A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_(cat)/K_m = 1000 ± 100 M^(−1) s^(−1) versus 700 ± 100 M^(−1) s^(−1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_(cat)/K_m = 80 ± 40 M^(−1) s^(−1)). In the presence of 3-HSA the K_(mapp) for O_2 was 100 ± 10 μM. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val^(367)–Val^(394)) could adopt two conformations differing by a rigid body rotation of 25° around Arg^(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids

An Example of Two-Dimensional Interpolation Using a Linear Combination of Bicubic B-Splines

The paper describes how a linear combination of bicubic B-splines can be effectively used in a two-dimensional interpolation. It is assumed that values of a function to be interpolated are evaluated at the uniformly located nodes of a corresponding rectangular grid. All formulae of importance have been derived step by step and are presented in a form convenient for computer implementations. To ensure clarity of considerations a short description of one-dimensional B-spline is also given in Appendix 1. The usefulness of the presented interpolation algorithm has been confirmed by the real engineering example of applications

Upper limb therapy in children with cerebral palsy (CP) – The Pirate Group

Introduction: Children with cerebral palsy (CP) in the form of spastic hemiplegia experience numerous difficulties concerning an affected upper limb such as reaching for objects, gripping or manipulating them. These limitations affect their everyday activity. Conducting an effective and simultaneously an interesting therapy aimed at meeting the child’s individual needs and improving upper limb function is a challenge for a physiotherapist. The aim of the study was to assess the effectiveness of upper limb therapy carried out within the project titled “The Pirate Group” based on Constraint-Induced Movement Therapy (CIMT) and Bimanual Training (BIT) conducted in a specially arranged environment. Material and methods: The research included 16 children with CP in the form of spastic hemiplegia. Mean age of the study participants was 4.23 years. The children underwent a two-week Constraint-Induced Movement Therapy (CIMT) combined with Bimanual Training (BIT). In order to evaluate the effects of the therapy, each child underwent the Assisting Hand Assessment (AHA) prior to the therapy and after its completion. Results: Statistical analysis revealed a significant difference (p<0.05) between the results of AHA prior to and after the therapy (t(14)=9.12, p<0.0001). An improvement in the affected upper limb function was noted in all the children participating in the research. Conclusions: The project titled “The Pirate Group”, based on CIMT and BIT is an effective therapeutic intervention which improves spontaneous activity of the affected upper limb in children with hemiplegia

The Reliability of Pelvic Floor Muscle Bioelectrical Activity (sEMG) Assessment Using a Multi-Activity Measurement Protocol in Young Women

The aim of the study was to determine the between-trial and between-day reliability of the Glazer protocol and our multi-activity surface electromyography (sEMG) measurement protocol for pelvic floor muscle (PFM) evaluation. The bioelectrical activity of PFM was collected using an endovaginal electrode in 30 young, Caucasian, nulliparous women (age 22&ndash;27, 168.6 &plusmn; 5.1 cm, 57.1 &plusmn; 11.8 kg). The between-trial and between-day reliability of the original Glazer protocol and the new multi-activity sEMG protocol were assessed during the following phases: pre-baseline rest, phasic (flick) contractions, tonic contractions, endurance contraction, and post-baseline rest. The Glazer protocol was characterized by poor and moderate measurement reliability. The time-domain parameters for the rise and fall of the signal amplitude and median frequency showed poor between-trial and between-day reliability. The mean and peak amplitudes indicated mainly good between-trial and moderate between-days reliability. Our protocol showed moderate to excellent reliability of both time-domain and quantitative parameters of muscle recruitment. In our protocol, the frequency-domain parameters describing muscle fatigue demonstrated much higher reliability than in the case of the Glazer protocol. The most important information obtained in this study was the significant improvement of diagnostic validity in PFM bioelectrical activity evaluation. The higher reliability of our sEMG protocol compared to original Glazer protocol allowed us to suggest that protocol modifications and changes in sEMG signal processing methods were effective in the improvement of PFM assessment quality. The new parameters calculated from the sEMG signal proposed in our sEMG protocol allowed us to obtain additional clinically important information about PFM dysfunctions regarding specific deficits of muscle contraction such as decrease in muscle strength; endurance or coordination related to, e.g., stress urinary incontinence; or pelvic floor muscle imbalance after childbirth