Evaluation of the vervet (Clorocebus aethiops) as a model for the assisted reproductive technologies

The vervet monkey was evaluated as a primate model for use in assisted reproductive technologies (ARTs). Eight adult female vervets were hormonally monitored for their potential use as egg donors and those six females displaying regular menstrual cycles were subjected to controlled ovarian stimulation with recombinant human gonadotropins. Three animals failed to respond while laparoscopic follicular aspiration was performed on the other three females at 27-30 h post-human chorionic gonadotropin administration. A total of 62, 40, and 18 oocytes was recovered from these three animals of which 30, 20, and 4, respectively, matured to the metaphase II stage and were subsequently inseminated using intracytoplasmic sperm injection. An average of 40+/-15% (SEM) of the inseminated oocytes were fertilized based on pronucleus formation and timely cleavage. One embryo from each of the two stimulated females developed into expanded blastocysts. Two adult male vervets were assessed as sperm donors. Neither adjusted well to the restraint and collection procedure required for penile electroejaculation. Samples collected via rectal electroejaculation were very low in sperm motility and concentration; however, cauda epididymal aspirations from one male yielded an adequate concentration of motile sperm. These results emphasize the need to establish species-specific ovarian stimulation protocols and semen collection techniques if vervets are to be considered for basic and applied (ARTs) research on primate gametes or embryos.     

Unreduced gametes and neopolyploids in natural populations of Achillea borealis (Asteraceae)

Polyploidy is a major mechanism of speciation and adaptation, yet little is known about the origins of polyploids in natural species. I investigated gametic nonreduction and neopolyploid formation in natural tetraploid populations of Achillea borealis (Asteraceae), an autopolyploid complex consisting of tetraploid and hexaploid cytotypes. Cytological analyses of tetraploid populations revealed the occurrence of reduced (n=2x) as well as unreduced 'big' (2n=4x) and 'jumbo' (4n=8x) pollen grains, which were clearly distinguished by size. Production of unreduced pollen was monitored in two tetraploid populations in 1997 and 1998. Mean population-level frequencies of unreduced pollen ranged from 0.030 to 0.538%, with as few as one-third and as many as one-half of sampled plants producing unreduced grains. Eight individuals were found to produce >1% unreduced pollen, with highest observed frequencies of 7.0, 13.2 and 15.8%. Experimental crosses using high unreduced pollen producers as male parents generated viable seeds. However, the frequency of neohexaploids in the progeny of experimental crosses (0.388%) was similar to that observed in progeny of randomly selected, open-pollinated control parents (0.465%). These results suggest that unreduced eggs are the most likely source of new polyploids. In spite of the inefficiency of unreduced pollen in unilateral sexual polyploidization, the overall rate of neohexaploid formation (one in 233) was several orders of magnitude greater than estimates of genic mutation rates.     

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.     

C-8 Modifications of 3-alkyl-1,8-dibenzylxanthines as inhibitors of human cytosolic phosphoenolpyruvate carboxykinase

New modifications on the C-8 4-aminobenzyl unit of the previously reported 3-alkyl-1,8-dibenzylxanthine inhibitors of cPEPCK are presented. The most active compound reported here is the 5-chloro-1,3-dimethyl-1H-pyrazole-4-sulfonic acid amide derivative 2 with an IC(50) of 0.29+/-0.08 microM. An X-ray analysis of a heteroaromatic sulfonamide is presented showing a new pi-pi interaction.     

Atlas of Mexican Triatominae (Reduviidae: Hemiptera) and vector transmission of Chagas disease

Chagas disease is one of the most important yet neglected parasitic diseases in Mexico and is transmitted by Triatominae. Nineteen of the 31 Mexican triatomine species have been consistently found to invade human houses and all have been found to be naturally infected with Trypanosoma cruzi. The present paper aims to produce a state-of-knowledge atlas of Mexican triatomines and analyse their geographic associations with T. cruzi, human demographics and landscape modification. Ecological niche models (ENMs) were constructed for the 19 species with more than 10 records in North America, as well as for T. cruzi. The 2010 Mexican national census and the 2007 National Forestry Inventory were used to analyse overlap patterns with ENMs. Niche breadth was greatest in species from the semiarid Nearctic Region, whereas species richness was associated with topographic heterogeneity in the Neotropical Region, particularly along the Pacific Coast. Three species, Triatoma longipennis, Triatoma mexicana and Triatoma barberi, overlapped with the greatest numbers of human communities, but these communities had the lowest rural/urban population ratios. Triatomine vectors have urbanised in most regions, demonstrating a high tolerance to human-modified habitats and broadened historical ranges, exposing more than 88% of the Mexican population and leaving few areas in Mexico without the potential for T. cruzi transmission.     

An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk‐dependent repression of macrophage cholesterol efflux

LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment‐quantitative mass spectrometry (PE‐QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand‐ and binding‐dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll‐like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro‐inflammatory and anti‐inflammatory pathways that results in repression of macrophage cholesterol efflux.     

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1‐checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. Biotechnol. Bioeng. 2015;112: 141–155. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.