Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells

Realizing the therapeutic potential of human induced pluripotent stem (iPS) cells will require robust, precise and safe strategies for genetic modification, as cell therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify human iPS cells at 'safe harbor' sites in the genome, which fulfill five criteria based on their position relative to contiguous coding genes, microRNAs and ultraconserved regions. We demonstrate that ～10% of integrations of a lentivirally encoded β-globin transgene in β-thalassemia-patient iPS cell clones meet our safe harbor criteria and permit high-level β-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. This approach, combining bioinformatics and functional analyses, should be broadly applicable to introducing therapeutic or suicide genes into patient-specific iPS cells for use in cell therapy.     

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

Hermansky–Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2–deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2–deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.     

Arf-like GTPases: not so Arf-like after all

ADP-ribosylation factor (Arf) GTP-binding proteins are among the best-characterized members of the Ras superfamily of GTPases, with well-established functions in membrane-trafficking pathways. A recent watershed of genomic and structural information has identified a family of conserved related proteins: the Arf-like (Arl) GTPases. The best-characterized Arl protein, Arl2, regulates the folding of beta tubulin, and recent data suggest that Arl1 and Arf-related protein 1 (ARFRP1) are localized to the trans-Golgi network (TGN), where they function, in part, to regulate the tethering of endosome-derived transport vesicles. Other Arl proteins are localized to the cytosol, nucleus, cytoskeleton and mitochondria, which indicates that Arl proteins have diverse roles that are distinct from the known functions of traditional Arf GTPases.     

Golgi localization of glycosyltransferases requires a Vps74p oligomer

The mechanism of glycosyltransferase localization to the Golgi apparatus is a long-standing question in secretory cell biology. All Golgi glycosyltransferases are type II membrane proteins with small cytosolic domains that contribute to Golgi localization. To date, no protein has been identified that recognizes the cytosolic domains of Golgi enzymes and contributes to their localization. Here, we report that yeast Vps74p directly binds to the cytosolic domains of cis and medial Golgi mannosyltransferases and that loss of this interaction correlates with loss of Golgi localization of these enzymes. We have solved the X-ray crystal structure of Vps74p and find that it forms a tetramer, which we also observe in solution. Deletion of a critical structural motif disrupts tetramer formation and results in loss of Vps74p localization and function. Vps74p is highly homologous to the human GMx33 Golgi matrix proteins, suggesting a conserved function for these proteins in the Golgi enzyme localization machinery.     

13-Hydroxyoctadeca-9,11-dienoic acid (13-HODE) inhibits thromboxane A2 synthesis, and stimulates 12-HETE production in human platelets

The effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE), a major lipoxygenase product of endothelial cell linoleic acid metabolism on thrombin-induced platelet thromboxane B2 (TxB2), and 12-hydroxyeico-satetraenoic acid (12-HETE) production was evaluated. 13-HODE inhibited thrombin-induced TxB2 production in human platelets in a concentration-dependent manner. At concentrations of 10 and 30 microM, 13-HODE inhibited TxB2 production by 28 +/- 8% (1SE, n = 5; P less than 0.05) and 48 +/- 6% (P less than 0.01) respectively. 13-HODE (30 microM) also inhibited the production of platelet hydroxyheptadecatrienoic acid (38 +/- 5%, P less than 0.01). A concomitant stimulation of 12-HETE production by 13-HODE was observed (25 +/- 5% and 49 +/- 22% over control values at 10 and 30 microM respectively, P less than 0.01). Our results demonstrate a differential effect of 13-HODE on thrombin stimulated platelet cyclooxygenase and lipoxygenase metabolites.     

Time-dependent inhibition of platelet cyclo-oxygenase by indomethacin is slowly reversible

Indomethacin has been charterized as a time-dependent, irreversible inhibitor of cyclo-oxygenase, yet its effects on human platelets have been found to be reversible . To understand this apparent contradiction, we have investigated the kinetics of recovery of platelet thromboxane production after a single dose of indomethacin. The inhibition of platelet thromboxane production was greater than would be expected from the levels of indomethacin found in the plasma suggesting that the time-dependent inhibition occurs . Yet recovery of platelet thromboxane production was faster than expected for the irreversible inhibitor, with 50% of control values being regained within 24 hours after ingestion of the drug. When platelets were isolated and resuspended in homologous drug-free plasma, slow recovery of thromboxane production was seen to occur with 50% of control activity regained in 100 minutes. This recovery was much slower than that seen from a competitive inhibitor of cyclo-oxygenase, ibuprofen. Ibuprofen-treated platelets recovered nearly completely immediately on being resuspended in drug-free plasma. When microsomes were isolated from platelets, then treated with indomethacin, no time-dependent recovery of activity was seen. The recovery of cyclo-oxygenase after indomethacin inhibition appears to be limited to the unperturbed enzyme in this natural milieu.     

Identification of QTLs conferring resistance to downy mildews of maize in Asia

Downy mildew is one of the most destructive diseases of maize in subtropical and tropical regions in Asia. As a prerequisite for improving downy mildew resistance in maize, we analyzed quantitative trait loci (QTLs) involved in resistance to the important downy mildew pathogens--Peronosclerospora sorghi (sorghum downy mildew) and P. heteropogoni (Rajasthan downy mildew) in India, P. maydis (Java downy mildew) in Indonesia, P. zeae in Thailand and P. philippinensis in the Philippines--using a recombinant inbred line population derived from a cross between Ki3 (downy mildew resistant) and CML139 (susceptible). Resistance was evaluated as percentage disease incidence in replicated field trials at five downy mildew 'hotspots' in the four countries. Heritability estimates of individual environments ranged from 0.58 to 0.75 with an across environment heritability of 0.50. Composite interval mapping was applied for QTL detection using a previously constructed restriction fragment length polymorphism linkage map. The investigation resulted in the identification of six genomic regions on chromosomes 1, 2, 6, 7 and 10 involved in the resistance to the downy mildews under study, explaining, in total, 26-57% of the phenotypic variance for disease response. Most QTL alleles conferring resistance to the downy mildews were from Ki3. All QTLs showed significant QTL x environment interactions, suggesting that the expression of the QTL may be environment-dependent. A strong QTL on chromosome 6 was stable across environments, significantly affecting disease resistance at the five locations in four Asian countries. Simple-sequence repeat markers tightly linked to this QTL were identified for potential use in marker-assisted selection.     

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.     

Effect of norgestrel on spermatogenesis & fertility of rats

The effect of noregstrel (d1-13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-estren-3-one) on spermatogenesis and fertility of rats was studied. Male albino rats received norgestrel .1 or .5 mg/rat im daily for 30, 48, or 96 days. Fertility performance tests were conducted and the animals were mated to coeval female of proven fertility. Treatment was discontinued after the 96th day and the animals were sacrificed after 30 days for reversibility studies. .1 mg was ineffective in causing spermatogenic inhibition when given for 30 days but was antispermatogenic when given for 48 or 96 days. Similar effects were seen with .5 mg after 30 and 48 days of treatment, however, partial restoration of spermatogenesis occurred in 50% of the animals even after 96 days of treatment. Androgen deficiency produced at 30 days caused ponderal depression and functional impairment of accessory genital organs. It is concluded that these results cast doubt on the usefulness of norgestrel as a male contraceptive since loss of libido and inconsistent effects on spermatogenesis appear to be major contraindications.