Agglutination Of Human Erythrocytes Research Articles

SCREENING OF AGGLUTINATION ACTIVITIES OF COMMON VEGETABLES IN BANGLADESH

Lectins are capable of cell agglutination by interacting with the cell surface exposed glycans. Lectins that agglutinate erythrocytes are called hemagglutinin. These compounds occur in various organisms including plants. In this study, twenty edible vegetables were subjected to screen for haemagglutination and lectin activity. Lectin activity was assessed by measuring the inhibition ability of haemagglutination by different sugars. PBS (Phosphate Buffer Saline) treated homogenate was tested for haemagglutination against erythrocytes suspension, and inhibition of agglutination was determined by serially diluted sugar solution. The results of this study demonstrated that one homogenate among the homogenates of 20 vegetables was found to be capable of human erythrocyte agglutination. The Ipomoea aquatica homogenate shown positive haemagglutination reaction against red blood cells (RBCs) with HA titer value of 4 HA-U/25uL, and four out of seven sugars were able to inhibit RBCs agglutination. Among the four sugars, two are potent inhibitors of agglutination. The neutralization of haemagglutination by four monosaccharides provides a suggestion that the lectin present in I. aquatica may be multivalent carbohydrate binding hemagglutinin. This is the first report to identify that Ipomoea aquatica contains lectin protein which is a polyvalent sugar binding lectin and specific for glucose, galactose, mannose and N-Acetyl-D-Glucosamine.

Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses.

Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary-K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B-mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary-K1, but not in human endothelial kidney 293-F cells.

Microscopy examination of red blood and yeast cell agglutination induced by bacterial lectins.

Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope.

Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein–Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A–mediated agglutination of human erythrocytes.

Genotypic identification and study some virulence factors of Aeromonas hydrophila isolated from children's diarrheal specimens in Baghdad

In this study, 266 watery diarrhea specimens were collected from children less than 12 years that suffering from gastroenteritis in Welfare teaching hospital and Teaching laboratories/ Medical city. Out of 266 specimens, 19 (7.14) were identified as Aeromonas hydrophila isolates. The bacteria were identified as Aeromonas hydrophila phenotypically by Api-20 E system. Genotypic identification of Aeromonas hydrophila isolates also done by detection 16S rRNA gene by PCR assay and the results showed the presence of 16S rRNA gene in all 19 (100%) Aeromonas hydrophila studied isolates and this confirm the identification of Aeromonas hydrophila isolates. The antimicrobial susceptibility of Aeromonas hydrophila isolates was determined and the results showed that all 19 Aeromonas hydrophila isolates were resistant to Ampicillin and Amoxicillin (100%), while all 19 isolates were sensitive to Cefotaxime, Cefteriaxon, Norfloxacin and Imipenem (100%). Some virulence factors of the Aeromonas hydrophila isolates were investigated, all 19 Aeromonas hydrophila isolates were tested to detect their ability to produce hemolysin (toxin), protease, lipase, agglutinate human erythrocytes groups and ability to bind to Congo red. Out of 19 isolates, 14(73.68%) Aeromonas hydrophila isolates were hemolysin (toxin) producer which showed a clear zone of lysis on blood agar plates, 11(57.9%) isolates gave a positive result for protease production and 5(26.3%) isolates were gave a positive result for lipase production. Also isolates of Aeromonas hydrophila were tested for their ability to agglutinate human erythrocytes groups (O, A, B, AB). In which 10(52.6%) isolates showed agglutination with group (A) human erythrocytes, 9(47.4%) isolates showed agglutination with group (B) human erythrocytes, 9(47.4%) isolates showed agglutination with group (AB) human erythrocytes, 12(63.2%) isolates showed agglutination with group (O) human erythrocytes and 8 (42.1%) isolates showed agglutination with human erythrocytes groups (O, A, B, AB). The results of the ability to bind to Congo red revealed that 13(68.4%) isolates were bind to Congo red dye, while 6(31.6%) isolates were failed to bind to Congo red.

Effects of Plant Lectins on Human Erythrocyte Agglutination

AbstractPlant lectins are carbohydrate binding proteins or phytohaemagglutinins present in most plants, especially seeds and tubers, which include cereals, potatoes and beans. Lectins have great significance in the diet because of their involvement in gastrointestinal difficulties and erythrocyte agglutination. Blood agglutination activity against A, B, AB and O groups was shown after exposing blood to extracts obtained from 55% of tested plants, while in 45% of plants, agglutination was absent. The results of our study have shown that in humans, 40% of plant extracts exhibited activity against A, 40% of plant extracts exhibited activity against B, and 50% of plant extracts exhibited activity against AB and O groups in humans. The concentration of plant lectins depends on the part of the plant. Lectins from the seeds of certain plants cause the greatest percentage of erythrocyte agglutination, while the lowest agglutination was caused by plant bulbs and leaves. However, lectins derived from all plant species of the family Fabaceae agglutinated erythrocytes of all blood types to some extent.

Agglutination of human erythrocytes by the interaction of Zn2+ion with histidine-651 on the extracellular domain of band 3

Clustering of band 3, chloride/bicarbonate exchanger, has been reported in Zn2+-treated human erythrocytes. However, the agglutination of human erythrocytes is also induced by the interaction of Zn2+ion with histidine on band 3. Identification of histidine that interacts with Zn2+ion remains to be determined. The Zn2+-induced agglutination of human erythrocytes was unaffected by chymotrypsin cleavage of the small loop region containing His-547 in the extracellular domain of band 3. On the other hand, papain digestion of the large loop region containing His-651 in band 3 inhibited such Zn2+-induced agglutination. Moreover, Zn2+-induced erythrocyte agglutination was inhibited by the peptide (ARGWVIHPLG) containing His-651, but not by the peptide such as ARGWVIRPLG, which His-651 was substituted by arginine. Among 10 kinds of animal erythrocytes tested, interestingly, no agglutination by Zn2+ions was observed in cow cells only that the forth amino acid in the upstream from His-669 on the large loop of cow band 3 is aspartate (Asp-665) instead of glycine. As expected, the agglutination of human erythrocytes by Zn2+ ions was inhibited in the presence of aspartate. These data indicate that the interaction of Zn2+ ion with His-651 residue of band 3 plays an important role in the Zn2+-induced agglutination of human erythrocytes.

Adrenalin modulated time of start of human erythrocyte agglutination depending on agglutination inducer, sex and stage of reproductive process in the case of women

Adrenalin modulated time of start of human erythrocyte agglutination depending on agglutination inducer, sex and stage of reproductive process in the case of women

Purification, partial characterization and antimicrobial activity of Lectin from Chenopodium Quinoa seeds

A novel lectin was isolated from the seeds of Chenopodium quinoa. To achieve this end, the crude extract from the quinoa was submitted to two purification steps, Sephadex G50 and Mono Q. The hemagglutinating activity showed that this lectin agglutinates human erythrocytes. Its activity is inhibited by glucose and mannose, and remained stable under a wide range of pH levels and temperatures. The quinoa lectin was found to be a heterodimeric lectin of approximately 60 kDa, consisting of two subunits of approximately 25 kDa and 35 kDa. This lectin had its antimicrobial activity tested against several bacteria strains and effectively inhibited three strains. These strains were all Gram-negative, making this lectin a promising antimicrobial tool.

Quantifying the bioadhesive properties of surface-modified polyurethane-urea nanoparticles in the vascular network

Nanomedicine research is currently requiring new standard methods to quantify the biocompatibility and bioadhesivity of emerging biomaterials designed to be used in contact with blood or soft tissues. In this study, we used biotinylated polyurethane-urea nanoparticles as a model to examine the applicabitility of an adapted hemagglutination assay to quantify the bioadhesive potential of these nanoparticles to red blood cells and, in turn, to extrapolate this data to vascular endothelial cells. We demonstrated that biotinylated nanoparticles adsorb to human erythrocytes and preferentially gather in erythrocyte contact areas. Moreover, these nanoparticles promoted a higher percentage of pig and human erythrocyte agglutination than naked polyurethane-urea nanoparticles in a biotin concentration-dependent manner. Conversely, pegylated nanoparticles were used as a negative control of the technique thus showing decreasing hemagglutination values as compared to naked nanoparticles until a minimum threshold. Furthermore, hemagglutination assay demonstrated an excellent positive correlation with bioadhesion quantification in human endothelial cells and the endothelial layer of pig aorta thus validating the hemagglutination assay described here as a useful method for predicting nanoparticle bioadhesivity to vascular endothelium. Therefore, the methodology described here is a versatile and straightforward method that allows evaluating the bioadhesive features of surface-modified polyurethane-urea nanoparticles in contact with blood and the vascular network and appears as a powerful tool to better design any drug delivery systems or implantable devices for biomedical applications.

The mechanism of change in the rate of agglutination of human erythrocytes under the influence of adrenaline

In the study of red blood cells of 80 men found that adrenaline (10(-10) - 10(-6) g/mL) and phenylephrine (10-(10) - 10(-6) g/mL) dose-dependently increase the speed of agglutination of red blood cells, according to the decrease in agglutination of the start time and ginipral (10(-10) - 10(-7) g/mL), on the contrary, decreases it. The effect of adrenaline and phenylephrine is blocked by nicergoline (10(-6) g/mL), increased obzidan (10(-6) g/mL) and does not change under the action ofyohimbine (10(-6) g/mL) and atenolol (10(-6) g/mL). These data indicate that the speed of agglutination increases with activation alpha1-adrenergic receptor (AR) and decreases in the activation of beta2-AR, while the activation of alpha2- and beta1-AR does not affect it. Trifluoperazine (10(-6) g/mL) as the calmodulin antagonist, barium chloride (10(-6) g/mL) as a blocked of Ca(2+)-dependent K(+)-channels and indomethacine (10(-6) g/mL) as an inhibitor of cyclooxygenase and phospholipase A2 inhibit the ability of adrenaline to increases the speed of agglutination of red blood cells. This suggests that the effect of adrenaline caused an increase in erythrocyte entry of Ca2+, activation of calmodulin, cyclooxygenase, phospholipase A2 and the release of K+ from red blood cell through the Ca(2+)-dependent K+ channels, which is regarded as a manifestation of eryptosis. Indirectly, this means that more efficient activation of alpha1-AR and beta2-AR, respectively, increases or, conversely, decreases the rate of eryptosis.

Mechanism of increasing the rate of agglutination of human erythrocytes under the influence of adrenaline and its relation to eryptosis

Mechanism of increasing the rate of agglutination of human erythrocytes under the influence of adrenaline and its relation to eryptosis

ОЧИЩЕННЯ І ХАРАКТЕРИСТИКА НОВОГО МАНОЗОСПЕЦИФІЧНОГО ЛЕКТИНУ ІЗ ЦИБУЛИН ГІАЦИНТИКА ГОСТРОЛОПАТЕВОГО (Hyacinthella аcutiloba K. Perss.)

A new lectin with 6.5 mg/kg yield was purified from fresh bulb Hyacinthella аcutiloba K. Perss. by combination of affinity chromathography on yeast mannan and by ion exchange chromatography on DEAE-toyopearl. The best lectin inhibitor among tested monoand disaccharides was D-turanose (Glcp α1-3 Fruf). Very powerful inhibitor of lectin activity was yeast mannan. The lectin revealed weak affinity to α-methyl-Dmannoside, D-fructose and 2-acetamido-D-galactopyranoside. The lectin interacted also with pig liver glycogen, starch and mannose-containing glycoproteins (ovoalbumin, ovomucoid and calf thyroglobulin), but don’t interacted with horsera dish peroxidase and calf intestine alkaline phosphatase. According to electrophoresis, in 20% SDS-PAAG containing SDS-Na the lectin consists with subunits of molecular weight 12 kDa. Molecular weight of the lectin is 48 kDa according to gel-chromatography on Toyopearl HW-55. The lectin agglutinated best of all rabbit erythrocytes and worse agglutinated guinea-pig, very weak — rat erythrocytes and did not agglutinate human and goat erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin did not lose hemaglutinating activity and endured heating to +65 °C during one hour.

Combined Fiber Modifications Both to Target αvβ6and Detarget the Coxsackievirus–Adenovirus Receptor Improve Virus Toxicity ProfilesIn Vivobut Fail to Improve Antitumoral Efficacy Relative to Adenovirus Serotype 5

Achieving high-efficiency tumor targeting after systemic delivery is a considerable challenge facing oncolytic gene therapists. Efficient retargeting should be combined with efforts to improve in vivo safety, reduce hepatotoxicity, minimize off-target interactions, and improve antitumoral potency and efficacy. We previously described the successful retargeting of adenovirus serotype 5 (Ad5) to α(v)β(6), an integrin that is highly overexpressed in numerous human carcinomas. In this study, we have further modified this construct by introducing mutations that ablate coxsackievirus-adenovirus receptor (CAR) binding and putative interactions with factor IX (FIX)/C4b-binding protein (C4BP). We have found that the resulting vector, Ad5-477dlTAYT(A20), displays a desirable in vivo safety profile. This vector does not agglutinate human erythrocytes, fails to cause thrombocytopenia after intravenous delivery, has limited induction of proinflammatory cytokines, and results in low-level toxicity (aspartate aminotransferase/alanine aminotransferase) when compared with Ad5-EGFP(WT). Furthermore, it has reduced accumulation in Kupffer cells (1 hr) and limited hepatocyte transduction at later time points (24 and 96 hr). The parental vector, Ad5-EGFP(A20), also displayed many of these desirable properties. As a result of the improved safety profile of both A20-modified vectors, we escalated the dose from 2×10(10) to 4×10(10) viral particles in an antitumoral efficacy study. We observed improvements in reducing percent tumor growth at early time points (96 hr) when compared with Ad5-EGFP(WT), although increasing the dose did not affect the therapeutic outcome beneficially. On completion of the experiment, we detected increased E1A staining in the tumors of all A20-treated groups and we determined that E1A expression was localized largely within α(v)β(6)(+) tumor cells. However, in spite of apparently efficient tumor transduction, this did not result in enhanced antitumoral efficacy as the virus failed to disseminate effectively throughout the tumor mass, presumably due to physical intratumoral restrictions. This highlights a remaining challenge that needs to be overcome before such vectors can be developed for future cancer gene therapy applications.

Isolation and Characterization of a Sex-Specific Lectin in a Marine Red Alga, Aglaothamnion oosumiense Itono

In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaothamnion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairlike trichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin responsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-D-galactosamine-specific protein was isolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and did not agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and degenerate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular mass of 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plants have this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to 15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia, suggesting its involvement in gamete binding.

Blood Group Typing Based on Digital Imaging of Sedimentation of Erythrocytes and Their Agglutinates

The efficiency of application of the digital photographic method to detection of human erythrocyte agglutination in vitro was improved using an ultrasonic standing wave. Analysis of the digital photograph brightness distribution along the depth of a cuvette filled with blood − agglutinating serum solution revealed a zone of mathematical processing in which the resolving power of the method is maximal. In the zone chosen for the analysis of the sample, the effect of sample preparation and incubation time on the resolving power was studied. The results of this work can be used to develop an optical method and a device supplied with ultrasonic intensification of erythrocyte agglutination for human blood group typing.

The contrasting properties of 2 xenogenic erythrocyte-reactive natural antibodies in commercial ducks

Sera obtained from commercial drakes on days 14 and 38 of age were tested by microtiter for the capacity to agglutinate and lyse rabbit (Rb) and human (HuO) erythrocytes. Three agglutination types, differing by strength, were recognized: HA1 (strong), HA2 (weak), and HA45 (very weak).Two degrees of lysis: L100 (complete) and L50 (partial), measured complement activity. Day 14 sera agglutinated Rb (average log2 titers: HA1 = 1.5, HA2 = 4.1) and lysed Rb (average log2 titers: L100 = 2, L50 = 2.5) but only 8/115 (˜9%) agglutinated HuO (HA45 = 0.4) while most (>80%) lysed HuO (average log2 titers: L100 = 1.3, L50 = 1.8). Both Rb and HuO agglutination and lysis titers were higher by d 38. At that age, all ducks lysed HuO and 50% of ducks acquired a capacity to agglutinate these cells with more strength. However, the quality of HuO agglutination could not be differentiated into HA1 or HA2 types. Average d 38 log2 titers of all measures were Rb: HA1 = 4, HA2 = 8.4, L100 = 3, and L50 = 4; and HuO: HA = 2.8, L100 = 3.9, and L50 = 1.2. The quality of the Rb agglutination suggested the participation of both IgM and standard-sized IgY antibodies. Lysis of Rb may occur by both classic and alternate complement pathways. The HuO lysis appears to depend primarily on the alternate complement pathway. It is suggested that multiple measurement systems such as these offer a practical way of obtaining information on immunity in experiments where the chief interest lies elsewhere.

Detection of chimpanzee polyomavirus-specific antibodies in captive and wild-caught chimpanzees using yeast-expressed virus-like particles

Chimpanzee polyomavirus (ChPyV) was originally detected in the faeces of a captive chimpanzee by random screening using broad-spectrum PCR. Its pathogenicity and the distribution among chimpanzees are unknown so far. Here, the major capsid protein VP1 of ChPyV was expressed in yeast cells. Virus-like particles (VLPs) with a diameter of approximately 45 nm were demonstrated although the efficiency of VLP formation was low as compared to monkey polyomavirus SV40-VLPs. The ChPyV-VLP preparation did not agglutinate human erythrocytes. Low cross-reactions between ChPyV- and SV40-VLP-specific sera were detected by immunoblotting, but not by ELISA. Testing of 163 sera derived from captive and wild-caught healthy chimpanzees using an ELISA based on the ChPyV-VLPs resulted in 11.7% positive results, ranging from 0% to 56% in different groups. The VLPs may be used in future to assess the distribution of ChPyV infections among other animal species and humans.

Human blood group typing based on digital photographs of RBC agglutination process

A method for the monitoring of the human erythrocyte agglutination reaction in vitro, which is the basis for determining the human blood type (group), is proposed. The method is based on a statistical analysis of digital photographs of the agglutination process. It is experimentally shown that this analysis of photographs makes it possible to determine the probability that the agglutination reaction of erythrocytes of the studied specimen of blood with corresponding isohemagglutinating sera does occur. To increase the rate of the agglutination reaction of erythrocytes and to improve the sensitivity of the method of monitoring, the bioobject under study is subjected to the action of ultrasonic waves, as was previously proposed by the authors, and the result of the erythrocyte agglutination reaction is read optically. It is shown that, in principle, the method of statistical processing of digital photographs can be used to develop devices for automatic human blood typing in the AB0 and Rh systems.

The importance of rhesus status

The history of haemolytic disease of the newborn, caused by incompatibility between the fetus and mother for the rhesus D (RhD) antigen, has been called one of the most brilliant successes achieved in medicine.1 Preventive treatment is so effective that it has decreased the number of intrauterine fetal deaths by more than 100 fold and the incidence of maternal alloimmunisation by more than 50 fold.2 The first dated case report of haemolytic disease of the newborn was in a set of twins born in the 1600s.3 However, it was not until 1940 that the link with the RhD antigen was made by Landsteiner and Weiner. They discovered an antibody that led to the agglutination of human erythrocytes after experiments in which rabbits were immunised with rhesus monkey erythrocytes.4 In 1969, a campaign of anti-D immunoglobulin prophylaxis was introduced. Deaths attributed to RhD alloimmunisation were reduced from 46 in 100 000 births to 1.6 in 100 000 births during the first 20 years of the campaign. However, haemolytic disease of the newborn and neonatal death continued to occur, and still do—owing in part to covert sensitising events during pregnancy, and in part to a lack of knowledge and compliance with guidelines.5 Erythrocytes carry many antigens on their surface. The most important are those belonging to the ABO system and to the rhesus system. The rhesus system comprises antigens c, C, D, e, and E. Of these, the D antigen is the most important. It is inherited on one gene, located on chromosome 1. It has two alleles: the dominant RhD positive, where the individual expresses the D antigen, and the recessive RhD negative, where there is no expression.6 The incidence of the rhesus D antigen varies between different ethnic groups.6 It is estimated that the prevalence …