Structural and biophysical studies of new L-asparaginase variants : lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amido­hydrolase

This work reports the results of random mutagenesis of the Escherichia coli class 2 l-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the l-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for l-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes

Mechanical thrombectomy in acute stroke – Five years of experience in Poland

Objectives Mechanical thrombectomy (MT) is not reimbursed by the Polish public health system. We present a description of 5 years of experience with MT in acute stroke in Comprehensive Stroke Centers (CSCs) in Poland. Methods and results We retrospectively analyzed the results of a structured questionnaire from 23 out of 25 identified CSCs and 22 data sets that include 61 clinical, radiological and outcome measures. Results Most of the CSCs (74%) were founded at University Hospitals and most (65.2%) work round the clock. In 78.3% of them, the working teams are composed of neurologists and neuro-radiologists. All CSCs perform CT and angio-CT before MT. In total 586 patients were subjected to MT and data from 531 of them were analyzed. Mean time laps from stroke onset to groin puncture was 250±99min. 90.3% of the studied patients had MT within 6h from stroke onset; 59.3% of them were treated with IV rt-PA prior to MT; 15.1% had IA rt-PA during MT and 4.7% – emergent stenting of a large vessel. M1 of MCA was occluded in 47.8% of cases. The Solitaire device was used in 53% of cases. Successful recanalization (TICI2b–TICI3) was achieved in 64.6% of cases and 53.4% of patients did not experience hemorrhagic transformation. Clinical improvement on discharge was noticed in 53.7% of cases, futile recanalization – in 30.7%, mRS of 0–2 – in 31.4% and mRS of 6 in 22% of cases. Conclusion Our results can help harmonize standards for MT in Poland according to international guidelines

The use of synthetic materials in the treatment of stress urinary incontinence

Stress urinary incontinence is a very serious problem which has been noticed by the WHO. This difficult medical condition poses a serious problem as it affects c.a. 20% of the female population and it increases up to 35% in the group of females over 60 years of age. Since there is no single standard surgical procedure which could solve this problem, numerous synthetic materials are used for the operations. It seems that the materials are effective as they improve the condition of women suffering from stress urinary incontinence. Unfortunately these materials have their shortcomings which might lead to certain post-operative complications. Stress urinary incontinence is a disease which affects the social life of the patients. It has a high percentage of recurrence and causes the patient substantial difficulties with keeping high standards of personal hygiene which is consequence makes it impossible to fulfill their social roles. The etiology of this disease is complex and calls for a cross-disciplinary approach to the problem. As there are no standardized or unanimous treatment methods of stress urinary incontinence, numerous sources based on the clinical experience of many medical centers suggest performing TVT and TOT procedures as the most effective treatment methods. The efficacy of the TOT procedure is about 90.8%

Structural and biophysical studies of new L -asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

This work reports the results of random mutagenesis of the Escherichia coli class 2 L-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the L-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for L-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes

Mechanical thrombectomy in acute stroke : five years of experience in Poland

Objectives: Mechanical thrombectomy (MT) is not reimbursed by the Polish public health system. We present a description of 5 years of experience with MT in acute stroke in Comprehensive Stroke Centers (CSCs) in Poland. Methods and results: We retrospectively analyzed the results of a structured questionnaire from 23 out of 25 identified CSCs and 22 data sets that include 61 clinical, radiological and outcome measures. Results: Most of the CSCs (74%) were founded at University Hospitals and most (65.2%) work round the clock. In 78.3% of them, the working teams are composed of neurologists and neuro-radiologists. All CSCs perform CT and angio-CT before MT. In total 586 patients were subjected to MT and data from 531 of them were analyzed. Mean time laps from stroke onset to groin puncture was 250 99 min. 90.3% of the studied patients had MT within 6 h from stroke onset; 59.3% of them were treated with IV rt-PA prior to MT; 15.1% had IA rt-PA during MT and 4.7% - emergent stenting of a large vessel. M1 of MCA was occluded in 47.8% of cases. The Solitaire device was used in 53% of cases. Successful recanalization (TICI2b–TICI3) was achieved in 64.6% of cases and 53.4% of patients did not experience hemorrhagic transformation. Clinical improvement on discharge was noticed in 53.7% of cases, futile recanalization - in 30.7%, mRS of 0–2 - in 31.4% and mRS of 6 in 22% of cases. Conclusion: Our results can help harmonize standards for MT in Poland according to international guideline