Frequency weighting derived from power absorption of fingers-hand-arm system under z(h)-axis vibration

The objectives of this study are to derive the frequency weighting from three vibration power absorption (VPA) methods (finger VPA, palm VPA, and total or hand VPA), and to explore whether these energy methods are better than the currently accepted acceleration method. To calculate the VPA weightings, the mechanical impedance of eight subjects exposed to a broadband random vibration spectrum in the z(h)-axis using 18 combinations of hand couplings and applied forces was measured. The VPA weightings were compared with the frequency weighting specified in ISO 5349-1 [2001. Mechanical Vibration--Measurement and Evaluation of Human Exposure to Hand--Transmitted Vibration--Part 1: General Requirements. International Organization for Standardization, Geneva, Switzerland]. This study found that the hand and palm VPA weightings are very similar to the ISO weighting but the finger VPA weighting for the combined grip and push action is much higher than the ISO weighting at frequencies higher than 25 Hz. Therefore, this study predicted that the total power absorption of the entire hand-arm system is likely to be correlated with psychophysical response or subjective sensation. However, if the ISO weighting method cannot yield good predictions of the vibration-induced disorders in the fingers and hand, the hand and palm energy methods are unlikely to yield significantly better predictions. The finger VPA is a vibration measure between unweighted and ISO weighted accelerations. The palm VPA method may have some value for studying the disorders in the wrist-arm system.     

FMRFamide-Like Peptides (FLPs) Enhance Voltage-Gated Calcium Currents to Elicit Muscle Contraction in the Human Parasite Schistosoma mansoni

Schistosomes are amongst the most important and neglected pathogens in the world, and schistosomiasis control relies almost exclusively on a single drug. The neuromuscular system of schistosomes is fertile ground for therapeutic intervention, yet the details of physiological events involved in neuromuscular function remain largely unknown. Short amidated neuropeptides, FMRFamide-like peptides (FLPs), are distributed abundantly throughout the nervous system of every flatworm examined and they produce potent myoexcitation. Our goal here was to determine the mechanism by which FLPs elicit contractions of schistosome muscle fibers. Contraction studies showed that the FLP Tyr-Ile-Arg-Phe-amide (YIRFamide) contracts the muscle fibers through a mechanism that requires Ca²⁺ influx through sarcolemmal voltage operated Ca²⁺ channels (VOCCs), as the contractions are inhibited by classical VOCC blockers nicardipine, verapamil and methoxyverapamil. Whole-cell patch-clamp experiments revealed that inward currents through VOCCs are significantly and reversibly enhanced by the application of 1 µM YIRFamide; the sustained inward currents were increased to 190% of controls and the peak currents were increased to 180%. In order to examine the biochemical link between the FLP receptor and the VOCCs, PKC inhibitors calphostin C, RO 31–8220 and chelerythrine were tested and all produced concentration dependent block of the contractions elicited by 1 µM YIRFamide. Taken together, the data show that FLPs elicit contractions by enhancing Ca²⁺ influx through VOCC currents using a PKC-dependent pathway.     

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.     

An ADAMTSL2 Founder Mutation Causes Musladin-Lueke Syndrome,a Heritable Disorder of Beagle Dogs,Featuring Stiff Skin and Joint Contractures

Background

Musladin-Lueke Syndrome (MLS) is a hereditary disorder affecting Beagle dogs that manifests with extensive fibrosis of the skin and joints. In this respect, it resembles human stiff skin syndrome and the Tight skin mouse, each of which is caused by gene defects affecting fibrillin-1, a major component of tissue microfibrils. The objective of this work was to determine the genetic basis of MLS and the molecular consequence of the identified mutation.

Methodology and Principal Findings

We mapped the locus for MLS by genome-wide association to a 3.05 Mb haplotype on canine chromosome 9 (CFA9 (50.11–54.26; p_raw <10⁻⁷)), which was homozygous and identical-by-descent among all affected dogs, consistent with recessive inheritance of a founder mutation. Sequence analysis of a candidate gene at this locus, ADAMTSL2, which is responsible for the human TGFβ dysregulation syndrome, Geleophysic Dysplasia (GD), uncovered a mutation in exon 7 (c.660C>T; p.R221C) perfectly associated with MLS (p-value = 10⁻¹²). Murine ADAMTSL2 containing the p.R221C mutation formed anomalous disulfide-bonded dimers when transiently expressed in COS-1, HEK293F and CHO cells, and was present in the medium of these cells at lower levels than wild-type ADAMTSL2 expressed in parallel.

Conclusions/Significance

The genetic basis of MLS is a founder mutation in ADAMTSL2, previously shown to interact with latent TGF-β binding protein, which binds fibrillin-1. The molecular effect of the founder mutation on ADAMTSL2 is formation of disulfide-bonded dimers. Although caused by a distinct mutation, and having a milder phenotype than human GD, MLS nevertheless offers a new animal model for study of GD, and for prospective insights on mechanisms and pathways of skin fibrosis and joint contractures.     

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells

The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA‐dependent DEAD‐box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP₆)‐bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure‐function analysis in S. cerevisiae and human (h) cells, and find that the high‐affinity Nup42‐Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy‐terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1‐Dbp5/hDDX19B interaction site. A nup42‐CTD/gle1‐CTD/Dbp5 trimeric complex forms in the presence of IP₆. Deletion of NUP42 abrogates Gle1‐Dbp5 interaction, and disruption of the Nup42 or IP₆ binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42‐CTD and IP₆ stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42‐hGle1B interaction.      

Identification of epidermal growth factor receptor-positive glioblastoma using lipid-encapsulated targeted superparamagnetic iron oxide nanoparticles in vitro

Background

Targeted superparamagnetic iron oxide (SPIO) nanoparticles have emerged as a promising biomarker detection tool for molecular magnetic resonance (MR) image diagnosis. To identify patients who could benefit from Epidermal growth factor receptor (EGFR)-targeted therapies, we introduce lipid-encapsulated SPIO nanoparticles and hypothesized that anti-EGFR antibody cetuximab conjugated of such nanoparticles can be used to identify EGFR-positive glioblastomas in non-invasive T₂ MR image assays. The newly introduced lipid-coated SPIOs, which imitate biological cell surface and thus inherited innate nonfouling property, were utilized to reduce nonspecific binding to off-targeted cells and prevent agglomeration that commonly occurs in nanoparticles.

Results

The synthesized targeted EGFR-antibody-conjugated SPIO (EGFR-SPIO) nanoparticles were characterized using dynamic light scattering, zeta potential assays, gel electrophoresis mobility shift assays, transmission electron microscopy (TEM) images, and cell line affinity assays, and the results showed that the conjugation was successful. The targeting efficiency of the synthesized EGFR-SPIO nanoparticles was confirmed through Prussian blue staining and TEM images by using glioblastoma cell lines with high or low EGFR expression levels. The EGFR-SPIO nanoparticles preferentially targeted U-251 cells, which have high EGFR expression, and were internalized by cells in a prolonged incubation condition. Moreover, the T₂ MR relaxation time of EGFR-SPIO nanoparticles could be used for successfully identifying glioblastoma cells with elevated EGFR expression in vitro and distinguishing U-251 cells from U-87MG cells, which have low EFGR expression.

Conclusion

These findings reveal that the lipid-encapsulated EGFR-SPIO nanoparticles can specifically target cells with elevated EGFR expression in the three tested human glioblastoma cell lines. The results of this study can be used for noninvasive molecular MR image diagnosis in the future.

     

High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry

Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids are achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples. .