Label-free multiple reaction monitoring, a promising method for quantification analyses of specific proteins in bacteria

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity-and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9-and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture

GENDER STEREOTYPES OF THE PERSONALITY AND FAMILY SETTINGS IN PATIENTS WITH RESISTANT ARTERIAL HYPERTENSION

Objective: To study the influence of gender stereotypes of personality (GSP) and family settings (FS) on the course of arterial hypertension (AH) in middle-aged patients. Methods: One hundred sixty patients examined with AH who were under observation for at least two months after in-patient treatment. Patients divided into 2 groups: I group – 80 people (men – 34, women – 46, average age 53.2±0.45 years) with resistant hypertension; Group II – 80 patients (men – 36, women – 44, average age 53.3±0.5 years) with controlled hypertension. In all patients, GSP studied for the index staple (IS) and CS for the family settings (FS) in comparison with adherence to treatment and cardiovascular risk. Results: Frequent GSP with AH was masculine and high-masculine, revealed in 71.3% of patients with resistant and in 52.7% of patients with controlled hypertension. A moderate positive relationship between IS and FS has been established (r=0.593852, p<0.05) and a decrease in IS values to areas of masculinity and high masculinity. In a comparison of 2 groups, this combination of gender characteristics associated with a significantly low adherence to treatment (2.1±0.1 and 1.5±0.1) and a high cardiovascular risk (3.0±0.1 and 3.4±0.1), more pronounced in patients with resistant AH. It is revealed that women with gender feature indicators of the relationship characterizing the severity of the course of hypertension is much more closely related, whereas in men this relationship is insignificant. Conclusion: The combination of the masculine type of personality with traditional FS in patients with resistant hypertension accompanied by a decrease in adherence to treatment and an increase in the total cardiovascular risk, that is, it reduces the effectiveness of antihypertensive therapy and promotes the progressive course of hypertension. The identification of predictors of the decrease in the effectiveness of antihypertensive therapy, taking into account the gender characteristics of the individual and family settings, is useful for identifying a highrisk group for the development of resistant AH

TREATMENT OF CHRONIC HEART FAILURE IN MIDDLE-AGED MEN WITH DEPRESSION

Objective: Comparative assessment of the efficiency of standard and combined antidepressant therapy of chronic heart failure (CHF), flowing depression in middle-aged men with mild and moderate arterial hypertension (AH). Methods: Evaluation of the effectiveness of CHF treatment in patients with absence (Group I) and the presence of depressive disorders (Group II) and combined with antidepressants (paroxetine) of standard therapy for CHF occurring with depression (Group III) was spent. Clinical indices and structural parameters of the myocardium and vessels are studied in the dynamics Results: In patients with depression not receiving antidepressants, the lack of depression is accompanied by low adherence to treatment and the meaningful by high BP. Despite the high efficacy of standard CHF treatment, the combination with an antidepressant (the importance of improving the structural parameters of the myocardium, reduction of depression and increased adherence to treatment), adherence to treatment is significantly lower than in patients with initial absence of depression Conclusion: In the absence of depression, standard CHF therapy in middle-aged men with hypertension significantly improves the hemodynamic parameters and the reduced severity of heart failure. Against the background of depression, the effectiveness of CHF treatment (without an antidepressant) is lower: the level of depression, the left ventricular myocardial mass index, and intima-media thickness are much higher, treatment adherence and the ratio of maximum speeds of early and late filling (E/A) are lower than in comparison groups. Combined with paroxetine, standard CHF therapy in middle-aged men with depression, mild depression, and CHF, increases adherence to treatment, improves the structural parameters of the heart. However, adherence to the treatment is meaningful low than in patients who initially lacked depression, which dictates the need to develop effective measures to prevent depressions in patients with the risk of developing CHF

The effect of Bacillus subtilis GM2 and GM5 probiotics on the growth and fodder digestibility of broiler chickens

The probiotic properties of the Bacillus subtilis GM2 and GM5 strains were studied on 90 Cobb 500 broiler chickens aged one day that were randomly divided into three groups: the control group with the standard diet and two experimental groups (30 chickens per group) with the diet supplemented with B. subtilis GM2 (group 1) and GM5 (group 2) spores. The addition of B. subtilis GM2 and GM5 spores at a concentration of 1·107 CFU/g to the ration of broiler chickens improved their growth rate, as well as increased the digestibility of nutrients and modulated the intestinal microflora in them. The use of probiotics stimulated an increase in the live weight of chickens by 6.30% and 13.78% (p = 0.05) as compared with the control group. The average daily weight gain in experimental groups 1 and 2 amounted to 52.82 ± 0.36 g and 56.54 ± 0.47 g per chicken, which is more than in the control group (49.69 ± 0.40 g) – by 6.30% and 13.79% (p = 0.05), respectively. The administration of probiotics by feeding favored an increase in the population of lactic acid bacteria in the small and large (to a lesser extent) intestine – bacteria belonging to the Enterobacteriaceae, Lactobacillaceae, and Clostridiaceae families were isolated from the intestinal contents and identified. The veterinary-sanitary examination found that the meat of broiler chickens from the experimental groups meets all the GOST requirements for organoleptic, physico-chemical, and bacterioscopic characteristics. Thus, probiotics based on the B. subtilis GM2 and GM5 strains have a positive effect on the growth and fodder digestibility of broiler chickens