Chemo-adoptive immunotherapy against a guinea-pig leukemia

Summary Strain-2 guinea-pigs bearing the transplantable L₂C leukemia were treated with cytoxan 24 h prior to receiving an injection of either allogeneic or syngeneic spleen cells from donors preimmunized to the leukemia. Treatment with the drug alone produced a remission period which lasted for 4–5 weeks before eventual relapse and death. An IP transfer of spleen or peritoneal exudate (PE) cells from syngeneic strain-2 guinea-pigs hyperimmunized to the leukemia greatly extended the survival times of drug-treated animals beyond that observed in animals receiving normal strain-2 cells. Long-term survivors were refractory to a subsequent challenge with a lethal inoculum of L₂C cells. A reduced tumor load was essential for an immunotherapeutic effect of adoptively transferred cells. The use of sensitized lymphocytes alone failed to control the established disease. Hyperimmune spleen cells from strain-13 and Hartley guinea-pigs also demonstrated a slight capacity to inhibit the proliferation of leukemia cells when injected into diseased animals previously treated with the drug. Due to inadequate drug suppression, however, the injection of allogeneic cells from either immune strain-13 or Hartley guinea-pigs did not prolong the latent period for the appearance of the leukemia to the same extent as either immune strain-2 spleen or PE cells. A marked delay in the onset of disease was noted when immune spleen cells from either syngeneic or allogeneic sources were mixed in vitro with L₂C leukemia cells at a ratio of 200:1 before injection back into normal strain-2 animals. However, an exposure of L₂C blast cells in vitro with heat-inactivated serum obtained from L₂C-immune strain-2 animals significantly enhanced the onset of disease.     

Effects of Cortisol and Dexamethasone on Insulin Signalling Pathways in Skeletal Muscle of the Ovine Fetus during Late Gestation

Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth.     

Developmental aspects of the tympanic membrane: Shedding light on function and disease

The ear drum, or tympanic membrane (TM), is a key component in the intricate relay that transmits air‐borne sound to our fluid‐filled inner ear. Despite early belief that the mammalian ear drum evolved as a transformation of a reptilian drum, newer fossil data suggests a parallel and independent evolution of this structure in mammals. The term “drum” belies what is in fact a complex three‐dimensional structure formed from multiple embryonic cell lineages. Intriguingly, disease affects the ear drum differently in its different parts, with the superior and posterior parts being much more frequently affected. This suggests a key role for the developmental details of TM formation in its final form and function, both in homeostasis and regeneration. Here we review recent studies in rodent models and humans that are beginning to address large knowledge gaps in TM cell dynamics from a developmental biologist's point of view. We outline the biological and clinical uncertainties that remain, with a view to guiding the indispensable contribution that developmental biology will be able to make to better understanding the TM.     

Seed quality and seed quantity in red maple depends on weather and individual tree characteristics

Under future climate change, plant species are expected to shift their ranges in response to increasing temperatures and altered precipitation patterns. As seeds represent the single opportunity for plants to move, it is critical to quantify the factors that influence reproduction. While total seed production is clearly important, seed quality is equally as critical and often overlooked. Thus, to quantify how environmental and tree‐level characteristics affect seed quality and quantity, the reproductive output of red maple (Acer rubrum) was measured along an elevation gradient in the Monongahela National Forest, WV. A variety of individual‐level characteristics were measured (e.g., DBH, canopy area, height, and tree cores were taken to quantify growth), and seed traps were placed under seed‐bearing trees to collect samaras and quantify total seed production. A random subsample of collected seeds from each tree was micro‐CT scanned to determine embryo volume, photographed for morphology measurements, and used for germination trials. The number of seeds produced was negatively affected by frost events during flowering, and stand density. The trees with the most seeds also showed reduced growth in recent years. Only 63% of scanned seeds showed embryo development, and of those seeds—only 23% germinated. The likelihood of embryo presence increased as growth rate decreased, while embryo size increased with tree height, smaller DBH, and in areas dominated by hemlock. Both larger embryo volume and larger overall seed size increased the likelihood of germination. The results highlight the importance of including seed quality in addition to seed quantity for a more complete representation of reproductive output.     

Generation of APOE knock-down SK-N-SH human neuroblastoma cells using CRISPR/Cas9: a novel cellular model relevant to Alzheimer’s disease research

APOE ε4 is the major genetic risk factor for Alzheimer’s disease (AD). A precise role for apolipoprotein E (apoE) in the pathogenesis of the disease remains unclear in part due to its expression in multiple cell types of the brain. APOE is highly expressed in astrocytes and microglia, however its expression can also be induced in neurons under various conditions. The neuron-like cell line SK-N-SH is a useful model in the study of the cellular and molecular effects of apoE as it can be differentiated with retinoic acid to express and secrete high levels of apoE and it also shows the same apoE fragmentation patterns observed in the human brain. We previously found that apoE is cleaved into a 25-kDa fragment by high temperature-requirement serine protease A1 (HtrA1) in SK-N-SH cells. To further understand the endogenous functions of apoE, we used CRISPR/Cas9 to generate SK-N-SH cell lines with APOE expression knocked-down (KD). APOE KD cells showed lower APOE and HTRA1 expression than parental SK-N-SH cells but no overt differences in neuritogenesis or cell proliferation compared with the CRISPR/Cas9 control cells. This research shows that the loss of apoE and HtrA1 has a negligible effect on neuritogenesis and cell survival in SK-N-SH neuron-like cells.     

Repeated horizontal gene transfer of GALactose metabolism genes violates Dollo’s law of irreversible loss

Dollo’s law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected multiple galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.     

Genetic analysis of Pycr1 and Pycr2 in mice

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.