Efficient secretory production of proline/alanine/serine (PAS) biopolymers in Corynebacterium glutamicum yielding a monodisperse biological alternative to polyethylene glycol (PEG).

BACKGROUND: PAS biopolymers are recombinant polypeptides comprising the small uncharged L-amino acids Pro, Ala and/or Ser which resemble the widely used poly-ethylene glycol (PEG) in terms of pronounced hydrophilicity. Likewise, their random chain behaviour in physiological solution results in a strongly expanded hydrodynamic volume. Thus, apart from their use as fusion partner for biopharmaceuticals to achieve prolonged half-life in vivo, PAS biopolymers appear attractive as substitute for PEG-or other poorly degradable chemical polymers-in many areas. As a prerequisite for the wide application of PAS biopolymers at affordable cost, we have established their highly efficient biotechnological production in Corynebacterium glutamicum serving as a well characterized bacterial host organism. RESULTS: Using the CspA signal sequence, we have secreted two representative PAS biopolymers as polypeptides with ~ 600 and ~ 1200 amino acid residues, respectively. Both PAS biopolymers were purified from the culture supernatant by means of a simple downstream process in a truly monodisperse state as evidenced by ESI-MS. Yields after purification were up to ≥ 4 g per liter culture, with potential for further increase by strain optimization as well as fermentation and bioprocess development. Beyond direct application as hydrocolloids or to exploit their rheological properties, such PAS biopolymers are suitable for site-specific chemical conjugation with pharmacologically active molecules via their unique terminal amino or carboxyl groups. To enable the specific activation of the carboxylate, without interference by the free amino group, we generated a blocked N-terminus for the PAS(1200) polypeptide simply by introducing an N-terminal Gln residue which, after processing of the signal peptide, was cyclised to a chemically inert pyroglutamyl group upon acid treatment. The fact that PAS biopolymers are genetically encoded offers further conjugation strategies via incorporation of amino acids with reactive side chains (e.g., Cys, Lys, Glu/Asp) at defined positions. CONCLUSIONS: Our new PAS expression platform using Corynex® technology opens the way to applications of PASylation® technology in multiple areas such as the pharmaceutical industry, cosmetics and food technology.

Treatment outcomes of proton beam therapy combined with retrograde intra-arterial infusion chemotherapy for locally advanced oral cancer in the elderly.

The aim of this study was to evaluate the therapeutic efficacy and safety of proton beam therapy combined with retrograde intra-arterial infusion chemotherapy in elderly patients with locally advanced oral cancer. Between February 2009 and October 2019, 42 oral cancer patients aged ≥75 years were treated with this therapy. Median age was 80 years (range 75-90 years) and the median follow-up duration was 39 months (range 2-106 months). Of the 42 patients, 34 (81%) were diagnosed with stage IV cancer. The 3-year overall survival, local control, progression-free survival, and disease-specific survival rates were 56%, 69%, 32%, and 67%, respectively. Regarding acute toxicities, grade 3 neutropenia was observed in six patients (14%), anaemia in five (12%), acute kidney injury in one (2%), and oral mucositis in 18 (42%). Late toxicities of grade 3 were observed in seven patients: dysphagia in six (14%) and osteonecrosis of the jaw in one (2%). This study showed that proton beam therapy combined with retrograde intra-arterial infusion chemotherapy was effective for elderly patients with oral cancer, and toxicities were tolerable and manageable. The study findings suggest that this therapy is a potential treatment option for elderly oral cancer patients with difficulty in surgery and systemic chemotherapy.

Range of motion exercise of temporo-mandibular joint with hot pack increases occlusal force in patients with Duchenne muscular dystrophy.

The purpose of this study is to evaluate whether the range of motion exercise of the temporo-mandibular joint (jaw ROM exercise) with a hot pack and massage of the masseter muscle improve biting disorder in Duchenne muscular dystrophy (DMD). The subjects were 18 DMD patients (21.3+/- 4.1 years old). The jaw ROM exercise consisted of therapist-assisted training (2 times a week) and self-training (before each meal every day). The therapist-assisted training consisted of the application of a hot pack on the cheek of the masseter muscle region (15 minutes), the massage of the masseter (10 minutes), and jaw ROM exercise (5 minutes). The self-training involved jaw ROM exercise by opening the mouth to the maximum degree, ten times. These trainings continued for six months. Outcomes were evaluated by measuring the greatest occlusal force and the distance at the maximum degree of mouth opening between an incisor of the top and that of the bottom. Six months later, the greatest occlusal force had increased significantly compared with that at the start of jaw ROM exercise (intermediate values: from 73.8N to 97.3N) (p = 0.005) as determined by the Friedman test and Scheffi's nonparametric test. The patients' satisfaction with meals increased. However, the maximum degree of mouth opening did not change after six months of jaw ROM exercise. Jaw ROM exercise in DMD is effective for increasing the greatest occlusal force.

Cellular locations of proteinases and association with vesicles in Porphyromonas gingivalis.

We found that locations of arginine-specific gingipain (RGP) in the cellular fractions in the crude extract, envelope, vesicles, and culture supernatants were 48%, 16%, 17%, and 31%, respectively, and the corresponding values of lysine-specific gingipain (KGP) were 47%, 10%, 7%, and 36%, respectively. Although the molecular mass of RGP in the culture supernatant had been determined as 43 kDa, and that of KGP had been as 48 kDa, molecular masses of both proteinases solubilized from the vesicles were estimated to be over 1,500 kDa, since they eluted in the void volume of the column in the gel filtration on Sephacryl S-300. There was no reduction of molecular size by the following treatment with SDS, high-concentration NaCl, or urea. Interestingly, the occurrence of the macromolecular forms could not observed in other enzymes tested such as monopeptidyl, dipeptidyl, and tripeptidyl peptidases, as well as alkaline phosphatase. Therefore, occurrence of the macromolecular forms may be restricted to the proteinases. When the vesicle and culture supernatants containing free RGP and KGP were mixed and incubated, neither RGP nor KGP seemed to bind to vesicles. RGP bound to the vesicle was found to be more stable to heat treatment than the free form, suggesting that association of RGP with the vesicle caused heat stability of this enzyme.

Porphyromonas gingivalis mutant defective in a putative extracytoplasmic function sigma factor shows a mutator phenotype.

INTRODUCTION: Porphyromonas gingivalis is implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis must possess the ability to tolerate stress signals outside the cytoplasmic membrane by transcriptional activation of genes encoding proteins involved in defense or repair processes. Some bacteria utilize a distinct subfamily of sigma factors to regulate extracytoplasmic function (hence termed the ECF subfamily). METHODS: To elucidate their role in P. gingivalis, a chromosomal mutant carrying a disruption of an ECF sigma factor PG1318-encoding gene was constructed. Hemagglutination and proteolytic activities were measured in the PG1318-defective mutant. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and southern blot analysis were used to assess transcription of kgp in the PG1318-defective mutant. Frequency of spontaneous mutation that conferred resistance to l-trifluoromethionine was measured in the PG1318-defective mutant. RESULTS: The PG1318-defective mutant formed non-pigmented colonies on blood agar plates at a relatively high frequency. Arginine-specific and lysine-specific proteinase activities of the non-pigmented variants were remarkably decreased compared with those of the parent strain and the pigmented variants. RT-PCR analysis showed that kgp was not transcribed in some non-pigmented variants and southern blot analysis revealed that there was a deletion in their kgp region. Frequency of mutation conferring resistance to l-trifluoromethionine was significantly higher in the PG1318-defective mutant than in the wild-type. CONCLUSION: These results suggest that PG1318 plays a role in the regulation of mutation frequency in the bacterium.

Sexual Dimorphism of Endocranial, Facial and Limb Measurements in the Yellow Baboon (Papio cynocephalus).

The present study focused on the sexual dimorphism of yellow baboon (Papio cynocephalus) to clarify its relationship with social behaviour. The degree of sexual dimorphism in the endocranial volume is the lowest among the investigated measurements. Among the facial measurements, the degree value of sexual dimorphism was the maximum (38.4%) for palate length and the minimum for palate breadth at the upper second molar (M2) (16.8%). Reduced major axis (RMA) regression analysis indicated that most positive allometry in relation to body mass was barely shown in the endocranial volume, palate length, palate breadth at M2 and mandibular ramal width. On the other hand, most negative allometry in relation to body mass was barely indicated in the bizygomatic breadth, skull length, humeral length and femoral length. The plate breadth at M2 in males was smaller than that in females in equivalent to body mass. The results of the present study suggest that more males have longer pointed muzzles than females, which is considered to create an impressive view of large canine teeth. This contributes to display among males and agonistic encounters rather than to necessity of increased facial size due to larger body size or dietary influences.

Oxygen consumption of respiratory muscles in patients with COPD.

We measured the oxygen consumption (VO2) of respiratory muscles in 8 COPD patients and 12 age-matched healthy subjects using a closed circuit device which allows a continuous increase in external dead space and is equipped with a 9-L Collins spirometer. Furthermore, we measured simultaneously mouth occlusion pressure at 0.1 s of inspiration (P0.1), minute ventilation (VE), and other ventilatory parameters during the measurement of total VO2 (VO2 tot). We found that the logarithm of VO2tot (logVO2tot) had a good correlation with VE in both groups. The mean slope of the regression line of logVO2tot and VE (delta logVO2tot/delta VE) of COPD patients was significantly higher than that of normal subjects (p < 0.001). However, the mean Y-intercept (metabolic VO2[VO2met]) of the regression lines did not differ between the two groups. The P0.1 in COPD patients was higher than that in normal subjects at the corresponding dead space loading. However, the VE did not differ between the two groups except for at rest and the first 1 min after dead space loading. These results suggest that the VO2 of respiratory muscles in patients with COPD is higher at given ventilation compared with that in age-matched normal subjects and that this increased VO2 partly may be due to an augmented ventilatory drive.

Inspiratory effort sensation to added resistive loading in patients with obstructive sleep apnea.

STUDY OBJECTIVES: Repeated episodes of upper-airway occlusion are the main characteristics of patients with obstructive sleep apnea (OSA) during sleep. It has been reported that an impairment in the sensation of detection and a depression of ventilatory compensation to added load could be observed in such patients. In this study, we examined patients with OSA to evaluate the inspiratory effort sensation (IES), ventilation, and mouth occlusion pressures during added resistive loading while awake and to determine whether they can be reversed by nasal continuous positive airway pressure (CPAP) treatment. DESIGN: A hospital-based case-control study. SETTING: A sleep laboratory of a medical unit in Japan. SUBJECTS: Seventeen patients with moderate to severe OSA and 10 control subjects were included in this study. MEASUREMENTS: All patients with OSA had undergone standard nocturnal polysomnography. Patients with OSA and control subjects were evaluated for IES measured by a modified Borg score, ventilation, and mouth occlusion pressure during control and inspiratory resistive loaded breathing. These tests were repeated in all patients with OSA after 2 weeks of nasal CPAP treatment. RESULTS: IES to inspiratory resistive loading was lower in patients with OSA than in control subjects. There were no differences in ventilation and mouth occlusion pressure between patients and control subjects during loaded breathing. After 2 weeks of nasal CPAP, the decreased IES was increased in patients with OSA. CONCLUSION: In patients with OSA, the decreased IES to inspiratory resistive loaded breathing is reversible with nasal CPAP. This could be one additional benefit of nasal CPAP in the treatment of OSA.

Total and local impedances of the chest wall up to 10 Hz.

To understand how bical mechanical chest wall (CW) properties are related to those of the CW as a whole, we measured esophageal and gastric pressures, CW volume changes (measured with a head-out body plethysmograph), and anteroposterior and transverse CW diameter changes (measured with magnetometers attached to the surface) during sinusoidal forcing at the mouth (2.5% vital capacity, 0.5-10 Hz) in four healthy subjects. Total CW resistance decreased sharply as frequency rose to 3-4 Hz and remained relatively constant at higher frequencies. Total CW reactance became less negative with increasing frequency but showed no tendency to change sign. Above 2 Hz, diameters measured at different locations changed asynchronously between and within the rib cage and abdomen. "Local pathway impedances" (ratios of esophageal or gastric pressure to a rate of diameter change) showed frequency dependence similar to that of the total CW less than 3 Hz. Local pathway impedances increased during contraction of respiratory muscles acting on the pathway. We conclude that 1) total CW behavior is mainly a reflection of its individual local properties at less than or equal to 3 Hz, 2) local impedances within the rib cage or within the abdomen can change independently in some situations, and 3) asynchronies that develop within the CW during forcing greater than 3 Hz suggest that two compartments may be insufficient to describe CW properties from impedance measurements.

Decrease in functional residual capacity during inspiratory loading and the sensation of dyspnea.

The purposes of the present study were to determine the changes in functional residual capacity (FRC) during inspiratory loading and to examine their mechanisms. We studied seven normal subjects seated in a body plethysmograph. In both graded inspiratory elastic (35, 48, and 68 cmH2O/l) and resistive (21, 86, and 192 cmH2O.l-1.s) loading, FRC invariably decreased from control FRC and phasic expiratory activity increased. The reduction in FRC was greater with greater loads. A single inspiratory effort against an inspiratory occlusion at three different target mouth pressures (-25, -50, and -75 cmH2O) and durations (1, 2, and 5 s) also resulted in a decrease in FRC with an increase in expiratory electromyogram activity in the following expiration. The decrease in FRC was greater with greater target pressure and duration. This decrease in FRC is qualitatively similar to that during inspiratory loaded breathing, and we suspect that the same mechanisms are at work. Because neither vagal nor chemoreceptor reflex can account for these responses, we suspect conscious awareness of breathing or behavioral control to be responsible. In an additional study, the sensation of discomfort of breathing during elastic loading decreased with a decrease in FRC. These results suggest that the reduced FRC may be due to behavioral control of breathing to reduce the sensation of dyspnea during inspiratory loading.

Chest wall impedance partitioned into rib cage and diaphragm-abdominal pathways.

We measured chest wall "pathway impedances" (ratios of pressure changes to rates of volume displacement at the surface) with esophageal and gastric balloons and inductance plethysmographic belts around the rib cage and abdomen during forced volume oscillations (5% vital capacity, 0.5-4 Hz) at the mouth of five relaxed, seated subjects. Volume displacements of the total chest wall surface, measured by summing the rib cage and abdominal signals, approximated measurements using volume-displacement, body plethysmography over the entire frequency range. Resistance (R) and elastance (E) of the diaphragm-abdomen pathway were several times greater than those of the rib cage pathway, except at the highest frequencies where diaphragm-abdominal E was small. R and E of the diaphragm-abdomen pathway and of the rib cage pathway showed the same frequency dependencies as that of the total chest wall: R decreased markedly as frequency increased, and E (especially in the diaphragm-abdomen) decreased at the highest frequencies. These results suggest that the chest wall can be reasonably modeled, over the frequency range studied, as a system with two major pathways for displacement. Each pathway seems to exhibit behavior that reflects nonlinear, rate-independent dissipation as well as viscoelastic properties. Impedances of these pathways are useful indexes of changes in chest wall mechanical behavior in different situations.

Protective efficacy of active and passive immunizations against experimental infection with Bacteroides gingivalis in ligated hamsters.

The protective efficacy of immunization against Bacteroides gingivalis infection was examined in hamsters. Whole cells or extracted hemagglutinin of B. gingivalis 381 was injected with incomplete Freund adjuvant into the inguinal regions of hamsters. Two weeks after the rats received a booster injection, cotton threads were tied coronally to the gingival margins of the mandibular first molars, and then a streptomycin-resistant B. gingivalis 381R' strain was inoculated into the rats' oral cavities. The subcutaneous immunizations resulted in slight reductions in the numbers of B. gingivalis on the ligature threads compared with the sham-immunized group. Peroral administration of whole cells of B. gingivalis to hamsters elicited salivary immunoglobulin responses, but no reduction of B. gingivalis 381R' colonization was found in this group. Repeated passive immunizations with rabbit antiserum to B. gingivalis into the oral cavities of the hamsters resulted in a reduction in the number of organisms in the periodontal region.

Susceptibility of Bacteroides gingivalis to bactericidal activity of human serum.

Susceptibility of Bacteroides gingivalis strains to the complement-mediated bactericidal activity of human serum was examined under anaerobic conditions. Serum containing high concentrations of IgG antibody to B. gingivalis enhanced the bactericidal activity. No enhancement was found in serum which did not contain the specific antibody or in serum absorbed with intact cells. The sensitivity to the killing by pooled serum differed among B. gingivalis strains. Cells of B. gingivalis activated the pooled human serum complement not only through the classic pathway but also through an alternative pathway. It was found that the susceptibility of B. gingivalis to the bactericidal activity was classic-pathway-dependent.

Breath sample collection through the nostril reduces false-positive results of 13C-urea breath test for the diagnosis of helicobacter pylori infection.

BACKGROUND: One of the disadvantages of '3C-urea breath test is possible interference by urease activity not related to Helicobacterpylori. AIMS: We design the simple and non-invasive modification to avoid the contamination of 13CO(2) produced in the mouth. PATIENTS AND METHODS: One hundred and twenty-nine patients who underwent diagnostic upper endoscopy were enrolled. Within 1 week of the endoscopic procedure, each patient received the modified 13C-urea breath test. Breath samples were collected at baseline and at 1, 3, 5, 10, 15, 20 and 30 min after ingestion of 100 mg 13C-urea solution through the mouth and the nostril at each time point. RESULTS: The breath delta13CO2 value through the nostril at 1 min was already higher in H. pylori-positive patients than in H. pylori-negative patients. Using 2.5% as the cut-off value, the sensitivity and specificity of the modified 13C-urea breath test at 20 min were both 100%, whereas the sensitivity and specificity of the standard 13C-urea breath test were 97.7 and 94%, respectively, using 3% as the cut-off value. CONCLUSIONS: The modified 13C-urea breath test in which breath samples are collected through the nostril provides an easy way of avoiding false-positive results for the detection of H. pylori infection.

A mild transient decrease of peripheral red blood cell counts induced by a suprapharmacological dose of pegylated human megakaryocyte growth and development factor in rats.

Previous studies have shown that pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) at suprapharmacological dose induces a mild transient decrease of red blood cell counts according to thrombopoiesis in normal mice. To unravel the mechanism underlying this mild transient decrease of red blood cells, we have studied the effect of PEG-rHuMGDF on the circulating plasma and blood volume, and the serum biochemical parameters of anaemia and splenectomy. Also, we have performed histological studies of the bone marrow and the spleen of PEG-rHuMGDF-treated rats. PEG-rHuMGDF (300 microg kg(-1)]) or vehicle was subcutaneously administered to rats once a day for up to five days. From day 6 after the start of PEG-rHuMGDF administration, the platelet counts and plateletcrit levels were significantly increased, reaching peak values on day 10, and recovering to normal by day 20. The red blood cell counts and the haematocrit levels were significantly decreased on day 6 to 13. The decreases in red blood cell levels and haematocrit produced by PEG-rHuMGDF treatment were mild and had recovered by day 15. The plasma and blood volumes were significantly increased on day 10 in PEG-rHuMGDF-treated rats. No alteration of the serum biochemical parameters for anaemia, iron or total bilirubin, were observed on day 10. The histological examination on day 10 revealed a marked increase in megakaryocytes and a slight decrease in erythropoiesis in the bone marrow of rats that received PEG-rHuMGDF (300 microg kg(-1)). There was also a slight increase in splenic megakaryocytes and erythropoiesis. The decrease of red blood cells by PEG-rHuMGDF was not affected by splenectomy. These results suggest that the mild transient decrease of red blood cells induced by PEG-rHuMGDF treatment for up to five days is based mainly on the increases in the plasma and blood volume. These events are secondary changes due to the regulation of the excess production of megakaryocytes in the marrow and the peripheral platelets.

Precision control of the surface structure of gelatin films using micropit plates.

We attempted to use micropit plates as a silicon mold in order to control the surface structures of gelatin films. The three-dimensional micropit plates, fabricated with micromachining technology, have uniform quadrangular or circular pits with side lengths or diameters from 25-400 microm and depths of 40 microm. The micropit plates were originally hydrophilic because of silanol groups on the surface. In order to facilitate peeling the aqueous gelatin film from the micropit plates, the plate was therefore modified with hexamethyldisilazane. Gelatin films with precisely microfabricated structures on the surfaces were obtained by using the modified micropit plates. The release behavior of the films with different surface structures was examined.

Deltopectoral skin flap as a free skin flap revisited: further refinement in flap design, fabrication, and clinical usage.

The deltopectoral skin flap is an axial flap; therefore, it can be fashioned as a free skin flap. Although color and texture of the skin are well suited for facial resurfacing, the structural features of inconsistent thickness of the skin, a short vascular pedicle, a minute caliber of the nutrient vessel, and donor site morbidity often preclude the use of this flap for this purpose. The deltopectoral skin flap fabricated as a free skin flap transferred by means of a microsurgical technique was used in 27 patients between 1985 and 1998 at our hospital. The anterior perforating branches of the internal mammary vessels were the primary nutrient vessels of the flap in seven instances. The external caliber of this artery varied between 0.6 mm and 1.2 mm, with an average size of 0.9 mm. The size of the accompanying vein varied between 1.5 mm and 3.2 mm, with an average of 2.3 mm. Coaptation of these vessels with those in the recipient site was technically difficult. Thrombosis occurred at the anastomotic site in three patients, requiring reoperation. Two flaps were saved. The flap failure was drastically reduced in the remaining 20 patients by including a segment of the internal mammary vessel when fabricating the vascular pedicle. The size of the internal mammary arterial segment averaged 2.1 mm, and the average size of the accompanying vein was 2.9 mm. The problem of a bulky flap was managed by surgical defatting/thinning of the flap at the time of flap fabrication and transfer. A V-to-Y skin flap advancement technique of wound closure was used in eight individuals. The flap donor-site morbidities were minimized with this method of wound closure.

Fertilizability and developmental capacity of individually cultured bovine oocytes.

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.

[Closure of median sternotomy incision with EPTFE sutures].

EPTFE (CV-0) suture was utilized for closure of median sternotomy incision in 100 patients under 6 years of age who underwent open heart surgery. During 3 years follow up, except for one dislocation of sternum, all patients have healed without complication. EPTFE suture is swanged to a needle that has the same diameter as the thread, which reduces bleeding from the needle hole. It is radiologically lucent, and therefore does not disrupt chest X-ray, angiography or MRI investigations, in contrast to sternal wires. In addition, in the case of life threatening postoperative complication, EPTFE suture allows rapid and simple access to the heart, without the need to cut wires. We concluded that EPTFE suture can be utilized for closure of median sternotomy in patients under the age of six, without an increased risk of postoperative or long-term complications.