The rockefeller university

To quantitatively understand chemosensory behaviors, it is desirable to present many animals with repeatable, well-defined chemical stimuli. To that end, we describe a microfluidic system to analyze Caenorhabditis elegans behavior in defined temporal and spatial stimulus patterns. A 2 cm x 2 cm structured arena allowed C. elegans to perform crawling locomotion in a controlled liquid environment. We characterized behavioral responses to attractive odors with three stimulus patterns: temporal pulses, spatial stripes and a linear concentration gradient, all delivered in the fluid phase to eliminate variability associated with air-fluid transitions. Different stimulus configurations preferentially revealed turning dynamics in a biased random walk, directed orientation into an odor stripe and speed regulation by odor. We identified both expected and unexpected responses in wild-type worms and sensory mutants by quantifying dozens of behavioral parameters. The devices are inexpensive, easy to fabricate, reusable and suitable for delivering any liquid-borne stimulus.

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although All has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGF beta-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGF beta signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTS12 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGF beta signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.

Mammalian RNA complexity is regulated through interactions of RNA-binding proteins (RBPs) with their target transcripts. High-throughput sequencing together with UV-crosslinking and immunoprecipitation (HITS-CLIP) is able to globally map RBPbinding footprint regions at a resolution of similar to 30-60 nucleotides. Here we describe a systematic way to analyze HITS-CLIP data to identify exact crosslink sites, and thereby determine protein-RNA interactions at single-nucleotide resolution. We found that reverse transcriptase used in CLIP frequently skips the crosslinked amino-acid-RNA adduct, resulting in a nucleotide deletion. Genome-wide analysis of these crosslinking-induced mutation sites (CIMS) in HITS-CLIP data for Nova and Argonaute (Ago) proteins in mouse brain tissue revealed deletions in similar to 8-20% of mRNA tags, which mapped to Nova and Ago binding sites on mRNA or miRNA. CIMS analysis provides a general and more precise means of mapping protein-RNA interactions than currently available methods and insight into the biochemical properties of such interactions in living tissues.

FMRP loss of function causes Fragile X syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here, we use high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results suggest that loss of a translational brake on the synthesis of a subset of synaptic proteins contributes to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD and suggest multiple targets for clinical intervention.

Histone H2B ubiquitylation was shown to be associated with actively transcribed genes in mammalian cells and has been suggested to be involved in transcriptional regulation. Despite the limited applicability of genetic tools to analyze H2B ubiquitylation in mammals, several biochemical and immunological approaches have been successfully implemented to study this modification. Here we describe several techniques to detect ubiquitylated H2B in mammalian cells and to dissect its genomic localization. (C) 2011 Elsevier Inc. All rights reserved.

Background: Methicillin resistant Staphylococcus hominis (MRSHo) are important human pathogens in immunocompromised patients. However, little is known regarding its population structure and staphylococcal chromosomal cassette mec (SCCmec) content. Methodology/Principal Findings: To assess the population structure and the SCCmec content of S. hominis, 34 MRSHo and 11 methicillin-susceptible S. hominis (MSSHo) from neutropenic patients collected over a 3-year period were studied. The genetic backgrounds of S. hominis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and SCCmec types were determined by PCR. Cassette chromosome recombinases (ccr) were characterized by PCR and ccrB sequencing. The 34 S. hominis isolates were classified into as many as 28 types and 32 subtypes (SID = 99.82%); clonal dissemination was occasionally observed. The main SCCmec structures identified were SCCmec type VI (4B) (20%), SCCmec VIII (4A) (15%), and a new SCCmec composed of mec complex A in association with ccrAB1 (38%); 27% of the isolates harbored non-typeable SCCmec. Overall, a high prevalence of mec complex A (73.5%), ccrAB1 (50%) and ccrAB4 (44%) were found. Importantly, ccrB1 and ccrB4 from both MRSHo and MSSHo showed a high nucleotide sequence homology with those found in S. aureus SCCmec I, VI and VIII respectively (>95%). Conclusions/Significance: The S. hominis population showed a limited clonality and a low genetic diversity in the allotypes of ccr and classes of mec complex. Moreover, our data suggest that S. hominis might have been a privileged source of mec complex A, ccrB1 and ccrB4, for the assembly of primordial SCCmec types.

The assembly of an HIV-1 particle is a complex, multistep process involving several viral and cellular proteins, RNAs and lipids. While many macroscopic and fixed-cell microscopic techniques have provided important insights into the structure of HIV-1 particles and the mechanisms by which they assemble, analysis of individual particles and their assembly in living cells offers the potential of surmounting many of the limitations inherent in other approaches. In this review, we discuss how the recent application of live-cell microscopic imaging techniques has increased our understanding of the process of HIV-1 particle assembly. In particular, we focus on recent studies that have employed total internal reflection fluorescence microscopy and other single-virion imaging techniques in live cells. These approaches have illuminated the dynamics of Gag protein assembly, viral RNA packaging and ESCRT (endosomal sorting complex required for transport) protein recruitment at the level of individual viral particles. Overall, the particular advantages of individual particle imaging in living cells have yielded findings that would have been difficult or impossible to obtain using macroscopic or fixed-cell microscopic techniques. (C) 2011 Elsevier Ltd. All rights reserved.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped similar to 2-fold less in the Pcsk9(-/-) -lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.-Rousselet, E., J. Marcinkiewicz, J. Kriz, A. Zhou, M. E. Hatten, A. Prat, and N. G. Seidah. PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke. J. Lipid Res. 2011. 52: 1383-1391.

Extracellular heat shock proteins (eHsps) in blood circulation have been associated with various diseases, including cancer. However, the lack of methods to enrich eHsps from serum samples has hampered the characterization of eHsps. This Letter presents our serendipitous finding that the monomeric avidin resin can serve as an affinity resin to enrich eHsps from blood serum. Biochemical mechanism of this eHsp enrichment as well as implications in biomarker discovery is discussed. (C) 2011 Elsevier Ltd. All rights reserved.