Granulosa Cell Endothelin-2 Expression is Fundamental for Ovulatory Follicle Rupture

Ovulation is dependent upon numerous factors mediating follicular growth, vascularization, and ultimately oocyte release via follicle rupture. Endothelin-2 (EDN2) is a potent vasoconstrictor that is transiently produced prior to follicle rupture by granulosa cells of periovulatory follicles and induces ovarian contraction. To determine the role of Edn2 expression, surgical transplant and novel conditional knockout mice were super-ovulated and analyzed. Conditional knockout mice utilized a new iCre driven by the Esr2 promoter to selectively remove Edn2. Follicle rupture and fertility were significantly impaired in the absence of ovarian Edn2 expression. When ovaries of Edn2KO mice were transplanted in wild type recipients, significantly more corpora lutea containing un-ovulated oocytes were present after hormonal stimulation (1.0 vs. 5.4, p = 0.010). Following selective ablation of Edn2 in granulosa cells, Esr2-Edn2KO dams had reduced oocytes ovulated (3.8 vs. 16.4 oocytes/ovary) and smaller litters (4.29 ± l.02 vs. 8.50 pups/dam). However, the number of pregnancies per pairing was not different and the reproductive axis remained intact. Esr2-Edn2KO ovaries had a higher percentage of antral follicles and fewer corpora lutea; follicles progressed to the antral stage but many were unable to rupture. Conditional loss of endothelin receptor A in granulosa cells also decreased ovulation but did not affect fecundity. These data demonstrate that EDN2-induced intraovarian contraction is a critical trigger of normal ovulation and subsequent fecundity

A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data

Analgesic properties of a peripherally acting and GalR2 receptor-preferring galanin analog in inflammatory, neuropathic, and acute pain models

ABSTRACT There are ongoing efforts to develop pain therapeutics with novel mechanisms of action that avoid common side effects associated with other analgesics. The anticonvulsant neuropeptide galanin is a potent regulator of neuronal excitability and has a well established role in pain modulation, making it a potential target for novel therapies. Our previous efforts focused on improving blood-brain-barrier penetration and enhancing the metabolic stability of galanin analogs to protect against seizures. More recently, we designed peripherally acting galanin analogs that reduce pain-related behaviors by acting in the periphery and exhibit preferential binding toward galanin receptor (GalR)2 over GalR1. In this study, we report preclinical studies of a monodisperse oligoethylene glycol-containing galanin analog, NAX 409-9 (previously reported as GalR2-dPEG 24 ), in rodent analgesic and safety models. Results obtained with NAX 409-9 in these tests were compared with the representative analgesics gabapentin, ibuprofen, acetylsalicylic acid, acetaminophen, and morphine. In mice that received intraplantar carrageenan, NAX 409-9 increased paw withdrawal latency with an ED 50 of 6.6 mg/kg i.p. NAX 409-9 also increased the paw withdrawal threshold to mechanical stimulation following partial sciatic nerve ligation in rats (2 mg/kg). Conversely, NAX 409-9 had no effect in the tail flick or hot plate assays (up to 24 mg/kg). Importantly, NAX 409-9 did not negatively affect gastrointestinal motility (4-20 mg/kg), respiratory rate (40-80 mg/kg), or bleed time (20 mg/kg). These studies illustrate that this nonbrain-penetrating galanin analog reduces pain behaviors in several models and does not produce some of the dose-limiting toxicities associated with other analgesics

Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2–lipid bilayer interactions

AbstractCYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood. To gain better insights of these complex protein–lipid interactions, we employed molecular dynamics (MD) simulations using a highly mobile membrane mimetic (HMMM) model that enabled multiple independent spontaneous membrane binding events to be captured. Simulations revealed that CYP2J2 engages with the membrane at the F-G loop through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were modeled from the truncated CYP2J2 construct (Δ34) which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Using the HMMM coordinates of CYP2J2, the simulations were extended to a full POPC membrane which showed a significant decrease in the depth of insertion for each of the F-G loop mutants. The CYP2J2 F-G loop mutants were expressed in E. coli and were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34. Notably, the functional data demonstrated that the double mutant, Δ34-I236D/F239H, maintained native-like enzymatic activity. The membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, the F-G loop mutants exhibited lower Trp quenching and membrane insertion. Taken together, the results suggest that the mutants exhibit a different membrane topology in agreement with the MD simulations and provide important evidence towards the involvement of key residues in the F-G loop of CYP2J2

Lipoprotein Nanoplatelets: Brightly Fluorescent, Zwitterionic Probes with Rapid Cellular Entry

Semiconductor nano­platelets (NPLs) are planar nanocrystals that have recently attracted considerable attention due to their quantum-well-like physics, atomically precise thickness, and unique photophysical properties such as narrow-band fluorescence emission. These attributes are of potential interest for applications in biomolecular and cellular imaging, but it has been challenging to colloidally stabilize these nanocrystals in biological media due to their large dimensions and tendency to aggregate. Here we introduce a new colloidal material that is a hybrid between a NPL and an organic nanodisc composed of phospho­lipids and lipo­proteins. The phospho­lipids adsorb to flat surfaces on the NPL, and lipo­proteins bind to sharp edges to enable monodisperse NPL encapsulation with long-term stability in biological buffers and high-salt solutions. The lipo­protein NPLs (L-NPLs) are highly fluorescent, with brightness comparable to that of wavelength-matched quantum dots at both the ensemble and single-molecule levels. They also exhibit a unique feature of rapid internalization into living cells, after which they retain their fluorescence. These unique properties suggest that L-NPLs are particularly well suited for applications in live-cell single-molecule imaging and multiplexed cellular labeling