A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement

Identification of novel enzymes from lignin degrading microorganisms will help to develop biotechnologies for biomass valorization and aromatic hydrocarbons degradation. Bacillus ligniniphilus L1 grows with alkaline lignin as the single carbon source and is a great candidate for ligninolytic enzyme identification. The first dioxygenase from strain L1 was heterologously expressed, purified, and characterized with an optimal temperature and pH of 32.5 °C and 7.4, respectively. It showed the highest activity with 3-ethylcatechol and significant activities with other substrates in the decreasing order of 3-ethylcatechol > 3-methylcatechol > 3-isopropyl catechol > 2, 3-dihydroxybiphenyl > 4-methylcatechol > catechol. It did not show activities against other tested substrates with similar structures. Most reported catechol 2,3-dioxygenases (C23Os) are Fe2+-dependent whereas Bacillus ligniniphilus catechol 2,3-dioxygenase (BLC23O) is more Mn2+- dependent. At 1 mM, Mn2+ led to 230-fold activity increase and Fe2+ led to 22-fold increase. Sequence comparison and phylogenetic analyses suggested that BL23O is different from other Mn-dependent enzymes and uniquely grouped with an uncharacterized vicinal oxygen chelate (VOC) family protein from Paenibacillus apiaries. Gel filtration analysis showed that BLC23O is a monomer under native condition. This is the first report of a C23O from Bacillus ligniniphilus L1 with unique substrate preference, metal-dependency, and monomeric structure.

In silico evaluation of the interaction between ACE2 and SARS-CoV-2 Spike protein in a hyperglycemic environment

The worse outcome of COVID-19 in people with diabetes mellitus could be related to the non-enzymatic glycation of human ACE2, leading to a more susceptible interaction with virus Spike protein. We aimed to evaluate, through a computational approach, the interaction between human ACE2 receptor and SARS-CoV-2 Spike protein under different conditions of hyperglycemic environment. A computational analysis was performed, based on the X-ray crystallographic structure of the Spike Receptor-Binding Domain (RBD)-ACE2 system. The possible scenarios of lysine aminoacid residues on surface transformed by glycation were considered: (1) on ACE2 receptor; (2) on Spike protein; (3) on both ACE2 receptor and Spike protein. In comparison to the native condition, the number of polar bonds (comprising both hydrogen bonds and salt bridges) in the poses considered are 10, 6, 6, and 4 for the states ACE2/Spike both native, ACE2 native/Spike glycated, ACE2 glycated/Spike native, ACE2/Spike both glycated, respectively. The analysis highlighted also how the number of non-polar contacts (in this case, van der Waals and aromatic interactions) significantly decreases when the lysine aminoacid residues undergo glycation. Following non-enzymatic glycation, the number of interactions between human ACE2 receptor and SARS-CoV-2 Spike protein is decreased in comparison to the unmodified model. The reduced affinity of the Spike protein for ACE2 receptor in case of non-enzymatic glycation may shift the virus to multiple alternative entry routes.

Stability of uniformly labeled (13C and 15N) cytochrome c and its L94G mutant

Cytochrome c (cyt c) is widely used as a model protein to study (i) folding and stability aspects of the protein folding problem and (ii) structure–function relationship from the evolutionary point of view. Databases of cyts c now contain 285 cyt c sequences from different organisms. A sequence alignment of all these proteins with respect to horse cyt c led to several important conclusions. One of them is that Leu94 is always conserved in all 30 mammalian cyts c. It is known that mutation L94G of the wild type (WT) horse cyt c is destabilizing and mutant exists as molten globule under the native condition (buffer pH 6 and 25 °C). We have expressed and purified uniformly labeled (13C and 15N) and unlabeled WT horse cyt c and its L94G mutant. We report that labeling does not affect the thermodynamic stability of proteins. To support this conclusion, the secondary and tertiary structure of each protein in labeled and unlabeled forms was determined by conventional techniques (UV–Vis absorption and circular dichroism spectroscopy).

Correlation between different instrumentation variants and the degree of destabilization in treating cervical spondylotic spinal canal stenosis by unilateral hemilaminectomy with bilateral decompression: a biomechanical investigation

Purpose Unilateral hemilaminectomy with bilateral decompression (BDZ) was proposed as an alternative decompressive procedure in cervical spondylotic myelopathy (CSM). Despite promising clinical results, the destabilizing effect is yet unknown. We therefore performed a biomechanical study to investigate whether lateral mass screw fixation should follow BDZ. Methods Six human C2–C7 cervical specimens were tested under various conditions: native, unilateral hemilaminectomy with bilateral decompression without/with fixation (BDZ/BDF), unilateral hemilaminectomy with bilateral decompression and unilateral foraminotomy without/with fixation (UFZ/UFF), unilateral hemilaminectomy with bilateral decompression and bilateral foraminotomy without/with fixation (BFZ/BFF), and laminectomy without/with fixation (LAZ/LAF). Instrumention was applied from C3–C6. For each condition, the three-dimensional kinematics of the cervical specimen were measured in three main loading directions with an ultrasonic motion analysis system. ANOVA was used to determine differences between the specific segment conditions to assess the parameter’s range of motion (ROM) and neutral zone (NZ). Results For flexion–extension, lateral bending and axial rotation, ROM of BDZ, UFZ, BFZ and LAZ remained at the level of the native condition (p > 0.74), whereas fixation reduced ROM significantly (p < 0.01). Between BDF, UFF, BFF and LAF, no significant differences in reduction in ROM were seen (p > 0.49). Results for NZ were equivalent to ROM in flexion–extension and lateral bending. For axial rotation, NZ remained almost constant on the native level for all tested conditions. Conclusion Bilateral decompression via a hemilaminectomy, even if combined with foraminotomy, could be a less invasive treatment option for multilevel CSM in patients with lordotic cervical alignment and absence of segmental instability.

Differential Impact of School Segregation in the Performance of Native and Non-Native Students in Spain

There is evidence of the impact of school segregation on students’ academic achievement, but it is debated whether the extent of this impact is dependent on students’ socioeconomic status, or on their native or non-native condition. This research addresses the problem in Spain, seeking to determine how immigrant and socioeconomic segregation affect the academic achievement of native and non-native students. With this aim, the PISA study database was specially exploited by means of two-tier Multilevel Models, estimating school segregation through the Hutchens Square Root Index. Specifically, the study estimates the influence of school segregation on students’ academic achievement in the subjects of Mathematics, Language and Science. The results confirm that school socioeconomical and immigrant segregation affect students’ academic achievement differently. Whereas socioeconomic segregation negatively affects both groups in all three subjects, immigrant segregation affects non-native students more strongly. Thus, data shows school segregation on socioeconomic grounds is always significant, and always has a considerable impact on achievement, regardless of students’ national origin. School segregation reproduces and accentuates conditions of social injustice. To counter its harmful effects, it is necessary to act first and foremost on socioeconomic segregation, as this causes the most devastating effects in education, particularly for non-native students.

Comment on “Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry”

Chorev et al. (Reports, 16 November 2018, p. 829) describe mass spectrometry on mitochondrial membrane proteins ionized directly from their native environment. However, the assignments made to measured masses are incorrect or inconclusive and lack experimental validation. The proteins are not in their “native” condition: They have been stripped of tightly bound lipids, and the complexes are fragmented or in physiologically irrelevant oligomeric states.

Evaluation of polysaccharide intercellular adhesion (PIA) and glycerol teichoic acid (Gly-TA) arisen antibodies to prevention of biofilm formation in Staphylococcus aureus and Staphylococcus epidermidis strains

Objective Staphylococcus aureus and S. epidermidis as opportunistic pathogens, notable for their frequency and severity of infections are recognized as the most usual reasons for medical device-associated infections that strike hospitalized patients and also immunocompromised individuals. In this study, the polysaccharide intercellular adhesion (PIA) and Glycerol teichoic acid) Gly-TA) as two major macromolecules in the biofilm formation process were purified under the native condition and their structure was analyzed by using colorimetric assays and Fourier Transform Infrared spectroscopy (FTIR). Afterward, the immune response of macromolecules and the mixture of them were assessed by measuring total IgG titers. Subsequently, biofilm inhibitory effects of raising antibodies to biofilm former S. aureus and S. epidermidis were evaluated. Results Obtained data were shown a significant rise in levels of antibodies in immunized mice with mentioned antibodies in comparison with the control group. According to the obtained findings, mentioned antibodies could eliminate S. aureus and S. epidermidis biofilm formation in vitro assays. This survey confirms the proposal that immunization of mice with a mixture of Gly-TA and PIA vaccine could be secure and protected against S. epidermidis and S. aureus infection.

Comparative analysis of PIA and rSesC mixture, arisen antibodies against the biofilm forming Staphylococcus aureus.

Background: Staphylococcus aureus as a causative agent of hospital-acquired infections, has been considered as the primary concern in biomaterial-related infections (BAIs). Following the purification of polysaccharide intercellular adhesion (PIA) as an efficient macromolecule in biofilm formation in the native condition, recombinant S . epidermidis surface exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed purified PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biofilm forming activity of arisen antibodies to a biofilm forming S. aureus strains was assessed by semi-quantitative micro-plate procedure. Results: Data showed that the total IgGs was boosted in mice immunized sera. By performing inhibition assay, biofilm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture significantly were more potent than PIA and rSesC, when comparing them in a biofilm inhibition assay. Conclusion: Immunization of mice with mentioned antigens especially a mixture of them, could eliminate the biofilm formation process in S. aureus . Hopefully, this study corresponds the suggestion that, the immunization of mice with PIA and rSesC candidate vaccine could protect against S. aureus infection.

Comparative analysis of PIA and rSesC mixture, as vaccine candidate against the biofilm forming Staphylococcus aureus.

Background: Staphylococcus aureus as a causative agent of hospital-acquired infections, has been considered as the primary concern in biomaterial-related infections (BAIs). Methods: Following the purification of polysaccharide intercellular adhesion (PIA) as an efficient macromolecule in biofilm formation in the native condition, recombinant S. epidermidis surface exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed purified PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biofilm forming activity of arisen antibodies to a biofilm forming S. aureus strains was assessed by semi-quantitative micro-plate procedure. Results: Data showed that the total IgGs was boosted in mice immunized sera. By performing inhibition assay, biofilm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture significantly were more potent than PIA and rSesC, when comparing them in a biofilm inhibition assay.