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.

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.

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.

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.

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.

Molecular characterization of the aryl hydrocarbon receptor (AhR) pathway in goldfish (Carassius auratus) exposure to TCDD: the mRNA and protein levels.

In bony fish or other aquatic vertebrates, the aryl hydrocarbon receptor (AhR) signaling pathway is initiated by exposure to polycyclic (or/and halogenated) aromatic hydrocarbons (PAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), which subsequently induces the up-regulated expression of a series of related genes (such as cytochrome P4501A (CYP1A)). However, a lack of applicable protein reagents hinders our further understanding of the AhR signaling pathway, which focuses only on gene-based investigations. The goldfish (Carassius auratus) is an ideal model for a study of environmental pollution in whole-Asian fresh water. Here, three sensitive and specific polyclonal antisera against goldfish AhR1, AhR2, and CYP1A proteins were developed. These antisera not only bound the in-vitro synthesized target proteins, but recognized the real proteins expressed in goldfish tissues, with minimal cross-reactivity to non-specific proteins. Together with the analysis of semi-quantitative RT-PCR and polyclonal-antibody-based sandwich ELISA, we confirmed that goldfish AhRs differed in the expression (mRNA and protein levels) patterns among test tissues. Importantly, the relative abundance of each AhR mRNA levels from the different tissues showed no obvious consistency with their protein levels. After exposure to TCDD, goldfish AhR2 showed a more sensitivity than AhR1, and stimulated CYP1A expression directly, similar with the other reported fish models. Overall, development of these antibodies in this study will allow valuable and versatile investigations to further understand the AhR signaling pathway, and different expression (mRNA and protein) patterns represent the first step in determining the regulatory mechanisms underlying the TCDD-exposed aquatic environment.

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.

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.

Analgesic effectiveness of nerve block in shoulder arthroscopy: comparison between interscalene, suprascapular and axillary nerve blocks.

PURPOSE: Postoperative pain in arthroscopic shoulder surgery cannot be easily controlled with analgesics and nerve blocks. This study shows the analgesic effect of interscalene block (ISB) and suprascapular nerve block and axillary nerve block (SSNB + ANB) in patients under patient controlled analgesia (PCA). METHODS: Sixty-one patients (26 men and 35 women) who underwent arthroscopic rotator cuff repair were selected and allocated non-randomly to one of three groups: PCA only-group, PCA with ISB-group and PCA with SSNB + ANB-group. Visual analogue scale (VAS) score, degree of satisfaction, PCA usage and incidence of nausea and vomiting were evaluated at the recovery room, 8, 16 and 24 postoperative hours. RESULTS: The VAS score of the PCA only-group was highest at the recovery room. The VAS score of the PCA with ISB-group was the lowest, however, with large fluctuations over time. Although the VAS score of the PCA with SSNB + ANB-group was higher than that of the PCA with ISB-group, it was steadily lower than the PCA-only group, without any fluctuations. The degree of satisfaction of the PCA with ISB-group was highest at the recovery room. The number of times the PCA was used at the 8-h postoperative evaluation was largest in the PCA only-group. CONCLUSIONS: The initial 24 h after surgery plays a key role in controlling pain after arthroscopic shoulder surgery. PCA with SSNB + ANB is a better anaesthetic choice than PCA with ISB or PCA only during the initial 24 h of the postoperative period. LEVEL OF EVIDENCE: Clinical study, Level II.

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.

Influence of divalent metal ions on E2-induced ER pathway in goldfish (Carassius auratus) hepatocytes.

Metal ions existing in the environment could influence the estrogen pathway in aquatic animal, but the detailed mechanism is still delusive. We here showed that in male Carassius auratus hepatocytes, copper (Cu) or cadmium (Cd), did not directly induce vitellogenin (VTG) expression. Interestingly, co-exposure with Cd²âº (or Cu²âº) and 17-ß-estradiol (E2) greatly increased the VTG level, comparing with single treatment of E2. Meanwhile, Cd²âº or Cu²âº (but not E2) triggers HSP70 expression. But, mixture of Cd²âº or Cu²âº with E2 did not obviously raise HSP70 level. E2 also had no obvious effect on reactive oxygen species. Co-treatment of Cd²âº and E2 showed no obvious increase compared to single treatment with Cd²âº. We further assume that Cd²âº-involved oxidative stress generates misfolded proteins, resulting in the competition of HSP70 proteins from a heterocomplex (with estrogen receptor). Thus, dissociation of the heterocomplex actives the receptor-ligand binding activity and promotes the E2-induced VTG expression.

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.

Persistent inflammation induces GluR2 internalization via NMDA receptor-triggered PKC activation in dorsal horn neurons.

Spinal cord GluR2-lacking AMPA receptors (AMPARs) contribute to nociceptive hypersensitivity in persistent pain, but the molecular mechanisms underlying this event are not completely understood. We report that complete Freund's adjuvant (CFA)-induced peripheral inflammation induces synaptic GluR2 internalization in dorsal horn neurons during the maintenance of CFA-evoked nociceptive hypersensitivity. This internalization is initiated by GluR2 phosphorylation at Ser(880) and subsequent disruption of GluR2 binding to its synaptic anchoring protein (GRIP), resulting in a switch of GluR2-containing AMPARs to GluR2-lacking AMPARs and an increase of AMPAR Ca(2+) permeability at the synapses in dorsal horn neurons. Spinal cord NMDA receptor-mediated triggering of protein kinase C (PKC) activation is required for the induction and maintenance of CFA-induced dorsal horn GluR2 internalization. Moreover, preventing CFA-induced spinal GluR2 internalization through targeted mutation of the GluR2 PKC phosphorylation site impairs CFA-evoked nociceptive hypersensitivity during the maintenance period. These results suggest that dorsal horn GluR2 internalization might participate in the maintenance of NMDA receptor/PKC-dependent nociceptive hypersensitivity in persistent inflammatory pain.

Estrogenic response in male bullfrog (Rana catesbeiana) hepatocytes after single or combined exposure to cadmium (Cd) and 17beta-estradiol (E2).

Contamination by heavy metals and sex hormones in a water environment is an important health issue. In this study, we investigated the estrogenic effects of cadmium (Cd) administration alone and in combination with 17beta-estradiol (E2) on the hepatocytes of male Bullfrog (Rana catesbeiana). Their vitellogenin (VTG) expression and reactive oxygen species (ROS) were analyzed upon exposure to Cd alone or to both Cd and E2. Our results suggest that the VTG levels induced by the co-treatment of 100 nM E2 and 100 nM CdCl(2) were significantly higher than those induced by 100 nM E2 alone (p < 0.05), and were comparable to vitellogenin induction observed with 1 µM E2. A similar result was observed by western blot analysis in the culture medium of hepatocytes. Meanwhile, Cd (but not E2) increased the ROS levels. These results suggest that Cd has a cooperative effect with E2 in the induction of VTG, thus acting as an estrogenic disruptor. Cd also causes oxidative stress that occurs with the enhanced vitellogenesis.

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.

Aromatic-participant interactions are essential for disulfide-bond-based trimerization in human heat shock transcription factor 1.

Heat shock transcription factor 1 (HSF1) is a central regulator in the heat shock response. However, its trimerization mechanism remains unclear. Here, we demonstrate that three conserved aromatic amino acids (Trp37, Tyr60, and Phe104) are essential for HSF1 trimerization. Point mutation and fluorescence spectroscopy experiments show that an intramolecular interaction between Tyr60 and alpha-helix 1 in the DNA-binding domain stabilizes the HSF1 structure upon heat stress. Furthermore, intermolecular aromatic-aromatic interaction between the Trp37 and Phe104 supports the approach with the Cys36 and Cys103. Thus, the existence of two differential interactions facilitates the formation of intermolecular disulfide bonds, leading to the heat-induced HSF1 trimerization.