Systematic implications of flavonoids in Hazardia

The flavonoid patterns in Hazardia species support species delimitations and relationships based on morphology and geography. The compounds thus far elucidated are glycosides of quercetin, kaempferol, isorhamnetin, luteolin, and apigenin, glycoflavones of apigenin, and methoxylated flavonol aglycones.     

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.     

Antibody Engineering and Therapeutics: December 8-12, 2013, Huntington Beach,CA

The 24^th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.     

Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.     

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

Background

Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.

Methods

TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.

Results

The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.

Conclusions

TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.     

Presentation of Sporothrix schenkii in pulmonary cytology specimens

Macrophages containing small ovoid intracytoplasmic yeast cells, surrounded by a slight "halo," were observed in Papanicolaou-stained smears of sputum submitted for cytologic examination from two patients with culture-confirmed pulmonary sporotrichosis. The cytologic findings are presented and discussed in relation to other pulmonary fungal infections and the appropriate stains to be used for their cytologic detection. The clinical symptoms of pulmonary sporotrichosis are nonspecific and may mimic those of tuberculosis or other diseases; the causative organism, Sporothrix schenkii, may remain undetected by routine microbiologic procedures unless there is cause to suspect the diagnosis. Sporotrichosis should be considered in the differential diagnosis when small ovoid intracellular yeast cells (2 microns to 4 microns in length) with an apparent "halo" are observed within macrophages in pulmonary cytology specimens; the recognition of these findings may aid in the diagnosis of this relatively rare disease.     

Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.     

Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D) and hypoglycemic group (D + IIH and C + IIH). α7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and α7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar dysfunction associated with hypoglycemia.     

Total cost estimation for implementing genome-enabled selection in a multi-level swine production system

Background

Determining an animal’s genetic merit using genomic information can improve estimated breeding value (EBV) accuracy; however, the magnitude of the accuracy improvement must be large enough to recover the costs associated with implementing genome-enabled selection. One way to reduce costs is to genotype nucleus herd selection candidates using a low-density chip and to use high-density chip genotyping for animals that are used as parents in the nucleus breeding herd. The objective of this study was to develop a tool to estimate the cost structure associated with incorporating genome-enabled selection into multi-level commercial breeding programs.

Results

For the purpose of this deterministic study, it was assumed that a commercial pig is created from a terminal line sire and a dam that is a cross between two maternal lines. It was also assumed that all male and female selection candidates from the 1000 sow maternal line nucleus herds were genotyped at low density and all animals used for breeding at high density. With the assumptions used in this analysis, it was estimated that genome-enabled selection costs for a maternal line would be approximately US$0.082 per weaned pig in the commercial production system. A total of US$0.164 per weaned pig is needed to incorporate genome-enabled selection into the two maternal lines. Similarly, for a 600 sow terminal line nucleus herd and genotyping only male selection candidates with the low-density panel, the cost per weaned pig in the commercial herd was estimated to be US$0.044. This means that US$0.21 per weaned pig produced at the commercial level and sired by boars obtained from the nucleus herd breeding program needs to be added to the genetic merit value in order to break even on the additional cost required when genome-enabled selection is used in both maternal lines and the terminal line.

Conclusions

By modifying the input values, such as herd size and genotyping strategy, a flexible spreadsheet tool developed from this work can be used to estimate the additional costs associated with genome-enabled selection. This tool will aid breeders in estimating the economic viability of incorporating genome-enabled selection into their specific breeding program.