Evaluating bio-physicochemical properties of raw powder prepared from whole larvae containing liquid silk of the domestic silkworm.

The domestic silkworm, Bombyx mori, has been widely used in silk production for centuries. It is also used as a bioreactor by the textile and pharmaceutical industries to mass produce recombinant bioactive proteins containing silk-based materials. Furthermore, silkworms are well-known as a source of food and have also been orally administered to prevent and treat several human disorders. In this study, we aimed to investigate the inherent bio-physicochemical properties of edible silkworms to accurately evaluate their clinical and nutritional potential. We prepared raw powder from whole larvae of silkworm. The yield rate of the powder derived from dried larvae was almost 100% (98.1-99.1% in replicates). As "percentage yield" translates to "Budomari" in Japanese, this raw powder was named "B100rw." We further prepared B100dn that was denatured through autoclaving. Thereafter, we examined whether B100rw sustained the original bio-physicochemical properties by comparing it with B100dn. There was no significant difference in nutritional content between B100rw and B100dn. B100rw contained proteins derived from silkworm larvae and mulberry leaves, whereas the proteins of B100dn were mostly degraded. On measuring the enzymatic activity of both powders using trehalase as an indicator enzyme, B100rw was found to maintain trehalase activity. B100rw also maintained a random coil conformation, similar to that of liquid silk. This suggested that B100rw sustained the unique bio-physicochemical properties of living larvae. These findings may facilitate the development of novel food products or orally administered vaccines.

Characterization of a novel format scFv×VHH single-chain biparatopic antibody against metal binding protein MtsA.

Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.

"Rich arginine and strong positive charge" antimicrobial protein protamine: From its action on cell membranes to inhibition of bacterial vital functions.

Protamine, an antimicrobial protein derived from salmon sperm with a molecular weight of approximately 5 kDa, is composed of 60-70 % arginine and is a highly charged protein. Here, we investigated the mechanism of antimicrobial action of protamine against Cutibacterium acnes (C. acnes) focusing on its rich arginine content and strong positive charge. Especially, we focused on the attribution of dual mechanisms of antimicrobial protein, including membrane disruption or interaction with intracellular components. We first determined the dose-dependent antibacterial activity of protamine against C. acnes. In order to explore the interaction between bacterial membrane and protamine, we analyzed cell morphology, zeta potential, membrane permeability, and the composition of membrane fatty acid. In addition, the localization of protamine in bacteria was observed using fluorescent-labeled protamine. For investigation of the intracellular targets of protamine, bacterial translation was examined using a cell-free translation system. Based on our results, the mechanism of the antimicrobial action of protamine against C. acnes is as follows: 1) electrostatic interactions with the bacterial cell membrane; 2) self-internalization into the bacterial cell by changing the composition of the bacterial membrane; and 3) inhibition of bacterial growth by blocking translation inside the bacteria. However, owing to its strong electric charge, protamine can also interact with DNA, RNA, and other proteins inside the bacteria, and may inhibit various bacterial life processes beyond the translation process.

Development of Benziodarone Analogues with Enhanced Potency for Selective Binding to Transthyretin in Human Plasma.

Transthyretin amyloidosis is a fatal disorder caused by transthyretin amyloid aggregation. Stabilizing the native structure of transthyretin is an effective approach to inhibit amyloid aggregation. To develop kinetic stabilizers of transthyretin, it is crucial to explore compounds that selectively bind to transthyretin in plasma. Our recent findings demonstrated that the uricosuric agent benziodarone selectively binds to transthyretin in plasma. Here, we report the development of benziodarone analogues with enhanced potency for selective binding to transthyretin in plasma compared to benziodarone. These analogues featured substituents of chlorine, bromine, iodine, a methyl group, or a trifluoromethyl group, at the 4-position of the benzofuran ring. X-ray crystal structure analysis revealed that CH···O hydrogen bonds and a halogen bond are important for the binding of the compounds to the thyroxine-binding sites. The bioavailability of benziodarone analogues with 4-Br, 4-Cl, or 4-CH3 was comparable to that of tafamidis, a current therapeutic agent for transthyretin amyloidosis.

Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine.

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm2C), respectively, in the anticodon of tRNAIle to decipher AUA codon. L and agm2C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAsIle recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N6-threonylcarbamoyladenosine (ct6A), another tRNA modification, and revealed a molecular basis how ct6A contributes to efficient decoding.

Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1.

In response to environmental changes, cells flexibly and rapidly alter gene expression through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is downregulated in response to an excess amount of boric acid in the environment through upstream open reading frames (uORFs) that consist of only AUG and stop codons. However, the molecular details of how this minimum uORF controls translation of the downstream main ORF in a boric acid-dependent manner have remained unclear. Here, by combining ribosome profiling, translation complex profile sequencing, structural analysis with cryo-electron microscopy and biochemical assays, we show that the 80S ribosome assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 on the 80S ribosome on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.

Direct visualization of ribosomes in the cell-free system revealed the functional evolution of aminoglycoside.

The rapid emergence of multi-drug-resistant bacteria has raised a serious public health concern. Therefore, new antibiotic developments have been highly desired. Here, we propose a new method to visualize antibiotic actions on translating ribosomes in the cell-free system under macromolecular crowding conditions by cryo-electron microscopy, designated as the DARC method: the Direct visualization of Antibiotic binding on Ribosomes in the Cell-free translation system. This new method allows for acquiring a more comprehensive understanding of the mode of action of antibiotics on the translation inhibition without ribosome purification. Furthermore, with the direct link to biochemical analysis at the same condition as cryo-EM observation, we revealed the evolution of 2-DOS aminoglycosides from dibekacin (DBK) to arbekacin (ABK) by acquiring the synthetic tailored anchoring motif to lead to stronger binding affinity to ribosomes. Our cryo-EM structures of DBK and ABK bound ribosomes in the cell-free environment clearly depicted a synthetic tailored γ-amino-α-hydroxybutyryl (HABA) motif formed additional interactions with the ribosome enhancing antibiotic bindings. This new approach would be valuable for understanding the function of antibiotics for more efficient drug development.

Resveratrol Derivatives Inhibit Transthyretin Fibrillization: Structural Insights into the Interactions between Resveratrol Derivatives and Transthyretin.

Hereditary ATTR amyloidosis is a disease caused by the deposition of amyloid fibrils formed by mutated transthyretin (TTR), a protein that binds to thyroid hormone in the serum, in the organs. The development of a small molecule that binds to and stabilizes TTR is a promising strategy for the treatment of ATTR amyloidosis. In the present study, we demonstrated that the resveratrol derivatives including pterostilbene available as a dietary supplement inhibit the fibrillization of V30M-TTR to the same extent as the approved drug tafamidis. Furthermore, based on a thermodynamic and X-ray crystallographic analysis, the binding of the resveratrol derivative to TTR was shown to be enthalpy-driven, with the binding enthalpy being acquired by hydrogen bonding to S117. Moreover, direct observation of hydrogen atoms by neutron crystallography provided details of the hydrogen bond network by S117 and emphasized the importance of the CH···π interaction by L110 in the ligand binding.

Comparison of the tidal volume by the recruitment maneuver combined with positive end-expiratory pressure for mechanically ventilated children.

The recruitment maneuver (RM) combined with PEEP to prevent atelectasis have beneficial effects. However, the change in tidal volume (VT) due to RM combined with PEEP in pediatric patients during the induction of general anesthesia is unknown. Therefore, we assessed the effects of RM combined with PEEP on VT. Pediatric patients were divided into three groups: infants, preschool children, and school children. The RM was performed by maintaining pressure control continuous mandatory ventilation (PC-CMV) with a 15 cmH2O and PEEP increase of 5 cmH2O. VT, respiratory function and hemodynamics were monitored before and after RM combined with PEEP. VT (mL) /ideal body weight (kg) before vs. after RM combined with PEEP were 9 vs 12 mL/kg (p < 0.05) in the infants, 9 vs 11 mL/kg (p < 0.05) in the preschool children, 8 vs 10 mL/kg (p < 0.05) in the school children, respectively. HR and BP before and after RM combined with PEEP increased by 2-3% and decreased by 4-7% in all groups. RM combined with PEEP resulted in an increase in VT per ideal body weight (1.1-1.2%). Therefore, this RM combined with PEEP method might improve the lung function in pediatric patients.

Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth.

Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.

Positional Change Used to Manage Postextubation Respiratory Failure in a Child With Cerebral Palsy.

Dental treatment for patients with cerebral palsy (CP) is often performed under general anesthesia due to involuntary movements that can render dental treatment difficult. Since CP is often accompanied by spasticity, care must be taken when positioning patients during general anesthesia. We report the management of a 14-year-old girl with CP and epilepsy undergoing general anesthesia for dental treatment who experienced respiratory failure due to acute thoracoabdominal muscle hypertonia after extubation. She had a history of cardiac arrest due to respiratory failure caused by acute muscle hypertonia and successful resuscitation. General anesthesia was induced after careful positioning of the patient to prevent spastic muscle stretching, and the dental treatment was completed without complications. However, upon awakening after extubation, the patient developed respiratory failure due to acute muscle hypertonia. The patient was resedated and repositioned from a supine to a sitting position, and her symptoms improved. There was no recurrence of muscle hypertonia, and she recovered fully without complications. In this case, respiratory failure associated with acute muscle hypertonia was successfully managed by position change after initial treatment with positive-pressure ventilation and propofol. It is important to be prepared for the possibility of respiratory failure associated with acute muscle hypertonia and its countermeasures when providing general anesthesia for patients with CP.

Singleton pregnancy using in vitro fertilization or intracytoplasmic sperm injection does not increase risk of bleeding in cesarean delivery: a retrospective cohort study.

PURPOSE: Several studies indicate that assisted reproductive technology (ART) including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) pregnancies carries increased risk of complications including postpartum hemorrhage (PPH). However, the association between IVF/ICSI and the bleeding risk particularly in cesarean delivery has not been systematically assessed. The aim of this study was to evaluate bleeding risk during and after cesarean delivery in parturients who conceived using IVF or ICSI. METHODS: This is a retrospective observational study analyzing data from 310 parturients who underwent cesarean delivery: 155 who had conceived using IVF or ICSI (IVF/ICSI group) and 155 age and year of delivery matched controls who had conceived spontaneously (control group). The primary outcome measure was the amount of blood lost during and within 24 h after cesarean delivery. Secondary outcome measure was the incidence of severe PPH. With 132 parturients in each group, we had 90% power to detect a 200 mL difference in the bleeding amount, at a 0.05 two-sided significance level. RESULTS: The amount of bleeding in the IVF/ICSI group was 1234 ± 669 mL, which was 124 mL (11.2%) greater than that in the control group (95% CI - 34 to 282; p = 0.12). The incidence of severe PPH in the IVF/ICSI group and in the control group was 23.9% and 16.8%, respectively (p = 0.16), and the unadjusted odds ratio was 1.6 (95% CI, 0.9-2.7; p = 0.12). No significant independent effect of IVF/ICSI on the bleeding amount and the incidence of severe PPH was observed in multivariable regression analyses (p = 0.22, p = 0.16). CONCLUSION: In this study, IVF and ICSI were not associated with increasing risk of bleeding in cesarean delivery.

The difference in rotation angle of the distal endotracheal tube through nasal approach.

BACKGROUND: Nasal intubation using a fiberoptic scope is a useful technique. In clinical practice, we have experienced difficulty in advancing the endotracheal tube (ETT) over the fiberoptic scope because of resistance to the passage of the ETT against rotation in the nasal cavity, when it gets hung up on structures of the laryngeal inlet. Several maneuvers have been proposed to overcome this difficulty. The gap between the tip of the ETT and the fiberoptic scope can be reduced using a thicker fiberoptic scope and a thinner ETT. Moreover, simultaneous rotation of the fiberoptic scope and ETT could lead to successful intubation by reducing impingement on the ETT. However, the discrepancy between these rotation angles is unclear. This observational prospective study aimed to investigate the discrepancy in the rotation angle between the ETT and fiberoptic scope during nasal intubation. METHODS: The patients (aged 20-80 years) who underwent nasal intubation for oral and maxillofacial surgery participated in three sizes of preformed nasal ETT and were intubated using a fiberoptic scope. They were divided into three groups; the ETT internal diameter (ID) 6.5 mm (6.5 group), ID 7.0 mm (7.0 group), and ID 7.5 mm (7.5 group). The ETT was then inserted through the nasal cavity into the pharynx. After the fiberoptic scope was advanced through the ETT above the glottis, simultaneous rotation by both the proximal end of the fiberoptic scope and ETT was performed in 90° and 180° in both right (clockwise) and left (counterclockwise) directions, and the rotation angle at the distal end of the ETT was monitored using a video laryngoscope (Pentax-AWS). RESULTS: A total of 39 patients were included in the study. When both the proximal end of the fiberscope and ETT were simultaneously rotated by 90°, in the 6.5 group (n = 13), the distal end of the ETT rotated by 47.8 ± 1.5°. In the 7.0 °group (n = 13), the distal end of the ETT rotated by 45.5 ± 1.0°. In the 7.5 group (n = 13), the distal end of the ETT rotated by 39.9 ± 1.0°. When the proximal end of the fiberscope and ETT were rotated by 180°, in the 6.5 group, the distal end of the ETT rotated by 166.2 ± 2.5°. In the 7.0 group, the distal end of the ETT rotated by 145.7 ± 2.2°. In the 7.5 group, the distal end of the ETT rotated by 115.1 ± 2.0°. All rotation angles in the distal end of the ETT were significantly lower than those in both the proximal end of the fiberscope and ETT (p < 0.05). Rotating right by 180° was significantly different among the three groups (p < 0.05), although rotating right by 90° was not significantly different. Similar results were obtained for the left rotation. CONCLUSION: Simultaneous rotation by the proximal end of the ETT and fiberscope above the glottis for the nasal approach induced significant differences in the distal end of the ETT. The larger tube lagged by the resistance of the nasal passages during rotation. Therefore, the ETT does not rotate as much as the rotation angle. TRIAL REGISTRATION: This prospective observational study was conducted after receiving approval from the Ethics Review Board of Kyushu University Hospital (Approval No. 30-447).

Rafoxanide, a salicylanilide anthelmintic, interacts with human plasma protein transthyretin.

Transthyretin (TTR) is a carrier protein for thyroid hormone thyroxine (T4 ) in plasma, placental cytosol, and cerebrospinal fluid. While the potential toxicity of small molecules that compete with T4 for binding to TTR should be carefully studied, these small molecules can also serve as anti-ATTR amyloidosis drugs by stabilizing the TTR structure. Here, we demonstrated that rafoxanide, an EU-approved anthelmintic drug for domesticated animals, binds to the T4 -binding site of TTR. An intrinsic fluorescence quenching assay showed that rafoxanide also binds to the thyroid hormone-related proteins, including serum albumin and thyroid hormone receptor ß. Rafoxanide strongly inhibited TTR amyloidogenesis in fibrillization assay, but the binding of rafoxanide to TTR was interfered with in human plasma, probably due to interactions with thyroid hormone-related proteins. Protein crystallography provided clues for the optimization of binding affinity and selectivity. Our findings emphasize the importance of considering rafoxanide as both a possible thyroid-disrupting chemical and a lead compound for the development of new ATTR amyloidosis inhibitors.

Bilateral video-assisted thoracic surgery for esophageal cancer with left inferior pulmonary vein invasion following chemoradiation therapy.

BACKGROUND: The surgical strategy for thoracic esophageal cancer that invades the lungs is controversial. In particular, invasion of the pulmonary vein is often regarded unresectable. We successfully applied bilateral video-assisted thoracic surgery (VATS) in esophagectomy for esophageal cancer with left inferior pulmonary vein invasion following induction chemoradiotherapy (CRT), with a favorable response. CASE PRESENTATION: A 64-year-old woman was diagnosed with squamous cell carcinoma of the lower third of the esophagus. Computed tomography (CT) revealed that the tumor was suspected to be invading the main trunk of the left lower pulmonary vein and left lower lung. We initiated induction CRT comprising 5-fluorouracil, cisplatin, and concurrent radiotherapy at 50.4 Gy/28Fr. CT revealed shrinkage of the tumor, and the main trunk of the left inferior pulmonary vein was released from the tumor invasion. We considered the tumor to be completely resectable. VATS esophagectomy is usually performed using a right-sided approach. However, the right-sided approach is inappropriate for evaluating tumors around the left inferior pulmonary vein. We started with left-sided VATS to determine tumor resectability and dissected between the esophagus and the main trunk of the left inferior pulmonary vein. We only needed to perform partial resection of the left lower lobe. We then performed a right-sided VATS esophagectomy and lymphadenectomy with partial en bloc resection of the left lower lobe. Following this, we performed hand-assisted laparoscopic proximal gastrectomy and reconstruction using the gastric remnant. The postoperative course was uneventful. The patient was discharged on postoperative day 14. Histopathological examination of the surgical specimen revealed a complete pathological response without any remnant tumor or lymph node metastasis. There were no signs of recurrence or metastasis at the 1-year follow-up. CONCLUSIONS: Curative resection for thoracic esophageal cancer that invades the pulmonary vein could be possible via the bilateral VATS approach following induction CRT with a favorable response.

Synthesis of epitope-targeting nanobody based on native protein-protein interactions for FtsZ filamentation suppressor.

Phage display and biopanning are powerful tools for generating binding molecules for a specific target. However, the selection process based only on binding affinity provides no assurance for the antibody's affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein-protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the nanobody, and a nonrelated CDR loop (in the grafted nanobody) was randomized to create a phage display library. In this construction of nanobodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence added functionality to the nanobody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein-protein interactions. To apply for biological tool with growth screening, model nanobodies with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) from Staphylococcus aureus were constructed and completely inhibited the polymerization of FtsZ as a function. Consequently, the expression of these nanobodies drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein-protein interactions for steady epitope-specific evolutionary engineering.

Benziodarone and 6-hydroxybenziodarone are potent and selective inhibitors of transthyretin amyloidogenesis.

Transthyretin amyloidosis is a progressive systemic disorder that is caused by the amyloid deposition of transthyretin in various organs. Stabilization of the native transthyretin is an effective strategy for the treatment of transthyretin amyloidosis. In this study we demonstrate that the clinically used uricosuric agent benziodarone is highly effective to stabilize the tetrameric structure of transthyretin. An acid-induced aggregation assay showed that benziodarone had strong inhibitory activity similar to that of tafamidis, which is currently used as a therapeutic agent for transthyretin amyloidosis. Moreover, a possible metabolite, 6-hydroxybenziodarone, retained the strong amyloid inhibitory activity of benziodarone. An ex vivo competitive binding assay using a fluorogenic probe showed that benziodarone and 6-hydroxybenziodarone were highly potent for selective binding to transthyretin in human plasma. An X-ray crystal structure analysis revealed that the halogenated hydroxyphenyl ring was located at the entrance of the thyroxine binding channel of transthyretin and that the benzofuran ring was located in the inner channel. These studies suggest that benziodarone and 6-hydroxybenziodarone would potentially be effective against transthyretin amyloidosis.