Internal initiation of translation of the TrkB mRNA is mediated by multiple regions within the 5′ leader

Translational regulation of the dendritically localized mRNA encoding for the neurotrophin receptor TrkB has important ramifications for synaptic function. We examined whether the TrkB mRNA is translated through an internal initiation entry site (IRES). The human TrkB 5′ leaders are derived from the use of alternative promoters and alternative splicing, but all 5′ leaders share a common exon. Insertion of a full-length 5′ leader, as well as the common exon into the intercistronic region of a dicistronic luciferase construct, yielded luciferase activity generated from the second cistron that was either equivalent or higher than that observed from the encephalomyocarditis virus IRES. Moreover, inhibiting cap-dependent translation ex vivo and in in vitro lysates had only a minimal effect on the translation of mRNA containing the TrkB 5′ leader. Dissecting the 5′ leader showed that the IRES is located in the exon common to all TrkB 5′ leaders. Moreover, six regions ranging from 2 to 25 nt were identified that either promoted or inhibited IRES activity. Taken together, these results suggest that the 5′ leader of the human TrkB mRNA contains multiple cis-elements that regulate internal initiation of translation and that this mechanism may contribute significantly to the translation of the TrkB mRNA in neuronal dendrites

Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex

A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone–protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP

Increased Lung Expression of Anti-Angiogenic Factors in Down Syndrome: Potential Role in Abnormal Lung Vascular Growth and the Risk for Pulmonary Hypertension

<div><p>Background and Aims</p><p>Infants with Down syndrome (DS) or Trisomy 21, are at high risk for developing pulmonary arterial hypertension (PAH), but mechanisms that increase susceptibility are poorly understood. Laboratory studies have shown that early disruption of angiogenesis during development impairs vascular and alveolar growth and causes PAH. Human chromosome 21 encodes known anti-angiogenic factors, including collagen18a1 (endostatin, ES), ß-amyloid peptide (BAP) and Down Syndrome Critical Region 1 (DSCR-1). Therefore, we hypothesized that fetal lungs from subjects with DS are characterized by early over-expression of anti-angiogenic factors and have abnormal lung vascular growth <i>in utero</i>.</p><p>Methods</p><p>Human fetal lung tissue from DS and non-DS subjects were obtained from a biorepository. Quantitative reverse transcriptase PCR (qRT-PCR) was performed to assay 84 angiogenesis-associated genes and individual qRT-PCR was performed for ES, amyloid protein precursor (APP) and DSCR1. Western blot analysis (WBA) was used to assay lung ES, APP and DSCR-1 protein contents. Lung vessel density and wall thickness were determined by morphometric analysis.</p><p>Results</p><p>The angiogenesis array identified up-regulation of three anti-angiogenic genes: <i>COL18A1</i> (ES), <i>COL4A3</i> (tumstatin) and <i>TIMP3</i> (tissue inhibitor of metallopeptidase 3) in DS lungs. Single qRT-PCR and WBA showed striking elevations of ES and APP mRNA (p = 0.022 and p = 0.001) and protein (p = 0.040 and p = 0.002; respectively). Vessel density was reduced (p = 0.041) and vessel wall thickness was increased in DS lung tissue (p = 0.033) when compared to non-DS subjects.</p><p>Conclusions</p><p>We conclude that lung anti-angiogenic factors, including <i>COL18A1</i> (ES), <i>COL4A3</i>, <i>TIMP3</i> and <i>APP</i> are over-expressed and fetal lung vessel growth is decreased in subjects with DS. We speculate that increased fetal lung anti-angiogenic factor expression due to trisomy 21 impairs lung vascular growth and signaling, which impairs alveolarization and contributes to high risk for PAH during infancy.</p></div

Clinical Response to Anti-CD47 Immunotherapy Is Associated with Rapid Reduction of Exhausted Bystander CD4+ BTLA+ T Cells in Tumor Microenvironment of Mycosis Fungoides

Cancer progression in mycosis fungoides, the most common form of cutaneous T-cell lymphoma, occurs in a predictable, sequential pattern that starts from patches and that evolves to plaques and later to tumors. Therefore, unlocking the relationship between the microarchitecture of mycosis fungoides and the clinical counterparts of that microstructure represents important steps for the design of targeted therapies. Using multispectral fluorescent imaging, we show that the progression of mycosis fungoides from plaque to tumor parallels the cutaneous expansion of the malignant CD4+ T cells that express TOX. The density of exhausted BTLA+ CD4+ T cells around malignant CD4+TOX+ cells was higher in tumors than it was in plaques, suggesting that undesired safeguards are in place within the tumor microenvironment that prevent immune activation and subsequent cancer eradication. Overriding the CD47 checkpoint with an intralesional SIRP&alpha;Fc fusion decoy receptor induced the resolution of mycosis fungoides in patients that paralleled an amplified expansion of NK and CD8+ T cells in addition to a reduction of the exhausted BTLA+ CD4+ T cells that were engaged in promiscuous intercellular interactions. These therapeutic benefits of the CD47 blockade were further unleashed by adjuvant interferon-&alpha;, which stimulates cytotoxic cells, underscoring the importance of an inflamed microenvironment in facilitating the response to immunotherapy. Collectively, these findings support CD47 as a therapeutic target in treating mycosis fungoides and demonstrate a synergistic role of interferon-&alpha; in exploiting these clinical benefits

A platform-independent framework for phenotyping of multiplex tissue imaging data.

Multiplex imaging is a powerful tool to analyze the structural and functional states of cells in their morphological and pathological contexts. However, hypothesis testing with multiplex imaging data is a challenging task due to the extent and complexity of the information obtained. Various computational pipelines have been developed and validated to extract knowledge from specific imaging platforms. A common problem with customized pipelines is their reduced applicability across different imaging platforms: Every multiplex imaging technique exhibits platform-specific characteristics in terms of signal-to-noise ratio and acquisition artifacts that need to be accounted for to yield reliable and reproducible results. We propose a pixel classifier-based image preprocessing step that aims to minimize platform-dependency for all multiplex image analysis pipelines. Signal detection and noise reduction as well as artifact removal can be posed as a pixel classification problem in which all pixels in multiplex images can be assigned to two general classes of either I) signal of interest or II) artifacts and noise. The resulting feature representation maps contain pixel-scale representations of the input data, but exhibit significantly increased signal-to-noise ratios with normalized pixel values as output data. We demonstrate the validity of our proposed image preprocessing approach by comparing the results of two well-accepted and widely-used image analysis pipelines

At low magnification (2x) a striking arrest of lung growth is seen in DS (middle) as characterized by decreased and enlarged alveoli in comparison with the normal lung (*alveolar spaces).

<p>At high magnification (40x), vascular developmental arrest in DS is characterized by persistence of double capillary layers (arrows) lining the alveolar spaces, which in contrast, have matured to a single layer in the control lung (top). At medium magnification (20x) arterial remodeling defect in DS lung showing a muscular pulmonary artery (A) with markedly thickened wall while intact remodeling results in thin-walled pulmonary arteries (a) in the control lung. DS lung pathologic features are strikingly similar to those of preterm infants with bronchopulmonary dysplasia (bottom).</p

<i>COL18A</i> and <i>APP</i> and <i>DSCR1</i> mRNA expression levels are elevated in human fetal DS lungs as measured by individual qPCR (upper panel, left), while array show significant increase in <i>COL18A</i> (upper panel, right).

<p>Fetal DS lung samples show increased <i>COL18A</i>, <i>APP</i> and <i>DSCR1</i> protein expression as measured by Western blot (lower panels). While <i>DSCR1</i> mRNA expression trended towards elevation, statistical significance was noted in <i>COL18A</i> and <i>APP</i>.</p

Anti-angiogenic genes are upregulated in human fetal DS lungs as shown by a volcano plot.

<p>Out of 84 human genes involved in the regulation of angiogenesis the expression of anti-angiogenic <i>COL18A1</i> (endostatin), <i>COL4A3</i> (tumstatin) and <i>TIMP3</i> (Tissue inhibitor of metallopeptidase 3) genes are significantly upregulated as measured by Human Angiogenesis RT2 ProfilerTM PCR Array (Qiagen PAHS- 024Z).</p