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.

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.

Influence of preparedness on caregiver burden, depression, and quality of life in caregivers of people with disabilities.

Introduction: Caregiver preparedness is defined as the perceived preparation of caregivers to care for the physical and emotional needs of the patient. Purpose: This study investigated caregiver preparedness and its influences on caregiver burden, depression, and quality of life (QoL) in caregivers of individuals with disabilities. Methods: We conducted a multicenter cross-sectional survey study on caregivers caring for patients with disabilities. Sociodemographic characteristics were collected via questionnaires. The Preparedness for Caregiving Scale (PCS), Burden Interview (BI), Center for Epidemiologic Studies Depression Scale (CES-D), and EuroQol-Visual Analogue Scale (EQ-VAS) were administered. Results: A total of 151 caregivers were enrolled. The mean age of caregivers was 53.7 ± 12.4 years, and 80.8% were female. The majority of participants were the main caregivers of patients with stroke, spinal cord injury, or traumatic brain injury. The mean PCS score was 2.1 ± 0.9, demonstrating significant relationships with BI (r = -0.512, p < 0.001), CES-D (r = -0.622, p < 0.001), and EQ-VAS (r = 0.441, p < 0.001). The CES-D was significantly associated with the PCS after controlling other variables. However, PCS did not show any correlation with the duration of caregiving or amount of time spent per day on caregiving. Discussion: The clinical implications of this study are that higher caregiver preparedness is a predictor of less caregiver burden and depression, and better QoL. However, preparedness did not increase as the duration or time spent on caregiving was extended. Therefore, efforts to enhance the caregivers' preparedness are required to reduce caregiver burden and improve health outcomes for both caregivers and patients.

Three-Dimensional Postoperative Results Prediction for Orthognathic Surgery through Deep Learning-Based Alignment Network.

To date, for the diagnosis of dentofacial dysmorphosis, we have relied almost entirely on reference points, planes, and angles. This is time consuming, and it is also greatly influenced by the skill level of the practitioner. To solve this problem, we wanted to know if deep neural networks could predict postoperative results of orthognathic surgery without relying on reference points, planes, and angles. We use three-dimensional point cloud data of the skull of 269 patients. The proposed method has two main stages for prediction. In step 1, the skull is divided into six parts through the segmentation network. In step 2, three-dimensional transformation parameters are predicted through the alignment network. The ground truth values of transformation parameters are calculated through the iterative closest points (ICP), which align the preoperative part of skull to the corresponding postoperative part of skull. We compare pointnet, pointnet++ and pointconv for the feature extractor of the alignment network. Moreover, we design a new loss function, which considers the distance error of transformed points for a better accuracy. The accuracy, mean intersection over union (mIoU), and dice coefficient (DC) of the first segmentation network, which divides the upper and lower part of skull, are 0.9998, 0.9994, and 0.9998, respectively. For the second segmentation network, which divides the lower part of skull into 5 parts, they were 0.9949, 0.9900, 0.9949, respectively. The mean absolute error of transverse, anterior-posterior, and vertical distance of part 2 (maxilla) are 0.765 mm, 1.455 mm, and 1.392 mm, respectively. For part 3 (mandible), they were 1.069 mm, 1.831 mm, and 1.375 mm, respectively, and for part 4 (chin), they were 1.913 mm, 2.340 mm, and 1.257 mm, respectively. From this study, postoperative results can now be easily predicted by simply entering the point cloud data of computed tomography.

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.