Stable Encapsulated Reference Nucleic Acid and Method of Making

Stable reference nucleic acid for use in all steps of molecular screening and diagnostic assays and method of making. A desired nucleic acid sequence is amplified, ligated into an expression vector, and used to transform a vehicle. A cellular vehicle is subsequently killed without affecting the encapsulated nucleic acid. The vehicle membrane is stabilized under controlled conditions to a stability substantially matching that of a test cell membrane. The recovered nucleic acid is used as a standard or control in molecular diagnostic and genetic testing assays

Novel roles of miR-199b in regulating fat and bone metabolism

Public Health Problem: The incidence of obesity has reached epidemic proportions worldwide and has contributed to an increase in the risk of numerous chronic disorders-type 2 diabetes-liver pathologies, dyslipidemia, and cardiovascular diseases. Obesity can have negative effects on bone remodeling-reduced mineral density-osteoporosis Imbalance between food intake and energy expenditure-obesity-accumulation of fat mass and energy storage in white adipose tissue (WAT).https://knowledgeconnection.mainehealth.org/lambrew-retreat-2021/1036/thumbnail.jp

Suppression of Spry4 enhances cancer stem cell properties of human MDA-MB-231 breast carcinoma cells

BACKGROUND: Cancer stem cells contribute to tumor initiation, heterogeneity, and recurrence, and are critical targets in cancer therapy. Sprouty4 (Spry4) is a potent inhibitor of signal transduction pathways elicited by receptor tyrosine kinases, and has roles in regulating cell proliferation, migration and differentiation. Spry4 has been implicated as a tumor suppressor and in modulating embryonic stem cells. OBJECTIVES: The purpose of this research was to test the novel idea that Spry4 regulates cancer stem cell properties in breast cancer. METHODS: Loss-of function of Spry4 in human MDA-MB-231 cell was used to test our hypothesis. Spry4 knockdown or control cell lines were generated using lentiviral delivery of human Spry4 or non-targeting control shRNAs, and then selected with 2 μg/ml puromycin. Cell growth and migratory abilities were determined using growth curve and cell cycle flow cytometry analyses and scratch assays, respectively. Xenograft tumor model was used to determine the tumorigenic activity and metastasis in vivo. Cancer stem cell related markers were evaluated using immunoblotting assays and fluorescence-activated cell sorting. Cancer stem cell phenotype was evaluated using in vitro mammosphere formation and drug sensitivity tests, and in vivo limiting dilution tumor formation assay. RESULTS: Two out of three tested human Spry4 shRNAs significantly suppressed the expression of endogenous Spry4 in MDA-MB-231 cells. Suppressing Spry4 expression increased MDA-MB-231 cell proliferation and migration. Suppressing Spry4 increased β3-integrin expression, and CD133(+)CD44(+) subpopulation. Suppressing Spry4 increased mammosphere formation, while decreasing the sensitivity of MDA-MB-231 cells to Paclitaxel treatment. Finally, suppressing Spry4 increased the potency of MDA-MB-231 cell tumor initiation, a feature attributed to cancer stem cells. CONCLUSIONS: Our findings provide novel evidence that endogenous Spry4 may have tumor suppressive activity in breast cancer by suppressing cancer stem cell properties in addition to negative effects on tumor cell proliferation and migration

Phosphatidylserine colocalizes with epichromatin in interphase nuclei and mitotic chromosomes

Cycling eukaryotic cells rapidly re-establish the nuclear envelope and internal architecture following mitosis. Studies with a specific anti-nucleosome antibody recently demonstrated that the surface (“epichromatin”) of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation. Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones. This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope

A biochemical approach to define the interactome for calpain2 in endothelial cells

Current repositories for protein-protein interactions and high throughput screening methods focus on individual gene products and do not consider the significance of calcium induced conformational changes. These limitations suggest the need for alternative strategies to better define the calpain2 interactome. Affinity capture coupled with LC-MS/MS and proteomic analysis of the recovered proteins provides a powerful approach to identify protein-protein interactions for the heterodimeric calpain2. CAPN2 (rat) was modified to be catalytically incompetent (C105A) and fused with a C-terminal 15 residue peptide optimized for biotinylation by the biotin protein ligase, BirA. The resulting CAPN2*, heterodimerized with truncated CAPNS1, was purified from E. coli, and biotinylated in vitro. Biotinylated calpain2* served as ‘bait’ for streptavidin affinity capture of calpain2 and its interacting proteins from lysates of bovine aortic (BAEC) and human umbilical vein (HUVEC) endothelial cells (ECs). Protein-calpain2 complexes were formed in the presence of calcium to allow EGTA elution of interacting proteins and LC-MS/MS analysis in the absence of an abundance of bait peptides. Capture of the well characterized calpain inhibitor protein calpastatin (CAST), and a known substrate, vimentin provide proof of concept and validates the conformational integrity of the bait calpain2*. Significant overlap between datasets (two from BAEC and one HUVEC) is also encouraging. Of numerous other proteins including several annexins, ANXA1 was confirmed as a substrate for calpain2. Findings are expected to contribute to continuing efforts in the field to better characterize calpain2’s selection of substrates and may reveal other important clues to calpain’s localization and regulation

Two novel human NUMB isoforms provide a potential link between development and cancer

We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer

Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1

<p>Abstract</p> <p>Background</p> <p>Activin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-β (TGF-β) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (<it>ACVRL1</it>) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of <it>ACVRL1</it>. Here, we have studied the different origins of <it>ACVRL1 </it>transcription, we have analyzed <it>in silico </it>its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of <it>ACVRL1</it>.</p> <p>Results</p> <p>We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of <it>ACVRL1 </it>transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of <it>ACVRL1 </it>(-1,035/+210) was analyzed <it>in silico</it>, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, <it>ACVRL1 </it>promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of <it>ACVRL1 </it>transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of <it>ACVRL1 </it>in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in <it>ACVRL1 </it>transcription, whereas <it>in vitro </it>hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of <it>ACVRL1</it>.</p> <p>Conclusions</p> <p>Our results describe two new transcriptional start sites in <it>ACVRL1 </it>gene, and indicate that Sp1 is a key regulator of <it>ACVRL1 </it>transcription, providing new insights into the molecular mechanisms that contribute to the expression of <it>ACVRL1 </it>gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of <it>ACVRL1 </it>gene transcriptional activity.</p