Systematic conformation-to-phenotype mapping via limited deep sequencing of proteins.

Non-native conformations drive protein-misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

Co-translational formation of disulfides guides folding of the SARS-CoV-2 receptor binding domain.

Many secreted proteins, including viral proteins, contain multiple disulfide bonds. How disulfide formation is coupled to protein folding in the cell remains poorly understood at the molecular level. Here, we combine experiment and simulation to address this question as it pertains to the SARS-CoV-2 receptor binding domain (RBD). We show that the RBD can only refold reversibly if its native disulfides are present before folding. But in their absence, the RBD spontaneously misfolds into a nonnative, molten-globule-like state that is structurally incompatible with complete disulfide formation and that is highly prone to aggregation. Thus, the RBD native structure represents a metastable state on the protein's energy landscape with reduced disulfides, indicating that nonequilibrium mechanisms are needed to ensure native disulfides form before folding. Our atomistic simulations suggest that this may be achieved via co-translational folding during RBD secretion into the endoplasmic reticulum. Namely, at intermediate translation lengths, native disulfide pairs are predicted to come together with high probability, and thus, under suitable kinetic conditions, this process may lock the protein into its native state and circumvent highly aggregation-prone nonnative intermediates. This detailed molecular picture of the RBD folding landscape may shed light on SARS-CoV-2 pathology and molecular constraints governing SARS-CoV-2 evolution.

Analgesic efficacy of preemptive pregabalin administration in arthroscopic shoulder surgery: a randomized controlled trial.

PURPOSE: The purpose of the current study was to evaluate the effects of pregabalin administration as an adjunct to fentanyl-based intravenous patient-controlled analgesia on opioid consumption and postoperative pain following arthroscopic shoulder surgery. METHODS: In this randomized controlled trial, 60 adult patients undergoing arthroscopic shoulder surgery were randomly assigned to receive either pregabalin 150 mg (Pregabalin group, n = 30) or placebo (Control group, n = 30) one hour before anesthetic induction. The primary outcome was the cumulative amount of fentanyl consumption during 48 hr postoperatively. Secondary outcomes were pain intensity, the number of rescue analgesics administered, and adverse effects related to the analgesic regimen, which were serially assessed during 48 hr postoperatively. RESULTS: The cumulative fentanyl consumption during 48 hr postoperatively was significantly lower in the Pregabalin group than in the Control group ([1,126.0 (283.6) µg vs 1,641.4 (320.3) µg, respectively; mean difference, 515.4 µg; 95% confidence interval [CI], 359.0 to 671.8; P = < 0.001). Numeric rating scores for pain (0 to 10) were significantly lower in the Pregabalin group at six hours (mean difference, 2.9; 95% CI, 1.8 to 4.0), 24 hr (mean difference, 2.9; 95% CI, 1.9 to 3.8), and 48 hr (mean difference, 1.5; 95% CI, 0.6 to 2.3). The incidence of adverse effects related to the analgesic regimens, including nausea, sedation, and dizziness, were similar between the two groups. CONCLUSION: A preoperative dose of pregabalin 150 mg administered before arthroscopic shoulder surgery resulted in significant analgesic efficacy for 48 hr in terms of opioid-sparing effect and improved pain intensity scores without influencing complications. This trial was registered at: http://cris.nih.go.kr , number CT0000578.

Pyrolysis of biomass harvested from heavy-metal contaminated area: Characteristics of bio-oils and biochars from batch-wise one-stage and continuous two-stage pyrolysis.

This study evaluates pyrolysis products obtained from biomasses (silver grass, pine, and acacia) harvested from heavy-metal-contaminated soil. To do so, we utilized two methods: a batch one-stage pyrolysis, and a continuous two-stage pyrolysis. The study results show that the yields and characteristics of bio-oils and biochars varied depending on the pyrolysis process and the type of biomass. The two-stage pyrolysis having two reactors (auger and fluidized bed reactors) appeared to be very suitable for specific chemicals production such as acetic acid, acetol, catechol, and levoglucosan. The biochar obtained from the fluidized-bed reactor of two-stage pyrolysis had high thermal stability, high crystallinity, high inorganic content, and a small number of functional groups. In contrast, the biochar obtained from the one-stage pyrolysis had low thermal stability, low crystallinity, a high carbon content, and a large number of functional groups. The biochar obtained from the two-stage pyrolysis appeared to be suitable as a material for catalyst support and as an adsorbent. The biochar obtained from one-stage pyrolysis appeared to be a suitable as a soil amendment, as an adsorbent, and as a precursor of activated carbon. All biochars showed a negative carbon footprint. In the end, this study, which was conducted using two different processes, was able to obtain the fact that products of pyrolysis biomass contaminated with heavy metals have different characteristics depending on the process characteristics and that their utilization plans are different accordingly. If the optimal utilization method proposed through this study is found, pyrolysis will be able to gain importance as an effective treatment method for biomass contaminated with heavy metals.

Kimchi and Leuconostoc mesenteroides DRC 1506 Alleviate Dextran Sulfate Sodium (DSS)-Induced Colitis via Attenuating Inflammatory Responses.

Ulcerative colitis (UC) is caused by inflammation only in the mucosa of the colon, and its incidence is increasing worldwide. The intake of probiotics is known to have a beneficial effect on the development of UC. In this study, we investigated the alleviating effects of kimchi (KC), a fermented food rich in probiotics, and Leuconostoc mesenteroides DRC 1506 (DRC) isolated from kimchi on UC. A freeze-dried kimchi suspension and DRC were orally given to mice at a dose of 1 × 109 CFU/day for 3 weeks. Furthermore, 3% dextran sulfate sodium (DSS) in drinking water was given to induce UC. The KC and DRC groups reduced symptoms of colitis, such as disease activity index, decrease in colon length, colon weight-to-length ratio, and pathological damage to the colon caused by DSS treatment. The KC and DRC groups decreased the levels of pro-inflammatory cytokine (TNF-α) and increased anti-inflammatory cytokine (IL-10) in the colon tissues. At the mRNA and protein expression levels in the colon tissue, KC and DRC groups downregulated inflammatory factors and upregulated tight junction-related factors. Therefore, DRC, as well as KC supplementation, are potent in alleviating UC by improving the inflammatory response and mucosal barrier function in the colon.

LAMMER Kinase Modulates Cell Cycle by Phosphorylating the MBF Repressor, Yox1, in Schizosaccharomyces pombe.

Lkh1, a LAMMER kinase homolog in the fission yeast Schizosaccharomyces pombe, acts as a negative regulator of filamentous growth and flocculation. It is also involved in the response to oxidative stress. The lkh1-deletion mutant displays slower cell growth, shorter cell size, and abnormal DNA content compared to the wild type. These phenotypes suggest that Lkh1 controls cell size and cell cycle progression. When we performed microarray analysis using the lkh1-deletion mutant, we found that only four of the up-regulated genes in the lkh1-deletion were associated with the cell cycle. Interestingly, all of these genes are regulated by the Mlu1 cell cycle box binding factor (MBF), which is a transcription complex responsible for regulating the expression of cell cycle genes during the G1/S phase. Transcription analyses of the MBF-dependent cell-cycle genes, including negative feedback regulators, confirmed the up-regulation of these genes by the deletion of lkh1. Pull-down assay confirmed the interaction between Lkh1 and Yox1, which is a negative feedback regulator of MBF. This result supports the involvement of LAMMER kinase in cell cycle regulation by modulating MBF activity. In vitro kinase assay and NetPhosK 2.0 analysis with the Yox1T40,41A mutant allele revealed that T40 and T41 residues are the phosphorylation sites mediated by Lkh1. These sites affect the G1/S cell cycle progression of fission yeast by modulating the activity of the MBF complex.

Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins.

Non-native conformations drive protein misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well-suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

The effect of transcutaneous electrical nerve stimulation on pain during venous cannulation.

BACKGROUND: The venous cannulation procedure was widely used in many clinical procedures; however, it is associated with pain or discomfort. OBJECTIVES: The purpose of this study was to investigate whether transcutaneous electrical nerve stimulation (TENS) could reduce pain during cannulation of vein. METHODS: One hundred patients were allocated randomly to 2 groups. In the active TENS group, TENS was delivered via 2 electrodes on the venous cannulation site (radial side of the wrist of dominant forearm) 20 minutes before venous cannulation, and the control group received placebo (no current) TENS. Venous cannulation with a 22-gauge cannula was performed. During venous cannulation, the pain intensity (0 = no pain, 10 = worst pain imaginable) was measured. Any side effects during study periods were recorded. RESULTS: The incidence of pain during venous cannulation was similar between the 2 groups. However, pain intensity was significantly lower in the active TENS group than placebo group (P < 0.01). There was no significant difference in the side effects such as erythema and itching between the groups. CONCLUSIONS: TENS may be a suitable option for reducing the pain intensity during venous cannulation. ClinicalTrials.gov identifier: NCT01607463.

Cotranslational formation of disulfides guides folding of the SARS COV-2 receptor binding domain.

Many secreted proteins contain multiple disulfide bonds. How disulfide formation is coupled to protein folding in the cell remains poorly understood at the molecular level. Here, we combine experiment and simulation to address this question as it pertains to the SARS-CoV-2 receptor binding domain (RBD). We show that, whereas RBD can refold reversibly when its disulfides are intact, their disruption causes misfolding into a nonnative molten-globule state that is highly prone to aggregation and disulfide scrambling. Thus, non-equilibrium mechanisms are needed to ensure disulfides form prior to folding in vivo. Our simulations suggest that co-translational folding may accomplish this, as native disulfide pairs are predicted to form with high probability at intermediate lengths, ultimately committing the RBD to its metastable native state and circumventing nonnative intermediates. This detailed molecular picture of the RBD folding landscape may shed light on SARS-CoV-2 pathology and molecular constraints governing SARS-CoV-2 evolution.

Association between Vascular Calcification and Esophagojejunal Anastomotic Complications after Total Gastrectomy for Gastric Cancer: A Propensity-Matched Study.

Esophagojejunal anastomosis (EJA) complications after total gastrectomy are related to significant morbidity and mortality. The aim of this study was to evaluate the association between arterial calcifications and EJA complications such as leak and stricture for gastric cancer. Between January 2014 and October 2019, 30 patients with EJA complications after total gastrectomy were enrolled and matched to 30 patients without complications through retrospective data review. Arterial calcification grade on preoperative computed tomography (CT) was reported in the abdominal aorta and superior mesenteric artery (SMA) as "absent", "minor", or "major", and in the jejunal vascular arcade (JVA) and left inferior phrenic artery (LIPA) as "absent" or "present". A Chi-square test was used to compare the variables between the two groups. p-Value < 0.050 was considered statistically significant. Among 30 patients, the numbers of patients with leak and stricture were 23 and seven, respectively. Aortic calcifications were not associated with EJA complications regardless of their grade (p = 0.440). Only major SMA calcifications were associated with EJA complications, as they were present in five patients (16.7%) in the complication group and absent in the non-complication group (p = 0.020). Major SMA calcifications were more related to anastomotic stricture than leak. Three (13.0%) out of 23 patients with leak and two (28.6%) out of seven with stricture had major SMA calcifications (p = 0.028). No calcifications were detected in the JVA or LIPA in any of the 60 patients. Major SMA calcifications were found to be associated with EJA complications, especially in stricture.

Study on Creep Damage of Ni-Based Superalloy Caused by Variable Load Conditions at Elevated Temperatures.

Higher fatigue and creep resistance at high temperatures are the essential properties for materials such as those used in gas turbines for power generation and aircraft turbines. Therefore, the nickel-based superalloy CMSX-4 was developed through single-crystal casting to satisfy these requirements. In this study, the CMSX-4 creep test results reported by previous researchers were used to mathematically derive an equation to estimate the amount of creep damage occurring under variable load conditions. In addition, low-cycle fatigue tests were performed, and the effect of creep damage occurring during fatigue on material failure was described.

Systemic analysis of a novel coxsackievirus gene delivery system in a mouse model.

In order to systemically investigate the possibility of using coxsackievirus B3 (CVB3) to deliver foreign genes in vivo, a recombinant strain of CVB3 encoding the renilla gene (CVB3- renilla) was constructed. The recombinant CVB3 resulted in extensive and transient expression of the renilla protein within mouse organs, especially the pancreas. The level of expression was generally dependent upon the viral titer present. Moreover, the CVB3-renilla strain was completely attenuated. Interestingly, the recombinant CVB3 vector was expressed much more strongly in mouse organs than was a comparable adenoviral vector. The CVB3-renilla strain did not express the renilla gene in mice with pre-existing coxsackievirus-specific neutralizing antibodies, but direct organ-specific administration of the virus during openperitoneum surgery was able to circumvent this immunity. This coxsackievirus vector may represent a useful means for delivering and expressing foreign genes in mouse models in an acute and extensive fashion.

Combined Vitrectomy With Macular Buckling In High Myopic Eyes With Macular Hole Retinal Detachment: A Pilot Study Of A Novel Snail-Tipped Exoplant.

PURPOSE: To evaluate the efficacy of a novel snail-tipped exoplant for macular buckling combined with vitrectomy in high myopic eyes with macular hole retinal detachment. PATIENT AND METHODS: A novel exoplant was simply prepared with a 5 × 3 mm silicone sponge strengthened in the center with a malleable titanium plate. One end was bent to make a rolled tip like a snail shell to be placed under the macula. Combined vitrectomy with macular buckling using this exoplant was performed in eight consecutive cases. The long arm of the exoplant was manipulated manually to fit the curvature of the eyeball and the length was trimmed appropriately after scleral suturing of the exoplant. RESULTS: Retinal reattachment was achieved in all cases (100%) and macular hole closure was confirmed in 6 eyes (75%). The mean best-corrected visual acuity improved from 1.53 ± 0.73 LogMAR preoperatively to 1.14 ± 0.59 LogMAR to postoperatively (p = 0.063). The mean pre- and postoperative AL was 28.44 ± 1.86 mm and 27.60 ± 1.83 mm, respectively (p = 0.016). The mean follow-up period was 15.4 months and no buckle-related complications such as diplopia, infection or exposure of the exoplant were noticed during the period. CONCLUSION: This exoplant could easily be prepared with readily available materials in the operating room and it was well tolerated with favorable anatomic results in high myopic eyes. Further studies of increased number of patients with long-term follow-up will be necessary.

Postoperative emergence agitation and intraoperative sevoflurane sedation under caudal block in children: a randomized comparison of two sevoflurane doses.

BACKGROUND: Sub-umbilical surgery under caudal block in conjunction with sevoflurane sedation may be safe in terms of maintaining spontaneous breathing and avoiding complications associated with general anesthesia. However, sevoflurane-induced emergence agitation (EA) continues to be a clinically important phenomenon in children. To compare the incidence of EA in children undergoing sub-umbilical surgery under caudal block with two different doses of sevoflurane. METHODS: Forty children (aged 1-5 years) scheduled to undergo inguinal hernia repair under caudal block with sevoflurane sedation via a face mask were randomized into either the low-dose (1.0%) end-tidal sevoflurane concentration group (Group LS) or the high-dose (2.5%) end-tidal sevoflurane concentration group (Group HS). We monitored EA episodes at 5 and 30 min in the post-anesthetic care unit (PACU) by using the fourpoint agitation scale and the Pediatric Anesthesia Emergence Delirium (PAED) scale. RESULTS: The four-point agitation scale scores and PAED scores were not different between the groups at 5 min. However, the agitation score was higher in Group HS than in Group LS at 30 min after arriving in the PACU. The time required to recover from sedation was longer in Group HS than in Group LS. CONCLUSIONS: Face-mask sedation with 1.0% sevoflurane in conjunction with caudal block may be more effective than that with 2.5% sevoflurane in preventing EA.

Multi-DOF (Degree of Freedom) Articulating Laparoscopic Instrument is an Effective Device in Performing Challenging Sutures.

Purpose: Although laparoscopic surgery had been performed in clinical practice for over 30 years, there has not been much improvement on instruments. Several articulating laparoscopic instruments have been developed including the robotic system. A new multi-degree of freedom (DOF) articulating laparoscopic device has been developed. We compared the ability to perform challenging sutures between the new device and the robotic system. Methods: Five experienced surgeons with over 100 laparoscopic surgery cases performed the suture task with both instruments. Everyone was new at articulating instruments including a robotic system. The suturing task consisted of two vertical sutures, downward and upward vertical direction. The duration of needle grabbing, first surgical tie, square tie, and the final reverse tie was measured. Results: When doing the downward suture, the median time to complete the suture was 127 vs. 136 seconds for ArtiSential® and the robot, respectively (p=0.754). Other measurements such as needle grabbing, first tie, second tie and final knot did not show any significant difference between the two instruments. Upward suture also did not show a significant difference. The total completion time was 127 vs. 112 seconds for for ArtiSential® and the robot, respectively (p=0.675). Time taken in each interval did not show any significant difference. Conclusion: Both instruments performed the suturing tasks with no difference in duration. ArtiSential® can be mixed up with usual instruments. Surgeons can choose any device, but when articulation is needed, ArtiSential® could be an alternative choice to the robotic system.

Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3.

Lactobacillus brevis OPK-3, having 84.292 mg/L/h of gamma-aminobutyric acid (GABA) productivity, was isolated from Kimchi, a traditional fermented food in Korea. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from the L. brevis OPK-3, using primers based on two highly conserved regions of GAD. A full-length GAD (LbGAD) clone was subsequently isolated through rapid amplification of cDNA ends (RACE) PCR. Nucleotide sequence analysis revealed that the open reading frame (ORF) consisted of 1401 bases and encoded a protein of 467 amino acid residues with a calculated molecular weight of 53.4 kDa and a pI of 5.65. The amino acid sequence deduced from LbGAD ORF showed 83%, 71%, and 60% identity to the Lactobacillus plantarum GAD, Lactococcus lactis GAD, and Listeria monocytogenes GAD sequences, respectively. The LbGAD gene was expressed in Escherichia coli strain UT481, and the extract of transformed E. coli UT481 contained an induced 53.4 kDa protein and had significantly enhanced GAD activity.

Production of yogurt with enhanced levels of gamma-aminobutyric acid and valuable nutrients using lactic acid bacteria and germinated soybean extract.

Yogurt with high levels of gamma-aminobutyric acid (GABA), free amino acids and isoflavones was developed using lactic acid bacteria (LAB) and germinated soybean extract. Fermented soya milk (GABA soya yogurt) produced with starter and substrate had the GABA concentration of 424.67 microg/gDW, whereas fermented milk produced by a conventional method had GABA less than 1.5 microg/gDW. The GABA soya yogurt also contained significantly high levels of free amino acids and isoflavones compared with other conventional yogurts. The results suggested that the Lactobacillus brevis OPY-1 and germinated soybean possessed a prospect to be applied in dairy and other health products with high nutritive values and functional properties.

Comparative evaluation of the effect of remifentanil and 2 different doses of esmolol on pain during propofol injection: A double-blind, randomized clinical consort study.

BACKGROUND: Propofol is associated with pain during injection, which is stressful to patients. The present study was designed to investigate the analgesic effect of pretreatment with remifentanil and esmolol in minimizing propofol injection pain, compared with placebo. METHODS: In a randomized, double-blind, prospective trial, 120 patients, scheduled for elective dental surgery under general anesthesia, were randomized to 1 of the 4 treatment arms (n = 30 each) receiving normal saline, remifentanil 0.35 µg/kg, esmolol 0.5 mg/kg, and 1 mg/kg before administration of propofol. During injection of 1% propofol 0.5 mg/kg, pain was evaluated by a 4-point score (0 = none, 1 = mild, 2 = moderate, 3 = severe). Any adverse effects such as hypotension and bradycardia were recorded during the perioperative periods. RESULTS: In all, 120 patients completed this study. There were no significant differences in terms of demographic data. The incidence of pain on injection of propofol was 11 (36.7%) with remifentanil 0.35 µg/kg, 12 (40%) with esmolol 0.5 mg/kg, and 11 (36.7%) with esmolol 1 mg/kg, compared with 25 (83.3%) with normal saline (respectively, P < 0.05). There were no significant differences in the incidence of pain between groups with remifentanil 0.35 µg/kg, and esmolol 0.5 mg/kg and 1 mg/kg. There were no emergence reactions such as hypotension and bradycardia in all groups. CONCLUSIONS: Pretreatment with esmolol 0.5 mg/kg and 1 mg/kg and remifentanil 0.35 µg/kg equally decreased pain during propofol injection.

[The effect of palonosetron on rocuronium-induced withdrawal movement]. / O efeito de palonosetron sobre o movimento de retração induzido por rocurônio.

BACKGROUND: Rocuronium causes pain and withdrawal movement during induction of anesthesia. In this study, palonosetron was investigated to have analgesic effect on the reduction of rocuronium-induced withdrawal movement. METHODS: 120 patients were randomly assigned to one of three groups to receive either saline, lidocaine 20mg, or palonosetron 0.075mg with a tourniquet applied two minutes before thiopental sodium (5mg.kg-1) was given intravenously. After loss of consciousness, rocuronium (0.6mg.kg-1) was injected and the withdrawal movement was estimated by 4-point scale in a double-blind manner. RESULTS: The overall incidence of rocuronium withdrawal movement was 50% with lidocaine (p=0.038), 38% with palonosetron (p=0.006) compared with 75% for saline. The incidence of no pain to mild pain was significantly lower in the lidocaine and palonosetron groups (85% and 92% respectively) than in the saline group (58%). However, there was no significant difference in withdrawal movement between the lidocaine and palonosetron groups. There was no severe movement with palonosetron. CONCLUSION: Pretreatment of palonosetron with venous occlusion may attenuate rocuronium-induced withdrawal movement as effective as the use of lidocaine. It suggested that peripheral action of palonosetron was effective to reduce rocuronium-induced withdrawal movement.

Meat Quality Traits of Pigs Finished on Food Waste.

Despite the benefits associated with the use of food waste (FW), there are mixed consumer perceptions regarding pork quality harvested from pigs fed FW. Twenty crossbred pigs were selected for the present study. Ten pigs were fed a conventional diet (control group), and the other 10 pigs were given a conventional diet and FW (FW group) during different growth stages. Meat quality in the FW group showed deteriorative qualities with higher lightness and yellowness synonymous to pale soft exudative meat. Drip loss in the experimental group was significantly higher than that in the control group (p<0.01). The contents of polyunsaturated fatty acids in the FW group were higher and those of saturated and monounsaturated fatty acids were lower than those in the control group. The contents of thiobarbituric acid were significantly different between the control and FW groups (p<0.05). There was also a significant difference between the control and FW groups in terms of off-flavor (p<0.05) after sensory evaluation. To conclude, the off-flavor noted, including other inferior pork quality traits, in the FW group implies that FW should not be used as swine feed.