Chemical profiling of Ocimum americanum using external flavonoids

A HPLC survey was undertaken of the external flavonoids in 111 herbarium specimens of Ocimum americanum L. (O. canum Sims), which were largely collected from their natural habitats throughout Africa and Asia. The purpose of this study was to establish the flavonoid profiles of this species over the full range of its geographic distribution in order to use these for authentication purposes. Six different external flavonoid chemotypes were found. The major chemotype, present in circa 80% of the specimens of both var. americanum and var. pilosum collected throughout the distribution area of the species, was characterised by very high levels of nevadensin, slightly lower levels of salvigenin and much lower levels of up to 15 other external flavones. Of the remaining five chemotypes, two were found in var. americanum and three in var. pilosum. All specimens belonging to these chemotypes were collected in South or East Africa and represented by only a few specimens. These samples contained much smaller levels of flavones than present in the major chemotype of O. americanum and all lacked nevadensin. Xanthomicrol, a compound absent from the main chemotype, was the dominant flavone in two of the minor chemotypes. The external flavonoid profiles found in the six chemotypes of O. americanum were compared with those of O. x citriodorum (11 herbarium specimens studied) and seven other closely related species of Ocimum. The main nevadensin/salvigenin pattern present in O. americanum was also found in O. x citriodorum, O. basilicum and some specimens of O. minimum, but there were strong quantitative differences in external flavonoids among these taxa. The other chemotypes of O. americanum showed some similarities in their external flavone profiles to those found in the closely related East African species O. fischeri, O. forskolei, O. kenyense and O. kilimandscharicum, which occur in the same geographic areas. This suggests that the uncommon chemotypes of O. americanum may have originated by an exchange of genes with other Ocimum species, e.g. by introgressive hybridisation. Despite some similarities in profiles, chemical differences were also found among the species, so that it should be possible to authenticate a large proportion of leaf samples of O. americanum on the basis of external flavonoid profiles.     

A borreliacidal factor in Amblyomma americanum saliva is associated with phospholipase A2 activity

Previous work in our laboratory described the in vitro killing of Borrelia burgdorferi when co-cultured with saliva from adult Amblyomma americanum. Borreliacidal activity was not evident using Ixodes scapularis saliva. Mixing trypsin with saliva eliminated the borreliacidal activity of A. americanum saliva, while incorporating a trypsin inhibitor restored all borreliacidal activity, indicating this factor was of protein or peptide origin. One-dimensional PAGE indicated at least 7 major protein differences between I. scapularis and A. americanum saliva. To determine the borreliacidal factor, A. americanum saliva was fractionated by gel filtration and subsequent killing of B. burgdorferi was associated with a single fraction. Two-dimensional gel analysis indicated protein and/or peptide(s) in borreliacidal fractions running between 38 and 64 kDa. Finally, admixing saliva with the phospholipase A₂ inhibitor oleyloxyethyl phosphorylcholine completely eliminated the ability of A. americanum saliva to kill B. burgdorferi. These studies indicate the borreliacidal activity found in A. americanum saliva is likely due to phospholipase A₂ enzymatic activity.     

基于判别分析预测马铃薯甲虫发生与出土时间

【目的】马铃薯甲虫是马铃薯生产过程中的毁灭性害虫。温度是影响马铃薯甲虫发生的重要因素,明确马铃薯甲虫越冬期及发生期的温度对其发生的影响,可为该害虫未来发生情况的预测和防治提供理论支持。【方法】采用逐步判别分析法对1994—2021年马铃薯甲虫越冬及发生期（上一年12月—当年9月）新疆察布查尔县马铃薯甲虫发生等级及出土时间进行判别分类,建立发生预测模型。【结果】在训练组中,马铃薯甲虫的发生等级、出土时间判别准确率分别为100.00%、80.00%;在预测组中,马铃薯甲虫的发生等级、出土时间总判别准确率分别为69.23%、76.92%,认为判别结果较可信。【结论】通过对影响发生程度、出土时间判别的因素筛选发现,察布查尔县马铃薯甲虫的出土和发生判别均受到4月温度的影响。     

浙江双栉蝠蛾发生与土壤关系的层次递进判别分析

为了揭示浙江双栉蝠蛾发生与土壤化学指标、土壤深度的关系,在福建省邵武市拿口镇三丰村选取春笋被浙江栉蝠蛾重度危害和轻度危害各2个样地进行调查,土壤由表及里依次分成3个层次,对每个层次测定pH值、全N含量等7个指标,采用判别分析法分别对每一层及不同层次的组合进行分析。结果表明: (1)各层独立分析,第1层单个指标中具有显著性的是有机质(X₇),判别最有效的组合是全N含量(X₂)和速效K(X₆);第2层中单个指标都不具有显著性,判别最有效的组合仍为全N含量 (X₂)和速效K(X₆);第3层单个指标中具有显著性的是有机质(X₇),判别最有效的组合是速效K(X₆)和有机质(X₇)。 (2)依据第1层判别函数能够完全正确判别分组第2层和第3层,第2层判别函数能够完全正确判别分组第3层,但第2层判别函数不能够完全正确判别分组第1层,第3层判别函数不能够完全正确判别分组第1层和第2层,判别函数具有随土壤层递进而正确判别的递进性,而不具有逆土壤层进行正确判别的逆向性。 (3)对第1层与第2层的组合、第2层与第3层的组合的判别分析,发现单个指标的显著性有所变化,但判别组合的指标没有变化,再次说明了土壤理化性状是由表及里的影响特性。 (4)与方差分析和多重比较的结果相比较,判别分析不仅能反映单个指标在分组中的显著性,而且反映出指标组合的重要性,表明单个指标的显著性不能构成对分组区别的决定性。在层次递进分析过程中,可以清晰地表明各指标在各层以及层次组合中的作用,反映出了层与层间的关系。 由此可以得出:土壤的理化性状是由表及里地影响,与浙江双栉蝠蛾发生危害程度关系密切的是全N含量 (X₂)和速效K(X₆),即全N含量 (X₂)的增加和速效K(X₆)的减少会使危害程度加重,其次为全P含量(X₃),全P含量的增加可使危害程度加重。判别分析法较方差分析和多重比较更全面深刻地反映了浙江双栉蝠蛾发生与土壤的关系。     

Allometry, sexual dimorphism, and phylogeny: A cladistic analysis of extant African papionins using craniodental data

This study conducts a phylogenetic analysis of extant African papionin craniodental morphology, including both quantitative and qualitative characters. We use two different methods to control for allometry: the previously described narrow allometric coding method, and the general allometric coding method, introduced herein. The results of this study strongly suggest that African papionin phylogeny based on molecular systematics, and that based on morphology, are congruent and support a Cercocebus/Mandrillus clade as well as a Papio/Lophocebus/Theropithecus clade. In contrast to previous claims regarding papionin and, more broadly, primate craniodental data, this study finds that such data are a source of valuable phylogenetic information and removes the basis for considering hard tissue anatomy “unreliable” in phylogeny reconstruction. Among highly sexually dimorphic primates such as papionins, male morphologies appear to be particularly good sources of phylogenetic information. In addition, we argue that the male and female morphotypes should be analyzed separately and then added together in a concatenated matrix in future studies of sexually dimorphic taxa. Character transformation analyses identify a series of synapomorphies uniting the various papionin clades that, given a sufficient sample size, should potentially be useful in future morphological analyses, especially those involving fossil taxa.     

Changes in the bacterial community structure in two-stage constructed wetlands with different plants for industrial wastewater treatment

This study focused on the diversity of bacterial communities from two series of two-stage constructed wetlands (CWs) treating tannery wastewater, under different hydraulic conditions. Series were separately planted with Typha latifolia and Phragmites australis in expanded clay aggregates and operated for 31 months. The effect of plant species, hydraulic loading and unit stage on bacterial communities was addressed through bacterial enumeration and denaturating gradient gel electrophoresis (DGGE). Diverse and distinct bacterial communities were found in each system unit, which was related in part to the type of plant and stage position (first or second unit in the series). Numerical analysis of DGGE profiles showed high diversity in each unit with an even distribution of species. No clear relation was established between the sample collection time, hydraulic loading applied and the bacterial diversity.     

Identification of candidate markers associated with agronomic traits in rice using discriminant analysis

Plant genetic mapping strategies routinely utilize marker genotype frequencies obtained from progeny of controlled crosses to declare presence of a quantitative trait locus (QTL) on previously constructed linkage maps. We have evaluated the potential of discriminant analysis (DA), a multivariate statistical procedure, to detect candidate markers associated with agronomic traits among inbred lines of rice (Oryza sativa L.). A total of 218 lines originating from the US and Asia were planted in field plots near Alvin, Texas, in 1996 and 1997. Agronomic data were collected for 12 economically important traits, and DNA profiles of each inbred line were produced using 60 SSR and 114 RFLP markers. Model-based methods revealed population structure among the lines. Marker alleles associated with all traits were identified by DA at high levels of correct percent classification within subpopulations and across all lines. Associated marker alleles pointed to the same and different regions on the rice genetic map when compared to previous QTL mapping experiments. Results from this study suggest that candidate markers associated with agronomic traits can be readily detected among inbred lines of rice using DA combined with other methods described in this report.N. Zhang and Y. Xu contributed equally to work and considered as first authors.Approved for publication by the Director of the Louisiana Agricultural Experiment Station as paper no. 04-14-0335.     

Homo floresiensis: a cladistic analysis

The announcement of a new species, Homo floresiensis, a primitive hominin that survived until relatively recent times is an enormous challenge to paradigms of human evolution. Until this announcement, the dominant paradigm stipulated that: 1) only more derived hominins had emerged from Africa, and 2) H. sapiens was the only hominin since the demise of Homo erectus and Homo neanderthalensis. Resistance to H. floresiensis has been intense, and debate centers on two sets of competing hypotheses: 1) that it is a primitive hominin, and 2) that it is a modern human, either a pygmoid form or a pathological individual. Despite a range of analytical techniques having been applied to the question, no resolution has been reached. Here, we use cladistic analysis, a tool that has not, until now, been applied to the problem, to establish the phylogenetic position of the species. Our results produce two equally parsimonious phylogenetic trees. The first suggests that H. floresiensis is an early hominin that emerged after Homo rudolfensis (1.86 Ma) but before H. habilis (1.66 Ma, or after 1.9 Ma if the earlier chronology for H. habilis is retained). The second tree indicates H. floresiensis branched after Homo habilis.     

A high-throughput method for microbial metabolome analysis using gas chromatography/mass spectrometry

An analytical high-throughput method based on gas chromatography/mass spectrometry (GC/MS) was developed for fast metabolome investigation. By parallelization and partial automation the time needed for the preanalytical steps could be reduced. In addition a strong decrease of the relative standard deviation of metabolite concentrations from independent samples on the same microtiter plate from 25 to 13% was achieved. Between different plates the relative standard deviation is comparable to the one observed in standard experiments with shaking flasks. Using a fast GC the time need for the full GC/MS-based metabolome analysis could be decreased from 60 to 18 min per run, allowing the measurement of 72 single samples per day and GC/MS machine. In samples of the model organism Corynebacterium glutamicum more than 1000 peaks in the total ion current could be detected in a single fast GC/MS run of which 650 were strong enough to be quantified. Approximately 150 compounds of these were identified using our metabolite MS-library. Correlation analysis of the concentration vectors of independent wild-type samples raised under the same conditions show very high correlations of 0.99+/-0.01 (logs). In conclusion this method allows screenings of large mutant libraries for genetically induced metabolic perturbations.     

Strain typing in Leishmania donovani by using sequence-confirmed amplified region analysis

To differentiate strains of Leishmania donovani, allelic markers at the DNA level were developed by sequence-confirmed amplified region analysis (SCAR). Homologous fragments from different strains of L. donovani were amplified by PCR using random primers and subsequently screened for single-strand conformation polymorphisms. Direct sequencing revealed 55 sequence polymorphisms in eight co-dominant DNA markers; 38 of them were single point mutations. Heterozygosity was evident for 69% and fixed heterozygosity for 25% of all polymorphisms. At most polymorphic sites one of the segregation genotypes was missing. Nineteen unique multilocus genotypes were identified among 29 strains of L. donovani. One genotype was represented by eight Sudanese strains; also two strains from Sudan as well as two strains from Kenya, respectively, shared identical genotypes. All other strains had individual multilocus genotypes. Calculation of genetic distances showed a correlation between multilocus genotypes and the geographical origin of these strains. African strains were found in one well-supported cluster with Kenyan and Sudanese strains clearly separated. SCAR markers seem to represent a random sample of neutral genetic variation present in natural populations. They are co-dominant because they can detect all possible allele combinations in a diploid organism and may, therefore, be very useful for population genetic analysis in Leishmania.     

Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis

A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.     

Multilocus sequence analysis provides basis for fast and reliable identification of Vibrio harveyi-related species and reveals previous misidentification of important marine pathogens

Vibrio harveyi and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry worldwide. Phenotypic tests and 16S rRNA gene analysis fail to discriminate species within the V. harveyi group because these are phenotypically and genetically nearly identical. This study used multilocus sequence analysis to identify 36 V. harveyi-like isolates obtained from a wide range of sources in Australia and to re-evaluate the identity of important pathogens. Phylogenies inferred from the 16S rRNA gene and five concatenated protein-coding genes (rpoA-pyrH-topA-ftsZ-mreB) revealed four well-supported clusters identified as V. harveyi, V. campbellii, V. rotiferianus and V. owensii. Results revealed that important V. campbellii and V. owensii prawn pathogens were previously misidentified as V. harveyi and also that the recently described V. communis sp. nov. is likely a junior synonym of V. owensii. Although the MLSA topologies corroborated the 16S rRNA gene phylogeny, the latter was less informative than each of the protein-coding genes taken singularly or the concatenated dataset. A two-locus phylogeny based on topA-mreB concatenated sequences was consistent with the five-locus MLSA phylogeny. Global Bayesian phylogenies inferred from topA-mreB suggested that this gene combination provides a practical yet still accurate approach for routine identification of V. harveyi-related species.     

Metabolic profiling of Dactylopius (Hemiptera: Dactylopiidae) species pigments by geographical origin and hosts using multivariate data analysis

The genus Dactylopius includes the group of insects historically used in Mexico as a source of natural red colorant, the cochineal color. Five species of Dactylopius collected in thirteen states of Mexico and two provinces of Argentina were analyzed using high-performance liquid chromatography with a photodiode array detector. This analysis allowed each species to be identified on the basis of differences in their metabolic profiles. Cluster analysis and principal component analysis differentiated the species by localities and host plant. These two multivariate data analysis techniques were complementary and confirmed the grouping of all analyzed Dactylopius samples. For all species, carminic acid, identified by reference to a commercial sample, was the major compound present in significant amounts, making all five species potential sources of colorant. In addition, each species could be differentiated by the presence of other minor compounds.     

A method for enzyme quenching in microbial metabolome analysis successfully applied to gram-positive and gram-negative bacteria and yeast

Although microbial metabolome analysis has now become a widely used method, no generally applicable quenching method has been published so far. Either the methods were established for only one defined organism or the metabolite coverage was quite low. In the current work, a novel, reliable, and robust quenching method for different types of organisms is described. Compared with the commonly used quenching procedure with 60% methanol (−50 °C), we obtained improved results for three examined organisms with different cell wall and membrane structures using a 40% ethanol/0.8% sodium chloride solution (−20 °C). Increased metabolite levels were achieved for 60-80% of all identified compounds. Moreover, the estimated standard error of the relative concentrations of 120-160 different substances was only 14 ± 4% compared with 17 ± 3% in unquenched samples and 24 ± 7% in samples quenched with methanol for the different tested organisms.     

Gene expression analysis of monospecies Salmonella typhimurium biofilms using differential fluorescence induction

Bacterial biofilm formation is an important cause of environmental persistence of food-borne pathogens, such as Salmonella Typhimurium. As the ensemble of bacterial cells within a biofilm represents different physiological states, even for monospecies biofilms, gene expression patterns in these multicellular assemblages show a high degree of heterogeneity. This heterogeneity might mask differential gene expression that occurs only in subpopulations of the entire biofilm population when using methods that average expression output. In an attempt to address this problem and to refine expression analysis in biofilm studies, we used the Differential Fluorescence Induction (DFI) technique to gain more insight in S. Typhimurium biofilm gene expression. Using this single cell approach, we were able to identify 26 genetic loci showing biofilm specific increased expression. For a selected number of identified genes, we confirmed the DFI results by the construction of defined promoter fusions, measurement of relative gene expression levels and construction of mutants. Overall, we have shown for the first time that the DFI technique can be used in biofilm research. The fact that this analysis revealed genes that have not been linked with Salmonella biofilm formation in previous studies using different approaches illustrates that no single technique, in casu biofilm formation, is able to identify all genes related to a given phenotype.     

Cold glycerol-saline: the promising quenching solution for accurate intracellular metabolite analysis of microbial cells

Microbial metabolomics has been seriously limited by our inability to perform a reliable separation of intra- and extracellular metabolites with efficient quenching of cell metabolism. Microbial cells are sensitive to most (if not all) quenching agents developed to date, resulting in leakage of intracellular metabolites to the extracellular medium during quenching. Therefore, as yet we are unable to obtain an accurate concentration of intracellular metabolites from microbial cell cultures. However, knowledge of the in vivo concentrations of intermediary metabolites is of fundamental importance for the characterization of microbial metabolism so as to integrate meaningful metabolomics data with other levels of functional genomics analysis. In this article, we report a novel and robust quenching method for microbial cell cultures based on cold glycerol-saline solution as the quenching agent that prevents significant leakage of intracellular metabolites and, therefore, permits more accurate measurement of intracellular metabolite concentrations in microbial cells.     

Preservation of viable Francisella tularensis for forensic analysis

As a preservation solution, (1%) ammonium chloride may be preferred over other conventionally used storage solutions because of its compatibility with analytical techniques such as Mass Spectrometry. In this study, ammonium chloride performed as well or better than phosphate buffered saline with Tween or Butterfields/Tween for preserving Francisella tularensis subsp. novicida.