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Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.
Sponges (Porifera) are ancient metazoans dating back more than 580 million years. They populate tropical and subtropical benthic marine habitats but are also found at higher latitudes and even in freshwater lakes and streams. So far an estimated 15,000 species have been described, but the true diversity is probably much higher (20). Particularly the tropical sponges are known for their colorful appearances and their morphological plasticity, encompassing encrusting, rope, ball, and vase shapes ranging in size from a few millimeters to >1 m. Sponges are diploblast metazoans that lack true tissues or organs. In spite of their simple organization, genome sequencing has revealed genes encoding functions that are highly homologous to those of their vertebrate analogs (4, 31, 32). As sessile filter feeders, they pump large volumes of water through a specialized canal system, termed the aquiferous system. The filtration capacities of sponges are remarkably efficient (up to 24,000 liters kg−1 day−1), leaving the expelled water essentially sterile (36, 39, 44, 54). Food particles such as unicellular algae and bacteria are removed from the seawater and translocated into the mesohyl interior. The mesohyl is a collagen scaffold that constitutes much of the sponge body. Single amoeboid sponge cells, termed archaeocytes, move freely through the mesohyl matrix and digest food particles by phagocytosis (3, 5).
The presence of large amounts of microorganisms within the mesohyl of many demosponges is well documented (45, 46, 51, 52, 53; for recent reviews, see references 19 and 24). Bacteria can contribute up to 40% of the sponge biomass (equal to about 108 to 109 bacteria g of tissue−1) and are probably permanently associated with the host sponge unless they are disturbed by external stress factors (13, 43, 48, 50). Several recent studies have sought to address the phylogenetic diversity of microbial communities associated with marine sponges by using 16S rRNA gene sequence analysis (1, 18, 27, 38, 49). A comprehensive analysis showed that sponges from different oceans contain phylogenetically complex, yet highly specific, microbial signatures (18, 19). In particular, representatives of the poorly characterized phyla Chloroflexi (formerly green nonsulfur bacteria), Acidobacteria, and Actinobacteria as well as Alpha-, Gamma-, and Deltaproteobacteria are abundant in gene libraries. It is noteworthy that none of these sponge-specific bacteria have so far been cultured or are related to cultured bacteria.
Very early on researchers noted that similar bacterial morphotypes occur in taxonomically different sponges (46, 53). One particular morphotype has gained attention because of a nucleoid-like structure within the microbial cell (16, 17). Six different morphological variants were described that resembled the general appearance of sponge-associated morphotype E according to Vacelet (45) and Friedrich et al. (12) and morphotype 4 according to Wilkinson (53). Prokaryotic cells do not generally have cell compartmentalization, with the only exception being members of the phylum Planctomycetes (26). Because of the presence of nucleoid-containing bacteria in the sponge mesohyl and because fluorescence in situ hybridization (FISH) yielded Planctomycetes-specific signals in tissue cryosections (13, 49), this study aimed to target these microorganisms by using molecular methods in order to provide insights into their phylogeny, abundance, and distribution among tropical and subtropical sponge species.
Sponge collection.The sponge Aplysina aerophoba was collected by scuba diving in Banyuls sur Mer, France (42°29′N, 03°08′E), at depths of 5 to 15 m in April 2000. The sponges Aplysina fistularis, Aplysina insularis, Aplysina lacunosa, Verongula gigantea, and Smenospongia aurea were collected by scuba diving offshore of Little San Salvador Island, Bahamas (24°34.39′N, 75°58.00′W), at depths of 5 to 15 m in July 2000. Individual specimens were placed separately into plastic bags and brought to the surface. After collection, sponge tissues were cut into pieces and stored at −80°C until use.
Amplification of 16S rRNA genes and library construction.Genomic DNAs were extracted from sponge tissues frozen in liquid nitrogen by using Fast DNA spin kits for soil (Q-Biogene, Heidelberg, Germany) according to the manufacturer's protocol. Amplification of the 16S rRNA genes was accomplished with the primers PLA46f (5′-GGA TTA GGC ATG CAA GTC-3′) and 1390r (5′-CGG GCG GTG TCT ACA A-3′) (34, 57). A second library was constructed from A. aerophoba, with PLA46f as a forward primer and PLA886 (5′-GGG AGT ATG GTC GCA AGG C-3′) as a reverse primer (34). The resulting sequences from this library are identified by the prefix "Q." PCR cycling conditions were as follows: initial denaturation (2 min at 96°C); 30 cycles of denaturation (1 min at 96°C), primer annealing (1 min at 56°C), and elongation (1.5 min at 72°C); and a final extension step (10 min at 72°C). Genomic DNA from Pirellula marina was used as a positive control. PCR products were ligated into the pGEM-T-Easy vector (Promega) and transformed into CaCl2-competent Escherichia coli DH5α. Plasmid DNAs were isolated by standard miniprep protocols. Correct insert sizes were verified by EcoRI restriction digestion and agarose gel electrophoresis (41).
Design and application of "Poribacteria"-specific PCR primers for screening.In order to determine whether the novel candidate phylum "Poribacteria" is specific to sponges, we designed the primers POR389f (5′ ACG ATG CGA CGC CGC GTG 3′) and POR1130r (5′ GGC TCG TCA CCA GCG GTC 3′) and used them to probe environmental samples from within the vicinity of sponges. Seawater was collected from both dive locations (Banyuls sur Mer and Little San Salvador Island) on the same dives but prior to the removal of sponges from the reef surface. In order to compensate for the estimated 100 to 1,000 times higher bacterial concentration in sponge tissues than in seawater, we collected 1 to 3 liters of seawater, prefiltered it through folded filter papers, and then filtered it through 0.2-μm-pore-size bottle-top SFCA membrane filters (Nalgene, Rochester, N.Y.). Genomic DNAs were extracted from the filters after thermal lysis in a boiling water bath for 10 min and subsequent precipitation in ethanol. Also, coarse and fine sediment samples taken within a 1-m radius of an A. fistularis sponge and tissues of a filter-feeding tunicate (Ecteinascidia turbinata) were probed for the presence of the candidate phylum "Poribacteria". Several DNA template dilutions (1:1, 1:10, 1:100, and 1:1,000) were added to PCRs that were performed at an annealing temperature of 68°C. As a positive control, genomic DNAs from frozen sponge tissues and "Poribacteria"-positive clones from 16S ribosomal DNA libraries were used. PCR amplification with the universal eubacterial primers 27f and 1492r was performed to validate the quality of the environmental DNAs. Furthermore, the resulting PCR amplicons were used in a nested PCR approach using the primers POR389f and POR1130r. As a negative control, 1 μl of H2O instead of DNA was always added to a PCR. The resulting PCR products were analyzed in 1% agarose gels.
In order to assess the distribution of the novel candidate phylum "Poribacteria," we probed several other sponges by PCR. These were Aplysina cavernicola from Elba (Italy) (43), Theonella swinhoei from the Western Caroline Islands (Palau) (18), Agelas wiedenmayeri from the Florida Keys (30), A. fistularis from offshore of San Diego, Calif. (unpublished), and Aplysina cauliformis, Aplysina archeri, Pseudoceratina crassa, Xestospongia muta, Chondrilla nucula, Agelas wiedenmayeri, Agelas cerebrum, Ptilocaulis sp., and Ectyoplasia ferox from around Little San Salvador Island.
Restriction fragment length polymorphism, sequencing, and phylogenetic analyses.PCR products were characterized by single digestion with the restriction endonucleases HaeIII and AvaI. Clones with similar restriction patterns were grouped together, and random clones from each group were chosen for sequence analysis. Sequencing was performed on a LiCor 4200 automated sequencer (LiCor Inc., Lincoln, Nebr.) and an ABI 377XL automated sequencer (Applied Biosystems). Sequence data were edited with ABI Prism Autoassembler, v. 2.1, software (Perkin-Elmer) and then checked for chimera formation (CHECK_CHIMERA online analysis of Ribosomal Database Project II [RDP-II] [6]). Phylogenetic trees including 16S rRNA gene sequences of approximately 1.3 kb were constructed with ARB software (http://www.arb-home.de). Trees were calculated by using the neighbor-joining (Jukes-Cantor correction), maximum parsimony, and maximum likelihood methods implemented in ARB. Partial sequences (about 600 bp) were analyzed with the basic local alignment search tool (BLASTn) algorithm for initial identification. Affiliations of partial sequences were confirmed by adding them to a tree consisting of full-length sequences only without changing the tree topology by use of the ARB "parsimony interactive" method.
FISH.The inner core (about 15 g) of freshly collected A. aerophoba was cut into pieces and disintegrated in 50 ml of ice-cold Ca2+- and Mg2+-free artificial seawater (400 mM NaCl, 27.6 mM Na2SO4, 2.3 mM NaHCO3, 8.9 mM KCl, 0.8 mM KBr, 0.4 mM H3BO3, 0.15 mM SrCl2, 0.07 mM NaF) (40) by use of a mortar and pestle. The suspension was vortexed for 10 min, prefiltered through folded filter papers to remove tissue fibers, and centrifuged at 4°C for 30 min at 100 × g to remove the remaining sponge cells. Bacteria were obtained from the supernatant by centrifugation at 4°C at 12,000 × g for 30 min to ensure complete microbial recovery and washed three times in ice-cold Ca2+- and Mg2+-free artificial seawater. The bacterium-containing fractions were fixed overnight in 0.2-μm-pore-size-filtered seawater containing 4% paraformaldehyde, resuspended in 50% ethanol-phosphate-buffered saline (PBS), and stored at −20°C.
For FISH analyses, aliquots were heat fixed at 40°C onto microscope slides, dehydrated in an aqueous ethanol series (50, 70, and 96%) for 3 min at each step, and air dried. FISH was performed in an isotonically equilibrated humid chamber at 46°C in hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 7.4], 0.01% sodium dodecyl sulfate) and various amounts of formamide (10 to 30%) for 4 h. Probes were used at a concentration of 2 ng/μl. The hybridization buffer was gently rinsed off and the slides were incubated at 48°C in wash buffer (0.7 M NaCl, 20 mM Tris-HCl [pH 7.4], 50 mM EDTA, 0.01% sodium dodecyl sulfate) for 20 min. The wash buffer was rinsed off with ice-cold distilled water, and the slides were air dried in the dark and mounted in Citifluor (Citifluor Ltd., London, United Kingdom). Visualization was achieved with an Axiolab microscope (MC 80; Zeiss). Each hybridization was performed at least five times in parallel and three times independently. The hybridization conditions for the new "Poribacteria"-specific probes were adjusted with an increasing formamide series and evaluated visually. Table 1 lists the FISH probes used for this study.
Transmission electron microscopy.A. aerophoba was fixed in 2.5% glutaraldehyde-PBS for 12 h, rinsed 3 times for 20 min each in 1× PBS, and postfixed in 2% osmium tetroxide-1× PBS for 12 h. The pieces were dehydrated in an ethanol series (30, 50, 70, and 90% and three times at 100%), incubated three times for 20 min each in propylene oxide, and polymerized in Epon 812 (Serva) for 4 days at 60°C. The embedded sponge pieces were sectioned with an ultramicrotome (OM U3; C. Reichert, Vienna, Austria). For contrast, 70- to 80-nm-thick sections were poststained with 0.5% uranyl acetate in methanol for 10 min and with Reynold's lead citrate for 5 min. The resulting sections were investigated by electron microscopy (EM 10; Zeiss).
Phylogenetic analysis.Amplification of the 16S rRNA genes from the total sponge-derived DNA yielded fragments of the expected size of 1,300 or 800 bp, depending on which PCR primer set was used. Altogether, 600 clones were compared by restriction fragment length polymorphism analyses, resulting in six major restriction patterns. A total of 157 representative clones were chosen for sequencing. Thirteen chimeric sequences were excluded from further analyses. A large portion (n = 41 [29%]) of the recovered sequences, with an average of only 74.6% 16S rRNA sequence similarity to members of known phyla, formed a novel evolutionary lineage branching deeply in the domain Bacteria in all treeing methods applied (Fig. 1a). Based on the phylogenetically distant position of this phylum, the candidate phylum "Poribacteria" was proposed. A moderate relationship of this evolutionary lineage to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of descent was suggested by the monophyletic grouping of these phyla in maximum likelihood and neighbor-joining trees, while parsimony trees showed no consistent branching pattern (Fig. 1a). All near-full-length sequences belonging to the candidate phylum "Poribacteria" were conserved in 13 of 15 (87%) signature nucleotides that are characteristic of the Planctomycetes (15, 25, 42, 55). The average in-cluster similarity (92.7%) from sponge-derived sequences is high compared to that for other phyla (9). The remaining 103 16S rRNA gene clones consisted of sequences affiliated with (i) the Deltaproteobacteria (n = 41 [28%]), (ii) the Chloroflexi (n = 51 [35%]), (iii) the Planctomycetes (n = 4 [3%]), and (iv) a subset of sequences with various phylogenetic positions (n = 7 [5%]) (Fig. 1b).
To determine the specificity of "Poribacteria" and their distribution within sponges, we applied "Poribacteria"-specific PCR primers to various environmental samples and to other sponge species. A positive PCR product amplified with the universal bacterial primers 27f and 1492r served as an internal control for the validity of each DNA sample. The seawater samples from both dive locations, coarse and fine sediments taken near a sponge colony, and a filter-feeding tunicate were PCR negative with the "Poribacteria"-specific primers POR389f and POR1130r in conventional and nested PCRs. Positive "Poribacteria"-specific PCR products were obtained from all tested sponges of the order Verongida (A. aerophoba, A. cavernicola [Elba, Italy], A. lacunosa, A. cauliformis, A. fistularis [San Diego, Calif.], A. insularis, A. archeri, V. gigantea, and Pseudoceratina crassa) independent of their geographic location. Positive PCR products amplified with "Poribacteria"-specific primers were also recovered from S. aurea (Dictyoceratida), X. muta (Haplosclerida), and T. swinhoei (Lithistida). Negative "Poribacteria"-specific PCR results were obtained with the sponges C. nucula (Hadromerida), Agelas wiedenmayeri and Agelas cerebrum (both Agelasida), Ptilocaulis sp. (Halichondria), and Ectyoplasia ferox (Poecilosclerida).
FISH.In order to visualize the presence of the "Poribacteria" and to assess their relative abundance within the microbial consortium of A. aerophoba, we designed a set of specific FISH probes, using the probe design and probe match tool of the ARB software package (Table 1). Hybridization with the EUB338 probe mix revealed a very high signal abundance. An estimation of the ratio of FISH signals to DAPI-stained cells revealed >98% detection when the EUB338 probe mix was applied (data not shown). The application of the bacterial probe mix EUBI-III together with the "Poribacteria"-specific probe POR1130 revealed a high abundance of this lineage within the microbial consortium of A. aerophoba (Fig. 2a). In fact, "Poribacteria"-specific signals were visibly more abundant than all previously applied specific FISH probes (12, 13).
The identity of the "Poribacteria" was further confirmed by the application of probes that were specific to the two major subgroups of this lineage. Cohybridization is visible when a red signal (Cy3) colocalizes with a green signal (fluorescein isothiocyanate [FITC]), resulting in a yellow signal. Cohybridization was indeed observed after the application of the probe POR1130 (FITC labeled) together with either probe POR389 or POR600 (Cy3 labeled) (Fig. 2b and c). Because these probes differ in their target sites on the corresponding 16S rRNA, their colocalization confirms the authenticity of the "Poribacteria" lineage. It is noteworthy that the FISH signals appeared in a ring-shaped manner that is consistent with the presence of a nucleoid in this particular cell type (Fig. 2b and c).
Because sequences from other lineages were also recovered from the gene libraries, cohybridization reactions were performed to assess the relative abundance of the different lineages. After cohybridization of the Chloroflexi I cluster-specific probe GNS934 with POR1130, green and red signals were visible as independent spots in about equal amounts (Fig. 2d). Cohybridization of the probe POR1130 with probe SRB385, targeting many Deltaproteobacteria, revealed that the "Poribacteria" are more abundant than the Deltaproteobacteria (data not shown). After the application of Planctomycetes-specific probes PLA46 and PLA886 to microbial preparations, signals were detected only rarely (data not shown).
Transmission electron microscopy.Electron microscopy was performed to provide insights into the morphology of the nucleoid-containing microorganisms of A. aerophoba. Altogether, six different morphotypes were found that closely resemble the different subtypes of Australian Great Barrier Reef sponges (Fig. 3; Table 2) (16, 17). Five of the morphotypes contain a membrane-bound organelle that occupies a central position in the cell. The organelle contains fibrillar DNA that is surrounded by an electron-dense riboplasm. The morphotypes vary in cell shape (short and long rods and D-shaped cells), membrane structure (gram-negative and S-layer types), and density of the cytoplasm. A sixth morphotype shows a reverse topology in that the nuclear material lies in the cytoplasm, whereas the membrane-bound compartment is DNA-free. Morphotype 6 can easily be distinguished by bleb-like structures of the outer cell membrane.
16S rRNA gene-based studies have enormously enhanced our knowledge about the phylogenetic diversity of environmental microbial communities. More than 40 different phyla are now recognized for the domain Bacteria, about half of which contain cultured representatives. Even though databases encompassing >78,000 rRNA gene sequences have been established (RDP-II preview [http://rdp.cme.msu.edu/html/analyses_preview.html]), the extent of prokaryotic diversity is still underestimated even at the phylum level. A striking example of novel microbial diversity is the description of an entirely new phylum, Gemmatimonadetes (formerly candidate phylum BD [33] or KS-B [29]), which was recently added to the domain Bacteria (56). Moreover, the OP candidate phylum from hot springs (23), the WS6, TM7, and SC1-SC4 candidate phyla from soil communities (9, 10, 11, 21, 22), and the BRC1 candidate phylum from rice roots (8) have been described in recent years. The term "candidate phylum" is reserved for phylogenetic lineages that show <75% sequence similarity to known phyla but that lack cultivated representatives or contain too few sequences for a precise phylogenetic placement.
In this study, we report the discovery of a novel, deeply rooting candidate phylum of bacteria in marine sponges. This study was originated because electron microscopic data suggested the presence of unusual bacteria with membrane-bound nuclear bodies in several sponges (12, 16, 17, 45). Because cell compartmentalization has so far only been observed in the phylum Planctomycetes (14, 26), the Planctomycetes-specific FISH probes Pla46 and Pla886 were used as PCR primers. After library construction and phylogenetic analysis, a novel lineage was identified that showed an average of 74.6% sequence similarity to any of the known bacterial phyla (Fig. 1a). The phylogenetic lineage was reproducibly monophyletic and stable after the application of different tree-building algorithms. Because of the lack of relatedness to known bacterial phyla and because bacterial cultivation has so far been unsuccessful (37), the status of candidate phylum and the name "Poribacteria" are proposed. This name was chosen to acknowledge the affiliation of these bacteria with sponges (phylum Porifera). The poribacterial lineage currently encompasses 12 nearly complete 16S rRNA gene sequences and 29 partial sequences from five tropical and one temperate verongid species. The average in-cluster similarity of this lineage is high (92.7%) compared to those of other published phyla (9). If this value reflects the limited resources that have been sampled so far (verongid sponges), then one would expect this value to increase as more sequences become available. Alternatively, if the "Poribacteria" lineage has been affiliated with sponges for evolutionarily long periods of time, reduced diversity as a result of adaptation would be expected.
The "Poribacteria" lineage is moderately related to the Planctomycetes, Chlamydia, and Verrucomicrobia lines of descent. Also, the lineage shows an affiliation with the soil clone PRR-1D, which was recovered in a PCR screening assay (the PV assay) that led to the discovery of considerable bacterial diversity in the Planctomycetes-Verrucomicrobia branch of the tree (8). The question arises regarding why the "Poribacteria" lineage has not been recovered in any previous environmental 16S rRNA gene library. One possible explanation is that there may be mismatches in the target region of the "Poribacteria" 16S rRNA genes in the universal primers 27f and/or 1492r, as has been reported for Planctomycetes (47) and some spirochetes (35). Similarly, the FISH probe EUB338, originally thought to target all bacteria, also did not hybridize with Planctomycetes and Verrucomicrobia, a flaw that has been compensated for by the construction of a more comprehensive probe mix, EUBI-III (7).
The 16S rRNA gene libraries made for this study also contained sequences of specific phylogenetic clusters of the Chloroflexi and Deltaproteobacteria that had previously been reported from marine sponges (Fig. 2b) (18, 49). Their amplification was most likely due to nonspecific primer binding, as there are three mismatches each between the Pla46 primer sequence and the corresponding 16S rRNA gene sequence of the closely related Chloroflexi and Deltaproteobacteria. However, their recovery is still noteworthy, as it extends their documented distribution to five additional tropical sponge species. Surprisingly, only 2 of 157 sequenced clones were affiliated with the Planctomycetes even though group-specific primers were used. We concluded, for the following reasons, that the Planctomycetes probably play a minor role in the microbial community structure of marine sponges: (i) with the exception of clones Q12 and Q32 from this study, planctomycete sequences have not been recovered from any sponge-derived 16S ribosomal DNA library to date; (ii) Planctomycetes have been cultivated from Aplysina sponges, but the application of FISH probes revealed that they are only minor components of the sponge-associated microbial community (37); and (iii) the application of the FISH probes Pla46 and Pla886 produced fluorescent signals on sponge tissue cryosections (13), but not when bacterial preparations were used (data not shown), indicating either nonspecific binding of the FISH probes to the host extracellular matrix or the lysis of planctomycetes during the bacterial extraction procedure.
In order to visualize members of the novel candidate phylum "Poribacteria" within sponge tissues, we designed a set of "Poribacteria"-specific FISH probes (Table 1). Cohybridization with the universal bacterial probe mix EUBI-III showed that the "Poribacteria" lineage is an abundant member of the sponge-associated microbial community (Fig. 2a). As expected, the application of the phylum-specific probe POR1130 with either the subphylum probe POR389 or POR600 resulted in the cohybridization of most cells (Fig. 2b and c). The remaining green and red signals reflect either a certain degree of nonspecific binding to nontarget microorganisms or the presence of as yet unrecognized poribacterial subgroups. It is also noteworthy that the resulting fluorescence forms a ring-shaped signal (Fig. 2b and c). This finding is consistent with the presence of a membrane-bound compartment in the target microorganisms (Table 2; Fig. 3). Simultaneous hybridization with a Chloroflexi-specific probe and a "Poribacteria"-specific probe resulted in more signal abundance than was reported previously for other phylum- and subphylum-level probes (Fig. 2d) (12).
Electron microscopic studies were performed to characterize the nucleoid-containing bacterial morphotypes (Fig. 2). The structural plasticity level was high with respect to cell shape, membrane structure, and cytosol density (Table 2). The cell shapes represented included short and long rods as well as D-shaped cells. The cell walls represented were typical cell walls of gram-negative bacteria but also the S-layer-type cell walls of Archaea. Morphotype 6 contains characteristic bleb-like structures. Previous reports showed that in morphotypes 1 to 5, the fibrillar DNA is located in the nucleoid, while in morphotype 6, the RNA is contained in the nucleoid and the DNA is located in the cytoplasm (17). Morphotype 6 shows some resemblance to Gemmatimonas aurantiaca, the first cultivated representative of the novel phylum Gemmatimonadetes (56). Both are rod-shaped cells with a gram-negative cell envelope. The characteristic outer cell wall is wavy, contains budding structures, and is separated from the cytosol by an atypically wide periplasm. Interestingly, Gemmatimonadetes 16S rRNA gene sequences have been recovered from the sponge T. swinhoei from Palau, suggesting that sponges may indeed be a reservoir for this novel bacterial phylum (56).
The results of this study did not permit us to correlate the electron microscopic data with the molecular analysis results with absolute certainty. However, the following lines of evidence suggest that the "Poribacteria" candidate phylum does represent the nucleoid-containing morphotypes. (i) The "Poribacteria" lineage is moderately related to the Planctomycetes lineage, which is the only other phylum known for cell compartmentalization. (ii) The abundance of FISH signals correlated well with the abundance of the nucleoid-containing morphotypes as examined by electron microscopy. In both studies, the "Poribacteria" amounted to an estimated one-fourth to one-third of the total microbial community. (iii) Of all the FISH probes tested in this and previous studies, only the "Poribacteria"-specific FISH probes fluoresced in a ring-shaped manner. This is consistent with immunogold labeling studies by Fuerst et al. (17), who showed that, at least for morphotypes 1 to 5, the RNA is absent from the nucleoid. Whether morphotype 6 belongs to the "Poribacteria" or to another lineage such as the Gemmatimonadetes remains to be investigated.
The candidate phylum "Poribacteria" appears to be specifically affiliated with marine sponges, as samples taken from the immediate environment (seawater, sediments, and a filter-feeding invertebrate) were PCR negative. However, these results do not necessarily mean that the "Poribacteria" are absent from the environment; it suggests, rather, that the "Poribacteria" are concentrated in sponges. Negative PCR results can be explained if the concentrations in the environment are exceedingly low, if bacteria are only seasonally present, or if other seawater phylogenetic lineages mask the amplification of the poribacterial 16S rRNA gene sequences. PCR screening with specific primers indicated the presence of the "Poribacteria" in other sponge species (X. muta and T. swinhoei) and in sponges from different geographic regions (Palau [T. swinhoei], offshore California [A. fistularis], and by electron microscopic evidence, the Great Barrier Reef [17]). However, not all sponges are equally well suited for the maintenance of the "Poribacteria," as some species gave negative PCR results (C. nucula, Agelas sp., and Ecytoplasia ferox).
The distribution of the candidate phylum "Poribacteria" is consistent with the general microbial pattern that has been described for several marine sponges (18, 19). Marine sponges have a unique microbial signature that consists of specific sequence clusters belonging to at least seven different known bacterial phyla. As a result of this study, the candidate phylum "Poribacteria" has been added to the list. To our knowledge, this is only the second report of cell compartmentalization for a prokaryotic lineage. With "Poribacteria"-specific PCR primers and FISH probes in hand, it is now possible to explore the abundance and distribution patterns of this elusive, sponge-specific candidate phylum.
We gratefully acknowledge the marine operations personnel at the Laboratoire Arago (Banyuls-sur-Mer, France) and the captain and crew of the R/V Seaward Johnson (HBOI, Fort Pierce, Fla.) for their expert help during sponge collection. We thank Christine Gernert (Universität Würzburg) for excellent technical assistance, J. Pawlik (UNC Wilmington) for cruise organization, and Feras Lafi (University of Queensland) for stimulating discussions during the 6th Sponge Conference.
Financial support for this work was provided by grant SFB567 to U.H. from the DFG.
- Received 25 November 2003.
- Accepted 23 February 2004.
- ↵*Corresponding author. Mailing address: Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931-312588. Fax: 49-931-312578. E-mail: ute.hentschel{at}mail.uni-wuerzburg.de.
- American Society for Microbiology


text:
Need of Transport System in Plants
Category : 7th Class
Plants are made up of mainly three parts, i.e. root, stem and leaves. Leaves develop in upper part of plants so that they remain in directly contact with atmosphere to trap \[C{{O}_{2}}\] and sunlight. Roots develop in lower part of plants and remain underground to absorb water and minerals from the soil. These raw materials, obtained by different parts of plant, need to be get together in the leaves for the process of photosynthesis. So for transporting these materials from one part to another part, plants need a transport system..
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subset: commoncrawl


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1 valoración en Stingray Bracelet Skull Head Black/Red
Can I just say what a relief to find someone who actually knows what theyre talking about on the internet. You definitely know how to bring an issue to light and make it important. More people need to read this and understand this side of the story. I cant believe youre not more popular because you definitely have the gift.


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River Rundown
Location: Lower Canyon
Weather: Sunny, slight breeze, air temp in the 70s
River Conditions: ~2400 cfs at Umtanum, ~3 feet of visibility, low 60s water temp. Water flow information here.
Hot Flies: Coffee Pat's (#6), Lightning Bug (#14-18), Frenchie (#14-16), Soft Hackle Caddis (#12-16), LaFontaine Sparkle Pupa (#16)
Bug Scene: THe Caddis are out, the stones are making a presence
The caddisflies are hatching late in the mornings and the hatch grows throughout the day. Fish start keying in on the surface in the early afternoon and begin to feed heavier around 7 p.m. or when the sun gets close to the canyon rim. Fish are eating caddis nymphs and emergers fished subsurface before the hatch. There are a few golden stones hatching higher in the river, as well as a few Yellow Sallies. There are some fish looking to the surface for larger bugs, but most of the stonefly game is going on subsurface right now. We are starting to see a few grasshoppers and drakes as well, so make sure to start packing a few larger dries!
Fishing Forecast: Nymph the mornings, focus on dries in the afternoon and evenings
Nymphing in the mornings is going to be your best bet. A Coffee Pat's (#6) 4-6 feet under an indicator trailed by a Gold Lightning Bug (#14-18) and a Soft Hackle Caddis (#12-16) has been producing. Focus on a bit heavier water, but play around to find where the fish are holding if you aren't finding them in a certain water type. Look around main currents and where there is structure.
The dry fly bite has picked up again in the Lower Canyon. Fish are looking up for caddis starting around 7 p.m. and are feeding heavily until dark. You can look for feeders during the day and anchor up to fish them, but it will not be consistent until later in the evening. Fishing an adult caddis like a Fluttering Caddis (#14-16) and trailing it with an emerger like a LaFontaine Sparkle Pupa (#16) has been productive with fish mainly eating the emerger. There are a few fish eating bigger bugs, so fishing a Purple Chubby Chernobyl (#6-10) trailed by a caddis emerger can be a productive rig as well. Larger attractors can also help you keep track of your smaller flies when it starts to get dark or when there are a lot of naturals on the water. Look to fish your caddis CLOSE to the bank. Fish are often eating right along the grass and trees that dip into the water. Fish will also look for caddis in foam lines and in soft pockets of water near heavy current. Anchoring on actively feeding fish is the best way to take advantage of the hatch. Be prepared to bring a few extra flies as you might be sending some into the trees!


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Bible Search
Passage Lookup: Job 18
Bible Navigator: of 42
21 verses
New International Version
Job 18
Chapter 181 Then Bildad the Shuhite replied:
2 "When will you end these speeches? Be sensible, and then we can talk.
3 Why are we regarded as cattle and considered stupid in your sight?
4 You who tear yourself to pieces in your anger, is the earth to be abandoned for your sake? Or must the rocks be moved from their place?
5 "The lamp of a wicked man is snuffed out; the flame of his fire stops burning.
6 The light in his tent becomes dark; the lamp beside him goes out.
7 The vigor of his step is weakened; his own schemes throw him down.
8 His feet thrust him into a net; he wanders into its mesh.
9 A trap seizes him by the heel; a snare holds him fast.
10 A noose is hidden for him on the ground; a trap lies in his path.
11 Terrors startle him on every side and dog his every step.
12 Calamity is hungry for him; disaster is ready for him when he falls.
13 It eats away parts of his skin; death's firstborn devours his limbs.
14 He is torn from the security of his tent and marched off to the king of terrors.
15 Fire resides[a] in his tent; burning sulfur is scattered over his dwelling.
16 His roots dry up below and his branches wither above.
17 The memory of him perishes from the earth; he has no name in the land.
18 He is driven from light into the realm of darkness and is banished from the world.
19 He has no offspring or descendants among his people, no survivor where once he lived.
20 People of the west are appalled at his fate; those of the east are seized with horror.
21 Surely such is the dwelling of an evil man; such is the place of one who does not know God."
21 verses
Copyright Statement:
New International Version
THE HOLY BIBLE, NEW INTERNATIONAL VERSION® NIV® Copyright © 1973, 1978, 1984, 2011 by Biblica, Inc.® Used by permission of Biblica, Inc.® All rights reserved worldwide.
These Scriptures are copyrighted and have been made available for your personal use only. Any other use including, but not limited to, copying, posting or reposting on the Internet is prohibited. These Scriptures may not be altered or modified in any form and must remain in their original context. These Scriptures may not be sold or otherwise offered for sale.
These Scriptures are not shareware and may not be duplicated.
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Biblica provides God's Word to people through translation, publishing and Bible engagement in Africa, Asia Pacific, Europe, Latin America, Middle East, and North America. Through its worldwide reach, Biblica engages people with God's Word so that their lives are transformed through a relationship with Jesus Christ.
Lectionary Calendar
Wednesday, October 16th, 2019
the Week of Proper 23 / Ordinary 28


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Secret File #029: Branding Starts With You
So you've probably heard about the phrase "Your vibe attracts your tribe" — probably a lot of times, especially if you are following a lot of women entrepreneurs on Instagram, on Facebook, and even watching them on YouTube. In this episode, we're going to discuss this phrase and what it means for your branding.
In this episode, you'll hear about:
How branding starts with you
People resonate with you
Be on-brand
Support your followers
Listen to the Episode below:
What do you think?
I'd love to know what you think of this episode. Do you plan on leaving your full-time job soon to start your business? If so, where are you now in that plan? Share with me in the comments below!
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Loving our podcast? I encourage you to use the hashtag #FEMPRENEURSECRETS to share with our community of Fempreneurs what you're inspired by. As a bonus, you'll get to go through all the posts to find women entrepreneurs who are in your similar industry — get inspired to collabore and go beyond competition. I'll be reposting some of my favourite posts too!
Thank you so much for listening!
Welcome to Fempreneur Secrets where business secrets are revealed. In today's episode, I am going to talk about your vibe and how it attracts your tribe. I'm sure you've heard of this before, so we're going to go down a little bit deeper into how branding starts with you.
So you've probably heard about the phrase "Your vibe attracts your tribe" — probably a lot of times, especially if you are following a lot of women entrepreneurs on Instagram, on Facebook, and even watching them on YouTube. First, really understand your vibe.
What is your vibe?
So your vibe is what you give off to other people when they meet you for the very first time. But what happens if they can't meet you? Nowadays, we are doing our businesses predominantly online, so probably 90% of the time the business is being run online. So how then do you deliver your vibe across to attract the right audience that you want and attract your tribe?
Why Your Vibe Attracts Your Tribe: How Branding Starts With You
1. People can resonate with you
The first thing that you need to know is that your vibe attracts your tribe because people can resonate and you can relate to you. So the fact that it can relate to you means that you have something to offer that they really believe in.
So when we talk about vibe, what we are referring to is that:
- ability to deliver a message across clearly; and the
- emotions associated with it.
Branding really starts with you and it really helps you attract who you are. So if you are a very fun, bubbly and very realistic person, you will start to attract those type of people into your business.
The more you are behaving like who you want to attract in your business, the more they're going to appear in your direction.
So the more you are behaving like who you want to attract in your business, your dream clients, your ideal business partners, your ideal customers, the more they're going to appear in your direction. If you're a very positive person, you will tend to attract more positive people as well. And if you are someone who is very empowered, you will tend to attract people who are feeling empowered as well. So, you attract who you are.
So you have to be the best version of yourself when you are delivering your message across online regardless of what platform.
And if you are delivering your message offline, then you would have to exude that kind of positivity, that kind of emotions and those values that you really and truly believe in even in your day to day conversations.
Be On-Brand & Don't Confuse Your Audience
Step number two is to be on-brand and avoid confusion. I have been guilty of this a few times as well, so I can really tell you that there is a lot of things that you can do to perfect yourself from being confused by other values.
I started off my brand completely related to who I am at that point in time. I am actually quite a religious person, but I decided not to portray that anymore in my branding because I want people to look at me beyond my religion and I want people to look at me based on my values that I carry through in my life.
So I did a little tweak of all the things that I've ever done and said in my business. So I really removed and got rid of all the things that are off-brand. It took a lot of work. But that effort of reducing the confusion from my audience really helped me attract a better pool of audience.
Of course, it doesn't necessarily mean that if today you change your brand, that instantly all these ideal clients will come in. It's not overnight, but they will start appearing and they will start believing in your message. And at the end of the day, that is what you want to do. And that's what you want to achieve.
Do you want your business to grow? So if you want to do that, you have to be on brand, you have to stay on your path and you really have to up your game by avoiding confusion in your message.
So they need to understand who you are. And you can just start off with the simple things like identifying five core values that you truly believe in and then focusing on that all throughout your messaging. So every single thing that you're going to talk about online, offline has to be related to those core values.
Support Your Followers
And the third step about branding and how it starts with you is to support your followers.
So what I mean by this is that your followers, I'm not talking about the numbers on social media, I'm talking about actual followers in your tribe. They will tend to support you far longer if you are able to support them as well.
So usually they're gonna come to you because you have a solution to a current problem that they are facing in their business or in their life. When you are offering that service, or that product that solves their problem, they will be more than happy to stick around.
The challenge here is to identify what is the solution that you provide and what is the problem? What is the pain point that they are facing? A lot of businesses struggle with identifying the pain points of your business. I mean, it's not an easy thing to identify.
And truth be told, when I was writing my business plan for the very first time, I probably didn't know what the pain point of my customers are.
But as you go along and as you grow your business, you start to see and start to tweak your business according to what those pain points are. More and more people will come towards you and they'll be part of your tribe. The more they are associated and the more they can relate with your tribe, the more they'll be willing to help you.
By helping you, it's not just about buying your products. They can also help you by giving feedback and I think feedback is really a gold mine that you really need to tap on. The feedback that they can give could be as simple as "I feel that this is my problem before working with you and I never knew I had this problem, but after I worked with you I realized that I had that problem solved".
So that is such a gold mine right there that if you do not tap on, you will be missing out in growth for your business. So your vibe really attracts your tribe and it can help you position and develop your business into the next level. So much growth that you can have potentially for your business when you reposition your message, clarify our message, and really attract the right tribe. If you want to attract the right tribe, it is time for you to fix your vibe.
Identify the cause that you believe in, and also straighten out and clarify your message.
If you love this episode, be sure to leave a review on apple podcast. It will help me know what type of content we produce for you. Also, if you have any questions that you would like me to answer in our podcasts, go on over to FempreneurSecrets.com/podcast to drop those questions.
Keep learning and keep believing in yourself because the one and inspiration and just like you, and I'll see you in the next episode of Fempreneur Secrets.
Access FREE Resources
Gain access to FREE Resources to help you start and grow your business
Some of the resources I've used to pick myself up in business are in the Secret Vault. I've created them and stored them in one location, just for you. Gain access now.
Till the next episode, This is Huda — Empowering Women Through Business.


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Travail non-trouvé
Désolé, nous n'avons pas pu trouver le travail que vous cherchiez.
Trouver les travaux les plus récents ici :
I need you to write a research article.
Looking for React developers to join a small team to work on project on React, GraphQL, Prisma, Apollo. Any experience are welcomed. Please answer the following questions when applying? 1. Why do you program? 2. How do you free up your brain RAM? 3. How do you structure your React apps? 4. What you do if you encounter a bug in a third-party library?
Hi,I want to hire a web developer who can design my new restaurant website. Kindly get back to me via my Email address at,email@example.com. Regards
This is the first of 30 buildings we require. Successful candidate will receive future work. Using illustrator, please draw a vector line art illustration of the buildings in the attached jpeg. There is also a sample of the style to copy. - Must be supplied within 2 days - Must be reasonable price, there are 30 more jobs to complete after this - Must be editable stroke (i.e. I can change stroke ...
#Research/List building - Must be able to research potential clients that meet our requirements. (Simple Google searches will suffice using keywords we provide). - Add potential clients to a list with details name, phone, email etc (excel to start with then into a CRM like HubSpot later). #Calling/Prospecting - Call potential clients to introduce the software, qualify that the software could be u...
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Hi,I want to hire a web developer who can design my new restaurant website. Kindly get back to me via my Email address at,firstname.lastname@example.com. Regards
Hello, I want a person who can shoot videos with voice over and also can make screen recorder videos. Its a long term project. You can also hid your face.


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1. Kuler
A creative an intuitive swatch color website from adobe. You can choose a color theme, save it to Adobe products and use them in there. You can also pick apart the colors on pictures uploaded to flickr. This is a must have website for web graphic designers and possibly print designers too.
2. Ma.Gnolia
Aside from featuring a design considerably better than Del.icio.us, this new social bookmarking site is much more intuitive, friendly, and manageable than its rivals. It offers DO-Follow links such for you SEO lovers out there I highly recommend this social booking marking site to you.
2. Bright Creative
"Bright Creative is a one-man design & web studio run by Dave Shea, of css Zen Garden and mezzoblue fame¹. Based in Vancouver, Bright Creative takes web and application design commissions from clients around the globe. "
4. Popmatik
The creative design portfolio of a web/interactive based designer. The reason this website made the list was it's decision to use an empty bottle of the focal point of the user's eye. Simple, clean, and easy to navigate. Popmatik manages to set itself aside in a world of design portfolios.
5. Davor Vaneijk
An online impression of Davor Vaneijk's work, information about who he is and some personally designed downloads. It's the layout that had me sold from the get-go with the creative use of typography in a web based system. The relationship between generally dull colors. (the lack of web 2.0 glossy buttons made it worthwhile too). Check out his work for inspiration.


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Construction works in and around Maastricht
In the coming years Maastricht will be working hard on the improvement of the city's infrastructure. In 2016 the A2 tunnel will open, the above ground refurbishment of the Groene Loper will continue, and we will see the commencement of the Noorderbrug diversion and the construction of the underground bicycle parking area at Maastricht central station. Thus making Maastricht and the region increasingly more accessible and contributing to a better living and working environment. In addition, we are ensuring that the city and the surrounding areas remain accessible during these construction works through the intelligent planning of road closures, detours and by offering great alternatives to travelling by car.


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Now Contributing to Philliedelphia.com!
A quick post to promote the fact that I am now a weekly contributor to the Phillies-themed blog, Philliedelphia.com. Rest assured, this activity is not replacing The Phillies Collector blog. Instead it will be another outlet for me to talk about Phillies memorabilia, and hopefully expose my writing to a audience that might not otherwise be seeking information on the topic. As someone who has always wanted to write material that people read, it is a great opportunity.
Here is the link to my contributions. Please note: all posts on Philliedelphia.com are "new content", not copied from this blog. Also, in case you want to check back, I usually upload my posts to the site on Sunday morning...
Thanks for the support!


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Flint Dille
director: Jim Gillespie
starring: Flint DilleMeagan GoodLaura Ramsey
Venom is a 2005 American voodoo horror-of-the-demonic horror film starring Agnes Bruckner, Jonathan ...
director: Simon WellsPhil Nibbelink
starring: Flint DilleAmy IrvingDom DeLuise
(This article is about the film. For the 1994 video game, see An American Tail: Fievel Goes West (vi...
Browse our database of best movies which you can watch on popular VODs platforms. You can find films for you by filters or right side of screen. You can also use search field on top of screen and find movie by title. Right now you see filtered titles, if you want see all you need reset filters.
Do you enjoy watching movies, but you do not know which movie to choose and where can you view it? Movs.io will help you find videos especially for you. In addition, you'll be able to check if the video is available on your favorite Video On Demand site (VOD)
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Love T-shirt
Freya Moses, who has hearing loss and tinnitus, has designed an eye-catching T-shirt that spells 'love' using the British Sign Language alphabet. She says: "I'm helping raise money for Action on Hearing Loss as a thank you for all of the support they have offered me, since getting hearing aids. You would be supporting an amazing cause."
This T-shirt is now available in rainbow colours to celebrate LGBT pride.
To get your T-shirt delivered for Brighton and Belfast Pride please order before Thursday 18 July.
You can buy this T-shirt for £14.99 plus postage and packaging.
Please note, you'll be sent to a third party supplier webpage to buy your T-shirt.
Pride Parade dates:
Glasgow Pride: 17-18 August, Brighton Pride: 2-4 August, Pride Cymru: 23-25 Aug, Belfast Pride: 3 Aug


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No comeback for Kasatkina as Svitolina wins again
• Top-seeded Elina Svitolina retained her Dubai Championships title on Saturday with a 6-4, 6-0 win over unseeded Daria Kasatkina, who was finally unable to make a comeback.
It was the 11th singles title for the 23-year-old Svitolina, who had won a tour-high five titles in 2017, and added Brisbane to that list earlier this year. The fourth-ranked Ukrainian improved to 14-2 for the year.
• Third-seeded Lucas Pouille remains on course for his second ATP title of the season after beating qualifier Ilya Ivashka 6-3, 7-6 (6) to reach the Open 13 final in Marseille, France. Pouille will face Karen Khachanov of Russia, who beat fourth-seeded Tomas Berdych 6-3, 6-2.
• American wild-card Francis Tiafoe reached his first career ATP final with a 7-5, 6-4 semifinal victory over Canada's Denis Shapovalov in the Delray Beach Open in Florida. The 91st-ranked Tiafoe will play Peter Gojowczyk of Germany, a 7-6 (3), 6-3 winner over American Steve Johnson in the afternoon.
• Spain's Fernando Verdasco and Argentina's Diego Schwartzman advanced to the Rio Open final in Brazil. The eighth-seed Verdasco beat Italy's Fabio Fognini 6-1, 7-5, and the sixth-seeded Schwartzman topped Chile's Nicolas Jarry 7-5, 6-2 in the clay-court event at Jockey Club Brasileiro.
College SPORTS
Arizona's Miller out against Oregon after wiretap report
• Arizona's Sean Miller did not coach against Oregon on Saturday night, a day after ESPN reported through anonymous sources that he was heard on an FBI wiretap discussing a $100,000 payment to current Wildcats freshman Deandre Ayton. The school said Ayton was eligible to play against the Ducks and associate head coach Lorenzo Romar would lead the No. 14 Wildcats.
• Shaquille O'Neal's son, Shareef, announced on Twitter that he is opening up his recruitment, citing the problems at Arizona. O'Neal is one of the nation's top high school recruits and previously committed to Arizona.
• Miles Bridges was cleared to play by the NCAA, allowing him to help No. 2 Michigan State chase championships. Documents in a report showed the star forward may have improperly accepted benefits from an agency implicated in a federal investigation involving college basketball corruption.
Other sports
Jaguars sign QB Bortles to three-year contract
• The Jacksonville Jaguars signed quarterback Blake Bortles to a three-year, $54 million deal that will keep him under contract through the 2020 season. The deal includes $26.5 million guaranteed.
• The Edmonton Oilers traded defenseman Brandon Davidson to the New York Islanders for a 2019 third-round draft pick. Davidson, 26, has three goals and two assists in 36 games with Edmonton and the Montreal Canadiens this season.
— ABJ/Ohio.com wires
Sports news roundup, Feb. 25: Svitolina keeps Dubai title


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External Door Furniture
This image gallery named as Custom Tri Fold Closet Doors Avie Home might be ideal solution for your decoration. As the author has insert this image into Custom Closet Doors post section, would ease you to find the perfect match of you decor desire.
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WHILE leaders Ponteland United and Percy Main were on cup duty, Alnwick Town and Harraby Catholic Club won and share second spot.
Unbeaten Alnwick won 6-2 at home to Gateshead Rutherford. Ben Keenan completed his hat-trick for the Northumbrians, whose other goals were shared by Dale Kennedy, James Swordy and Stephen Hogg.
Rutherford's Isaac Mitchell opened the scoring with their second from Michael Jeffares.
Harraby extended an unbeaten run to six games by beating hosts Shankhouse 2-0, he home side twice denied by the woodwork, but soon trailing to Mark Worrall's two early goals for the Cumbrians.
Worrall claimed his first after only eight minutes with a shot across home goalkeeper Tony Deakin, under pressure from Stuart Moffat and Daniel Balmer.
Then, midway through the half, Worrall doubled Harraby's lead, beating Deakin with a swirling effort from the corner of the area.
Worrall was close to completing his hat-trick on the hour, but his chip was disallowed for a handball.
After drawing two games on the trot, Blyth Town proved unstoppable in their 7-3 home success against Heaton Stannington.
Michael Connor's side were four goals to the good at half-time and there was no let-up on the resumption.
Jonnie Mann nabbed a hat-trick for the winners and his colleague Gary Day bagged a brace.
The Town's other marksmen were Jeff Fuller and Ian Herron, who had just been signed from Morpeth Town.
Heaton had inflicted a first defeat on Percy Main in midweek, but were second best this time despite Adrian Watson, Kevin Carr and Michael Waggott netting.
Ashington Colliers recorded an impressive 3-1 victory at Wark, where their counters were shared by Michael Hogg, Dave Brown and Jamie Patterson with Stuart Brown responding for the losers.
Walker Central overcame Killingworth Sporting 1-0 at Monkchester where Liam Mulligan scored their first-half winner from the spot.
A red card for Liam Callaghan on the hour did not help Killy.
Stocksfield improved their position in the basement by beating visitors Murton 3-1.
Liam Wintrip was Murton's scorer, but their hosts sealed the points with replies from Graham Hudspith and Jack Wanless (2) – who was making his debut after moving to Stocksfield from Wark.
Carlisle City and their visitors Seaton Delaval had suffered from a loss of form before meeting up at Sheepmount, with City made to work hard for a 2-1 result.
Stephen Bell gave the home side a 17th-minute opener, holding off two defenders before squeezing his shot past Delaval goalkeeper Dave Herrington.
City's lead was doubled in the 34th minute with a cross-cum-shot from Jamie Heath, which went in off the back post.
The visitors were always threatening but had to be content with a reply on the hour from John Fitzpatrick, whose header came off Carlisle's Lee Otway on its way into the net.
Ponteland United remained two points clear at the head of the table but the holders lost 2-1 to Percy Main in their Challenge Cup preliminary round clash at the Leisure Centre Ground – both the victors' goals claimed by Tony Browell. He ended 52 minutes of stalemate with his headed opener from Dean Ellis's corner kick and bagged his second only two minutes later.
From another flag kick, this time curled in by Pierre-Luc Coiffait, Browell forced the ball home from close range.
Richard Kent replied in the 85th minute for United from Gavin Wood's cross before Dean Gate almost bagged an equaliser, his drive striking Rob Rodgerson without the Main goalkeeper knowing much about it.


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Cescon Barrieu is one of Brazil's leading law firms. The lawyers stand out for their strong commitment to the representation of clients' interests and for their work in highly sophisticated, groundbreaking matters. The firm's goal is to be the clients' law firm of choice for their most complex legal matters and most strategic transactions and disputes.
Main areas of practice
Corporate law and governance, mergers and acquisitions, and private equity: the firm is often retained by potential targets and boards of directors for representation in connection with mergers, acquisitions, tender offers, de-listings and other corporate transactions, as well as matters involving antitrust and anti-corruption laws. The M&A and private equity practices are recognized as Brazil's leading practices by any measure. The clients come to the firm whenever the scope of a transaction, no matter how large or small, is strategic and the stakes are high.
Capital markets and investments funds: the capital markets team is recognized as Brazil's most experienced and far-reaching. It represents the world's leading financial institutions, public and private corporations, hedge funds, asset managers and entrepreneurs, in connection with their most complex domestic and cross-border securities transactions, including IPOs and other equity offers, tender offers, debt-offerings, derivatives, structured products, mutual funds, receivables funds and securitization.
Its vast experience with the Brazilian Securities Commission (CVM) provides the firm with insight into important regulatory aspects of registered offers in Brazil that may be critical in successfully completing a transaction.
Banking and finance: the firm advises a wide range of clients in the roles of lenders, borrowers, trustees, rating agencies and others in connection with syndicated bank facilities, trade financing, leveraged investments, structured financing, DIP financing and derivatives. The firm also regularly represents clients who trade in banks and other financings in the secondary market, such as hedge funds and other asset managers.
Insolvency and restructuring: the team has wide-ranging experience in assisting clients in connection with complex debt workouts, corporate restructurings and bankruptcy proceedings, with a particular focus on representing financial creditors. The firm has vast experience in credit recovery, enforcement of collateral and other court-assisted proceedings in a wide range of industries.
Litigation and Arbitration: the disputes team is recognized for its breadth of experience and talent at simplifying even the most complicated cases. We are retained by clients from all industries to litigate their most critical cases.
Our lawyers are specialized in high-stakes litigation involving M&A transactions, commercial contract disputes, environmental cases, and technology and IP litigation. We represent clients in international and domestic arbitrations. We have handled several landmark corporate arbitration cases and some of the most complex arbitrations in construction, infrastructure, M&A, technology, and other matters. Our lawyers are also retained to act as arbitrators, expert witnesses, and co-counsel in multi-jurisdiction arbitrations.
Our client list includes multinationals, private and public corporations, investment funds, financial institutions, and entrepreneurs who come to us for assistance with a variety of disputes.
Project finance: the project finance practice is recognized as a market leader in Brazil. The firm's team has advised on groundbreaking transactions, such as the first greenfield limited recourse, cross-border project finance, the first non-recourse oil production-based project finance, the first cross-border project bond and the first cross-border oil royalty-backed bonds.
Infrastructure and project finance: the firm has a first-class practice in the development of infrastructure projects across all its sectors, such as energy, telecom, oil and gas, water and wastewater, ports and navigation, airports, railways, mining and toll roads. The firm provides advice on all phases of a project, from its conception to its financing, assisting companies in structuring projects, participating in bidding procedures and in claims with the regulatory agencies and granting authorities. The firm has a multidisciplinary team specialized in drafting and negotiating project contracts, such as EPC contracts, offtake contracts and O&M agreements, as well as in EPC contract management, assisting clients with managing claims during the construction.
Mining: the team has a comprehensive knowledge of the sector's entire production chain and in-depth expertise in dealing with its main players. We provide regulatory and negotiation counsel to mining and financing companies, in addition to regulatory and business advice to mining companies within M&A, project finance and capital markets transactions. The Cescon Barrieu team also advises autonomously on regulatory matters and contracts with joint venture partners, suppliers, or service providers.
The expertise of our mining lawyers, as well as their integration with all of our practice areas, enables our partners and associates to have a broader view of their projects, identifying possible structures and tailor made solutions for our clients and deals, always adhering to the highest quality standards.
Government relations: the firm combines corporate experience and strategic location in Bras?lia to provide legal advice to clients in the dialogue between companies and the public sector. The firm's objective is to translate to clients the main legislative issues and public policies and their respective impact over the economy and on the business sector. Clients include multinationals, domestic and foreign corporations, investment funds, financial institutions and entrepreneurs in several sectors such as health, pharmaceutical, mining, fertilizers and technology.
Antitrust: the firm combines broad experience in strategic corporate transactions with complex cartel investigations to form one of Brazil's leading antitrust practice groups. The firm works closely with its M&A, private equity and litigation groups to represent clients in a wide variety of domestic and cross-border matters across all industries.
Compliance and anti-corruption: the firm advises on a wide range of compliance and anti-corruption matters, including investigations involving anti-corruption and anti-money laundering laws. The compliance and anti-corruption lawyers help the firm's clients identify and anticipate tendencies in regulatory standards that may be critical to their business.
Technology and intellectual property: the firm's practice is recognized for advising clients in connection with complex IP and technology transactions. The team is also active in advising clients in a wide variety of regulatory matters faced by companies in the tech sector, including related to data protection and the legal framework for fintechs. Clients include technology and media companies, startups, retailers, venture capital and private equity funds, financial institutions, electronic platforms, serial investors and entrepreneurs.
Tax: tax has been the cornerstone of the firm's practice since its inception. The team of experienced tax lawyers advises on a broad range of domestic and international tax matters in financings, acquisitions, mergers, investment funds and international trade, as well as financial products.
Labor: clients come to the firm's labor law department for complex labor law issues rather than their day-to-day labor claims. The firm has over the years assisted clients with strategic and complex lawsuits, created compensation and incentive programs and assisted them in dealing with trade unions.
Real estate: the real estate practice is at the forefront of the marketplace. The firm advises a myriad of clients on some of the most complex and innovative real estate transactions, including land acquisitions, development, financing, joint ventures and restructurings.
Other areas of practice
Environmental, agribusiness, insurance and reinsurance, regulated industries such as power, oil and gas, renewable energy, maritime, transportation, logistics and shipping.
- Partners
- 40
- Other fee-earners: 185
- Languages
- English
Above material supplied by Cescon, Barrieu, Flesch & Barreto Advogados.


text: Christian Le headed a late winner after being picked out by a Josh Watson delivery as Town triumphed 2-1 at Melton Town last Saturday. Huntingdon hit the front on the half-hour when Richard Baines was left with the simple task of finishing a Herbie Panting cross. They were pegged back on the stroke of half-time and that is the way it stayed until Le struck with three minutes to go. Brattan said: Both goalkeepers made good saves and both teams missed good chances in the first half. Melton then had more of the possession and created the better opportunities in the second half. We set out to frustrate them and to try to hit them on the counter-attack when we could. It paid off in the end with the winner. Hats off to my three boys at the back Liam Rodden, Odie Wason and Josh who were superb and they stood tall when we had to weather a storm at times. Were enjoying a decent run and it was pleasing to bounce back from a disappointing result at Lutterworth Town the previous Saturday. Now Huntingdon have to avoid slipping on a potential banana-skin this Saturday when they entertain rock-bottom S & L Corby at Jubilee Park, 3pm. The basement side are yet to pick up a point this season, but Town were made to sweat before winning 1-0 in the reverse fixture last month thanks to a stoppage time goal. We made hard work of winning at their place, admitted Brattan. S & L will try to fight and scrap for their first points. Theyve only been losing by the odd goal lately which suggests they are improving and they certainly cant be taken lightly. Before then, Town go to higher-level St Ives in a Hunts Senior Cup quarter-final tomorrow night (Tuesday). Defenders James Seymour and Callum Walter are available after serving suspensions. Midfielder Chris Down made a first start against Melton since collapsing in an August game at home to Buckingham. Town have appointed Nather Al-Khatib as assistant boss to Brattan. He initially joined the club as a coach earlier in the season. Al-Khatib also coaches in the Cambridge United youth set-up.


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- By Southern Times --
- Jul05,2019 --
- 0 Comments
By Penelope Paliani-Kamanga
DEDZA - Malawi's President Peter Mutharika this week vehemently dismissed claims that he is in hiding after his victory in the most disputed May 21 presidential race, insisting he has no reason to hide.
There have been reports that Mutharika had been staying in Chikoko Bay state house in the lakeshore district Mangochi, avoiding his official residence at Kamuzu Palace in Lilongwe because he was fearful of protesters questioning his victory as declared by the Malawi Electoral Commission (MEC) chairperson Jane Ansah on May 27 this year.
But Mutharika, speaking at the commissioning of the 800-hectare Bwanje Valley Irrigation Scheme and Dam at Nsekeni Primary School ground in Mtakataka, Dedza, said he would never abandon Malawi and that he feared no one.
"Some people are alleging that I have fled the country to Mozambique. That is not true. I fear no one in this country," said Mutharika.
The country's immediate past vice-president, Saulos Chilima of UTM Party, and Malawi Congress Party (MCP) president Lazarus Chakwera, are challenging results of the presidential election claiming that they were rigged in favour of Mutharika who got 38 percent of the vote.
The results announced by the MEC show that Chakwera trailed Mutharika by 35 percent while Chilima, who was until May this year vice-president to Mutharika, finished third with 20 percent of the votes.
Currently, Chilima and Chakwera are in court as first and second petitioners, respectively, seeking nullification of presidential election results over alleged irregularities, especially in the results management system.
A panel of five judges of the High Court of Malawi, sitting as the Constitutional Court comprising Healey Potani, Dingiswayo Madise, Ivy Kamanga, Redson Kapindu and Mike Tembo, set July 29 as the date for the hearing of the substantive case.
Ever since his victory declaration, Mutharika has only been to Lilongwe once, and spent two nights, before leaving for Chikoko Bay state lodge in Monkey Bay, Mangochi, soon after delivering a State of the Nation Address (SONA) in Parliament on June 21.
MCP legislators booed the president and walked out on him as he delivered the SONA, saying they did not recognise his legitimacy.
Mutharika amassed a majority of his votes from the Southern Region while Chakwera dominated in the Central Region and Chilima was the favourite in the sparsely-populated Northern Region.
During his address this week, the President said the election proved his earlier declaration that he would be re-elected, drawing applause from his partisan audience predominantly clad in the Democratic Progressive Party (DPP) blue cloth.
Mutharika claimed his challengers failed to dislodge him because they lacked the vision to take the country on the development path.
However, Public Affairs Committee (PAC) and the European Union Election Observer Mission expressed some reservations. The European Union Election Observer Mission also faulted the results management system.
Just a day before he was to address the National Assembly when he was coming from Chikoko Bay, his first trip to the capital city since getting re-elected in the May 21 elections, a group of people blocked the M1 road forcing, President Mutharika to use the Lilongwe West bypass road as he was going to Kamuzu Palace.
Mutharika had been expected to address his DPP supporters at Malangalanga traffic lights in the city.
However, his plan was ruined by suspected MCP supporters who were involved in running battles with police around Biwi Triangle along the M1 road.
The people were coming from the Lilongwe High Court for the Malawi election case and at Biwi Triangle, the group burned wood and tree branches on the M1 Road.
Police officers fired teargas to disperse the crowd and clear the road.
Following the fracas, Mutharika changed plans and used the bypass to travel to Kamuzu Palace.
Meanwhile, the Human Rights Defenders Coalition (HRDC) has been holding protests to force MEC chairperson Jane Ansah to resign.
Protesters held vigils in Lilongwe while in Mzuzu, Zomba and in 14 other districts, the protests took the form of street demonstrations.
But MEC chair Ansah has said she will not resign as demanded by the protestors and rebuffed allegations made by Chilima that she favoured Mutharika in the May 21 to the disadvantage of his challengers.


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Current Budgeting Practices in U.S. Industry: The State of the Art
Quorum Books, Jan 1, 1987 - Business & Economics - 176 pages
Umapathy, in conjunction with the National Association of Accountants, surveyed over 400 companies nationwide to compile a profile of budgeting practices and budget manager qualifications. He also sought to determine whether there are significant variations in practices in different industries, different management philosophies and between financially successful and unsuccessful companies. In the last two chapters he provides a number of suggestions for improving budgetary practices by examining those used by successful companies and exploring the implications of the research for managers and academics. He briefly describes a model of effective planning and control systems taht combines the perspectives of accounting, organizational beharior, and strategic policy. Accountants in industry will find this an interesting work with a lot of useful information to help appraise the relative state of their companies' budgetary practices. Academic accountants will encounter much interesting data and a good syntheses of recent research on the subject. "Journal of Accountancy"
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Research Objectives and Methodology
Characteristics of the Participants
Management Philosophy and Budgeting
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The Home Affordable Modification Program, or HAMP, is ending on December 31, 2016. Learn more about what HAMP is, who it's for and how YOU can benefit.
History of HAMP
Many factors contributed to the 2008 Foreclosure Crisis. The key factor, by far, was that lenders were offering unusually low but temporary interest rates in an effort to get more people into homes. But once those low interest rates increased and other issues arose , such as loss of income, several homeowners found that they owed more on their homes than they were worth and could no longer afford their payments. They were upside down on their mortgages without any sign of relief.
Lenders panicked with the historical influx of potential foreclosures on the horizon. They were simply ill-equipped to provide relief to the large amount of potential foreclosures, and focused on their collections and foreclosure processes, instead. This placed even more of a burden on homeowners – so the Home Affordable Modification Program was introduced.
What is the Home Affordable Modification Program?
First, it's a voluntary program that needs to be applied for. The program application is also known as the "Request for Mortgage Assistance" or RMA. In response to the most recent foreclosure crisis, the Obama Administration sought to provide financially-struggling homeowners with a way to keep their homes. So, the program provides a way for homeowners to keep their homes and work with lenders to continue payments, but at a lesser amount, to deal with either loss of income or no income at all. The program was designed to modify mortgage conditions by extending homeowners' loan terms, fixing or lowering their interest rates or providing a payment deferral. In some instances, deferments were made so that borrowers would not continue to get behind on their mortgage payments.
Second, homeowners must meet the current requirements and criteria in order to be eligible. You may be eligible for HAMP if you meet the following basic criteria:
- You are struggling to make your mortgage payments due to financial hardship.
- You are delinquent or in danger of falling behind on your mortgage.
- You obtained your mortgage on or before January 1, 2009.
- Your property has not been condemned.
- You owe up to $729,750 on your primary residence or one-to-four unit rental property (loan limits are higher for two- to four-unit properties).
There are other considerations for homeowners interested in applying for this program. For example, when they are calculating their mortgage payments, they should take into account all of their expenses. Proof of gross income is also necessary. In the instance of a self-employed homeowner, their adjusted gross income is used.
What are the Main Benefits of HAMP?
- HAMP achieves a more affordable payment by adjusting your interest rate, extending your term, and reducing or forbearing your principal.
- HAMP homeowners typically save about $500 per month.
- Through HAMP, you can get help with your primary residence or rental property.
- If you owe significantly more than your home is worth (>115 Loan-To-Value), you're automatically evaluated for principal reduction.
- Just for making timely payments, you could earn up to $10,000, which would be used to reduce your principal balance.
Why HAMP is Ending and What it Means for You
The RMA was designed in 2009 as a temporary program to help homeowners in a time of a crisis, which, largely, no longer exists. This is evidenced by the decreasing number of new borrowers who have entered the program.
The program has helped an incredible 5 million homeowners avoid foreclosure through mortgage assistance programs. And the current timeframe still allows homeowners to take advantage of the program. If you are in need of mortgage modifications, now is the time because the HAMP program ends on December 31, 2016.
Find Out if You're Eligible
At Vokshori Law Group, we have made it our mission to help homeowners like you to keep your home through loan modifications. If you're struggling to keep up your payments, contact us TODAY for a free consultation.


text: How is seismic discovery important to us? Astronomy Our Solar System The Earth 1 Answer SCooke Feb 16, 2018 Answer: Seismic surveys of the earth tell us a grae deal aboutits structure and composition. Explanation: Seismic tests reveal the types of materials making up the crust, mantle, inner and outer cores of the planet. Related questions What's the Earth's "absolute" speed? i.e. in relation to the central Sun, the first-order star, or ... How does density affect earth's layers? How fast does the earth rotate? How does this compare to the rotation of other planets? At what speed does the earth travel on its orbit around the sun? What would happen if the earth's rotation was faster or slower? How fast does earth rotate near the equator? How fast does the earth rotate per second? How fast is the earth traveling along its orbit? How fast does the earth rotate on its axis? How fast does the earth rotate in one minute? What is the formula to determine this? See all questions in The Earth Impact of this question 225 views around the world You can reuse this answer Creative Commons License


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This is Jeff Bayer, and I don't update this site very often. If you'd like to listen to my current movie podcast you can find it at MovieBS.com.
All tagged Identity Thief
Cast: Jason Bateman, Melissa McCarthy, Eric StonestreetRunning Time: 1 hrs 52 mins Rating: R Release Date: February 8, 2013
Final Score: 4/10
Reviewed by Jeff Bayer


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Marc by Marc Jacobs new backpack: Casting Call Packrat
Marc by Marc Jacobs released the new nylon fabricated backpack the Casting Call Packrat which features the embroidered logo of a cigarette and lipstick forming a cross shape with the words "Marc Jacobs 1984"; a historic year for the brand. In front, there are double zip exteriors below the logo. Marc by Marc Jacobs Casting Call Packrat Is now available at Marc Jacobs online