Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells.

Introduction: Basic fibroblast growth factor (bFGF, FGF2) and integrin α6ß1 are important for maintaining the pluripotency of human pluripotent stem cells (hPSCs). Although bFGF-integrin binding contributes to biofunctions in cancer cells, the relationship in hPSCs remains unclear. Methods: To investigate the relationship between bFGF and integrin in human induced pluripotent stem cells (hiPSCs), we generated recombinant human bFGF wild-type and mutant proteins, that do not bind to integrin, FGFR, or both. We then cultured hiPSCs with these recombinant bFGF proteins. To evaluate the abilities of recombinant bFGF proteins in maintaining hPSC properties, pluripotent markers, ERK activity, and focal adhesion structure were analyzed through flow cytometry, immunofluorescence (IF), and immunoblotting (IB). Result: We identified an interaction between bFGF and integrin α6ß1 in vitro and in hiPSCs. The integrin non-binding mutant was incapable of inducing the hPSC properties, such as proliferation, ERK activity, and large focal adhesions at the edges of hiPSC colonies. Signaling induced by bFGF-FGFR binding was essential during the first 24 h after cell seeding for maintaining the properties of hPSCs, followed by a shift towards intracellular signaling via the bFGF-integrin interaction. The mixture of the two bFGF mutants also failed to maintain hPSC properties, indicating that bFGF binds to both FGFR and integrin. Conclusion: Our study demonstrates that the integrin-bFGF-FGFR ternary complex maintains the properties of hPSCs via intracellular signaling, providing insights into the functional crosstalk between bFGF and integrins in hiPSCs.

Extracting time series matching a small-angle X-ray scattering profile from trajectories of molecular dynamics simulations.

Solving structural ensembles of flexible biomolecules is a challenging research area. Here, we propose a method to obtain possible structural ensembles of a biomolecule based on small-angle X-ray scattering (SAXS) and molecular dynamics simulations. Our idea is to clip a time series that matches a SAXS profile from a simulation trajectory. To examine its practicability, we applied our idea to a multi-domain protein ER-60 and successfully extracted time series longer than 1 micro second from trajectories of coarse-grained molecular dynamics simulations. In the extracted time series, the domain conformation was distributed continuously and smoothly in a conformational space. Preferred domain conformations were also observed. Diversity among scattering curves calculated from each ER-60 structure was interpreted to reflect an open-close motion of the protein. Although our approach did not provide a unique solution for the structural ensemble of the biomolecule, each extracted time series can be an element of the real behavior of ER-60. Considering its low computational cost, our approach will play a key role to identify biomolecular dynamics by integrating SAXS, simulations, and other experiments.

Prediction Models for Two Year Overall Survival and Amputation Free Survival After Revascularisation for Chronic Limb Threatening Ischaemia.

OBJECTIVE: The aim of this study was to create prediction models for two year overall survival (OS) and amputation free survival (AFS) after revascularisation in patients with chronic limb threatening ischaemia (CLTI). METHODS: This was a retrospective analysis of prospectively collected multicentre registry data (JAPAN Critical Limb Ischaemia Database; JCLIMB). Data from 3 505 unique patients with CLTI who had undergone revascularisation from 2013 to 2017 were extracted from the JCLIMB for the analysis. The cohort was randomly divided into development (2 861 patients) and validation cohorts (644 patients). In the development cohort, multivariable risk models were constructed to predict two year OS and AFS using Cox proportional hazard regression analysis. These models were applied to the validation cohort and their performances were evaluated using Harrell's C index and calibration plots. RESULTS: Kaplan-Meier estimates of two year OS and AFS post-revascularisation in the whole cohort were 69% and 62%, respectively. Strong predictors for OS consisted of age, activity, malignant neoplasm, chronic kidney disease (CKD), congestive heart failure (CHF), geriatric nutritional risk index (GNRI), and sex. Strong predictors for AFS included age, activity, malignant neoplasm, CKD, CHF, GNRI, body temperature, white blood cells, urgent revascularisation procedure, and sex. Prediction models for two year OS and AFS showed good discrimination with Harrell's C indexes of 0.73 (95% confidence interval [CI] 0.69 - 0.77) and 0.72 (95% CI 0.68 - 0.76), respectively CONCLUSION: Prediction models for two year OS and AFS post-revascularisation in patients with CLTI were created. They can assist in determining treatment strategies and serve as risk adjustment modalities for quality benchmarking for revascularisation in patients with CLTI at each facility.

Overall structure of fully assembled cyanobacterial KaiABC circadian clock complex by an integrated experimental-computational approach.

In the cyanobacterial circadian clock system, KaiA, KaiB and KaiC periodically assemble into a large complex. Here we determined the overall structure of their fully assembled complex by integrating experimental and computational approaches. Small-angle X-ray and inverse contrast matching small-angle neutron scatterings coupled with size-exclusion chromatography provided constraints to highlight the spatial arrangements of the N-terminal domains of KaiA, which were not resolved in the previous structural analyses. Computationally built 20 million structural models of the complex were screened out utilizing the constrains and then subjected to molecular dynamics simulations to examine their stabilities. The final model suggests that, despite large fluctuation of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions. Thus, our integrative approach provides a useful tool to resolve large complex structures harboring dynamically fluctuating domains.

Deuteration Aiming for Neutron Scattering.

The distinguished feature of neutron as a scattering probe is an isotope effect, especially the large difference in neutron scattering length between hydrogen and deuterium. The difference renders the different visibility between hydrogenated and deuterated proteins. Therefore, the combination of deuterated protein and neutron scattering enables the selective visualization of a target domain in the complex or a target protein in the multi-component system. Despite of this fascinating character, there exist several problems for the general use of this method: difficulty and high cost for protein deuteration, and control and determination of deuteration ratio of the sample. To resolve them, the protocol of protein deuteration techniques is presented in this report. It is strongly expected that this protocol will offer more opportunity for conducting the neutron scattering studies with deuterated proteins.

Intraoperative collection of autologous platelet-rich plasma from the cardiopulmonary bypass circuit upon initiation of extracorporeal circulation.

OBJECTIVES: The aim of this study is to evaluate the possibility of the autologous platelet-rich plasma (PRP) collection from the cardiopulmonary bypass (CPB) circuit and to evaluate its effect on the aggregative function. METHODS: For seventy-two patients undergoing cardiac surgery with CPB, an autologous PRP was prepared using the Haemonetics Component Collection System® by drawing blood from the CPB circuit immediately after CPB was established. The blood samples were taken at three points for examination, A: beginning of surgery, B: immediately after heparin reversal with protamine following discontinuation of CPB, C: after the collected autologous PRP was returned to the patient. Platelet count and platelet aggregation ability were analyzed. RESULTS: The mean platelet count in autologous PRP was 5.5 (range: 3-14) units. Platelet count decreased by 115.0 (±27.3) × 1000/µl from A to B and increased by 27.3 ± 17.2 (× 1000/µl) from B to C. When platelet aggregation was measured by Adenosine Diphosphate (ADP) 3.0 µM, it decreased by 42.6% ± 12.1% from A to B and increased by 8.7% ± 7.4% from B to C. CONCLUSIONS: Autologous PRP can be safely collected by drawing blood from the CPB circuit, platelet count and aggregation ability significantly decreased after CPB including autologous PRP collection. Some improvement was detected in the number of the platelets count and platelet aggregation ability by administrating an autologous PRP even if autologous PRP is collected from CPB circuit. TRIAL REGISTRATION: UMI-CTR, UMIN000023776 . Registered 1 October 2016.

Treatment of ventricular septal perforation caused by blunt trauma using the right ventricle approach: A case report.

Traumatic ventricular septal perforation (VSP) is a rare condition that can occur following chest trauma and can lead to heart failure. Herein, a case of VSP caused by blunt chest trauma successfully closed using the double-patch technique via the right ventricle after medial sternotomy is presented. This case report highlights the necessity of emergency surgery in the acute phase of traumatic VSP if heart failure is difficult to control. This technique was useful for acute surgery. .

[Initial Clinical Experience of the Herbal Medicine Inchinkoto for Refractory Hyperbilirubinemia Following Open-heart Surgery].

We retrospectively assessed our initial clinical experience of the herbal medicine Inchinkoto for refractory hyperbilirubinemia following open-heart surgery. Six patients developed hyperbilirubinemia in an acute phase after surgery and their maximum total bilirubin levels were 6.4~26.4 mg/dl( mean:13.1± 8.2 mg/dl). They were initially treated with ursodeoxycholic acid and/or Stronger Neo-Minophagen C containing monoammonium glycyrrhizinate, glycine, aminoacetic acid, and L-cysteine hydrochloride hydrate. These treatments, however, were ineffective, and Inchinkoto was introduced at 5~34 day (mean:13.3±11.3 days) after surgery. Hyperbilirubinemia improved in all patients after the introduction of Inchinkoto:1 day after in 1 case, 2 days after in 2 cases, 3 days after in 2 cases, and 4 days after in 1 case. These results indicate the potential of Inchinkoto to attenuate refractory hyperbilirubinemia following cardiac surgery with cardiopulmonary bypass.

Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria.

The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB-KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB-KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB-KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay.

ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system.

The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB-KaiC interaction; however, its relationship with the KaiA-KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.

Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

Positioning for Endovenous Laser Ablation: Comparative Study with Thigh Stripping.

We performed a comparative study of surgical outcomes and venous functions between endovenous laser ablation with a 980-nm diode laser (EV group) and thigh stripping (ST group). There were no severe complications and initial success rates were 100% in both groups. In the EV group, preoperative symptoms improved in 94.3% of cases, the venous occlusion rate was 98%, and endovenous heat induced thrombosis had occurred in 11.9% (Class 3: 0.7%) at 12 months after the operation. Although comparative study of postoperative venous function by air plethysmography showed significant improvement in both groups, there was less recovery of postoperative venous function in the EV than in the ST group. (This article is a translation of J Jpn Coll Angiol 2015; 55: 13-20.).

Long-Term Results of Obturator Bypass.

OBJECTIVE: We evaluated the long-term outcomes of obturator bypass. MATERIAL AND METHODS: A total of 16 patients (13 males and 3 females; 17 limbs) who underwent obturator bypass surgery at our department between April 1995 and March 2008 were included. RESULTS: Their ages ranged from 50 to 90 with a mean of 74 years. Inguinal infections observed in the 16 patients consisted of vascular graft infections in 13 patients, hemostatic device infections following endovascular therapy in two patients, and femoral artery infections following coronary angiography in one patient. The cumulative patency rate was 69% for 3 years and 43% for 5 years. The cumulative survival rate was 64% for 3 years and 55% for 5 years. CONCLUSION: Obturator bypass surgery was successfully performed with favorable results for arterial infections and vascular graft infections in the inguinal region.

Ankle-Brachial Index, Toe-Brachial Index, and Pulse Volume Recording in Healthy Young Adults.

OBJECTIVE: To clarify the characteristics of ankle-brachial index (ABI), toe-brachial index (TBI), and pulse volume recording (PVR) of the ankle with brachial-ankle pulse wave velocity (baPWV) in healthy young adults. MATERIAL AND METHODS: We analyzed ABI, TBI, baPWV, and PVR in the ankle of healthy adults aged 20 to 25 years (median, 20 years) using an automatic oscillometric device between 2002 and 2013. The ABI, baPWV, and PVR in 1282 legs of 641 subjects (301 men and 340 women) and the TBI in 474 toes of 237 subjects (117 men and 120 women) were evaluated. RESULTS: The measured values showed no bilateral differences. ABI and baPWV were higher in men than in women, but TBI was similar in both sexes. ABI <1.0 was observed in 18.1% of the legs in men and in 25.6% in women. TBI <0.7 was observed in 16.2% of the toes in men and 19.1% in women. For ankle PVR, the % mean arterial pressure was higher in women than in men. The upstroke time was <180 ms in most subjects. CONCLUSIONS: For young people, ABI <1.0 or TBI <0.7 may not always indicate vascular abnormalities. When evaluating circulatory indexes, age and sex should be considered.

Bypass vs. Endovascular Therapy of Infrapopliteal Lesions for Critical Limb Ischemia.

OBJECTIVE: This study was conducted to determine whether to perform endovascular intervention or bypass surgery as a treatment option for critical limb ischemia (CLI) with lesions in the popliteal artery or below. SUBJECTS AND METHODS: A total of 150 patients (164 limbs) with CLI underwent endovascular intervention or bypass surgery for lesions in the popliteal artery or below at our department between May 1995 and June 2011. Therapeutic outcomes were examined by surgical technique. An indication for endovascular intervention was established with the combination of (1) poor general condition, and (2) a stenotic or occlusive lesion ≤5 cm. RESULTS: The bypass group (group B) comprised 119 patients (99 males, 20 females) with 131 affected limbs at 46 to 89 years of age (mean: 70 years). The endovascular intervention group (group E) comprised 31 patients (25 males, 6 females) with 33 affected limbs at 47 to 89 years of age (mean: 72 years). There was no significant difference in patient demography between the two groups. Regarding preoperative complications, hypertension was observed in 54% and 61% of the subjects in groups B and E, respectively, diabetes in 36% and 55%, renal dysfunction in 29% and 58%, ischemic heart disease in 27% and 32%, and cerebrovascular disorder in 18% and 23%; renal dysfunction accounted for a significantly higher percentage in group E. As for early postoperative complications, subjects in group B experienced wound infections (6 patients), hemorrhage (2), thrombosis (2), pneumonia (1), and another complication (1), and those in group E experienced wound infections (1) and another complication (1). The hospital mortality rate was 0.8% (1 patient) for group B and 0% for group E. The 3-year cumulative primary patency rate was 72% for group B and 54% for group E; the rate was significantly higher for group B. The 3-year secondary patency rate was 82% for group B and 60% for group E. The 3-year limb salvage rate was 86% for group B and 82% for group E; there was no significant difference between the two groups. The 5-year survival rate was 57% for group B and 42% for group E; the survival rate was significantly lower for group E. CONCLUSION: For the study population of CLI patients with lesions in the popliteal artery or below, the patency rate was higher for the bypass group than for the endovascular intervention group, whereas there was no difference in the limb salvage rate. Based on the findings in prognosis for survival, the indication for endovascular intervention at our department is believed to be appropriate. (English translation of Jpn J Vasc Surg 2013; 22: 715-718).

Collective therapy and therapeutic strategy for critical limb ischemia.

OBJECTIVE: To determine a treatment strategy based on the outcomes of various previous interventions for critical limb ischemia in arteriosclerosis obliterans (ASO). MATERIAL AND METHODS: We examined outcomes of 292 ASO patients who had had critical limb ischemia between May 1995 and July 2009. Patients underwent the following procedures in 167 cases: aortofemoral bypass (n = 14), femorofemoral crossover bypass (n = 29), femoropopliteal bypass (n = 104) and femorotibial bypass (n = 40). Other procedures included bypass only (n = 147), bypass combined with thromboendarterectomy (n = 10), bypass combined with endovascular therapy (n = 6), bypass combined with lumbar sympathectomy (n = 2), endovascular therapy combined with thromboendarterectomy (n = 4), endovascular therapy (n = 19), lumbar sympathectomy (n = 6), conservative therapy (n = 65), and major amputation (n = 31). We also calculated P3 risk scores and measured transcutaneous oxygen pressure (tcPO2) and skin perfusion pressure (SPP) before and after therapy. RESULTS: The limb salvage rate was 87% at 2 years in the arterial reconstruction group. In the low-risk group (a P 3 risk score of 3), the 1-year amputation-free survival rate was 96%. In the medium-risk group (a P 3 risk score of 4-7), the 1-year amputation-free survival rate was 88%. In the high-risk group (a P 3 risk score of 8), the 1-year amputation-free survival rate was 66%. The hospital death rate in the arterial reconstruction group was 3.2%, all of whom were patients who underwent bypass. The survival rate at 5 years was 65% and 36% in the conservative therapy only group. Ulcers healed in 140 out of 144 patients. The 4 patients with unhealed infections had tcPO2 or SPP values of more than 30 mmHg after treatment. Major amputations were performed in 4 of 5 patients who had tcPO2 or SPP values from 20 to 30 mmHg after treatment. Major amputations were performed in all 6 patients who had tcPO2 or SPP values of less than 20 mmHg after treatment. CONCLUSION: In cases with tcPO2 or SPP values of more than 30 mmHg, an ulcer will probably heal, except in infected cases. We suggest that, if these values are less than 30 mmHg, complete revascularization should be performed. The P3 risk score was useful in predicting limb salvage in the current series. Hybrid therapy in bypass and endovascular therapy must be performed in cases where patients are in a generally poor condition. It is important to attempt amelioration in limb salvage and to control the operative mortality rate with sufficient perioperative control. (English Translation of Jpn J Vasc Surg 2011;20:905-911).

Assessment of the characteristics and detectability of skin perfusion pressure measured using a thermostatic heating probe.

OBJECTIVES: To assess the characteristics of skin perfusion pressure (SPP) measured using a thermostatic heating probe and whether a thermostatic heating probe improves SPP detection. METHODS: We studied 8 feet of healthy young subjects and 31 feet of elderly patients suspected to have severe limb ischemia. We measured SPP at the dorsum and plantar aspects of each foot using a plain laser Doppler probe and a thermostatic heating probe heated at 44°C. Results were expressed as median. Comparisons were analyzed using a non-parametric test. RESULTS: In the healthy subjects, the SPP values at both the dorsum and the plantar aspect were not significantly different after heating. The thermostatic heating probe did not improve the SPP detection rates. In the patients with ischemic limb, the SPP values at both the dorsum and the plantar aspect significantly increased after heating (p <0.001 for both). The SPP detection rate at the dorsum remained at 96.8%; however, it was improved from 87.1% to 100% at the plantar aspect after heating. CONCLUSION: The thermostatic heating probe was shown to be useful for improving the detectability of SPP in the ischemic limbs. An SPP increase after heating may be considered as a parameter of limb ischemia.

Total aortic arch replacement for a patient with poor left ventricular function and patent coronary artery bypass grafts: how to establish effective myocardial protection.

A 66-year-old man who had previously undergone coronary artery bypass grafting (CABG) was admitted to our institution for surgical treatment of a ruptured aortic arch aneurysm. He had three patent bypassed grafts including the left internal thoracic artery (LITA) to the left anterior descending artery (LAD), complicated by left ventricular dysfunction. Coronary angiography performed 1 year after the initial surgery revealed total occlusion of the LAD. In addition, the aneurysm was located next to the LITA; therefore, there was a significant risk of injury to the LITA during intraoperative dissection. For such a complicated and challenging case, we successfully performed a total aortic arch replacement using a Y-shaped composite saphenous vein graft (SVG) for the administration of cardioplegic solution to establish effective myocardial protection. This procedure, by which effective myocardial protection can be achieved, is a useful treatment option for aortic arch surgery after CABG with a patent LITA graft.

Colon carcinoma after thymectomy for myasthenia gravis: report of a case.

A 74-year-old Japanese man was admitted to our hospital with anemia, 4 years after a thymectomy for thymoma associated with myasthenia gravis. A diagnosis of sigmoid colon carcinoma was confirmed, followed by surgical resection. This case is presented to reinforce that physicians should bear in mind the possibility of extrathymic malignancies in patients with thymoma.