A novel family VII esterase with industrial potential from compost metagenomic library

<p>Abstract</p> <p>Background</p> <p>Among the vast microbial genomic resources now available, most microbes are unculturable in the laboratory. A culture-independent metagenomic approach is a novel technique that circumvents this culture limitation. For the screening of novel lipolytic enzymes, a metagenomic library was constructed from compost, and the clone of <it>estCS2 </it>was selected for lipolytic properties on a tributyrin-containing medium.</p> <p>Results</p> <p>The <it>estCS2 </it>sequence encodes a protein of 570 amino acid residues, with a predicted molecular mass of 63 kDa, and based on amino acid identity it most closely matches (45%) the carboxylesterase from <it>Haliangium ochraceum </it>DSM 14365. EstCS2 belong to family VII, according to the lipolytic enzyme classification proposed by Arpigny and Jaeger, and it retains the catalytic triad Ser₂₄₅-Glu₃₆₃-His₄₆₆that is typical of an α/β hydrolase. The Ser₂₄₅residue in the catalytic triad of EstCS2 is located in the consensus active site motif GXSXG. The EstCS2 exhibits strong activity toward <it>p</it>-nitrophenyl caproate (C6), and it is stable up to 60°C with an optimal enzymatic activity at 55°C. The maximal activity is observed at pH 9, and it remains active between pH 6-10. EstCS2 shows remarkable stability in up to 50% (v/v) dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). The enzyme has the ability to cleave sterically hindered esters of tertiary alcohol, as well as to degrade polyurethanes, which are widely used in various industries.</p> <p>Conclusions</p> <p>The high stability of EstCS2 in organic solvents and its activity towards esters of ketoprofen and tertiary alcohols, and in polyurethane suggests that it has potential uses for many applications in biotransformation and bioremediation.</p

Insulin Facilitates the Recovery of Myocardial Contractility and Conduction during Cardiac Compression in Rabbits with Bupivacaine-Induced Cardiovascular Collapse

Bupivacaine inhibits cardiac conduction and contractility. Insulin enhances cardiac repolarization and myocardial contractility. We hypothesizes that insulin therapy would be effective in resuscitating bupivacaine-induced cardiac toxicity in rabbits. Twelve rabbits were tracheally intubated and midline sternotomy was performed under general anesthesia. Cardiovascular collapse (CVC) was induced by an IV bolus injection of bupivacaine 10 mg/kg. The rabbits were treated with either saline (control) or insulin injection, administered as a 2 U/kg bolus. Internal cardiac massage was performed until the return of spontaneous circulation (ROSC) and the time to the return of sinus rhythm (ROSR) was also noted in both groups. Arterial blood pressure, and electrocardiography were continuously monitored for 30 min and plasma bupivacaine concentrations at every 5 min. The ROSC, ROSR and normalization of QRS duration were attained faster in the insulin-treated group than in the control group. At the ROSC, there was a significant difference in bupivacaine concentration between two groups. Insulin facilitates the return of myocardial contractility and conduction from bupivacaine-induced CVC in rabbits. However, recovery of cardiac conduction is dependent mainly on the change of plasma bupivacaine concentrations

Transcriptome analysis of skeletal muscle in dermatomyositis, polymyositis, and dysferlinopathy, using a bioinformatics approach

BackgroundPolymyositis (PM) and dermatomyositis (DM) are two distinct subgroups of idiopathic inflammatory myopathies. Dysferlinopathy, caused by a dysferlin gene mutation, usually presents in late adolescence with muscle weakness, degenerative muscle changes are often accompanied by inflammatory infiltrates, often resulting in a misdiagnosis as polymyositis.ObjectiveTo identify differential biological pathways and hub genes related to polymyositis, dermatomyositis and dysferlinopathy using bioinformatics analysis for understanding the pathomechanisms and providing guidance for therapy development.MethodsWe analyzed intramuscular ribonucleic acid (RNA) sequencing data from seven dermatomyositis, eight polymyositis, eight dysferlinopathy and five control subjects. Differentially expressed genes (DEGs) were identified by using DESeq2. Enrichment analyses were performed to understand the functions and enriched pathways of DEGs. A protein–protein interaction (PPI) network was constructed, and clarified the gene cluster using the molecular complex detection tool (MCODE) analysis to identify hub genes.ResultsA total of 1,048, 179 and 3,807 DEGs were detected in DM, PM and dysferlinopathy, respectively. Enrichment analyses revealed that upregulated DEGs were involved in type 1 interferon (IFN1) signaling pathway in DM, antigen processing and presentation of peptide antigen in PM, and cellular response to stimuli in dysferlinopathy. The PPI network and MCODE cluster identified 23 genes related to type 1 interferon signaling pathway in DM, 4 genes (PDIA3, HLA-C, B2M, and TAP1) related to MHC class 1 formation and quality control in PM, and 7 genes (HSPA9, RPTOR, MTOR, LAMTOR1, LAMTOR5, ATP6V0D1, and ATP6V0B) related to cellular response to stress in dysferliniopathy.ConclusionOverexpression of genes related to the IFN1 signaling pathway and major histocompatibility complex (MHC) class I formation was identified in DM and PM, respectively. In dysferlinopathy, overexpression of HSPA9 and the mTORC1 signaling pathway genes was detected

Congenital muscular dystrophy type 1A with residual merosin expression

Congenital muscular dystrophy type 1A (MDC1A) is an autosomal recessive disorder characterized by hypotonia, elevated serum creatine kinase level, delayed motor milestones, white matter changes observed by brain magnetic resonance imaging, and normal intelligence. A mutation in the laminin α2 (LAMA2) gene, located at 6q22-23, is a genetic cause of MDC1A. Patients have merosin (laminin α2)-deficient skeletal muscles. However, the degree of merosin expression ranges from total absence to partial reduction. Patients with residual merosin expression have more variable and milder phenotypes than those with absolute merosin deficiency. We observed a Korean girl with MDC1A with residual merosin expression. Clinical presentation of this patient was typical except for late onset of the disease and external capsule involvement. Immunohistochemical staining of muscle fibers including merosin, is important to evaluate patients with hypotonia, delayed motor development, and abnormal white matter changes

Functional Benefit after Modification of Radial Forearm Free Flap for Soft Palate Reconstruction

ObjectivesTo compare the velopharyngeal function, swallowing and speech of the conventional and modified radial forearm free flap (RFFF) for soft palate reconstruction.MethodsRetrospective clinical study. Twenty-eight patients who underwent oropharyngeal reconstruction with RFFF were divided into two groups: 10 patients had conventional folded RFFF and 18 patients underwent modified method.ResultsThe average speech intelligibility score in modified RFFF group was 8.0±2.4, and 6.2±2.2 in conventional RFFF group (P<0.05). The nasalance was 27.4±7.8% in modified group and 38.6±2.7% in conventional group during no nasal passage reading and 43.6±7.3% in modified group, 55.2±7.6% in conventional group during high nasal passage reading (P<0.05). The subjective swallowing functional score was 2.8 in modified group and 2.1 in conventional group.ConclusionThe speech assessment and nasalance demonstrate a more favorable outcome in modified group than conventional group

EFFECTS OF LIQUID SWIRLING ON GAS-TO-LIQUID MASS TRANSFER IN THREE-PHASE FLUIDIZED BEDS

The swirling flow mode of liquid phase was adopted to promote the gas-to-liquid mass transfer in three-phase(gas-liquid-solid) fluidized beds. Effects of gas(0.01-0.09m/s) and liquid(0.035-0.172m/s) velocities, particle size(1.7-6.0mm) and swirling ratio of liquid phase(0-0.5) on the volumetric gas-to-liquid mass transfer coefficient in the bed were examined. The mass transfer coefficient increased up to 70% by adjusting the swirling flow of liquid phase, especially when the gas velocity is relatively low range. The value of gas-to-liquid mass transfer coefficient was well correlated in terms of dimensionless groups which were derived from the dimensional analysis on the mass transfer system

Anti-metastatic effect of GV1001 on prostate cancer cells; roles of GnRHR-mediated Gαs-cAMP pathway and AR-YAP1 axis

Background Gonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi. However, the precise mechanism underlying the functions of Gαs signal in prostate cancer cells is still unclear. We have previously identified that GV1001, a fragment of the human telomerase reverse transcriptase, functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway. Here, we tried to reveal the potential mechanisms of which GV1001-stimulated Gαs-cAMP signaling pathway reduces the migration and metastasis of prostate cancer (PCa) cells. Methods The expression of epithelial-mesenchymal transition (EMT)-related genes was measured by western-blotting and spheroid formation on ultra-low attachment plate was detected after GV1001 treatment. In vivo Spleen-liver metastasis mouse model was used to explore the inhibitory effect of GV1001 on metastatic ability of PCa and the transwell migration assay was performed to identify whether GV1001 had a suppressive effect on cell migration in vitro. In order to demonstrate the interaction between androgen receptor (AR) and YAP1, co-immunoprecipitation (co-IP), immunofluorescence (IF) staining, chromatin immunoprecipitation (ChIP) were performed in LNCaP cells with and without GV1001 treatment. Results GV1001 inhibited expression of EMT-related genes and spheroid formation. GV1001 also suppressed in vivo spleen-liver metastasis of LNCaP cells as well as cell migration in vitro. GV1001 enhanced the phosphorylation of AR and transcription activity of androgen response element reporter gene through cAMP/protein kinase A pathway. Moreover, GV1001 increased Ser-127 phosphorylation of YAP1 and its ubiquitination, and subsequently decreased the levels of AR-YAP1 binding in the promoter region of the CTGF gene. In contrast, both protein and mRNA levels of NKX3.1 known for tumor suppressor gene and AR-coregulator were upregulated by GV1001 in LNCaP cells. YAP1 knockout using CRISPR/Cas9 significantly suppressed the migration ability of LNCaP cells, and GV1001 did not affect the cell migration of YAP1-deficient LNCaP cells. On the contrary, cell migration was more potentiated in LNCaP cells overexpressing YAP5SA, a constitutively active form of YAP1, which was not changed by GV1001 treatment. Conclusions Overall, this study reveals an essential role of AR-YAP1 in the regulation of PCa cell migration, and provides evidence that GV1001 could be a novel GnRHR ligand to inhibit metastasis of PCa via the Gαs/cAMP pathway.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Kim HS, 2018M3A9C8021792, Kang KW, 2021R1A2C2093196)

Rupture of endotracheal tube cuff during robot-assisted endoscopic thyroidectomy -A case report-

We encountered a case of a rupture of an endotracheal tube cuff during robot-assisted thyroid surgery in a 35-year-old male patient. Two hours after commencing surgery, the bellows of the ventilator were not filled and a rupture of the endotracheal tube cuff was suspected. Once the robot-manipulator is engaged, the position of the operating table cannot be altered without removing it from the patient. Reintubation with direct laryngoscopy was performed with difficulty in the narrow space between the patient's head and robot-manipulator without moving the robot away from the patient. The rupture of the endotracheal tube cuff was confirmed by observing air bubbles exiting from the balloon in water. The patient was discharged 3 days after surgery without complications. In robot-assisted thyroid surgery, a preoperative arrangement of the robot away from the patient's head to obtain easy access to the patient is essential for safe anesthetic care

Flexor Tenorrhaphy Using Absorbable Suture Materials

BackgroundNonabsorbable sutures are favorable for repairing flexor tendons. However, absorbable sutures have performed favorably in an animal model.MethodsTwo-strand sutures using the interlocking modified Kessler method with polydioxanone absorbable sutures 4-0 were used to repair completely ruptured flexor tendons in 55 fingers from 41 consecutive patients. The medical records of average 42 follow up weeks were analyzed retrospectively. The data analyzed using the chi-squared test, and Fisher's exact test was used for postoperative complications. The results were compared with those of other studies.ResultsAmong the index, middle, ring, and little fingers were injured in 9, 17, 16, and 13 fingers, respectively. The injury levels varied from zone 1 to 5. Of the 55 digits in our study, there were 26 (47%) isolated flexor digitorum profundus (FDP) injuries and 29 (53%) combined FDP and with flexor digitorum superficialis injuries. Pulley repair was also conducted. Concomitant injuries of blood vessels and nerves were found in 17 patients (23 fingers); nerve injuries occurred in 5 patients (10 fingers). Two patients had ruptures (3.6%), and one patient had two adhesions (3.6%). Using the original Strickland criteria, all the patients were assessed to be excellent or good. Also, fibrosis and long-term foreign body tissue reactions such as stitch granuloma were less likely occurred in our study. Compared to the Cullen's report that used nonabsorbable sutures, there was no significant difference in the rupture or adhesion rates.ConclusionsTherefore, this study suggests that appropriate absorbable core sutures can be used safely for flexor tendon repairs