Structure function differences in nonpeptide CCR1 antagonists for human and mouse CCR1

A useful strategy for identifying ligand binding domains of G protein-coupled receptors has been the exploitation of species differences in antagonist potencies. We have used this approach for the CCR1 chemokine receptor with a novel series of antagonists, the 4-hydroxypiperidines, which were discovered by high throughput screening of human CCR1 and subsequently optimized. The structure-activity relationships for a number of different 4-hydroxypiperidine antagonists for human and mouse CCR1 were examined by receptor binding and functional assays. These compounds exhibit major differences in their rank order of potency for the human and mouse chemokine receptor CCR1. For example, the initial lead template, BX 510, which was a highly potent functional antagonist for human CCR1 (K(i) = 21 nM) was >400-fold less active on mouse CCR1 (K(i) = 9150 nM). However, increasing the length of the linker between the piperidine and dibenzothiepine groups by one methylene group generated a compound, BX 511, which was equipotent for both human and mouse CCR1. These and other analogs of the lead template BX 510, which have major differences in potency for human and mouse CCR1, are described, and a model for their interaction with human CCR1 is presented.     

A human TERT C-terminal polypeptide sensitizes HeLa cells to H2O2-induced senescence without affecting telomerase enzymatic activity

We have constructed a 27-kDa hTERT C-terminal polypeptide (hTERTC27) devoid of domains required for telomerase activity and demonstrated that it is capable of nuclear translocation/telomere-end targeting. Here we showed that expression of a low level of hTERTC27 renders hTERT positive HeLa cells sensitive to H(2)O(2)-induced oxidative stress and subsequent cell senescence. The senescence-associated gene, the cyclin/cdk inhibitor p21(Waf1), was up-regulated. This occurs without changing the expression of endogenous hTERT, causing significant telomere shortening or inhibiting telomerase activity. Results from this study suggest for the first time that in addition to telomerase activity, the C-terminus of hTERT also plays a role in hTERT-mediated cellular resistance to oxidative stress.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

Phylogenetic relationships of the ubiquitous coral reef crab subfamily Chlorodiellinae (Decapoda,Brachyura, Xanthidae)

The xanthid subfamily Chlorodiellinae is one of the most ubiquitous coral reef crab taxa in the Indo‐West Pacific region. Many species are common in coral rubble and rocky shores from Hawaii to eastern Africa, often dominating reef cryptofauna in terms of biomass. Phylogenetic analyses of mitochondrial (COX1, 12S rRNA and 16S rRNA) and nuclear (histone H3) gene sequences of 202 specimens indicate that the Chlorodiellinae is polyphyletic as presently defined. Three genera, Pilodius, Cyclodius and Chlorodiella, and two previously undescribed lineages were recovered as a well‐supported clade. In combination with morphological data, the subfamily is redefined and restricted to this clade. Two new genera, Soliella gen. n., and Luniella gen. n., are described based on features of the carapace, male thoracic sternum and male gonopods. The remaining chlorodielline genera and members of the Etisinae, a subfamily with supposedly close morphological affinities to the Chlorodiellinae, were recovered at various positions throughout the xanthid phylogeny, although with relatively low support values. These results reiterate the unresolved status of xanthid subfamilial relationships, but nevertheless provide progress for xanthid systematics.     

Platoon Interactions and Real-World Traffic Simulation and Validation Based on the LWR-IM

Platoon based traffic flow models form the underlying theoretical framework in traffic simulation tools. They are essentially important in facilitating efficient performance calculation and evaluation in urban traffic networks. For this purpose, a new platoon-based macroscopic model called the LWR-IM has been developed in [1]. Preliminary analytical validation conducted previously has proven the feasibility of the model. In this paper, the LWR-IM is further enhanced with algorithms that describe platoon interactions in urban arterials. The LWR-IM and the proposed platoon interaction algorithms are implemented in the real-world class I and class II urban arterials. Another purpose of the work is to perform quantitative validation to investigate the validity and ability of the LWR-IM and its underlying algorithms to describe platoon interactions and simulate performance indices that closely resemble the real traffic situations. The quantitative validation of the LWR-IM is achieved by performing a two-sampled t-test on queues simulated by the LWR-IM and real queues observed at these real-world locations. The results reveal insignificant differences of simulated queues with real queues where the p-values produced concluded that the null hypothesis cannot be rejected. Thus, the quantitative validation further proved the validity of the LWR-IM and the embedded platoon interactions algorithm for the intended purpose.     

Radiation dose of digital radiography (DR) versus micro-dose x-ray (EOS) on patients with adolescent idiopathic scoliosis: 2016 SOSORT- IRSSD “John Sevastic Award” Winner in Imaging Research

Background

Patients with adolescent idiopathic scoliosis (AIS) frequently receive x-ray imaging at diagnosis and subsequent follow monitoring. The ionizing radiation exposure has accumulated through their development stage and the effect of radiation to this young vulnerable group of patients is uncertain. To achieve the ALARA (as low as reasonably achievable) concept of radiation dose in medical imaging, a slot-scanning x-ray technique by the EOS system has been adopted and the radiation dose using micro-dose protocol was compared with the standard digital radiography on patients with AIS.

Methods

Ninety-nine participants with AIS underwent micro-dose EOS and 33 underwent standard digital radiography (DR) for imaging of the whole spine. Entrance-skin dose was measured using thermoluminescent dosimeters (TLD) at three regions (i.e. dorsal sites at the level of sternal notch, nipple line, symphysis pubis). Effective dose and organ dose were calculated by simulation using PCXMC 2.0. Data from two x-ray systems were compared using independent-samples t-test and significance level at 0.05. All TLD measurements were conducted on PA projection only. Image quality was also assessed by two raters using Cobb angle measurement and a set of imaging parameters for optimization purposes.

Results

Entrance-skin dose from micro-dose EOS system was 5.9–27.0 times lower at various regions compared with standard DR. The calculated effective dose was 2.6?±?0.5 (μSv) and 67.5?±?23.3 (μSv) from micro-dose and standard DR, respectively. The reduction in the micro-dose was approximately 26 times. Organ doses at thyroid, lung and gonad regions were significantly lower in micro-dose (p?<?0.001). Data were further compared within the different gender groups. Females received significantly higher (p?<?0.001) organ dose at ovaries compared to the testes in males. Patients with AIS received approximately 16–34 times lesser organ dose from micro-dose x-ray as compared with the standard DR. There was no significant difference in overall rating of imaging quality between EOS and DR. Micro-dose protocol provided enough quality to perform consistent measurement on Cobb angle.

Conclusions

Entrance-skin dose, effective dose and organ dose were significantly reduced in micro-dose x-ray. The effective dose of a single micro-dose x-ray (2.6 μSv) was less than a day of background radiation. As AIS patients require periodic x-ray follow up for surveillance of curve progression, clinical use of micro-dose x-ray system is beneficial for these young patients to reduce the intake of ionizing radiation.

     

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.     

Clinical analysis of the related factors in acute appendicitis

BACKGROUND: In order to determine reliable clues for early diagnosis of acute appendicitis, this study was conducted to examine the related factors in patients with clinically suspected acute appendicitis. METHODS: We retrospectively analyzed 282 patients with the clinical diagnosis of acute appendicitis at China Medical College Hospital in Taiwan from January to December 2000. To study the significant related factors of acute appendicitis, the t-test, chi-square analysis, and multivariate logistic regression analysis were used. RESULTS: There were 153 males (54.3 percent) and 129 females (45.7 percent). The mean age was 30.3+/-17.4 years (range 1 to 81). The diagnostic rate of acute appendicitis was 86.2 percent. If the combination of elevated C-reactive protein, leukocytosis and elevated neutrophil ratio was used, satisfactory specificity and positive predictive value were achieved in diagnosing acute appendicitis. After controlling for the other covariates, the multivariate logistic regression analysis showed that the significant related factors of acute appendicitis were male sex (odds ratio = 3.4; 95 percent confidence interval = 1.6 to 7.3; p <0.01) and elevated neutrophil ratio (odds ratio = 4.6; 95 percent confidence interval = 2.0 to 10.6; p <0.001). CONCLUSIONS: If an elevated neutrophil ratio was observed, the probability of acute appendicitis was increased in patients with clinically suspected acute appendicitis. Thus, neutrophil ratio appears to be a good parameter for diagnosis of acute appendicitis in primary healthcare settings.