Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

In vivo surveillance and elimination of teratoma-forming human embryonic stem cells with monoclonal antibody 2448 targeting annexin A2

This study describes the use of a previously reported chimerised monoclonal antibody (mAb), ch2448, to kill human embryonic stem cells (hESCs) in vivo and prevent or delay the formation of teratomas. ch2448 was raised against hESCs and was previously shown to effectively kill ovarian and breast cancer cells in vitro and in vivo. The antigen target was subsequently found to be Annexin A2, an oncofetal antigen expressed on both embryonic cells and cancer cells. Against cancer cells, ch2448 binds and kills via antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-drug conjugate (ADC) routes. Here, we investigate if the use of ch2448 can be extended to hESC. ch2448 was found to bind specifically to undifferentiated hESC but not differentiated progenitors. Similar to previous study using cancer cells, ch2448 kills hESC in vivo either indirectly by eliciting ADCC or directly as an ADC. The treatment with ch2448 post-transplantation eliminated the in vivo circulating undifferentiated cells and prevented or delayed the formation of teratomas. This surveillance role of ch2448 adds an additional layer of safeguard to enhance the safety and efficacious use of pluripotent stem cell-derived products in regenerative medicine. Thereby, translating the use of ch2448 in the treatment of cancers to a proof of concept study in hESC (or pluripotent stem cell [PSC]), we show that mAbs can also be used to eliminate teratoma forming cells in vivo during PSC-derived cell therapies. We propose to use this strategy to complement existing methods to eliminate teratoma-forming cells in vitro. Residual undifferentiated cells may escape in vitro removal methods and be introduced into patients together with the differentiated cells.     

Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35–100.00%); 100.00% sensitivity for T18 (71.51–100.00%); and 100.00% sensitivity for T13 analyses (66.37–100.00%). The specificities were >99% for each trisomy (99.7% (99.01–99.97%) for T21; 99.5% (98.62–99.85%) for T18; 99.7% (99.03–99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.     

Total synthesis and biological evaluation of methylgerambullone

First total synthesis of methylgerambullone (MGB, 1) isolated from Glycosmis angustifolia was completed via a convergent route. The effect of MGB on the contractile responses of the isolated guinea-pig ileum induced by acetylcholine was investigated. As a result, it showed a potent relaxation rate (78.66 ± 4.30% at 100 mg/L) in a concentration-dependent manner on longitudinal smooth muscle contraction of isolated guinea-pig ileum induced by 1 μM acetylcholine.     

Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation

There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes with specificity. Such molecules would be useful to test hypotheses for telomere length regulation, and may have therapeutic potential. The versatility and modular nature of the zinc finger motif makes it an ideal candidate for engineering G-quadruplex-binding proteins. Phage display technology has previously been widely used to screen libraries of zinc fingers for binding to novel duplex DNA sequences. In this study, a three-finger library has been screened for clones that bind to an oligonucleotide containing the human telomeric repeat sequence folded in the G-quadruplex conformation. The selected clones show a strong amino acid consensus, suggesting analogous modes of binding. Binding was found to be both sequence dependent and structure specific. This is the first example of an engineered protein that binds to G-quadruplex DNA, and represents a new type of binding interaction for a zinc finger protein.     

Venalstonine and dioxokopsan derivatives from Kopsia fruticosa

Two new venalstonine derivatives, viz., venacarpines A and B, and one new dioxokopsan derivative, kopsorinine, in addition to the kopsifolines A-F, and 11 other known alkaloids, were isolated from a Malayan Kopsia species. The structures of the new alkaloids were determined using NMR and MS analysis.     

Lipid raft proteome reveals ATP synthase complex in the cell surface

Since detergent-resistant lipid rafts are involved in pathogen invasion, cholesterol homeostasis, angiogenesis, neurodegenerative diseases and signal transduction, protein identification in the rafts could provide important information to study their function. Here, we analyzed detergent-resistant raft proteins isolated from rat liver by capillary liquid chromatography-tandem mass spectrometry. Out of 196 proteins identified, 32% belonged to the raft or plasma membrane, 24% to mitochondrial, 20% to microsomal, 7% to miscellaneous, and 17% are unknown proteins. For example, membrane-bound receptors, trimeric GTP-binding proteins, ATP-binding cassette transporters, and glycosylphosphatidylinositol-anchored proteins were identified in this analysis. Unexpectedly, there were many mitochondrial proteins, raising a new issue for the presence of mitochondrial rafts or the localization of mitochondrial proteins into plasma membrane rafts. We confirmed that ATP synthase alpha and beta were expressed on the surface of the plasma membrane in HepG2 hepatocytes by immunofluorescence, cell surface biotinylation, and cellular fractionation. They had two distinct biochemical properties, detergent insolubility and low density, suggesting that the ATP synthase complex might be located in plasma membrane rafts as well as in the mitochondria.     

Recent applications of Hidden Markov Models in computational biology

This paper examines recent developments and applications of Hidden Markov Models (HMMs) to various problems in computational biology, including multiple sequence alignment, homology detection, protein sequences classification, and genomic annotation.