Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.     

Medicinal plants used for the treatment of sexually transmitted infections by lay people in northern Maputaland,KwaZulu–Natal Province,South Africa

This ethnobotanical study on plants used for the treatment of sexually transmitted infections was undertaken to document the knowledge by lay people in a rural community in northern Maputaland, South Africa. The focus was on the medicinal plants which are growing in and around the immediate vicinity of the homesteads. Thirty three plant species were recorded as being used for the treatment of sexually transmitted infections such as gonorrhoea (drop or ugcusulu), external and internal sores caused by sexually transmitted infections, genital warts (cauliflower or umhluma) and syphilis. Nine plants (Bridelia cathartica subsp. cathartica, Cladostemon kirkii, Erianthemum dregei, Euphorbia hypericifolia, Ipomoea batatas, Krauseola mosambicina, Mimusops caffra, Opuntia stricta and Sarcophyte sanguinea subsp. sanguinea) were recorded for the first time in the literature world wide as a treatment for sexually transmitted infections. Five new vernacular names were documented for B. cathartica subsp. cathartica, Bryophyllum pinnatum, Clematis brachiata, E. hypericifolia and Pyrenacantha kaurabassana. The 33 plant species are used in 23 different combinations of two or more plants per herbal remedy. The three most frequently used plant species in the study area for the treatment of sexually transmitted infections are; Hypoxis hemerocallidia, Senecio serratuloides and Ranunculus multifidus. Roots are mostly prepared, as a decoction which is taken orally or used as an enema. All eighty of the interviewees preferred traditional medicine as the first therapeutic choice for treating sexually transmitted infections. The wide variety of plants that are used to treat sexually transmitted infections in this area emphasises the importance that medicinal plants can have in the primary health care system of the rural people in northern Maputaland (KwaZulu–Natal).     

A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1

Background

Urothelial carcinoma (UC) is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations.

Methodology/Principal Findings

We performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%.

Conclusions/Significance

Our data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC.     

Isolation and functional analysis of denitrifiers in an aquifer with high potential for denitrification

Aquifers are among the main freshwater sources. The Raigón aquifer is susceptible to contamination, mainly by nitrate and pesticides, such as atrazine, due to increasing agricultural activities in the area. The capacity of indigenous bacteria to attenuate nitrate contamination in different wells of this aquifer was assessed by measuring denitrification rates with either acetate plus succinate or nitrate amendments. Denitrification activity in nitrate-amended assays was significantly higher than in unamended assays, particularly in groundwater from wells where nitrate concentration was 33.5 mg L⁻¹ or lower. Furthermore, groundwater denitrifiers capable of using acetate or succinate as electron donors were isolated, identified by 16S rRNA gene sequencing and evaluated for functional denitrification genes (nirS, nirK and nosZ). Phylogenetic affiliation of 54 isolates showed that all members belonged to nine different genera within the Proteobacteria (Bosea, Ochrobactrum, Azospira, Zoogloea, Acidovorax, Achromobacter, Vogesella, Stenotrophomonas and Pseudomonas). In addition, isolate AR28 that clustered separately from validly described species could potentially belong to a new genus. The majority of the isolates were related to species belonging to previously reported denitrifying genera. However, the phylogeny of the nirS and nosZ genes revealed new sequences of these functional genes. To our knowledge, this is the first isolation and sequencing of the nirS gene from the genus Vogesella, as well as the nosZ gene from the genera Acidovorax and Zoogloea. The results indicated that indigenous bacteria in the Raigón aquifer had the capacity to overcome high nitrate contamination and exhibited functional gene diversity.     

Antiviral potential of Bulgarian medicinal plants

Medicinal plants have been widely used to treat a variety of infectious and non-infectious diseases. Bulgarian flora includes 4,300 plant species, over 500 of which are rare or endemic to the country or the Balkan region. The aim of the present work is to summarize comprehensively the investigations on the antiviral activity of Bulgarian medicinal plants from the past three decades. The effect of different extracts derived from in vitro propagated plants has been examined as well. The phytochemical composition and its influence on specific steps of the viral life cycle have been discussed in this paper. The review includes the following families: Amaryllidaceae, Fabaceae, Geraniaceae, Lamiaceae, Onagraceae, Ranunculaceae, Rosaceae, Scrophulariaceae and Rhodophyta. Special attention has been paid to viruses as important human pathogens.     

MALDI-TOF Mass Spectrometry: A Powerful Tool for Clinical Microbiology at H?pital Principal de Dakar,Senegal (West Africa)

Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, “Necropsobacter massiliensis.” The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries.     

Nuclear factors: Roles related to mitochondrial deafness

Hearing loss (HL) is a common disorder with mitochondrial dysfunction as one of the major causes leading to deafness. Mitochondrial dysfunction may be caused by either mutations in nuclear genes leading to defective nuclear-encoded proteins or mutations in mitochondrial genes leading to defective mitochondrial-encoded products. The specific nuclear genes involved in HL can be classified into two categories depending on whether mitochondrial gene mutations co-exist (modifier genes) or not (deafness-causing genes). TFB1M, MTO1, GTPBP3, and TRMU are modifier genes. A mutation in any of these modifier genes may lead to a deafness phenotype when accompanied by the mitochondrial gene mutation. OPA1, TIMM8A, SMAC/DIABLO, MPV17, PDSS1, BCS1L, SUCLA2, C10ORF2, COX10, PLOG1and RRM2B are deafness-causing genes. A mutation in any of these deafness-causing genes will directly induce variable phenotypic HL.     

Involvement of recF, recO, and recR Genes in UV-Radiation Mutagenesis of Escherichia coli

The recF, recO, and recR genes were originally identified as those affecting the RecF pathway of recombination in Escherichia coli cells. Several lines of evidence suggest that the recF, recO, and recR genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations in recF, recO, or recR greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp⁻ (trpE65) to a Trp⁺ phenotypes. Introduction of the defective lexA51 mutation [lexA51(Def)] and/or UmuD′ into recF, recO, and recR mutants failed to restore normal UVM in the mutants. On the other hand, the presence of recA2020, a suppressor mutation for recF, recO, and recR mutations, restored normal UVM in recF, recO, and recR mutants. These results indicate an involvement of the recF, recO, and recR genes and their products in UVM, possibly by affecting the third role of RecA in UVM.     

Ethnobotany in the Cumbres de Monterrey National Park, Nuevo León, México

The archives of Flora Medicinal, an ancient pharmaceutical laboratory that supported ethnomedical research in Brazil for more than 30 years, were searched for plants with antimalarial use. Forty plant species indicated to treat malaria were described by Dr. J. Monteiro da Silva (Flora Medicinal leader) and his co-workers. Eight species, Bathysa cuspidata, Cosmos sulphureus, Cecropia hololeuca, Erisma calcaratum, Gomphrena arborescens, Musa paradisiaca, Ocotea odorifera, and Pradosia lactescens, are related as antimalarial for the first time in ethnobotanical studies. Some species, including Mikania glomerata, Melampodium divaricatum, Galipea multiflora, Aspidosperma polyneuron, and Coutarea hexandra, were reported to have activity in malaria patients under clinical observation. In the information obtained, also, there were many details about the appropriate indication of each plant. For example, some plants are indicated to increase others' potency. There are also plants that are traditionally employed for specific symptoms or conditions that often accompany malaria, such as weakness, renal failure or cerebral malaria. Many plants that have been considered to lack activity against malaria due to absence of in vitro activity against Plasmodium can have other mechanisms of action. Thus researchers should observe ethnomedical information before deciding which kind of screening should be used in the search of antimalarial drugs.     

Evolutionary relationships between aquatic anamorphs and teleomorphs: Tricladium and Varicosporium

Tricladium, with 21 accepted species, is the largest genus of aquatic hyphomycetes. It encompasses species with dematiaceous as well as mucedinaceous colonies. Conidiogenesis is thalloblastic; conidiogenous cells proliferate percurrently or sympodially. Conidia have typically two alternate primary lateral branches. Fontanospora and Variocladium are segregates of Tricladium, differing by conidial branching. Varicosporium comprises nine species, one not well known. Conidiogenesis is blastic or thalloblastic, conidiogenous cells proliferate sympodially or are determinate; conidia regularly produce primary and secondary branches and often fragment into part conidia. Molecular analyses on the 28S rDNA of 86 isolates, including 16 species of Tricladium, five species of Varicosporium, two species of Fontanospora and one species of Variocladium, place these hyphomycetes within Helotiales. Tricladium is polyphyletic and placed in six clades; Varicosporium is polyphyletic and placed in three clades; Fontanospora is polyphyletic within a single clade. Variocladium is placed with poor support as a sister taxon to Varicosporium giganteum, Hymenoscyphus scutula and Torrendiella eucalypti.     

Enantiomeric trans β-aryl-δ-iodo-γ-lactones derived from 2,5-dimethylbenzaldehyde induce apoptosis in canine lymphoma cell lines by downregulation of anti-apoptotic Bcl-2 family members Bcl-xL and Bcl-2

For many years, studies focused on developing new natural or synthetic compounds with antineoplastic activity have attracted the attention of researchers. An interesting group of such compounds seem to be those with both lactone moiety and an aromatic ring which, in addition to antimicrobial or antiviral activity, also exhibit antitumor properties. The study shows antitumor activity of two enantiomeric trans isomers of 5-(1-iodoethyl)-4-(2′,5′-dimethylphenyl)dihydrofuran-2-one. Our aim was to determine their antitumor activity manifested as an ability to induce apoptosis in selected canine cancer cell lines as well as to evaluate differences in their strength depending on the configuration of their stereogenic centers. The enantiomers (+)-(4R,5S,6R)-1 and (?)-(4S,5R,6S)-2 were found to induce classical caspase-dependent apoptosis through downregulation of the expression of anti-apoptotic proteins Bcl-xL and Bcl-2. Although the mechanism of apoptosis induction was the same for both enantiomers, they differed in their strength, as stronger antineoplastic activity in vitro was exhibited by isomer (+)-(4R,5S,6R)-1.     

Interactions of Insecticidal Toxin Gene Products from Xenorhabdus nematophilus PMFI296

Four genes on a genomic fragment from Xenorhabdus nematophilus PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species. Each gene was expressed individually and in combinations in Escherichia coli, and the insecticidal activity of the lysates was determined. The combined four genes (xptA1, xptA2, xptB1, and xptC1), in E. coli, showed activity towards Pieris brassicae, Pieris rapae, and Heliothis virescens. The genes xptA1, xptB1, and xptC1 were involved in expressing activity towards P. rapae and P. brassicae, while the genes xptA2, xptB1, and xptC1 were needed for activity towards H. virescens. When each of these three genes was expressed individually in E. coli and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level. If the genes xptB1 and xptC1 were expressed in the same E. coli cell and this cell lysate was mixed with cells expressing xptA1, activity was restored to P. rapae and P. brassicae. Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards H. virescens. Individual gene disruptions in X. nematophilus PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either xptA1 or xptA2 for their respective disruptions or E. coli expressing both xptB1 and xptC1 for individual disruptions of either of these genes. The genes xptA2, xptC1, and xptB1 were expressed as an operon in PMFI296 and inactivation of xptA2 or xptC1 resulted in silencing of downstream gene(s), while xptA1 was expressed as a single gene. Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.     

Comprehensive expression analysis of all Wnt genes and their major secreted antagonists during mouse limb development and cartilage differentiation

Wnt signalling plays important roles in patterning and outgrowth of the vertebrate limb. Different mutations in Wnt genes, their antagonists or (co-)receptors result in patterning and outgrowth defects as well as chondrocyte and bone phenotypes in mouse and human. Understanding Wnt activity during mouse limb development and chondrogenesis requires a temporal and spatial overview of Wnt signalling key factor expression. Here we present a comparative expression analysis of all 19 Wnt genes and their major secreted antagonists of the Dickkopf (Dkk), Wisp and the secreted frizzled related protein (Sfrp) families during mouse limb development. Our study reveals new domains of expression for Wnt2, Wnt2b, Wnt5b, Wnt6, Wnt7b, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16, in the limb. We also identified novel expression domains for the Wnt antagonists Sfrp1, Sfrp3, Sfrp5, Wisp1 as well as Dkk2 and Dkk3. We provide a full expression pattern for Wif1 in limb development, for which no limb expression had been documented so far.     

Biodiversity of Vibrios

Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years.     

Pathogenicity of Pratylenchus coffeae,Scutellonema bradys,Meloidogyne incognita,and Rotylenchus reniformis on Dioscorea rotundata

Low populations (200 specimens per plant) of Pratylenchus cofl''eae, Scutellonema bradys, Meloidogyne incognita, and Rotylenchulus reniformis stimulated the development of tops, roots, and tubers of Dioscorea rotundata "Guinea" yam. We demonstrated experimentally that P. coffeae was responsible for the deterioration in quality of the yam tuber in Puerto Rico, a condition known as a dry rot of yam. Initial populations of 600 P. coffeae, S. bradys, or M. incognita, and populations of 1,000 P. coffeae or S. bradys per plant were high enough to induce dry rot of the yam tubers. P. coffeae and S. bradys were pathogenic to yam cultivar Guinea, but M. incognita and R. reniformis did not cause necrosis or cracking of the tuber cortex in our experiments.     

Biodiversity of Vibrios

Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years.