Acidic Domain Research Articles

Nomenclature of factor XI and the contact system

Nomenclature of factor XI and the contact system

ATP binds nucleic-acid-binding domains beyond RRM fold

It has remained a mystery why cells maintain ATP concentrations of 2–12 mM, much higher than required for its known functions, until ATP is decoded to act as a hydrotrope to non-specifically control protein homeostasis above 5 mM. Unexpectedly, our NMR studies further reveal that by specific binding, ATP also mediates liquid-liquid phase separation in a two-stage style and inhibits fibrillation of RRM domains of FUS and TDP-43, implying that ATP might have a second category of functions previously unknown. So can ATP also bind nucleic-acid-binding proteins without RRM fold? Here we characterized the interaction between ATP and SYNCRIP acidic domain (AcD), a non-canonical RNA-binding domain with no similarity to RRM fold in sequence and structure. The results reveal that ATP does bind AcD at physiologically-relevant concentrations with the affinity determinants generally underlying protein-nucleic acid interactions. Therefore, at concentrations above mM, ATP might bind most, if not all, nucleic-acid-binding proteins.

Andexanet Alpha Differentially Neutralizes the Anticoagulant, Antiprotease and Thrombin Generation Inhibitory Effects of Unfractionated Heparin, Enoxaparin and Fondaparinux

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

Efficient production of levan using a recombinant yeast Saccharomyces cerevisiae hypersecreting a bacterial levansucrase.

Levan is a fructose polymer with diverse applications in the food and medical industries. In this study, levansucrase from Rahnella aquatilis (RaLsrA) was hyper-secreted using a Saccharomyces cerevisiae protein secretion system. An optimal secretion signal, a translation fusion partner (TFP) containing an N-terminal 98 amino acid domain from a mitochondrial inner membrane protein, UTH1, was employed to secrete approximately 50U/mL of bioactive RaLsrA into culture media with 63% secretion efficiency by fed-batch fermentation. Although the purified RaLsrA was useful for enzymatic conversion of high-molecular-weight levan of approximately 3.75 × 106Da, recombinant yeast secreting RaLsrA could produce levan more efficiently by microbial fermentation. In a 50-L scale fermenter, 76-g/L levan was directly converted from 191-g/L sucrose by recombinant yeast cells, attaining an 80% conversion yield and 3.17-g/L/h productivity. Thus, we developed a cost-effective and industrially applicable production system for food-grade levan.

Molecular characterization of yolk proteins in the female crab Neptunus pelagicus (A. Milne-Edwards, 1861) from the Mediterranean Sea of Alexandria, Egypt

This study aimed to characterize the morphological changes in the ovary of the female crab Neptunus pelagicus and to identify specific fractions of vitelloginin and vitelline molecules during primary and secondary vitellogenesis. Samples of the blue crab were collected from the Mediterranean Sea of Alexandria monthly during 2017. Ovaries and oocytes in primary and secondary vitellogenesis were detached and treated for histological test. Native polyacrylamide gel electrophoresis (PAGE) Bis-Tris Gels was applied to identify vitelloginin (VN) and vitelline (VL) molecules. Protein Analyses were done by PAGE-SDS. The initial degenerate primers were built regarding the conserved amino acid domains of the yolk proteins. Primary and secondary vitellogeneses consisted of 8 phases. Lipoprotein fraction with molecular weight 550 kDa was identified in the hemolymph in secondary vitellogenesis. Two protein fractions (VLI & VLII) were identified in secondary vitellogenic oocytes. The electrophoresis performed with extract of stage I oocyte showed two protein fractions with molecular weights 550 kDa and 460 kDa. In stage II and III oocyte, 4 subunits were presented of 180, 195, 140 and 120 kDa in VLI and 2 subunits with molecular weight of 110 kDa and 95 kDa in VLII. Another two fractions in stage V oocyte presented with molecular weights of 380 kDa and 360 kDa. Western blot analysis proved that both fractions were of four major polypeptide subunits with molecular weight of 180, 125, 90 and 85 kDa in each of the two VLs. The hybridization signal obtained by the Northern blot was detected in the hepatopancreas during ovarian cycle and in the ovary during secondary vitellogenesis. The result of the reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNA that encodes the C-terminal region of the VN cDNA was found in the ovary in secondary vitellogenesis and in the hepatopancreas.

Dantrolene prevents ventricular tachycardia by stabilizing the ryanodine receptor in pressure- overload induced failing hearts

Aberrant Ca2+ release from cardiac ryanodine receptors (RyR2) has been shown to be one of the most important causes of lethal arrhythmia in various types of failing hearts. We previously showed that dantrolene, a specific agent for the treatment of malignant hyperthermia, inhibits Ca2+ leakage from the RyR2 by correcting the defective inter-domain interaction between the N-terminal (1–619 amino acids) and central (2000–2500 amino acids) domains of the RyR2 and allosterically enhancing the binding affinity of calmodulin to the RyR2 in diseased hearts. In this study, we examined whether dantrolene inhibits this Ca2+ leakage, thereby preventing the pharmacologically inducible ventricular tachycardia in ventricular pressure-overloaded failing hearts. Ventricular tachycardia (VT) was easily induced after an injection of epinephrine in mice after 8 weeks of transverse aortic constriction-induced pressure-overload. Pretreatment with dantrolene almost completely inhibited the pharmacologically inducible VT. In the presence of dantrolene, the occurrence of both Ca2+ sparks and spontaneous Ca2+ transients was inhibited, which was associated with enhanced calmodulin binding affinity to the RyR2. These results suggest that dantrolene could be a new potent agent in the treatment of lethal arrhythmia in cases of acquired heart failure.

Genes acting in longevity-related pathways in the endoparasitoid, Pteromalus puparum.

Among insects, lifespans vary over a broad range, from the short-lived mayflies to the 17-year periodical cicadas. Generally, lifespans are determined by a phase in life, the reproductive lifespan, which varies among species. Numerous pathways, such as the insulin/insulin-like growth factor signaling pathway, the target of rapamycin pathway and the mitogen-activated protein kinase/extracellular signal-regulated kinases pathways, influence aging and lifespan. Components of these pathways were identified as lifespan-related genes, including genes mediating growth, metabolism, development, resistance, and other processes. Many age-related genes have been discovered in fruit flies, honeybees, and ants among other insect species. Studies of insect aging and longevity can help understand insect biology and develop new pest management technologies. In this paper, we interrogated the new Pteromalus puparum genome, from which we predicted 133 putative lifespan-related genes based on their homology with known lifespan-related genes of Drosophila melanogaster. These genes function in five signaling pathways and three physiological processes. The conserved domain structures of these genes were predicted and their expression patterns were analyzed. Amino acid sequence alignments and domain structure analysis indicate that most components remain conserved across at least six insect orders. The data in this paper will facilitate future work on parasitoid lifespans, which may have economic value in biocontrol programs.

Liquefied Microcapsules as Dual-Microcarriers for 3D+3D Bottom-Up Tissue Engineering.

Cell encapsulation systems must ensure the diffusion of molecules to avoid the formation of necrotic cores. The architectural design of hydrogels, the gold standard tissue engineering strategy, is thus limited to a microsize range. To overcome such a limitation, liquefied microcapsules encapsulating cells and microparticles are proposed. Microcapsules with controlled sizes with average diameters of 608.5 ± 122.3 µm are produced at high rates by electrohydrodynamic atomization, and arginyl-glycyl-aspartic acid (RGD) domains are introduced in the multilayered membrane. While cells and microparticles interact toward the production of confined microaggregates, on the outside cell-mediated macroaggregates are formed due to the aggregation of microcapsules. The concept of simultaneous aggregation is herein termed as 3D+3D bottom-up tissue engineering. Microcapsules are cultured alone (microcapsule1 ) or on top of 2D cell beds composed of human umbilical vein endothelial cells (HUVECs) alone (microcapsule2 ) or cocultured with fibroblasts (microcapsule3 ). Microcapsules are able to support cell encapsulation shown by LiveDead, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphofenyl)-2H-tetrazolium (MTS), and dsDNA assays. Only microcapsule3 are able to form macroaggregates, as shown by F-actin immunofluorescence. The bioactive 3D system also presented alkaline phosphatase activity, thus allowing osteogenic differentiation. Upon implantation using the chick chorioallontoic membrane (CAM) model, microcapsules recruit a similar number of vessels with alike geometric parameters in comparison with CAMs supplemented with basic fibroblast growth factor (bFGF).

A new crustin is involved in the innate immune response of shrimp Litopenaeus vannamei

Crustin is an antimicrobial peptide (AMP) that plays a key role in the innate immunity of crustaceans. This study cloned a new crustin from Pacific white shrimp Litopenaeus vannamei, which we designated as LvCrustinB, using rapid amplification of cDNA ends (RACE). The full-length cDNA of LvCrustinB is 751 bp with an open reading frame (ORF) of 591 bp encoding a peptide of 196 amino acids that includes a putative signal sequence. LvCrustinB is a type II crustin that has a glycine-rich region and a single whey acidic protein domain (WAP) domain. The mRNA transcript of LvCrustinB was detected in all examined tissues and was found to be most abundantly expressed in the epithelium and muscle. The expression of LvCrustinB in hemocytes was significantly upregulated after L. vannamei was challenged with LPS, Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). When LvCrustinB was knocked down with RNAi, the mortality rate of L. vannamei significantly increased after V. parahaemolyticus or WSSV infection. Recombinant LvCrustinB was produced using Pichia pastoris GS115 and was shown to bind to 2 g-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and 2 g-negative bacteria (Escherichia coli and V. parahaemolyticus) via polysaccharides, which included PGN, LTA, and LPS. In vivo, the recombinant LvCrustinB remarkably protected L. vannamei from V. parahaemolyticus infection. These results suggest that LvCrustinB plays an important role in innate immunity and may be potentially utilized as antibacterial agents in shrimp.

First identification and characterization of a triple WAP domain containing protein in Procambarus clarkii provides new insights into the classification and evolution of WAP proteins in crustacean

Whey acidic protein domain (WAPD) is a usual motif in crustaceans, and is found mainly in the immune-related proteins. In the present study, a protein containing three tandem WAPDs was identified in red swamp crayfish Procambarus clarkii and designated as PcTWD. This is the first report of a protein of such domain architecture in crustaceans. Introducing the WAPDs of PcTWD into phylogenetic analysis led to the classification of crustacean WAP proteins into classical crustins and proteins containing solely WAPDs. PcTWD was widely expressed in multiple tissues, including hemocytes, gills, hepatopancreas, heart, stomach and intestine. Its expression could be significantly induced by Staphylococcus aureus or Aeromonas hydrophila challenge. Knockdown PcTWD expression by RNAi suppressed host resistance against A. hydrophila, while exogenous recombinant PcTWD could enhance the host immunity. The three WAPDs showed a labor division. The first two domains were responsible for the protease inhibitory activity, and the third domain contributed to the antimicrobial activity. Thus PcTWD was found as an important protein in crayfish antibacterial immunity.

A Maternal Transcription Factor, Junction Mediating and Regulatory Protein is Required for Preimplantation Development in the Mouse.

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. The actin-regulatory activity of JMY is based on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate actin filaments directly and promote nucleation of the Arp2/3 complex. In addition to these activities, we examined the activity of JMY generation in early embryo of mice carrying mutations in the JMY gene by CRISPR/Cas9 mediated genome engineering. We demonstrated that JMY protein shuttled expression between the cytoplasm and the nucleus. Knockout of exon 2, CA (central domain and Arp2/3-binding acidic domain) and NLS-2 (nuclear localization signal domain) on the JMY gene by CRISPR/Cas9 system was effective and markedly impeded embryonicdevelopment. Additionally, it impaired transcription and zygotic genome activation (ZGA)-related genes. These results suggest that JMY acts as a transcription factor, which is essential for the early embryonic development in mice.

Specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers

Given the highly contagious and acute nature of porcine epidemic diarrhea (PED), especially in piglets, there is an urgent need for the development of rapid and sensitive diagnostic assays. The diagnostic potentials of specific porcine epidemic diarrhea virus (PEDV) accessory and nonstructural proteins, if any, have not yet been investigated. In order to determine and compare which of the viral proteins may be useful as diagnostic antigens, whole virus (WV) particles and a panel of structural and nonstructural PEDV proteins [spike subunit 1 (S1), the C-terminal part of ORF3 (ORF3C), envelope (E), nonstructural protein 1 (Nsp1), Nsp2, Ac (acidic domain of Nsp3), and ADRP (ADP-ribose-1-monophosphatase domain of Nsp3), expressed individually in bacterial and/or mammalian cells] were tested for reactivity with sera from PEDV-infected pigs by ELISA and/or western blot analysis. According to western blots, serum antibody interactions with the S1 protein were relatively more sensitive and specific than ORF3C, E and Ac. Furthermore, a total of 851 serum samples from diarrheal pigs of different ages were analyzed by ELISA, with most showing immune-reactivity towards the WV, S1, ORF3C, and E proteins. The earliest IgG antibody response was observed in the one-week-old piglets, with similar antibody ontogeny and patterns of seroconversion for S1, ORF3C, E, and WV antigens. In addition, the pattern of neutralizing antibody was more similar to that of IgA in weaning piglets after PEDV infection. Collectively, these data provide more reliable information on the host immune response to different viral proteins, which will be useful for development of novel serological assays and for design of vaccines that better stimulate protective immunity.

TOM40 Targets Atg2 to Mitochondria-Associated ER Membranes for Phagophore Expansion

SUMMARYDuring autophagy, phagophores grow into doublemembrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or nonvesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.

Myeloid Zinc Finger 1 (MZF1) Maintains the Mesenchymal Phenotype by Down-regulating IGF1R/p38 MAPK/ERα Signaling Pathway in High-level MZF1-expressing TNBC cells

Signaling regulation of myeloid zinc finger 1 (MZF1) has been implicated in the progression of many human malignancies; however, the mechanistic action of MZF1 in triple-negative breast cancer (TNBC) progression remains elusive. In this study, the aim was to investigate the molecular mechanisms of MZF1 and its functional role in TNBC cellular migration and invasion. Hs578T and MDA-MB-231 cells were transfected to stably express the acidic domain of MZF1 (MZF160-72), or were transfected with MZF1-specific or ELK1-specific short hairpin RNA (shRNA). Changes in cell morphology and distributions of cellular proteins were observed and subsequently migration and invasion were measured by wound healing and transwell assays. Expression levels of epithelial-mesenchymal transition (EMT)-related genes were carried out using immunoblotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Data of transcriptional regulation were obtained from promoter-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Herein, we found that MZF1 in high-level MZF1-expressing TNBC cells is associated with cell migration, invasion, and mesenchymal phenotype. MZF1 interacted with the promoter region of insulin-like growth factor 1 receptor (IGF1R) to drive invasion and metastasis of high-level MZF1-expressing TNBC cells. Exogenous expression of the acidic domain of MZF1 repressed the binding of endogenous MZF1 to IGF1R promoter via blocking the interaction with ETS-like gene 1 (ELK1). This blockage not only caused MZF1 protein degradation, but also restrained ELK1 nuclear localization in high-level MZF1-expressing TNBC cells. MZF1, but not ELK1, was necessary for the retention of mesenchymal phenotype by repressing IGF1R promoter activity in TNBC cells expressing high levels of MZF1. Activation of the IGF1R-driven p38MAPK-ERα-slug-E-cadherin signaling axis mediated the conversion of mesenchymal cell to epithelial phenotype, caused by MZF1 destabilization. These results suggest that MZF1 is an oncogenic inducer. Blocking of the MZF1/ELK1 interaction to reduce MZF1 protein stability by saturating the endogenous MZF1/ELK1 binding domains might be a promising therapeutic strategy for the treatment of high-level MZF1-expressing TNBC.

An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice.

Lateral meningocele syndrome (LMS), a genetic disorder characterized by meningoceles and skeletal abnormalities, is associated with NOTCH3 mutations. We created a mouse model of LMS (Notch3tm1.1Ecan ) by introducing a tandem termination codon in the Notch3 locus upstream of the proline (P), glutamic acid (E), serine (S) and threonine (T) domain. Microcomputed tomography demonstrated that Notch3tm1.1Ecan mice exhibit osteopenia. The cancellous bone osteopenia was no longer observed after the intraperitoneal administration of antibodies directed to the negative regulatory region (NRR) of Notch3. The anti-Notch3 NRR antibody suppressed the expression of Hes1, Hey1, and Hey2 (Notch target genes), and decreased Tnfsf11 (receptor activator of NF Kappa B ligand) messenger RNA in Notch3tm1.1Ecan osteoblast (OB) cultures. Bone marrow-derived macrophages (BMMs) from Notch3tm1.1Ecan mutants exhibited enhanced osteoclastogenesis in culture, and this was increased in cocultures with Notch3tm1.1Ecan OB. Osteoclastogenesis was suppressed by anti-Notch3 NRR antibodies in Notch3tm1.1Ecan OB/BMM cocultures. In conclusion, the cancellous bone osteopenia of Notch3tm1.1Ecan mutants is reversed by anti-Notch3 NRR antibodies.

Genome of the Malawi golden cichlid fish (Melanochromis auratus) reveals exon loss of oca2 in an amelanistic morph

The tropical freshwater fish family Cichlidae is famous for its record-breaking rates of speciation and diversity in colors and color patterns. Here, we sequenced the genome of the Lake Malawi cichlid Melanochromis auratus to study the genetic basis of an amelanistic morph of this species that lacks the typical melanic stripes and markings. Genome sequencing of the amelanistic and wild-type morph revealed the loss of the second exon of the known pigmentation gene oculocutaneous albinism II (oca2), also known as p(ink-eyed dilution) gene or melanocyte-specific transporter gene. Additional genotyping confirms the complete association with this recessive Mendelian phenotype. The deletion results in a shorter transcript, lacking an acidic di-leucine domain that is crucial for trafficking of the Oca2 protein to melanosomes. The fact that oca2 is involved in a wide range of amelanistic morphs across vertebrates demonstrates its highly conserved function.

2233-PUB: Clinical Features and Biomarkers of Nonalbuminuric and Albuminuric Patterns of Chronic Kidney Disease in Subjects with Type 2 Diabetes

The Aim: to define the phenotypes of non-albuminuric and albuminuric chronic kidney disease (NA-CKD and A-CKD) in subjects with type 2 diabetes (T2D). Methods: One hundred and eighty four T2D patients were included in the study. Patients with urinary albumin to creatinine ratio (UACR) <3 mg/mmol and estimated glomerular filtration rate (eGFR) <60 ml/min×1.73 m2 were classified as NA-CKD group (n=111). Individuals with persistent UACR >3 mg/mmol and eGFR <60 ml/min×1.73 m2 were considered as A-CKD patients (n=73). Urinary nephrin and podocin, as podocyte injury markers, and whey acidic protein four-disulfide core domain protein 2 (WFDC-2), which is considered as a marker of tubulointerstitial involvement, were assessed by ELISA and compared to control (20 nondiabetic subjects). Results: The NA-CKD pattern was associated with female sex (p=0.04), reduced HbA1c levels (p=0.04) and diuretic use (p=0.001). Patients with A-CKD, as compared to NA-CKD one, demonstrated lower body weight (p=0.02), waist-to-hip ratio (p=0.009), HDL-cholesterol (p=0.01), and lymphocyte count (p=0.002). Age, diabetes duration, the prevalence of diabetic retinopathy, coronary artery disease, myocardial infarction, chronic heart failure and stroke did not differ between the groups. The nephrin and podocin concentrations, adjusted to urinary creatinine, were increased significantly in subjects with A-CKD only (p=0.002 and 0.04 respectively). The excretion of WFDC-2 was elevated in both NA-CKD and A-CKD groups (p=0.01 and p=0.0003 respectively). Conclusion: In T2D subjects, female gender, acceptable glycemic control and treatment with diuretics are associated with NA-CKD, whereas abdominal obesity, poor glycemic control, and elevated urinary excretion of podocyte markers are the features of A-CKD. Urinary WFDC-2 excretion is increased in both CKD phenotypes. Disclosure A.I. Korbut: None. V. Klimontov: Advisory Panel; Self; Sanofi. Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Sanofi.

The pepper AP2 domain-containing transcription factor CaDRAT1 plays a negative role in response to dehydration stress

APETALA2 (AP2) domain-containing transcription factors play critical regulatory roles in plant growth, development, and response to abiotic stresses, including dehydration stress. In Capsicum annuum (pepper), data are lacking regarding AP2 domain-containing transcription factors and their precise functions. Here, we identified six putative AP2 domain-containing transcription factors in pepper plants; the expression levels of these transcription factors were induced or repressed by dehydration stress. We named the gene having the highest fold change as Capsicum annuumDehydration Responsive AP2 domain-containing Transcription factor 1 (CaDRAT1). CaDRAT1 belongs to the ERF subfamily B-4, but the roles of these ERF proteins in dehydration responses are not well known. CaDRAT1 transcript levels were significantly induced after exposure to abscisic acid (ABA), mannitol, low temperature, and H2O2. The acidic domain in the CaDRAT1 C-terminal region had a transactivation effect on the reporter gene in the yeast GAL4 system. CaDRAT1-silenced pepper plants displayed a dehydration-resistant phenotype characterized by enhanced stomatal closure and low levels of transpirational water loss. Moreover, under dehydration stress conditions, expression levels of stress-related genes including CaNCED3 were higher in CaDRAT1-silenced pepper plants than in control plants and ABA content was also higher. Taken together, our findings suggest that CaDRAT1 functions as a negative regulator of dehydration resistance through modulation of ABA biosynthesis and signaling.

Functional characterization of BmOVOs in silkworm, Bombyx mori

BackgroundIn our previous study, we identified four isoforms of the Bmovo gene, Bmovo-1, Bmovo-2, Bmovo-3 and Bmovo-4 from the silkworm ovary and verified that ovarian development was regulated by the BmOVO proteins. Results: To understand the regulatory mechanisms of ovarian development, the regulation of four BmOVO isoforms on the B. mori ovarian tumor (Bmotu) promoter activity was investigated with luciferase reporter assays. The results showed the Bmotu promoter activity was positively regulated by BmOVO-1, BmOVO-2, BmOVO-3 and BmOVO-4 in a dose-dependent manner, of which BmOVO-2 had the highest transcriptional activation. However, the first (A1) and third acidic domains (A3) at the N-terminus of BmOVO-1 are transcriptional repression domains, while the fourth (A4) and fifth acidic domains (A5) are transcriptional activation domains. A recombinant BmOVO zinc-finger domain was found to bind to the GTACCGTTGTA sequence located at the Bmotu promoter. Furthermore, the Bmotu promoter activity was negatively regulated by ‘Tal-like’ peptide, which can trigger BmOVO-1 degradation at the N-terminus.ConclusionsThese results will help us to further understand the regulatory mechanisms of BmOVO isoforms on Bmotu promoter activity and ovarian development in the silkworm.

Arabidopsis thionin-like genes are involved in resistance against the beet-cyst nematode (Heterodera schachtii)

Plants express various antimicrobial peptides including thionins to protect themselves against pathogens. It was recently found that, in addition to four thionin genes, Arabidopsis contains 67 thionin-like (ThiL) genes including six pseudogenes. It is known that thionins have antimicrobial activity and are part of the plant defense system, however, nothing is known about ThiL genes.In this study, we present a bioinformatic analysis of the (ThiL) gene family in Arabidopsis. We identified 15 different motifs which positioned the ThiL peptides in four groups. A comparison of amino acid sequences showed that the ThiL peptides are actually more similar to the acidic domain of thionin proproteins than to the thionin domain.We selected 10 ThiL genes to study the expression and possible function in the Arabidopsis plant. RT-PCR and promoter:GUS fusions showed that most genes were expressed at a very low level but in several organs and at different developmental stages. Some genes were also expressed in syncytia induced by the beet cyst nematode Heterodera schachti in roots while others were downregulated in syncytia. Some overexpression lines supported lower number of nematodes that developed on the roots after inoculation. Two of the genes resulted in a strong hypersensitive response when infiltrated into leaves of Nicotiana benthamiana. These results indicate that ThiL genes might be involved in the response to biotic stress. ThiL genes have been expanded in the Brassicales and specifically the Brassicaceae. The most extreme example is the CRP2460 subfamily that contains 28 very closely related genes from Arabidopsis which are mostly the result of tandem duplications.