Marsupials and Eutherians reunited: genetic evidence for the Theria hypothesis of mammalian evolution

The three living monophyletic divisions of Class Mammalia are the Prototheria (monotremes), Metatheria (marsupials), and Eutheria (`placental' mammals). Determining the sister relationships among these three groups is the most fundamental question in mammalian evolution. Phylogenetic comparison of these mammals by either anatomy or mitochondrial DNA has resulted in two conflicting hypotheses, Theria and Marsupionta, and has fueled a ``genes versus morphology' controversy. We have cloned and analyzed a large nuclear gene, the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), from representatives of all three mammalian groups, including platypus, echidna, opossum, wallaby, hedgehog, mouse, rat, rabbit, cow, pig, bat, tree shrew, colugo, ringtail lemur, and human. Statistical analysis of this nuclear gene unambiguously supports the morphology-based Theria hypothesis that excludes monotremes from a clade of marsupials and eutherians. The M6P/IGF2R was also able to resolve the finer structure of the eutherian mammalian family tree. In particular, our analyses support sister group relationships between lagomorphs and rodents, and between the primates and Dermoptera. Statistical support for the grouping of the hedgehog with Feruungulata and Chiroptera was also strong. Received: 8 December 2000 / Accepted: 01 February 2001     

Process‐relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics

The biopharmaceutical industry has invested considerably in the implementation of single‐use disposable bioreactors in place of or in addition to their stainless steel‐counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene‐film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035–0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label‐free LC‐MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547–1558, 2016     

Orphanin FQ/Nociceptin Modulation of Mesolimbic Dopamine Transmission Determined by Microdialysis

Orphanin FQ has been reported to suppress extracellular dopamine levels in the nucleus accumbens after intracerebroventricular administration. This study sought to provide evidence for an intra-ventral tegmental site of action for this effect using a dual-probe microdialysis experimental design. Orphanin FQ was applied to the ventral tegmental area of anesthetized rats by reverse dialysis while extracellular dopamine was sampled with a second dialysis probe in the nucleus accumbens. Orphanin FQ at a probe concentration of 1 mM (but not at 0.1 mM) significantly reduced nucleus accumbens dialysate dopamine levels. The receptor-inactive analogue, des-Phe1-orphanin FQ (1 mM), produced a small but significant increase in nucleus accumbens dialysate dopamine levels. Simultaneous measurement of ventral tegmental area dialysate amino acid content revealed significant increases in both GABA and glutamate during infusion of orphanin FQ (1 mM). To determine if increased GABA overflow mediates the action of orphanin FQ on mesolimbic neurons, orphanin FQ (10 nmol) was microinjected directly into the ventral tegmental area in the presence or absence of the GABA(A) receptor antagonist, bicuculline (1 nmol). Bicuculline transiently blocked the suppressive action of orphanin FQ on accumbens dialysate dopamine levels. These data indicate that orphanin FQ decreases dopamine transmission in the nucleus accumbens by inhibiting dopamine neuronal activity in the ventral tegmental area through a mechanism that may involve an increased overflow of GABA.     

Analysis of Mice with Single and Multiple Copies of Transgenes Reveals a Novel Arrangement for the λ5-VpreB1 Locus Control Region

The murine λ5-V_preB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both λ5 and V_preB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3′ of the λ5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a λ5 gene in pre-B cells even when it is integrated into centomeric γ-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.     

Effects of histidine on tissue zinc distribution in rats

Histidine has been reported to affect body zinc status by increasing urinary zinc excretion. The effects of experimental histidinemia on distribution of⁶⁵Zn in anesthetized rats were studied. Infusion ofl-histidine at a rate sufficient to raise plasma concentrations to approximately 2mm for 6h starting 48 h after a single intraperitoneal⁶⁵Zn injection did not alter⁶⁵Zn activities in a variety of tissues when compared with anesthetized uninfused animals. However, plasma⁶⁵Zn and erythrocyte⁶⁵Zn were decreased, and liver⁶⁵Zn was increased. If⁶⁵Zn was injected intravenously during histidine infusion, net accumulation of zinc by some tissues was increased, but uptake by others was reduced relative to uninfused animals. In all cases, however, uptake expressed relative to plasma⁶⁵Zn levels was increased when allowance was made for the more rapid fall in plasma⁶⁵Zn during histidine infusion. Similar infusions ofd-histidine produced quantitatively similar effects. Since enzymatic mechanisms and amino acid carriers would be expected to show stereoselectivity, such processes are unlikely to be involved in the zinc distribution changes described. The possibility of zinc transport by a hitherto unidentified carrier is discussed. These experiments confirm that histidinemia can affect zinc status, but any associated changes in urinary zinc excretion do not seem adequate to account for the tissue changes found.     

Nest orientation and hatching success in the tree pipit Anthus trivialis

Many open-nesting bird species orient their nests relative to surrounding vegetation to avoid adverse environmental conditions. In eastern England, tree pipit Anthus trivialis nests predominantly faced east, an orientation that provided shelter from prevailing WSW winds, allowed warming by the morning sun and which, together with relatively high field layer cover, shaded nest contents from the midday sun. Mean orientation moved from south to north through the breeding season as temperatures increased, suggesting that solar radiation was a key influence on nest placement for tree pipits in the study area. Tree pipit nests with eastward orientations experienced better hatching success than those in less-preferred orientations and it is suggested that this was due to their thermal advantages. In contrast, nest orientation had no influence on nestling survival.     

Glutamate Concentration in the Medial Prefrontal Cortex Predicts Resting-State Cortical-Subcortical Functional Connectivity in Humans

Communication between cortical and subcortical regions is integral to a wide range of psychological processes and has been implicated in a number of psychiatric conditions. Studies in animals have provided insight into the biochemical and connectivity processes underlying such communication. However, to date no experiments that link these factors in humans in vivo have been carried out. To investigate the role of glutamate in individual differences in communication between the cortex – specifically the medial prefrontal cortex (mPFC) – and subcortical regions in humans, a combination of resting-state fMRI, DTI and MRS was performed. The subcortical target regions were the nucleus accumbens (NAc), dorsomedial thalamus (DMT), and periaqueductal grey (PAG). It was found that functional connectivity between the mPFC and each of the NAc and DMT was positively correlated with mPFC glutamate concentrations, whilst functional connectivity between the mPFC and PAG was negatively correlated with glutamate concentration. The correlations involving mPFC glutamate and FC between the mPFC and each of the DMT and PAG were mirrored by correlations with structural connectivity, providing evidence that the glutamatergic relationship may, in part, be due to direct connectivity. These results are in agreement with existing results from animal studies and may have relevance for MDD and schizophrenia.     

CGCDB: a web-based resource for the investigation of gene coexpression in CHO cell culture

Coexpression analysis is a powerful, widely used methodology for the investigation of underlying patterns in gene expression data. This "guilt-by-association" approach aims to find groups of genes with closely correlated expression profiles. Observation of consistent correlations across phenotypically diverse samples indicates that these genes have a shared function. We have recently described the application of weighted gene coexpression network analysis (WGCNA) to a 295 sample production CHO cell line microarray dataset and elucidated groups of genes related to growth rate and cell-specific productivity (Qp). In this study, we present the CHO gene coexpression database (CGCDB), a web-based system, designed specifically for researchers in the CHO community to provide user-friendly access to these gene-gene coexpression patterns. In addition to correlation between genes, the direct correlations between probesets and either growth rate or Qp are provided. Results are presented to the user via an interactive network diagram and in a downloadable tabular format. It is hoped that this resource will allow researchers to prioritize cell line engineering and/or biomarker candidates to enhance CHO-based cell culture for the production of biotherapeutics. Availability: www.cgcdb.org.     

Therapeutic implications of GIPC1 silencing in cancer

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers.