Cytochrome P450: taming a wild type enzyme

Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly ‘evolvable’ – they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability

Deep Seawater flow Characteristics Around the Manganese Nodule Collecting Device

AbstractFlow field characteristics with outflow discharge from a collecting device in deep seawater while gathering manganese nodules have been analyzed by CFD. Numerical model is used for the analysis with CFD program of FLUENT. It is assumed that the collecting device is 4.5×5.4×6.7m with outflow speed = 1.75 m/s and the current speed = 0.1m/s.Overall seawater flow field characteristics are largely influenced by the outflow discharge from the collecting device and manganese nodule particle behavior. The outflow discharge effect reaches to about few times of the collecting device in back. As simulation results, flow velocity and streamline distributions are compared including turbulence kinetic energyvariation. This study will be useful for optimal design for manganese nodule collecting device system in deep sea

Differential Genomic Imprinting and Expression of Imprinted microRNAs in Testes-Derived Male Germ-Line Stem Cells in Mouse

BACKGROUND: Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings. METHODS AND FINDINGS: Using mouse as a model system, here we show that, similar to sperm, expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher (P<0.001), while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower (P<0.001) in GS cells than in control embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA. CONCLUSIONS: Our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells

Surgical management of pilon fractures with large segmental bone defects using fibular strut allografts: a report of two cases

We present two patients with open pilon fractures with large bone defects treated successfully with fibular strut allografts. The patients were initially treated by massive irrigation, wound debridement, and temporary external fixation. After complete wound healing, the bone defects were managed. Because autologous iliac crest or fibular bone grafts were impossible to be harvested due to multiple fractures, the bone defects were reconstructed with fibular strut allografts. Fixation was performed with a periarticular distal tibia locking plate. At 2 months postoperatively, the patients ambulated with partial weight-bearing; at 6 months, they had full range of motion of the ankle joint and full weight-bearing

Enhanced cardiac expression of two isoforms of matrix metalloproteinase-2 in experimental diabetes mellitus.

BackgroundDiabetic cardiomyopathy (DM CMP) is defined as cardiomyocyte damage and ventricular dysfunction directly associated with diabetes independent of concomitant coronary artery disease or hypertension. Matrix metalloproteinases (MMPs), especially MMP-2, have been reported to underlie the pathogenesis of DM CMP by increasing extracellular collagen content.PurposeWe hypothesized that two discrete MMP-2 isoforms (full length MMP-2, FL-MMP-2; N-terminal truncated MMP-2, NTT-MMP-2) are induced by high glucose stimulation in vitro and in an experimental diabetic heart model.MethodsRat cardiomyoblasts (H9C2 cells) were examined to determine whether high glucose can induce the expression of the two isoforms of MMP-2. For the in vivo study, we used the streptozotocin-induced DM mouse heart model and age-matched controls. The changes of each MMP-2 isoform expression in the diabetic mice hearts were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical stains were conducted to identify the location and patterns of MMP-2 isoform expression. Echocardiography was performed to compare and analyze the changes in cardiac function induced by diabetes.ResultsQuantitative RT-PCR and immunofluorescence staining showed that the two MMP-2 isoforms were strongly induced by high glucose stimulation in H9C2 cells. Although no definite histologic features of diabetic cardiomyopathy were observed in diabetic mice hearts, left ventricular systolic dysfunction was determined by echocardiography. Quantitative RT-PCR and IHC staining showed this abnormal cardiac function was accompanied with the increases in the mRNA levels of the two isoforms of MMP-2 and related to intracellular localization.ConclusionTwo isoforms of MMP-2 were induced by high glucose stimulation in vitro and in a Type 1 DM mouse heart model. Further study is required to examine the role of these isoforms in DM CMP

The relationships between clinical variables and renal parenchymal disease in pediatric clinically suspected urinary tract infection

Purpose : To evaluate the significance of clinical signs and laboratory findings as predictors of renal parenchymal lesions and vesicoureteral reflux (VUR) in childhood urinary tract infection (UTI). Methods : From July 2005 to July 2008, 180 patients admitted with a first febrile UTI at the Pediatric Department of Konkuk University Hospital were included in this study. The following were the clinical variables: leukocytosis, elevated C-reactive protein (CRP), positive urine nitrite, positive urine culture, and fever duration both before and after treatment. We evaluated the relationships between clinical variables and dimercaptosuccinic acid (DMSA) scan and voiding cystourethrography (VCUG) results. Results : VCUG was performed in 148 patients&#59; of them, 37 (25.0&#37;) had VUR: 18 (12.2&#37;) had low-grade (I-II) VUR, and 19 (10.5&#37;) had high-grade (III-V) VUR. Of the 95 patients who underwent DMSA scanning, 29 (30.5&#37;) had cortical defects, of which 21 (63.6&#37;) had VUR: 10 (30.3&#37;), low-grade (I-II) VUR&#59; and 11 (33.3&#37;), high-grade VUR. Of the 57 patients who were normal on DMSA scan, 8 (14.0&#37;) had low-grade VUR and 6 (10.5&#37;) had high-grade VUR. The sensitivity, specificity, and positive and negative predictive values of the DMSA scan in predicting high-grade VUR were 64.7&#37;, 69.9&#37;, 33.3&#37;, and 89.5&#37;, respectively. Leukocytosis, elevated CRP, and prolonged fever (?#243;6 hours) after treatment were significantly correlated with the cortical defects on DMSA scans and high-grade VUR. Conclusion : Clinical signs, including prolonged fever after treatment, elevated CRP, and leukocytosis, are positive predictors of acute pyelonephritis and high-grade VUR

Endometrial Stromal Sarcoma Presenting as Prevesical Mass Mimicking Urachal Tumor

Endometrial stromal sarcoma (ESS) is a mesenchymal neoplasm that usually occurs as a primary tumor of the uterine corpus, but rarely arises in other sites, such as the ovary, pelvic cavity, mesentery, omentum and intestine. Herein, we present a rare case of low-grade ESS presented as prevesical mass. A 60-yr-old woman who had undergone total hysterectomy for endometriosis eleven years ago was presented with incidentally detected prevesical pelvic mass. Since malignant transformation of urachal remnants was possible, the mass was suspected to be a urachal tumor. Extraction of the mass was performed, and the histopathologic diagnosis was low-grade ESS. In summary, prevesical tumor is rare but in patients with endometriosis, we suggest endometriosis and its possible malignant changes should be taken into account in the differential diagnosis of prevesical mass

Structure-Guided Directed Evolution of Highly Selective P450-Based Magnetic Resonance Imaging Sensors for Dopamine and Serotonin

New tools that allow dynamic visualization of molecular neural events are important for studying the basis of brain activity and disease. Sensors that permit ligand-sensitive magnetic resonance imaging (MRI) are useful reagents due to the noninvasive nature and good temporal and spatial resolution of MR methods. Paramagnetic metalloproteins can be effective MRI sensors due to the selectivity imparted by the protein active site and the ability to tune protein properties using techniques such as directed evolution. Here, we show that structure-guided directed evolution of the active site of the cytochrome P450‐BM3 heme domain produces highly selective MRI probes with submicromolar affinities for small molecules. We report a new, high‐affinity dopamine sensor as well as the first MRI reporter for serotonin, with which we demonstrate quantification of neurotransmitter release in vitro. We also present a detailed structural analysis of evolved cytochrome P450‐BM3 heme domain lineages to systematically dissect the molecular basis of neurotransmitter binding affinity, selectivity, and enhanced MRI contrast activity in these engineered proteins

A Clinical Pilot Study Showing the Safety and Efficacy of Intramuscular Injection of Atelocollagen for Prevention of Paraspinal Muscle Atrophy after Spine Surgery

Objective The objective of this study was to assess the safety and efficacy of intramuscular injection of atelocollagen for the prevention of paraspinal muscle atrophy after spine surgery. Atelcollagen has been widely used as an intradermal filler to restore soft tissue defect. Many studies demonstrated that atelocollagen provides good therapeutic results by promoting cell proliferation and enhances the healing effect on injured connective tissues such as tendons and fasciae, while causing few complications. Methods A total of 118 patients who underwent single level of posterior lumbar interbody fusion (PILF) between December 2017 and April 2019 were retrospectively reviewed. In the study group of 60 patients, 3 mL of gel-type 3% atelocollagen solution was prepared and injected into the multifidus muscle during wound closure. Clinical efficacy was evaluated by the improvement of back pain, elevation of a muscle enzyme, and inflammatory markers. Radiologic efficacy was evaluated with a comparison of density and cross-sectional area (CSA) of multifidus and erector spinae muscle in CT images. Results Visual analogue scale (VAS) scores for back pain was not significantly lower in the study group postoperatively compared with the control group. The reduction of postoperative paraspinal muscle density and CSA was significantly lower in the study group. The serum level of muscle enzyme and inflammatory markers were significantly lower in the study group. No major procedure-related complications were observed during the follow-up period. Conclusion Intramuscular injection of atelocollagen is safe and feasible for the prevention of paraspinal muscle atrophy after spine surgery. This novel method seems advantageous for accelerating wound healing without causing inflammation