Rh Family Research Articles

Dihydroartemisinin alleviates ammonia toxicity and autophagy in zebrafish (Danio rerio) gill

Ammonia is highly toxic to aquatic organisms and has become a consistent pollutant in intensive fish farming conditions. Dihydroartemisinin (DhA) has potential value in the treatment of ammonia poisoning in aquaculture. However, the application investigation of DhA is insufficient in the field of aquaculture. In the present study, 540 zebrafish with an average body weight of (1.00 ± 0.01) g were randomly assigned into 9 groups. The zebrafish were fed a basal diet supplemented with or without 0.25% or 0.5% DhA for 1 week. The zebrafish were then exposed to water supplemented with or without 40 or 80 mg/L ammonium acetate (NH4AC) for 4 days. The results showed that dietary supplementation with 0.5% DhA attenuated mortality and ammonia accumulation in zebrafish exposed to 80 mg/L NH4AC. In addition, the alleviative effects of 0.5% DhA were associated with enhanced mRNA levels for ammonia transporter (Rhesus-associated glycoprotein, Rh family C glycoprotein 1, Na+/K+-ATPase, carbonic anhydrase), anti-inflammatory cytokines (transforming growth factor β), as well as the down-regulation of mRNA expression of inflammatory factors (interleukin 1β), hypoxia-inducible factors (hypoxia-inducible factor 1 αb, hypoxia-inducible factor 2α), and protein abundances of autophagy (autophagy-related gene 5, BCL-2-interacting myosin-like coiled-coil protein-1, microtubule-associated protein 1 light chain 3 Ⅱ, cathepsin B, cathepsin L). Taken together, DhA has great potential in aquaculture as a defence against ammonia toxicity.

Comprehensive Analysis to Identify Rh Family C Glycoprotein (RHCG) as the Causative Gene for Psoriasis and Search for Alternative Treatment Modalities.

Psoriasis is a complex autoimmune disease. Frequent interactions between epidermal and immune cells are likely to be responsible for the strong heterogeneity of psoriasis. Therefore, our work aims to build on current knowledge and further search for new molecular mechanisms related to psoriasis pathogenesis in order to develop new targeted drugs. Data from psoriasis samples were obtained from the Gene Expression Omnibus (GEO) database, and batch effects were corrected using the "Combat" algorithm in the "SVA" package. Functional annotation of differential genes in psoriasis was performed by Gene set enrichment analysis (GSEA). Core functional modules were identified using the Multiscale Embedded Gene Co-Expression Network Analysis (MEGENA) algorithm for selection from the differential gene interaction network. The expression and potential function of Rh Family C Glycoprotein (RHCG) was predicted in single cell data by the "Seurat" package and validated in psoriasis samples by multiplex immunofluorescence. In addition, the regulatory function of HOP Homeobox (HOPX) on RHCG in keratinocytes was confirmed using RNA interference. Using immune infiltration analysis, RHCG and DC cells were analyzed for their association. Finally, the molecular mechanisms of treatment of psoriasis using Tripterygii Radix (TR) and Cinnamomi Ramulus (CR) were explored through network pharmacology and experimental validation. Immune response (represented by C1_2) and collagen matrix formation (represented by C1_3) were identified as two important pathogenic factors in psoriasis and helped to define new biological subtypes of psoriasis. One important psoriasis hub gene, RHCG, was obtained and found to be closely associated with keratinocyte differentiation as well as DC cell maturation. And RHCG was regulated by HOPX in keratinocytes. In addition, the mechanism of action of CR and TR in the treatment of psoriasis was tentatively confirmed to be related to TRPV3, NFKB2, and YAP1. Our study identifies a new causal disease gene (RHCG) and offers potential alternatives for the treatment of psoriasis.

CircATIC inhibits esophageal carcinoma progression and promotes radiosensitivity by elevating RHCG through sponging miR-10-3p.

BackgroundCircular RNAs (circRNAs) are implicated in the progression and radiosensitivity of human cancers, including esophageal carcinoma (ESCA). In this study, we aimed to explore the functions of circRNA 5‐aminoimidazole‐4‐carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (circATIC) in ESCA progression.MethodsCircATIC expression, miR‐10b‐3p and Rh family C glycoprotein (RHCG) were examined via quantitative real‐time polymerase chain reaction (qRT‐PCR), western blot assay or immunohistochemistry (IHC) assay. 5′‐ethynyl‐2′‐deoxyuridine (EdU), wound‐healing, transwell, and cell colony formation assays and flow cytometry analysis were conducted to evaluate cell proliferation, migration, invasion, radiosensitivity and apoptosis, respectively. Dual‐luciferase reporter assay and RNA pulldown assay were conducted to analyze the relationships among circATIC, miR‐10b‐3p and RHCG. A murine xenograft model assay was performed to explore the functions of circATIC in tumor formation and radiosensitivity in vivo.ResultsCircATIC was decreased in ESCA. CircATIC overexpression suppressed cell proliferation, migration and invasion and promoted radiosensitivity and apoptosis in ESCA cells in vitro and repressed tumor formation and radioresistance in vivo. Functionally, circATIC served as the sponge for miR‐10b‐3p, which directly targeted RHCG. MiR‐10b‐3p elevation reversed circATIC‐mediated effect on ESCA cell progression. Moreover, miR‐10b‐3p inhibition suppressed cell growth and metastasis and enhanced radiosensitivity in ESCA cells by targeting RHCG.ConclusionsOverexpression of circATIC hampered ESCA progression and promoted radiosensitivity depending on the regulation of miR‐10b‐3p and RHCG.

Epidemiologic Characterization of Transfusion Recipients with Red Blood Cell Alloantibodies from Four US Regions: Evidence from the REDS-III Recipient Database

Introduction: Red blood cell (RBC) alloimmunization is one of the most common complications of RBC transfusion. Despite the fact that alloimmunization impacts many transfusion recipients, the lack of standardized alloimmunization registries in the US has prevented large studies from being completed. Through the development of a uniform electronic research database, we investigated alloimmunization rates and diagnoses in inpatients and outpatients across the US.Methods: The REDS-III recipient database, established in 2013, houses demographic, diagnostic, transfusion, laboratory, and other information on inpatients and outpatients at 12 hospitals in 4 geographic regions. For this study, an antibody “responder” (R) was defined as an individual who had a putative transfusion (or pregnancy)-associated red cell antibody identified by screen at any point during the study period, independent of documented RBC exposure or pregnancy during the study period. An antibody “non-responder” (NR) was defined as an individual who was transfused with at least one unit of RBCs during the study period, and had a negative antibody screen at least 15 days post-transfusion. Anti-D antibodies were excluded from studied females, given an inability to distinguish passively administered anti-D in the database from authentic alloantibodies. Demographics, blood type, ICD9/10 codes, and antibody screen results were compared for R and NRs. Odds ratios were calculated for the likelihood of being a R or NR, based on the presence or absence of select chronic disease states.Results: 612,417 patients were recorded in the REDS recipient database from 2013-2016. 318,918 unique patients in the database had an RBC antibody screen during the study period. Of these, 6597 had at least one transfusion-associated RBC alloantibody identified by routine antibody screen (overall alloimmunization rate among all screened patients independent of RBC exposure = 2.07%). 63.8% of the R were female (alloimmunization rate 2.97%) and 36.2% were male (alloimmunization rate 1.35%). 30,468 unique patients (49.8% female and 50.2% male) were NR; the mean RBC exposure during the study period was similar between R and NR (mean, s.d., and median of 8.8, 18.2, and 4 units versus 9.3, 14.3, and 5 units). Approximately 60-70% of the antibodies in R were pre-existing, and the remainder were generated during the study period. Few children under 10 years of age were R compared to older age groups; an equation of R/R+NR = 26/26 + 467 = 5.3%. There were no differences in R vs NR by A/B/O blood type, though Rh negative patients (R/R+NR of 1175/1175 + 3901 = 23.1%) were significantly more likely to be R than Rh positive patients (R/R + NR of 5365/5365 + 26544 = 16.8%). .A total of 8892 clinically significant antibodies were detected, including 4749 (53.4%) in the Rh family (2588 anti-E, 780 anti-C, 556 anti-c, 105 anti-e, 525 anti-D), 1937 (21.8%) in the K family, 912 (10.3%) in the Kidd family, and 669 (7.5%) in the Duffy family, among others. Patients with sickle cell disease (n=554, OR 2.6, p<0.0001), sickle cell trait (n=117, OR 1.8, p=0.02), 'coronary atherosclerosis or heart disease (n=12,403, OR 1.2, p<0.0001), lupus (n=969, OR 1.3, p=0.002), rheumatoid arthritis (n=1213, OR 1.2, p=0.01), and myelodysplastic syndrome (n=1162, OR 1.3, p=0.0008) were more likely to be R than NR. In contrast, patients with leukemia (n=2801, OR 0.5, p<0.0001), solid tumors (n=17,513, OR 0.6, p<0.0001), and solid organ transplant (n=1491, OR 0.7, p=0.0001) were less likely to be R than NR.Conclusions: Data collected in this multi-center recipient database provide the largest RBC alloimmunized patient cohort ever studied in the US. The 2.07% overall rate of alloimmunization is similar to that reported in other retrospective studies, with females having higher alloimmunization rates than males. Rh negative individuals were significantly more likely to be alloimmunized than Rh positive patients, despite the exclusion of anti-D antibodies in females. Other unexpected findings include the predisposition to alloimmunization observed in patients with sickle cell trait and coronary artery disease. Ongoing studies are investigating the timing of RBC exposure to new antibody formation in this patient cohort, as well as potential physiologic explanations for the chronic disease associated findings. DisclosuresNo relevant conflicts of interest to declare.

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH3/NH4+) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid-base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH4+ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

Next-generation RNA Sequencing–based Biomarker Characterization of Chromophobe Renal Cell Carcinoma and Related Oncocytic Neoplasms

BackgroundRenal cell carcinomas (RCCs) are a heterogeneous group of neoplasms. Recent sequencing studies revealed various molecular features associated with histologic RCC subtypes, including chromophobe renal cell carcinoma (ChRCC). ObjectiveTo characterize the gene expression and biomarker signatures associated with ChRCC. Design, setting, and participantsWe performed integrative analysis on RNA sequencing data available from 1049 RCC specimens from The Cancer Genome Atlas and in-house studies. Our workflow identified genes relatively enriched in ChRCC, including Forkhead box I1 (FOXI1), Rh family C glycoprotein (RHCG), and LINC01187. We assessed the expression pattern of FOXI1 and RHCG protein by immunohistochemistry (IHC) and LINC01187 mRNA by RNA in situ hybridization (RNA-ISH) in whole tissue sections representing a cohort of 197 RCC cases, including both primary and metastatic tumors. Outcome measurements and statistical analysisThe FOXI1 and RHCG IHC staining, as well as the LINC01187 RNA-ISH staining, was evaluated in each case for intensity, pattern, and localization of expression. Results and limitationsAll primary and metastatic classic ChRCCs demonstrated homogeneous positive labeling for FOXI1, RHCG proteins, and LINC01187 transcript. Unclassified RCC with oncocytic features, oncocytoma, and hybrid oncocytic tumor, as well as all but two cases of eosinophilic ChRCC also stained positive. Importantly, metastatic and primary RCC of all other subtypes did not demonstrate any unequivocal staining for FOXI1, RHCG, or LINC01187. In normal kidney, FOXI1, RHCG, and LINC01187 were detected in the distal nephron segment, specifically in intercalated cells. Two cases of eosinophilic ChRCC with focal expression of FOXI1 and LINC01187, and Golgi-like RHCG staining were found to contain MTOR gene mutations upon DNA sequencing. ConclusionsWe demonstrate a pipeline for the identification and validation of RCC subtype–specific biomarkers that can aid in the confirmation of cell of origin and may facilitate accurate classification and diagnosis of renal tumors. Patient summaryFOXI1, RHCG, and LINC01187 are lineage-specific signature genes for chromophobe renal cell carcinoma.

Targeted metagenomic recovery of four divergent viruses reveals shared and distinctive characteristics of giant viruses of marine eukaryotes.

Giant viruses have remarkable genomic repertoires—blurring the line with cellular life—and act as top–down controls of eukaryotic plankton. However, to date only six cultured giant virus genomes are available from the pelagic ocean. We used at-sea flow cytometry with staining and sorting designed to target wild predatory eukaryotes, followed by DNA sequencing and assembly, to recover novel giant viruses from the Pacific Ocean. We retrieved four ‘PacV’ partial genomes that range from 421 to 1605 Kb, with 13 contigs on average, including the largest marine viral genomic assembly reported to date. Phylogenetic analyses indicate that three of the new viruses span a clade with deep-branching members of giant Mimiviridae, incorporating the Cafeteria roenbergensis virus, the uncultivated terrestrial Faunusvirus, one PacV from a choanoflagellate and two PacV with unclear hosts. The fourth virus, oPacV-421, is phylogenetically related to viruses that infect haptophyte algae. About half the predicted proteins in each PacV have no matches in NCBI nr (e-value < 10−5), totalling 1735 previously unknown proteins; the closest affiliations of the other proteins were evenly distributed across eukaryotes, prokaryotes and viruses of eukaryotes. The PacVs encode many translational proteins and two encode eukaryotic-like proteins from the Rh family of the ammonium transporter superfamily, likely influencing the uptake of nitrogen during infection. cPacV-1605 encodes a microbial viral rhodopsin (VirR) and the biosynthesis pathway for the required chromophore, the second finding of a choanoflagellate-associated virus that encodes these genes. In co-collected metatranscriptomes, 85% of cPacV-1605 genes were expressed, with capsids, heat shock proteins and proteases among the most highly expressed. Based on orthologue presence–absence patterns across the PacVs and other eukaryotic viruses, we posit the observed viral groupings are connected to host lifestyles as heterotrophs or phototrophs.This article is part of a discussion meeting issue ‘Single cell ecology’.

Differences in acidosis-stimulated renal ammonia metabolism in the male and female kidney.

Renal ammonia excretion is a critical component of acid-base homeostasis, and changes in ammonia excretion are the predominant component of increased net acid excretion in response to metabolic acidosis. We recently reported substantial sex-dependent differences in basal ammonia metabolism that correlate with sex-dependent differences in renal structure and expression of key proteins involved in ammonia metabolism. The purpose of the present study was to investigate the effect of sex on the renal ammonia response to an exogenous acid load. We studied 4-mo-old C57BL/6 mice. Ammonia excretion, which was less in male mice under basal conditions, increased in response to acid loading to a greater extent in male mice, such that maximal ammonia excretion did not differ between the sexes. Fundamental structural sex differences in the nonacid-loaded kidney persisted after acid loading, with less cortical proximal tubule volume density in the female kidney than in the male kidney, whereas collecting duct volume density was greater in the female kidney. To further investigate sex-dependent differences in the response to acid loading, we examined the expression of proteins involved in ammonia metabolism. The change in expression of phosphoenolpyruvate carboxykinase and Rh family B glycoprotein with acid loading was greater in male mice than in female mice, whereas Na+-K+-2Cl- cotransporter and inner stripe of the outer medulla intercalated cell Rh family C glycoprotein expression were significantly greater in female mice than in male mice. There was no significant sex difference in glutamine synthetase, Na+/H+ exchanger isoform 3, or electrogenic Na+-bicarbonate cotransporter 1 variant A protein expression in response to acid loading. We conclude that substantial sex-dependent differences in the renal ammonia response to acid loading enable a similar maximum ammonia excretion response.

RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-β1

Cervical cancer is the second commonest cancer among women in the worldwide, and the majority cause of death in various countries, highlighting the importance of investigating new therapeutic targets. Rh family, C glycoprotein (RHCG) belongs to the Rhesus (Rh) family and was first identified as Rh blood group antigens. It has been confirmed to function in cancer progression, including prostate cancer and esophageal squamous cell carcinoma. However, its role in cervical cancer has never been explored. The present study indicated that RHCG was down-regulated in cervical cancers compared to that in normal cervical tissues, and further decreased in cervical cancer cell lines. Functionally, RHCG overexpression reduced cervical cancer cell proliferation and migration, as evidenced by the decreased transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-2 and MMP-9 expressions in cancer cells; however, an opposite effect was observed when RHCG was knocked down. Further, increase of RHCG markedly induced apoptosis in cervical cancer cells by improving the cleavage of Caspase-3 and poly (ADP-Ribose) polymerase (PARP). And cells transfected with RHCG siRNA exhibited a notable reduction of cleaved Caspase-3 and PARP. Moreover, nucleus nuclear factor-κB (NF-κB) and whole cell xIPA expressions were markedly reduced by over-expressing RHCG. Conversely, suppressing RHCG elevated NF-κB activation and xIPA expression in cervical cancer cells. Notably, we found that TGF-β1 treatment could abolish the effects of RHCG over-expression on the reduction of cell migration and enhancement of apoptosis in cervical cancer cells. Over-expressing RHCG-reduced NF-κB activation and xIPA expression were also abrogated by TGF-β1 pre-treatment. Additionally, enhancing NF-κB activity could restore xIPA expressions and decrease apoptotic response in cervical cancer cells over-expressing RHCG. In vivo, we also found that RHCG over-expression reduced cervical tumor growth through the same signaling pathways as we found in vitro. Therefore, RHCG may be a potential prognostic biomarker and therapeutic target for human cervical cancer.

Computer Vision for High-Throughput Quantitative Phenotyping: A Case Study of Grapevine Downy Mildew Sporulation and Leaf Trichomes.

Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.

Modulating O2 Permeability of the Central Pore of RH50 by in Silico Site Directed Mutagenesis

The Rhesus (Rh) family of integral membrane proteins exhibit ubiquitous expression among animals and their structural homology to ammonium transport (Amt) proteins suggests their function as ammonia/ammonium transporters. They have also been implicated in facilitating permeation of gaseous species, such as dioxygen and carbon dioxide, across the cell membrane. Rh50 proteins are expressed in erythrocytes, as well as in epithelial tissues of organs such as the kidneys where critical nitrogen processing occurs. Using the solved Rh50 structure from bacterial homologue NeRh50 found in Nitrosomonas europaea, we employed a combination of molecular dynamics and free energy approaches including implicit ligand sampling, explicit ligand sampling, umbrella sampling, and in silico mutagenesis to characterize O2 permeation pathway, mechanism, and energetics. Free-energy calculations reveal high-energy barriers both in the monomeric pore as well as the pore formed in the trimerization domain similar to those previously reported for CO2 and NH3. Based on the the free-energy calculation results, we identified key amino acids potentially responsible for the high barrier. Bioinformatic analysis of these sites shows that some of the residues lining the high-energy region of the pore exhibit a high degree of conservation only for members of the Rh50 family, with the exception of NeRh50. Residues Thr199 in Arg202, which are localized near the pore opening, exist as hydrophobic amino acids in the protein's eukaryotic counterparts and Leu206 in NeRh50 presents itself primarily as an isoleucine. Mutation of these residues to the conserved amino acids results in a reduction in the energy barrier. This suggests a permeation pathway for gaseous species not observable in the native NeRh50 structure which can be verified by functional characterization of the mutants.

Analysis of Mad Honey (Grayanotoksin) Cases Admitted to Duzce University School of Medicine Emergency Department

Background: Mad honey is the type of honey made by bees that are nourished from the plants of Rhododendron (Rh.) family, and it contains grayanotoksin (GTX). The most significant types of Rh family, which contain GTX, are Rh. The purpose of this study was to analyze the cases which came to the emergency room because of GTX intoxication in detail in order to help diagnosis, treatment, follow-up and prognosis in daily emergency medicine practice. A total of 36 patients with the symptoms and diagnosis of intoxication after consuming mad honey, were included in the study. Ten healthy individuals who did not have a history of taking mad honey were included in the control group. The study is designed as a prospective case control study. The average GTX level of the group who ate mad honey (studygroup) was 7.88 ng/ml (min: 0.00/max: 30.47). At control group, GTX level was 0 ng/ml. There was no statistically significant relationship between the symptoms (dizziness, nausea, vomiting, weakness, deterioration, angina) and blood GTX level in the study group. Also statistic value reflects relationship between kidney function tests, troponin and control troponin levels in case group. The patients who have absolute decrease in blood pressure and pulse should be questioned by emergency medicine doctors about consuming mad honey. Further studies are needed to demonstrate the relationship between symptoms and GTX poisoning.

Mechanism of NH4+ Recruitment and NH3 Transport in Rh Proteins

In human cells, membrane proteins of the rhesus (Rh) family excrete ammonium and play a role in pH regulation. Based on high-resolution structures, Rh proteins are generally understood to act as NH3 channels. Given that cell membranes are permeable to gases like NH3, the role of such proteins remains a paradox. Using molecular and quantum mechanical calculations, we show that a crystallographically identified site in the RhCG pore actually recruits NH4(+), which is found in higher concentration and binds with higher affinity than NH3, increasing the efficiency of the transport mechanism. A proton is transferred from NH4(+) to a signature histidine (the only moiety thermodynamically likely to accept a proton) followed by the diffusion of NH3 down the pore. The excess proton is circulated back to the extracellular vestibule through a hydrogen bond network, which involves a highly conserved and functionally important aspartic acid, resulting in the net transport of NH3.

Common Genetic Variants Associated with Resting Oxygenation in Chronic Obstructive Pulmonary Disease

Hypoxemia is a major complication of chronic obstructive pulmonary disease (COPD) that correlates with disease prognosis. Identifying genetic variants associated with oxygenation may provide clues for deciphering the heterogeneity in prognosis among patients with COPD. However, previous genetic studies have been restricted to investigating COPD candidate genes for association with hypoxemia. To report results from the first genome-wide association study (GWAS) of resting oxygen saturation (as measured by pulse oximetry [Spo2]) in subjects with COPD, we performed a GWAS of Spo2 in two large, well characterized COPD populations: COPDGene, including both the non-Hispanic white (NHW) and African American (AA) groups, and Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE). We identified several suggestive loci (P < 1 × 10(-5)) associated with Spo2 in COPDGene in the NHW (n = 2810) and ECLIPSE (n = 1758) groups, and two loci on chromosomes 14 and 15 in the AA group (n = 820) from COPDGene achieving a level of genome-wide significance (P < 5 × 10(-8)). The chromosome 14 single-nucleotide polymorphism, rs6576132, located in an intergenic region, was nominally replicated (P < 0.05) in the NHW group from COPDGene. The chromosome 15 single-nucleotide polymorphisms were rare in subjects of European ancestry, so the results could not be replicated. The chromosome 15 region contains several genes, including TICRR and KIF7, and is proximal to RHCG (Rh family C glyocoprotein gene). We have identified two loci associated with resting oxygen saturation in AA subjects with COPD, and several suggestive regions in subjects of European descent with COPD. Our study highlights the importance of investigating the genetics of complex traits in different racial groups.

Coordinate Action of the Circadian Clock Protein Per1 and the Mineralocorticoid Hormone Aldosterone

Many metabolic and physiological processes, including renal function and blood pressure, exhibit a circadian pattern of variation. The molecular mechanism underlying the regulation of these processes by the circadian clock is not understood. We have previously shown that the circadian clock protein Per1 positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (αENaC); and negatively regulates the expression of Claudin 1 (Cldn1), a component of epithelial tight junctions; and endothelin‐1 (ET‐1), a blood pressure‐regulating peptide hormone. In the present study we found that Per1 also positively regulates Rh family C glycoprotein (RhCG), an ammonia transporter in the kidney. The sodium‐regulating hormone aldosterone has been well established to regulate the transcription of αENaC, and was also found to upregulate the levels of Cldn1, ET‐1, and RhCG mRNA. Co‐immunoprecipitation of Per1 from nuclear extracts obtained from mouse renal cortex showed an interaction between Per1 and the glucocorticoid receptor (GR), a nuclear receptor that can bind aldosterone. Taken together, these results suggest a coordinate regulation of several genes by Per1 and aldosterone.

Targeting Sialic Acid Dependent and Independent Pathways of Invasion in Plasmodium falciparum

The pathology of malaria is a consequence of the parasitaemia which develops through the cyclical asexual replication of parasites in a patient's red blood cells. Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Plasmodium invasion of the human red cell. Two major malaria ligand families have been implicated in these variable ligand-receptor interactions used by Plasmodium falciparum to invade human red cells: the micronemal proteins from the Erythrocyte Binding Ligands (EBL) family and the rhoptry proteins from the Reticulocyte binding Homolog (PfRH) family. Ligands from the EBL family largely govern the sialic acid (SA) dependent pathways of invasion and the RH family ligands (except for RH1) mediate SA independent invasion. In an attempt to dissect out the invasion inhibitory effects of antibodies against ligands from both pathways, we have used EBA-175 and RH5 as model members of each pathway. Mice were immunized with either region II of EBA-175 produced in Pichia pastoris or full-length RH5 produced by the wheat germ cell-free system, or a combination of the two antigens to look for synergistic inhibitory effects of the induced antibodies. Sera obtained from these immunizations were tested for native antigen recognition and for efficacy in invasion inhibition assays. Results obtained show promise for the potential use of such hybrid vaccines to induce antibodies that can block multiple parasite ligand-red cell receptor interactions and thus inhibit parasite invasion.

Localized 5f electrons in superconducting PuCoIn5: consequences for superconductivity in PuCoGa5

The physical properties of the first In analog of the PuMGa5 (M = Co, Rh) family of superconductors, PuCoIn5, are reported. With its unit cell volume being 28% larger than that of PuCoGa5, the characteristic spin-fluctuation energy scale of PuCoIn5 is three to four times smaller than that of PuCoGa5, which suggests that the Pu 5f electrons are in a more localized state relative to PuCoGa5. This raises the possibility that the high superconducting transition temperature Tc = 18.5 K of PuCoGa5 stems from the proximity to a valence instability, while the superconductivity at Tc = 2.5 K of PuCoIn5 is mediated by antiferromagnetic spin fluctuations associated with a quantum critical point.

A Novel Murine Model of Pregnancy and Transfusion Induced Anti-Kell RBC Alloimmunization

Abstract 715 Introduction:Pregnancy and transfusion induced RBC alloimmunization can be clinically significant, leading to difficulties with future transfusions and also leading to hemolytic disease of the fetus and newborn (HDFN). Maternal anti-Kell alloimmunization is one of the most common causes of non ABO-mediated HDFN. To date, there are few treatments to prevent HDFN due to RBC antibodies outside of the Rh(D) family, and there are few existing animal models of pregnancy induced RBC alloimmunization. Herein, we describe a novel murine model in which Kell RBC alloimmunization occurs following pregnancy or transfusion. Materials and Methods:Mice with RBC specific expression of the K2 allele of the human Kell glycoprotein (subsequently referred to as “K2” mice) were generated utilizing constructs containing the human K2 sequence expressed by beta-globin regulatory elements. For the transfusion induced alloimmunization experiments, C57BL/6 female recipients were transfused 3 times with K2 RBCs in the presence of poly (I:C), with anti-Kell glycoprotein antibodies measured after each transfusion by flow cytometric crossmatch using K2 targets and subtype specific antibodies. For the pregnancy induced alloimmunization experiments, control naïve C57BL/6 females were mated with K2 males three times; females immunized following transfusion were mated with K2 males as well. After the final pregnancy, anti-Kell glycoprotein antibodies were also measured by flow cytometric. Number and percentage of K2 positive pups were noted in a subset of pregnancies, and deceased pups were genotyped by PCR. Results:Anti-Kell glycoprotein antibodies were detectable in C57BL/6 recipients following either K2 RBC transfusion or following repeat pregnancies with K2 positive fetuses. In 5 experiments, 30/30 transfused mice had anti-Kell antibody titers that increased with subsequent transfusion, with IgM, IgG1, IgG2b, IgG2c, and IgG3 anti-Kell being detectable. After 2–3 pregnancies with K2 mates, 11/12 C57BL/6 females developed anti-Kell glycoprotein antibodies, with IgM and all IgG anti-Kell subtypes being present. Regardless of the mechanism of immunization (e.g. transfusion or prior pregnancy), live births and pups surviving beyond day of life #2 were lower in Kell immunized mothers compared with control non-immunized mothers (approximately 2–4 pups compared to 6–10 pups). At least one K2 stillborn pup had severe edema resembling hydrops. Discussion:This is the first animal model of Kell RBC alloimmunization, in which anti-Kell can be generated either through prior pregnancy or transfusion and appears to be detrimental to pups. This model mimics that of pregnancy or transfusion induced anti-Rh(D), in that the described antigen (K2) is largely foreign to the C57BL/6 recipients (who lack human K1 or K2 altogether). Ongoing studies are investigating whether a particular method of immunization (e.g. pregnancy vs transfusion) is more detrimental to fetuses and pups. As expected, the anti-Kell antibody appears more detrimental to K2 positive than negative fetuses, with immunized mothers having smaller litters of predominantly K2 negative fetuses; however, K2 negative pups may also have higher rates of demise following birth to immunized compared to non-immunized mothers. In sum, this model will allow for further investigation of pregnancy and transfusion induced anti-Kell RBC alloimmunization, and may also serve to increase the understanding of the pathogenesis and prevention of HDFN. Disclosures:Zimring:Immucor: Funds from an Immucor sponsored project (unrelated to the current project) were utilized in part to generate the KEL transgenic animals.

A family study of the Chinese Rhnull individual of the regulator type: a novel single missense mutation identified in RHAG gene

Rh(null) is a rare autosomal recessive disorder, and Rh(null) of the regulator type may result from mutation of the RHAG gene, which encodes RhAG glycoprotein and modulates Rh antigen expression. This study described the molecular genetic analysis of a Chinese Rh(null) family and identified a novel mutation in the RHAG gene. RBCs from the Rh(null) family members were analyzed for Rh phenotype by standard methods. DNA sequences of all 10 exons of RHAG gene were analyzed using genomic DNA by polymerase chain reaction and direct DNA sequencing. Genomic DNA analyses showed that a 672C>A mutation in Exon 5 of the RHAG gene was present at the homozygous state in DH and at the heterozygous state in the other members of the Rh(null) family. The 672C>A missense mutation converted serine into arginine at Position 224 in the Transmembrane Segment 7 of RhAG glycoprotein. These findings provide evidence that the 672C>A missense mutation in the RHAG gene could result in Rh(null) of the regulator type, and the single-amino-acid change (Ser to Arg) might be critical for assembly of the Rh antigen complex within the membrane.

Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a

The Plasmodium falciparum reticulocyte-binding-like protein homologue (RH) and erythrocyte binding-like (EBL) protein families play important roles during invasion, though their exact roles are not clear. Both EBL and RH proteins are thought to directly bind different receptors on the surface of the erythrocyte, and the binding properties for a number of EBLs and RHs have been described. While P. falciparum RH1 (PfRH1) and PfRH4 have been shown to act directly in two alternative invasion pathways used by merozoites, the functions of PfRH2a and PfRH2b during invasion are less defined. Here, using monoclonal antibodies raised against a unique region of PfRH2a, we show that PfRH2a moves from the rhoptry neck to the moving junction during merozoite invasion. The movement of PfRH2a to the junction is independent of the invasion pathway used by the merozoite, suggesting an additional function of the protein that is independent of receptor binding. We further show that PfRH2a is processed both in the schizont and during invasion, resulting in proteins with different erythrocyte binding properties. Our findings suggest that PfRH2a and, most likely, the other members of the RH family, depending on their processing stage, can engage different receptors at different stages of the invasion process.