Prevalence of, and risk factors for, carriage of carbapenem-resistant Enterobacteriaceae among hospitalized patients in Japan.

BACKGROUND: The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has been reported to be lower in Japan than in many other countries. However, extensive surveillance for CRE carriage has not been performed in Japan. AIM: To investigate the prevalence of CRE carriage in Japan among convalescent patients considered to be at high risk of being CRE carriers using an improved selective culture medium. METHODS: A cross-sectional survey was conducted in 22 acute care hospitals (ACHs) and 21 long-term care hospitals (LTCHs) in northern Osaka from December 2015 to January 2016. Patients who used incontinence aids, an enteral feeding tube or a urinary catheter were enrolled. Faecal specimens were examined using the newly developed M-ECC for imipenemase (IMP)-producing CRE, which is the most prevalent form of CRE in Japan. The positive isolates were analysed by polymerase chain reaction and sequencing. Risk factors associated with carriage were analysed by logistic regression. FINDINGS: Among 1507 patients, 184 (12.2%) carried CRE. The percentage of positive patients was significantly higher in LTCHs (14.9%) than in ACHs (3.6%) (P<0.001). Risk factors for CRE carriage were longer hospital stay [odds ratio (OR) 2.59; 95% confidence interval (CI) 1.87-3.60], enteral feeding (OR 3.03, 95% CI 2.08-4.42) and antibiotic exposure (OR 2.00, 95% CI 1.40-2.87). Among the 233 CRE isolates identified, 223 were IMP producers; the remaining isolates did not produce carbapenemase. CONCLUSIONS: This is the first Japanese report to demonstrate the significant spread of CRE in both ACHs and LTCHs using an improved selective medium. A coordinated regional approach may help to prevent further spread.

Development of an improved RT-LAMP assay for detection of currently circulating rubella viruses.

Rubella virus is the causative agent of rubella. The symptoms are usually mild, and characterized by a maculopapular rash and fever. However, rubella infection in pregnant women sometimes can result in the birth of infants with congenital rubella syndrome (CRS). Global efforts have been made to reduce and eliminate CRS. Although a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of rubella virus has been reported, the primers contained several mismatched nucleotides with the genomes of currently circulating rubella virus strains. In the present study, a new RT-LAMP assay was established. The detection limit of this assay was 100-1000PFU/reaction of viruses for all rubella genotypes, except for genotype 2C, which is not commonly found in the current era. Therefore, the new RT-LAMP assay can successfully detect all current rubella virus genotypes, and does not require sophisticated devices like TaqMan real-time PCR systems. This assay should be a useful assay for laboratory diagnosis of rubella and CRS.

The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells.

Collectins are a family of C-type lectins that have collagen-like sequences and carbohydrate recognition domains (CRD). They are involved in host defense through their ability to bind to carbohydrate antigens of microorganisms. The scavenger receptors type A and MARCO are classical type scavenger receptors that have internal collagen-like domains. Here we describe a new scavenger receptor that is a membrane-type collectin from placenta (collectin placenta 1 (CL-P1)), which has a typical collectin collagen-like domain and a CRD. The cDNA has an insert of about 2.2 kilobases coding for a protein containing 742 amino acid residues. The deduced amino acid sequence shows that CL-P1 is a type II membrane protein, has a coiled-coil region, a collagen-like domain, and a CRD. It resembles type A scavenger receptors because the scavenger receptor cysteine-rich domain is replaced by a CRD. Northern analyses, reverse transcription-polymerase chain reaction, and immunohistochemistry show that CL-P1 is expressed in vascular endothelial cells but not in macrophages. By immunoblotting and flow cytometry CL-P1 appears to be a membrane glycoprotein of about 140 kDa in human umbilical vein or arterial endothelial cells, placental membrane extracts, and CL-P1 transfected Chinese hamster ovary cells. We found that CL-P1 can bind and phagocytose not only bacteria (Escherichia coli and Staphylococcus aureus) but also yeast (Saccharomyces cerevisiae). Furthermore, it reacts with oxidized low density lipoprotein (OxLDL) but not with acetylated LDL (AcLDL). These binding activities are inhibited by polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate) and OxLDL but not by polycationic ligands (polyadenylic acid or polycytidylic acid), LDL, or AcLDL. These results indicate that CL-P1 might play important roles in host defenses that are different from those of soluble collectins in innate immunity.

A clinical study of urine basic fetoprotein and urine polyamine as tumor markers in epithelial cancer of the urinary tract.

BACKGROUND: The reliability of a new screening test for epithelial cancer of the urinary tract was evaluated and the results were compared with those obtained employing urinary cytology in routine use. METHODS: The subjects consisted of 187 cases selected randomly from among the patients who attended Toho University Ohashi Hospital during a period of 1 year from January 1998. The values for urine basic fetoprotein (BFP) and polyamine and urinary cytology were examined. RESULTS: Urine BFP is considered useful for screening and monitoring urinary tract epithelial cancer as is urinary cytology. Urine BFP showed a statistically significant difference (P < 0.05) for G1 compared with urinary cytology and a significantly high level compared with urinary cytology as to the positive rate in the low stage group (P < 0.05). The positive rate of urine BFP was high in patients with urinary tract infection. CONCLUSION: Determining urine BFP, when combined with urinary cytology, is considered very useful for diagnosing patients with urinary tract epithelial cancer. This study suggests the possibility of urine BFP being superior to urinary cytology for screening early cancer and also as an indicator for observations on the clinical course.

Penile schwannoma.

Schwannoma of the penis is extremely rare. A 65-year-old man presented with a subcutaneous tumor of penile shaft without any other symptoms. Histopathologic examination of the excised tumor revealed benign schwannoma. No recurrence has been observed over the 6 months since the surgery.

Single-tube multiplex PCR for rapid and sensitive diagnosis of subgenus B and other subgenera adenoviruses in clinical samples.

We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non-nested polymerase chain reaction (PCR). Sequences of the conserved hexon-coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single-tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non-Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty-seven out of 71 (94%) Sub B Ad culture-positive samples, and 15 out of 19 (79%) Sub C or E-positive samples amplified products of the expected size. Two of 20 (10%) culture-negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non-Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.

p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting directly with the N-terminal A/B domains.

Estrogen receptor (ER) alpha and beta mediate estrogen actions in target cells through transcriptional control of target gene expression. For 17beta-estradiol-induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required. Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown. To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/CREB binding protein, steroid receptor coactivator-1/transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein- 205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function. We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ERalpha and human ERbeta. Direct interactions of p300 with the A/B domains of ERalpha and ERbeta were observed in an in vitro glutathione S-transferase pull-down assay in accordance with the interactions in yeast and mammalian two-hybrid assays. Furthermore, mutations in the p300 binding sites (56-72 amino acids in ERalpha and 62-72 amino acids in ERbeta) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ERalpha and ERbeta. Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ERalpha and ERbeta.

Human mannan-binding lectin inhibits the infection of influenza A virus without complement.

Mannan-binding lectin (MBL) is a C-type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagen-like sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro-organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV-A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti-human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non-damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.

Molecular cloning of a novel human collectin from liver (CL-L1).

Collectins are a C-lectin family with collagen-like sequences and carbohydrate recognition domains. These proteins can bind to carbohydrate antigens of microorganisms and inhibit their infection by direct neutralization and agglutination, the activation of complement through the lectin pathway, and opsonization by collectin receptors. Here we report the cloning of a cDNA encoding human collectin from liver (CL-L1 (collectin liver 1)) that has typical collectin structural characteristics, consisting of an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain. The cDNA has an insert of 831 base pairs coding for a protein of 277 amino acid residues. The deduced amino acid sequence shows that this collectin has a unique repeat of four lysine residues in its C-terminal area. Northern blot, Western blot, and reverse transcription-polymerase chain reaction analyses showed that CL-L1 is present mainly in liver as a cytosolic protein and at low levels in placenta. More sensitive analyses by reverse transcription-polymerase chain reactions showed that most tissues (except skeletal muscle) have CL-L1 mRNA. Zoo-blot analysis indicated that CL-L1 is limited to mammals and birds. A chromosomal localization study indicated that the CL-L1 gene localizes to chromosome 8q23-q24.1, different from chromosome 10 of other human collectin genes. Expression studies of fusion proteins lacking the collagen and N-terminal domains produced in Escherichia coli affirmed that CL-L1 binds mannose weakly. CL-L1 and recombinant CL-L1 fusion proteins do not bind to mannan columns. Analysis of the phylogenetic tree of CL-L1 and other collectins indicated that CL-L1 belongs to a fourth subfamily of collectins following the mannan-binding protein, surfactant protein A, and surfactant protein D subfamilies including bovine conglutinin and collectin-43 (CL-43). These findings indicate that CL-L1 may be involved in different biological functions.

Rapid detection and identification of two lineages of influenza B strains with monoclonal antibodies.

Monoclonal antibodies (Mabs) against influenza B virus were obtained by immunizing mice with B/Nagasaki/1/87, one of the strains of the B/Victoria group. Immunoprecipitation analysis revealed that individual Mabs precipitated the nucleoprotein (NP), the matrix protein (M) or the hemagglutinin protein (HA). By using these Mabs by the peroxidase-antiperoxidase (PAP) staining method, a rapid detection and identification method for influenza B virus was established. Monolayers of Madin-Darby canine kidney cells in microplates were infected with each-strain and incubated for about 24 h, and then were subjected to the PAP staining method using the Mabs as the first antibody. Influenza B virus strains are classified into two major phylogenetic trees, the B/Victoria group and the B/Yamagata group. When anti-NP and anti-M antibodies were used in the PAP staining method, all 13 influenza B virus strains isolated from clinical specimens between 1940 and 1994 were detected regardless of the antigenic drift of the influenza virus. On the other hand, several anti-HA Mabs which reacted specifically with the strains of the B/Victoria group, did not react with any strain of the B/Yamagata group. In the 1996/97 influenza season in Osaka Prefecture in Japan, two antigenically distinct groups of influenza B virus strains were isolated. They belonged to different phylogenetic trees and were clearly distinguishable by the PAP staining method with anti-HA Mabs.

High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells.

We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 microg/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MBL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans.

Purification and characterization of a trehalose synthase from the basidiomycete grifola frondosa

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from D-glucose and alpha-D-glucose 1-phosphate (alpha-D-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to D-glucose and alpha-D-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35 degreesC and 35 to 37.5 degreesC, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only alpha-D-glucose 1-P served as a donor substrate with D-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the alpha-D-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with D-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5 degreesC, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO4 to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.

Characterization of truncated human mannan-binding protein (MBP) expressed in Escherichia coli.

Mannan-binding protein (MBP) is a calcium-dependent mammalian serum lectin important in first-line host defense. MBP belongs to the collectin family, which is characterized by an NH2-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain (CRD). We have expressed a recombinant human MBP, consisting of the short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain, and the CRD, in Escherichia coli. The truncated MBP was capable of forming trimers by association of the neck domain and could bind sugar with a specificity similar to that of the native form. Results of hemagglutination inhibition (HI) assay of influenza A virus showed that the truncated MBP inhibited hemagglutination less strongly, although the native MBP induced the HI phenomenon. These results suggest that an oligomeric structure is an advantage for MBP to have full biological activity against influenza A virus.

Molecular and biological characterization of rabbit mannan-binding protein.

Mannan-binding protein (MBP) is a member of the collectin family of protein. There are two types of MBP, MBP-A and MBP-C, which were found in rodent (rats and mice), rhesus monkey, and cynomolgus monkey, while chimpanzee and human have only one MBP. It was considered that the loss of one MBP gene occurred during hominoid evolution. In this article two rabbit MBP, a liver and serum MBP, were characterized biologically and genetically. Analyses by SDS-PAGE under reduced condition and their amino acid sequences of both MBPs showed that they have a same molecular weight of 32 kDa and their amino acid sequences were identical. A serum MBP has a higher ability to activate complement than does a liver MBP; however, a liver MBP inhibits hemagglutination by influenza virus as strongly as a serum MBP does. cDNA clones encoding the rabbit MBP were isolated from a rabbit cDNA liver library using whole cDNA of mouse MBP-C as a probe. The cDNA carried an insert of 744 bp coding for a protein of 247 acid residues with a signal peptide of 22 residues. The deduced amino acid sequence of the cDNA was identical to that of amino acid sequences of the 32 kDa proteins determined here. Northern blot analysis showed that mRNA transcripts of about 0. 9 and 3.0 kb were expressed only in the liver. The analysis of the phylogenetic tree of rabbit and bovine MBPs and other collectins indicates that the loss of MBP gene occurred not only during hominoid evolution but also at some points after the separation of birds and mammals.

Healing of tibial fractures is not impaired after acute hindlimb ischemia in rats.

The influence of transient circulatory arrest on the healing of closed tibial fractures was investigated in rats by the use of a hindlimb tourniquet technique. Twenty-four animals were randomly divided into three groups. In all animals, the left lower leg was fractured and fixed with an intramedullary nail system. In the ischemic group, complete acute transient ischemia for 4.5 h and neurapraxia of the sciatic and femoral nerves were induced prior to fracture. In the neurapraxia group, the sciatic and femoral nerves were crushed with forceps before fracture. In the control group, no other intervention than fracture was made. The rats of the control group ambulated normally 3-4 days after the operation. The animals of the ischemic and neurapraxia groups resumed normal weight-bearing after about 3 weeks. After 6 weeks, all animals were killed, and mechanical strength and bone mineral turnover of the healing tibia as well as blood flow of the bone and musculature were evaluated. The weight of the tibia and the corresponding anterior tibial muscle in the ischemic and neurapraxia animals were reduced compared with the control rats. Bone mineral turnover was found to be lower in the ischemic group. There were no differences between the groups in mechanical strength nor in blood circulation of bone and muscle. In conclusion, complete, acute hindlimb ischemia for 4.5 h in rats did not cause delayed healing of closed tibial fractures.

Training in pregnant women: effects on fetal development and birth.

OBJECTIVE: The effects of high- and medium-intensity exercise on the fetus and on the onset and length of labor, birth weight, and Apgar score were studied in healthy athletes who performed a high level of exercise before conception. STUDY DESIGN: Forty-two women were recruited to the study by newspaper ads and through acquaintances. They elected to follow either a high- or a medium-intensity exercise program throughout pregnancy until 6 weeks after delivery. Documentation of their intensity of activity before conception (retrospectively), during pregnancy, and after delivery was obtained. RESULTS: There were no differences between the high- and medium-intensity exercise group in duration of labor, birth weight, or 1- and 5-minute Apgar scores. The higher level of exercise correlated with a significantly greater maternal weight gain during pregnancy and significantly earlier onset of labor for those women who gave birth to girls but not for those who gave birth to boys. CONCLUSION: Our results indicate that healthy and well-conditioned women may take part in exercise during pregnancy without compromising fetal growth and development as judged by birth weight or complicating the course of pregnancy or labor.

Detection of HSV mRNA using reverse transcription-polymerase chain reaction for diagnosis in murine herpetic keratitis model.

Reverse transcription polymerase chain reaction (RT-PCR) was applied in the detection of herpes simplex virus-1 (HSV-1) mRNA from tear film and corneal epithelium in a murine herpetic keratitis model. The diagnostic value of this new technique for acute herpetic keratitis was evaluated in comparison with direct PCR for genomic DNA and viral culture. On day 2 postinfection (PI) of HSV, all mice showed dendritic keratitis, and PCR, RT-PCR, and viral culture were positive in all samples. On day 8 PI, no dendritic keratitis was observed in any mouse, PCR was positive in all samples, while RT-PCR was positive in only 5 of 12 samples and viral culture in only 2 of 12. The sensitivity of RT-PCR was lower than that of PCR, and approximately the same as viral culture; however, the findings of RT-PCR more closely concurred with clinical observations than the findings of PCR. These results show the potential of RT-PCR for rapid, specific diagnosis of acute herpetic keratitis.

Application of subtype-specific monoclonal antibodies for rapid detection and identification of influenza A and B viruses.

We established a rapid method for the identification of influenza A and B virus strains: the peroxidase-antiperoxidase (PAP) staining method with two subtype-specific murine monoclonal antibodies, C179 (H1 and H2 specific) and F49 (H3 specific), and an anti-influenza B virus rabbit polyclonal serum. The types and subtypes of 160 strains were examined, and 158 strains were identified to be the same by the hemagglutination-inhibition (HI) test and the PAP method. In contrast to the results by the HI test, two strains were revealed to be a mixture of two subtypes (H1 and H3) by the PAP method, which was confirmed by plaque cloning. We further analyzed clinical specimens by the PAP method by directly inoculating specimens into Madin-Darby canine kidney cells in microplates. After 40 h of incubation, the types and subtypes of viruses in 52 of 152 specimens were clearly identified. Since the reactivities of the two monoclonal antibodies are not influenced by the antigenic drift of influenza virus, the newly developed method should be applicable not only for rapid diagnosis but also for the epidemiological study of influenza.

100-TW sub-20-fs Ti:sapphire laser system operating at a 10-Hz repetition rate.

We developed a compact three-stage Ti:sapphire amplifier laser system that produced peak power in excess of 100 TW for a pulse duration of less than 19 fs and an average power of 19 W at a 10-Hz repetition rate. A final 40-mm-diameter Ti:sapphire amplifier is pumped by a Nd:YAG master-oscillator-power-amplifier system that produces ~7-J output of 532-nm radiation. The spatial beam quality is approximately 2 times diffraction limited for the full amplified compressed output pulse. With f/3 optics, this system should therefore be capable of producing a focused intensity of ~3x10(20) W/cm(2) .

Characterization of recombinant bovine conglutinin expressed in a mammalian cell.

We describe here the successful expression of recombinant bovine conglutinin in CHO cells as well as its physical and biological characteristics. Geneticin-resistant transformants harboring bovine conglutinin cDNA in the expression vector pNOW/CMV-A were screened by Western blot analysis for secretion into media of recombinant conglutinin. A four-day amplification of the transgene with increasing concentrations of methotrexate resulted in a dose-dependent increase in the production of recombinant conglutinin to a final concentration of 18.6 microg/ml of media. Recombinant conglutinin purified from this media by affinity column chromatography on mannan-agarose had a migration pattern similar to that of native conglutinin on polyacrylamide gel electrophoresis under reducing, nonreducing, and native conditions. The recombinant conglutinin exhibited sugar binding, conglutination, hemagglutination inhibition, and neutralization of influenza A virus, activities engaged in by the native conglutinin. This is the first report describing a high level of expression of a serum cruciform collectin with the full range of biological activity.