Transcriptome Analysis of Gelatin Seed Treatment as a Biostimulant of Cucumber Plant Growth.

The beneficial effects of gelatin capsule seed treatment on enhanced plant growth and tolerance to abiotic stress have been reported in a number of crops, but the molecular mechanisms underlying such effects are poorly understood. Using mRNA sequencing based approach, transcriptomes of one- and two-week-old cucumber plants from gelatin capsule treated and nontreated seeds were characterized. The gelatin treated plants had greater total leaf area, fresh weight, frozen weight, and nitrogen content. Pairwise comparisons of the RNA-seq data identified 620 differentially expressed genes between treated and control two-week-old plants, consistent with the timing when the growth related measurements also showed the largest differences. Using weighted gene coexpression network analysis, significant coexpression gene network module of 208 of the 620 differentially expressed genes was identified, which included 16 hub genes in the blue module, a NAC transcription factor, a MYB transcription factor, an amino acid transporter, an ammonium transporter, a xenobiotic detoxifier-glutathione S-transferase, and others. Based on the putative functions of these genes, the identification of the significant WGCNA module and the hub genes provided important insights into the molecular mechanisms of gelatin seed treatment as a biostimulant to enhance plant growth.

Relationship of the dietary phytochemical index to weight gain, oxidative stress and inflammation in overweight young adults.

BACKGROUND: Phytochemicals are bioactive nutrients that help reduce disease risk. A high intake of these compounds is important for optimal health and prevention of disease, but quantification of these nutrients in vivo is costly and time consuming. The present examined whether an alternative, simple 'phytochemical index' (PI) ratio calculation (PI = the ratio of the energy from high-nutrient phytochemical-rich foods to overall daily energy consumed [kJ phytochemical rich foods/total kJ consumed]) was related to several precursors of future disease: annual weight gain, adiposity, oxidative stress and inflammation. METHODS: This was a cross-sectional, quantitative, descriptive study (n = 54, age range 18-30 years). Participants were stratified into normal weight and overweight groups. Three-day dietary records were analysed for food items, food groups, energy and the PI score at repeated time points. Blood plasma samples were analysed by colorimetric or an enzyme-linked immunoabsorbent assay for cholesterol subfractions, glycated haemoglobin, total antioxidant status, lipid hydroperoxides, cytokines (interleukins-1beta and -6) and C-reactive protein). RESULTS: PI values were higher in the overweight-obese group. Correlation values between the PI score and body mass index, waist circumference, waist-to-hip ratio and plasma oxidative stress were significant. The PI score did not correlate with any cytokine levels. The PI score was a significant contributor to yearly weight gain. CONCLUSIONS: The PI is inversely related to adiposity and oxidative stress in healthy young adults, and is responsive to body weight changes. This simple, easy to administer index might be useful as a dietary target for appropriate proportion consumption of nutrient-rich foods in weight reduction or management programmes.

Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line.

NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.

By 1023/SK&F 96022: biochemistry of a novel (H+ + K+)-ATPase inhibitor.

The mechanism by which the substituted benzimidazole sulphoxide BY 1023/SK&F 96022 inhibited the (H+ + K+)-ATPase, the enzyme responsible for hydrogen ion secretion in the stomach, was studied in a variety of in vitro preparations. In gastric preparations that were capable of active hydrogen ion transport with consequent lumenal acidification, BY 1023/SK&F 96022 inhibited with high potency and in a time-dependent manner consistent with the acid-induced conversion of the parent benzimidazole sulphoxide to a covalent inhibitor (cyclic sulphenamide). The following IC50 values were obtained for the inhibition of aminopyrine accumulation: intact gastric glands stimulated with 1 mM dibutyryl cAMP, 1.0 microM; permeabilized gastric glands stimulated with 5 mM ATP, 0.42 microM; intact gastric vesicles stimulated with 150 mM KCl, 9 microM valinomycin and 2 mM MgATP, 3.5 microM. In a preparation that could not generate pH gradients, lyophilized gastric vesicles at pH 7.4, BY 1023/SK&F 96022 inhibited K(+)-stimulated ATPase activity with relatively low potency, 70 microM, indicating its good chemical stability at neutral pH. As assessed by ATPase inhibition, this stability was three times greater than that of omeprazole. Inhibition by BY 1023/SK&F 96022 was not reversed by dilution in either permeabilized gastric glands or intact gastric vesicles. Inhibition could, however, be completely reversed by subsequent incubation with 20 mM beta-mercaptoethanol (intact gastric glands) or 100 mM dithiothreitol (intact gastric vesicles) suggesting a disulphide link between inhibitor and enzyme. The concentration of glutathione needed to protect against inhibition by BY 1023/SK&F 96022 was 10,000 times higher in intact, compared with lyophilized, gastric vesicles indicating an interaction with the lumenal (extra-cellular) face of the (H+ + K+)-ATPase. BY 1023/SK&F 96022 and omeprazole were also found to inhibit acidification in purified kidney lysosomes with IC50 values of 194 and 75 microM, respectively. Protection by 10 microM glutathione suggested that this did not result from intralysosomal activation of these inhibitors. Thus, BY 1023/SK&F 96022 has the combined properties of good chemical stability at neutral pH and effective conversion to the cyclic sulphenamide at acidic pH. In this way the activation to the cyclic sulphenamide may be optimally restricted to the parietal cell canaliculus.

Diagnosis of Legionella pneumophila infections by means of formolised yolk sac antigens.

Formolised yolk sac antigens of Legionella pneumophila serogroups 1-6 were used to test 1792 serum specimens from 1431 patients with respiratory illness of serological evidence of Legionnaires' disease (LD). Thirty-five patients showed titres against the serogroup 1 antigen diagnostic for LD. Only two further cases were considered to have non-serogroup I infections (both serogroup 4) indicating that such infections are rare. Titres of greater than 1/16 against the serogroup 1 antigen occur in only 3% of subjects without LD and thus the demonstration of such a titre in patients with pneumonia during the early phase of illness can alert the clinician to the likelihood of LD. The supply of serogroup 1 antigen from the Division of Microbiological Reagents and Quality Control to routine diagnostic laboratories will be continued and monovalent serogroup 2-6 antigens will continue to be made available to reference laboratories.

Comparison of the Vi indirect fluorescent antibody test with the Widal agglutination method in the serodiagnosis of typhoid fever.

The indirect fluorescent antibody test using a whole Salmonella typhi Vi suspension as the antigen has been evaluated for the diagnosis of typhoid fever. Results using sera from 140 patients with S typhi infections proved on culture show the test to have good sensitivity. The test appears to be highly specific, although it has not yet been investigated with respect to typhoid vaccination or in the context of infections due to salmonellas other than S typhi.

A rapid microagglutination test for the diagnosis of Legionella pneumophila (serogroup 1) infection.

A rapid microagglutination test has been developed which can be performed in 30 minutes. Ninety-seven percent of 96 patients diagnosed as having Legionella pneumophila (serogroup 1) infection by indirect immunofluorescence were also detected by the rapid microagglutination test.

Anti-gamma haemolysin as a diagnostic test in staphylococcal osteomyelitis.

The anti-alpha-haemolysin test is widely used in the diagnosis of staphylococcal osteomyelitis. An additional test would be welcomed because raised antibodies to this antigen are not seen in all proven cases. This communication reports that anti-gamma-lysin was present in the serum of most of the 19 patients with proven staphylococcal osteomyelitis studied here. In two cases followed up after chemotherapy was initiated both tests indicated the satisfactory progress of the patient.

Evaluation of sensitivity of two serological tests for diagnosing pneumonia caused by Legionella pneumophila serogroup 1.

The sensitivity of an indirect immunofluorescence antibody test (IFAT) and a rapid microagglutination test (RMAT) for the diagnosis of Legionnaires' disease caused by Legionella pneumophila serogroup 1 was evaluated using serum specimens from 119 patients with bacteriologically confirmed infections. The sensitivity of both assays was found to be about 80%. In addition, antibody titres suggestive of L pneumophila infection were found in 40% of patients in the first week after admission to hospital. These data show that both assays can be used with confidence in the early diagnosis of Legionnaires' disease.

Infection of total hip prostheses by Peptococcus magnus: an immunofluorescence and ELISA study of two cases.

In two cases of infected total hip replacements, Peptococcus magnus was isolated in pure culture from the implant when it was removed. Fluorescent antibody and ELISA studies have shown that both patients developed an antibody response to this anaerobic coccus soon after the replacement operation. These results suggest that the organism is a true infective agent, which was probably responsible for the failure of the arthroplasty operation.

Serological tests in the differentiation of staphylococcal and tuberculous bone disease.

The haemagglutination test for antileucocidin is frequently positive in cases of bone tuberculosis in the absence of obvious staphylococcal infection. This test is therefore of little practical use in the differentiation of staphylococcal and tuberculous bone disease, and its use has been discontinued at the Royal National Orthopaedic Hospital. The antigamma haemolysin test in bone tuberculosis appears to give rise to few false positive results. Our observations confirm that the anti-alpha haemolysin and antigamma haemolysin tests used together reveal about 80 percent of cases of staphylococcal bone infection on first presentation or relapse.

Evaluation of urinary antigen ELISA for diagnosing Legionella pneumophila serogroup 1 infection.

The enzyme linked immunosorbent assay (ELISA) described was developed to detect a soluble antigen in the urine of patients with Legionnaires' disease caused by Legionella pneumophila serogroup 1 (L.pn 1). The assay was evaluated and showed good specificity (100%) and intra-assay reproducibility. Antigen was detected in the urine of 93 (77%) of 120 patients, overall, and in 86% of patients from whom a specimen obtained within seven days of onset of illness was available. On all but one occasion the first urine sample taken from a patient for whom a positive ELISA result was obtained, was itself positive. In one case antigen was not detected at four days but was present on the fifth day after onset of symptoms. In two patients urinary antigen was detectable as early as two days after onset of symptoms. In another the antigen persisted for at least 60 days. More than half the patients, however, had stopped producing detectable antigen within 14 days of onset of symptoms. It is therefore important that where Legionnaires' disease is suspected urine is collected as early as possible in the course of the disease.

Monoclonal antibodies show Listeria monocytogenes in necropsy tissue samples.

Stable mouse monoclonal hybridoma cell lines secreting antibodies against Listeria monocytogenes were produced. Antibodies from two of these cell lines (designated CL2 and CL17) have been partially characterised. The specificities of these antibodies were assessed using indirect immunofluorescence antibody tests and L monocytogenes (166 strains) grown in vitro, other species of Listeria (21 strains), and bacteria from 14 other genera (87 strains). The antibodies were found to be specific for Listeria, and when used in combination, reacted with almost all strains of L monocytogenes. A simple and rapid direct immunofluorescence technique was developed, and the presence of L monocytogenes was shown in necropsy tissue from three patients where listeriosis had been confirmed by isolation of the bacterium. Bacteria were also confirmed using one of these antibodies in necropsy tissue from one further patient in whom listeriosis was suspected, but not confirmed by the cultivation of L monocytogenes.

Phylogenic homogeneity of Coxiella burnetii strains as determinated by 16S ribosomal RNA sequencing.

DNA coding for the 16S rRNA of six strains of the obligate intracellular bacterium Coxiella burnetii was directly amplified from lysed host cells using the polymerase chain reaction. The amplification product was sequenced using a linear-PCR procedure and compared with other published 16S rRNA sequences. The results of this analysis confirm the position of C. burnetii in the gamma subgroup of the proteobacteria. The data show that all of the C. burnetii strains are highly related (> 99%) on the basis of 16S rRNA sequences although they had different geographic origins and phenotypic characteristics. The data support a phylogenetic homogeneity of the genus Coxiella with only one species which is C. burnetii.

Taxonomic considerations of Bartonella bacilliformis based on phylogenetic and phenotypic characteristics.

The 16S-rRNA gene of Bartonella bacilliformis was amplified using the polymerase-chain reaction (PCR). The amplification product was sequenced using a linear-PCR procedure and compared with other published 16S-rRNA sequences. The results of this analysis placed B. bacilliformis in the alpha subgroup of the proteobacteria, and more specifically demonstrated its close phylogenetic relationship to Rochalimaea quintana. This relationship is supported by similarities in the size and mean base composition of the genomes of the two species, and by shared phenotypic characteristics.

The evaluation of a phage-typing system for Listeria monocytogenes for use in epidemiological studies.

A typing system for strains of Listeria monocytogenes based on the lytic properties of 28 phages has been evaluated with a set of strains isolated in the UK and tested in a blind trial. The system was highly reproducible and discriminatory, and 64% of all the strains tested could be typed.

Aspects of the epidemiology of human Listeria monocytogenes infections in Britain 1967-1984; the use of serotyping and phage typing.

Strains of Listeria monocytogenes from 475 cases of human listeriosis collected during 1967-1984, belonged to one of three serogroups (1/2, 3 or 4). They were phage typed with a set of 28 phages to investigate three aspects of the epidemiology of listeriosis. Three patients each had two episodes of listeriosis, 3 months to 2 years apart, with strains of the same serogroup and indistinguishable by phage typing. Ten episodes of possible cross-infection between pairs of neonates in the same hospital occurred; the first baby was ill at or within 1 day of birth, and the second baby became ill 8-12 days after contact with the first. In each pair the L. monocytogenes strains were of the same serogroup and indistinguishable by phage typing. In three clusters of cases there may have been a common source of infection. L. monocytogenes strains from 10 of 11 cases of listeriosis in the Carlisle area in Jul.-Dec. 1981 were of the same serogroup; nine strains were non-phage-typable. The second cluster involved four adults treated at one hospital and the third a pair of neonates who were ill shortly after birth. In each cluster, strains were of the same serogroup, and were indistinguishable by phage typing. These last two clusters occurred during a short period when an unusually high proportion of strains from all cases of human listeriosis in Britain were indistinguishable by phage typing from the cluster strains, suggesting the possibility of common source infection.

A comparison of probes for restriction fragment length polymorphism (RFLP) typing of Legionella pneumophila serogroup 1 strains.

The use of probes derived from rRNA sequences to detect restriction fragment length polymorphisms (RFLPs) associated with the ribosomal RNA genes for epidemiological typing (ribotyping) is a powerful and readily applicable tool. Different probes and enzymes for ribotyping were compared for a series of 73 unrelated Legionella pneumophila serogroup 1 strains. The probes compared were cDNAs, transcribed from L. pneumophila or Escherichia coli rRNA subunits, and a cloned L. pneumophila rRNA gene. The cloned rRNA gene probe gave the best discrimination and this probe was further compared with cloned probes comprised of randomly selected (non-rRNA) parts of the L. pneumophila chromosome. In this instance the greatest discrimination was achieved when one of the non-ribosomal RNA gene probes was employed. The overall discrimination of RFLP typing was enhanced by combining the data obtained with both rRNA and non-rRNA probes.

Further evidence that genotypically closely related strains of Legionella pneumophila can express different serogroup specific antigens.

The relationship between serogroup and genotype of Legionella pneumophila strains was investigated by restriction fragment length polymorphism (RFLP) typing with a previously standardised method. Of the 51 RFLP types identified, 19 comprised strains of more than one serogroup. Several RFLP types included strains of five or more serogroups. To determine if sharing the same RFLP type indicates that strains are genotypically indistinguishable or merely that they are superficially similar, 31 strains were selected for further analysis with an extended range of restriction endonucleases and nucleic acid probes. In some cases, strains of a particular RFLP type were indistinguishable, while in others the restriction fragment patterns showed minor differences. It is possible that in the latter case the strains are diverging representatives of a parent clone. We conclude that analysis of restriction fragment patterns, either probed or unprobed, provides a more accurate measure of the ancestral relationship between strains than can be obtained with serological methods.

A method for typing strains of Legionella pneumophila serogroup 1 by analysis of restriction fragment length polymorphisms.

A restriction fragment length polymorphism (RFLP) typing method for Legionella pneumophila serogroup 1 was developed. The method depended upon the use of cloned EcoR1 fragments from L. pneumophila (Knoxville-1) probing Nci1 restriction fragments of chromosomal DNA. Examination of strains of L. pneumophila which were apparently unrelated showed that inter-strain RFLPs were common, and these formed the basis of the typing scheme. The technique was found to be highly reproducible and discriminatory. When the RFLP data were compared to that obtained by monoclonal antibody (MAb) subgrouping both methods of strain differentiation gave consistent results. The isolates examined by either method were also sub-divided by the alternative technique. The analysis of RFLPs by cloned probes should be of considerable epidemiological value.