Prognostic Value of Electroneuronography in Severe Cases of Facial Palsy.

OBJECTIVE: To examine the prognostic value of electroneuronography (ENoG) in predicting functional recovery in severe cases of acute facial palsy. METHODS: Patients with severe degrees of facial palsy (initial House-Brackmann [HB] grades IV to VI) with available electrodiagnostic studies conducted 2-4 weeks after symptom onset were reviewed retrospectively. The patients were categorized into "good recovery" and "poor recovery" groups, with the former showing mild to no dysfunction (HB I to III) and the latter exhibiting moderate to severe dysfunction (HB IV to VI) on follow-up evaluation, 2 months after onset. ENoG amplitudes in four facial muscles (frontalis, nasalis, orbicularis oculi, and orbicularis oris), as well as age, sex, affected side, disease etiology, comorbidities, and laboratory findings, were compared between the two groups. RESULTS: Thirty-seven patients were included. Twenty-nine of the patients showed "good recovery," and eight showed "poor recovery" at 2 months after symptom onset. Univariate analysis yielded no significant difference in age, sex, affected side, disease etiology, comorbidities, and laboratory findings between the two groups. Preserved ENoG amplitudes (individual, average, and trimmed means) were significantly higher in the good recovery group than in the poor recovery group (p<0.005). Sex (p=0.038) and the ENoG of the nasalis muscle, acquired 2-4 weeks from symptom onset (p=0.004), showed significant differences in multivariate regression analysis. CONCLUSION: This study suggests that the female sex and lower ENoG of the nasalis muscle, acquired 2-4 weeks from symptom onset, have negative prognostic value for the 2-month functional outcome of severe facial palsy cases.

Effect of Intrathecal Baclofen Pump on Scoliosis in Children With Cerebral Palsy: A Meta-Analysis.

OBJECTIVE: To systematically review the effect of intrathecal baclofen pump insertion in children with cerebral palsy (CP) with respect to scoliosis. METHODS: A systematic literature search was conducted in PubMed, Embase, Cochrane Library, and Google Scholar databases up to June 2022. The inclusion criteria were as follows: (1) studies with a quantitative study design; (2) studies with a study group of children with CP; (3) studies comparing scoliosis in children with and without an intrathecal baclofen pump; and (4) studies with Cobb's angle as a parameter. RESULTS: Of the 183 studies found, four studies, all of which were retrospective comparative studies, met the aforementioned inclusion criteria. All studies were homogeneous (I2=0%, p=0.53) and intrathecal baclofen pump insertion accelerated the progression of scoliosis (standard mean difference=0.27; 95% confidence interval=0.07-0.48). CONCLUSION: Intrathecal baclofen pumps have been used to alleviate spasticity in children with CP, thus aiding their daily activities and movements. However, their advantages and disadvantages should be reviewed after sufficient time considering the pumps' negative effect on the course of scoliosis.

Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system.

BACKGROUND: Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-sized gene clusters for heterologous as well as homologous expression. RESULTS: A versatile Escherichia coli-Streptomyces shuttle bacterial artificial chromosomal (BAC) conjugation vector, pSBAC, was used along with a cluster tandem integration approach to carry out homologous and heterologous overexpression of a large 80-kb polyketide biosynthetic pathway gene cluster of tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T cell-specific immunosuppressant. Unique XbaI restriction sites were precisely inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing Streptomyces sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then rescued as a single giant recombinant pSBAC by XbaI digestion of the chromosomal DNA as well as subsequent self-ligation. Next, the recombinant pSBAC construct containing the entire TMC cluster in E. coli was directly conjugated into model Streptomyces strains, resulting in rapid and enhanced TMC production. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type Streptomyces sp. CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 14-fold and 5.4-fold enhanced TMC productivities, respectively. CONCLUSIONS: The 80-kb TMC biosynthetic gene cluster was isolated in a single integration vector, pSBAC. Introduction of TMC biosynthetic gene cluster in TMC non-producing strains has resulted in similar amount of TMC production yield. Moreover, over-expression of TMC biosynthetic gene cluster in original producing strain and recombinant S. coelicolor dramatically increased TMC production. Thus, this strategy can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes.

Domain Characterization of Cyclosporin Regio-Specific Hydroxylases in Rare Actinomycetes.

Cytochrome P450 hydroxylase (CYP) in actinomycetes plays an important role in the biosynthesis and bioconversion of various secondary metabolites. Two unique CYPs named CYP-sb21 and CYP-pa1, which were identified from Sebekia benihana and Pseudonocardia autotrophica, respectively, were proven to transfer a hydroxyl group at the 4(th) or 9(th) N-methyl leucine position of immunosuppressive agent cyclosporin A (CsA). Interestingly, these two homologous CYPs showed different CsA regio-selectivities. CYP-sb21 exhibited preferential hydroxylation activity at the 4(th) position over the 9(th) position, whereas CYP-pa1 showed the opposite preference. To narrow down the CYP domain critical for CsA regio-selectivity, each CYP was divided into four domains, and each domain was swapped with its counterpart from the other CYP. A total of 18 hybrid CYPs were then individually tested for CsA regioselectivity. Although most of the hybrid CYPs failed to exhibit a significant change in regioselectivity in the context of CsA hydroxylation, hybrid CYP-pa1 swapped with the second domain of CYP-sb21 showed a higher preference for the 9th position. Moreover, hybrid CYPsb21 containing seven amino acids from the 2nd domain of CYP-pa1 showed higher preference for the 4(th) position. These results imply that the 2nd domain of CsA-specific CYP plays a critical role in CsA regio-selectivity, thereby setting the stage for biotechnological application of CsA regio-selective hydroxylation.

A novel regio­specific cyclosporin hydroxylase gene revealed through the genome mining of Pseudonocardia autotrophica.

The regio-specific hydroxylation at the 4th N-methyl leucine of the immunosuppressive agent cyclosporin A (CsA) was previously proposed to be mediated by a unique cytochrome P450 hydroxylase (CYP), CYP-sb21 from the rare actinomycetes Sebekia benihana. Interestingly, a different rare actinomycetes species, Pseudonocardia autotrophica, was found to possess a different regio-selectivity, the preferential hydroxylation at the 9th N-methyl leucine of CsA. Through an in silico analysis of the whole genome of P. autotrophica, we describe here the classification of 31 total CYPs in P. autotrophica. Three putative CsA CYP genes, showing the highest sequence homologies with CYPsb21, were successfully inactivated using PCR-targeted gene disruption. Only one knock-out mutant, ΔCYP-pa1, failed to convert CsA to its hydroxylated forms. The hydroxylation activity of CsA by CYP-pa1 was confirmed by CYP-pa1 gene complementation as well as heterologous expression in the CsA non-hydroxylating Streptomyces coelicolor. Moreover, the cyclosporine regio-selectivity of CYP-pa1 expressed in the ΔCYP-sb21 S. benihana mutant strain was also confirmed unchanged through cross complementation. These results show that preferential regio-specific hydroxylation at the 9th N-methyl leucine of CsA is carried out by a specific P450 hydroxylase gene in P. autotrophica, CYP-pa1, setting the stage for the biotechnological application of CsA regioselective hydroxylation.