Associations between White Matter Hyperintensities and β Amyloid on Integrity of Projection,Association, and Limbic Fiber Tracts Measured with Diffusion Tensor MRI

The goal of this study was to assess the relationship between Aβ deposition and white matter pathology (i.e., white matter hyperintensities, WMH) on microstructural integrity of the white matter. Fifty-seven participants (mean age: 78±7 years) from an ongoing multi-site research program who spanned the spectrum of normal to mild cognitive impairment (Clinical dementia rating 0–0.5) and low to high risk factors for arteriosclerosis and WMH pathology (defined as WMH volume >0.5% total intracranial volume) were assessed with positron emission tomography (PET) with Pittsburg compound B (PiB) and magnetic resonance and diffusion tensor imaging (DTI). Multivariate analysis of covariance were used to investigate the relationship between Aβ deposition and WMH pathology on fractional anisotropy (FA) from 9 tracts of interest (i.e., corona radiata, internal capsule, cingulum, parahippocampal white matter, corpus callosum, superior longitudinal, superior and inferior front-occipital fasciculi, and fornix). WMH pathology was associated with reduced FA in projection (i.e., internal capsule and corona radiate) and association (i.e., superior longitudinal, superior and inferior fronto-occipital fasciculi) fiber tracts. Aβ deposition (i.e., PiB positivity) was associated with reduced FA in the fornix and splenium of the corpus callosum. There were interactions between PiB and WMH pathology in the internal capsule and parahippocampal white matter, where Aβ deposition reduced FA more among subjects with WMH pathology than those without. However, accounting for apoE ε4 genotype rendered these interactions insignificant. Although this finding suggests that apoE4 may increase amyloid deposition, both in the parenchyma (resulting in PiB positivity) and in blood vessels (resulting in amyloid angiopathy and WMH pathology), and that these two factors together may be associated with compromised white matter microstructural integrity in multiple brain regions, additional studies with a longitudinal design will be necessary to resolve this issue.     

Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.     

Pelagostrobilidium liui n. sp. (Ciliophora,Choreotrichida) from the Coastal Waters of Northeastern Taiwan and an Improved Description of Pelagostrobilidium minutum Liu et al., 2012

The number of somatic kineties in Pelagostrobilidium ranges from 4 to 6 according to the present state of knowledge. This study investigates Pelagostrobilidium liui n. sp. using live observation, protargol stain, and small subunit rDNA data sequencing. Pelagostrobilidium liui n. sp. is characterized by having a spherical‐shaped body, four somatic kineties, with kinety 2 spiraled around the left side of body, about six elongated external membranelles, and invariably no buccal membranelle. It differs from its most similar congener, Pelagostrobilidium minutum Liu et al., 2012 , in (i) cell shape; (ii) macronucleus width; (iii) oral apparatus; (iv) anterior orientation of kinety 2; (v) location where kinety 2 commences; (vi) arrangement of kinety 1; (vii) distance between the anterior cell end and the locations where kineties commence; and (viii) the presence of 12 different bases (including two deletions) in the small subunit rDNA sequences. The diagnosis of P. minutum Liu et al., 2012 is also improved to include the following new characteristics: invariably four somatic kineties; kineties 2 and 4 alone commence at the same level; kinety 2 originates from right anterior cell half on ventral side, extends sinistrally posteriorly, over kinety 1, around left posterior region, terminates near posterior cell end on dorsal side; kinety 1 commences below anterior third of kinety 2.     

Role of cardiopulmonary baroreflexes during dynamic exercise

To examine the role of cardiopulmonary (CP) mechanoreceptors in the regulation of arterial blood pressure during dynamic exercise in humans, we measured mean arterial pressure (MAP), cardiac output (Q), and forearm blood flow (FBF) during mild cycle ergometer exercise (77 W) in 14 volunteers in the supine position with and without lower-body negative pressure (LBNP). During exercise, MAP averaged 103 +/- 2 mmHg and was not altered by LBNP (-10, -20, or -40 mmHg). Steady-state Q during exercise was reduced from 10.2 +/- 0.5 to 9.2 +/- 0.5 l/min (P less than 0.05) by application of -10 mmHg LBNP, whereas heart rate (97 +/- 3 beats/min) was unchanged. MAP was maintained during -10 mmHg LBNP by an increase in total systemic vascular resistance (TSVR) from 10.3 +/- 0.5 to 11.4 +/- 0.6 U and forearm vascular resistance (FVR) from 17.5 +/- 1.9 to 23.3 +/- 2.6 U. The absence of a reflex tachycardia or reduction in arterial pulse pressure during -10 mmHg LBNP supports the hypothesis that the increase in TSVR and FVR results primarily from the unloading of CP mechanoreceptors. Because CP mechanoreceptor unloading during exercise stimulates reflex circulatory adjustments that act to defend the elevated MAP, we conclude that the elevation in MAP during exercise is regulated and not merely the consequence of differential changes in Q and TSVR. In addition, a major portion of the reduction in FBF in our experimental conditions occurs in the cutaneous circulation. As such, these data support the hypothesis that CP baroreflex control of cutaneous vasomotor tone is preserved during mild dynamic exercise.     

The RBP-Jκ Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

Kaposi''s sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman''s disease (MCD). However, the development of KSHV-mediated lymphoproliferative disease is not fully understood. Here, we generated two recombinant KSHV viruses deleted for the first RBP-Jκ binding site (RTA_1st) and all three RBP-Jκ binding sites (RTA_all) within the RTA promoter. Our results showed that RTA_1st and RTA_all recombinant viruses possess increased viral latency and a decreased capability for lytic replication in HEK 293 cells, enhancing colony formation and proliferation of infected cells. Furthermore, recombinant RTA_1st and RTA_all viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA_1st and RTA_all recombinant viruses infected both T and B cells and all three viruses efficiently infected T and B cells in a time-dependent manner early after infection. Also, the capability of both RTA_1st and RTA_all recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA_1st and RTA_all recombinant viruses showed greater infectivity for CD3+ T cells up to 7 days. Furthermore, studies in Telomerase-immortalized human umbilical vein endothelial (TIVE) cells infected with KSHV corroborated our data that RTA_1st and RTA_all recombinant viruses have enhanced ability to persist in latently infected cells with increased proliferation. These recombinant viruses now provide a model to explore early stages of primary infection in human PBMCs and development of KSHV-associated lymphoproliferative diseases.     

Risk Factors of Pneumothorax after Endobronchial Ultrasound-Guided Transbronchial Biopsy for Peripheral Lung Lesions

Background

The risk of endobronchial ultrasound-guided transbronchial biopsy-related pneumothorax is a major concern and warrants further studies. The aim of our study was to estimate the risk of pneumothorax after this procedure and identify its risk factors.

Methods

From 2007 to 2011, 399 patients who underwent endobronchial ultrasound-guided transbronchial biopsy for peripheral lung lesions were included in this study. The variables analyzed included patient factors, lesion factors and procedure factors. Multivariate logistic regression analysis was used to identify independent risk factors for pneumothorax.

Results

The incidence of pneumothorax was 3.3% (13/399). Chest tube placement was required for 31% (4/13) of pneumothoraces. Independent risk factors for pneumothorax included pulmonary emphysema (OR, 55.09; 95% CI, 9.37–324.03; p<0.001) and probe position adjacent to the lesion (OR, 17.01; 95% CI, 2.85–101.64; p = 0.002). The number of biopsy specimens, age, sex, history of prior lung surgery and lesion size, location and character did not influence the risk of pneumothorax in our analyses.

Conclusions

The risk of pneumothorax after endobronchial ultrasound-guided transbronchial biopsy is low. To further reduce the risk of pneumothorax, every effort should be made to advance the endobronchial ultrasound probe into the bronchus where it is imaged within the target lesion before embarking on transbronchial biopsy.     

Pivotal role of ADP-ribosylation factor 6 in Toll-like receptor 9-mediated immune signaling

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.     

Design, synthesis and biological evaluation of pyridine-phenylpiperazines: a novel series of potent and selective alpha1a-adrenergic receptor antagonist

Beginning from the screening hit and literature alpha1-adrenergic compounds, a hybridized basic skeleton A was proposed as the pharmacophore for potent and selective alpha1a-AR antagonists. Introduction of a hydroxy group to increase the flexibility afforded B which served as the screening model and resulted in the identification of the second-generation lead 1. Using the Topliss approach, a number of potent and selective alpha1a-AR antagonists were discovered. In all cases, binding affinity and selectivity at the alpha1a-AR of S-hydroxy enantiomers were higher than the R-hydroxy enantiomers. As compared to the des-hydroxy analogues, the S-hydroxy enantiomers displayed comparable potency and better selectivity at alpha1a-AR. The S-hydroxy enantiomer 17 (Ki = 0.79 nM; alpha1b/alpha1a = 800; alpha1d/alpha1a = 104) was slightly less potent but much more selective at alpha1a-AR than tamsulosin (Ki = 0.13 nM, alpha1b/alpha1a = 15, alpha1d/alpha1a = 1.4). Compound 17 displayed higher selectivity in inhibiting rat prostate contraction over rat aorta contraction and also exhibited a higher degree of uroselectivity than tamsulosin in the anesthetized dog model.