A Stepwise Approach Using Metastatic Lymph Node Ratio-Combined Thyroglobulin for Customization of [18F]FDG-PET/CT Indication to Detect Persistent Disease in Patients with Papillary Thyroid Cancer.

We investigated whether an indication for [18F]FDG-PET/CT to detect FDG-avid persistent disease (PD) could be identified precisely using the extent of metastatic lymph nodes (MLNs) and serum thyroglobulin (Tg) in papillary thyroid cancer (PTC) patients. This retrospective study included 429 PTC patients who underwent surgery and radioactive iodine (RAI) therapy. [18F]FDG-PET/CT and serum Tg were evaluated just before RAI therapy. The MLN ratio (LNR) was defined as the ratio of the number of MLNs to the number of removed LNs. To derive the LNR-combined criteria, different Tg cut-off values for identifying the PET/CT-indicated group for PD detection were applied individually to subgroups initially classified based on LNR cut-off values. The cut-off values for serum Tg, the number of MLNs, and LNR for a PET/CT indication were 6.0 ng/mL, 5, and 0.51, respectively. Compared to a single parameter (serum Tg, total number of MLNs, and LNR), the LNR-combined criteria showed significantly superior diagnostic performance in detecting FDG-avid PD (p < 0.001). The diagnostic performance of PET/CT in detecting FDG-avid PD was significantly improved when the PET/CT-indicated group was identified through the LNR-combined criteria in a stepwise manner; this can contribute to a customized PET/CT indication in PTC patients.

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different "Nabi1" constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz.

Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential.

ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al., 2012; Han et al., 2013). Sequence alignment of voltage-sensing phosphatases from different species revealed conserved polar and charged residues at 7 aa intervals in the S1-S3 transmembrane segments of the voltage-sensing domain, suggesting potential coil-coil interactions. The contribution of these residues to the voltage-induced optical signal was tested using a cassette mutagenesis screen by flanking each transmembrane segment with unique restriction sites to allow for the testing of individual mutations in each transmembrane segment, as well as combinations in all four transmembrane segments. Addition of a counter charge in S2 improved the kinetics of the optical response. A double mutation in the S4 domain dramatically reduced the slow component of the optical signal seen in ArcLight. Combining that double S4 mutant with the mutation in the S2 domain yielded a probe with kinetics <10 ms. Optimization of the linker sequence between S4 and the fluorescent protein resulted in a new ArcLight-derived probe, Bongwoori, capable of resolving action potentials in a hippocampal neuron firing at 60 Hz. Additional manipulation of the voltage-sensing domain could potentially lead to fluorescent sensors capable of optically resolving neuronal inhibition and subthreshold synaptic activity.

Synthesis and biological evaluation of (±)-3-(2-(2-fluorobenzyloxy) naphthalen-6-yl)-2-aminopropanoic acid derivatives as novel PTP1B inhibitors.

A series of novel nonphosphonate-based pTyr mimetics comprised (±)-3-(2-(2-fluorobenzyloxy)naphthalen-6-yl)-2-aminopropanoic acid derivatives were identified as reversible and competitive PTP1B inhibitors via a structure-based design approach. Among the compounds studied, 12h was found to have the best in vitro inhibition activity against PTP1B (IC(50) = 1.25 ± 0.24 µM) and the best selectivity (3-fold) between PTP1B and TCPTP. These results should provide suitable druglike lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

Molecular cloning and characterization of clyA genes in various serotypes of Salmonella enterica.

Cytolysin A (ClyA) is a pore-forming hemolytic protein encoded by the clyA gene. It has been identified in Salmonella enterica serovars Typhi and Paratyphi A. To identify and characterize the clyA genes in various Salmonella enterica strains, 21 different serotypes of strains isolated from clinical specimens were presently examined. Full-length clyA genes were found in S. enterica serovar Brandenburg, Indiana, Panama, and Schwarzengrund strains by polymerase chain reaction amplification. The ClyA proteins from these four strains showed >97% amino acid identity to that of S. enterica serovar Typhi. Although all four serovars expressed detectable levels of ClyA as determined by Western blot analysis, they did not show a strong hemolytic effect on blood agar, indicating that ClyA may not be efficiently expressed or secreted. Escherichia coli transformed with clyA genes from the four serovars enhanced production of ClyA proteins and hemolytic activities to a level similar to S. enterica serovar Typhi ClyA. The present results suggest that ClyA may play a role in the pathogenesis of S. enterica serovar Brandenburg, Indiana, Panama and Schwarzengrund.

Immunological responses induced by asd and wzy/asd mutant strains of Salmonella enterica serovar Typhimurium in BALB/c mice.

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate beta-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer's patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 10(6) higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1x10(10)CFU) or i.p. (1x10(7) CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD(50) (5x10(6) CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.

Expression of c-Myc is related to host cell death following Salmonella typhimurium infection in macrophage.

It has been known that ornithine decarboxylase (ODC) induced by the binding of c-Myc to odc gene is closely linked to cell death. Here, we investigated the relationship between their expressions and cell death in macrophage cells following treatment with Salmonella typhimurium or lipopolysaccharide (LPS). ODC expression was increased by bacteria or LPS and repressed by inhibitors against mitogen-activated protein kinases (MAPKs) in Toll-like receptor 4 (TLR4) signaling pathway. In contrast, c-Myc protein level was increased after treatment with bacteria, but not by treatment with LPS or heat-killed bacteria although both bacteria and LPS increased the levels of c-myc mRNA to a similar extent. c-Myc protein level is dependent upon bacterial invasion because treatment with cytochalasin D (CCD), inhibitors of endocytosis, decreased c-Myc protein level. The cell death induced by bacteria was significantly decreased after treatment of CCD or c-Myc inhibitor, indicating that cell death by S. typhimurium infection is related to c-Myc, but not ODC. Consistent with this conclusion, treatment with bacteria mutated to host invasion did not increase c-Myc protein level and cell death rate. Taken together, it is suggested that induction of c-Myc by live bacterial infection is directly related to host cell death.