Detection of microRNAs in dried serum blots

We observed the preservation of microRNAs in unrefrigerated dried serum blots. Preservation was not adversely affected by drying or storing at 37, 45, or 60 °C instead of room temperature, but it was harmed when blots were dried incompletely before storage. Preservation of microRNAs in serum was not diminished if, instead of being kept frozen at −80 °C, it was stored as dried blots at room temperature for 5 months or at 37 °C for 4 weeks. Thus, dried blots can be a convenient and safer way to save, transport, and store serum for microRNA assays.     

The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand–receptor interaction (CD58–CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-ζ chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.     

Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene-inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion.

Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. In addition, a UDPGT isoform which is active toward 4-nitrophenol and is induced by 3-methylcholanthrene (3-MC) in normal rats, is produced in a nonfunctional truncated form in Gunn rats due to the deletion of a single guanosine residue in the coding region of its mRNA. The hepatic concentration of bilirubin-UDPGT mRNA was lower in Gunn rats than in congeneic normal rats. However, bilirubin-UDPGT mRNA was of apparently normal length and was induced by clofibrate, a known inducer of bilirubin-UDPGT activity. 3' regions of bilirubin- and 3-MC-inducible UDPGT mRNAs have identical nucleotide sequences; the single base deletion in the 3-MC-inducible UDPGT in Gunn rats occurs within this region. Using oligonucleotide primers corresponding to the identical and unique regions of the two mRNAs, and polymerase chain reaction, we amplified segments of mRNAs for the bilirubin- and 3-MC-inducible UDPGTs from normal and Gunn rat livers. Both amplified DNAs in Gunn rats lacked the restriction site for BstNI. Nucleotide sequence determination revealed that bilirubin- and 3-MC-inducible UDPGT mRNAs in Gunn rats contain an identical frame-shift deletion of a single guanosine residue within the common region of their coding sequences.     

The Synthetic Tryptanthrin Analogue Suppresses STAT3 Signaling and Induces Caspase Dependent Apoptosis via ERK Up Regulation in Human Leukemia HL-60 Cells

Tryptanthrin is a natural product which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signaling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK which led to the activation of intrinsic and extrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of tyrosine 705 and serine 727 p-STAT3. As IL-6 is the main driver of the STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, PD98059 significantly reduced the apoptotic effects of TBr, thus showing the direct involvement of p-ERK in TBr mediated cell death. It was further shown that apoptotic protein Bax silencing in HL-60 cells resists TBr mediated ERK dependent apoptosis. In summary, for the first time we report the mechanism of TBr mediated cell death in human leukemia cell lines by targeting STAT3 and ERK pathways.     

Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.     

White Spot Syndrome Virus infection in Penaeus monodon is facilitated by housekeeping molecules

White Spot Syndrome Virus (WSSV) is a major pathogen in shrimp aquaculture, and its rampant spread has resulted in great economic loss. Identification of host cellular proteins interacting with WSSV will help in unravelling the repertoire of host proteins involved in WSSV infection. In this study, we have employed one-dimensional and two-dimension virus overlay protein binding assay (VOPBA) followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the host proteins of Penaeus monodon that could interact with WSSV. The VOPBA results suggest that WSSV interacted with housekeeping proteins such as heat shock protein 70, ATP synthase subunit β, phosphopyruvate hydratase, allergen Pen m 2, glyceraldehyde-3-phosphate dehydrogenase, sarcoplasmic calcium-binding protein, actin and 14-3-3-like protein. Our findings suggest that WSSV exploits an array of housekeeping proteins for its transmission and propagation in P. monodon.     

Signalling transduction of O-GlcNAcylation and PI3K/AKT/mTOR-axis in prostate cancer

Hexosamine biosynthetic (HBP) and PI3K/AKT/mTOR pathways are found to predominate the proliferation and survival of prostate cancer cells. Both these pathways have their own specific intermediates to propagate the secondary signals in down-stream cascades and besides having their own structured network, also have shared interconnecting branches. These interconnections are either competitive or co-operative in nature depending on the microenvironmental conditions. Specifically, in prostate cancer HBP and mTOR pathways increases the expression and protein level of androgen receptor in order to support cancer cell proliferation, advancement and metastasis. Pharmacological inhibition of a single pathway is therefore insufficient to stop disease progression as the cancer cells manage to alter the signalling channel. This is one of the primary reasons for the therapeutic failure in prostate cancer and emergence of chemoresistance. Inhibition of these multiple pathways at their common junctures might prove to be of benefit in men suffering from an advanced disease state. Hence, a thorough understanding of these cellular intersecting points and their significance with respect to signal transduction mechanisms might assist in the rational designing of combinations for effective management of prostate cancer.     

Biomineralization of 2,3,4,6-Tetrachlorophenol by Bacillus sp. and Staphylococcus sp. Isolated from Secondary Sludge of Pulp and Paper Mill

Three 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)-mineralizing bacteria were isolated from the secondary sludge of a pulp and paper industry. The isolates used 2,3,4,6-TeCP as a source of carbon and energy and were capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on 16S rRNA gene sequence analysis, the bacteria were identified as Bacillus megaterium (CL3), Staphylococcus suciri (CL10), and Bacillus thuringensis (CL11). High-performance liquid chromatography (HPLC) analysis revealed that these isolates were able to degrade 2,3,4,6-TeCP at higher concentrations (600 mg/L or 2.5 mM). A consortia of the isolates completely removed 2,3,4,6-TeCP from the sludge obtained from a pulp and paper mill within 2 weeks when supplemented at a rate of 100 mg/L or 0.43 mM. A bacterial consortium also significantly reduced absorbable organic halogen (AOX) and extractable organic halogen (EOX) by 63% and 68%, respectively, from the sludge. These isolates have a high potential to remove 2,3,4,6-TeCP and may be used for remediation of pulp paper mill waste containing 2,3,4,6-TeCP.