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Nanoscopic investigation of bacterial cells is essential to reveal their physiological status, impacting all cellular functions. Currently, this requires labeled probes or targeted staining procedures. Herein, we report a new bacterial feature, intracellular dynamics-resolved Rayleigh scattering (IDRS), that visualizes spatiotemporal cytoplasmic transitions in unlabeled bacteria and characterizes their real-time physiological status in 10 s. From single-bacterium IDRS signals, we discovered unique spatial patterns and their multiple transitions in Gram-negative and Gram-positive bacteria. The magnitude of IDRS signal variation highly correlated with the metabolic status of bacteria, differentiating persistent subpopulations. This is also the first report demonstrating distinct real-time metabolic conditions of unlabeled drug-resistant bacteria that are exposed to different doses of antibiotics. Our strategy opens up a way to simultaneously trace in situmetabolic and antibiotic resistance statuses, which can be applied in single-cell level control of bacterial metabolism and efficacy with a heterogeneous nature.

The purpose of this study was to investigate the correlation of primary tumor FDG uptake to clinicopathological prognostic factors in invasive ductal carcinoma of the breast. We retrospectively reviewed 136 of 215 female patients with pathologically proven invasive ductal breast cancer from January 2008 to December 2011 who underwent F-18 FDG PET/CT for initial staging and follow-up after curative treatment with analysis of estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (HER2). The maximum standardized uptake value (SUVmax) of the primary breast tumor was measured and compared with hormonal receptor and HER2 overexpression status. The high SUVmax of primary breast tumors is significantly correlated with the clinicopathological factors: tumor size, histologic grade, TNM stage, negativity of ER, negativity of PR, HER2 overexpression and triple negativity. The recurrent group with non-triple negative cancer had a higher SUVmax compared with the non-recurrent group, though no significant difference in FDG uptake was noted between the recurrence and non-recurrent groups in subjects with triple-negative cancer. Lymph node involvement was the independent risk factor for cancer recurrence in the multivariate analysis. In conclusion, high FDG uptake in primary breast tumors is significantly correlated with clinicopathological factors, such as tumor size, histologic grade, TNM stage, negativity of the hormonal receptor, HER2 overexpression and triple negativity. Therefore, FDG PET/CT is a helpful prognostic tool to direct the further management of patients with breast cancer.

BACKGROUND: The International Liaison Committee on Resuscitation (ILCOR) recommends that strategies should be implemented that promote cardiopulmonary resuscitation (CPR) training in the workplace. Non-medical employees at a hospital were therefore trained to conduct basic life support (BLS). Subject background information, test results and survey findings were examined and factors affecting BLS skill acquisition were studied. METHODS: Of 1432 non-medical employees at a hospital trained to conduct BLS, 880 agreed to participate in the survey. The training course consisted of a single session of 3 h of lectures, practice and testing. Skill acquisition was assessed using a 13-item skill checklist and a 5-point overall competency scale. The effects of age, gender, type of job, educational status, a previous history of CPR training and level of subject-perceived training difficulty were examined. RESULTS: According to total checklist scores, subjects achieved a mean (SD) score of 8.66 (3.57). 22.3% performed all 13 skills. Based on 5-point overall competency ratings, 43.7% of subjects were rated as "competent", "very good" or "outstanding". Age (<40 years and 40 years) was the only factor that significantly affected skill acquisition (skill acquisition by those 40 years of age was poorer than by those aged <40 years). CONCLUSION: Traditional BLS training is less effective in individuals aged 40 years.

De novo development of anti–human leukocyte antigen (HLA) antibodies after transplantation is associated with increased rejection and decreased graft survival. In this study, the effect of posttransplant HLA antibodies on clinical outcome was evaluated in patients treated with tacrolimus by means of flow cytometric crossmatches (FCXm).

Forward slip is an important parameter often used in rolling-speed control models for tandem hot strip rolling mills. In a hot strip mill, on-line measurement of strip speed is inherently very difficult. Therefore, for the set-up of the finishing mill, a forward slip model is used to calculate the strip speed from roll circumferential velocity at each mill stand. Due to its complexity, most previous researches have used semi-empirical methods in determining values for the forward slip. Although these investigations may be useful in process design and control, they do not have a theoretical basis. In the present study, a better forward slip model has been developed, which provides for a better set-up and more precise control of the mill. Factors such as neutral point, friction coefficient, width spread, shape of deformation zone in the roll bite are incorporated into the model. Implementation of the new forward slip model for the control of a 7-stand hot strip tandem rolling mill shows significant improvement in roll speed set-up accuracy.

Forward slip is an important parameter often used in rolling-speed control models for tandem hot strip rolling mills. In a hot strip mill, on-line measurement of strip speed is inherently very difficult. Therefore, for the set-up of the finishing mill, a forward slip model is used to calculate the strip speed from roll circumferential velocity at each mill stand. Due to its complexity, most previous researches have used semi-empirical methods in determining values for the forward slip. Although these investigations may be useful in process design and control, they do not have a theoretical basis. In the present study, a better forward slip model has been developed, which provides for a better set-up and more precise control of the mill. Factors such as neutral point, friction coefficient, width spread, shape of deformation zone in the roll bite are incorporated into the model. Implementation of the new forward slip model for the control of a 7-stand hot strip tandem rolling mill shows significant improvement in roll speed set-up accuracy.

Antidiuretic hormone modulates the water permeability (Pf) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel Pf(Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel Pf, we compared the Pf values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [gamma-32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the Pf (0.16 +/- 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 +/- 0. 3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 +/- 8% of the phosphate has been removed (Pf 0.14 +/- 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's Pf. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments.

The in situ state of assembly of the human erythrocyte anion carrier (band 3) has been investigated by applying target size analysis on the radiation-induced inactivation of anion flux and degradation of polypeptide band 3. Irradiation with a high energy electron beam resulted in inactivation of carrier-mediated anion flux in intact cells, in inside-out vesicles devoid of cytoskeletal and cytoplasmic proteins, and in inside-out vesicles whose band 3 protein has been partially proteolyzed, with little change in leak pathway. The inactivation showed a single exponential function of radiation dose from which the target size of the anion carrier was estimated to be 210,000, 220,000, and 98,000 daltons in intact cells, in the inside-out vesicles, and in the vesicles after a limited proteolysis, respectively. Irradiation also resulted in degradation of the band 3 of the inside-out vesicles, as detected on sodium dodecyl sulfate-gel electrophoresis, with a dose dependence characteristic of a target size of 220,000 daltons. It is suggested that the human erythrocyte anion carrier exists in situ as a dimer of band 3.

Antidiuretic hormone (ADH) regulates renal water excretion by altering the permeability of the collecting duct to water. ADH-responsive epithelial cells are the major cell type lining kidney tubules in the inner medulla and papilla. ADH modulates apical membrane water permeability by the insertion and removal of vesicles containing aquaporin collecting duct water channel protein (now termed AQP-2). To identify and characterize proteins responsible for trafficking of AQP-2-containing vesicles, we utilized antibody and cDNA probes to synaptobrevin b (also termed VAMP-2, for vesicle-associated membrane protein 2), a protein that mediates synaptic vesicle exocytosis in the brain and whose structural homologs are now considered to be components of a complex responsible for intracellular vesicle fusion in all cells. We now report that rat kidney inner medulla and papilla contain abundant synaptobrevin protein. Only light endosomes, one of two types of purified papillary AQP-2-containing endosomes, possess synaptobrevin. Light endosomes fuse in vitro by means of an ATP-dependent process that is significantly inhibited when endosomes are preincubated with either anti-synaptobrevin antibody or tetanus toxin. These data define a functional role for a synaptobrevin protein in the fusion of endosomes in vitro. The presence of abundant synaptobrevin proteins in endosomes containing AQP-2 water channels, as well as insulin-sensitive glucose transporters [Cain, C. C., Trimble, W. S. & Lienhard, G. E. (1992) J. Biol. Chem. 267, 11681-11684], and in cells of Malpighian tubules responsible for urine formation in insects [Chin, A. S., Burgess, R. W., Wong, B. R., Schwartz, T. L. & Scheller, R. H. (1993) Gene 131, 175-181] suggests a specialized role for synaptobrevin in vesicle-mediated membrane transport modulated by peptide hormones.

We have previously shown that the sulfonylureas increase insulin-stimulated glucose transport in adipocytes mainly by enhancing the insulin-induced recruitment of glucose transporter from its intracellular storage pool to the plasma membrane (Jacobs, D. B., and Jung, C. Y. (1985) J. Biol. Chem. 260, 2593–2596). In order to determine if this sulfonylurea effect is mediated by a specific membrane-associated sulfonylurea-binding protein, in the present report we measured exact dose dependence of the transport enhancement activities of different sulfonylureas in adipocytes in primary culture and equilibrium binding affinities of these agents to various adipocyte membrane fractions. Glycuride was found to increase the insulin-stimulated, 3-O-methyl-D-glucose equilibrium exchange in cultured rat adipocytes by up to 60% with little effect in the absence of insulin. The effect developed gradually reaching the maximum level at 24 h of incubation. The effect was concentration dependent showing a simple, one-to-one stoichiometry and an apparent activation constant (Ka) of approximately 1 εM. Glypizide, tolazamide, and tolbutamide also enhanced the insulin-stimulated hexose transport by up to 60%, but with Kaof approximately 2, 11, and 25 εM, respectively. HB-699 and ciglitazone, non-sulfonylureas, were without effect under the same condition. In equilibrium binding experiments, [3H]glyburide was found to bind to adipocyte membranes at two or more protein-specific, saturable sites, with similar apparent dissociation constants (KD) ranging 1–3 εM. These protein-specific glyburide bindings were displaced not only by tolazamide and tolbutamide, but also by ciglitazone and HB-699, with indicated KDof 11–16, 80–85, 20–25, and 85–95 εM, respectively. However, with the plasma membrane fraction, the displacements by ciglitazone and HB-699 were partial and did not exceed 56–61% at maximum. Based on these findings, we propose that there is a sulfonylurea-specific-binding protein in the plasma membrane of adipocytes, and that this sulfonylurea-binding protein may play a key role in the enhancement of insulin-stimulated hexose transport by sulfonylureas, probably via potentiation of the insulin-induced recruitment of glucose transporter.

ABSTRACTFifty-three rotavirus-positive fecal specimens from children with diarrhea admitted to a Rio de Janeiro, Brazil, children's hospital between January 1997 and December 1998 were characterized for P and G types by using reverse transcription-PCR. Genotype P[4]G2 accounted for 21% of isolates, while uncommon genotypes P[8]G9, P[6]G9, and P[4]G9 accounted for 13% of the isolates.

The isoquinolinetriones (I) react with the trialkyl phosphites (II), producing the isomeric isoquinolinedion‐4‐ylidenes (III) and (IV).