A Multiplexed Assay for Exon Recognition Reveals that an Unappreciated Fraction of Rare Genetic Variants Cause Large-Effect Splicing Disruptions

1. Download high-res image (142KB)
2. Download full-size image

     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.     

Overexpression of SR proteins and splice variants modulates chondrogenesis

Fibronectin alternative exon EIIIA is largely included in undifferentiated mesenchymal cells of the developing limb bud, whereas the exon is excluded in differentiated chondrocytes. Inclusion of exon EIIIA in chondrocytic cells is increased by overexpression of SRp40, and, to a lesser extent, SRp75, but not SRp55. RT-PCR analysis using real-time PCR revealed that the levels of the mRNAs for these three proteins did not vary significantly in chick chondrocytes versus mesenchymal cells of the developing limb bud. However, a variant spliced form of SRp40, termed, SRp40LF, is detected preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. Overexpression of SRp40LF, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis and are consistent with earlier results that implicated exon EIIIA-containing isoforms of fibronectin in formation of chondrogenic condensations.     

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation

1. Download high-res image (181KB)
2. Download full-size image

     

HICLAS: a taxonomic database system for displaying and comparing biological classification and phylogenetic trees

MOTIVATION: Numerous database management systems have been developed for processing various taxonomic data bases on biological classification or phylogenetic information. In this paper, we present an integrated system to deal with interacting classifications and phylogenies concerning particular taxonomic groups. RESULTS: An information-theoretic view (taxon view) has been applied to capture taxonomic concepts as taxonomic data entities. A data model which is suitable for supporting semantically interacting dynamic views of hierarchic classifications and a query method for interacting classifications have been developed. The concept of taxonomic view and the data model can also be expanded to carry phylogenetic information in phylogenetic trees. We have designed a prototype taxonomic database system called HICLAS (HIerarchical CLAssification System) based on the concept of taxon view, and the data models and query methods have been designed and implemented. This system can be effectively used in the taxonomic revisionary process, especially when databases are being constructed by specialists in particular groups, and the system can be used to compare classifications and phylogenetic trees. AVAILABILITY: Freely available at the WWW URL: http://aims.cps.msu.edu/hiclas/ CONTACT: pramanik@cps.msu.edu; lotus@wipm.whcnc.ac.cn     

Evidence for acyl homoserine lactone signal production in bacteria associated with marine sponges

We report for the first time the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges. Given the involvement of AHLs in bacterial colonization of many higher organisms, we speculate that such quorum sensing signals could play a part in interactions between sponges and the dense bacterial communities living within them.     

A distinctive class of spermidine synthase is involved in chilling response in rice

A cDNA for a putative 42 kD spermidine synthase (OsSPDS2) was cloned from rice. The deduced OsSPDS2 sequence showed highest similarity with Arabidopsis AtSPDS3. Phylogenetic analysis revealed that OsSPDS2 and AtSPDS3 form a distinctive subclass in the spermidine synthase family in plants. OsSPDS2 mRNA accumulated in roots during long term exposure to chilling temperature (12 degrees C). In contrast, no such induction of the paralogous OsSPDS1 was observed during the chilling treatment. ABA treatment up-regulated OsSPDS2, whereas salt stress did not change OsSPDS2 levels significantly. Data suggested a distinct function of OsSPDS2 in chilling response in rice.