Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in <it>E. coli </it>frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant <it>E. coli</it>. Here, we describe the successful use of the immobilized folding machineries for <it>in vitro </it>refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO).</p> <p>Results</p> <p>We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL) and two foldases (DsbA and human peptidyl-prolyl <it>cis-trans </it>isomerase) by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively.</p> <p>Conclusion</p> <p>The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of <it>E. coli </it>inclusion bodies in high yield with biological function.</p

Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

<p>Abstract</p> <p>Background</p> <p>Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. <it>Candida magnoliae </it>JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from <it>C. magnoliae </it>JH110.</p> <p>Results</p> <p>The gene encoding a novel ER was isolated from an osmophilic yeast <it>C. magnoliae </it>JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from <it>Trichosporonoides megachiliensis </it>SNG-42. The intact coding region of ER from <it>C. magnoliae </it>JH110 was cloned, functionally expressed in <it>Escherichia coli </it>using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the <it>C. magnoliae </it>JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the <it>C. magnoliae </it>JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.</p> <p>Conclusions</p> <p>A novel ER enzyme and its corresponding gene were isolated from <it>C. magnoliae </it>JH110. The <it>C. magnoliae </it>JH110 ER with high activity and catalytic efficiency would be very useful for <it>in vitro </it>erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.</p

Effects of exercise on obesity-induced mitochondrial dysfunction in skeletal muscle

Obesity is known to induce inhibition of glucose uptake, reduction of lipid metabolism, and progressive loss of skeletal muscle function, which are all as- sociated with mitochondrial dysfunction in skeletal muscle. Mitochondria are dy- namic organelles that regulate cellular metabolism and bioenergetics, including ATP production via oxidative phosphorylation. Due to these critical roles of mitochon- dria, mitochondrial dysfunction results in various diseases such as obesity and type 2 diabetes. Obesity is associated with impairment of mitochondrial function (e.g., decrease in O2 respiration and increase in oxidative stress) in skeletal muscle. The bal- ance between mitochondrial fusion and fission is critical to maintain mitochondrial homeostasis in skeletal muscle. Obesity impairs mitochondrial dynamics, leading to an unbalance between fusion and fission by favorably shifting fission or reducing fusion proteins. Mitophagy is the catabolic process of damaged or unnecessary mito- chondria. Obesity reduces mitochondrial biogenesis in skeletal muscle and increases accumulation of dysfunctional cellular organelles, suggesting that mitophagy does not work properly in obesity. Mitochondrial dysfunction and oxidative stress are reported to trigger apoptosis, and mitochondrial apoptosis is induced by obesity in skeletal muscle. It is well known that exercise is the most effective intervention to protect against obesity. Although the cellular and molecular mechanisms by which exercise protects against obesity-induced mitochondrial dysfunction in skeletal mus- cle are not clearly elucidated, exercise training attenuates mitochondrial dysfunction, allows mitochondria to maintain the balance between mitochondrial dynamics and mitophagy, and reduces apoptotic signaling in obese skeletal muscle

Outcome prediction of pediatric drowning

Purpose Despite the well-known mortality of pediatric drowning, there is a paucity of evidence on the implications of an initial evaluation on the relevant outcome of drowning. This study aimed to investigate the association of initial clinical findings with outcome of children undergoing drowning. Methods This retrospective study was conducted using the medical records of 56 children undergoing drowning who visited 3 Korean academic hospitals from January 2000 through May 2020. We analyzed information regarding the prehospital resuscitation, drowning time, a 4-tiered chest radiographic grade, and the baseline characteristics. The grade was defined based on the findings of initial chest radiographs. The poor outcomes were defined as the need for intensive care unit care or death aftercare. We analyzed the association of the prehospital resuscitation, submersion time, and the radiographic grade with the poor outcomes using binary logistic regression. Results Among the 56 children, 31 (55.4%) were aged 1-4 years. Prehospital resuscitation and 1-5 minutes of submersion time were noted in the 25 (44.6%) and 30 children (53.6%), respectively. The chest radiographic grades 1 through 4 accounted for 17 (30.4%), 20 (35.7%), 12 (21.4%), and 3 children (5.4%), respectively. Poor outcomes occurred in 17 children (30.4%), including 3 deaths (5.4%). The association with the poor outcomes was noted in the submersion time of longer than 5 minutes (adjusted odds ratio, 21.49; 95% confidence interval, 1.11-415.73; compared with < 1 minute) while not in the submersion time and chest radiographic grade. Conclusion This study confirms that submersion time is an outcome predictor of drowning

Factors associated with administration of analgesics for children with forearm fracture

Purpose Analgesia is essential for the treatment of children’s fracture. We aimed to investigate the factors associated with administration of analgesics in children with forearm fracture. Methods We retrospectively reviewed medical records of children (< 20 years) with forearm fracture who visited 2 tertiary hospital emergency departments from 2014 to 2015. We analyzed factors, such as gender, age, whether the mother accompanied the visit, visiting time and route, mechanism of injury, duration of symptoms, complicated fracture, manual reduction, surgery, and type and route of analgesics. We also performed logistic regression analysis to identify the factors associated with administration of analgesics. Results Of 179 children with forearm fracture, 48 (26.8%) were administered analgesics. These children showed older age, shorter duration of symptoms, and more frequent visit with their mothers, visit during the day, use of emergency medical services, and surgery. After logistic regression analysis, we found use of emergency medical service (adjusted odds ratio [OR], 8.73; 95% confidence interval [CI], 3.16-24.08; P < 0.001), visit with the mother (OR, 6.23; 95% CI, 1.68-23.09; P = 0.006), age (OR, 1.18; 95% CI, 1.05-1.32; P = 0.004), and duration of symptoms (OR, 0.99; 95% CI, 0.986-0.999; P = 0.035) as the factors associated with administration of analgesics. Conclusion The factors associated with administration of analgesics might be communicating skill-related factors, such as older age and shorter duration of symptoms. Children with poor communicating skill may need more aggressive analgesia in the emergency department

Cortical Magnetic Resonance Imaging Findings in Patients With Posttraumatic Olfactory Dysfunction: Comparison According to the Interval Between Trauma and Evaluation

ObjectivesPatients with smell loss after craniocerebral trauma are known to have some brain abnormalities, but there was no study to analyze the findings according to the time interval between injury and evaluation. We aimed to identify whether the time interval may influence on the findings in the brain.MethodsMedical records of 19 patients with posttraumatic olfactory dysfunction were reviewed. All of them underwent a magnetic resonance imaging and olfactory function tests. The patients were divided into early (n=10) and delayed (n=9) groups according to the time interval.ResultsMagnetic resonance imaging was taken at a mean time of 2.2 and 59.6 months after trauma in the early and delayed groups, respectively. Abnormal findings in the brain were found in 6 and 8 patients in the early and delayed groups, respectively. The olfactory bulb and orbitofrontal cortex were commonly affected olfactory pathways in both groups. In the early group, the abnormalities were brain tissue defect, hemorrhage, and focal edema whereas tissue defect was the only finding in the delayed group. In the early group, 5 of 6 patients with severe olfactory dysfunction showed brain abnormality while 1 of 4 patients with mild dysfunction had abnormality. In the delayed group, all the patients had severe dysfunction and 8 of 9 patients showed brain abnormality.ConclusionMost patients with traumatic olfactory dysfunction had abnormality in the brain, and brain abnormality might be different according to the timing of evaluation. Furthermore, there might be an association between the severity of olfactory dysfunction and radiological abnormalities

Exercise Training Attenuates Ovariectomy-Induced Alterations in Skeletal Muscle Remodeling, Apoptotic Signaling, and Atrophy Signaling in Rat Skeletal Muscle

Purpose The effects of aerobic exercise training on soleus muscle morphology, mitochondria-mediated apoptotic signaling, and atrophy/hypertrophy signaling in ovariectomized rat skeletal muscle were investigated. Methods Female Sprague-Dawley rats were divided into control (CON), ovariectomy (OVX), and ovariectomy plus exercise (OVX+EX) groups. After ovarian excision, exercise training was performed using a rat treadmill at 20 m/min, 50 min/day, 5 days/week for 12 weeks. Protein levels of mitochondria-mediated apoptotic signaling and atrophy/hypertrophy signaling in the skeletal muscle (soleus) were examined through western immunoblot analysis. Results The number of myocytes and myocyte cross-sectional area (CSA) were increased and the extramyocyte space was decreased in the OVX group compared to those in the CON group. However, aerobic exercise training significantly increased myocyte CSA and decreased extramyocyte space in the OVX+EX group compared to those in the OVX group. The protein levels of proapoptotic signaling and muscle atrophy signaling were significantly increased, whereas the protein levels of muscle hypertrophy signaling were significantly decreased in the OVX group compared to that in the CON group. Aerobic exercise training significantly decreased the protein levels of proapoptotic signaling and increased the protein level of antiapoptotic protein in the OVX+EX group compared to that in the OVX group. Aerobic exercise training significantly increased the protein levels of hypertrophy signaling and decreased protein levels of atrophy signaling in the OVX+EX group compared to those in the OVX group. Conclusions Treadmill exercise improved estrogen deficiency-induced impairment in skeletal muscle remodeling, mitochondria-mediated apoptotic signaling, and atrophy/hypertrophy signaling in skeletal muscle

Effects of Acute Exercise on Mitochondrial Function, Dynamics, and Mitophagy in Rat Cardiac and Skeletal Muscles

Purpose This study aimed to investigate the effects of single-bout exercise on mitochondrial function, dynamics (fusion, fission), and mitophagy in cardiac and skeletal muscles. Methods Fischer 344 rats (4 months old) were randomly divided into the control (CON) or acute exercise (EX) group (n=10 each). The rats performed a single bout of treadmill exercise for 60 minutes. Mitochondrial function (e.g., O2 respiration, H2O2 emission, Ca2+ retention capacity), mitochondrial fusion (e.g., Mfn1, Mfn2, Opa1), mitochondrial fission (e.g., Drp1, Fis1), and mitophagy (e.g., Parkin, Pink1, LC3II, Bnip3) were measured in permeabilized cardiac (e.g., left ventricle) and skeletal (e.g., soleus, white gastrocnemius) muscles. Results Mitochondrial O2 respiration and Ca2+ retention capacity were significantly increased in all tissues of the EX group compared with the CON group. Mitochondrial H2O2 emissions showed tissue-specific results; the emissions showed no significant differences in the left ventricle or soleus (type I fibers) but was significantly increased in the white gastrocnemius (type II fibers) after acute exercise. Mitochondrial fusion and fission were not altered in any tissues of the EX group. Mitophagy showed tissue-specific differences: It was not changed in the left ventricle or white gastrocnemius, whereas Parkin and LC3II were significantly elevated in the soleus muscle. Conclusions A single bout of aerobic exercise may improve mitochondrial function (e.g., O2 respiration and Ca2+ retention capacity) in the heart and skeletal muscles without changes in mitochondrial dynamics or mitophagy