Statistical Analysis of the Role of Cavity Flexibility in Thermostability of Proteins.

Conventional statistical investigations have primarily focused on the comparison of the simple one-dimensional characteristics of protein cavities, such as number, surface area, and volume. These studies have failed to discern the crucial distinctions in cavity properties between thermophilic and mesophilic proteins that contribute to protein thermostability. In this study, the significance of cavity properties, i.e., flexibility and location, in protein thermostability was investigated by comparing structural differences between homologous thermophilic and mesophilic proteins. Three dimensions of protein structure were categorized into three regions (core, boundary, and surface) and a comparative analysis of cavity properties using this structural index was conducted. The statistical analysis revealed that cavity flexibility is closely related to protein thermostability. The core cavities of thermophilic proteins were less flexible than those of mesophilic proteins (averaged B' factor values, -0.6484 and -0.5111), which might be less deleterious to protein thermostability. Thermophilic proteins exhibited fewer cavities in the boundary and surface regions. Notably, cavities in mesophilic proteins, across all regions, exhibited greater flexibility than those in thermophilic proteins (>95% probability). The increased flexibility of cavities in the boundary and surface regions of mesophilic proteins, as opposed to thermophilic proteins, may compromise stability. Recent protein engineering investigations involving mesophilic xylanase and protease showed results consistent with the findings of this study, suggesting that the manipulation of flexible cavities in the surface region can enhance thermostability. Consequently, our findings suggest that a rational or computational approach to the design of flexible cavities in surface or boundary regions could serve as an effective strategy to enhance the thermostability of mesophilic proteins.

Evaluation of Robotic Systems on Cytotoxic Drug Preparation: A Systematic Review and Meta-Analysis.

Background and Objectives: With the increased prevalence of patients with cancer, the demand for preparing cytotoxic drugs was increased by health-system pharmacists. To reduce the workload and contamination of work areas in pharmacies, compounding robots preparing cytotoxic drugs have been introduced, and the use of the robots has been expanded in recent years. As reports on the comprehensive and quantitative evaluation of compounding robots remain lacking, a systematic review and meta-analysis were conducted to provide descriptive and quantitative evaluations of the accuracy of preparing injectable cytotoxic drugs. Materials and Methods: A systematic review and meta-analysis were conducted using published studies up to 2020. To identify eligible studies, PubMed, EMBASE, and Cochrane Library were used. All studies reporting the outcomes relevant to drug-compounding robots such as accuracy, safety, and drug contamination were included. Outcomes from included studies were descriptively summarized. Drug contamination by the robot was quantitatively analyzed using the odds ratio (OR) with a 95% confidence interval (CI). The risk of bias was assessed using the Risk of Bias Assessment tool for Non-randomized Studies (RoBANS). Results: A total of 14 compounding robot studies were eligible for review and 4 studies were included in the meta-analysis. Robotic compounding showed failure rates of 0.9-16.75%, while the accuracy range was set at 5%. Two studies reported that robotic compounding needed more time than manual compounding, two reported that robotic compounding needed less time, and one just reported preparation time without a control group. In a meta-analysis regarding the contamination of the compounding area, manual compounding was associated with lower contamination, although the result was not statistically significant (OR 4.251, 95% CI 0.439-51.772). For the contamination of infusion bags, the robot was associated with lower contamination (OR 0.176, 95% CI 0.084-0.365). Conclusions: Robotic compounding showed better accuracy than manual compounding and, without control groups, showed a high accuracy rate and also reduced the risk of drug contamination and compounding workload. The preparation time of the robot was not consistent because the type of robot and introduced system were different. In conclusion, robotic compounding showed mixed results compared to the manual compounding of drugs, so the system should be introduced considering the risks and benefits of robots.

Gibberellic acid sensitive dwarf encodes an ARPC2 subunit that mediates gibberellic acid biosynthesis, effects to grain yield in rice.

The plant hormone gibberellic acid (GA) is important for plant growth and productivity. Actin-related proteins (ARPs) also play central roles in plant growth, including cell elongation and development. However, the relationships between ARPs and GA signaling and biosynthesis are not fully understood. Here, we isolated OsGASD, encoding an ARP subunit from rice (Oryza sativa), using the Ac/Ds knockout system. The osgasd knockout (Ko) mutation reduced GA3 content in shoots as well as plant growth and height. However, GA application restored the plant height of the osgasd Ko mutant to a height similar to that of the wild type (WT). Rice plants overexpressing OsGASD (Ox) showed increased plant height and grain yield compared to the WT. Transcriptome analysis of flag leaves of OsGASD Ox and osgasd Ko plants revealed that OsGASD regulates cell development and the expression of elongation-related genes. These observations suggest that OsGASD is involved in maintaining GA homeostasis to regulate plant development, thereby affecting rice growth and productivity.

Prenatal diagnosis of de novo DMD duplication by multiplex ligation-dependent probe amplification (MLPA) after noninvasive prenatal screening (NIPS) at 11 gestational weeks.

OBJECTIVE: Dystrophinopathy is an X-linked recessive muscular dystrophy caused by mutations in the DMD gene. Herein we describe the prenatal detection of DMD gene mutations in a patient with no family history, by multiplex ligation-dependent probe amplification (MLPA) after noninvasive prenatal screening (NIPS). CASE REPORT: A 41-year-old woman underwent NIPS owing to an advanced maternal age. A copy number variation was detected in the maternal X chromosome, and uninformative results were obtained for the fetal sex chromosomes. Following amniocentesis, a duplication was identified in exons 1-29 of the dystrophin gene by MLPA. After interviewing her family members it was confirmed that the patient is a de novo carrier of DMD duplications, and her daughter is a carrier of the same mutation. CONCLUSION: his is the first case report to describe the prenatal diagnosis of duplications in the DMD gene by MLPA following NIPS in a patient with no family history.

Characterization of dwarf and narrow leaf (dnl-4) mutant in rice.

Height and leaf morphology are important agronomic traits of the major crop plant rice (Oryza sativa). In previous studies, the dwarf and narrow leaf genes (dnl1, dnl2 and dnl3) have identified in rice. Using the Ac/Ds knockout system, we found a new dwarf and narrow leaf (dnl) mutant and identified mutated gene. The dnl-4 mutant showed reduced plant height and leaf blade width compared to the wild type, and increased leaf inclination. The morphological defects of the mutant were caused by the suppressed expression of the DNL-4 gene, which encodes a pfkB carbohydrate kinase protein. These results suggest that DNL-4 expression is involved in modulating plant height and leaf growth. Furthermore, DNL-4 expression also affects productivity in rice: the dnl-4 mutant exhibited reduced panicle length and grain width compared with the wild type. To understand DNL-4 function in rice, we analyzed the expression levels of leaf growth-related genes, such as NAL1, NAL7, and CSLD4, in the dnl-4 mutant. Expression of NAL1 and NAL7 was downregulated in the dnl-4 mutant compared to the wild type. The observation that DNL-4 expression corresponded with that of NAL1 and NAL7 is consistent with the narrow leaf phenotype of the dnl-4 mutant. These results suggest that DNL-4 regulates plant height and leaf structure in rice.

Drug use evaluation of opioid analgesics in pain management among patients with hematopoietic stem cell transplantation.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) patients usually experience mucositis, musculoskeletal pain associated with high-dose chemotherapy, radiation, post-HSCT infection, or graft-versus-host disease. Pain management is important for the patients' quality of life. We evaluated appropriate opioid analgesic use in HSCT patients to propose effective pain management strategies. METHODS: A retrospective analysis was conducted using electronic medical records of adult patients with HSCT treated with opioids for moderate to severe pain at Seoul National University Bundang Hospital. The numeric rating scale (NRS) was used in pain management. NRS scores of 4‒10 correspond to moderate to severe pain. Appropriate opioid analgesic use was evaluated following published cancer pain management guidelines. RESULTS: In total, 119 cases were evaluated, including 369 episodes of moderate to severe pain. Mucositis-related, musculoskeletal, and headache pain occurred in 62.6%, 25.8%, and 6.0% of episodes, respectively. Frequently used opioids were intravenous tramadol (84.9%), fentanyl patch (73.9%), and intravenous morphine sulfate (68.9%). Intravenous and topical administrations were used for mucosal pain. In total, 95.0% of patients received appropriate short-acting opioids for initial pain management, 80.5% received appropriate doses of short-acting opioids, appropriate opioids dose adjustment was done after first assessment in 95.5% of patients, and 85.6% were converted to appropriate long-acting opioids. CONCLUSION: Short-acting opioid analgesic use for initial pain management and dose adjustment after assessment were appropriate. However, initial and conversion dosages recommended by guidelines may be difficult to implement considering the severity of HSCT patients. Pain management guidelines specific for HSCT patients should be developed in the future.

Efficacy of parenteral glutamine supplementation in adult hematopoietic stem cell transplantation patients.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) patients need parenteral nutrition because of nausea, vomiting, and mucositis caused by conditioning regimens. The demand for glutamine increases during the HSCT period. We evaluated the effects of glutamine-containing parenteral nutrition on the clinical outcomes of HSCT patients. METHODS: In this retrospective analysis, we reviewed HSCT patients from Seoul National University from August 2013 to July 2017. Depending on their glutamine supplementation status, 91 patients were divided into 2 groups: glutamine group (N=44) and non-glutamine group (N=47). We analyzed the rate of weight change, infection (clinically/microbiologically documented), complications (duration of mucositis and neutropenia, acute graft versus host disease), and 100-days mortality in each group. RESULTS: Regarding the clinical characteristics of the patients, there were no significant differences between the 2 groups except that there was a larger proportion of myeloablative conditioning regimen in the glutamine group (P=0.005). In the glutamine group, the average number of days of glutamine use, parenteral nutrition, and mucositis was 7.6±1.4, 14.6±9.9, and 13.3±9.5, respectively. Furthermore, multivariate analysis revealed odds ratios of 0.37 (95% CI, 0.14-0.96; P=0.042) and 0.08 (95% CI, 0.01-0.98; P=0.048) for clinically documented infection and 100-days mortality, respectively, in the glutamine group. CONCLUSION: Results showed that the glutamine group had less clinically documented infection and 100-days mortality than the non-glutamine group, but the other outcomes did not show significant differences. The extended duration of glutamine supplementation according to the period of total parenteral nutrition and mucositis should be considered.

Rice OsMYB5P improves plant phosphate acquisition by regulation of phosphate transporter.

Myeloblastosis (MYB) transcription factors play central roles in plant developmental processes and in responses to nutrient deficiency. In this study, OsMYB5P, an R2R3-MYB transcription factor, was isolated and identified from rice (Oryza sativa L. 'Dongjin') under inorganic phosphate (Pi)-deficient conditions. OsMYB5P protein is localized to the nucleus and functions as a transcription activator in plant development. Overexpression of OsMYB5P in rice and Arabidopsis (Arabidopsis thaliana Col-0) increases tolerance to phosphate starvation, whereas OsMYB5P knock-out through RNA interference increases sensitivity to Pi depletion in rice. Furthermore, shoots and roots of transgenic rice plants overexpressing OsMYB5P were longer than those of wild plants under both normal and Pi-deficient conditions. These results indicate that OsMYB5P is associated with the regulation of shoot development and root- system architecture. Overexpression of OsMYB5P led to increased Pi accumulation in shoots and roots. Interestingly, OsMYB5P directly bound to MBS (MYB binding site) motifs on the OsPT5 promoter and induced transcription of OsPT5 in rice. In addition, overexpression of OsMYB5P in Arabidopsis triggered increased expression of AtPht1;3, an Arabidopsis Pi transporter, in shoots and roots under normal and Pi-deficient conditions. Together, these results demonstrate that overexpression of OsMYB5P increases tolerance to Pi deficiency in plants by modulating Pi transporters at the transcriptional level in monocots and dicots.

Enhancing the activity of Bacillus circulans xylanase by modulating the flexibility of the hinge region.

Enzymes undergo multiple conformational changes in solution, and these dynamics are considered to play a critical role in enzyme activity. Hinge-bending motions, resulting from reciprocal movements of dynamical quasi-rigid bodies, are thought to be related to turnover rate and are affected by the physical properties of the hinge regions. In this study, hinge identification and flexibility modification of the regions by mutagenesis were conducted to explore the relationship between hinge flexibility and catalytic activity. Bacillus circulans xylanase was selected for the identification and mutation of the hinge regions. As a result, turnover rate (V(max)) was improved approximately twofold in mutants that have more rigid hinge structure, despite the decrease in K(m) and V(max)/K(m). This result indicates that the rigidly mutated hinge has positive effects on B. circulans xylanase activity.

Activity enhancement of Candida antarctica lipase B by flexibility modulation in helix region surrounding the active site.

The activity of Candida antarctica lipase B was improved by mutation of the area surrounding the active site. We changed the edges of four helices surrounding the active site to flexible amino acids. Two mutants, V139E and I255E, obtained as a result of Pichia pastoris expression, showed enhanced specific activity of 9.9 and 8.1 U/mg while that of wild type was 2.3 U/mg for p-nitrophenyl caprylate hydrolysis. It was nearly 5.4-fold and 3.5-fold, respectively. The stability of both mutants on organic solvent was slightly decreased but almost similar with that of wild type. In the kinetic assay, k(cat) values were shown as dominant factor for the enhancement of catalytic efficiency, k(cat)/K(m), since it was 4.1-fold and 3.8-fold, respectively.

Continuous biodiesel production using in situ glycerol separation by membrane bioreactor system.

Biodiesel is one of the most promising renewable fuel sources. Candida antarctica lipase B (CalB) has been used for biodiesel production because of its high activity and stability. However, CalB can only be utilized in industrial biodiesel production if the enzyme deactivation by methanol and the negative effects of glycerol can be mitigated. Methanol inhibition can be avoided by utilizing a stepwise addition of methanol, but there is no suitable method to reduce the glycerol effect. This study aims to use a membrane bioreactor system to remove glycerol during biodiesel production. In addition, methanol inhibition can be reduced by continuously feeding methanol through the membrane system. This continuous membrane bioreactor system can be used for efficient biodiesel production.

Activation of RhoA and FAK induces ERK-mediated osteopontin expression in mechanical force-subjected periodontal ligament fibroblasts.

The precise mechanism by which Rho kinase translates the mechanical signals into OPN up-regulation in force-exposed fibroblasts has not been elucidated. Human periodontal ligament fibroblasts (hPLFs) were exposed to mechanical force by centrifuging the culture plates at a magnitude of 50 g/cm(2) for 60 min. At various times of the force application, they were processed for analyzing cell viability, trypan blue exclusion, and OPN expression at protein and RNA levels. Cellular mechanism(s) of the force-induced OPN up-regulation was also examined using various kinase inhibitors or antisense oligonucleotides specific to mechanosensitive factors. Centrifugal force up-regulated OPN expression and induced a rapid and transient increase in the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and Elk1. Pharmacological blockade of RhoA/Rho-associated coiled coil-containing kinase (ROCK) signaling markedly reduced force-induced FAK and ERK1/2 phosphorylation. Transfecting hPLFs with FAK antisense oligonucleotide diminished ERK1/2 activation and force-induced OPN expression. Further, ERK inhibitor inhibited significantly OPN expression, Elk1 phosphorylation, and activator protein-1 (AP-1)-DNA binding activation, but not FAK phosphorylation, in the force-applied cells. These results demonstrate that FAK signaling plays critical roles in force-induced OPN expression in hPLFs through interaction with Rho/ROCK as upstream effectors and ERK-Elk1/ERK-c-Fos as downstream effectors.