Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Current perspectives on biosimilars

In this work, an overview of the biosimilars market, pipeline and industry targets is discussed. Biosimilars typically have a shorter timeline for approval (8 years) compared to 12 years for innovator drugs and the development cost can be 10–20% of the innovator drug. The biosimilar pipeline is reviewed as well as the quality management system (QMS) that is needed to generate traceable, trackable data sets. One difference between developing a biosimilar compared to an originator is that a broader analytical foundation is required for biosimilars and advances made in developing analytical similarity to characterize these products are discussed. An example is presented on the decisions and considerations explored in the development of a biosimilar and includes identification of the best process parameters and methods based on cost, time, and titer. Finally factors to consider in the manufacture of a biosimilar and approaches used to achieve the target-directed development of a biosimilar are discussed.

     

The diatom flora of the Salton Sea, California

We report on diatom species of the Salton Sea, a highly saline (43 g l^–1) inland lake in California. We identified and photographed all diatom taxa encountered in the phytoplankton and benthos of the Salton Sea and its immediate tributaries. Ninety-four taxa were distinguished based on their morphological features using light- and electron microscopy. In the Salton Sea, there are four general categories of diatom assemblages related to their habitats: (1) A planktonic assemblage composed of Chaetoceros muelleri var. subsalsum, Cyclotella choctawhatcheeana, Cyclotella sp., Cylindrotheca closterium, Pleurosigma ambrosianum, Thalassionema sp.; (2) a benthic assemblage with diatoms that live on the bottom (e.g. genera Caloneis, Diploneis, Entomoneis, Gyrosigma, Plagiotropis, Pleurosigma, Surirella and Tryblionella), or in algal mats (Proschkinia bulnheimii, several species of Navicula and Seminavis gracilenta); (3) an epiphytic community attached to the macroscopic green algae which grow on the rocks and other hard surfaces near shore (e.g. Achnanthes brevipes, Licmophora ehrenbergii, Tabularia parva); and (4) a freshwater assemblage composed of species that get washed in by the rivers and other inflows discharging into the Sea (e.g. Achnanthidium minutissimum, Cocconeis pediculus, Cyclotella atomus, C. scaldensis, Nitzschia elegantula, T. weissflogii). The most striking feature of the phytoplankton is the abundance of species formerly known only from marine environments; this is not surprising given the high salinity and the peculiar history of the lake.     

Changes in environmental temperature influence leptin responsiveness in low- and high-fat-fed mice

Loss of body fat in leptin-treated animals has been attributed to reduced energy intake, increased thermogenesis, and preferential fatty acid oxidation. Leptin does not decrease food intake or body fat in leptin-resistant high-fat (HF)-fed mice, possibly due to a failure of leptin to activate hypothalamic receptors. We measured energy expenditure of male C57BL/6 mice adapted to low-fat (LF) or HF diet and infused them for 13 days with PBS or 10 mug leptin/day from an intraperitoneal mini-osmotic pump to test whether leptin resistance prevented leptin-induced increases in energy expenditure and fatty acid oxidation. There was no effect of low-dose leptin infusions on either of these measures in LF-fed or HF-fed mice, even though LF-fed mice lost body fat. Experiment 2 tested leptin responsiveness in LF-fed and HF-fed mice housed at different temperatures (18 degrees C, 23 degrees C, 27 degrees C), assuming that the cold would increase and the hot environment would inhibit food intake and thermogenesis, which could potentially interfere with leptin action. LF-fed mice housed at 23 degrees C were the only mice that lost body fat during leptin infusion, suggesting that an ability to modify energy expenditure is essential to the maintenance of leptin responsiveness. HF-fed mice in cold or warm environments did not respond to leptin. HF-fed mice in the hot environment were fatter than other HF-fed mice, and, surprisingly, leptin caused a further increase in body fat, demonstrating that the mice were not totally leptin resistant and that partial leptin resistance in a hot environment favors positive energy balance and fat deposition.     

Novel PTEN germline mutation in a family with mild phenotype: Difficulties in genetic counseling

PTEN gene (phosphatase and tensin homolog deleted on chromosome ten, MIM 601628) is a tumor suppressor gene implicated in PTEN hamartoma tumor syndromes (PHTS) including Cowden syndrome, Bannayan–Riley–Ruvalcaba syndrome and Proteus-like syndrome. PTEN mutations have been more recently reported in children with macrocephaly and autism spectrum disorders or mental retardation, without other symptoms of PHTS. Although tumor risk has not been evaluated in these patients and their relatives, the same surveillance as for Cowden syndrome is usually proposed. We report a family including patients carrying a novel PTEN mutation and presenting with a mild phenotype consisting of macrocephaly, hypotonia during the first year of life and mild learning disabilities, without autistic features. None of these patients exhibited PTHS-related symptoms such as tumors, lipomas, vascular malformations or pigmented macules of the glans penis. This report raises the question of extending the indications of PTEN mutation screening to familial macrocephaly with learning disabilities. Detection of a mutation in this family led to difficult questions about surveillance, genetic counseling and familial information since the mother declined tumor screening and disclosure of genetic risk information to at-risk relatives.     

Biogeochemical Contributions of Tree Islands to Everglades Wetland Landscape Nitrogen Cycling During Seasonal Inundation

In the Florida Everglades, tree islands are conspicuous heterogeneous elements in the herbaceous wetland landscape. We characterized the biogeochemical role of a seasonally flooded tree island during wet season inundation, specifically examining hydrologically mediated flows of nitrogen (N) and N retention by the tree island. We estimated ecosystem N standing stocks and fluxes, soil and litter N transformation rates, and hydrologic fluxes of N to quantify the net ecosystem N mass flux. Results showed that hydrologic sources of N were dominated by surface water loads of nitrate (NO₃) and ammonium (NH₄). Nitrate immobilization by soils and surficial leaf litter was an important sink for surface water dissolved inorganic N (DIN). We estimated that the net annual DIN retention by a seasonally flooded tree island was 20.5 ± 5.0 g m⁻² during wet season inundation. Based on the estimated tree island surface water DIN loading rate, a seasonally flooded tree island retained 76% of imported DIN. As such, seasonally flooded tree islands have the potential to retain 55% of DIN entering the marsh landscape via upstream canal overland flow in the wet season. By increasing reactive surface area and DOC availability, we suggest that tree islands promote convergence of elements that enhance DIN retention. Tree islands of this region are thus important components of landscape-scale restoration efforts that seek to reduce sources of anthropogenic DIN to downstream estuaries.     

The lens in focus: a comparison of lens development in Drosophila and vertebrates

The evolution of the eye has been a major subject of study dating back centuries. The advent of molecular genetics offered the surprising finding that morphologically distinct eyes rely on conserved regulatory gene networks for their formation. While many of these advances often stemmed from studies of the compound eye of the fruit fly, Drosophila melanogaster, and later translated to discoveries in vertebrate systems, studies on vertebrate lens development far outnumber those in Drosophila. This may be largely historical, since Spemann and Mangold's paradigm of tissue induction was discovered in the amphibian lens. Recent studies on lens development in Drosophila have begun to define molecular commonalities with the vertebrate lens. Here, we provide an overview of Drosophila lens development, discussing intrinsic and extrinsic factors controlling lens cell specification and differentiation. We then summarize key morphological and molecular events in vertebrate lens development, emphasizing regulatory factors and networks strongly associated with both systems. Finally, we provide a comparative analysis that highlights areas of research that would help further clarify the degree of conservation between the formation of dioptric systems in invertebrates and vertebrates.     

Yeast as a tractable genetic system for functional studies of the insulin-degrading enzyme

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.     

Grape powder extract attenuates tumor necrosis factor α-mediated inflammation and insulin resistance in primary cultures of human adipocytes

Grapes are rich in phenolic phytochemicals that possess anti-oxidant and anti-inflammatory properties. However, the ability of grape powder extract (GPE) to prevent inflammation and insulin resistance in human adipocytes caused by tumor necrosis factor α (TNFα), a cytokine elevated in plasma and white adipose tissue (WAT) of obese, diabetic individuals, is unknown. Therefore, we examined the effects of GPE on markers of inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes treated with TNFα. We found that GPE attenuated TNFα-induced expression of inflammatory genes including interleukin (IL)-6, IL-1β, IL-8, monocyte chemoattractant protein (MCP)-1, cyclooxygenase (COX)-2 and Toll-like receptor (TLR)-2. GPE attenuated TNFα-mediated activation of extracellular signal-related kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) and activator protein-1 (AP-1, i.e., c-Jun). GPE also attenuated TNFα-mediated IκBα degradation and nuclear factor-kappa B (NF-κB) activity. Finally, GPE prevented TNFα-induced expression of protein tyrosine phosphatase (PTP)-1B and phosphorylation of serine residue 307 of insulin receptor substrate-1 (IRS-1), which are negative regulators of insulin sensitivity, and suppression of insulin-stimulated glucose uptake. Taken together, these data demonstrate that GPE attenuates TNFα-mediated inflammation and insulin resistance in human adipocytes, possibly by suppressing the activation of ERK, JNK, c-Jun and NF-κB.