Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin.

Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae.

Clinical Outcomes and Factors Associated With Aortic Shrinkage After Thoracic Endovascular Aortic Repair for Aneurysmal Chronic Aortic Dissection.

PURPOSE: The effectiveness of thoracic endovascular aortic repair (TEVAR) for chronic aortic dissection (AD) with aneurysmal degeneration remains controversial. We retrospectively investigated clinical outcomes and assessed predictors of aortic shrinkage after TEVAR for chronic aneurysmal AD. MATERIALS AND METHODS: Between January 2010 and December 2021, 70 patients with double-barrel-type chronic AD were enrolled. Major intimal tears in thoracic aorta were covered by stent graft. Early and late clinical outcomes, and diameter change of downstream aorta during follow-up period were reviewed. Subsequently, factors associated with aortic shrinkage were assessed by logistic regression analysis. RESULTS: Mean age was 63 (interquartile range [IQR]: 54-68) years, 54 (80%) men, median duration from AD onset was 4 (IQR: 1-10) years, and maximum aortic diameter was 53 (IQR: 49-58) mm. Supra-aortic debranching procedure was required in 57 (81%) patients. Early aorta-related death occurred in 2 (3%) patients. Both stroke and spinal cord ischemia occurred in 1 (2%) patient. Five-year freedom rates from aorta-related death and reintervention were 96% and 51%, respectively. Sixty-four patients underwent follow-up computed tomography (84%) 1 year after TEVAR, with 33 (52%) achieving aortic shrinkage. In multivariable analysis, duration from AD onset (per year) (odds ratio [OR]: 0.82, 0.70-0.97; p=0.017) and maximum aortic-diameter ratio between aortic arch and descending aorta (per 0.1) (morphologic index; OR: 1.34, 1.04-1.74; p=0.023) were independent aortic shrinkage predictors. CONCLUSIONS: Thoracic endovascular aortic repair for chronic AD with aneurysmal degeneration achieved satisfactory survival outcomes, but with a considerable reintervention rate. Duration from AD onset and preoperative aortic morphology could affect post-TEVAR aortic shrinkage. Earlier intervention could lead to better aortic shrinkage. CLINICAL IMPACT: Thoracic endovascular aortic repair for chronic aortic dissection with aneurysmal degeneration showed low incidence of early and late aorta-related death. By contrast, aortic shrinkage rate was low with high incidence of reintervention to the residual downstream aorta. According to the assessment of preoperative variables, chronicity and aortic morphology could predict postoperative aortic shrinkage.

Impact of Patent Lumbar Arteries on Aneurysm Sac Enlargement with Type II Endoleak after Endovascular Aneurysm Repair.

OBJECTIVE: This study aimed to investigate the impact of the number of patent lumbar arteries (LAs) on sac enlargement after endovascular aneurysm repair (EVAR). METHODS: This was a retrospective cohort single centre registry study. Between January 2006 and December 2019, 336 EVARs were reviewed using a commercially available device excluding type I or type III endoleaks during a follow up of ≥ 12 months. Patients were divided into four groups based on the pre-operative patency of the inferior mesenteric artery (IMA) and high (≥ 4) or low (≤ 3) number of patent LAs: Group 1, patent IMA and high number of patent LAs; Group 2, patent IMA and low number of patent LAs; Group 3, occluded IMA and a high number of patent LAs; Group 4, occluded IMA and low number of patent LAs. RESULTS: Groups 1, 2, 3, and 4 included 124, 104, 45, and 63 patients, respectively. The median follow up duration was 65.1 months. Significant differences in the incidence of overall type II endoleak (T2EL) at discharge between Group 1 and Group 2 (59.7% vs. 36.5%, p < .001) and between Group 3 and Group 4 (33.3% vs. 4.8%, p < .001) were observed. In patients with a pre-operatively patent IMA, the rate of freedom from aneurysm sac enlargement was significantly lower in Group 1 than in Group 2 (69.0% vs. 81.7% five years after EVAR, p < .001). In patients with a pre-operatively occluded IMA, the freedom rate from aneurysm sac enlargement was not significantly different between Groups 3 and Group 4 (95.0% vs. 100% five years after EVAR, p = .075). CONCLUSION: A high number of patent LAs seemed to have a significant role in sac enlargement with T2EL when the IMA was patent pre-operatively, whereas a high number of patent LAs seemed to have limited influence on sac enlargement when the IMA was occluded pre-operatively.

Impact of the Patency of Inferior Mesenteric Artery on 7-Year Outcomes After Endovascular Aneurysm Repair.

PURPOSE: The impact of preoperative patent inferior mesenteric artery (IMA) on late outcomes following endovascular aneurysm repair (EVAR) remains unclear. This study aimed to investigate the specific influence of IMA patency on 7-year outcomes after EVAR. MATERIALS AND METHODS: In this retrospective cohort study, 556 EVARs performed for true abdominal aortic aneurysm cases between January 2006 and December 2019 at our institution were reviewed. Endovascular aneurysm repairs performed using a commercially available device with no type I or type III endoleak (EL) during follow-up and with follow-up ≥12 months were included. A total of 336 patients were enrolled in this study. The cohort was divided into the patent IMA group and the occluded IMA group according to preoperative IMA status. The late outcomes, including aneurysm sac enlargement, reintervention, and mortality rates, were compared between both groups using propensity-score-matched data. RESULTS: After propensity score matching, 86 patients were included in each group. The median follow-up period was 56 months (interquartile range: 32-94 months). The incidence of type II EL at discharge was 50% in the patent IMA group and 19% in the occluded IMA group (p<0.001). The type II EL from IMA and lumbar arteries was significantly higher in the patent IMA group than in the occluded IMA group (p<0.001 and p=0.002). The rate of freedom from aneurysm sac enlargement with type II EL was significantly higher in the occluded IMA group than in the patent IMA group (94% vs 69% at 7 years; p<0.001). The rate of freedom from reintervention was significantly higher in the occluded IMA group than in the patent IMA group (90% vs 74% at 7 years; p=0.007). Abdominal aortic aneurysm-related death and all-cause mortality did not significantly differ between groups (p=0.32 and p=0.34). CONCLUSIONS: Inferior mesenteric artery patency could affect late reintervention and aneurysm sac enlargement but did not have a significant impact on mortality. Preoperative assessment and embolization of IMA might be an important factor for improvement in late EVAR outcomes. CLINICAL IMPACT: The preoperative patency of the inferior mesenteric artery was significantly associated with a higher incidence of sac enlargement and reintervention with type II endoleak following endovascular aneurysm repair, even after adjustment for patient background. Preoperative assessment and embolization of inferior mesenteric artery might be an important factor for improvement in late EVAR outcomes.

Risk Analysis of Aneurysm Sac Enlargement Caused by Type II Endoleak after Endovascular Aortic Repair.

BACKGROUND: Although the preoperative risk factors associated with the occurrence of type II endoleak (ETII) after endovascular aortic repair (EVAR) have gradually become more evident, the preoperative risk factors associated with aneurysm sac enlargement caused by ETII remain unclear. This study aimed to determine the preoperative risk factors associated with aneurysm sac enlargement caused by ETII after EVAR. METHODS: This retrospective cohort study reviewed 519 EVARs performed for true abdominal aortic aneurysm between January 2006 and December 2018 at our institution. EVARs using commercially available bifurcated devices with no type I or III endoleaks during follow-up and with ≥12 months follow-up were included. A total of 320 patients were enrolled in the study. To identify the preoperative risk factors of sac enlargement after EVAR, Cox regression analysis was used to assess preoperative data. RESULTS: The median follow-up period was 60.8 months. Overall, 135 of 320 patients (42%) had ETII during follow-up, and 47 of 135 patients (35%) developed aneurysm sac enlargement. Multivariate analysis revealed that chronic kidney disease (CKD) stage ≥4 (hazard ratio [HR], 4.65; 95% confidence interval [CI], 2.13-10.15; P = 0.001), patent inferior mesenteric artery (IMA) (HR, 17.85; 95% CI, 2.46-129.73; P< 0.001), and number of patent lumbar arteries (LAs) (HR, 1.37; 95% CI, 1.13-1.68; P= 0.002) were risk factors of aneurysm sac enlargement caused by ETII. CONCLUSIONS: CKD stage ≥4, patent IMA, and number of patent LAs were independent risk factors for aneurysm sac enlargement after EVAR. In particular, patent IMA had the highest HR and seemed to have the greatest impact on long-term aneurysm sac enlargement. Hence, taking preoperative measures to address a patent IMA appears to be important in reducing the incidence of sac enlargement.

Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane ß-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane ß-hairpin region. KEY POINTS: • Cry46Ab mutants were constructed by targeting the putative transmembrane ß-hairpin region. • Charged residues within the ß-hairpin control the flux of ions through channel pores. • Channel-pore cation selectivity is correlated with insecticidal activity.

A single amino acid gates the KcsA channel.

The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel.

Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane.

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non-cargo proteins during COPII vesicle formation using single-molecule microscopy combined with an artificial planar lipid bilayer. Single-molecule analysis showed that the Sar1p-Sec23/24p-cargo complex, but not the Sar1p-Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non-cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non-cargo proteins from the COPII vesicles.

Random Mutational Analysis Targeting Residue K155 within the Transmembrane ß-Hairpin of the Mosquitocidal Mpp46Ab Toxin.

Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane ß-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane ß-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab.

Successful surgery for secondary aortoduodenal fistula based on 18F-fluorodeoxyglucose positron emission tomography/computed tomography findings.

Secondary aortoduodenal fistula (sADF) is a critical late complication of abdominal aortic repair, requiring complete excision of the infected prosthesis. However, this is a highly invasive procedure for the elderly. We describe a case of sADF repair in a 76-year-old woman. Through 18F (fluorine-18)-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography mapping, focal high FDG uptake at the sADF site, right medial limb, and ligated left lateral limb of the prosthesis was detected. The duodenal and prosthetic grafts were partially resected. The proximal and distal anastomotic segments, with no FDG uptake, were retained. The abdominal aorta was reconstructed using a bovine pericardium roll and femorofemoral bypass. Thus, FDG positron emission tomography/computed tomography mapping of the infection site could help in such cases.

Challenging endovascular treatment of a thoracic saccular aneurysm in extensive coral reef aorta.

Coral reef aorta (CRA) is characterized by heavily calcified obstructive lesions in the aorta. Thoracic endovascular aortic repair (TEVAR) is an established, less invasive procedure for aortic diseases; however, aortic occlusive diseases are commonly treated with conventional open surgery, and there are no reports of TEVAR in patients with a saccular aneurysm in CRA. We present a 72-year-old frail woman with a descending thoracic saccular aneurysm in CRA; therefore, we performed TEVAR. Although we had difficulty in advancing the stent graft system because it was caught in the severely calcified aorta, we finally succeeded in excluding the aneurysm.

Impact of radiation to the eye of operators during endo-cardiovascular surgery and the importance of protection.

OBJECTIVE: To verify the amount of radiation exposure to the eye of operators during endocardiovascular surgery (ECVS) in hybrid operating room (HOR), which increases the risk of cataracts in surgeons. METHODS: Fifty cases of ECVS (including 36 transcatheter aortic valve implantation and 14 thoracic endovascular repair) using the transfemoral approach performed from February 2020 to July 2020 were included. A measurement device was attached to the left side of the head of the operators and their assistants to measure the cumulative dose (CD) of intraoperative radiation exposure. The subjects were divided into the control group (Group C, n = 26), received conventional protection using the protective curtain in HOR and the protected group (Group R, n = 24), received conventional protection and protection sheet. The normalized CD by dose area product (CD/DAP) was evaluated. RESULTS: The CD/DAP of the surgeons was significantly decreased in Group R, averaging at 5.97 µSV/Gy cm2 in Group C group and 4.40 µSV/Gy cm2 in Group R (p < 0.01). Moreover, CD/DAP of the assistant was significantly reduced in the Group R, with an average of 1.87 µSV/Gy cm2 in the Group C and 1.01 µSV/Gy cm2 in Group R (p < 0.01). CONCLUSIONS: The radiation exposure to the surgeon's eye could be significantly reduced by protection sheet.

Kastor and Polluks polypeptides encoded by a single gene locus cooperatively regulate VDAC and spermatogenesis.

Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility.

Role of the KcsA channel cytoplasmic domain in pH-dependent gating.

The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domain's ability to sense and respond to pH.

Rearrangements in the KcsA cytoplasmic domain underlie its gating.

A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (P(o)) was approximately 1 at pH 4 and approximately 0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.

Direct manipulation of a single potassium channel gate with an atomic force microscope probe.

Ion channels are membrane proteins that regulate cell functions by controlling the ion permeability of cell membranes. An ion channel contains an ion-selective pore that permeates ions and a sensor that senses a specific stimulus such as ligand binding to regulate the permeability. The detailed molecular mechanisms of this regulation, or gating, are unknown. Gating is thought to occur from conformational changes in the sensor domain in response to the stimulus, which results in opening the gate to permit ion conduction. Using an atomic force microscope and artificial bilayer system, a mechanical stimulus is applied to a potassium channel, and its gating is monitored in real time. The channel-open probability increases greatly when pushing the cytoplasmic domain toward the membrane. This result shows that a mechanical stimulus at the cytoplasmic domain causes changes in the gating and is the first to show direct evidence of coupling between conformational changes in the cytoplasmic domain and channel gating. This novel technology has the potential to be a powerful tool for investigating the activation dynamics in channel proteins.

Channels formed by amphotericin B covalent dimers exhibit rectification.

Amphotericin B (AmB) is a widely used antifungal antibiotic with high specificity for fungi. We previously synthesized several covalently conjugated AmB dimers to clarify the AmB channel structure. Among these dimers, that with an aminoalkyl linker was found to exhibit potent hemolytic activity. We continue this work by investigating the channel activity of the dimer, finding that all channels comprised of AmB dimers show rectification. The direction of the dimer channel in the membrane depended on the electric potential at which the dimer channel was formed. On the other hand, only about half the monomer channels showed rectification. In addition, these channels were easily switched from a rectified to a nonrectified state following voltage stimulation, indicating instability. We propose a model to describe the AmB channel structure that explains why AmB dimer channels necessarily show rectification.

Development of an automated system to measure ion channel currents using a surface-modified gold probe.

Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes' hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

Simultaneous optical and electrical single channel recordings on a PEG glass.

Single molecule imaging of working ion-channels is much more difficult than that of water-soluble proteins because of the fragile nature of membranes and lateral diffusion of particles in the membranes, which does not allow fluorescent contamination for optical single channel recording. In this report, we reconstituted maxi-potassium channels from porcine uterine smooth muscle into artificial planar bilayers formed on poly(ethylene glycol) (PEG) modified glass and performed simultaneous optical and electrical recording of the single channels. The channels were immobilized in the membranes by anchoring to PEG molecules on the glass. The technique developed in this study should pave the way for single molecule pharmacology of ion-channels.

Single channel properties of lysenin measured in artificial lipid bilayers and their applications to biomolecule detection.

Single channel currents of lysenin were measured using artificial lipid bilayers formed on a glass micropipette tip. The single channel conductance for KCl, NaCl, CaCl(2), and Trimethylammonium-Cl were 474 ± 87, 537 ± 66, 210 ± 14, and 274 ± 10 pS, respectively, while the permeability ratio P(Na)/P(Cl) was 5.8. By adding poly(deoxy adenine) or poly(L-lysine) to one side of the bilayer, channel currents were influenced when membrane voltages were applied to pass the charged molecules through the channel pores. Current inhibition process was concentration-dependent with applied DNA. As the current fluctuations of α-hemolysin channels is often cited as the detector in a molecular sensor, these results suggest that by monitoring channel current changes, the lysenin channel has possibilities to detect interactions between it and certain biomolecules by its current fluctuations.