Hybrid-fuel bacterial flagellar motors in Escherichia coli

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H⁺ or Na⁺ ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H⁺- and Na⁺-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na⁺ concentration ([Na⁺]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na⁺ and H⁺ stator components expressed at high and medium levels, respectively, Na⁺ stator units dominate at high [Na⁺] and are replaced by H⁺ units when Na⁺ is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H⁺ and Na⁺ stator components sharing a common rotor.Molecular motors are tiny machines that perform a wide range of functions in living cells. Typically each motor generates mechanical work using a specific chemical or electrochemical energy source. Linear motors such as kinesin on microtubules or myosin on actin filaments and rotary motors such as F₁-ATPase, the soluble part of ATP-synthase, run on ATP, whereas the rotary bacterial flagellar motor embedded in the bacterial cell envelope is driven by the flux of ions across the cytoplasmic membrane (1–4). Coupling ions are known to be either protons (H⁺) or sodium ions (Na⁺) (5, 6).The bacterial flagellar motor consists of a rotor ∼50 nm in diameter surrounded by multiple stator units (7–10). Each unit contains two types of membrane proteins forming ion channels: MotA and MotB in H⁺ motors in neutrophiles (e.g., Escherichia coli and Salmonella) and PomA and PomB in Na⁺ motors in alkalophiles and Vibrio species (e.g., Vibrio alginolyticus) (1, 11). Multiple units interact with the rotor to generate torque independently in a working motor (9, 10, 12, 13). The structure and function of H⁺ and Na⁺ motors are very similar, to the extent that several functional chimeric motors have been made containing different mixtures of H⁺- and Na⁺-motor components (11). One such motor that runs on Na⁺ in E. coli combines the rotor of the H⁺-driven E. coli motor with the chimeric stator unit PomA/PotB, containing PomA from V. alginolyticus and a fusion protein between MotB from E. coli and PomB from V. alginolyticus (14).In most flagellated bacteria, motors are driven by ion-specific rotor–stator combinations. However, some species (e.g., Bacillus subtilis and Shewanella oneidensis) combine a single set of rotor genes with multiple sets of stator genes encoding both H⁺ and Na⁺ stator proteins, and it has been speculated that these stator components may interact with the rotor simultaneously, allowing a single motor to use both H⁺ and Na⁺. An appealing hypothesis that the mixture of stator components is controlled dynamically depending on the environment has arisen from the observation that the localization of both stator components depends upon Na⁺ (15). However, despite some experimental effort there is as yet no direct evidence of both H⁺ and Na⁺ stator units interacting with the same rotor (16).The rotation of single flagellar motors can be monitored in real time by light microscopy of polystyrene beads (diameter ∼1 μm) attached to truncated flagellar filaments (17). Under these conditions, the E. coli motor torque and speed are proportional to the number of stator units in both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB (17–19) motors. The maximum number of units that can work simultaneously in a single motor has been shown to be at least 11 by “resurrection” experiments, in which newly produced functional units lead to restoration of motor rotation in discrete speed increments in an E. coli strain lacking functional stator proteins (19). Stator units are not fixed permanently in a motor: Each dissociates from the motor with a typical rate of ∼2 min⁻¹, exchanging between the motor and a pool of diffusing units in the cytoplasmic membrane (20). Removal of the relevant ion gradient inactivates both H⁺ and Na⁺ stator units, most likely leading to dissociation from the motor into the membrane pool (2, 21, 22).Here we demonstrate a hybrid-fuel motor containing both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB stator components, sharing a common rotor in E. coli. We control the expression level of each stator type by induced expression from plasmids, and the affinity of Na⁺-driven stator units for the motor by external [Na⁺]. Units of each type compete for spaces around the rotor, and the motor torque is simply the sum of the independent contributions, with no evidence of direct interaction between units. Thus, we demonstrate the possibility of modularity in the E. coli flagellar motor, with ion selectivity determined by the choice of stator modules interacting with a common rotor. Our artificial hybrid motor demonstrates that species with multiple types of stator gene and a single set of rotor genes could contain natural hybrid motors that work on a similar principle (15, 16, 23).     

Midurethral autologous fascial sling surgery with reconstruction of the lower abdominal wall using the tensor fascia lata muscle flap for post‐hemipelvectomy stress urinary incontinence

Hemipelvectomy is surgery for pelvic bone neoplasms. In the case of pubic bone osteosarcoma, the distal end of the rectus abdominis muscle is severed from the pubic and ischium bones, and the pelvic floor muscles are resected en bloc with the bone, which leads to stress urinary incontinence. Cancer control is prioritized over complications, and stress urinary incontinence is generally disregarded. A 25‐year‐old woman presented with stress urinary incontinence. She had undergone a hemipelvectomy for left pubic bone osteosarcoma, and stress urinary incontinence appeared and persisted since the surgery. We carried out a reconstruction of the tissue deficit of the rectus abdominis using the tensor fascia lata muscle flap simultaneously with a midurethral autologous fascial sling anchoring to the tensor fascia lata flap. Stress incontinence was successfully improved without morbidity. This is the first reported case of midurethral suspension with reconstruction of the lower abdominal wall with the tensor fascia lata flap for post‐hemipelvectomy stress urinary incontinence.     

High-dose concurrent chemo–proton therapy for Stage III NSCLC: preliminary results of a Phase II study

The aim of this report is to present the preliminary results of a Phase II study of high-dose (74 Gy RBE) proton beam therapy (PBT) with concurrent chemotherapy for unresectable locally advanced non-small-cell lung cancer (NSCLC). Patients were treated with PBT and chemotherapy with monthly cisplatin (on Day 1) and vinorelbine (on Days 1 and 8). The treatment doses were 74 Gy RBE for the primary site and 66 Gy RBE for the lymph nodes without elective lymph nodes. Adapted planning was made during the treatment. A total of 15 patients with Stage III NSCLC (IIIA: 4, IIIB: 11) were evaluated in this study. The median follow-up period was 21.7 months. None of the patients experienced Grade 4 or 5 non-hematologic toxicities. Acute pneumonitis was observed in three patients (Grade 1 in one, and Grade 3 in two), but Grade 3 pneumonitis was considered to be non-proton-related. Grade 3 acute esophagitis and dermatitis were observed in one and two patients, respectively. Severe ( ≥ Grade 3) leukocytopenia, neutropenia and thrombocytopenia were observed in 10 patients, seven patients and one patient, respectively. Late radiation Grades 2 and 3 pneumonitis was observed in one patient each. Six patients (40%) experienced local recurrence at the primary site and were treated with 74 Gy RBE. Disease progression was observed in 11 patients. The mean survival time was 26.7 months. We concluded that high-dose PBT with concurrent chemotherapy is safe to use in the treatment of unresectable Stage III NSCLC.     

Correction to: Intermittent application of external positive pressure helps to preserve organ viability during ex vivo perfusion and culture

Journal of Artificial Organs - article Intermittent application     

DNA-daunorubicin complexes specifically suppress in vitro spontaneous anti-DNA antibody production in lymphocytes of patients with systemic lupus erythematosus

Elevated production of anti-DNA antibody in patients with systemic lupus erythematosus (SLE) is a central problem in the pathogenesis of tissue injury. In the present study, we attempted to manipulate anti-DNA antibody production through the antigen-cytotoxic drug conjugates, DNA-daunorubicin complexes. The effect of DNA-daunorubicin complexes was determined by examining SLE lymphocytes for spontaneous in vitro production of anti-DNA antibody. These complexes, at 2 μg/ml, suppressed anti-DNA antibody production, but not total IgG production, which suggests that specific suppression of anti-DNA antibody production was achieved at this concentration. We believe that the DNA-daunorubicin complexes affected mainly B cells, since such suppression was obtained by treating B cells, as well as B plus T cells. Furthermore, the complexes had no effect on the proliferative responses of SLE T cells to DNA, phytohemagglutinin, or concanavalin A. These results indicate that DNA-daunorubicin complexes may have the potential for selectively suppressing anti-DNA antibody production in patients with SLE.     

Increased serum amiodarone concentration in hypertriglyceridemic patients: Effects of drug distribution to serum lipoproteins

Amiodarone and its main metabolite, desethylamiodarone (DEA), are highly distributed to serum lipoproteins such as very‐low‐density lipoprotein (VLDL) and low‐density lipoprotein (LDL), which are the carriers of triglyceride and cholesterol. This study aimed to investigate the association of serum concentrations of amiodarone and DEA with the levels of serum lipids in terms of drug distribution to lipoprotein fractions in patients with hyperlipidemia. Total serum concentrations of amiodarone and DEA were examined in 116 patients receiving amiodarone for tachyarrhythmias. The concentration‐to‐dose (C/D) ratio of amiodarone positively correlated with the level of serum triglyceride (r_s  = 0.541, p < 0.001) and was higher in the hypertriglyceridemic state than in normotriglyceridemic state (479 ± 211 vs. 320 ± 161, p < 0.001). No correlation was found between the C/D ratio of DEA and serum triglyceride levels (r_s  = 0.272), although higher values were observed in the hypertriglyceridemic state (322 ± 125 vs. 285 ± 143, p < 0.001). In the hypertriglyceridemic state, the distribution of amiodarone increased in LDL/VLDL fraction and decreased in high‐density lipoprotein and albumin fractions. The ratio of serum amiodarone to serum DEA, a metabolic ratio of amiodarone, positively correlated with serum triglyceride levels (r_s  = 0.572, p < 0.001) and was higher in the hypertriglyceridemic state, suggesting that amiodarone metabolism decreased in hyperlipidemia. The results of this study reveal that serum concentrations of amiodarone increase in the hypertriglyceridemic state through the increased lipoprotein‐binding and decreased metabolism of amiodarone.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Lipoproteins can carry not only serum lipids but also certain lipophilic compounds such as amiodarone. Changes in the lipoprotein‐binding of amiodarone may lead to highly variable pharmacokinetics and poor concentration–effect relationships of the drug.

• WHAT QUESTION DID THIS STUDY ADDRESS?

This study addressed the association between serum amiodarone concentration and serum lipid levels in patients with arrhythmia, as well as the lipoprotein‐binding and metabolism of amiodarone in the hyperlipidemic state.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Serum amiodarone concentration is increased in patients with hypertriglyceridemia and it is positively correlated with serum triglyceride levels. These results are attributable to an increase in the circulating lipoprotein‐bound form of amiodarone and decreased metabolism of the drug in the hypertriglyceridemic state.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

Changes in the lipoprotein‐binding of amiodarone can help explain differences in the pharmacokinetics and pharmacodynamics of amiodarone related to normotriglyceridemic and hypertriglyceridemic states.     

Automated quantification of left ventricular function by the automated contour tracking method

The automated contour tracking (ACT) method has been developed for the automated measurement of area volume using the energy minimization method without tracing a region of interest. The purpose of this study was to compare the ACT method and left ventriculography (LVG) for the measurement of left ventricular (LV) function in the clinical setting. An apical four-chamber view was visualized by two-dimensional echocardiography and recorded for off-line analysis in 14 patients with high-quality images who underwent LVG. The ACT method automatically traces the endocardial border from the recorded images and calculates LV volumes (end-diastole and end-systole) and ejection fraction (EF). Both ACT and LVG were compared by linear regression analysis for the measurement of EF. EF determined by the ACT method agreed well with that by LVG (r = 0.96, y = 0.94x + 4.6, standard error of the estimate = 3.9%). The mean difference between the ACT and LVG was -1.4%+/- 7.3%.In conclusion, the ACT method is reliable for noninvasive estimation of EF in high-quality images. This suggests that this new technique may be useful in the automated quantification of LV function.     

Autoantigenic epitopes on dna topoisomerase i

Objective. To elucidate the clinical and immunogenetic associations with reactivity to autoantigenic epitopes on DNA topoisomerase I (topo I) recognized by sera from patients with systemic sclerosis (SSc). Methods. Autoantigenic epitopes on topo I were identified by screening an epitope library constructed from topo I complementary DNA restriction fragments using autoimmune anti–topo I–positive sera as a probe. Epitope reactivities of sera from 43 anti–topo I–positive SSc patients were surveyed by immunoblotting, and associations with clinical symptoms and HLA–DR types were examined. Results. Four different epitope regions were identified on the topo I molecule. Immunoreactivity to the region encompassing amino acid residues 658–700, termed ER4, was found to be associated with diffuse cutaneous SSc, progressive pulmonary interstitial fibrosis, and poor prognosis for 15-year survival. SSc patients with ER4 reactivity frequently displayed the DR2/DRw52 phenotype. Conclusion. Molecular analysis of precise antigenic epitopes on topo I is helpful in classifying clinical subsets of SSc.     

Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in japanese patients with inflammatory muscle disease

Objective. To elucidate the profile and clinical significance of autoantibodies to small nuclear and small cytoplasmic ribonucleoproteins in Japanese patients with polymyositis and dermatomyositis (PM/DM). Methods. Radioimmunoprecipitation assays were performed with sera from 91 patients with various inflammatory muscle diseases and from 254 control patients with other rheumatic diseases. Patients with PM/DM were categorized according to autoantibody specificities, and clinical comparisons were made. Results. Antibodies to aminoacyl transfer RNA (tRNA) synthetases and the signal recognition particle (SRP) were found to be specific for PM/DM. PM/DM patients with antibodies to 3 aminoacyl-tRNA synthetases (histidyl, threonyl, and glycyl) appeared to form a distinct clinical subset, associated with chronic interstitial lung disease, polyarthritis, and myositis, while 3 patients with antibodies that bound the SRP had severe myositis that was resistant to corticosteroid therapy. In general, the frequency and clinical correlations of anti—aminoacyl-tRNA synthetase antibodies in Japanese patients were similar to those in North American populations. In contrast, among this Japanese cohort, the occurrence of anti-PM/ScI appeared to be reduced in frequency, whereas anti-Ku and anti—U2 RNP antibodies were accentuated in patients with overlap syndromes. Conclusion. We conclude that racial groups differ somewhat in their characteristic autoimmune responses to individual autoantigens. These differences could reflect variation in immunogenetic background or different etiologic mechanisms which might occur at different geographic locations.