Zambian Invertebrate Scoring System (ZISS): A macroinvertebrate-based biotic index for rapid bioassessment of southern tropical African river systems

An extensive survey of tropical rivers, conducted during 2009–2012 throughout Zambia, collected 151 samples of benthic macroinvertebrates, located on 95 rivers in six of the nine freshwater ecoregions. Associated data for physico-chemistry, human activities and ecosystem stressors were collected. Data were used to develop and test a new Rapid Bioassessment Protocol (the Zambian Invertebrate Scoring System: ZISS) for assessment of water quality and river condition in both wadeable and non-wadeable rivers. ZISS, which is based on the South African Scoring System (SASS), includes a total of 85 taxa, of which 79 are shared with SASS. Assignment of sensitivity weightings to new ZISS taxa was based on sensitivity weightings of closely related SASS families; known life-history modes and anatomical adaptations; and correlation of occurrence to impact ratings. The ability of the ZISS to measure impacts was assessed by determining the relationships between ZISS metrics and impacts. ZISS data for the Kafue River demonstrated the efficacy of the ZISS for detecting moderate to high impacts on water quality and river condition. ZISS represents a major step in developing a user-friendly, widely applicable, macroinvertebrate-based biotic index, which can provide easily interpretable assessments of river condition for southern tropical African rivers.     

A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice

The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.     

5-Hydroxy-2-(2-phenylethyl)chromone (5-HPEC): A novel non-nitrogenous ligand for 5-HT2B receptor

Chromones are a class of natural products found in almost every known terrestrial plant with over 4000 naturally occurring derivatives having been isolated and structurally elucidated. Recently, 5-hydroxy-2-(2-phenylethyl)chromone (5-HPEC), isolated from Imperata cylindrical, showed neuroprotective activity against glutamate induced excitotoxicity in primary cultures of rat cortical cells. In comparison to other naturally occurring neuroprotective chromones, 5-HPEC contains fewer hydroxyl groups. Here we report our most recent characterization on this interesting natural product against a number of CNS receptors for the purpose to identify the potential molecular targets that may be related to its biological activity. Based on our studies, including radiobinding assays, calcium flux functional assays and molecular modeling studies, 5-HPEC may represent a type of novel nonnitrogenous ligands to the 5-HT2B receptor.     

The Pampean Diatom Index (IDP) for assessment of rivers and streams in Argentina

The rivers and streams of the Pampean plain are subjected to the impact of agriculture, cattle-raising and industrial activities. The largest urban center of Argentina is located here. The most important stresses on rivers and streams in the Pampean plain are organic enrichments (discharge of insufficiently treated sewage), nutrients, heavy metals, pathogenic agents, pesticides, herbicides and physical changes produced by dredging and canalisation. The epipelic community is suitable for biomonitoring purposes because it allows for comparing similar substrates along the rivers and streams. A total of 164 samples of epipelic diatoms were collected during 1995-1999 from Pampean rivers and streams. The analysis of these samples resulted in the development of a specific biotic index: the Pampean Diatom Index (IDP). The results were correlated with the main chemical water characteristics and with other biotic indices. This study suggests that the IDP is integrating organic pollution and eutrophication and can be applied for monitoring the biological quality of rivers and streams in the Pampean plain.     

Surface-enhanced laser desorption ionization/time-of-flight (SELDI-TOF) mass spectrometry (MS) as a phenotypic method for rapid identification of antibiotic resistance

Based on experiments with 10 defined strains of Escherichia coli, we present a new method for bacterial phenotyping using SELDI-TOF mass spectrometry. Changes in bacterial protein profiles in the context of the time of cultivation and the antibiotic environment were minimal. Proteom subprofiling may further distinguish between strains with specific susceptibility to antimicrobials. Mass spec-based methods may become common in the future of bacterial pathogen identification in clinical microbiology diagnostics.     

The protective role of 5-hydroxymethyl-2-furfural (5-HMF) against acute hypobaric hypoxia

Our previous study showed that pretreatment with 5-hydroxymethyl-2-furfural (5-HMF) led to protection against hypoxic injury via a p-ERK-mediated pathway in vitro. Whether the protection of 5-HMF against hypoxia is effective in vivo is unknown. The present study is aimed to verify the role of 5-HMF in acute hypobaric hypoxia using Kunming mice as an in vivo model and further investigate the underlying mechanisms. Mice pretreated with or without 5-HMF for 1 h were exposed to acute hypobaric hypoxic condition for 6 h and then the survival time, the survival rate, the permeability of blood–brain barrier (BBB), the histological analysis in hippocampus and cortex, and the phosphorylation level of mitogen-activated protein kinases (ERK, JNK, and p38) were investigated. The results showed that 5-HMF significantly increased the survival time and the survival rate of mice. Accordingly, pretreatment with 5-HMF markedly attenuated acute hypobaric hypoxia-induced permeability of BBB (P < 0.01). In addition, the cellular damage extent of the hippocampus and the cortex induced by hypoxia for 6 h was also attenuated by pretreatment with 5-HMF, especially in the hippocampus CA1 region. Furthermore, the activation of ERK rather than JNK and p38 was involved in the protection of 5-HMF against acute hypobaric hypoxia. In summary, 5-HMF enhanced the survival capability of mice and decreased acute hypoxic damage to the brain, which may be associated with the effects on BBB and p-ERK.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.     

ORP2 Delivers Cholesterol to the Plasma Membrane in Exchange for Phosphatidylinositol 4, 5-Bisphosphate (PI(4,5)P2)

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The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis

Summary The oligopeptide permease is encoded by at least four genes which are transcribed as a single operon. We cloned and characterized this operon from Salmonella typhimurium, as well as the flanking genes, tonB, ana and a new gene, cwd, which affects cell wall synthesis. We correlated the physical map of opp DNA with a detailed genetic map of the opp operon and the individual opp genes were accurately located with respect to various restriction sites by Southern blotting. The region of the chromosome near opp was found to be highly unstable with deletions arising at a high frequency. The operon also contains hot-spots for IS1 and IS5 insertions.     

A rapid bioluminescent enzyme immunoassay (BLEIA) for the detection of Shiga toxin types 1 and 2.

In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx). Traditional culture-based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous. In this work, we established a toxin-detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device. The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pg/ml. In our study of previously characterized Shigatoxigenic and all non-Shigatoxigenic E. coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non-Shigatoxigenic E. coli and other bacterial species to be negative. This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef. For clinical stool samples we noted a tendency for the system to give unexpectedly high background level. Our results suggest the feasibility of using BLEIA methodology for the simple, rapid and sensitive detection of toxins from culture supernatant, various foods and clinical samples.     

The Ca2+-Transport System of Yeast (Endomyces magnusii) Mitochondria: Independent Pathways for Ca2+ Uptake and Release

Some features of the Ca²⁺-transport system in mitochondria of the yeast Endomyces magnusii are considered. The Ca²⁺ uniporter was shown to be specifically activated by low concentrations of physiological modulators such as ADP, NADH, spermine, and Ca²⁺ itself. The Na⁺-independent system responsible for Ca²⁺ release from Ca²⁺-preloaded yeast mitochondria was characterized. The rate of the Ca²⁺ release was proportional to the Ca²⁺ load, insensitive to cyclosporin A and to Na⁺, inhibited by La³⁺, TPP⁺, Pi, and nigericin, while being activated by spermine. We conclude that Ca²⁺ release from preloaded E. magnusii yeast mitochondria is mediated by a Na⁺-independent pathway, very similar to that in mitochondria from nonexcitable mammalian tissues. A scheme describing an arrangement of the Ca²⁺ transport system of yeast mitochondria is proposed.     

Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83) in human plasma

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83; I) directly and its 5′-glucuronide metabolite (5-chloro-2′,3′-dideoxy-5′-O-β- -glucopyranuronosyl-3′-fluorouridine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with β-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from −5.5 to 7.1% during the validation and from −6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-μl sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.     

The caddisfly fauna (Insecta, Trichoptera) of the rivers of the Black Sea basin in Kosovo with distributional data for some rare species

Adult caddisflies were collected from 12 stations in the Black Sea basin in Kosovo using UV light traps. Sixty-five of the seventy-six species reported in this paper are first records for the Kosovo caddisfly fauna. The unexpected discovery of several species during this investigation: Agapetus delicatulus McLachlan, 1884, Psychomyia klapaleki Malicky, 1995, Tinodes janssensi Jacquemart, 1957, Hydropsyche emarginata Navas, 1923, Drusus botosaneanui Kumanski, 1968, Potamophylax rotundipennis (Brauer, 1857), Potamophylax schmidi Marinković-Gospodnetić, 1970, Ceraclea albimacula (Rambur, 1842), Helicopsyche bacescui Orghidan & Botosaneanu, 1953, Adicella filicornis (Pictet, 1834), Beraea maurus (Curtis, 1834) and Beraeamyia hrabei Mayer, 1937 illustrates that collections from poorly investigated areas in Europe will almost certainly revise the existing knowledge on the distribution of these and other species.     

A sieving method for rapid determination of size-frequency distribution of small gastropods. Example of the mud snail Hydrobia ventrosa (Gastropoda: Prosobranchia)

A sieving system using sieves of predefined mesh size has been developed for rapid size–frequency distribution analysis as a basis for the assessment of the secondary production of the hydrobiid species Hydrobia ventrosa Montagu of Lake Ichkeul in northern Tunisia (Mediterranean). The relationship between shell length (SL) and shell width (SW) SL = e^0.5005 SW^1.4306 calculated by a geometric mean regression is very highly significant allowing the application of sieves in mesh intervals of 0.1 mm for the determination of size–frequency distribution. The mean difference between sieved and measured size distributions, for different distribution patterns, only slightly exceeds 1%. The regression between shell length and ash-free dry mass (AFDM) fits an exponential model AFDM = e^{(-2.6591 + 0.9194 SL)}. Mean difference between estimated and observed biomass is in the 10% range. The elementary composition (C, H and N) of the ash-free dry mass is size-dependent. As a result, the proposed sieving method reduces by at least 20 times the data acquisition time (extraction, sorting and size-measuring), avoiding subsampling without significant loss of accuracy for secondary production studies of small gastropods.     

The choice of a method of isolating human IgG F(ab)2 fragments for determining antibodies to staphylococcal protein A]

The comparative study of two methods of the proteolysis of IgG with the aim of obtaining F(Ab)2-fragments from the blood sera of patients and healthy donors has been made. Different methods for the isolation of F (ab)2-fragments, such as gel filtration on Sephadex G-200, affinity chromatography on protein A-BrCN-sepharose, reprecipitation with zinc sulfate, have been analyzed. For further work the method of the peptic decomposition of whole serum with subsequent salting out with ammonium sulfate and gel filtration on Sephadex G-200 has been chosen.     

一种珍稀蝴蝶大帛斑蝶Idea leuconoe(Eriehson)的人工饲养方法

【目的】针对现有大帛斑蝶Idea leuconoe(Eriehson)饲养方法中无法有效回避高温造成的成虫大量死亡,卵空壳或干瘪失水的问题,本研究发明了一种新的繁殖网纹室来规模化人工饲养大帛斑蝶的方法。【方法】本研究测定了在新发明和普通繁殖网纹室的饲养下,大帛斑蝶的存活、交配成功率、产卵量和卵孵化率,并对新发明和普通繁殖网纹室之间的参数进行了对比。【结果】采用新发明繁殖网纹室饲养的大帛斑蝶幼虫化蛹率为90.75%±1.12%,蛹羽化率为91.36%±2.43%,均维持在90%以上,均极显著高于普通繁殖网纹室;从幼虫发育到成虫的存活率为82.93%±2.55%,也极显著高于普通繁殖网纹室;在15日龄内,相对于普通繁殖网纹室,新发明繁殖网纹室饲养的存活的成虫,取食频率较高,活跃性较高,虫体比较健壮,且此时成虫存活率显著较高,为81.22%±2.07%;利用新发明繁殖网纹室饲养的成虫,其雌蝶交配成功率为81.67%±4.77%,每雌产卵量为(269.32±9.85)粒,卵孵化率为85.88%±1.93%,均极显著高于普通繁殖网纹室;本研究新发明的繁殖网纹室,可以有效的营造一个温度显著低于空气温度的小环境,供大帛斑蝶躲避高温灼伤。【结论】通过在本研究新发明繁殖网纹室的饲养,大帛斑蝶存活率、交配成功率和繁殖力均维持在较高的水平上,显著高于普通繁殖网纹室,能够保证为市场提供充足的大帛斑蝶虫源,避免农民养殖户的损失。     

The gene for domains rearranged methyltransferase (DRM2) in Arabidopsis thaliana plants is methylated at both cytosine and adenine residues

The methylation patterns of cytosine and adenine residues in the Arabidopsis thaliana gene for domains rearranged methyltransferase (DRM2) were studied in wild-type and several transgene plant lines containing antisense fragments of the cytosine DNA-methyltransferase gene METI under the control of copper-inducible promoters. It was shown that the promoter region of the DRM2 gene is mostly unmethylated at the internal cytosine residue in CCGG sites whereas the 3'-end proximal part of the gene coding region is highly methylated. The DRM2 gene was found to be also methylated at adenine residues in some GATC sequences. Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the DRM2 gene are variable between wild-type and different transgenic plants. The induction of antisense METI constructs with copper ions in transgene plants in most cases leads to further alterations in the DRM2 gene methylation patterns.