7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ∼20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.     

MBASED: allele-specific expression detection in cancer tissues and cell lines

Allele-specific gene expression, ASE, is an important aspect of gene regulation. We developed a novel method MBASED, meta-analysis based allele-specific expression detection for ASE detection using RNA-seq data that aggregates information across multiple single nucleotide variation loci to obtain a gene-level measure of ASE, even when prior phasing information is unavailable. MBASED is capable of one-sample and two-sample analyses and performs well in simulations. We applied MBASED to a panel of cancer cell lines and paired tumor-normal tissue samples, and observed extensive ASE in cancer, but not normal, samples, mainly driven by genomic copy number alterations.     

Dystocia following prolonged retention of a dead fetus in an Asian elephant (Elephas maximus)

A 32-year-old nulliparous female Asian elephant (Elephas maximus) showed signs of parturition 8 months later than predicted from the breeding records. However, while serosanguineous fluid, necrotic tissue and pieces of amnion were expelled, second-stage labor did not progress. Since the fetus was not found during an endoscopic examination of the vestibule, it was assumed that the elephant had calved unseen and she was returned to the forest to recuperate. Twelve months later, the elephant showed clear signs of second-stage labor accompanied by a bulge in the perineum and passage of keratinized nail through the vulva. A 35 cm episiotomy incision was made in the perineum just below the anus, via which chains were attached to the forelimbs of the fetus. Traction on the forelimbs alone proved insufficient to achieve delivery because the fetal head kept rotating and impacting in the pelvis. However, traction applied via a hook inserted behind the mandibular symphysis allowed the head to be elevated and extended, and the fetus to be delivered. The episiotomy wound was sutured in two layers and although the skin did not heal during primary closure it subsequently healed uneventfully by second intention. Retrospective evaluation of the elephant's serum progestagens profile demonstrated a fall to baseline at the suspected onset of parturition, supporting the supposition that the fetus was retained in the uterus for 12 months after parturition began. It is suggested that serum progestagens concentrations should be monitored regularly in mated elephant cows to verify the establishment of pregnancy and to better estimate the expected timing, and the onset of calving.     

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.     

Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle

Duchenne muscular dystrophy (DMD) is an X-linked, lethal, degenerative disease that results from mutations in the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4(+) and CD8(+) T cells in skeletal muscle tissue, while Foxp3(+) regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.     

Building proteomic tool boxes to monitor MHC class I and class II peptides

Major histocompatibility complex Class I (MHCI) and Class II (MHCII) presented peptides powerfully modulate T cell immunity and play a vital role in generating effective anti‐tumor and anti‐viral immune responses in mammals. Characterizing these MHCI or MHCII presented peptides can help generate therapeutic treatments, afford information on T cell mediated biomarkers, provide insight into disease progression, and reduce adverse anti‐drug side effects from engineered biotherapeutics. Here, we explore the tools and techniques commonly employed to discover both MHCI‐ and MHCII‐presented peptides. We describe complementary strategies that enhance the characterization of these peptides and the informatics tools employed for both predicting and characterizing MHCI‐ and MHCII‐presented epitopes. The evolution of methodologies for isolating MHC‐presented peptides is discussed, as are the mass spectrometric workflows that can be employed for their characterization. We provide a perspective on where this field is headed, and how these tools may be applicable to the discovery and monitoring of epitopes in a variety of scenarios.     

Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells

Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach’s potential to engineer cell function is demonstrated in HIV infection studies.