Convergent evolution of processivity in bacterial and fungal cellulases.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

[Incidence of Antiresorptive Agent-Related Osteonecrosis of the Jaw in Urologic Cancers].

Antiresorptive agent-related osteonecrosis of the jaw (ARONJ) is a serious adverse event of bone resorption inhibitors (BRIs), such as zoledronic acid and denosumab. Based on the results of phase 3 clinical trials for BRIs, the frequency of ARONJ is 1 to 2%, but the actual frequency is presumed to be higher. We studied 143 patients with urologic cancers with bone metastases who were treated with zoledronic acid or denosumab at our hospital between April 2007 and March 2020. ARONJ occurred in 24 patients (16.8%) ; that is, 14 of the 113 patients (12.4%) who received zoledronic acid alone, 8 of the 24 patients (33.3%) who received denosumab alone, and 2 of the 6 patients (33.3%) who sequentially switched from zoledronic acid to denosumab. ARONJ was cured in 8 patients (33.3%), improved in 3 patients (12.5%), unchanged in 4 patients (16.7%), and worsened in 9 patients (37.5%). The frequency of ARONJ increased as the duration of BRI administration prolonged. Time-to-ARONJ was shorter in patients treated with denosumab than in patients treated with zoledronic acid. The occurrence of ARONJ may be underestimated; therefore, further studies are needed to investigate the actual frequency of ARONJ in Japan.

Structural properties determining low K+ affinity of the selectivity filter in the TWIK1 K+ channel.

Canonical K+ channels are tetrameric and highly K+-selective, whereas two-pore-domain K+ (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na+ and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K+-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K+ ions, WT and both variants exhibit an amide-I band at 1680 cm-1 This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K+, a feature very similar to that of the canonical K+ channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K+ affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm-1 band upon changes from K+ to Rb+ or Cs+ conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K+ and Na+ solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K+ channels.

Nylon mesh cryodevice for bovine mature oocytes, easily removable excess vitrification solution.

The aim of this study was to determine pore size of nylon mesh (NM) device suitable for cryosurvival of bovine mature oocytes and to apply the device to vitrification of large quantities of the oocytes. Ten to twelve oocytes were loaded onto an NM device (a square opening 37-, 57- or 77-µm on a side length). After removal of the excess volume of vitrification solution by paper absorption, the oocytes were vitrified-warmed, fertilized and cultured in vitro. Oocyte recovery and morphological survival were comparable among the three groups. However, blastocyst yield in the 37-µm group (39%) was higher than that in the 77-µm group (28%), and the yield in the 57-µm group (31%) was the intermediate. The 37-µm NM device was applicable for increased oocyte number >40 (blastocyst yield, 33%). These results suggest that 37-µm-pore sized NM can serve as cryodevice to vitrify large quantities of in vitro-matured bovine oocytes.

Expression Profiles of NOS Isoforms in Dental Pulp and Odontoblasts in nNOS Knockout Mice.

Active oxygen and free radicals are involved in metabolism in cells and tissues. Immunohistological studies of related enzymes are few, and the morphological dynamics of these enzymes in dental pulp and odontoblasts remain to be elucidated. Nitric oxide synthase (NOS) has 3 isoforms: nNOS, iNOS, and eNOS. The aim of this study was to investigate the profiles of NOS isoforms in the absence of nNOS in dental pulp and odontoblasts. Five-week-old male C57BL/6 and nNOS knockout (KO) mice were sacrificed and expression of nNOS, iNOS, and eNOS determined immunohistochemically. Expression of nNOS was positive, whereas that of iNOS was negative and eNOS weakly positive in the dental pulp and odontoblasts of the control mice. In nNOS KO mice, expression of iNOS was positive in dental pulp and strongly positive in odontoblasts, whereas that of eNOS was stronger in fibroblasts, endothelial cells in the vicinity of blood vessels in the dental pulp, and odontoblasts. Expression of nNOS was negative in the nNOS KO mice. This suggests that iNOS and eNOS compensate for nNOS deficiency in vascular endothelial cells and fibroblasts in the dental pulp and odontoblasts.

Lymphangiogenesis and NOS Localization in Healing Process after Tooth Extraction in Akita Mouse.

Type I diabetes, an autoimmune disease, induces insulin deficiency, which then disrupts vascular endothelial cell function, affecting blood and lymphatic vessels. Nitric oxide (NO) is an immune-induced destructive mediator in type I diabetes, and inhibition of its production promotes arteriosclerosis. In this study, lymphangiogenesis and expression of NO synthase (NOS) during the healing process after tooth extraction were investigated immunohistochemically in control (C57BL) and Akita mice as a diabetes model. Between 1, 4, and 10 days after extraction, expression of NOS, vascular endothelial growth factor-C (VEGF-C), VEGF receptor-3 (VEGFR-3), and von Willebrand factor was strongest during the granulation tissue phase. This suggests that severe inflammation triggers regulation of NOS and these other angiogenic and lymphangiogenic factors. During the callus phase, a few days after extraction, induced osteoblasts were positive for VEGF-C and VEGFR-3 in both the control and Akita mice, suggesting that bone formation is active in this period. Bone formation in the Akita group exceeded that in the controls. Bone tissue formation was disrupted under hyperglycemic conditions, however, suggesting that such activity would be insufficient to produce new bone.

Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.

Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.

Analysis of heterogeneous catalysis at an interface is difficult because of the variety of reaction sites and the difficulty of observing the reaction. Enzymatic hydrolysis of cellulose by cellulases is a typical heterogeneous reaction at a solid/liquid interface, and a key parameter of such reactions on polymeric substrates is the processivity, i.e., the number of catalytic cycles that can occur without detachment of the enzyme from the substrate. In this study, we evaluated the reactions of three closely related glycoside hydrolase family 7 cellobiohydrolases from filamentous fungi at the molecular level by means of high-speed atomic force microscopy to investigate the structure-function relationship of the cellobiohydrolases on crystalline cellulose. We found that high moving velocity of enzyme molecules on the surface is associated with a high dissociation rate constant from the substrate, which means weak interaction between enzyme and substrate. Moreover, higher values of processivity were associated with more loop regions covering the subsite cleft, which may imply higher binding affinity. Loop regions covering the subsites result in stronger interaction, which decreases the velocity but increases the processivity. These results indicate that there is a trade-off between processivity and hydrolytic velocity among processive cellulases.

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses.

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.

Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation.

This study evaluated how differences in the surface properties of prefillable syringe barrels and in-solution sampling methods affect micron aggregates and protein adsorption levels. Three syringe types (glass barrel with silicone oil coating [GLS/SO+], glass barrel without silicone oil coating [GLS/SO-], and cyclo-olefin polymer [COP] barrel syringes) were tested with 3 therapeutic proteins (adalimumab, etanercept, and infliximab) using 2 sampling methods (aspiration or ejection). After quiescent incubation, solutions sampled by aspiration exhibited no significant change in micron aggregate concentration in any syringes, whereas those sampled by ejection exhibited increased micron aggregates in both GLS syringe types. Micron aggregate concentration in ejected solutions generally increased with increasing density of adsorbed proteins. Notably, COP syringes contained the lowest micron aggregate concentrations, which were independent of the sampling method. Correspondingly, the adsorbed protein density on COP syringes was the lowest at 1-2 mg/m2, which was much less compared with that on GLS syringes and was calculated to be equivalent to only 1-2 protein layers, as visually confirmed by high-speed atomic force microscopy. These data indicate that low-adsorption prefillable syringes should be used for therapeutic proteins because protein aggregate concentration in the ejected solution is elevated by increased protein adsorption to the syringe surface.

Preclinical evaluation of a paclitaxel-incorporated nanoparticle-coated balloon in rabbit and porcine models.

BACKGROUND: The main drawback of current available drug coated balloons (DCB) is that a certain percentage of the coated drug is lost in the bloodstream during its delivery to the target lesion. We integrated the nanoparticle-mediated drug delivery technology and polydimethylsiloxane (PDMS) as a new excipient to facilitate an efficient drug delivery and uptake by endothelial cells. The present study aimed to evaluate the efficacy of the new DCB. METHOD AND RESULTS: The novel DCB were coated with 5.6mg of paclitaxel-incorporated nanoparticles using PDMS. The efficacy of the new DCB was examined in rabbit iliac stent model (n=12) and in the swine in-stent restenosis model (n=8) by quantitative coronary angiography (QCA) and optical coherence tomography (OCT). At 28days follow-up in the swine in-stent restenosis model, the area stenosis was significantly lower in DCB group as compared with that of the control group in OCT analysis (0.31±0.05 vs 0.49±0.06, p=0.04) though there was no significant differences observed in the rabbit iliac stent model in QCA and OCT analysis. CONCLUSION: The study results indicated that the paclitaxel-incorporated nanoparticle-coated balloon using PDMS has an inhibitory effect for the proliferation of smooth muscle cell in a swine coronary in-stent restenosis model.

Calcium phosphate cement-based vertebroplasty compared with conservative treatment for osteoporotic compression fractures: a matched case-control study.

OBJECT: Few studies have been conducted to compare vertebroplasty and conservative treatment for osteoporotic vertebral compression fractures (OVCFs). To investigate the effects of calcium phosphate cement (CPC)-based vertebroplasty on relief of pain and augmentation of the fractured vertebral body (VB), the authors compared the results of CPC-assisted vertebroplasty with those of conservative treatment alone. METHODS: Two groups of patients were examined: the vertebroplasty group (30 consecutive patients with primary OVCF) and the control group (30 patients matched for age, sex, interval from injury to treatment, and grade of the posterior wall defects of the fractured VB). Outcome measures included the visual analog scale (VAS) score of back pain and analgesic requirements, and the radiographically documented rate of the VB kyphosis. The follow-up duration was more than 12 months (mean 17 months). The mean VAS score at 12 months after injury was 0.67 cm in the vertebroplasty group and 1.97 cm in the control group, and the mean improvement rates in the VAS scores were 91.6 and 73.6%, respectively (p < 0.0001). The mean duration of analgesic requirement was 8.3 days in the vertebroplasty group and 62.2 days in the control group (p = 0.0005). The mean kyphosis rate at 12 months after injury was 72.9% in the vertebroplasty group and 58% in the control group, and the mean recovery rate of kyphosis was +8.4 and -21%, respectively (p < 0.0001). CONCLUSIONS: The authors conclude that CPC-assisted vertebroplasty provides better clinical and radiological results than conservative treatment for primary OVCF.

Muscle-fiber characteristics in adult mouse-tongue muscles.

To clarify functions of the mouse-tongue muscles, proteins such as myocin heavy chain (MHC) 2a and MHC-2b, which are isoforms of the fast-twitch fiber type myosin heavy chain, in the lateral margin of the tongue were observed by reverse transcription polymerase chain reaction and immunohistochemical analyses. The main MHC isoform in the superior longitudinal muscle of the tongue was MHC-2b, with the fastest function and the main MHC isoform in the transverse muscle of the tongue was MHC-2a. These findings suggested that the fastest function is necessary for the superior longitudinal muscle of the tongue, which is useful for moving the tongue in and out of the mouth in the sagittal direction, showing different cellular biological properties of the myofibers from those of the transverse muscle of the tongue.

Percutaneous transpedicular vertebroplasty with calcium phosphate cement in the treatment of osteoporotic vertebral compression and burst fractures.

OBJECT: Osteoporotic vertebral fractures occasionally lead to late-onset collapse, kyphosis, persistent back pain, and disability. The authors describe a series of patients in whom they performed percutaneous vertebroplasty by using calcium phosphate cement (CPC) to obtain early pain relief and improve the integrity of the osteoporotic vertebral body (VB). METHODS: Between August 2000 and February 2001, they performed 17 percutaneous transpedicular CPC-assisted vertebroplasty procedures in 16 patients who harbored thoracic or lumbar osteoporotic vertebral fractures. Following repositioning and curettage of the pathological soft tissues, CPC-assisted vertebroplasty was percutaneously performed in four patients with osteoporotic burst fracture and pseudarthrosis (Procedure A). In situ CPC-assisted vertebroplasty was performed in 12 patients with fresh vertebral compression fractures due to osteoporosis (Procedure B). Back pain and low-back pain were evaluated using a visual analog scale (VAS). The deformity index of the VB was measured on a lateral radiograph as the ratio of the VB's height (sum of measurements at anterior, middle, and posterior regions) to its longitudinal diameter. Based on VAS scores, pain was decreased in all patients immediately after surgery, and pain relief was maintained at the last follow up. The mean preoperative deformity index score of the VB was 1.43 in Procedure A and 1.67 in Procedure B; postoperatively scores improved to 1.59 and 1.93, respectively. At the 6-month follow-up examination, the mean deformity index score rebounded to 1.52 in Procedure A and 1.79 in Procedure B. Bone union was documented in all patients. Complications, such as a temporary respiratory insufficiency and a small amount of CPC leakage into the spinal canal, were observed in patients who underwent Procedure B. CONCLUSIONS: Percutaneous transpedicular CPC-assisted vertebroplasty is a minimally invasive procedure that provides early relief of pain and prevents vertebral collapse and pseudarthrosis in patients with osteoporotic vertebral fracture.

Temporomandibular joint ankylosis: a case report.

This report is based on a case of temporomandibular joint ankylosis discovered in a cadaver during routine student dissection in the Department of Anatomy at Tokyo Dental College. Extensive osseous ankylosis in the left temporomandibular joint was evident in this case which exhibited a distinctive bird-like facial deformity caused by a mandibular growth disorder. This resulted in the underdevelopment of the mental area in particular. Furthermore, abnormally enlarged antegonal notching was present along the inferior border of the mandible anterior to the angle. Both these manifestations indicated the likelihood that the ankylosis had contributed to the abnormalities and had commenced at an early stage of skeletal development.

Microcatheter-facilitated reverse wire technique for side branch wiring in bifurcated vessels: an in vitro evaluation.

AIMS: To assess when and how the microcatheter-facilitated reverse wire technique should be applied to cross the guidewire into side branches in coronary bifurcations. METHODS AND RESULTS: Three interventional cardiologists with different levels of experience performed in vitro bench testing using an originally developed coronary bifurcation simulator which had six coronary bifurcations. The bifurcation angles were 90, 105, 120, 135, 150 and 165 degrees (°). Experiment 1 was conducted to assess in what coronary bifurcation the reverse wire technique is required. Antegrade guidewire advancement was conducted with two different guidewires: the spring coil guidewire SION blue and the polymer-jacket hydrophilic guidewire Fielder FC. Experiment 2 was conducted to determine what the optimal guidewire selection and the optimal guidewire shape for the reverse wire technique would be. Assessment of the guidewire crossability into the highly angulated side branch was performed, and then the balloon crossability was assessed. A total of four guidewire types were compared in experiment 2. In experiment 1, guidewire crossing was impossible by conventional antegrade wiring when the bifurcation angle became 150° or more. In experiment 2, guidewire crossing of more than 150° of bifurcation angle was achievable independent of the guidewire types and shape. Balloon deliverability was best when using a polymer-jacket hydrophilic guidewire with a round shape 3 cm from the guidewire tip. CONCLUSIONS: Although the guidewire crossing into the side branch was impossible by conventional antegrade methods when the bifurcation angle became 150° or more, the guidewire crossing into such a highly angulated side branch was easily possible using the reverse wire technique. The optimal guidewire selection for the reverse wire technique is the polymer-jacket hydrophilic guidewire with a round shape 3 cm from the guidewire tip.

Angioedema: 6 years experience with fourteen cases.

OBJECTIVE: To study the difference in the findings between the causes of angioedema and the characteristics of angioedema induced by angiotensin receptor II blockers (ARBs), and to investigate whether laboratory examinations for acute phase inflammatory markers can aid in diagnosis and predict airway risk. METHODS: We retrospectively reviewed fourteen cases of patients with angioedema that were treated from 2000 to 2006. Data were collected regarding age, sex, location of the edema, cause, time course of resolution and laboratory examinations (leukocyte counts, serum C-reactive protein (CRP) level, complement function and the activity of C1 esterase inhibitor). RESULTS: The causes of angioedema were ACEIs in six patients (42.9%), candesartan (ARB) in three (21.4%), HAE (types 1 and 2) in two, and unknown in three. Of these patients, 71.4% exhibited edema in the floor of the mouth, irrespective of the cause. Two patients with edema induced by candesartan exhibited both lingual and laryngeal edemas. The remaining one with candesartan-induced edema exhibited edema in the neck and mediastinum and pleural effusion. The average time to resolution was 4.1 days, ranging from one to twelve days. The edema in eleven patients resolved with conservative therapy, while three patients underwent tracheotomy. In two patients with candesartan-induced edema, although the edemas resolved completely after cessation of candesartan administration, the edemas reappeared in the same locations, two and thirty days after the cessation of candesartan for each patient. None of the patients with angioedema induced by ACEIs exhibited elevation of serum CRP levels. No significant differences were found for leukocyte counts and serum CRP levels between patients with angioedemas induced by ACEIs, ARB and those of unknown cause. No significant differences were observed in the above findings between the patients who underwent tracheotomy and those who did not. Two patients exhibited low C4 levels, and one of the two exhibited no activity of C1 esterase inhibitor. CONCLUSION: Consistent with previous reports, angioedema in the floor of the mouth extending to the tongue should be considered as a possible risk factor for airway compromise. Laboratory examinations for acute phase inflammatory markers are not useful for diagnosis and are not predictive for airway intubation and tracheotomy. Angioedema induced by candesartan can present in anomalous sites and reappear following drug cessation even if the edema has resolved completely.