The shRNAmir libraries containing plasmid DNA were arrayed in 96-well plates such that each well contained one unique and identifiable shRNAmir. The library matrix was introduced into RE-luc2P-HEK293 GS-1101 price cells using a high-throughput transfection method: 100–200 ng shRNA plasmid DNA was incubated at RT for 20 min in 20 μl serum-free MEM containing 600 nl TransIT-Express reagent (MirusBio, Pittsburgh, PA) and transfected into 2×104 HEK293 cells in 100 μl DMEM/10% FBS. Approximately 30 h after transfection, culture media was replaced with DMEM/10% FBS containing 1 μg ml-1 puromycin. After 72 h of selection, during which >80% of the mock-transfected cells died, the selection media was removed, cells

were washed with PBS, and then re-suspended in 200 μl serum-free DMEM containing 1 μg ml-1 trypsin. The cell suspension (50 μl) was aliquoted to four white, clear bottom replica plates containing 50 μl DMEM/20% FBS. Cells were incubated 24h at 37°C prior to bacterial infection. For a more precise estimation of multiplicity of infection (MOI), one of the replica plates was used to calculate the number of host cells with the Cell Titer-Glo assay (Promega, Fitchburg, WI). A standard curve that correlates the ALUs to cell number (5000, 10000, 15000, 20000, 25000, and 30000 cells per well) was determined for every batch of substrate.

Two of the three remaining replica plates were infected with Y. enterocolitica WA at MOI 5 by addition of bacteria in 5 μl DMEM/10% FBS, followed by centrifugation selleck chemicals llc at 200 g for 5 min at RT. The remaining replica plate was used as a reference control (MOI 0). After 1h at 37°C, 20 μl DMEM/10% FBS containing 800 μg ml-1 of the bacteriostatic antibiotic chloramphenicol was added to each well in the plates to limit further Y. enterocolitica growth and to avoid activation of apoptotic pathways. Applying Cell Titer-Glo (Promega), we determined that the HEK293 cells infected with Y. enterocolitica at MOI 5 exhibited maximal inhibition of NF-κB-driven gene expression in response to TNF-α stimulation with no or minimal cellular toxicity. At 5 h post-infection, 25 μl DMEM/10% FBS containing

50 nM TNF-α was added to all culture plates. The screen was run once in duplicate plates. At 20h post-infection, the Cell Titer-Glo assay was used to normalize NF-κB-driven luciferase activity however to the cell titer. Arbitrary luciferase units (ALUs) were measured using the Synergy2 Multi-Mode Microplate Reader (BioTec, Winooski, VT). The relative percentage of NF-κB inhibition (R%I) by Yersinia infection was determined using the formula, R%I = [1-(ALU:MOI 5/ALU:MOI 0)]×100, where ALU:MOI 5 corresponds to the luciferase activity in bacteria-infected cells relative to ALU:MOI 0, the luciferase activity in no infection control. Hit selection criteria and validation assays Genes with at least two shRNAmir constructs that resulted in ≥40% (≥ 2 SD) decrease in R%I of NF-κB reporter gene activity were chosen for further validation.

The cells were pre-treated with lysozyme (0.7 mg ml−1 final concentration) and incubated at 37 °C for 30 min. DNase I was added to the cells prior to lysis and the pressing was conducted at a cell pressure of 2.9 MPa in an Aminco French pressure cell. The pressing was repeated for maximum lysis. The lysate was loaded onto a 15 %/40 % (wt/wt) sucrose step gradient and centrifuged in a Beckman Ti 45 rotor for 10 h at 57,000×g at 4 °C. The intracytoplasmic membrane fraction was harvested from the interface Dabrafenib clinical trial and further treated to concentrate the membranes by diluting out the sucrose with 10 mM HEPES pH 7.4 buffer and centrifuging in a Beckman

Ti 45 rotor for 2 h at 125,000×g at 4 °C. The membrane pellet was re-suspended in a small volume, typically 1 ml of 10 mM HEPES pH 7.4 buffer, and frozen at −20 °C for further use. The membrane pellet obtained from sucrose gradient centrifugation were solubilised with n-dodecyl-beta-D-maltoside (β-DDM, Glycon) at a final concentration of 59 mM, and a final OD of the membrane sample of ~60 at 875 nm. The mixture was stirred at 4 °C in the dark for 90 min. Non-solubilised click here material was removed by centrifugation (in a Beckman Ti 45 rotor for 2 h at 125,000×g), and the supernatant was loaded onto Chelating Sepharose

Fast Flow Ni–NTA column (GE Healthcare) equilibrated with 10 mM HEPES pH 7.4, 500 mM NaCl, 10 mM Imidazole, 0.59 mM β-DDM buffer. A gradient of 10–400 mM imidazole was applied and the main peak, which contains pure His12-RC-LH1-PufX, appeared when the concentration of imidazole reached ~300 mM. Eluted protein was concentrated (Vivaspin 500 spin-concentrator, Sartorius) and dialyzed against 10 mM HEPES pH 7.4, 50 mM NaCl, 0.59 mM β-DDM buffer. Then, the RC-His12-LH1-PufX protein was loaded onto a DEAE-Sepharose (Sigma) ion-exchange column equilibrated with 10 mM HEPES Smoothened pH 7.4, 50 mM NaCl, 0.59 mM β-DDM buffer. A gradient of 50–300 mM NaCl was applied with the main peak of pure protein appearing at NaCl concentration of ~280 mM. The best fractions

judged from the peak absorbance ratio of 875–280 nm were pooled (A 880/A 280 ~ 1.9). The protein was again concentrated and dialyzed against 10 mM HEPES pH 7.4, 50 mM NaCl, 0.59 mM β-DDM buffer and applied to a HPLC column (Phenomenex BioSep) and eluted at a flow rate of 0.3 ml min−1 in order to separate the monomeric and dimeric RC-His12-LH1-PufX complexes. The second elution peak (corresponding to the monomeric fraction of RC-His12-LH1-PufX) was collected, concentrated to a final concentration of 15 μM in 10 mM HEPES pH 7.4, 50 mM NaCl, 0.59 mM β-DDM buffer and stored at −80 °C for further use. Cyt c 2-His6 The gene encoding cyt c 2 was amplified from genomic DNA from Rba. sphaeroides 2.4.

Lastly, these PmBR zeocinR KanS SmR conjugants were screened by PCR using Platinum® Pfx DNA Polymerase (Invitrogen™) with the primers P7 (5′-TTG AGC ACG ACC AAC AGC AAC GTC-3′) and P8 (5′-CCA ATG CGG TCG AAT GAT TGC C-3′), which led to the identification of the mutant strain DD503.boaA. These primers yielded a PCR product of 1.3-kb in B. pseudomallei DD503 and a larger amplicon of 1.8-kb in the mutant. The primers P9 (5′-TAT CGC AAG GTT TGG AAC AAG GCG-3′) and P10 (5′-ACG CCG AAT ACC CTT GAT AGC TG-3′) were also used to further confirm gene replacement in the B. pseudomallei mutant strain. These primers amplified selleck inhibitor DNA fragments of 5-kb in the parent strain

DD503 and of 5.5-kb in the isogenic boaA mutant. After the conjugative transfer of plasmid pKASboaAZEO into the B. mallei strain

ATCC23344, colonies shown to be PmBR, zeocinR and KanS were screened by PCR with P7 and P8 as described above to identify the mutant strain ATCC23344.boaA. Of note, the boaA genes of both isogenic mutant strains DD503.boaA and ATCC23344.boaA were amplified and sequenced in their entirety to verify proper allelic exchange and successful disruption of boaA. Construction of a boaB B. pseudomallei isogenic mutant strain The plasmid pSLboaB was digested with NheI to remove a 162-bp fragment internal to the boaB ORF, treated with the End-It™ DNA End Repair Kit and ligated with the 0.45-kb zeocinR marker to yield the construct pSLboaBZEO. This plasmid was digested with BamHI and Nutlin-3 datasheet a 6.2-kb fragment, which corresponds to the boaB ORF disrupted with the zeocinR cassette, was purified from agarose gel slices, subcloned into the suicide plasmid pKAS46 and

introduced into B. pseudomallei DD503 by conjugation as described above. Conjugants shown to be PmBR zeocinR KanS SmR were screened by PCR using Platinum® Pfx DNA Polymerase (Invitrogen™) with primers P11 (5′-AGG TGG CGAC TCA AAT AGA ACC GT-3′) and P12 (5′-GTT CGT GTT GTT GGC TAC GGC AAT-3′) to identify the mutant strain DD503.boaB. These primers amplified a PCR product of 1.7-kb in B. pseudomallei DD503 and of 2.0-kb in the mutant. The primers P13 (5′-AGG TGG CGA CTC AAA TAG AAC CGT-3′) and P10 were also used to further confirm gene replacement in the B. pseudomallei mutant strain. Suplatast tosilate These primers generated amplicons of 5.2-kb and 5.5-kb in strains DD503 and DD503.boaB, respectively. Additionally, the boaB gene of DD503.boaB was amplified and both strands of the PCR product were sequenced to verify allelic exchange. Construction of a B. pseudomallei boaA boaB double mutant strain A 0.8-kb PCR product, which corresponds to a region located within the 5′end of the B. pseudomallei DD503 boaB ORF, was amplified with Platinum® Pfx DNA Polymerase (Invitrogen™) using primers P14 (5′-CTC GGG CTC AAT AAC ATG GC-3′) and P15 (5′-CGG AAT TCC GGT TCG TGT TGT TGG CT-3′; EcoRI site underlined).

Back in Germany in 1955, Menke resumed his studies on the chemical composition, structure and function of the photosynthetic apparatus, mainly chloroplasts. Having had already seen lamellar structures in chloroplasts from Nicotiana, Spinacia and Aspidistra in the laboratory of Manfred von Ardenne in 1940 (Menke 1940) and also in Anthoceros (Menke and Koydl 1939) before World War II, he finally understood the inner structure of the chloroplast as a system of stacked and unstacked

flattened vesicles surrounded this website by a membrane made of proteins and—besides pigments—lipids, mainly galactolipids, as A. Benson, J.F.G.M. Wintermans and R. Wiser were later able to demonstrate (1959). He called them thylakoids, a Greek term for “sac-like” δνλαχοειδής (Menke 1961). The original publication is in German (Menke 1961, translation in Gunning et al. 2006); however, many authors

cite his review in this context, namely the 1962 article in Annual Review of Plant Physiology (Menke 1962). Together with his research group, Menke made many efforts to elucidate the structure and chemical composition of chloroplasts. Thylakoids were investigated by means of small angle X-ray scattering (Kreutz and Menke 1960a, b). Pigments, lipids and proteins www.selleckchem.com/products/yap-tead-inhibitor-1-peptide-17.html were isolated from thylakoids (“lamellar systems”), separated from each other, quantified and eventually characterized in their localization and function by means of specific antisera (for literature which he himself considered worth citing, see Menke 1990). The introduction of immunological methods into botanical research was one of his important contributions Fossariinae (Berzborn et al. 1966). In 1972, Menke elegantly summarized the results of his efforts concerning the elucidation of chloroplast structure in an article in the annual report of the Max-Planck-Gesellschaft: “40 Jahre Versuche zur Aufklärung der molekularen Struktur der Chloroplasten” (Menke 1972). Over the years, several investigations on thylakoid membrane structure, using specific antibodies directed against different chloroplast components, have shown that the thylakoid membrane also has a “mosaic”

structure and is not made of two separate layers of protein (external) and lipids (internal), as was originally suggested by Menke (1966a, b). This was concluded from observations that certain components of the photosynthetic apparatus were accessible to antibodies from the stromal as well as from the luminal side of the thylakoid membrane (Koenig et al. 1977; Schmid et al. 1978). Spectroscopy was one of Menke’s scientific hobbies. Fork (1996) shows him together with C. Stacey French working with a derivative spectrophotometer, both smoking cigars. At the Botanical Institute of Cologne University and later at the Max-Planck-Institut für Züchtungsforschung in Cologne, we could always locate him by the smell of smoke from his cigar.

suis in accordance to results reported for S. aureus[15]. By this we identified persister cell formation in three different S. suis strains, suggesting that this phenomenon may be a general trait among this species. Though this has to be further confirmed by testing more

S. suis strains and antibiotics that are of higher clinical relevance to treat S. suis infections in pigs and humans, persister cells should be considered in the future in cases of ineffective antibiotic treatments or when studying antibiotic tolerance of S. suis. In line with several previous studies [3, 14, 22, 46] the number of persisters observed was higher during stationary growth of S. suis when compared to exponential grown bacteria. Type I persisters were found to be the main

source of antibiotic tolerance in our experiments. Among other stress signals, nutrient limitation in stationary growth is thought to be a trigger Tyrosine Kinase Inhibitor Library inducing down-regulation of the metabolic activity and bacterial dormancy in energy-deprived cells which can protect the bacteria from antibiotic Deforolimus concentration killing. We found some hints for involvement of the catabolic enzyme system ADS, since approximately two log-fold higher levels of persister cells were found in the exponential growth phase of an arginine deiminase knock-out strain (10ΔAD) as compared to its wild type strain. In S. suis the arginine deiminase system metabolizes arginine as a substrate to produce energy in form of ATP [38]. The diminished ATP levels

may lead to reduced general metabolic activity of strain 10ΔAD that might explain the slower growth rate (see Additional file 2: Figure S1) and enhanced number of antibiotic tolerant persister cells. Furthermore, the ccpA deficient strain exhibited lower numbers of persister cells in the stationary growth phase when compared to the wild type. This is in agreement with studies in S. gordonii showing that a ccpA knock-out resulted in an increased sensitivity of the bacteria to penicillin treatment [47]. Since CcpA is a pleiotropic regulator that is important for a balanced metabolic flux in the central carbon metabolism, the alteration of central metabolic processes may influence persister cell formation of S. suis. Accordingly, an interplay between carbohydrate consumption and formation of persisters has recently been demonstrated for E. coli[12]. Methisazone Further studies are needed to clarify the mechanisms involved in CcpA and/or arginine deiminase dependent changes in antibiotic tolerance of S. suis. When using antibiotics with varying modes of action, resulting killing profiles were quite different, ranging from pronounced biphasic killing patterns to nearly plane curves, at least for exponential grown S. suis. These findings seem to be highly dependent on the type of antibiotic used, which is also emphasized by the fact that treatment with the β-lactam antibiotics amoxicillin and penicillin resulted in similar killing curves.

sYJ20 was previously identified by Vogel et al. in E. coli as SroA [5], encoded by a sequence downstream of yabN (encoding SgrR, a transcriptional regulator in E. coli[33]) and upstream of tbpA (encoding the thiamine-binding check details periplasmic protein, homologous to thiB in E. coli) (Figures 2C (ii) and 5A). Figure 5 The chromosomal location of the sYJ20 (SroA) encoding region and its encoding sequence. sYJ20 is encoded upstream of the tbpA-yabK-yabJ operon, and the shared

TSS of sYJ20 and tbpA as determined by 5’ RACE analysis is represented by the dark-black arrow. The DNA sequence of sYJ20 (SroA) is shown in bold letters, which is also the region that was deleted in YJ104 and used for TargetRNA prediction (Table 1). The THI-box sequence is underlined. The start codon of tbpA is displayed at larger size as GTG, where the first G is considered +1 in the numbering system. sYJ5, sYJ20 (SroA) and sYJ118 are all highly conserved within the different members of Enterobacteriaceae, although the coding sequences of sYJ5, sYJ20 and sYJ118 are also found in other families of bacteria (such as sYJ5 and sYJ118 in Prevotella ruminicola,

sYJ20 in Marinobacter aquaeolei VT8), in plants (such as sYJ20 and sYJ118 in Zea mays cultivar line T63) and in animals (sYJ118 in Gryllus bimaculatus). In contrast, sYJ75 is only found in Salmonella, Enterobacter, Photorhabdus and Citrobacter. sYJ20 (SroA), sYJ5, sYJ75 and sYJ118 in other species and relevance to other drug classes We proceeded PF-562271 in vitro to determine whether the increased expression of these sRNAs would be Salmonella specific or drug-class specific. Hence, we assessed the levels of our sRNA candidates (sYJ5, sYJ20 and sYJ118) in other members of Enterobacteriaceae (Klebsiella pneumoniae and Escherichia coli) when challenged with sub-inhibitory Atorvastatin levels of tigecycline (sYJ75 was not included since it is

not encoded in the tested species). Additionally, in order to determine whether these sRNAs are upregulated solely as a result of tigecycline challenge or are generally upregulated as a result of sub-inhibitory antibiotic challenge, S. Typhimurium SL1344 was challenged with either half the MIC of ampicillin (1 μg/ml) or ciprofloxacin (0.0156 μg/ml). As shown in Figure 3B, none of the four tested sRNAs were upregulated in response to ciprofloxacin exposure, whilst three (sYJ5, sYJ75 and sYJ118) were found to be upregulated in the presence of ampicillin. Interestingly, E. coli did not upregulate the expression of the three candidate sRNAs (sYJ5, sYJ20 and sYJ118) in response to challenge at half the MIC of tigecycline. However, sYJ118 exhibited an elevated level of expression in K. pneumoniae in the presence of tigecycline (Figure 3B). Of note, although the sYJ20 (SroA) coding sequence is present in K. pneumoniae, two transcripts were detected after hybridisation.

Pflugers Archive 1978, 376:55–65.CrossRef 10. Fabiato A, Fabiato F: Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. J Physiol 1978, FDA approved Drug Library cost 276:233–235.PubMed 11. Mannion AF, Jakeman PM, Dunnett M, Harris RC, Willian PL: Carnosine and anserine concentrations in the quadriceptsfemoris muscle of healthy humans. Eur J Appl Physiol 1992, 64:47–50.CrossRef 12. Bate-Smith EC: The buffering of muscle in rigour: protein, phosphate and carnosine. J Physiol 1938, 92:336–343. 13. Harris RC, Marlin DJ, Dunnett M, Snow DH, Hultman E: Muscle buffering capacity and dipeptide content in the thoroughbred horse, greyhound dog and man.

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pseudotuberculosis. As G. mellonella possesses an innate immune system with structural and functional similarities to the mammalian innate immune system, it is a useful alternative to the traditional murine yersiniosis infection model, to examine virulence in vivo,

especially as unlike the C. elegans model, G. mellonella can be incubated at 37°C [42, 54]. Previous studies with Y. pseudotuberculosis comparing G. mellonella and the murine model, showed that G. mellonella could reflect infection in mammals and therefore could be useful as a check details higher throughput screen of mutants, before a more in depth analysis was undertaken in the murine model [42]. In this study the G. mellonella model demonstrated a role for Ifp in the pathogenesis of Y. pseudotuberculosis, in particular in concert with invasin, as the double mutant showed a significant increase in survival compared to the wild type (Figure 7). There also appeared to be mild attenuation in virulence

with both of the single mutants. This suggests that Ifp, together with invasin, does have a role in virulence of Y. pseudotuberculosis in this infection model. Conclusions We have shown the presence of a novel functional adhesin in Y. pseudotuberculosis that has been mutated with an IS element and is presumably non-functional in Y. pestis. Ifp is expressed during late log to early stationary phase at 37°C and demonstrates an ability to bind to HEp-2 cells in vitro, which can be disrupted by mutation of the gene, or even a single cysteine residue. Together

with invasin and intimin, Ifp is a new member of a family of outer membrane adhesins that is activated at 37°C and may act at a later stage than invasin during infection. Selleckchem MK-2206 Acknowledgements We are grateful to G. Frankel, Imperial College, London, UK for the intimin advice; E. Carniel, Institut Pasteur, Paris, France for the Y. pseudotuberculosis strain IP32953 and the pKOBEG vector; A. Darwin, NYU School of Medicine, New York, USA for the pAJD434 plasmid; and R. Isberg, Tufts University, Boston, USA Methocarbamol for the gift of the anti-invasin monoclonal antibody. We thank DSTL for financial support for this project. Electronic supplementary material Additional file 1: Amino acid alignment of Ifp from the four currently sequenced genomes of Y. pseudotuberculosis. Utilising the ClustalW program, the amino acid sequences of Y. pseudotuberculosis strains IP32953, IP31758, PB1 and YPIII were aligned. (DOC 38 KB) Additional file 2: Growth curves from the temporal expression of Ifp and invasin assay. Within the Anthos Lucy1 combined photometer and luminometer, OD readings at 600 nm were taken at 30 minute intervals and used to construct these growth curves. Cultures were incubated at (A) 24°C (B) 28°C and (C) 37°C. (PPT 96 KB) References 1. Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A, Carniel E: Yersinia pestis , the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis . Proc Natl Acad Sci USA 1999,96(24):14043–14048.

: A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium. Cell 2009,139(7):1268–1278.PubMedCrossRef 17. Pfarr K, Hoerauf A: The annotated genome of Wolbachia from the filarial nematode Brugia malayi: what it means for progress in antifilarial medicine. PLoS Med 2005,2(4):e110.PubMedCrossRef 18. Zabalou S, Riegler M, Theodorakopoulou M, Stauffer C, Savakis C, Bourtzis K: Wolbachia -induced cytoplasmic incompatibility as a means for insect pest population control. Proc Natl Acad Sci U S A 2004,101(42):15042–15045.PubMedCrossRef 19. Beard CB, Durvasula RV, Richards FF: Bacterial symbiosis in arthropods

and the control of disease transmission. Emerg Infect Dis 1998,4(4):581–591.PubMedCrossRef 20. McMeniman CJ, Lane RV, Cass BN, Fong AW, Sidhu M, Wang YF, O’Neill SL: Stable introduction of a life-shortening Wolbachia infection into the mosquito Aedes aegypti. Science find more 2009,323(5910):141–144.PubMedCrossRef 21. Xi Z, Khoo CC, Dobson SL: Wolbachia establishment and invasion in an Aedes aegypti laboratory population. Science 2005,310(5746):326–328.PubMedCrossRef 22. Bourtzis K: Wolbachia -based technologies for insect pest population control. Adv Exp Med Biol 2008, 627:104–113.PubMedCrossRef 23. Welburn SC, Fevre EM, Coleman PG, Odiit M, Maudlin I: Sleeping sickness: a tale of two diseases.

Trends Parasitol 2001,17(1):19–24.PubMedCrossRef Selleckchem AUY-922 24. Cattand P: The scourge of human African trypanosomiasis. Afr Health 1995,17(5):9–11.PubMed 25. Kioy D, Jannin J, Mattock N: Human African trypanosomiasis. Nat Rev Microbiol 2004,2(3):186–187.PubMedCrossRef 26. Simarro PP, Diarra A, Ruiz Postigo JA, Franco JR, Jannin JG: The human African trypanosomiasis control

and surveillance programme of the world health organization 2000–2009: the way forward. PLoS Negl Trop Dis 2011,5(2):e1007.PubMedCrossRef 27. Aksoy S: Sleeping sickness elimination in sight: time to celebrate and reflect, but not relax. PLoS Negl Trop Dis 2011,5(2):e1008.PubMedCrossRef 28. Zabalou S, Apostolaki A, Livadaras I, Franz G, Robinson AS, Savakis C, Bourtzis K: Incompatible insect technique: incompatible males from a Ceratitis capitata genetic Sucrase sexing strain. Entomologia Experimentalis Et Applicata 2009,132(3):232–240.CrossRef 29. Bourtzis K, Robinson AS: Insect pest control using Wolbachia and/or radiation. In Insect Symbiosis 2. Edited by: Bourtzis K, Miller TA. Florida, USA: CRC Press, Talylor and Francis Group, LLC; 2006:225–246.CrossRef 30. Apostolaki A, Saridaki A, Livadaras I, Savakis C, Bourtzis K: Transinfection of the olive fruit fly with a Wolbachia CI inducing strain: a promising symbiont-based population control strategy? Journal of Applied Entomology 2011. 10.1111/j.1439–0418.2011.01614.x 31. Cheng Q, Aksoy S: Tissue tropism, transmission and expression of foreign genes in vivo in midgut symbionts of tsetse flies. Insect Mol Biol 1999,8(1):125–132.PubMedCrossRef 32.

05, ** P < 0.001. Significance of each transition was determined using Fisher’s sign tests Afforestation: grassland to plantation and shrubland to plantation Afforestation of natural grasslands and shrublands resulted in a decrease in species richness and

diversity in all but two cases. The two cases where species richness increased were both in the shrubland to plantation category and were from a publication on the effects of afforestation of a highly endemic but naturally species poor ultramafic grassland in Italy (Chiarucci Selleck BMN-673 and Dedominicis 1995). While overall species richness increased with plantation establishment, specialist and endemic species richness decreased; as such, the increase in total species richness can be attributed to the expansion

of generalist or exotic species (Chiarucci and Dedominicis 1995). Although few afforestation publications reported this measure, those that did reported a decline in richness of narrow/endemic/specialist species (species noted by author as restricted to a particular habitat type), with an average decrease of 47% (±15%) in the three grassland cases where it was reported and 38% (±11; n = 4; P < 0.05) Hedgehog antagonist in shrubland afforestation (Table 1). Exotic species richness was unaffected in one grassland to plantation case (Cremene et al. 2005) while in the other case reported it increased by 470% (O’Connor 2005). Exotic species richness increased in all five shrubland to plantation cases (mean = 36%), but not significantly so (P = 0.41; Fig. 3). Native species richness,

in contrast, decreased in all afforestation cases and significantly so in the shrubland to plantation category (55% decrease, n = 4, P < 0.05; Table 1). Fig. 3 Change in exotic species richness with plantation establishment by category of land use change. *P < 0.05. •Boxplot outliers Primary forest and secondary forest to plantation Monoiodotyrosine Species richness was lower in plantations than in primary forest in 24 of 27 cases with a mean decrease of 35% across all observations (Table 1; Fig. 2; P < 0.001). Eight of the 27 cases were direct comparisons, meaning that plantations replaced natural forests, while 19 of the cases involved an intermediate land use whereby plantations were established on previously deforested land that had been used for another purpose, most often grazing (Appendix 1 includes details on the intermediate land use for each case). Overall, plantations replacing primary forests were 39% (±8%) less species rich than paired primary forest (P < 0.05), while those with an intermediate land use were 33% (±8%) less species rich than paired primary forests (P < 0.01). Likewise, native species richness significantly decreased by 65% (±10%) (P < 0.05) in the five cases reported in the primary forest.