Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Severe pulmonary edema occurred during endobronchial ultrasound under monitored anesthesia care - A case report.

BACKGROUND: Endobronchial ultrasound (EBUS) is widely used to diagnose lung cancer. Monitored anesthesia care (MAC) can enhance patient comfort and procedural conditions during EBUS. EBUS under MAC is usually safe but can lead to various complications. CASE: A 34-year-old male who had increased sputum for two months showed an enlarged paratracheal lymph node and planned for lymph node biopsy by EBUS. During EBUS under MAC, an unexpected oxygen saturation decline required intervention. After intubation, copious frothy fluid was suctioned from the bronchi, and oxygenation was recovered. A narrowed trachea and the EBUS bronchoscope might have resulted in upper airway obstruction, and suction performed under these conditions might have caused pulmonary edema. The patient received non-invasive ventilation and high-flow nasal cannula and recovered without complications. CONCLUSIONS: When there is an expected risk of upper airway obstruction during EBUS, careful preoperative evaluation and preparation are essential to prevent negative pressure pulmonary edema.

Anesthetic management of an elderly COVID-19 patient with pulmonary thromboembolism for hip surgery - A case report.

BACKGROUND: COVID-19 and delayed hip surgery are well-known risk factors for thromboembolism in elderly patients. CASE: We report the case of an 88-year-old female patient with COVID-19 and pulmonary thromboembolism (PTE) who underwent delayed hip surgery 21 days after the injury. Heparinization and inferior vena cava filters were used to treat and prevent PTE. Transesophageal echocardiography and extracorporeal membrane oxygenation (ECMO) sheaths were inserted as a precaution in case of emergencies during surgery; the procedure was performed without any specific event. CONCLUSIONS: COVID-19-infected patients suffering from a hip fracture have a high risk of thromboembolism, and therefore, require utmost attention for appropriate evaluation and prevention.

Catatonia associated with prolonged stupor after general anesthesia in a patient with multiple neuropsychiatric disorders -a case report.

BACKGROUND: Delayed emergence after general anesthesia may significantly affect a patient's condition. We present the case of a patient who experienced prolonged delayed recovery of consciousness, language, and motor response due to catatonia after eight hours of total elbow arthroplasty under general anesthesia. CASE: A 68-year-old woman with neuropsychiatric disorders and Parkinson's disease did not respond adequately during recovery after more than eight hours of general anesthesia. Following the operation, the patient was semi-comatose and appeared to have nonconvulsive status epilepticus upon awakening from anesthesia. However, subsequent examinations did not reveal any organic causes. The patient was subsequently diagnosed with catatonia, treated, and discharged following gradual improvement. CONCLUSIONS: Although rare, patients taking psychiatric drugs for an extended period may experience delayed emergence after prolonged general anesthesia without identifiable causes. Catatonia should be considered in the differential diagnoses of these patients.

Prophylactic use of donut-shaped cushion to reduce sacral pressure injuries during open heart surgery.

Background: Pressure injuries are likely to develop in the operating room due to the high temperature and humidity underneath the patients. This study was designed to reduce sacral pressure injuries using donut-shaped cushions on patients undergoing open heart surgery in a supine position for more than three hours. Materials and Methods: Patients undergoing open heart surgery for more than three hours were randomly allocated. Depending on the allocation, either the donut-shaped cushion (donut group) or hydrophilic foam dressing (control group) was applied before draping. Patients were evaluated for the development of pressure injuries, National Pressure Ulcer Advisory Panel (NPUAP) stage, and injury size immediately after surgery, 48 hours, and seven days after surgery. Results: Forty-five patients were enrolled in this study. Twenty-two were assigned to the donut group and 23 were assigned to the control group. Three patients developed pressure injuries of NPUAP stage I or higher. All injuries occurred in the control group, but there was no statistically significant difference (P = 0.083). Conclusions: Patients who underwent cardiac surgery for more than three hours and used a donut-shaped cushion did not develop pressure injuries, although no statistical difference was noted. Specific preventative measures in the operating room may play a crucial role in preventing pressure injuries, and further research should be pursued.

Two cases of late-onset cardiovascular toxicities after a single injection of local anesthetics during supraclavicular brachial plexus block - A report of two cases.

BACKGROUND: Local anesthetics systemic toxicity (LAST) is a grave complication of regional anesthesia that usually occurs immediately after local anesthetics injection. Here, we report on rare late-onset toxicity cases after supraclavicular brachial plexus blocks. CASE: Two patients underwent surgery for radius fractures. We used lidocaine 100 mg and ropivacaine 150 mg for blocking and infused dexmedetomidine for intraoperative sedation. The 63-year-old male patient's blood pressure dropped to 87/60 mmHg after 3 h 15 min after blocking. Ventricular fibrillation occurred 10 min later. After five defibrillations, electrocardiography showed ventricular tachycardia that was normalized through one cardioversion. The 54-year-old female patient's heart rate decreased to 35 beats/min 2 h 30 min after blocking. Her vital signs returned to normal after administering atropine, ephedrine, epinephrine, and lipid emulsion. CONCLUSIONS: Physicians should remember that LAST may occur long after local anesthetic injection and be aware of factors that may adversely affect the course of LAST.

Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolated from salmonid fish in Gangwon Province, Korea.

The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed <60% cumulative mortality in Atlantic salmon and coho salmon. The expression of G genes in the kidney was higher than that in the spleen-infected fish, with the highest expression observed in the kidneys of rainbow trout. The relative expression levels of innate immunity genes were higher in rainbow trout than in Atlantic salmon and coho salmon. The expression level of immunoglobulin M increased until day 7, and the expression of type I interferon was higher in the spleen than in other tissues. The expression of Mx-1 was higher in the kidney and liver than other tissues. These results indicate that IHNV isolates from Gangwon province show host-specific virulence in rainbow trout and that their virulence and replication were higher in JRt-Shizuoka type than in JRt-Nagano type isolates.

Successful anesthetic management of a giant lower lip hemangioma patient using high flow nasal cannula -a case report.

BACKGROUND: Giant lip hemangioma is a rare disease that may cause difficulty in preoxygenation and ventilation when using face masks and intubation during general anesthesia induction. CASE: A laparoscopic cholecystectomy was planned for a 77-year-old woman. The patient had a giant lower lip hemangioma that was 12 x 5 x 5 cm, which made preoxygenation and ventilation through a face mask impossible and put her at risk of hemangioma rupture. We preoxygenated her through a high-flow nasal cannula (HFNC). Following propofol and succinylcholine administration, we intubated the patient with a video laryngoscope without desaturation, hemangioma rupture, or CO2 retention. CONCLUSIONS: HFNC is a useful tool when difficult intubation is expected in patients who have problems using conventional face masks.

Epidural analgesia versus intravenous analgesia after minimally invasive repair of pectus excavatum in pediatric patients: a systematic review and meta-analysis.

BACKGROUND: Postoperative pain control after the minimally invasive repair of pectus excavatum (MIRPE) is essential, but there is a controversy about a better analgesic method between epidural and intravenous (IV) analgesia. This systematic review and meta-analysis aimed to compare the effect of epidural versus IV analgesia following MIRPE. METHODS: We searched PubMed, MEDLINE, EMBASE, Cochrane Central Register, and ClinicalTrials.gov for randomized controlled trials (RCTs) dated up to 31st May 2021. The primary outcome was the area under the curve (AUC) of the weighted mean visual analog scale (VAS) after MIRPE. The secondary outcomes were postoperative nausea, operation time, total operating room time, and postoperative length of hospital stay. RESULTS: Four RCTs involving 243 patients were finally included in this meta-analysis. The AUC of the weighted mean VAS was 343.62 in the epidural group and 375.24 in the IV group. The epidural group showed lower VAS than the IV group at 12 to 48 h after the surgery. Postoperative nausea, operation time and length of hospital stay was not different between two groups. The epidural group had a significantly longer total operating room time due to epidural catheter insertion time. CONCLUSIONS: Epidural analgesia after the MIRPE had a better analgesic effect than IV analgesia. However, IV analgesia may also be a viable option, and physicians should wisely choose analgesic modalities after MIRPE.

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds.

Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

Blood clot formation in reinfusion bag with CATSmart: Two case reports.

Autologous blood cell salvage reduces the need for postoperative allogeneic blood transfusion and alleviates immunologic reactions, so the technique is commonly used in cardiac surgery. The continuous autotransfusion system is a type of blood cell salvage device. Although the processing program of continuous autotransfusion system includes filtering of several materials from suctioned blood, such as clots, leukocytes, cytokines, and complement, we identified some unexpected blood clots in the reinfusion bag. Pathologic examination revealed that the clots were composed of fibrin, red blood cell aggregates, and histiocytes. We report two cases of these abnormal findings during the use of CATSmart in cardiac surgery.

Anticholinergic premedication-induced fever in paediatric ambulatory ketamine anaesthesia.

OBJECTIVE: A randomized, double-blind, prospective study to evaluate the effect of anticholinergic drugs on thermoregulation in paediatric patients undergoing ambulatory anaesthesia with ketamine. METHODS: Patients were randomized to receive either 0.005 mg/kg glycopyrrolate or the equivalent volume of normal saline (placebo) at 30 min before ketamine anaesthesia. Body temperature was measured tympanically at baseline and at 0, 30, 60 and 90 min postoperatively. The quantity of saliva prodiced during surgery and incidence of fever were recorded. RESULTS: Body temperature was significantly higher in the glycopyrrolate group (n = 42) than the placebo group (n = 42) at 30, 60 and 90 min after surgery, and higher than baseline at 0, 30, 60 and 90 min after surgery. In the placebo group, body temperature was significantly higher than baseline at 0 and 30 min after surgery. Saliva secretion was significantly lower in the glycopyrrolate group than the placebo group. CONCLUSION: Routine premedication with adjunctive anticholinergics should not be considered in paediatric patients receiving ketamine sedation due to the increased risk of fever.Trial registration number, Clinicaltrials.gov: NCT02430272.

Postpartum spinal subdural hematoma: irrelevant epidural blood patch: a case report.

We report a healthy patient with postpartum headache and neck stiffness which were diagnosed as symptoms of pseudoaneurysm of vertebral artery. She had received a Cesarean section under the spinal anesthesia, and complaint of headache and neck stiffness. Epidural blood patches were done twice, but symptoms persisted. Eight days later, she experienced sensory disturbance and emergent laminectomy was done. When persistent postpartum headache occurs after epidural blood patch, more precise differential diagnosis should be made and considering other possible pathologies.

Influence of Ketamine on Early Postoperative Cognitive Function After Orthopedic Surgery in Elderly Patients.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a serious and frequent complication after surgery, especially in elderly patients. Ketamine is an N-methyl D-aspartic acid receptor antagonist with demonstrated neuroprotective effects. An intravenous bolus of a sub-anesthetic dose (0.5 mg/kg) of ketamine can reduce postoperative delirium (POD) and POCD after cardiac surgery. But, the influence of ketamine on early POCD after non-cardiac surgery is unclear. OBJECTIVES: The current study aimed to evaluate the influence of ketamine on early postoperative cognitive function after orthopedic surgery in elderly patients. PATIENTS AND METHODS: Fifty six elderly patients (> 60-years-old), scheduled for elective orthopedic surgery during general anesthesia (duration of anesthesia > two hours) were enrolled. Patients received intravenous bolus, a total of 3 mL mixed with 0.9% normal saline and 0.5 mg/kg ketamine (K group) or 3 mL of 0.9% normal saline (N group). Three neurocognitive function tests (mini-mental status examination, trail-making test, digit substitution test), and c-reactive protein (CRP) concentration were determined before surgery and on postoperative day one (POD 1) and postoperative day six (POD 6). RESULTS: The two groups had similar demographic characteristics except for the gender. Surgical and anesthetic data were not significantly different. A statistically significant difference was observed in comparison of trail-making test score. Trail-making test score increased more in the N group (52.5 points) than the K group (13 points) at POD 1 (P = 0.047) compared with baseline scores. There were no significant differences in the mini-mental status examination, digit substitution test and CRP concentration at POD 1 and POD 6 between the two groups. POCD (the two Z-scores in more than two tests or the combined Z-score was 1.96 or more) was present in one patient (4%) in the K group at POD 6 (P = 0.98). CONCLUSIONS: The incidence of POCD was not significantly influenced by a bolus dose of ketamine (0.5 mg/kg) after orthopedic surgery in elderly patients. There were no negative effects of ketamine on early POCD.

protonation behavior of histidine during HSF1 activation by physiological acidification.

The expression of eukaryotic molecular chaperones (heat shock proteins, HSPs) is triggered in response to a wide range of environmental stresses, including: heat shock, hydrogen peroxide, heavy metal, low-pH, or virus infection. Biochemical and genetic studies have clearly shown the fundamental roles of heat shock factor 1 (HSF1) in stress-inducible HSP gene expression, resistance to stress-induced cell death, carcinogenesis, and other biological phenomena. Previous studies show that acidic pH changes within the physiological range directly activate the HSF1 function in vitro. However, the detailed mechanism is unclear. Though computational pKa-predications of the amino acid side-chain, acidic-pH induced protonation of a histidine residue was found to be most-likely involved in this process. The histidine 83 (His83) residue, which could be protonated by mild decrease in pH, causes mild acidic-induced HSF1 activation (including in-vitro trimerization, DNA binding, in-vivo nuclear accumulation, and HSPs expression). His83, which is located in the loop region of the HSF1 DNA binding domain, was suggested to enhance the intermolecular force with Arginine 79, which helps HSF1 form a DNA-binding competent. Therefore, low-pH-induced activation of HSF1 by the protonation of histidine can help us better to understand the HSF1 mechanism and develop more therapeutic applications (particularly in cancer therapy). J. Cell. Biochem. 116: 977-984, 2015. © 2015 Wiley Periodicals, Inc.

Identification, tissue distribution and characterization of two heat shock factors (HSFs) in goldfish (Carassius auratus).

Heat shock proteins (HSPs) are synthesized rapidly in response to a variety of physiological or environmental stressors, whereas the transcriptional activation of HSPs is regulated by a family of heat shock factors (HSFs). In vertebrates, multiple HSFs (HSF1-4) have been reported to have different roles in response to a range of stresses. This paper reports the cDNA cloning of two goldfish (Carassius auratus) HSF gene families, HSF1 and three isoforms of HSF2. Both HSF1 and HSF2s showed high homology to the known HSFs from other organisms, particularly the zebrafish. Different patterns of HSF1 and HSF2 mRNA expression were detected in several goldfish tissues, highlighting their distinct roles. In cadmium (Cd)-treated tissues, the responses of HSP70 showed less difference. However, the increase in HSF1 and HSF2 in these tissues differs considerable. In particular, HSF2 was induced strongly in the heart and liver. On the other hand, in heart tissue, HSF1 showed the smallest increment. These results suggest the potential role of HSF2 in assisting HSF1 in these tissues. In another in vitro experiment of hepatocyte cultures, Cd exposure caused similar patterns of goldfish HSF1 and HSF2 mRNA expression and induction of the HSP70 protein. On the other hand, an examination of the characterization of recombinant proteins showed that HSF1 undergoes a conformation change induced by heat shock above 30 °C and approaches each other in the trimer, whereas HSF2 could not sense thermal stress directly. Furthermore, immune-blot analysis of HSFs showed that both monomers and trimmers of HSF1 were observed in cadmium-induced tissues, whereas HSF2 were all in monomeric. These results show that HSF1 and HSF2 play different roles in the transcription of heat shock proteins.

Production of disulfide bond-rich peptides by fusion expression using small transmembrane proteins of Escherichia coli.

Recombinant expression in Escherichia coli allows the simple, economical, and effective production of bioactive peptides. On the other hand, the production of native peptides, particularly those rich in disulfide bonds, is a major problem. Previous studies have reported that the use of carrier proteins for fusion expression can result in good peptide yields, but few are folded correctly. In this study, two transmembrane small proteins in E. coli, YoaJ and YkgR, which both orientate with their N-termini in cytoplasm and their C-termini in periplasm, were used for fusion expression. The recombinant production of two peptides, asteropsin A (ASPA) and ß-defensin (BD), was induced in the periplasm of E. coli using a selected carrier protein. Both peptides were expressed at high levels, at yields of approximately 5-10 mg/L of culture. Mass spectrometry showed that the resulting peptide had the same molecular weight as their natural forms. After purification, single peaks were observed by reversed phase high-performance liquid chromatography (RP-HPLC), demonstrating the absence of isoforms. Furthermore, cytoplasmically expressed fusion proteins with a carrier at their C-termini did not contain disulfide bonds. This study provides new carrier proteins for fusion expression of disulfide bond-rich peptides in E. coli.

Functional expression of Carassius auratus cytochrome P4501A in a novel Shewanella oneidensis expression system and application for the degradation of benzo[a]pyrene.

Cytochrome P4501A (CYP1A) in fish has attracted an increasing interest because of its important roles in the metabolic activation of certain xenobiotics such as aromatic hydrocarbons. CYPs are reported to be expressed in yeast, insect cells and Escherichia coli, but are critical for high-level expression. Besides, this study found that the purification of recombinant goldfish CYP1A would result in a loss of enzyme activity. Because large quantities of functional CYP1A are required, it is necessary to find a suitable host with high-level expression. In the present study, a novel expression system using Shewanella oneidensis was established successfully for the production of goldfish CYP1A. A signal peptide in the expression vector leads to the high-level expression in the periplasmic space of Shewanella. The recombinant CYP1A in Shewanella reached up to 1µmol per liter of culture, and showed the typical P450 hemoprotein spectra. An ethoxyresorufin O-deethylase assay revealed the amount of functional proteins in Shewanella to be almost ten times more than those in other expression systems. Furthermore, the CYP1A-mediated degradation of benzo[a]pyrene was also observed by (1)H NMR spectroscopy. These results indicate the possible application of periplasmic fractions to obtain sufficient quantities of functional CYP1A for further studies.

Potential role of HSP90 in mediating the interactions between estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) signaling pathways.

The estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) are ligand-activated transcription factors involved in estrogen or xenobiotic exposure, whereas the 90-kDa heat shock protein (HSP90), which is a ubiquitously expressed molecular chaperone, is involved in the signal transduction process. Although the interactions between these pathways have been under investigation, the mechanisms are unclear and the potential role of HSP90 in these interactions has not been reported. The results of goldfish primary hepatocytes showed that exposure to PCB77 and 17ß-estradiol (E2) alone induced significant protein expression of cytochrome P450 1A (CYP1A) and vitellogenin (VTG), respectively. On the other hand, the combined exposure to PCB77 and E2 led to the reduction of CYP1A and VTG compared to the single treatments. Although the AhRs and ERs were naturally induced during the co-treatment, the total amount of HSP90 binding to the receptors was not changed. Furthermore, while the HSP90 chaperon activity was blocked by the specific inhibitor (geldanamycin), reciprocal inhibition between AhR and ER pathways was not observed. These findings indicate a potential role of HSP90 where competition between AhR and ER for binding to HSP90 can occur and cause reciprocal inhibition.