Long-term prognostic value of inducible and resting perfusion defects detected by single-photon emission computed tomography in the era of wide availability of coronary revascularization.

PURPOSE: To assess the long-term prognostic value of various types of perfusion defects detected by single-photon emission computed tomography (SPECT) in patients with stable angina. METHODS: Seven hundred and thirty two patients (299 men, mean age 57 ± 9 years) with suspected or known stable coronary artery disease underwent rest/exercise SPECT protocol using 99mTc-methoxyisobutylisonitrile (MIBI). All patients completed clinical follow-up regarding cardiac events (cardiac death, sudden cardiac death, acute coronary syndrome, revascularization, cardiac hospitalization) for a mean period of 58 ± 8 months. Event rates were analysed in subgroups defined according to the presence of fixed or inducible myocardial perfusion defects. RESULTS: During the follow-up, 15 cardiac deaths were recorded, 13 of which were qualified as a sudden cardiac death, 59 acute coronary syndromes, 65 revascularizations and 209 cardiac hospitalizations. The presence of inducible perfusion defects on SPECT was associated with a high risk for occurrence of all analysed end points: sudden cardiac death (HR = 3·96, P = 0·01), cardiac hospitalization (HR = 1·5, P = 0·004), coronary syndrome (HR = 2·33, P = 0·001) and coronary revascularization (HR = 2·76, P = 0·0002), except for the cardiac death (P = 0·27). Resting perfusion defects were highly predictive for cardiac death (HR = 7·45; P = 0·001), but not for other cardiac events (P = NS). The presence of any (inducible or resting) perfusion defects was associated with a high risk of all cardiovascular complications. CONCLUSIONS: In long-term follow-up, SPECT proved highly predictive of cardiac events in patients with suspected or known CAD. In the revascularization era, cardiac death is most accurately related to the presence of resting perfusion defects, but all other cardiac events were better predicted by inducible perfusion defects.