Tetracycline resistance of Escherichia coli isolated from water, human stool, and fish gills from the Lake Sevan basin.

The ecological state of Lake Sevan, the largest drinking water reservoir for the South Caucasus, formed under the influence of climatic and social changes. This study assesses the bacteriological quality of water in the rivers of the Lake Sevan basin and tetracycline-resistant bacteria isolated from fish and people living near the rivers of the Lake Sevan basin in Armenia in autumn 2019 and spring 2020. No differences have been shown for the tetracycline resistance of the investigated E. coli isolated from the human gut and the Masrik, Argichi, and Gavaraget Rivers. Horizontal gel electrophoresis revealed the same plasmid bands in most of the investigated E. coli with the same tetracycline resistance from the different sources of the Argichi River (obtained from people/fish/water sources where the fish were caught). The results also showed that most of the waters carried Edwardsiella spp., Erwinia spp., Morganella spp., and Proteus spp. in addition to E. coli; the coliform index did not exceed the standard level of 5 × 104 CFU mL-1 there. These findings highlight the importance of multidisciplinary studies of bacteria from "interacting" ecosystems, which might serve as a basis for the suggestion of microbial antibiotic resistance as another indicator of water pollution.

Blood protein polymorphisms and the gut bacteria: impact of probiotic Lactobacillus acidophilus Narine on Salmonella carriage in sheep.

Related to previous reports on correlations between an animal's genotype, its commensal microbiota, and the ability to resist infections, the aim of the current study was to investigate the associations between sheep genotype and 5-methylcytosine (5-mC) DNA methylation patterns, sheep genotype and cell surface hydrophobicity of sheep gut commensal bacteria. In addition, the effect of the probiotic Lactobacillus acidophilus strain INMIA 9602 Er 317/402 (probiotic formulation Narine) on Salmonella carriage in sheep at Armenian farms was also investigated. Allelotypes and genotypes of different pathogen-sensitive sheep breeds from Armenian farms were studied based on genetic markers of blood transferrin, albumin, and ceruloplasmin. Additionally, the differences between the breeds of Mazekh, Balbas, and Mazekh/Balbas hybrids were reported. The relationship between host sheep blood transferrin and albumin polymorphisms and cell surface hydrophobicity/5-mC DNA methylation patterns from the predominant gut commensal bacteria was shown. The Narine probiotic eliminates Salmonella from the sheep gut microbiota. At the same time, no significant changes in the percentage of 5-mC DNA methylation of predominant gut bacteria after probiotic administration were observed. The evaluation of bacterial cell surface hydrophobicity, the most significant factor affecting bacterial adhesion, as well as 5-mC DNA methylation, might be used for specific sheep husbandry/breeding programs. This study suggests that the commercial probiotic Narine could potentially be used to reduce Salmonella carriage in sheep.

Method of preparation, visualization and ultrastructural analysis of a formulation of probiotic Bacillus subtilis KATMIRA1933 produced by solid-phase fermentation.

Probiotic preparations are used in medical treatment and in agricultural practice. They modulate numerous activities in eukaryotic hosts, such as: inhibition of pathogenic microbiota; stimulation of immunological responses; and production of antioxidants, anti-mutagens, and DNA protectors. Also, probiotic bacteria are used as a preventive measure to prevent bacterial diseases of the gastrointestinal tract. Solid-phase fermentation is reported as being used in the production of probiotic formulations where a solid substratum, such as soy and oil meal, is utilized for the growth of beneficial microorganisms. However, there are insufficient reports in the literature related to methodological approaches enabling evaluation of the final products of solid-phase fermentation. We suggest a novel method enabling evaluation of probiotic solid-state fermentation dry powders and observation of their morphology, ultrastructure, and elucidation of the quantitative distribution of probiotic microorganisms in solid substrates using electron microscopy. •The method is intended for ultrastructure microphotography of dry substances - for example, ultrastructure of solid-phase fermentation products.•The method allows preserving the ultrastructure of substrates that are damaged when soaking.•The method does not require additional equipment and reagents and can be used in all laboratories using electron microscopy.

Effect of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 on the productivity, reproductive aging, and physiological characteristics of hens and roosters.

The study aims at investigating the effect of preparations of two bacilli strains on laying hens and roosters. Preparations were based on the strains Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895. Several groups of roosters and hens received a preparation based on either strain, or a mixture of both, from the first day to the last day of poultry in production. These preparations improved egg production, quality of sperm production, quality/hatchery of eggs, and slowed down the reproductive aging of hens. These observations were confirmed by the mathematical model proposed herein. At the molecular level, the slowing down of aging was confirmed by a decrease in the amount of mitochondrial DNA damage. Monitoring the physiological parameters of the experimental and control groups of birds showed that live weight gain in all experimental groups was higher than in the control group, and the reproductive organs of hens were more developed. There was also an improvement in the biochemical parameters of blood, the quality of the sperm of roosters, the laying of laying hens, and the morphological and biochemical parameters of the eggs. One of the most significant results is an increase in egg fertilization and a decrease in embryo death during the first 7 days of incubation.

Methodology and effects of repeated intranasal delivery of DNSP-11 in awake Rhesus macaques.

BACKGROUND: To determine if the intranasal delivery of neuroactive compounds is a viable, long-term treatment strategy for progressive, chronic neurodegenerative disorders, such as Parkinson's disease (PD), intranasal methodologies in preclinical models comparable to humans are needed. NEW METHOD: We developed a methodology to evaluate the repeated intranasal delivery of neuroactive compounds on the non-human primate (NHP) brain, without the need for sedation. We evaluated the effects of the neuroactive peptide, DNSP-11 following repeated intranasal delivery and dose-escalation over the course of 10-weeks in Rhesus macaques. This approach allowed us to examine striatal target engagement, safety and tolerability, and brain distribution following a single 125I-labeled DNSP-11 dose. RESULTS: Our initial data support that repeated intranasal delivery and dose-escalation of DNSP-11 resulted in bilateral, striatal target engagement based on neurochemical changes in dopamine (DA) metabolites-without observable, adverse behavioral effects or weight loss in NHPs. Furthermore, a 125I-labeled DNSP-11 study illustrates diffuse rostral to caudal distribution in the brain including the striatum-our target region of interest. COMPARISON WITH EXISTING METHODS: The results of this study are compared to our experiments in normal and 6-OHDA lesioned rats, where DNSP-11 was repeatedly delivered intranasally using a micropipette with animals under light sedation. CONCLUSIONS: The results from this proof-of-concept study support the utility of our repeated intranasal dosing methodology in awake Rhesus macaques, to evaluate the effects of neuroactive compounds on the NHP brain. Additionally, results indicate that DNSP-11 can be safely and effectively delivered intranasally in MPTP-treated NHPs, while engaging the DA system.

Research priorities for conservation and natural resource management in Oceania's small-island developing states.

For conservation science to effectively inform management, research must focus on creating the scientific knowledge required to solve conservation problems. We identified research questions that, if answered, would increase the effectiveness of conservation and natural resource management practice and policy in Oceania's small-island developing states. We asked conservation professionals from academia, governmental, and nongovernmental organizations across the region to propose such questions and then identify which were of high priority in an online survey. We compared the high-priority questions with research questions identified globally and for other regions. Of 270 questions proposed by respondents, 38 were considered high priority, including: What are the highest priority areas for conservation in the face of increasing resource demand and climate change? How should marine protected areas be networked to account for connectivity and climate change? What are the most effective fisheries management policies that contribute to sustainable coral reef fisheries? High-priority questions related to the particular challenges of undertaking conservation on small-island developing states and the need for a research agenda that is responsive to the sociocultural context of Oceania. Research priorities for Oceania relative to elsewhere were broadly similar but differed in specific issues relevant to particular conservation contexts. These differences emphasize the importance of involving local practitioners in the identification of research priorities. Priorities were reasonably well aligned among sectoral groups. Only a few questions were widely considered answered, which may indicate a smaller-than-expected knowledge-action gap. We believe these questions can be used to strengthen research collaborations between scientists and practitioners working to further conservation and natural resource management in this region.

Effects of errors and gaps in spatial data sets on assessment of conservation progress.

Data on the location and extent of protected areas, ecosystems, and species' distributions are essential for determining gaps in biodiversity protection and identifying future conservation priorities. However, these data sets always come with errors in the maps and associated metadata. Errors are often overlooked in conservation studies, despite their potential negative effects on the reported extent of protection of species and ecosystems. We used 3 case studies to illustrate the implications of 3 sources of errors in reporting progress toward conservation objectives: protected areas with unknown boundaries that are replaced by buffered centroids, propagation of multiple errors in spatial data, and incomplete protected-area data sets. As of 2010, the frequency of protected areas with unknown boundaries in the World Database on Protected Areas (WDPA) caused the estimated extent of protection of 37.1% of the terrestrial Neotropical mammals to be overestimated by an average 402.8% and of 62.6% of species to be underestimated by an average 10.9%. Estimated level of protection of the world's coral reefs was 25% higher when using recent finer-resolution data on coral reefs as opposed to globally available coarse-resolution data. Accounting for additional data sets not yet incorporated into WDPA contributed up to 6.7% of additional protection to marine ecosystems in the Philippines. We suggest ways for data providers to reduce the errors in spatial and ancillary data and ways for data users to mitigate the effects of these errors on biodiversity assessments.

Behavioral factors in the placebo response.

Given its presence in almost every clinical trial, the placebo is the most frequently studied substance in clinical research. Demonstration of treatment efficacy demands that the target (active) agent must be shown to be statistically significantly superior to an inert substance (placebo) not believed to be a specific therapy for the target condition. In clinical practice, enhancing the non-specific factors that contribute to an enhanced treatment outcome is desirable to maximize the likelihood of therapeutic benefit. Variables affecting the impact of placebo on clinical research and practice remain poorly understood, however, as they have not been systematically studied. The present article will discuss behavioral factors that have been found to be relevant in placebo mechanisms.

Canonical WNT signalling determines lineage specificity in Wilms tumour.

Wilms tumours (WTs) have two distinct types of histology with or without ectopic mesenchymal elements, suggesting that WTs arise from either the mesenchymal or epithelial nephrogenic lineages. Regardless of the presence or absence of CTNNB1 mutations, nuclear accumulation of beta-catenin is often observed in WTs with ectopic mesenchymal elements. Here, we addressed the relationship between the WNT-signalling pathway and lineage in WTs by examining CTNNB1 and WT1 mutations, nuclear accumulation of beta-catenin, tumour histology and gene expression profiles. In addition, we screened for mutations in WTX, which has been proposed to be a negative regulator of the canonical WNT-signalling pathway. Unsupervised clustering analysis identified two classes of tumours: mesenchymal lineage WNT-dependent tumours, and epithelial lineage WNT-independent tumours. In contrast to the mesenchymal lineage specificity of CTNNB1 mutations, WTX mutations were surprisingly observed in both lineages. WTX-mutant WTs with ectopic mesenchymal elements had nuclear accumulation of beta-catenin, upregulation of WNT target genes and an association with CTNNB1 mutations in exon 7 or 8. However, epithelial lineage WTs with WTX mutations had no indications of active WNT signalling, suggesting that the involvement of WTX in the WNT-signalling pathway may be lineage dependent, and that WTX may have an alternative function to its role in the canonical WNT-signalling pathway.

Integration of behavioural techniques into clinical practice.

Most clinicians agree that biobehavioural factors are important considerations in the assessment and treatment of headache patients. Attention to psychological and behavioural issues may become even a greater concern as the frequency of a patient's headaches increases, there is increased disability secondary to headaches and/or there is inadequate response to usually effective treatment. The present article will highlight biobehavioural factors that should be considered in the assessment process involving headache patients and will provide a model for integration of behavioural treatment into clinical practice.

Novel urothelium specific gene expression identified by differential display reverse transcriptase-polymerase chain reaction.

PURPOSE: Understanding the molecular basis of differential gene expression among different tissues at various developmental stages and in neoplastic transformation is an important biological goal. The potential clinical applications of this improved understanding are more precise diagnosis of disease, prediction of prognosis, novel targeted therapies and prediction of response to therapy. MATERIALS AND METHODS: Differential display reverse transcriptase-polymerase chain reaction was used to compare gene expression in bovine urothelium to that in autologous lung, esophagus, liver and spleen. Products that appeared to have urothelial specific expression were sequenced and assessed for homology with known sequences. Ribonuclease protection assays were used to further confirm the expression pattern. RESULTS: A total of 32 discrete cDNAs were identified, including 3 products from genes known to be urothelium specific in their expression, 16 with significant homology to bovine, human or mouse expressed sequence tags and 5 with no sequence homology to any currently available sequence. Urothelium specific mRNA expression was confirmed for 3 genes by ribonuclease protection assays and one (Udd06) was further characterized as a urea transporter. CONCLUSIONS: The use of differential display reverse transcriptase-polymerase chain reaction and other complementary techniques for parallel gene expression analysis will permit the complete characterization of the urothelial transcriptome and help identify potential molecular targets for rationally targeted therapy.

A pilot study comparing the purity, functionality and isoform composition of alpha-1-proteinase inhibitor (human) products.

OBJECTIVE: Alpha-1-proteinase deficiency predisposes affected individuals to early onset pulmonary emphysema, and is treated with an alpha-1-proteinase inhibitor (A1-PI) from pooled human plasma. The objective of this pilot study was to assess analytical parameters of the three A1-PI products (Aralast, Prolastin, Zemaira) that may impact on clinical efficacy, safety, and convenience. These included: purity of the preparation; nature of impurities; functionality; and isoform composition. METHODS: Purity was evaluated using reverse phase and size exclusion chromatography high performance liquid chromatography (RP-HPLC and SEC-HPLC), capillary zone electrophoresis (CZE), sodium dodecyl sulfate polyacrylamide gel electrophoresis, sodium dodecyl sulfate capillary gel electrophoresis and Western blot analysis. The identity of protein impurities was determined by immunonephelometry; functionality by calculating the ratio of mg active A1-P1 present (by anti-neutrophil elastase activity assay) to the mg antigenic A1-PI (by immunonephelometry); and normality of the A1-PI isoform pattern by isoelectric focusing (IEF). Three samples of Zemaira and one sample each of Aralast and Prolastin were available for analysis. RESULTS: Zemaira had the highest specific activity. Using RP-HPLC analysis Zemaira averaged 99% purity, Aralast 70% and Prolastin less than 62%. Using SEC-HPLC Zemaira was 95.98% monomeric, Prolastin 79.00% and Aralast 63.55%. Prolastin had lower activity/mg antigenic A1-PI than the other two products. A shift in isoforms in Aralast was suggested by the results of CZE, and was confirmed by IEF. CONCLUSIONS: Zemaira demonstrated greater purity compared with Aralast and Prolastin. Prolastin had more inactive A1-PI than Zemaira or Aralast. Isoform ratios appeared to be altered in Aralast. The results from this pilot study warrant further investigation.

Transcriptional control of the human urothelial-specific gene, uroplakin Ia.

The transcriptional control elements of tissue-specific genes may be exploited in the design of therapeutic constructs for use in human gene therapy. The uroplakins are a family of four proteins which form the asymmetric unit membrane of the urothelium. We have cloned the human uroplakin Ia gene and defined its genomic structure and transcriptional start site. Using quantitative RT-PCR in an extended panel of normal tissues, we have demonstrated highly urothelial-specific expression of this gene. A Dual-Luciferase assay was used to assess the transcriptional activity of a variety of promoter fragments of the human uroplakin Ia gene. A highly specific promoter fragment (consisting of 2147 bp of 5'-flanking sequence, intron 1 and the 5' UTR) was identified which regulated urothelial-specific expression in vitro. The human uroplakin Ia promoter identified has potential use in future gene therapy strategies to restrict transgene expression to the urothelium.

MMPI personality profiles in patients with primary chronic daily headache: a case-control study.

We assessed the psychological profile of a large sample of patients with chronic daily headache (CDH) seen in tertiary care. We used a case-control design to study 791 patients who fell into the following categories: ARH group, chronic migraine with analgesic overuse (analgesic rebound headache, ARH), n=399; CM group, chronic migraine (CM) without analgesic overuse, n=158; and new daily persistent headache (NDPH) group, n=69. These groups were compared to two control groups: 1, migraine, n=100; 2, chronic posttraumatic headache (CPTH), n=65. We assessed personality and psychopathology with the Minnesota multiphasic personality inventory (MMPI)-2. The number of patients with Tscores > or =65 and < or =40 were analyzed by the two-sided Fischer's exact test. The ARH and CM groups had a higher number of subjects with T-scores > or =65, when compared to the migraine group, on the following scales: 1 (hypochondrias), 2 (depression), 8 (schizophrenia) and 0 (social introversion). No differences were observed between the NDPH and migraine groups. Considering CPTH as the control group, the pattern we found was quite the opposite of that described above: NDPH group presented a higher number of subjects with T-scores > or =65 on the following scales: 1, 2, 7 (psychasthenia) and 8. ARH and CM groups had significantly higher T-scores for scale 7 alone. NDPH showed T-scores < or =40 in scale 9 when compared to both control groups. We conclude that: (1) psychopathological factors are common in CDH patients, and appear to be a consequence of the chronification process; (2) low scores on scale 9 (hypomania) may relate to the development of NDPH; (3) psychopathological profiles differ among the subgroups of CDH and the MMPI-2 is reliable in identifying such patterns; and (4) psychological assessment is an essential step in the evaluation and treatment of patients with CDH.

Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes.

BACKGROUND: Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. RESULTS: HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre-treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In addition, the constructs with the 3' deletion were more responsive to stimulation by polyamine addition than those with the 5' deletion. CONCLUSIONS: The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting.

Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors.

In this paper we describe the synthesis and characterization of a series of simple spermine/amino acid conjugates, some of which potently inhibit the uptake of spermidine into MDA-MB-231 breast cancer cells. The presence of an amide in the functionalized polyamine appeared to add to the affinity for the polyamine transporter. The extensive biological characterization of an especially potent analogue from this series, the Lys-Spm conjugate (31), showed this molecule will be an extremely useful tool for use in polyamine research. It was shown that the use of 31 in combination with DFMO led to a cytostatic growth inhibition of a variety of cancer cells, even when used in the presence of an extracellular source of transportable spermidine. It was furthermore shown that this combination effectively reduced the cellular levels of putrescine and spermidine while not affecting the levels of spermine. These facts together with the nontoxic nature of 31 make it a novel lead for further anticancer development.

Comparison of auditory, somatosensory, and visually instructed and internally generated finger movements: a PET study.

We sought to determine how the pattern of cerebral activation, and in particular in frontal motor areas, during the performance of conditional motor tasks is dependent upon the modality of instruction (visual, auditory, or somatosensory). Regional cerebral blood flow (rCBF) changes with externally instructed movements were also compared with internally generated, self-paced, movements. We used positron emission tomography (PET) with the tracer H2(15O) to measure rCBF in 22 healthy volunteers. External stimuli consisted of the randomized presentation of single or double impulses using a single modality for each condition. In the movement scans, the subjects used the index and middle fingers of their right hands to press a left button for a single and a right button for a double impulse, respectively. In the control scans, subjects were required to covertly distinguish a single from a double stimulus without a motor response. Data were analyzed using conventional subtraction techniques with a statistical threshold of Z > 2.33 with corrections for multiple comparisons. When the activation differences between the three externally instructed movement conditions were statistically compared, nonsignificant trends toward increased rCBF in the sensory cortex of the modality of the cue were observed but no differential activity in cortical motor areas. Internally generated movements, when compared to externally triggered movements, were associated with enhanced activation in bilateral medial and lateral premotor, dorsolateral prefrontal and superior parietal regions, largely confirming previous reports. The data indicate that, on a regional level, modality-specific processing in a conditional motor task does not occur in frontal motor areas and is probably confined to sensory areas.

Effects of efrapeptin and destruxin, metabolites of entomogenous fungi, on the hydrolytic activity of a vacuolar type ATPase identified on the brush border membrane vesicles of Galleria mellonella midgut and on plant membrane bound hydrolytic enzymes.

The brush border membrane of the insect midgut is an initial site for interaction of insecticidal proteins. We have investigated the possibility that it may contain a target site for two insecticidal fungal toxins, destruxin and efrapeptin, both of which are ATPase inhibitors. We have studied the effects of the toxins on the hydrolytic activity of a vacuolar type ATPase (V-ATPase) that we have identified from Galleria mellonella midgut columnar cell brush border membrane vesicles (BBMV) by its cation and pH dependence, sensitivity to proton pump inhibitors and K(m) (0.49 mM ATP). Efrapeptin strongly inhibited the BBMV V-ATPase but destruxin had little effect. We compared the effects of the inhibitors on known plant membrane hydrolytic enzymes, and although the vacuolar pyrophosphatase and plasma membrane ATPase were not inhibited by the toxins, the V-ATPase from mung bean, but not barley, was inhibited (50%) by 10 microM concentrations of both compounds. Different forms of the toxins were tested on the ATPases and destruxin B and efrapeptin F were the most effective. Kinetic analysis showed that the purified forms of both compounds inhibited the V-ATPases uncompetitively and modelling of data for inhibition of the BBMV V-ATPase by efrapeptin at concentrations of 0.06--12 microM yielded a K(i) of 0.125 microM.

Differentiating the efficacy of 5-HT(1B/1D) agonists.

OBJECTIVE: To examine, for a set of published clinical trials of serotonin (5-HT(1B/1D)) agonists as acute treatments for migraine, whether transformation of efficacy data into therapeutic gain (TG) or number needed to treat (NNT) is useful. BACKGROUND: Pivotal clinical trials of 5-HT(1B/1D) agonists in migraine use a primary end point of change in pain score from 3 or 2 to 1 or 0. Placebo response rates among such studies are variable. Meta-analytic comparisons of 5-HT(1B/1D) agonists often employ TG and NNT as efficacy measures. METHODS: Data from US product labeling or published sources were converted into TG (TG = active response rate [%] - placebo response rate [%]) and NNT (NNT = 1/TG). Pivotal clinical trial data were compared before and after transformation. RESULTS: Therapeutic gain ranged from 17.5% to 51%. The transformation of TG into NNT yielded no clinically significant difference in efficacy estimate for the range of 17.5% to 47% (N = 29 clinical trials). However, NNT and TG had a nonlinear relationship for some secondary end points. When the relationship between the standard primary and secondary end points was compared, the correlation of TG with clinical disability (Pearson coefficient R = 0.93) was stronger than for NNT. Placebo response rates correlated more strongly with NNT (R = 0.66) than active response rates (R = 0.42; N = 29 clinical trials), although both TG and NNT were sensitive to placebo response rate. CONCLUSIONS: Transforming efficacy rates into TG or NNT adds no new information to placebo-controlled trials. The variables, TG and NNT, should not be used to compare members of this class of drugs. Migraine therapies can only be compared using well-designed head-to-head studies and not by meta-analysis. Broader measures of efficacy should be used to describe and compare 5-HT(1B/1D) efficacy.