§ 2-509.5 Members not to hold other office.
Editor's Note: This chapter was renumbered from § 9-305.5 to Ch. 2-509 4-9-2013 by Ord. No. 3-2013.
The Harrisburg Environmental Advisory Council (hereinafter "EAC"), heretofore created for the purpose of discussing and disseminating information on environmental issues and concerns in the City, is hereby reorganized to better serve this purpose.
The reorganized EAC shall consist of seven volunteer members who are residents of the City of Harrisburg. The initial members shall be chosen among the membership and/or agents and employees of any local commercial and institutional organization or shall be chosen due to their interest in environmental protection issues, with a particular emphasis on waste management and recycling matters. Each initial member of the reorganized EAC shall be appointed by the Mayor and Council; three initial appointments by the Mayor and four members shall be chosen by Council for a term of three years. Thereafter, Mayoral appointments shall be made by the Mayor and Councilmanic appointments shall be made by the City Council for a term of three years unless otherwise removed.
The initial members of the reorganized EAC shall prepare, approve, revise and implement bylaws to govern their function and duties.
The EAC shall meet monthly or at its discretion at a time and place as shall be determined by the EAC, and such meetings shall be open to the public.
The EAC shall, from time to time, appoint a Chairperson, a Secretary and other such agents as it may deem advisable.
The EAC may, from time to time, authorize one or more of its members to execute any document or documents and shall include in its minutes the name or names of the member or members so authorized. Notwithstanding the above, the EAC may not execute or otherwise bind the City or any of its boards or agencies to any agreement or to any fiduciary obligation.
The EAC or its delegate shall maintain and keep such records as are or may be required by any applicable law, regulation or ruling and shall provide for the preparation and filing of such forms or reports as may be required by any governmental agency or department and/or to any other person or entity who or which may be entitled to such information.
In addition to the EAC members, a representative of the City and the operator of City facilities, or his or her designee, may be present at all meetings to provide an operations report and other status reports concerning the City facilities. The County Host Municipality Inspector, or his or her designee, a representative of the Lancaster County Solid Waste Management Authority (LCSWMA), as operator of the Susquehanna Resource Management Complex, and a representative from the State Department of Environmental Protection may be requested to be present to provide an operations report and other status reports concerning the City facilities.
Any and all applications for grants to any federal, state, municipal or nonprofit governmental or corporate agency must be approved by resolution of Council prior to submission to such agency.
Voting members of the EAC shall hold no other office of employment in the government of the City. The EAC may allow nonvoting City personnel and officials to participate in Board matters.
Environmental Advisory Council members failing to attend three consecutive meetings shall forfeit their membership, unless the Chairperson of the EAC receives written notification from the member involved that the absence was for good cause shown. Should any EAC member fail to attend three consecutive meetings without providing notification as set forth above, the Chairperson of the EAC may remove such member.
Meetings of the EAC shall be advertised once per year in a local newspaper of general circulation and may be advertised up to twice per year as a monthly bill insert in all City residents' utility bills, and notice of upcoming meetings shall be maintained on the City's website.
At the request of the EAC and to the extent the EAC provides the written copy for publication, biannually, the City may publish educational information about the City's recycling programs to all residents of the City in furtherance of its obligations under § 1501 of Act 101 of 1988 by inserting the copy in all City sanitation billings. The EAC may also utilize the City's monthly calendar of events to provide such educational information.
At the request of Council, the EAC shall provide reports of its activities to the City and Council.
The City Clerk shall forward all environmental notices received by the City to the EAC within 10 days of their receipt. The City Clerk shall also forward all notices of leaking underground storage tanks and submission of site characterization and/or remedial action reports under the Department of Environmental Protection's corrective action regulations and the Land Recycling and Environmental Remediation Standards Act, Act 2 of 1995.
Editor's Note: See 35 P.S. § 6026.101 et seq.


What other outside funding possibilities are involved when talking about charter schools?
Because public charter schools are nonprofits, they can receive funding from philanthropic organizations, or form foundations that support the mission and goals of the school. They can even apply for grants specifically for public charter schools.


In 1997, we represented a real estate seller who had taken back a mortgage. The mortgage required monthly payments and a balloon payment of more than $300,000 in 2002. The buyer refused to pay certain sums due and owing on the mortgage and claimed that the seller owed him funds relating to the closing. Indeed, the seller did owe the buyer some money from the closing. We held several meetings and the judge tried to mediate the negotiation of a settlement. Did the ego of the buyer or the buyer's lawyer get in the way? It is impossible to say. The net amount the buyer owed us was less than $10,000; more than what we owed him at the critical time period. We took the case to trial and won. The amount due was therefore accelerated per the agreement, and the buyer was forced to make the $300,000 balloon payment to us within 90 days after the trial and pay our legal fees.


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View ORCID ProfileEugenio Mancera, Allison M. Porman, Christina A. Cuomo, Richard J. Bennett and Alexander D. Johnson
G3: Genes, Genomes, Genetics May 1, 2015 vol. 5 no. 5 849-856; https://doi.org/10.1534/g3.115.017566
Eugenio Mancera
*Department of Microbiology and Immunology and
ORCID record for Eugenio Mancera
Allison M. Porman
‡Department of Microbiology and Immunology, Brown University, Providence, Rhode Island 02912
Christina A. Cuomo
§Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142
Richard J. Bennett
Alexander D. Johnson
†Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94158
Fungi from the genus Candida are common members of the human microbiota; however, they are also important opportunistic pathogens in immunocompromised hosts. Several morphological transitions have been linked to the ability of these fungi to occupy the different ecological niches in the human body. The transcription factor Efg1 from the APSES family plays a central role in the transcription circuits underlying several of these morphological changes. In Candida albicans, for example, Efg1 is a central regulator of filamentation, biofilm formation, and white-opaque switching, processes associated with survival in the human host. Orthologs of Efg1 are present throughout the Candida clade but, surprisingly, the genome sequence of Candida tropicalis failed to uncover a gene coding for Efg1. One possibility was that the paralog of Efg1, Efh1, had assumed the function of Efg1 in C. tropicalis. However, we show that this gene has only a minor role in the morphological transitions mentioned above. Instead, we report here that C. tropicalis does have an ortholog of the EFG1 gene found in other Candida species. The gene is located in a different genomic position than EFG1 in C. albicans, in a region that contains a gap in the current genome assembly of C. tropicalis. We show that the newly identified C. tropicalis EFG1 gene regulates filamentation, biofilm formation, and white-opaque switching. Our results highlight the conserved role of Efg1 in controlling morphogenesis in Candida species and remind us that published genome sequences are drafts that require continuous curation and careful scrutiny.
Candida morphogenesis
filamentation
phenotypic switch
transcriptional regulation
Several species of the Candida genus belong to a monophyletic clade of ascomycetous fungi that translate the CTG codon as serine instead of leucine (Butler et al. 2009). Members of this CTG clade include species that are commensals of the human microbiota with no known environmental reservoirs outside of animals. However, these commensals can also cause mucosal disease in healthy individuals as well as systemic infections in immunocompromised hosts (Calderone 2002). Members of the CTG clade, therefore, are important fungal human pathogens, both in terms of their prevalence and their mortality rate.
Although they are most commonly encountered in the yeast form (unicellular spherical cells), most CTG species are able to undergo a variety of changes in cell and colony morphology. The best studied of these is the ability to switch between the yeast and hyphal (filamentous) forms, a transition that is closely linked to pathogenesis and biofilm formation (Sudbery 2011). Biofilms are communities of cells associated with a biologic or inert surface. In the case of Candida species, these communities are typically composed of several layers of yeast cells and hyphae that are embedded within an extracellular matrix (Finkel and Mitchell 2011; Nobile et al. 2012). Other morphological transitions include the phenomenon of heritable phenotypic switching. In C. albicans, this includes the white-opaque switch, in which cells transition between two different cell types that generate either shiny and dome-shaped colonies (white) or dull and flat colonies (opaque) (Slutsky et al. 1987; Lohse and Johnson 2009). White and opaque cells are morphologically distinct, have different metabolic preferences, and differ in their ability to mate (Slutsky et al. 1987; Lan et al. 2002; Miller and Johnson 2002). Several lines of evidence suggest that morphological transitions allow C. albicans to adapt to different ecological niches in the human host (Kvaal et al. 1997; Lohse and Johnson 2009; Pande et al. 2013; Tao et al. 2014). For example, filamentous cells are more effective at invading epithelia, whereas biofilms often form on indwelling medical devices and confer tolerance to antifungal drugs (Cutler 1991; Donlan 2001; Donlan and Costerton 2002; Kojic and Darouiche 2004; Sudbery 2011). White and opaque cells are also known to differ in their ability to colonize different anatomical locations and are distinct in terms of their virulence (Kvaal et al. 1997; Lachke et al. 2003; Lohse and Johnson 2009). Therefore, elucidating the molecular mechanisms underlying phenotypic changes will help determine how these fungi are able to colonize and infect multiple niches in the human body.
At the molecular level, the morphological changes described above have been extensively investigated in C. albicans. Filamentation, biofilm formation, and white-opaque switching are regulated by specific sets of transcription factors that control the expression of large numbers of target genes (Kadosh and Johnson 2005; Nobile et al. 2012; Hernday et al. 2013). Interestingly, the transcription factor Efg1 is unique in being common to all three transcription circuits. This transcription factor was originally identified as a regulator of hyphae formation and a member of the fungal-specific APSES family of DNA binding proteins (Stoldt et al. 1997). In C. albicans it acts as an activator or repressor of hyphae formation depending on the environmental conditions (Lo et al. 1997; Stoldt et al. 1997; Tebarth et al. 2003). Efg1 is also one of the six core transcription factors that control biofilm formation in C. albicans. Its deletion has the strongest effect on biofilm formation in vitro among the six core biofilm transcription factors (Nobile et al. 2012). In white-opaque switching, Efg1 is a critical transcription factor for formation of the white phenotypic state (Sonneborn et al. 1999; Srikantha et al. 2000; Zordan et al. 2007). Efg1 binds to the regulatory sequences of the five other transcription factors of the circuit and represses the expression of the master regulator of the opaque state, Wor1 (Zordan et al. 2007; Hernday et al. 2013). In addition to its role in these phenotypes and possibly as a consequence, Efg1 plays an important role in mediating colonization of the gastrointestinal tract (Pierce and Kumamoto 2012; Pande et al. 2013; Hirakawa et al. 2014). Together, these studies establish the central role of Efg1 in regulating multiple facets of C. albicans biology, including both commensalism and pathogenicity.
Relatively little is known about the role of EFG1 orthologs in other CTG species. In Candida parapsilosis, Efg1 is involved in biofilm formation and also regulates a colony morphology switch, although the morphologies appear distinct from C. albicans white and opaque states (Connolly et al. 2013). Interestingly, the role of Efg1 as a regulator of morphological change appears to be conserved even in species outside the CTG clade. For example, the ortholog of Efg1 in the model yeast Saccharomyces cerevisiae, Sok2, controls pseudohyphal growth (Pan and Heitman 2000), whereas in the filamentous ascomycetes Neurospora crassa and Aspergillus nidulans, the corresponding orthologs regulate ascospore maturation and conidiosphore development, respectively (Aramayo et al. 1996; Dutton et al. 1997).
Given the central role that EFG1 plays in adaptation of C. albicans to the human host and in morphological transitions across multiple fungal species, it was surprising that the genome sequence of C. tropicalis appeared to be missing an ortholog of this gene (Butler et al. 2009; Maguire et al. 2013). C. tropicalis is a CTG species related to C. albicans [approximately 55 million years since their last common ancestor (Mishra et al. 2007)] and is a common commensal and pathogen of humans (Butler et al. 2009). C. tropicalis is also known to undergo the yeast-to-hyphae switch, to form biofilms, and to exhibit white-opaque switching (Porman et al. 2011; Xie et al. 2012; Porman et al. 2013). Recent experiments have revealed that the C. tropicalis white-opaque switch involves a third stable state, termed the "intermediate" state, and therefore exhibits tristable rather than bistable switching (A. M. Porman, M. Anderson, N. Wang, E. Mancera, and R. J. Bennett, unpublished data). Among all CTG species whose genomes have been sequenced, C. tropicalis is the only one apparently missing an ortholog of EFG1 (Maguire et al. 2013).
One possibility to explain the absence of EFG1 in C. tropicalis is that its functional role has been assumed by the APSES paralog EFH1, but we show here that EFH1 plays only a minor role, if at all, in the morphological transitions mentioned above. Instead, we report here that the genome of C. tropicalis does contain an ortholog of EFG1, but it was not incorporated in the final assembly of the genome sequence. We first uncovered the gene by PCR amplification from genomic DNA using primers designed on EFG1 orthologs from related CTG species. We subsequently determined the sequence and genomic location of C. tropicalis EFG1. The C. tropicalis gene lies in a different genomic location than that of its ortholog in C. albicans and yet clearly represents a true EFG1 ortholog. Deletion of both alleles of the gene revealed that it is critical for filamentation, biofilm formation, and white-opaque switching in C. tropicalis, similar to its role in C. albicans. Overall, our results stress the conserved role of EFG1 in regulating physical transitions in fungi and provide a cautionary note for the analysis of published genome sequences.
Strain/plasmid construction
Transformations were performed as previously described for C. tropicalis (Porman et al. 2011). Nutritional gene deletions were constructed using the SAT1 flipper strategy (Reuss et al. 2004) in two different C. tropicalis genetic backgrounds: MYA3404 (the strain used by the genome sequencing project) and AM2005/0093. Plasmids to delete HIS1, LEU2, and ARG4 were made as described (Noble and Johnson 2005). For the deletion of EFH1 and EFG1, the 5′ and 3′ ∼300-bp regions flanking the ORFs were PCR-amplified. HIS1, LEU2, and ARG4 auxotrophic markers were PCR-amplified from plasmids pSN52, pSN40, and pSN69, respectively. Fusion PCR was then performed to fuse 5′ and 3′ flanks to nutritional markers. This PCR product was transformed into auxotrophic strains lacking HIS1 and LEU2 or HIS1 and ARG4 (Supporting Information, Table S2) and the process repeated to delete the second copy of the ORF. When using strains that were only leu minus, the first allele was deleted using the LEU2 marker from plasmid pEM002 and the second allele using the HYG marker from plasmid pYM70 (Basso et al. 2010). PCR was used to confirm correct genomic integration of the markers and homozygous deletion of the genes. Two independent isolates were generated for each deleted gene in each genetic background (Table S2).
Cloning EFG1 from C. tropicalis
The amplification of the first fragment of EFG1 was performed by PCR using degenerate primers designed against the portion of the EFG1 sequence that is most conserved from C. albicans to C. parapsilosis (Table S1, primers EMO348 and EMO352). C. tropicalis genomic DNA from the EFH1 deletion mutant was used as template and annealing temperatures of between 40° and 50° were used. Based on the sequence of the amplified fragment and conserved regions further out on the C. albicans gene, more primer pairs were designed to further extend the amplified region by PCR (Table S1).
For the final cloning of the gene, two flanking ∼300-bp segments in the center of the EFG1 sequence identified above were PCR-amplified from genomic DNA to be used as homology for integration. These two regions were PCR-fused to the edges of a segment containing the SAT1 marker, the CamR marker, and an E. coli origin of replication that was PCR-amplified from plasmid pSFS2A (Reuss et al. 2004). The resulting cassette (5′ EFG1 homology—drug markers and origin of replication—3′ EFG1 homology) was transformed into C. tropicalis cells as previously described. Transformants with the correct insertion at the center of the endogenous EFG1 sequence were selected on YEPD plates containing 400 μg/ml of nourseothricin and verified by colony PCR. Genomic DNA from the transformants was extracted, digested with XhoI and NheI separately, circularized by ligation, and electroporated into E. coli XL1-Blue Electroporation-Competent Cells (Agilent Technologies) following the manufacturer's protocol. Clones containing the EFG1 gene with the SAT1- CamR cassette were selected on LB plates containing 34 μg/ml chloramphenicol. Sequencing of the clones was performed by primer walking.
Characterization of hyphae formation
Similarly to what has been previously described (Lackey et al. 2013), cells from a YEPD, 30°, overnight culture, were washed twice with water and used to inoculate SD medium supplemented with 0.75% glucose and 50% fetal bovine serum at an OD600 of 1.5. This culture was grown with shaking at 37°. Samples were taken at the time of inoculation (0-hr time point) and at 2, 4, and 22 hr after inoculation and were visualized by differential interference contrast (DIC) microscopy.
Characterization of biofilm formation
Biofilm formation was assayed by confocal scanning laser microscopy and by determining biomass dry weight as previously described (Nobile et al. 2012). Briefly, overnight YEPD cultures grown at 30° were used to inoculate silicon squares pretreated with adult bovine serum at an OD600 of 0.5. Cells were left to adhere to the silicon square in Spider medium without mannitol and with 1% glucose at 37° and shaking at 200 rpm for 90 min. The squares were then washed with PBS and transferred to fresh Spider 1% glucose medium to be incubated for 48 hr at 37° and shaking at 200 rpm. Biofilms formed on the squares were stained for 1 hr with 50 mg/ml of concanavalin A Alexa Fluor 594 conjugate and visualized on a confocal microscope using a 40×/0.80W Nikon objective. For the determination of biomass dry weights, biofilms were grown in the same way, but directly on the bottom of six-well polystyrene plates. After 48 hr of growth at 37°, the medium was aspirated, biofilms were scraped from the bottom of each well, and they were dried on top of preweighed 0.8-μm nitrocellulose filter papers. Five replicate biofilms grown in separate wells were used for each strain.
Phenotypic switching assays on C. tropicalis mutant strains
Strains were grown overnight at 30° in Spider medium. Cells were diluted in water and plated onto Spider medium at a concentration of ∼100 colonies per plate. Plates were incubated for 7–10 days at 30° and colonies were examined for sectors. Cellular morphology was assessed by DIC microscopy of cells from the resulting colonies. Starting cells for each strain were in the corresponding default state: white, intermediate, and opaque for the wild-type, efg1Δ/EFG1, and efg1Δ/efg1Δ strains, respectively. For the heterozygous and homozygous mutants, we were not able to obtain stable populations in the white state.
The role of EFH1 in C. tropicalis morphogenesis
Given the apparent absence of an ortholog of EFG1 in C. tropicalis, it could be hypothesized that the role of EFG1 had been assumed by its APSES paralog, EFH1. To test this, we generated deletion mutants of this gene in C. tropicalis and evaluated filamentation, biofilm, and white-opaque switching phenotypes. Filamentation was assayed by growth at 37° in SD medium supplemented with 0.75% glucose and 50% fetal bovine serum. This medium has been previously reported to be optimal for the induction of C. tropicalis filamentation (Lackey et al. 2013). Hyphae formation was monitored at 2, 4, and 22 hr of growth in filamentation-inducing medium. No filamentation defect was observed in the efh1Δ/efh1Δ mutant compared to the wild-type strain at any of the examined time points. Biofilms were generated by growth of C. tropicalis cells on silicon or polystyrene surfaces for 48 hr (see Materials and Methods). Similar to what has been observed in C. albicans (Nobile et al. 2012), biofilms formed by C. tropicalis efh1Δ/efh1Δ deletion mutants were indistinguishable from those formed by the wild-type strain when grown on a silicon surface and visualized by confocal microscopy. When grown on polystyrene plates, efh1Δ/efh1Δ biofilms did appear different from the wild-type in terms of their structure but showed similar overall thickness and biomass (10.87 mg ± 0.5 in efh1Δ/efh1Δ vs. 11.62 mg ± 0.4 in WT). The deletion of EFH1 in C. tropicalis also had no significant effect on the frequency with which this species switches from the white to the opaque state (A. M. Porman, M. Anderson, N. Wang, E. Mancera, and R. J. Bennett, unpublished data). This is in agreement with previous reports showing that the expression levels of EFH1 in C. tropicalis are similar between white and opaque cells (Porman et al. 2011). Taken together, these results indicate that the role of EFG1 in C. tropicalis has not simply been assumed by its paralog EFH1.
Identification of an ortholog of EFG1 in C. tropicalis
To test whether C. tropicalis has an EFG1 gene that was missed by sequencing analysis, we performed PCR on C. tropicalis genomic DNA using degenerate primers designed to anneal to conserved regions of EFG1 orthologs from the CTG clade (Table S1). We used low annealing temperatures to allow for mismatches between primers and template, and we used genomic DNA from the efh1Δ/efh1Δ mutant to avoid PCR amplification of the paralog gene. This approach allowed us to PCR amplify a ∼260-bp sequence that was sequenced and shown to be more similar to C. albicans EFG1 than to C. tropicalis EFH1. Additional rounds of PCR using primers that anneal to the amplified region or to the C. albicans EFG1 gene allowed us to extend the sequenced region to 900 bp. This 900-bp region did not match any sequences in the assembled C. tropicalis genomic sequence that would allow placement of the gene in the genome.
To identify the complete sequence of the C. tropicalis EFG1 gene and to map its location in the genome, we used a targeting plasmid to integrate into the endogenous EFG1 locus. The plasmid contained the CamR drug marker selectable in Escherichia coli and the SAT1 marker selectable in Candida species. From this plasmid we generated a PCR cassette that contained the two selectable markers and an E. coli origin of replication, together with flanking arms with homology to the sequenced region of C. tropicalis EFG1. This cassette was transformed into C. tropicalis cells, where it inserted at the endogenous EFG1 gene by homologous recombination. Genomic DNA from these transformants was fragmented, circularized by ligation, and transformed into E. coli. Clones containing the putative C. tropicalis EFG1 gene were selected and sequencing of these plasmids revealed an ORF of 1701 bp, found to map to Supercontig 3.1 of the C. tropicalis genome sequence. Notably, the ORF spans a gap in the Supercontig beginning at 887,643 bp and ending at 888,943 bp. The ORF starts 13 bp upstream of the gap and ends 342 bp downstream of it (see Figure 1). Based on the neighboring genes, the genomic location of EFG1 in C. tropicalis is different from that of EFG1 in C. albicans (Figure 1). This is highlighted by the fact that the C. tropicalis orthologs of the genes neighboring EFG1 in C. albicans are located on a different Supercontig (Figure 1).
Genomic and phylogenetic position of EFG1 in C. tropicalis. (A) Schematic depiction of the genomic location of EFG1 in C. albicans and the corresponding genomic location in C. tropicalis. (B) As (A), but for C. tropicalis EFG1 and the corresponding location in C. albicans. In (A and B) the upper panel depicts C. albicans chromosomes (green background) and the lower panel depicts C. tropicalis chromosomes (blue background). Coordinates are in kb. One-to-one orthologs defined by CGOB are shown using the same color and are connected by dashed gray lines if in the same panel. The prefixes "orf19." and "CTRG_" for C. albicans and C. tropicalis ORF names, respectively, were omitted. The white segment at the bottom of the C. tropicalis EFG1 gene depicts the location of the sequence gap. (C) Gene phylogeny of the EFG1 and EFH1 genes in the CTG clade and S. cerevisiae. Ortholog sequences were obtained from CGOB (Maguire et al. 2013), aligned using MUSCLE, and a phylogenetic tree was generated using PhyML in Seaview (Gouy et al. 2010). Branch support values are SH-like approximate likelihood ratios and the branch-length scale bar represents substitutions per site. Generating the tree by bootstrapping (100 trees) gave the same tree topology. The species relationships depicted in the cladogram (lower right) were obtained from (Maguire et al. 2013).
Sequence analysis of the EFG1 ortholog in C. tropicalis
Translation of the C. tropicalis EFG1 ORF results in a polypeptide of 567 amino acids that has 54% sequence identity and 60% sequence similarity to the C. albicans Efg1 protein. The two Efg1 proteins also represent the best reciprocal BLAST hit among the genes of the C. tropicalis and C. albicans genomes. As can be observed in Figure 1, the identified gene is at the precise position in the phylogenetic tree where it would be expected given the species evolutionary relationships. It groups with the Efg1 orthologs in the CTG clade, closest to C. albicans and Candida dubliniensis, and in a distinct part of the tree from the EFH1 orthologs. C. tropicalis Efg1 has a single conserved domain between amino acids 190 and 296 that matches the helix-turn-helix DNA binding domain characteristic of the APSES transcription factors (Stoldt et al. 1997). The domain in the C. tropicalis gene is almost identical to the APSES domain of C. albicans Efg1, differing only by two amino acids (sequence identity of 98%). Thus, by several criteria, the C. tropicalis EFG1 is an ortholog of the family of EFG1 genes from related CTG species.
We were able to reconstruct the two alleles of the C. tropicalis EFG1 gene by comparing differences between sequenced clones (Sequence accession numbers KP314278 and KP314279). The two alleles differ by seven nucleotides, five of which are nonsynonymous. None of the allelic differences lie within the APSES DNA binding domain of the protein. These are the two alleles present in the C. tropicalis strain MYA3404, which was the strain used for the genome sequencing project (Butler et al. 2009). Although we also used a strain of another genetic background to confirm our functional results (described below), all of the findings presented here are for the MYA3404 strain background.
The upstream intergenic region of EFG1 in C. tropicalis is strikingly long (10,110 bp compared with an average intergenic region in the C. tropicalis genome of 902 bp (Butler et al. 2009) and, in this sense, resembles the control region of C. albicans EFG1 (9,831 bp or 10,077 bp if the tRNA in the intergenic region is excluded). In C. albicans it is believed that the extended control region helps explain the complex regulation of EFG1 expression (Lachke et al. 2003; Tebarth et al. 2003; Nobile et al. 2012; Hernday et al. 2013).
C. tropicalis EFG1 is involved in filamentation, biofilm formation, and white-opaque switching
We generated deletion mutants of C. tropicalis EFG1 to characterize the role of the gene in morphogenesis (Table S2). Two independent isolates of the deletion mutant were tested to ensure that the phenotypes observed were not due to off-target effects. The deletion mutants were assayed for filamentation as described above for the EFH1 deletion mutants. As can be seen in Figure 2, the deletion of EFG1 in C. tropicalis had a strong effect on hyphae formation, with cells remaining in the yeast form even after 22 hr in the filamentation-inducing conditions. In contrast, cells from the wild-type strain showed hyphal projections even at the 2-hr time point. This phenotype is in agreement with the observed phenotype in C. albicans when EFG1 is deleted (Lo et al. 1997).
EFG1 is involved in filamentation and biofilm formation in C. tropicalis. (A) Formation of filamentous cells by wild-type and EFG1 deletion strains of C. tropicalis in the MYA3404 genetic background. Representative DIC microscopy images are shown for each time point. Scale bars are 5 μm. (B) Images of representative biofilms for EFG1 deletion mutants in C. albicans and C. trpicalis (two independent isolates) grown in wells of six-well polystyrene plates. (C) Biomass dry weights of the same strains shown in (B), grown in the same conditions. Error bars represent the SD of five replicates.
To assess whether EFG1 is involved in biofilm formation in C. tropicalis, we determined the ability of the deletion mutant to form biofilms on two different in vitro surfaces, silicon and polystyrene, both pretreated with bovine serum. Biofilm accumulation was determined by confocal microscopy and dry weight measurements. After testing a variety of media, we performed assays in Spider medium with mannitol replaced by 1% glucose, because this medium was optimal for C. tropicalis biofilm formation. The ability to form biofilms by C. tropicalis was severely impaired in the efg1Δ/efg1Δ mutant compared to the wild-type strain (Figure 2). Using confocal microscopy, biofilms formed by the efg1Δ/efg1Δ mutant were composed only of layers of yeast cells with very few filamentous cells. Also, the few filamentous cells that were present resembled pseudohyphae rather than true hyphae. In contrast, wild-type strains formed biofilms that are multilayered with a thick layer of hyphal cells on top of basal layers of yeast cells. The biofilm deficiency in the efg1Δ/efg1Δ mutant was also evident in the overall biomass of the biofilm (Figure 2). As shown in Figure 2, the biofilm defect observed in the C. tropicalis deletion mutant resembles the C. albicans phenotype when the orthologous gene is deleted.
To test whether EFG1 plays a role in C. tropicalis white-opaque switching, we plated single colonies on Spider medium and grew them at 30° for 7 days. Phenotypic switching in C. tropicalis involves transitions between white, opaque, and a third phenotypic state that is intermediate to both the white and opaque forms (A. M. Porman, M. Anderson, N. Wang, E. Mancera, and R.J. Bennett, unpublished data). As shown in Figure 3, deletion of EFG1 had a major effect on the phenotypic states in C. tropicalis. The white state was stable in the wild-type background, but heterozygous efg1Δ/EFG1 and homozygous efg1Δ/efg1Δ strains, respectively, formed the "intermediate" or the opaque state as the default state. In terms of switching frequencies, white isolates from wild-type strains showed a low percentage of switching events, with only 4% of colonies showing sectoring to the "intermediate" phenotypic state. In contrast, isolates from heterozygous or homozygous strains showed only switching between the intermediate and opaque states (Figure 3B). These experiments establish that the role of EFG1 in C. tropicalis is similar to that of its ortholog in C. albicans, where it acts to promote stable formation of the white state (Srikantha et al. 2000; Zordan et al. 2007). We also generated efg1Δ/efg1Δ strains in a C. tropicalis genetic background different from the background used for the genome sequencing project and observed qualitatively the same phenotypes in biofilm formation and white-opaque switching (data not shown).
EFG1 is essential to maintain the white state in C. tropicalis. (A) Photos of representative colony and cellular morphologies of the default states of the wild-type, heterozygous efg1Δ/EFG1, and homozygous efg1Δ/efg1Δ strains in C. tropicalis MYA3404 genetic background. Scale bars are 5 μm. w, white; i, intermediate; and o, opaque cell types are indicated. (B) Phenotype frequencies of the same strains starting from a population of white, intermediate, or opaque cells. Cells in these states were used because these were the default states for wild-type, efg1Δ/EFG1, and efg1Δ/efg1Δ cells, respectively. Error bars are the SD of five independent experiments.
In summary, the phenotypes of the C. tropicalis EFG1 deletion mutants establish that this gene plays a key role in filamentation, biofilm formation, and white-opaque switching. These findings are consistent with a conserved role for EFG1 in regulating morphological transitions in both C. tropicalis and C. albicans.
The absence of an ortholog of EFG1 in the assembled genome of C. tropicalis was surprising given the conservation of this gene among other CTG species and its importance for regulating several key morphological transitions in C. albicans. Here, we report that C. tropicalis does indeed contain a true EFG1 ortholog. The C. tropicalis gene is located on the homologous chromosome to the C. albicans EFG1 gene (chromosome R), but it occupies a different position on this chromosome (Figure 1). Despite its different genomic location, the gene sequence reveals a shared evolutionary history with EFG1 genes from other species. It is difficult to recreate the exact genomic rearrangements that led to repositioning of EFG1, but the fact that the C. albicans and C. tropicalis EFG1 orthologs are on the same chromosome is consistent with the idea that most genomic rearrangements in the CTG clade are intrachromosomal (Butler et al. 2009).
We demonstrate, through gene knockouts, that C. tropicalis EFG1 regulates filamentation, biofilm formation, and white-opaque switching, indicating functional conservation with its ortholog in C. albicans. In contrast, analysis of the EFG1 paralog, EFH1, revealed a relatively minor role, if any, in each of these processes in both C. tropicalis (this work) and C. albicans (Doedt et al. 2004). EFG1 also regulates biofilm formation and a phenotypic switch in C. parapsilosis, although the latter appears distinct from that of the white-opaque switch (Connolly et al. 2013; Holland et al. 2014). It is therefore evident that this master regulator of morphological transitions has a conserved function between several of the most pathogenic Candida species. The molecular mechanisms underlying filamentation, biofilm formation, and white-opaque switching have been extensively studied in C. albicans, but little is known about these mechanisms in C. tropicalis. Identification of the ancient transcription factor EFG1 in C. tropicalis is a key step forward in understanding the strategies used by this species to adapt to the human host.
To gain insight into why the C. tropicalis EFG1 gene was not present in the published version of the genomic sequence (Butler et al. 2009), we examined the assembly process used for C. tropicalis. Initially, the genomic sequence was assembled using Arachne with standard parameters (Jaffe et al. 2003; Butler et al. 2009). However, it was found that many of the genes produced by this assembly were interrupted with stop codons caused by collapsing both haplotypes into a consensus haploid sequence. To address this problem, a new assembly was generated targeting highly polymorphic regions of the genome for iterative reassembly to minimize disagreement in the consensus and phase through a single haplotype for a given region (Butler et al. 2009). This strategy solved the problem of stop codon interruptions but reduced the total size of the assembly (8 kb smaller) and created new breaks in three scaffolds. Thus, assembling the C. tropicalis genome was a compromise between completeness, contiguity, and accuracy, made particularly challenging by the high number of allelic polymorphisms in the genome. In retrospect, we found that EFG1 was present in the initial assembly (where many genes were interrupted by stop codons), but was lost in the subsequent assembly that was published. Overall, our results stress the fact (often overlooked by investigators, including us) that published genome sequences are drafts in various stages of completeness. As was the case with the C. albicans genome sequence, further curation by the scientific community is critical to complement the computational assembly of genome sequences. This is an especially important and timely reminder in light of the exponential generation of new genomic sequences.
We thank Matthew B. Lohse and Katarzyna Oktaba for critical comments on the manuscript. This work was supported by a Human Frontier Science Program grant (to E.M.) and National Institutes of Health grants AI083311 (to A.D.J.), AI081704 (to R.J.B.) and F31DE022703 (to A.M.P.). This project has been funded in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272200900018C.
Supporting information is available online at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.115.017566/-/DC1
Sequence accession numbers: KP314278 and KP314279.
Communicating editor: B. J. Andrews
Received January 8, 2015.
Accepted March 5, 2015.
Copyright © 2015 Mancera et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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text:
Q: What does "Functions can only be declared at the top level of a scope" mean? Strict mode introduces some kinds of breaking changes. one of them is :

Functions can only be declared at the top level of a scope.

I am not able to understand this. Can anyone please describe it with an example?

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April 10, 2015 - February U.S. cutting tool consumption totaled $179.3 million, according to the U.S. Cutting Tool Institute (USCTI) and AMT – The Association For Manufacturing Technology. This total, as reported by companies participating in the Cutting Tool Market Report (CR) collaboration, was down 2.3% from January's total and up 1.6% from February 2014.
These numbers and all data in this report are based on the totals actually reported by the companies participating in the CR program. The totals here represent about 80 percent of the U.S. market for cutting tools.


Pin Sopheak of the anti-economic crime department, and Srey Veasna of the anti-drug department were demoted by National Police chief Neth Savoeun from captain to first lieutenant for breaching "National Police discipline", the announcement said.
The anti-drug department's deputy bureau chief In Song said on Sunday that Veasna was found to be inactive and careless in his duty. He frequently ignored assignments from superiors, Song said, citing instances where he failed to fulfil his duties during the Water Festival in late November, as well as the 40th anniversary of the founding of the Kampuchean United Front for National Salvation on December 2.
"He was often absent [from work] and ignored tasks assigned by his superior at the department," Song added.
In Sopheak's case, the anti-economic crime department deputy director Phan Sophal said on Sunday that the officer was often absent when assigned to maintain public order and security during major national festivals.


Join Ajna as she battles through forgotten temples in search of her tapir, Roti!
This game requires an NES emulator. If you're new to emulation, details for getting set up are included in the download.
By the way - with my comment i mean Kasumi`s NES-version of the game. The video shows a completely other version of the game, which i haven`t played until now.
Very good Jump and Run Game. Nice graphics, great playability, good sound. Superb !!!
Hey, thanks! And sorry for the late reply, itch seems to have not notified me of a new comment!
Indivisible is the 3rd game we review. Please Enjoy!
I may be the wrong person to share this with. I just made the NES version as a fan game. I'm not affiliated with the team who made the versions you reviewed.


Most popular GNXP post of all time?
Stupid thing to post about….
Perhaps the 'Genghis Khan haplotype' is not Genghis Khan's?
Not a scientific piece. But it helped me understand the political climate. And put some context on your future pieces about tribalism.
tells inconvenient truth was a classic. I would divide into perennial and reflecting on recent events/science/etc which is often prone to revision.
They are the first things I ever put up on the internet and have a lot of auto-biographical information I would like to preserve.
Is all of gnxp before August, 2002 lost? When was your first post? What about comments, how many have been lost?
You had a layman's man post that I have been unable to find time to find- lol, I have been wanting to share it with family and friends as an intro for them. An intro to intro to intros for those unfamiliar with science. It was very short and precise, possibly myth busting and or about admixtures. I might be sticking a few posts together..
Side note I see that a Stephen Pinker book is on your rec list and that's disheartening. That dude is mainstream backwards neuroscience.
he's a cognitive scientist, not a neuroscientist.
in, general i think he's a great public intellectual.
don't know, but that's literally 2 months of posts. i started blogging in april 2002, but those posts before gnxp are gone.
i have all the comments in mysql.
It would be great if you could do a rewrite, updated with new information.
This was my favourite: The Civilization Before History. And not even a year old!
I imagine Uruk as the last civilisation, before AD 1984, destroyed by Gozer The Traveler.
I remember a lot of great posts, e.g. the ones on the basques were a big surprise to many friends I spoke to.
I'm coming to Austin for the AAAS meeting. I land on Wednesday 14 Feb and fly back to Stockholm on Sunday eve 18 Feb.
Do you have time for a coffee or beer during that stay?
The post omar mentioned (February 2, 2018 at 10:39 am) was very good. And gets extra points for saying that more than 7 years ago.
sure. i don't work far from the venue so that should be fine.
https://www.gnxp.com/WordPress/2017/03/23/your-ancestry-inference-is-precise-and-accurateish/ is very useful for Northern Europeans interpreting or thinking of getting ancestry results.


Come rain or shine, whether you are a seasoned grower, or a complete novice, here is an idea of what you can get up to in your greenhouse all year round.
The gardening calendar starts off quietly and the beginning of the year is a good time to snuggle down in your favourite armchair with a selection of gardening books and seed catalogues to gain inspiration as to what you want to grow. No doubt you will discover many new varieties that you will want to experiment with but do remember that the plants you decide to grow must be able to happily co-exist side by side – a multitude of plants all requiring widely different temperature, light and humidity levels are unlikely to thrive, unless the greenhouse has been designed from the outset to have separate partitioned zones in which different growing environments can be maintained.
This quiet time ahead of the main growing season provides the perfect opportunity to check over the greenhouse and make sure that all is functioning as it should, particularly once you have had your greenhouse for a while. Our 'Clean & Care' service is the ideal way to ensure your greenhouse is in tip-top condition and looking at her best for the spring – please contact us on 01730 826900 for further information.
Frost-sensitive plants do not need a lot of heat in the winter months. A good rule of thumb is to aim for a minimum temperature of 5 - 7°C (42 - 45°F) – certainly there is no need to maintain temperatures higher than 12 - 15°C (55 - 60°F). During the night you may well find it necessary to cover frost-sensitive specimens with a horticultural fleece or even straw or newspaper if night frosts are forecast. Installing a min-max thermometer will help you keep track of temperature and ensure that any heaters are working as they should.
To protect plants from disease inside the greenhouse, keep the atmosphere dry and water sparingly unless plants are in flower. It is good practice to water early in the day and keep water directed to the roots where it is needed – don't splash the floor, benching or foliage with excess water as this will only increase humidity inside the greenhouse and the risk of pests and mould. Now is the best time to spray to combat grey mould or whitefly should either of these be present. Keep an eye on plants and remove any dead flowers, as well as yellowing or diseased leaves.
Despite the colder weather, the greenhouse will still need to be ventilated but do take care – keep the side vents closed on damp, foggy days and use them only on dry and sunny days. Open them in the morning and close mid-afternoon so as to conserve the heat of the sun before nightfall.
If you have early sowings of sweet peas or onions, now is a good time to pinch them out and plant them into larger pots. This is also a good month to take cuttings of tender plants. Make sure any compost used for sowing or potting is brought into the greenhouse to warm up several days before it is used to prevent seeds and young plants being chilled.
This month sees the days start to lengthen gradually and therefore you can slowly begin to increase the frequency with which plants are watered; nonetheless the soil should be kept on the dry side through the whole of the period from November to February. Wherever possible, plants should be watered by standing the pots in a bowl of water and leaving them until the surface of the compost glistens; the pots can then be removed and allowed to drain before returning them to the bench top. If watering plants from above, place the spout of the watering can under the leaves and try to water before midday.
Alpine houses can be well ventilated now, but for all other plants continue to maintain a dry atmosphere inside the greenhouse and ventilate on bright days only. The outside temperature may be improving compared to December and January but try to avoid exposing plants to fluctuations in temperature and aim to keep a relatively narrow temperature range of 5 - 15°C (42 - 60°F) inside the greenhouse. Keep frost-sensitive plants covered if you find it hard to maintain a minimum temperature of 5°C (42°F), especially on cold nights.
You can sow a bit more this month to get a head start before spring – good ideas ready for spring picking are early carrots, salad leaves and spinach. February is also the time to get potatoes chitting. Remember to give all your seed trays and pots a thorough scrub and make sure they are sterilised before use with a plant safe garden disinfectant. Sow seeds thinly in damp (not wet) compost and keep the container covered until germination has taken place; our propagator is the ideal location. Don't forget to label all your seed trays clearly too – an all too easy thing to overlook!
If you have a soil bed inside your greenhouse, prepare this well by digging in good quality garden compost or very well rotted farmyard manure.
Over-wintered fuchsias and pelargoniums can also be kick-started this month if the greenhouse is protected against frost – pot them on, increase the watering and start to feed them to encourage growth.
Now is also the time to prune conservatory climbers such as bougainvillea and passiflora.
This is traditionally the month that comes "In like a lion and out like a lamb" and greenhouse gardeners know this better than anyone! Although this is a great month for the greenhouse when things really get going with plenty to do, early spring can bring wide temperature fluctuations which can be a problem – a bright sunny day at the end of the month can result in the greenhouse overheating. Aim to keep the air temperature at around 7 - 18°C(45 - 65°F); this will involve heating the greenhouse at night and also ventilating and damping down the structure on cloudless days if required. Keeping the door and vents open more frequently as the month progresses helps to keep humidity levels down and prevent disease.
If your greenhouse is fitted with Bayliss roof vent openers, they may seem erratic as they do take time to adjust to the temperature fluctuations at this time of year. If you are concerned, please give us a call on 01730 826900.
You can also find plenty of useful advice from John Wood, Head Gardener at National Trust Hinton Ampner in Hampshire, about sowing seeds, as no doubt this will be your main focus in the greenhouse this month. Large-seeded, large-leaved vegetables such as cucumbers and courgettes can be planted into their own individual pots and placed in a propagator. In almost a month from now, the pots can be moved into the main body of the greenhouse for an early July harvest.
Seedlings need good light but young ones will still need protecting from the midday sun. If you do not have shades fitted to your greenhouse and find you need some – or additional ones – please contact us and we will be happy to advise you further.
Growing plants should be fed with a liquid fertiliser but take care not to overfeed your plants. A balanced formula should be used for leaf growth whilst a high potash feed should be used for flowering plants. Remember to keep actively growing plants damp but not soaking wet. The Hydroleca on your bench top helps to retain moisture.
Insects can start to be a problem this month – keep watch for greenfly, whitefly and red spider mite in particular. If evidence of bugs is found, spray with a greenhouse insecticide or investigate organic alternatives before the problem gets out of hand, as organic predators need to be ordered during early spring.
Pollinating insects are not yet present, so nectarine, peach and strawberry flowers must be pollinated by hand. Try to do this in the middle of the day and repeat for a few days to ensure success.
March is a good time to prepare hanging baskets for setting out in late May or June for a vibrant splash of colour. Fuchsias and pelargoniums can have their growing tips pinched out this month to encourage strong bushy plants.
Hopefully this month brings warmer weather with it, in which case you will need to keep your greenhouse ventilated. A good aim is to keep the temperature inside the greenhouse between 7 - 21°C (45 - 70°F). The roof vents should provide the majority of the ventilation, with the side vents only being used once the outside air is warmer. On sunny days with no clouds in the sky, you may well find that it is necessary to use your shades. If your greenhouse is not already fitted with shades, do give us a call and we will be happy to advise you as to the cost and the best location for shades for your particular structure.
As the temperature increases, your plants will become thirstier and you will need to check regularly to see if watering is needed. Plants that are actively growing may well need watering several times a week but remember over-watering can be as much of a hazard as under-watering! Actively growing plants will also need feeding but again you will need to take care - always follow the instructions on the packaging and remember 'little and often' is the golden rule, as overfeeding can cause distortion, leaf damage or root scorch.
Continue to keep an eye out for pests, particularly greenfly, whitefly, vine weevil and red spider mite, as these are all active at this time of year. Slugs are also out in force, and they can rapidly destroy a trayful of carefully nurtured seedlings.
Depending on the plants you are growing, the warmer temperatures may mean that the need to heat the greenhouse is reduced this month. Remember, however, that young plant growth is especially susceptible to low temperatures so check that heaters in the greenhouse are working whenever a frost is forecast.
As well as continuing to sow half-hardy annuals, now is a good month to start taking cuttings to increase your plant collection. If you are growing fruit, hand pollinate at midday by transferring pollen using either a soft brush or a ball of cotton wool and repeat over a period of several days to ensure success.
Your greenhouse is more than likely to be packed with vibrant healthy plants this month, so you can look forward to spending more time in your greenhouse to stay on top of watering and ventilation to keep them in tip-top condition.
As the weather continues to warm up, the main focus is to keep the temperature inside the greenhouse below 24 - 29°C (75 – 85°F) during sunny spells. Often this will mean active use of shades and monitoring their use every day according to the weather.
Ventilation is also paramount this month. Make full use of both the side and roof vents if the temperature is over 24°C (75°F) and supplement further by hooking the door open if necessary. In sunny periods you can also damp down the floor and benching. This not only lowers the air temperature as the water evaporates but also raises the relative humidity.
Whilst it should not be necessary to heat the greenhouse during the day in most parts of the UK, keep a check on night time temperatures as night time frosts are still a possibility.
Plants are actively growing this month and staking and tying should be done as plants grow. Unlike in the garden, remember that roots cannot extend out into open soil to seek out extra food. Regular feeding and watering is therefore important this month to ensure healthy, vigorous growth. 'Little and often' remains the golden rule when feeding and for general purposes a balanced formula with nitrogen, phosphates and potash in roughly equal proportions should be used. If you wish to stimulate leaf growth, a high-nitrogen feed should be used and for plants in flower or fruit use a high-potash fertiliser. You will also need to water plants regularly and thoroughly and must check the dryness of the compost every day – some plants will need daily watering.
With a greenhouse full of healthy specimens, space may become limited inside the greenhouse. Now is the ideal time to make full use of your cold frames if you have them and start the hardening-off of the early sowings.
Last but not least, this month heralds the start of the social season with the RHS Chelsea Flower Show traditionally taking place in the third week of May. If you have already ordered your tickets from the RHS why not pop along and visit our stand whilst gathering inspiration and replacing your seed and bulb stocks at the world's greatest garden show? We'd be delighted to see you!
The weather may well be hotting up but now is the time to keep your cool!
With the sun high in the sky, sun scorch can be a problem this month. Tell-tale signs are growth slowing down, flowers losing their colour and leaves showing signs of scorch. To combat this, you will need to make full use of your shades.
Ventilation is now also essential. Open roof and side vents to their maximum and the door as required to keep the temperature down inside the greenhouse. On warm days, ventilation should be carried out in the mornings and evening and it may even be necessary to keep the side vents open overnight. If you are due to be away on holiday and there is nobody to look after the greenhouse whilst you are away, we recommend the side vents are left open and the blinds left down. Automatic roof vents (including Bayliss) will continue to open and close as they adjust to the day and night time temperatures and are a great aid in keeping the greenhouse as cool as possible in such circumstances.
In hot spells, damping down inside the greenhouse regularly is a must and should be carried out in the morning or early afternoon by spraying the floor and benching. As well as monitoring the air and cooling the greenhouse using shades and vents in combination, damping down has the added benefit of discouraging red spider mite - at this time of year it can be a particular menace.
If your greenhouse is unheated, summer crops should be doing well. Melons and cucumbers should now be being trained and side shoots on tomato plants should be pinched out. Other crops such as chillies, aubergines and salad should also be growing well but do keep a watchful eye out for pests.
As an alternative to vegetables, your greenhouse can be used as a flowering display house. Ideal plants for this purpose include: coleus, pelargoniums, foliage begonias, small sunflowers, celosis – the list is endless!
Plants destined for the garden are moved out this month. Make sure that the danger of frosts has passed before putting out half-hardy plants and also make sure that specimens grown inside the greenhouse have had a period of hardening- off before they are moved into the garden. Some greenhouse plants, especially citrus, can be moved into the garden during the period between the end of June and early October. This not only improves their health but also gives you more space for growing under glass.
As you move more and more of your plants outdoors, give the greenhouse a good tidy and clean up. Do not leave dirty pots, discarded plants or dead leaves lying around – these can all harbour dirt and disease leading to problems in the future.
As summer reaches its peak, the main challenge facing greenhouse gardeners this month is preventing the greenhouse from getting too hot. Ventilation is vitally important and you will have to make sure there is good air circulation inside the structure during the night as well as during the day. If the weather is very hot, even opening all the vents and keeping the door hooked open may not be enough to keep the temperature comfortable. More than likely, you will have to make full use of shades and continue to damp down inside the greenhouse. Both of these will help but you may even need to bring in an air circulating fan to provide forced ventilation and keep the temperature down. Our Edwards heater may be just the answer if you do not already have one – details can be found here.
As in June, scorch from the sun can be damaging and some form of shading is essential for a wide variety of plants covering a diverse range such as flowering pot plants, rooted cuttings or cucumbers in flower. Blinds on the inside of a greenhouse can be difficult to use if you are growing tall plants such as tomatoes and cucumbers and can also have a tendency to collect debris, such as pests and leaves. Externally mounted shades - such as those we offer – avoid these drawbacks and are very easy to use. We would be delighted to discuss the options with you if you would like to add shading to your structure or even supplement existing shades that you have.
High air temperatures will cause soil to dry out quickly. Watering must be carried out without fail and it is essential that compost is checked every day to ensure it does not dry out.
Remember to watch like a hawk for pests and diseases. Red spider mite, greenfly, whitefly, thrips, grey mould and mildew are all prevalent at this time of year and combatting them is a major focus for the greenhouse gardener. Spray promptly if you notice signs of their presence or investigate biological alternatives and remember that hanging good old sticky yellow fly catchers also has its place in your armoury! Other garden visitors such as butterflies looking for places to lay their eggs and even birds can find their way into the greenhouse through open vents and doors, bringing a different array of problems with them. You may wish to consider adding bug-screens to your greenhouse – if you give us a call on 01730 826900 we will be happy to advise you what is possible.
This month is a good time for taking cuttings as roots will form quickly in the warm conditions. With most plants now growing vigorously, don't forget to establish routine feeding. A liquid feed at fortnightly intervals is suitable as a basic treatment, but fruit-bearing plants such as cucumbers and tomatoes need feeding every 7 - 10 days.
The battle against pests and disease continues this month. Keep an eye out in particular for grey mould. If leaves, fruit and flowers should succumb, destroy the affected parts, spray with a systemic fungicide and take steps to improve ventilation inside the greenhouse. To help prevent disease, take care when watering to ensure leaves are not splashed in the evening and never leave old leaves and fruit lying about. Dead-heading flowering plants regularly helps to combat disease and extends the flowering season.
Keeping the greenhouse ventilated remains vital. Keep the vents and door open on warm days, though as the temperature begins to drop over the course of the month, close the side vents before nightfall so that only the roof vents remain open during the night.
Plants will continue to require regular watering and vigorous plants in small containers will still need to be soaked daily. Continue to routinely feed actively growing plants too, as this remains a necessity.
A potash-rich feed will improve fruiting and flowering; 10 - 14 days should be left between feeds.
As in July, August is a good month for taking cuttings for plants in the garden. Cuttings typically fall into 3 groups – alpines, such as Armeria, shrubs such as Weigela, and half-hardy types such as Pelargonium. Don't take more cuttings than you will be able to handle but it is a good idea to strike a few extra – failures can occur, despite a healthy dose of positive thinking!
Now is also the time to plant bulbs. Make sure you have an adequate supply of pots, bowls and bulb fibre.
With plants in flower and salad crops at their peak, August provides the ideal opportunity to take stock and reflect on your successes (and not so successes!) and learn from these when making next year's plans. You can also use this part of the year to collect and store some seed from your own plants over the next few weeks.
Thinking ahead, now is a good time to contact Alitex to ask us to quote for an annual Clean and Care service of your greenhouse – we can book you in for either this autumn or next spring to best fit in with the greenhouse gardening season.
This month can be of mixed fortune, bringing a mix of warm and colder weather and you will need to control the temperature inside your greenhouse carefully as a result. Aim to keep the temperature in the 10 - 21°C (50 - 70°F) range and close the vents in the early evening to retain some of the sun's warmth overnight.
Cooler temperatures will also reduce the need to damp down. Only spray the floor and benching on warm days and, even then, make sure the job is completed by midday.
Plant growth also becomes less active as temperatures fall and compost stays moister for longer; you can therefore reduce the frequency with which you water. Keep watch for fungal diseases however, as they tend to appear this month.
If you are growing shade-loving plants such as ferns, these will need to continue to be kept shaded. For most plants however the prime concern is achieving maximum light levels rather than controlling heat and you should be using your shades less and less as the month progresses.
Salad days and the joys of eating the fruits of your labours al fresco are now drawing to a close. The end of September or early October is the time when tomatoes and cucumbers are cleared out of the greenhouse and frost-sensitive plants from outside are brought in. There may be a gap between the two and, if so, this is an excellent opportunity to fumigate the greenhouse with a fungicidal smoke.
Don't wait too long before bringing in tender specimens as one unexpected sharp frost is all that is needed to kill off a beloved plant! Do make sure that pots of chrysanthemums, fuchsia etc are free from pests before bringing them into the safety of the greenhouse.
You may find this month that you will need to introduce heat into your greenhouse as the outside temperature starts to drop. As a good rule of thumb, you should aim to maintain a minimum night-time temperature of 5 - 7°C (42 - 45°F) if you intend to keep your greenhouse frost-free. If frost is forecast, you may need to protect tender plants with a horticultural fleece or newspaper.
The extra humidity that was so vital in the warmer months is now no longer required and you should stop damping down inside the greenhouse as moist air can cause a problem by encouraging the growth of grey mould. Continue to ventilate the structure a little each day between mid-morning and early afternoon by using the roof vents in isolation – the goal is to bring fresh and moving air into the greenhouse rather than to reduce the inside temperature. Do not ventilate on damp or foggy days and make sure the vents are closed at night.
Watering may also be eased off this month and you should check that each plant requires watering before applying it. Be careful when watering too – do not splash the water about and try to keep it off the floor, benching, leaves and crown of the plants. You may find it helpful to water plants before midday so that any inadvertent splashes have time to dry out before sunset.
Inspect plants for evidence of pests and disease – diseased or yellowing leaves should be removed and spray if grey mould or whitefly has become a problem; spraying should be carried out in the morning. Slugs and snails may also be present and measures to combat these should be taken if you spot their tell-tale slime trails or evidence of nibbled leaves! Some snails hibernate and these may be attracted to the warmer climes of a greenhouse, as may slugs if the outside temperature dips below -5°C (42°F).
Late autumn is an ideal opportunity to think about your plant collection for next year and start collecting seeds – you will have plenty in your own garden and all for free! If you have like-minded gardening friends, why not get together and swap? Store any seed you collect in clearly marked paper bags over the winter – memories are likely to be fallible come spring! Click here for John Wood, Head Gardener at National Trust Hinton Ampner's top tips advice.
As winter approaches, make sure that all half-hardy plants have been brought into the greenhouse. In colder parts of the UK you may wish to insulate your greenhouse using bubble wrap. It may not be necessary to carry out this task until November or December in milder parts of the country.
With plants safely brought inside, the emphasis this month is on maintaining a healthy environment inside the greenhouse.
Just as last month, it is important to maintain a minimum temperature of 5 – 7°C (42 – 45°F) to protect frost-sensitive plants. If your greenhouse is unheated, it will be necessary to cover tender plants with fleece or newspaper at night if a frost is forecast.
Try to keep the air inside dry. On dry and sunny days side vents can be opened if they are away from wind but they should be closed mid-afternoon so the sun's heat will be conserved before nightfall. Vents should be kept closed on damp or foggy days.
All plants (except flowering plants) should be watered sparingly and you should avoid splashing the floor, benching or foliage when watering, as this will also help keep the atmosphere dry inside the greenhouse.
Pots should be spaced out on the bench top to ensure plants receive maximum light. Continue to check for signs of grey mould and whitefly and treat if either is present. Flowers should be dead-headed and diseased or yellowing leaves should be removed as a further measure to reduce the spread of disease.
November is a good time to give the greenhouse its annual clean and tidy up – the other opportune time is spring. Choose a bright, dry day and allow a day for the task as there will be lots to do! Place pots in a safe place in the garden and move tender plants indoors. If you have soil beds inside your greenhouse, now is the time to dig these over. Clear rubbish from inside the greenhouse and then scrub the benching and shelves with a garden disinfectant. Clean the frame and glass next, both inside and out, and take this time to check that gutters and roof valleys are free of fallen leaves and debris, as a build-up of these can cause blockages and encourage algae growth which can lead to leaks over time. Remember to bring your plants back into the greenhouse once the big tidy up is complete – sounds obvious but it is easy to overlook as you are admiring your new shiny greenhouse after all your hard work!
Sarah Wain's blog on our website is full of handy tips and tricks on cleaning your greenhouse – her enthusiasm for maintain a spotless greenhouse is boundless!
If the task is just too daunting or time is too limited, give us a call and see if we can help by carrying out a Clean and Care service for you. This is also an ideal opportunity to check over your greenhouse to see if you would like to make any additions to your fixtures, fittings or accessories in time for next year's season.
Continue to monitor the temperature carefully this month. Frost-free greenhouses should be kept at a minimum night time temperature of 5 - 7°C (42 - 45°F) and a maximum temperature of 13 - 16°C (55 - 60°F) during the day – any warmer may result in weak and lanky plant growth.
Some ventilation will be necessary to remove condensation and to keep the air moving inside the greenhouse. Do tackle this carefully though to avoid creating draughts that are prone to causing dangerous drops in temperature and killing plants; the advice given for the months of October and November will help you.
If plants are not in flower, the soil must be kept rather dry at this time of year. This is particularly true for cacti, succulents and pelargonium. Only damp down the floor if it is really necessary and be sure to do this job mid-morning so the greenhouse has ample time to dry out.
Pot cyclamen will now be approaching their best but be sure to only water when the top of the compost is dry to the touch.
Onion seed can be sown in a little heat towards the end of the month – your reward will be large onions next summer.
Cut down the last of the winter flowering chrysanthemums and store them in their pots underneath the benching. This month is also the time to start taking the first batch of chrysanthemum cuttings.
Continue to keep an eye out for pests and diseases and treat if necessary. Dead-heading and removal of yellowing or diseased leaves should also be a key focus this month.
You may be dreaming of a white Christmas and if snow is forecast, be sure to keep the shades on your greenhouse tightly rolled up on the ridge if you have these fitted to avoid damage to the shade from fallen snow.
All should be relatively calm and peaceful inside the greenhouse this month, fitting the mood of the season. With this year's hard work done, you can look forward to all being merry and bright next year!


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Charlie Coyle Chicago Blackhawks Coyle Dany Heatley Darcy Kuemper defense Erik Haula goalies Granlund Ilya Bryzgalov Jared Spurgeon Jason Pominville Jason Zucker Jonas Brodin Josh Harding Justin Fontaine Keith Ballard Kyle Brodziak lines lineup Marco Scandella Mathew Dumba Matt Cooke Mikael Granlund Mike Rupp Mike Yeo Mikko Koivu Niklas Backstrom Nino Niederreiter Olympics Parise playoffs power play Preseason Ryan Suter Sabres shootout Thomas Vanek Torrey Mitchell trade training camp Wild Yeo Zach Parise Zucker
Reaction from Minnesota Wild after 7-2 win over Tampa Bay Lightning
Niklas Backstrom would have preferred a win, but had fun in net after long road back
Thomas Vanek frustrated with the start to his Minnesota Wild career
Posts Tagged ' postgame '
Postgame reaction from the Wild's 3-2 shootout loss to the Kings
3 October //
Posted in Uncategorized //
Tags : Kyle Brodziak, Matt Cooke, Mikael Granlund, Nino Niederreiter, postgame
Comments Off on Postgame reaction from the Wild's 3-2 shootout loss to the Kings
The first of (at least) 82 Wild games is in the books. After an impressive first two periods to open the season, the Wild surrendered a late goal to Jeff Carter and lost in a shootout.
The Wild were the better team for two periods, but weren't able to light the lamp in the second despite a 13-3 shot advantage in the frame. It started with the second line. They were good and they created plenty of scoring chances — maybe more than any other line. But the chances didn't convert to more than a power play late in the first period.
The Wild took away one point from the season-opener after falling in the shootout.
"It's funny what the extra point makes you feel [like] at the end of the game," coach Mike Yeo said.
There were a couple ways of looking at the season-opener Thursday.
Take the positive side first — the Wild skated with one of the Stanley Cup favorites. That game could have gone either way and the Wild played much better than the eight-seeded team they were last season. They outshot the Kings and, frankly, outchanced them.
"Overall, it's really hard to be disappointed in the game from a performance standpoint," Yeo said. "If we play like that night in and night out we're going to win a lot of hockey games."
Or you can look at it the other way — the Wild were the better team for most of the game and once again couldn't find a way to finish off a better team with a key goal late. Talking about taking the next step is great, but taking the next step involves winning these types of games. With a 2-1 lead, the Wild couldn't find a way to tally their third of the night.
"You know that that next goal is important," Yeo said. "Obviously extending the lead makes a huge difference … but at the same time, we were playing pretty strong. When you're playing like that — it didn't work out tonight — but you should have the confidence to bring it home."
So which view is more depictive of the 2013-14 Wild? Stick around for the next 81 games to find out. What — you thought you were going to get answers on opening night?
On with the rewind (a new postgame blog format for this season).
Three things I think after tonight's game:
1) The 2011-12 Kyle Brodziak is back. After a down season last season where it seemed he was never in the right place at the right time and couldn't buy a goal, Brodziak was all over the ice Thursday. Unless something comes up, I'll have more on Brodziak in Saturday's paper. But he was everywhere in the opener and set up (read: caused) Matt Cooke's goal 64 seconds into the game.
"I didn't really change anything," Brodziak said. "Last year was obviously a weird year with the lockout — just whatever reason, multiple reasons. It was just a tough year and I wanted to start fresh and that's all I cared about. I felt like i was playing better than I did last year. No particular reason though, just felt good out there — strong and confident."
2) Matt Cooke has had a positive impact on Mikael Granlund. The 21-year-old was blocking shots, going in front of the net, and drew a late penalty in the third period. Perhaps it's not giving Granlund enough credit to say part of the change has to do with Cooke. Whatever the reason, Granlund looked good Thursday night.
3) Nino Niederreiter can be a top-6 forward in this league. He's been through about as much on the ice as any 21-year-old. Yet with his first chance on a new team in a big role, Niederreiter provided scoring chances. His backhanded pass to Jonas Brodin late in the first period set up the Wild's second goal. He's a big body winger that can fit well alongside Charlie Coyle and Dany Heatley on the second line. He wasn't perfect, of course, but he had flashes.
"He's looked good," Yeo said. "I don't see any reason why he shouldn't get anything but better. It's our job to kep helping him and obviously we have to keep getting him on the ice, but we've got to keep working with him. He's a very receptive kid. He's come a long way since Day 1 of training camp, so that's a really encouraging sign."
Backcheck: The Wild finished the preseason 4-2, which doesn't matter even in the slightest. After those games, though, the Wild's top line said they felt like they weren't at game speed yet. At times Thursday night, it looked like they were. But it was far from smooth sailing for the top unit.
"Offensively at times — and let's give LA some credit here, they're a really good team for a reason," Yeo said. "But I've got reason to believe that if those guys keep getting more games in them that more good things will continue to happen."
Forecheck: The Wild host the Ducks Saturday at 7 p.m. in St. Paul. Anaheim was toasted in their first game of the season in a 6-1 beatdown at the hands of the Avalanche.
That's it from a long day at Xcel Energy Center. I'll be at practice in the morning. Follow me on Twitter for updates. In the meantime, enjoy that the NHL is back.


These SAS macros provide basic information to characterize withdrawal/dropout information in the data set which can be the preliminary step of the missing data analyses. The descriptive summary statistics outputs provide visitwise percentages of patients with at least one post-baseline observation that have data at subsequent visits as well as visitwise response means by treatment for patients that dropout versus those that continue. The plot outputs include Kaplan-Meier plot of time to dropout by treatment and plot of visitwise response mean changes from baseline by treatment and dropout status (dropout or continue).


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On Pres. Duterte's recent pronouncements on Mary Jane Veloso
by Campaign for Human Rights Philippines | 16 Sep 2016 | Press Releases
On Pres. Duterte's recent pronouncements on Mary Jane Veloso:
Spare the Peddled, Punish the Peddlers
We believe it is quite politically palatable to plead for the life of one of the poor Filipinos whose ill fates made President Duterte say in exasperation, "My God, I hate drugs." Is it not because of them that we precisely abhor the drug menace, of which we must rid society (in the right way)?
Indeed, it satiates overwhelming appetites for both justice and mercy to ask for clemency at the right time on top of cogent humanitarian grounds. Beyond divergent language and semantics, asking for compassion for the victimized does not quarrel with hitting hard on the victimizer.
We urge the President not to confound who Mary Jane truly is: a poor, young mother of two young boys whose vulnerability was taken advantage of and exploited for the criminal purpose of drug smuggling, which she had no knowledge of doing. Despite being convicted and put on death row, she is in truth not the criminal but the victim, the peddled and not the peddler, not only of drug trafficking but also of trafficking in persons.
Simply put, the context is: Mary Jane is a victim of dire poverty, of lack of real opportunities for a decent job, of pernicious drug and human trafficking. The law may be the law but it should not be blind or deaf to reality.
At any rate, her final conviction, temporary reprieve and now indefinite suspension of execution, and the issue of possible clemency are matters all within the ambit of the laws of Indonesia in the same way that the ongoing trial of the cases against her recruiters and the plea for clemency are also matters in accordance with the laws of the Philippines. They do not cancel each other out.
As a matter of fact, Indonesian and Philippine domestic laws as well as international law, when duly applied, prohibit punishment and guarantee protection for trafficked persons like Mary Jane. There is thus nothing inconsistent with "following the law" in pleading for clemency at the right juncture and through the proper channel for Mary Jane.
As the leader of this nation and as the pater familias of all Filipinos, President Duterte is expected to rise to his bounden duty and fight for her, and fight hard as he does for all victims of this transnational infection.
In lieu of passive acquiescence towards her fate, Mary Jane needs compassion as a victim of the drug menace while we run after the real perpetrators. We thus call on the President and his government to not give up on Mary Jane, in the same way that the people refused to break their vigil at the time her life was almost snuffed out for a crime she did not commit. # (je)
Atty. Edre U. Olalia
NUPL Secretary-General
Atty. Josalee S. Deinla
Assistant Secretary-General for Education
National Secretariat
National Union of Peoples' Lawyers (NUPL)
3F Erythrina Bldg., Maaralin corner Matatag Sts. Central District,Quezon City, Philippines
Telefax no.920-6660
Email addresses: nupl2007@gmail.com and nuplphilippines@yahoo.com
Follow us on twitter @nuplphilippines and facebook @https://www.facebook.com/nuplphilippines
Visit the NUPL website at http://www.nupl.net/
"By calling yourselves the 'people's lawyer,' you have made a remarkable choice. You decided not to remain in the sidelines. Where human rights are assaulted, you have chosen to sacrifice the comfort of the fence for the dangers of the battlefield. But only those who choose to fight on the battlefield live beyond irrelevance."
– Supreme Court Chief Justice Reynato S. Puno, in his message at the NUPL Founding Congress, September 15, 2007
"After long years of experience as a people's lawyer, I can honestly say it has been a treasured journey of self-fulfillment and rewarding achievement. I know it will be the same for all others who choose to tread this path."
– Atty. Romeo T. Capulong, NUPL founding chairperson, in his keynote address at the Fifth Conference of Lawyers in Asia Pacific ( COLAP V), September 18, 2010


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Asterix och britterna (fr: Astérix chez les Bretons) är det åttonde i en serie av klassiska seriealbum, skrivna av René Goscinny och illustrerade av Albert Uderzo, båda fransmän, vars huvudperson är den modige gallern Asterix. Serierna publicerades ursprungligen (i Pilote) 9 september 1965–17 mars 1966 och som seriealbum 1966. Den första svenska utgåvan kom 1971. 1980 utgavs en ny översättning av Ingrid Emond med ny textning.

Handling
När Julius Caesar överger den lilla gallerbyn och istället satsar på att anfalla Britannien blir Obelix missnöjd, men det är först när Asterix' kusin Fixfax (i den senare översättningen Jollytorax) kommer med bud om att deras lilla by, som fortfarande håller ut mot de romerska arméerna, behöver hjälp, som Asterix och Obelix kommer till britternas undsättning med en tunna trolldryck.

Dessvärre lyckas ett fartyg med romare förvarna trupperna på ön, innan trion kommer fram med tunnan, och en allmän order ges om att alla tunnor ska konfiskeras. Och tunnan med trolldryck blir också konfiskerad. Snart inser romarna vilket kap de har, men varken de eller Asterix lyckas förhindra att tunnan blir stulen - av en vanlig tjuv.

Anspelningar
I Britannien ser Asterix och Obelix en kvartett trubadurer som är väldigt lika Beatles, vilka åren innan albumet kom fick stora framgångar internationellt.

Övrigt
I det här albumet lanseras idén om att det var Asterix som gav britterna téet. Innan dess drack britterna varmt vatten med mjölk.
Obelix tycker att det är opraktisk att behöva korsa Engelska kanalen på ytan, han föreslår att man bygger en tunnel under vattnet istället.
I det antika London, Londonium, sköts naturligtvis lokaltrafiken av dubbeldäckade oxkärror.
Britternas hövding heter Bigboss.

Se även
Asterix och britterna (filmen från 1986)

Asterixalbum
Seriealbum 1966
Serieåret 1965
Serieåret 1966

redpajama_set_name: RedPajamaWikipedia


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Q: how can i change the workflow name link in sharepoint I would like to change the custom action "Workflow" not the workflow name.
Just the link to the workflow when you create one from sharepoint designer.I'm using 2010.
Any ideas? Thanks in advance

A: I changed it via Sharepoint Designer custom actions.
Go to sharepoint designer and open up the list look right below and you will see  custom actions..Click New
Cheers


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Full List By Area
Casualties with Wills Date Added
Full List By Country
Local Memorials
Local Institutions
Armagh Dead in the Great War Ballygawley War Dead Book Drumglass Boys National School, Dungannon Fivemiletown War Dead Book Football Match 11 December 1915 Holdfast Loyal Orange Lodge No 1620 Queens University Belfast Royal School Dungannon Book Royal School Dungannon WW2 Stevenson and Son, Moygashel Union Place National School, Dungannon
Dates of Interest
On this day This month
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11163 Sergeant James McGuffin
Dated added: 30/12/2015 Last updated: 31/05/2020
Regiment/Service: 15th Battalion, Lancashire Fusiliers (British Army)
Died: 13/09/1917 (Died of Wounds)
James McGuffin was the son of Robert McGuffin and Margaret Cooke. Robert was born in Belfast at 76 Canmore St. on 18th April 1888. The family hailed from Dungannon. James McGuffin enlisted in Salford, Lancashire. Sergeant James McGuffin was serving with the 15th Battalion of the Lancashire Fusiliers when he died of wounds on 13th September 1917. He was 29 years old. Sergeant McGuffin is buried in Zuydcoote Military Cemetery, near Dunkirk in France. In the autumn of 1917, while the XV Corps was holding the Nieuport section, the 34th and 36th Casualty Clearing Stations were posted at Zuydcoote.
Cemetery Details
Cemetery: Zuydcoote Military Cemetery
Zuydcoote, Nord
Grave Ref: Grave I-B-14
CWGC Casualty 617697
Zuydcoote is a village in the Department of the Nord about 10 kilometres north-east of Dunkirk. The Military Cemetery is west of the village and about 550 metres east of Zuydcoote Halte.
Local Memorial Details
No Memorial Name Townland
1 Dungannon War Memorial - WWI - Panel 1 Dungannon
No Link Reference Map Doc
1 Drogheda Great War Memorial Lists SGT. J.MC GUFFIN LANC. FUS
2 National Archives UK Medal Card can be purchased here
3 War Graves Photographic Project Photo of James McGuffin's headstone can be purchased here.
Dungannon District's War Dead Acknowledgements 2015-2021


"I don't think I tolerate corruption, I don't think I picked anybody that I know will embarrass my government. But if you have any evidence about any of my ministers, I accept responsibility for the 36 ministers that I have. I don't think I took anybody among the ministers who has got a case in court. Tell me one out of the 36. I don't think I will deliberately make that mistake", he said. Asked if he would sack any of the ministers if he or she is charged to court for corruption, he said: "No, I will insist that the case go through the courts." Again,the president, Monday, gave a clean bill of health to his ministers, appealing to discerning Nigerians to ignore orchestrated attempts to destroy their integrity. The president also extended the same clearance to senior government officials, who were being tarred with the brush of corruption without any concrete evidence.
He spoke against the background of reports, particularly by online media, that the immediate past Group Managing Director of the Nigerian National Petroleum Corporation (NNPC), who is also Minister of State for Petroleum, Dr Ibe Kachikwu, was being investigated over crude oil swap deals and gas liftings during his tenure as GMD of the petroleum corporation. In a statement by his media aide, Mr. Femi Adesina, President Buhari appealed for decent and civilized comments, particularly when it has to do with the integrity of those who are serving the country.
"Terrible and unfounded comments about other people's integrity are not good. We are not going to spare anybody who soils his hands, but people should please wait till such individuals are indicted," the President said. President Buhari reiterated the administration's commitment to probity, accountability and integrity, promising that transparency remained a watchword that defined his administration.